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Research  in  Progress 

in  the  Howard  Hughes  Medical  Institute,  1992-1993 


Founded  in  1953  and  incorporated  in  Delaware,  the  Howard  Hughes  Medical  Institute  is  a  nonprofit  scientific 
and  philanthropic  organization.  It  is  dedicated  to  the  promotion  of  human  knowledge  within  the  basic  sciences, 
principally  medical  research  and  medical  education,  and  the  use  of  such  knowledge  for  the  benefit  of  humanity. 
The  Institute  is  directly  engaged  in  medical  research  and  is  qualified  as  a  medical  research  organization  under  the 
U.S.  tax  code.  It  is  an  equal  opportunity  employer. 


Howatxl  Hughes  Medical  Institute 
6701  Rockledge  Drive 
Bethesda,  Maryland  20817 
(301)  571-0200 


After  February  8,  1993: 

Howard  Hughes  Medical  Institute 

4000  Jones  Bridge  Road 

Chevy  Chase,  Maryland  20815-6789 

(301)  215-5000 


Developing  eye  of  the  common  fruit  fly  Drosophila  meianogaster,  as  imaged  by  a  computer-based 
confocal  microscope.  Red  and  yellow  represent  the  highest  concentrations  of  Notch,  a  cell-surface 
protein  that  is  necessary  for  the  cell-to-cell  interactions  that  decide  the  developmental  fate  of  each 
cell  within  the  eye. 

From  Pehon,  R.G.,  Johansen,  K.,  Rebay,  I.,  and  Artavanis-Tsakonas,  S.  1991.  J  Cell  Biol 
1 13:657-669.  Copyright  permission  by  the  Rockefeller  University  Press. 


Copyright  ©  1992  by  the  Howard  Hughes  Medical  Institute 

Ail  rights  reserved.  Address  correspondence  to 

Dr.  W.  Maxwell  Cowan 

Vice  President  and  Chief  Scientific  Officer 

Howard  Hughes  Medical  Institute 

Printed  in  the  United  States  of  America 


Howard  Hughes  Medical  Institute 


Research 
in  Progress 
1992-1993 


Contents 


xix  Foreword 

Purnell  W.  Choppin,  M.D. 

xxi  Trustees,  Officers,  and  Principal  Staff  Members 

xxiii  Medical  Advisory  Board 

xxiv  Scientific  Review  Board 

xxvi  Locations  of  Howard  Hughes  Medical  Institute  Laboratories 

xxix  Introduction 

xxix  Cell  Biology  and  Regulation  Program 

XXXix  Genetics  Program 

xlvi  Immunology  Program 

liv  Neuroscience  Program 

Ixi  Structural  Biology  Program 

W.  Maxwell  Cowan,  M.D.,  Ph.D. 

1     Electrical  Activity  of  Nerve  Cells 
Paul  R.  Adams,  Ph.D. 

3      Three-Dimensional  Macromolecular  and  Cellular  Structure 

David  A.  Agard,  Ph.D. 

5     Molecular  Mechanisms  of  Ion  Channel  Function 
Richard  W.  Aldrich,  Ph.D. 

7     Divergent  Members  of  the  SRY  Family  of  Transcriptional  Regulators 
Bind  an  Insulin-Responsive  Element,  IRE-A 

Maria  C.  Alexander-Bridges,  M.D.,  Ph.D. 

9     Genetic  Mechanisms  Involved  in  the  Generation  of  the 
Antibody  Repertoire 

Frederick  W.  Alt,  Ph.D. 

11      Control  of  Cell  Fate  During  Vertebrate  Neuronal  Development 

David  J.  Anderson,  Ph.D. 

13      Cell  Fate  Choices  in  the  Nervous  System  and  Elsewhere 

Spyridon  Artavanis-Tsakonas,  Ph.D. 

15     The  Complement  System 

John  P.  Atkinson,  M.D. 

1 7     The  Molecular  Biology  of  Smell 

Richard  Axel,  M.D. 

19     Mammalian  Developmental  Genetics 

Gregory  S.  Barsh,  M.D.,  Ph.D. 

21      Cell  Cycle  Control 

David  H.  Beach,  Ph.D. 


Hi 


Contents 


23      Genetic  Control  of  Morphogenesis  in  Drosophila 

Philip  A.  Beachy,  Ph.D. 

25     Molecular  Studies  of  Human  Genetic  Disease 

Arthur  L.  Beaudet,  M.D. 

27     Molecular  Genetics  of  Diabetes  Mellitus 

Graeme  I.  Bell,  Ph.D. 

29     Development  of  the  Drosophila  Peripheral  Nervous  System 
Hugo  J.  Bellen,  D.V.M.,  Ph.D. 

31      Genetic  Manipulation  of  Hematopoietic  Stem  Cells 

John  W.  Belmont,  M.D.,  Ph.D. 

33     Proteins  of  the  Spectrin- based  Membrane  Skeleton 

G.  Vann  Bennett,  M.D.,  Ph.D. 

35      TNF  and  the  Molecular  Pathogenesis  of  Shock 

Bruce  A.  Beutler,  M.D. 

37     Cytotoxic  T Lymphocyte  Recognition 

Michael  J.  Bevan,  Ph.D. 

39     Vascular  Endothelium  in  Inflammation  and  Metastasis 

Michael  P.  Bevilacqua,  M.D.,  Ph.D. 

41     Structural  Studies  of  Molecules  Involved  in  the  Immune  Recognition 
of  Infected  Cells 

Pamela  J.  Bjorkman,  Ph.D. 

43     Mechanisms  of  Insulin  Action 

Perry  J.  Blackshear,  M.D.,  D.Phil. 

45     Intracellular  Protein  Traffic  and  Nuclear  Organelles 
Giinter  Blobel,  M.D.,  Ph.D. 

47     Immunity  and  Pathogenesis  of  Third  World  Diseases:  Leprosy 
and  Tuberculosis 

Barry  R.  Bloom,  Ph.D. 

49     Molecular  Biology  of  the  Extracellular  Matrix 

Jeffrey  F.  Bonadio,  M.D. 

51     Functional  Heterogeneity  in  CD4-bearing  T  Lymphocytes 

H.  Kim  Bottomly,  Ph.D. 

53     Retroviral  Replication 

Patrick  O.  Brown,  M.D.,  Ph.D. 

55     Regulation  of  Cellular  Processes  by  Protein- Tyrosine  Phosphorylation 

Joan  S.  Brugge,  Ph.D. 


iv 


Contents 


57     Computational  Structural  Biology 

Axel  T.  Briinger,  Ph.D. 

59     Biophysical  Studies  of  Eukaryotic  Gene  Regulation  and 
Molecular  Recognition 

Stephen  K.  Burley,  M.D.,  D.Phil. 

61     Molecular  Studies  of  Calcium  Channels  and  the  Dystrophin- 
Glycoprotein  Complex 
Kevin  P.  Campbell,  Ph.D. 

63      Gene  Targeting 

Mario  R.  Capecchi,  Ph.D. 

65     Genetic  Control  of  Pattern  Formation  in  Drosophila 

Sean  B.  Carroll,  Ph.D. 

69     Human  Disease  Gene  Identification  and  Correction 

C.  Thomas  Caskey,  M.D. 

71     Enzymatic  RNA  Molecules  and  the  Structure  of  Chromosome  Ends 

Thomas  R.  Cech,  Ph.D. 

73     Molecular  and  Cellular  Physiology  of  Acute  Inflammatory  Cytokines 
David  D.  Chaplin,  M.D.,  Ph.D. 

75     Hormonal  Regulation  of  Gene  Expression 
William  W.  Chin,  M.D. 

77     Technologies  for  Genome-sequencing  Projects 

George  M.  Church,  Ph.D. 

79     Molecular  Genetics  of  Limb  Development  in  Drosophila 

Stephen  M.  Cohen,  Ph.D. 

81      Tracking  Genes  That  Cause  Human  Disease 

Francis  S.  Collins,  M.D.,  Ph.D. 

83     Development  of  the  Immune  System 

Max  D.  Cooper,  M.D. 

85     Structure  and  Function  of  RNA  Polymerase  II 

Jeffry  L.  Corden,  Ph.D. 

87     Mechanically  Activated  Ion  Channels 

David  P.  Corey,  Ph.D. 

89     Genetic  Regulatory  Mechanisms  in  Cellular  Differentiation 

Gerald  R.  Crabtree,  M.D. 

91      The  Mechanism  of  a  Bacterial  Transposition  Reaction 

Nancy  L.  Craig,  Ph.D. 


V 


Contents 


93     Mechanisms  of  Antigen  Processing 

Peter  Cresswell,  Ph.D. 

95     Regulation  of  Human  Retroviral  Gene  Expression 
Bryan  R.  Cullen,  Ph.D. 

"97     Mechanism  of  Retrovirus  Infection 

James  M.  Cunningham,  M.D. 

99      The  Nuclear  Pore  Complex 

Laura  I.  Davis,  Ph.D. 

1 01     Molecular  Approaches  to  T  Lymphocyte  Recognition  and  Differentiation 

Mark  M.  Davis,  Ph.D. 

103     Signal  Transduction  by  the  Epidermal  Growth  Factor  Receptor 

Roger  J.  Davis,  Ph.D. 

105      Traffic  of  Synaptic  Vesicle  Proteins  in  Neurons  and  Endocrine  Cells 

Pietro  De  Camilli,  M.D. 

107     Three-Dimensional  Structures  of  Biological  Macromolecules 

Johann  Deisenhofer,  Ph.D. 

109     Molecular  Mechanisms  of  Lymphocyte  Differentiation 

Stephen  V.  Desiderio,  M.D.,  Ph.D. 

113      Transcription  Control  During  Early  Drosophila  Development 

Claude  Desplan,  Ph.D. 

115     Immune  Evasion  by  Parasites  Causing  Tropical  Diseases 
John  E.  Donelson,  Ph.D. 

11 7     Post-transcriptional  Regulation  of  Gene  Expression,  RNA-Protein 
Complexes,  and  Nuclear  Structures 

Gideon  Dreyfuss,  Ph.D. 

119     Genetic  Basis  of  Hearing  Loss 

Geoffrey  M.  Duyk,  M.D.,  Ph.D. 

121     Molecular  Genetics  of  Intracellular  Protein  Sorting 

Scott  D.  Emr,  Ph.D. 

123     Mechanisms  Involved  in  Preventing  Unwanted  Blood  Clots 

Charles  T.  Esmon,  Ph.D. 

125     Molecular  Genetics  of  Steroid  and  Thyroid  Hormone  Receptors 

Ronald  M.  Evans,  Ph.D. 

129     Molecular  Mechanisms  Involved  in  the  Actions  of  Calcium-mediated 
Hormones 

John  H.  Exton,  M.D.,  Ph.D. 


Contents 


131      Tumor-Suppressor  Genes 

Andrew  P.  Feinberg,  M.D.,  M.P.H. 

133     Genetics,  Structure,  and  Function  of  Histocompatibility  Antigens 

Kirsten  Fischer  Lindahl,  Ph.D. 

137     Genetic  Approaches  to  Immune  Function  and  Tolerance 

Richard  A.  Flavell,  Ph.D. 

139     Biophysical  Genetics  of  Protein  Structure  and  Folding 

Robert  O.  Fox,  Ph.D. 

141     Molecular  Basis  of  Genetic  Diseases  and  Chromosome  Mapping 

Uta  Francke,  M.D. 

143     Molecular  Biology  of  Obesity  and  Diabetes 

Jeffrey  M.  Friedman,  M.D.,  Ph.D. 

147     Regulation  of  Keratin  Expression  During  Differentiation  and 
Development  in  Human  Skin 

Elaine  Fuchs,  Ph.D. 

149      The  Molecular  Basis  of  Viral  Replication  and  Pathogenesis 

Donald  E.  Ganem,  M.D. 

151      Second  Messengers  and  Cell  Regulation 

David  L.  Garbers,  Ph.D. 

153     Molecular  Genetics  of  the  Major  Histocompatibility  Complex 

Jan  Geliebter,  Ph.D. 

155     The  Decoding  Code  in  mRNA 

Raymond  F.  Gesteland,  Ph.D. 

157     Molecular  Analysis  of  Proteins  Involved  in  Human  Disease 
Mary-Jane  H.  Gething,  Ph.D. 

159     Signal  Transduction  Pathways  in  B  lymphocytes 

Sankar  Ghosh,  Ph.D. 

161     Molecular  Genetics  of  Blood  Coagulation 

David  Ginsburg,  M.D. 

163      Uncovering  the  Molecular  Basis  of  X-linked  Disorders 

Jane  M.  Gitschier,  Ph.D. 

165     Membrane  Lipids  and  Cell  Regulation 

John  A.  Glomset,  M.D. 

167     Determination  and  Maintenance  of  Cell  Type 

Richard  H.  Gomer,  Ph.D. 

169     Growth  Cone  Guidance  and  Neuronal  Recognition  in  Drosophila 

Corey  S.  Goodman,  Ph.D. 


vii 


Contents 


1 71     Mechanisms  of  Immunological  Self-Tolerance  and  Autoimmunity 

Christopher  C.  Goodnow,  B.V.Sc,  Ph.D. 

1 73     Developmental  Control  of  Gene  Expression 
Rudolf  Grosschedl,  Ph.D. 

175     Polypeptide  Hormone  Gene  Regulation 

Joel  F.  Habener,  M.D. 

1 77     Structural  Studies  of  Macromolecular  Assemblies 

Stephen  C.  Harrison,  Ph.D. 

1 79      Control  of  Gene  Expression  During  the  Cell  Cycle  and  Development 
of  the  Mammalian  Cerebellum 

Nathaniel  Heintz,  Ph.D. 

181      Structural  Biology  of  CD4  and  CDS  Involvement  in  the  Cellular 
Immune  Response 

Wayne  A.  Hendrickson,  Ph.D. 

183      Variegated  Position  Effects  in  Drosophila 

Steven  Henikoff,  Ph.D. 

185     Biological  Roles  and  Expression  of  Complement  Receptors 

V.  Michael  Holers,  M.D. 

187     Genetic  Control  of  Nematode  Development 

H.  Robert  Horvitz,  Ph.D. 

189     Protein  Folding  in  Vivo 

Arthur  L.  Horwich,  M.D. 

191     Molecular  Mechanisms  in  the  Regulation  of  Synaptic  Transmission 

Richard  L.  Huganir,  Ph.D. 

193     Molecular  Aspects  of  Signal  Transduction  in  the  Visual  System 

James  B.  Hurley,  Ph.D. 

195      The  Molecular  Basis  of  Cell  Adhesion  in  Normal  and 
Pathological  Situations 

Richard  O.  Hynes,  Ph.D. 

197     Molecular  Genetics  of  Intracellular  Microorganisms 

Ralph  R.  Isberg,  Ph.D. 

201      Genetic  Approaches  to  the  Control  of  Mycobacterial  Diseases 

William  R.  Jacobs,  Jr.,  Ph.D. 

203     Mechanisms  of  Neurotransmitter  Storage  and  Release 
Reinhard  Jahn,  Ph.D. 

205     Molecular  Studies  of  Voltage-Sensitive  Potassium  Channels 
Lily  Y.Jan,  Ph.D. 


via 


Contents 


207     Neural  Development  in  Drosophila 

Yuh  Nungjan,  Ph.D. 

205^     Activation  ofCD4  T  Cells 

Charles  A.  Janeway,  Jr.,  M.D. 

211      Control  of  Cell  Pattern  in  the  Developing  Nervous  System 
Thomas  M.  Jessell,  Ph.D. 

213     Energy-transducing  Membrane  Proteins 

H.  Ronald  Kaback,  M.D. 

215     Control  of  the  Immunoglobulin  Heavy-Chain  Gene 
Thomas  R.  Kadesch,  Ph.D. 

21 7     Genetic  Control  of  Hemoglobin  Synthesis 
Yuet  Wai  Kan,  M.D.,  D.Sc. 

219     Cell  Biological  Studies  of  Memory 
Eric  R.  Kandel,  M.D. 

221      The  T  Cell  Repertoire 

John  W.  Kappler,  Ph.D. 

223      The  Genetic  Control  of  Morphogenesis 

Thomas  C.  Kaufman,  Ph.D. 

225     Protein  Folding  and  Macromolecular  Recognition 

Peter  S.  Kim,  Ph.D. 

227     RNA  Viral  Genetics 

Karla  A.  Kirkegaard,  Ph.D. 

229     Adrenergic  Receptor  Structure  and  Function 
Brian  K.  Kobilka,  M.D. 

231     Molecular  Genetics  of  Lymphocyte  Development  and  Neoplasia 

Stanley  J.  Korsmeyer,  M.D. 

233     Molecular  Genetics  of  Neuromuscular  Disease 
Louis  M.  Kunkel,  Ph.D. 

235     Structural  Studies  on  DNA-Replication  Enzymes,  src -related  Oncogene 
Products,  and  Oxidoreductases 

John  Kuriyan,  Ph.D. 

237     Molecular  Analysis  of  Down  Syndrome 
David  M.  Kurnit,  M.D.,  Ph.D. 

239     Replication  and  Pathogenesis  of  RNA  Viruses 

Michael  M.-C.  Lai,  M.D.,  Ph.D. 

241     Molecular  Biology  of  Human  Papillomaviruses 

Laimonis  A.  Laimins,  Ph.D. 


ix 


Contents 


243      Genetic  Studies  in  Cardiovascular  Disease 

Jean-Marc  Lalouel,  M.D.,  D.Sc. 

245     Structure  and  Replication  of  Influenza  Virus  and  Paramyxoviruses 

Robert  A.  Lamb,  Ph.D.,  Sc.D. 

2?7     Cancer  and  Genetic  Modification 
Philip  Leder,  M.D. 

249     From  Molecular  Biology  to  Therapy  of  Human  Disease 

Fred  D.  Ledley,  M.D. 

251     Molecular  Biology  of  Hormone  and  Drug  Receptors  in  Health 
and  Disease 

Robert  J.  Lefkowitz,  M.D. 

253     Axis  Formation  and  Germline  Determination  in  Drosophila 

Ruth  Lehmann,  Ph.D. 

255     Regulation  of  Gene  Expression  During  Cellular  Differentiation 
and  Activation 

Jeffrey  M.  Leiden,  M.D.,  Ph.D. 

257     Chemical  Communication 

Michael  R.  Lerner,  M.D.,  Ph.D. 

259     Structural  Determinants  of  Human  a-Globin  Gene  Expression 

Stephen  A.  Liebhaber,  M.D. 

261      The  Heat-Shock  Response 
Susan  L.  Lindquist,  Ph.D. 

263      T  Cell  Surface  Glycoproteins  in  Development  and  Viral  Infections 

Dan  R.  Littman,  M.D.,  Ph.D. 

265     The  Biology  of  T  Lymphocyte  Development 

Dennis  Y.-D.  Loh,  M.D. 

267     Molecular  Genetics  of  Mammalian  Glycosyltransferases 

John  B.  Lowe,  M.D. 

269     Mechanisms  of  Embryonic  Induction  in  Vertebrates 

Richard  L.  Maas,  M.D.,  Ph.D. 

271      Cell  Cycle  Control 

James  L.  Mailer,  Ph.D. 

273      The  Role  of  T  Cells  in  Health  and  Sickness 
Philippa  Marrack,  Ph.D. 

275     Cell  Regulation  by  Transforming  Growth  Factors 
Joan  Massague,  Ph.D. 


Contents 


277     Structural  Basis  of  Interactions  Within  and  Between  Macromolecules 
Brian  W.  Matthews,  Ph.D.,  D.Sc. 

279     What  Viruses  Are  Telling  Us  About  Gene  Regulation  in  Mammalian  Cells 

Steven  Lanier  McKnight,  Ph.D. 

281     Fundamental  Mechanisms  of  Ion  Channel  Proteins 
Christopher  Miller,  Ph.D. 

283     Neural  Foundations  of  Vision 

J.  Anthony  Movshon,  Ph.D. 

285     Human  Retroviral  Gene  Expression  and  Cellular  Transcription 
Gary  J.  Nabel,  M.D.,  Ph.D. 

287     Molecular  Analysis  of  Muscle  Development  and  Function 
Bernardo  Nadal-Ginard,  M.D.,  Ph.D. 

289      The  Genomic  Response  to  Growth  Factors 

Daniel  Nathans,  M.D. 

291     Molecular  Biology  of  Visual  Pigments 

Jeremy  Nathans,  M.D.,  Ph.D. 

293      Gene  Regulation  in  Animal  Cells 

Joseph  R.  Nevins,  Ph.D. 

295     Molecular  Genetics  of  X-linked  Disease 
Robert  L.  Nussbaum,  M.D. 

297     Function  of  Oncogenes  in  Early  Embryogenesis 

Roel  Nusse,  Ph.D. 

25^5^     Molecular  Mechanisms  That  Regulate  B  Cell  Development 

Michel  C.  Nussenzweig,  M.D.,  Ph.D. 

301     Mechanism  of  DMA  Replication 

Michael  E.  O'Donnell,  Ph.D. 

303     Large-Scale  Analysis  of  Yeast  and  Human  DNA 

Maynard  V.  Olson,  Ph.D. 

305     Molecular  Genetic  Studies  of  Hematopoietic  Cells 
Stuart  H.  Orkin,  M.D. 

307     Albinism  and  Tyrosinase 
Paul  A.  Overbeek,  Ph.D. 

309     Structural  Studies  of  DNA-binding  Proteins 
Carl  O.  Pabo,  Ph.D. 

313      The  X  and  Y  Chromosomes  in  Mammalian  Development 

David  C.  Page,  M.D. 


xi 


Contents 


315     Mammalian  Development  and  Disease 

Richard  D.  Palmiter,  Ph.D. 

31 7     Regulation  of  Gene  Expression  in  Steroid  Hormone  Biosynthesis 

Keith  L.  Parker,  M.D.,  Ph.D. 

319     Molecular  Neuroimmunology 

Donald  G.  Payan,  M.D. 

321     Molecular  Basis  of  Lymphocyte  Signaling 

Roger  M.  Perlmutter,  M.D.,  Ph.D. 

323     Genetic  Dissection  of  a  Signal  Transduction  Pathway  in 
Drosophila  melanogaster 

Norbert  Perrimon,  Ph.D. 

325     Gene  Regulation  and  Immunodeficiency 

B.  Matija  Peterlin,  M.D. 

327     Mechanism  of  Action  of  Polypeptide  Growth  Factors 
Linda  J.  Pike,  Ph.D. 

329     Protein  Structures,  Molecular  Recognitions,  and  Functions 

Florante  A.  Quiocho,  Ph.D. 

331     Molecular  Approaches  to  Olfaction 

Randall  R.  Reed,  Ph.D. 

333      The  Molecular  Basis  of  Hereditary  Diseases  of  the  Kidney 

Stephen  T.  Reeders,  M.D. 

335     Extracellular  Factors  Affecting  Neuron  Development 

Louis  F.  Reichardt,  Ph.D. 

33  7     Molecular  Genetics  of  RNA  Processing  and  Behavior 

Michael  Rosbash,  Ph.D. 

339     Molecular  Mechanisms  of  Transcription,  Regulation,  and  Development 
of  the  Neuroendocrine  System 

Michael  G.  Rosenfeld,  M.D. 

341     Development  of  the  Drosophila  Visual  System 

Gerald  M.  Rubin,  Ph.D. 

345      The  Regulation  of  Blood  Coagulation 

J.  Evan  Sadler,  M.D.,  Ph.D. 

347     Molecular  Mechanism  of  Transmembrane  Signal  Transduction  by 
G  Protein-coupled  Receptors 
Thomas  P.  Sakmar,  M.D. 

349     Molecular  Genetics  of  Development  in  Drosophila 

Shigeru  Sakonju,  Ph.D. 


Contents 


351      Generating  a  Repertoire  of  Antigen-Specific  Receptors 

David  G.  Schatz,  Ph.D. 

353     Intracellular  Protein  Transport 

Randy  W.  Schekman,  Ph.D. 

355     Development  and  Function  of  the  Synapse 
Richard  H.  Scheller,  Ph.D. 

357     Molecular  Pathogenicity  Studies  of  Enteric  Bacteria 

Gary  K.  Schoolnik,  M.D. 

361      Three-Dimensional  Structure  of  Eukaryotic  Chromosomes 

John  W.  Sedat,  Ph.D. 

363     A  Molecular  Basis  of  Familial  Hypertrophic  Cardiomyopathy 

Jonathan  G.  Seidman,  Ph.D. 

367     Computational  Neurobiology  of  Sensory  Representations 

TerrenceJ.  Sejnowski,  Ph.D. 

369     Adenovirus  as  a  Model  for  Control  of  Gene  Expression 

Thomas  E.  Shenk,  Ph.D. 

371      Growth  Control  of  Myeloid  Cells 

Charles  J.  Sherr,  M.D.,  Ph.D. 

373      The  Role  of  Second  Messengers  in  Ion  Channel  Regulation 

Steven  A.  Siegelbaum,  Ph.D. 

375     Chemistry  of  Cellular  Regulation 

Paul  B.  Sigler,  M.D.,  Ph.D. 

377     The  Mitochondrial  Genome  of  Trypanosomes 

Larry  Simpson,  Ph.D. 

379     Regulation  of  Gene  Activity  During  B  Cell  Development 

Harinder  Singh,  Ph.D. 

381     Regulation  of  Gene  Expression  in  Developing  Lymphocytes 

Stephen  T.  Smale,  Ph.D. 

383     Developmental  Genetics 

Philippe  M.  Soriano,  Ph.D.,  D.Sc. 

385     Understanding  How  Eggs  Work 
Allan  C.  Spradling,  Ph.D. 

387     Structural  Studies  of  Regulatory  Proteins 

Stephen  R.  Sprang,  Ph.D. 

389     Insulin  and  the  Islets  of  Langerhans 
Donald  F.  Steiner,  M.D. 


xiii 


Contents 


391     Autoantibody  Probes  for  Mammalian  Gene  Expression 

Joan  A.  Steitz,  Ph.D. 

393     Structural  Studies  of  Protein-Nucleic  Acid  Interactions 

Thomas  A.  Steitz,  Ph.D. 

397     Pattern  Formation  and  Neuronal  Cell  Recognition  in  the  Drosophila 
Visual  System 
Hermann  Steller,  Ph.D. 

399     Molecular  Genetics  of  Nematode  Development  and  Behavior 

Paul  W.  Sternberg,  Ph.D. 

401      Why  Do  We  Drink  Coffee  and  Tea? 
Charles  F.  Stevens,  M.D.,  Ph.D. 

403     Morphogen  Gradients  and  the  Control  of  Body  Pattern  in  Drosophila 

Gary  Struhl,  Ph.D. 

405     Secretory  Pathways  in  Neurons 

Thomas  C.  Siidhof,  M.D. 

407     Transcription  Factors  in  Cell  Growth  and  Kidney  Differentiation 

Vikas  P.  Sukhatme,  M.D.,  Ph.D. 

409     Structure  and  Function  of  Voltage-Dependent  Calcium  Channels 

Tsutomu  Tanabe,  Ph.D. 

411      The  Molecular  Biology  of  Liver  Regeneration 

Rebecca  A.  Taub,  M.D. 

413     Protein- Tyrosine  Phosphatases  and  the  Control  of  Lymphocyte 
Activation 

Matthew  L.  Thomas,  Ph.D. 

415     Molecular  Regulation  of  Lymphoid  Cell  Growth  and  Development 

Craig  B.  Thompson,  M.D. 

41 7     The  Molecular  Basis  of  Metamorphosis 
Carl  S.  Thummel,  Ph.D. 

419     The  Regulation  of  Mammalian  Development 

Shirley  M.  Tilghman,  Ph.D. 

421     Mechanisms  of  Gene  Regulation  in  Animal  Cells 
Robert  Tjian,  Ph.D. 

423     Studies  on  T  Lymphocytes  and  Mammalian  Memory 

Susumu  Tonegawa,  Ph.D. 

427     Molecular  Engineering  Applied  to  Cell  Biology  and  Neurobiology 

Roger  Y.  Tsien,  Ph.D. 


xiv 


Contents 


429     Genetic  Defects  in  the  Metabolic  Pathways  Interconnecting  the  Urea 
and  Tricarboxylic  Acid  Cycles 
David  L.  Valle,  M.D. 

431     Human  Molecular  Genetics  in  Two  X-linked  Diseases 
Stephen  T.  Warren,  Ph.D. 

433      The  MyoD  Gene  Family:  A  Nodal  Point  During  Specification  of  Muscle 
Cell  Lineage 

Harold  M.  Weintraub,  M.D.,  Ph.D. 

435     Structural  and  Functional  Studies  of  the  T  Cell  Antigen  Receptor 
Arthur  Weiss,  M.D.,  Ph.D. 

437     Following  the  Life  History  of  Lymphocytes 

Irving  L.  Weissman,  M.D. 

439     Function  and  Regulation  of  the  Cystic  Fibrosis  Transmembrane 
Conductance  Regulator 

Michael  J.  Welsh,  M.D. 

441     Identification  of  the  Gene  Responsible  for  Adenomatous  Polyposis 

Raymond  L.  White,  Ph.D. 

443     Mechanisms  of  the  Biological  Activities  of  Membrane  Glycoproteins 

Don  C.  Wiley,  Ph.D. 

445     Studies  of  Blood  Cell  Formation 

David  A.  Williams,  M.D. 

447     Growth  Factor-stimulated  Cell  Proliferation 

Lewis  T.  Williams,  M.D.,  Ph.D. 

449     Somatic  Cell  Gene  Transfer 
James  M.  Wilson,  M.D.,  Ph.D. 

453     Normal  and  Abnormal  Lymphocyte  Growth  Regulation 

Owen  N.  Witte,  M.D. 

455     Translational  Regulation 

Sandra  L.  Wolin,  M.D.,  Ph.D. 

457     Molecular  Genetics  and  Studies  Toward  Gene  Therapy 
for  Metabolic  Disorders 

Savio  L.  C.  Woo,  Ph.D. 

455^     Paracrine  Control  of  Blood  Vessel  Function:  Role  of  the  Endothelins 

Masashi  Yanagisawa,  M.D.,  Ph.D. 

461     Mechanism  of  Phototransduction  in  Retinal  Rods  and  Cones 

King-Wai  Yau,  Ph.D. 

463     Molecular  Mechanisms  of  Ion  Channel  Function 
Gary  Yellen,  Ph.D. 


XV 


Contents 


465     Drosophila  Behavior  and  Neuromuscular  Development 

Michael  W.  Young,  Ph.D. 

469      Control  of  Transcription  by  Transmembrane  Signals 

Edward  B.  Ziff,  Ph.D. 

471      Cell-Cell  Interactions  Determine  Cell  Fate  in  the  Drosophila  Retina 

S.  Lawrence  Zipursky,  Ph.D. 

4  73     Molecular  Genetics  of  Sensory  Transduction 

Charles  S.  Zuker,  Ph.D. 

475     Investigators  by  Location 

481     International  Research  Scholars 

483     Molecular  Biology  and  Epidemiology  for  Control  of  Rotavirus  Diarrhea 

Carlos  F.  Arias,  Ph.D. 

485     Molecular  Genetics  of  Normal  and  Leukemic  Hematopoiesis 

Alan  Bernstein,  Ph.D. 

487     Molecular  Biology  of  Two  Enteropathogenic  Bacteria 

Edmundo  Calva,  Ph.D. 

489     Functional  Heterogeneity  in  Prolactin- secreting  Cells 

Gabriel  Cota,  Ph.D. 

491      Ionic  Channels  in  Sea  Urchin  Sperm  Physiology 

Alberto  Darszon,  Ph.D. 

493     Host-Pathogen  Interactions  in  Microbial  Pathogenesis 

B.  Brett  Finlay,  Ph.D. 

45^5     Mechanisms  of  Transcriptional  Regulation 

Jack  Greenblatt,  Ph.D. 

497     Ionic  Homeostasis  in  White  Blood  Cells 

Sergio  Grinstein,  Ph.D. 

499     Genetic  Basis  of  Multidrug  Resistance 

Philippe  Gros,  Ph.D. 

501      Control  of  Bacterial  Protein  Synthesis  During  Viral  Infection 

Gabriel  Guarneros  Pena,  Ph.D. 

503     Molecular  Genetics  of  Photosynthesis  and  Carbon  Assimilation  in  Plants 

Luis  R.  Herrera-Estrella,  Ph.D. 

507     Gene  Pattern  Expression  in  Early  Embryogenesis 
Alexandra  L.  Joyner,  Ph.D. 

509     Diagnostic  Use  of  UNA  Replication  in  Infectious  Diseases 
Paul  M.  Lizardi,  Ph.D. 

xvi 


Contents 


511     Lineage-Specific  Gene  Expression  in  Caenorhabditis  elegans 

James  D.  McGhee,  Ph.D. 

513      Cytokine  Regulation  of  Effector  Functions  in  Immune  Responses 

Tim  R.  Mosmann,  Ph.D. 

515     Cellular  and  Molecular  Basis  of  Variability  in  Entamoeba  histolytica 

Esther  Orozco,  Ph.D. 

51 7     Phosphorylation  and  Protein-Protein  Interactions  in 
Signal  Transduction 

Tony  Pawson,  Ph.D. 

519     Chemical  and  Functional  Characterization  of  Scorpion  Toxins 

Lourival  Domingos  Possani,  Ph.D. 

521     Protein  Crystallography  in  the  Study  of  Infectious  Diseases 
Randy  J.  Read,  Ph.D. 

523     Representations  and  Transformations  of  Tactile  Signals  in  Somatic 
and  Frontal  Motor  Cortices 
Ranulfo  Romo,  M.D.,  Ph.D. 

525     Anterior-Posterior  Patterning  in  the  Early  Mammalian  Embryo 
Janet  Rossant,  Ph.D. 

527     Response  of  the  Cerebral  Cortex  to  Spatial  Information 

Jean-Pierre  Roy,  M.D. 

529     Molecular  Studies  on  Neuronal  Calcium  Channels 

Terry  P.  Snutch,  Ph.D. 

531      Cystic  Fibrosis,  Gene  Expression  in  the  Mammalian  Lens,  and  Mapping 
of  Chromosome  7 

Lap-Chee  Tsui,  Ph.D. 

533  Index 


xvii 


THE  Howard  Hughes  Medical  Institute  (HHMI)  presents  the  seventh  volume  of  Research 
in  Progress,  a  series  begun  in  1986  to  describe  the  research  activities  of  the  Institute. 
Early  in  1992  the  scientists  associated  with  HHMI  at  that  time  were  asked  to  provide 
statements  in  nontechnical  terms  of  their  past  accomplishments,  current  work,  and  plans  for 
the  future,  in  order  to  provide  a  concise  but  informative  picture  of  the  Institute's  research 
in  progress.  You  will  read  their  personal  essays  in  the  pages  that  follow.  We  are  pleased  that 
this  annual  publication  has  come  to  be  a  useful  source  of  information  for  scientists  and 
nonscientists  interested  in  biomedical  research,  as  well  as  for  the  members  of  the  HHMI  family. 

The  Institute  also  publishes  as  a  companion  volume  the  formal  Annual  Scientific  Report. 
This  is  the  official  archival  record  of  the  research  of  each  HHMI  laboratory  and  includes 
yearly  bibliographies  as  well  as  descriptions  of  other  HHMI  scientific  activities.  A  general 
Annual  Report  of  the  Howard  Hughes  Medical  Institute  describes  the  various  programs  for 
a  lay  audience  and  gives  a  summary  of  the  Institute's  financial  data.  With  the  recent 
publication  of  From  Egg  to  Adult,  which  describes  the  exciting  process  of  biological 
development,  the  Institute  has  expanded  its  series  of  reports  on  subjects  of  current  scientific 
interest  written  for  a  general  audience.  This  booklet  joins  the  widely  acclaimed  Finding  the 
Critical  Shapes  and  Blazing  a  Genetic  Trail  that  have  been  in  great  demand  by  a  broad 
spectrum  of  readers,  especially  teachers  who  have  used  these  reports  in  classrooms  across 
this  country  and  abroad. 

In  recognition  of  the  fact  that  the  boundaries  of  biomedical  science  are  not  constrained  by 
national  borders,  the  Institute  launched  its  International  Research  Scholars  Program  in  the 
spring  of  1991-  This  is  a  limited  and  experimental  program  with  the  purpose  of  providing 
research  support  in  selected  countries  for  promising  scientists  whose  careers  are  in  a 
developing  phase  but  who  have  already  made  significant  contributions  to  biomedical 
research.  The  international  program  is  separate  from  the  Institute's  ongoing  medical  research 
program,  described  in  the  first  section  of  this  volume,  in  which  scientists  join  HHMI  as  full- 
time  employee-investigators  and  are  supported  through  the  Institute's  operation  as  a  Medical 
Research  Organization  (MRO).  The  Scholars  do  not  join  the  HHMI  staff,  but  instead  each 
receives  research  support  through  a  grant. 

For  the  initial  awards  in  this  program,  selected  scientists  in  the  countries  that  are  our 
immediate  neighbors,  Canada  and  Mexico,  were  invited  to  compete.  Twenty-four  outstanding 
investigators  received  five-year  awards  (14  from  Canada  and  10  from  Mexico).  You  will  read 
their  descriptions  of  the  research  in  their  laboratories  in  the  second  section  of  this  volume. 
For  awards  in  1993,  the  Institute  has  turned  to  scientists  in  the  United  Kingdom,  Australia, 
and  New  Zealand.  Those  selected  for  grant  support  will  be  announced  in  late  1992,  and  their 
work  will  be  included  in  the  next  volume  of  Research  in  Progress. 

The  Institute  has  a  large  grants  program  that  complements  its  MRO  activities.  In  addition  to 
the  International  Research  Scholars  initiative,  the  grants  program  supports  education  in  the 
biomedical  and  related  sciences  at  the  precollege,  college  undergraduate,  graduate,  and 
postdoctoral  levels.  New  in  1992  were  grants  awarded  to  certain  science  museums,  science 
and  technology  centers,  children's  museums,  and  natural  history  museums  to  assist  in 
education  and  outreach  programs  for  elementary  school  children  and  their  families.  The 
Institute's  annual  publication  Grants  for  Science  Education  details  these  initiatives. 

A  very  important  event  for  HHMI  occurred  in  the  spring  of  1991,  when  construction  began 
on  the  buildings  that  will  become  our  new  headquarters  and  conference  center  complex  in 
Chevy  Chase,  Maryland.  At  this  writing,  the  construction  is  entering  its  final  phases,  and  the 
attractiveness  of  the  buildings  of  this  large  facility  and  how  they  fit  into  the  gentle  hills  and 
valleys  of  the  lovely  site  are  exceeding  our  original  expectations.  We  expect  to  occupy  the 
buildings  in  early  1993-  The  facility  provides  the  administrative  focus  for  the  Institute's  222 
investigators  (as  of  July  1,  1992)  whose  laboratories  are  located  at  53  institutions  across 
this  country. 

We  invite  you  to  share  the  excitement  of  the  research  of  the  HHMI  investigators  whose 
work  forms  the  core  of  the  Institute's  activities  and  of  the  studies  of  our  International 


Foreword 


Research  Scholars  that  have  further  enriched  our  scientific  program.  We  are  especially 
pleased  that  the  Institute's  research  on  problems  of  medical  concern  to  developing  countries 
is  extended  through  the  basic  studies  of  a  number  of  these  Scholars.  The  advances  toward 
understanding  biological  processes  and  disease  mechanisms  that  are  described  herein  move 
us  further  tovv'ard  our  goal  of  the  betterment  of  the  human  condition. 

Purnell  W.  Choppin,  M.D. 
President 


XX 


Trustees,  Officers,  and  Principal  Staff  Members 


Trustees 


Alexander  G.  Beam,  M.D. 
Adjunct  Professor 
The  Rockefeller  University 
Professor  Emeritus  of  Medicine 
Cornell  University  Medical  College 
Former  Senior  Vice  President 
Merck  Sharp  &  Dohme,  International 

Helen  K.  Copley 

Chairman  of  the  Corporation  and 

Chief  Executive  Officer 
The  Copley  Press,  Inc. 

Frank  William  Gay 
Former  President  and  Chief 

Executive  Officer 
SUMMA  Corporation 

James  H.  Gilliam,  Jr.,  Esq. 
Executive  Vice  President 
Beneficial  Corporation 

Hanna  H.  Gray,  Ph.D. 
President 

The  University  of  Chicago 


William  R.  Lummis,  Esq. 
Chairman  of  the  Board  of  Directors 
SUMMA  Corporation 

Irving  S.  Shapiro,  Esq.,  Chairman 
Of  Counsel 

Skadden,  Arps,  Slate,  Meagher  &  Flom 
Former  Chairman  and  Chief 

Executive  Officer 
E.I.  du  Pont  de  Nemours  and  Company 

George  W.  Thorn,  M.D., 
Chairman  Emeritus 
Professor  Emeritus 
Harvard  Medical  School 

James  D.  Wolfensohn 
President 

James  D.  Wolfensohn  Incorporated 


Officers 


Purnell  W.  Choppin,  M.D. 
President 

W.  Maxwell  Cowan,  M.D.,  Ph.D. 

Vice  President  and  Chief  Scientific  Officer 

Graham  O.  Harrison 

Vice  President  and  Chief  Investment  Officer 


Joseph  G.  Perpich,  M.D  ,  J.D. 
Vice  President  for  Grants  and 
Special  Programs 

Jose  E.  Trias,  Esq. 

Vice  President  and  General  Counsel 
Robert  C.  White 

Vice  President  and  Chief  Financial  Officer 


Trustees,  Officers,  and  Principal  Staff  Members 


Principal  Staff  Members 


Craig  A.  Alexander,  Esq. 
Associate  General  Counsel 

Stephen  A.  Barkanic 
Grants  Program  Officer 

W.  Emmett  Barkley,  Ph.D. 
Director  of  Laboratory  Safety 

Lillian  H.  Blucher 

Managing  Director — Investments 

WinfredJ.  Clingenpeel 
Director  of  Purchasing 

David  Davis-Van  Atta 
Grants  Program  Officer 

Barbara  Filner,  Ph.D. 
Grants  Program  Officer 

Patricia  S.  Gage,  Esq. 
Associate  General  Counsel 

James  R.  Gavin  III,  M.D.,  Ph.D. 
Senior  Scientific  Officer 

Donald  H.  Harter,  M.D. 
Senior  Scientific  Officer  and 
Director,  HHMI-NIH  Research 
Scholars  Program 

Patricia  J.  Hoben,  Ph.D. 
Grants  Program  Officer 
(through  September  4,  1992) 

John  A.  Jones 

Director  of  Computer  Services 


David  W.  Kingsbury,  M.D. 
Senior  Scientific  Officer 

Laura  A.  Kumin,  Esq. 
Associate  General  Counsel 

Joan  S.  Leonard,  Esq. 
Associate  General  Counsel 

Robert  H.  McGhee 

Director  of  Research  Facilities  Planning 

Alan  E.  Mowbray 

Director  of  Management  Services 

Robert  C.  Mullins 
Director  of  Internal  Audit 

Edward  J.  Palmerino 
Assistant  Controller 

Robert  A.  Potter 

Director  of  Communications 

Donald  C.  Powell 

Director  of  Human  Resources 

Ellen  B.  Safir 

Managing  Director — Investments 

Mark  W.  Smith 
Controller 

Claire  H.  Winestock,  Ph.D. 
Senior  Scientific  Officer 


Medical  Advisory  Board 


Michael  S.  Brown,  M.D. 
Paul  J.  Thomas  Professor  of  Medicine  and 
Genetics 

Director,  Center  for  Genetic  Disease 
University  of  Texas  Southwestern  Medical 
Center  at  Dallas 

William  A.  Catterall,  Ph.D. 

Professor  and  Chair 

Department  of  Pharmacology 

University  of  Washington  School  of  Medicine 

John  E.  Dowling,  Ph.D. 

(through  December  31,  1992) 
Maria  Moors  Cabot  Professor  of  Natural 

Science 

Department  of  Cellular  and  Developmental 

Biology 
Harvard  University 

Thomas  J.  Kelly,  Jr.,  M.D.,  Ph.D. 

( effective  January  1,  1993 ) 
Chairman 

Department  of  Molecular  Biology  and 
Genetics 

The  Johns  Hopkins  University  School  of 
Medicine 

Hugh  O.  McDevitt,  M.D. 

( through  December  3 1,  1992 ) 
Burt  and  Marion  Avery  Professor  of 

Immunology 
Professor  of  Microbiology,  Immunology, 

and  Medicine 
Stanford  University  School  of  Medicine 

Dinshaw  Patel,  Ph.D. 

(effective  January  1,  1993) 
Abby  Rockefeller  Mauze  Chair  in 

Experimental  Therapeutics 
Member,  Cellular  Biochemistry  and 

Biophysics  Program 
Memorial  Sloan-Kettering  Cancer  Center 

William  E.  Paul,  M.D. 

Chief,  Laboratory  of  Immunology 

National  Institute  of  Allergy  and  Infectious 

Diseases 
National  Institutes  of  Health 


Frederic  M.  Richards,  Ph.D. 

( through  December  31,  1992 ) 
Sterling  Professor  Emeritus  of  Molecular 

Biophysics  and  Biochemistry 
Yale  University 

Janet  D.  Rowley,  M.D. 

Blum-Riese  Distinguished  Service  Professor 
Departments  of  Medicine  and  Molecular 

Genetics  and  Cell  Biology 
The  University  of  Chicago 

David  D.  Sabatini,  M.D.,  Ph.D. 

Frederick  L.  Ehrman  Professor  and  Chairman 

Department  of  Cell  Biology 

New  York  University  School  of  Medicine 

Phillip  A.  Sharp,  Ph.D. 

John  D.  MacArthur  Professor  of  Biology  and 

Head,  Department  of  Biology 
Massachusetts  Institute  of  Technology 

Melvin  I.  Simon,  Ph.D. 

(effective  January  1,  1993) 
Biaggini  Professor  of  Biology 
California  Institute  of  Technology 

William  S.  Sly,  M.D. 

(through  December  31,  1992) 
Alice  A.  Doisy  Professor  and  Chairman 
Edward  A.  Doisy  Department  of 

Biochemistry  and  Molecular  Biology 
St.  Louis  University  School  of  Medicine 

Lloyd  H.  Smith,  Jr.,  M.D.,  Chairman 
Professor  of  Medicine  and  Associate  Dean 
University  of  California,  San  Francisco, 
School  of  Medicine 

Jonathan  W.  Uhr,  M.D. 

(effective  January  1,  1993) 
Professor  and  Chairman 
Department  of  Microbiology 
University  of  Texas  Southwestern  Medical 

Center  at  Dallas 


xxiii 


Scientific  Review  Board 


Cell  Biology  and  Regulation 

Alfred  G.  Oilman,  M.D.,  Ph.D. 
Professor  and  Chairman 
Department  of  Pharmacology 
University  of  Texas  Southwestern  Medical 
Center  at  Dallas 

Tony  Hunter,  Ph.D. 

Professor  of  Molecular  Biology  and  Virology 
The  Salk  Institute 

Stuan  Kornfeld,  M.D. 

Professor  of  Medicine 

Washington  University  School  of  Medicine 

David  J.  L.  Luck,  M.D.,  Ph.D. 
Professor  of  Cell  Biology 
The  Rockefeller  University 

Thomas  D.  Pollard,  M.D. 
Professor  and  Director 
Department  of  Cell  Biology  and  Anatomy 
The  Johns  Hopkins  University  School 
of  Medicine 

Lucille  Shapiro,  Ph.D. 

Joseph  D.  Grant  Professor  in  the  School  of 
Medicine  and  Chairman,  Department  of 
Developmental  Biology 

Stanford  University  School  of  Medicine 

Genetics 

David  Botstein,  Ph.D. 

Professor  and  Chairman 

Department  of  Genetics 

Stanford  University  School  of  Medicine 

Ira  Herskowitz,  Ph.D. 
Professor  and  Chairman 
Department  of  Biochemistry  and  Biophysics 
Head,  Division  of  Genetics 
University  of  California,  San  Francisco, 
School  of  Medicine 

Anthony  P.  Mahowald,  Ph.D. 

Louis  Block  Professor  and  Chairman 

Department  of  Molecular  Genetics 

and  Cell  Biology 
The  University  of  Chicago 


Thomas  P.  Maniatis,  Ph.D. 

Professor  of  Biochemistry  and  Molecular 

Biology 
Harvard  University 

Malcolm  A.  Martin,  M.D. 

Chief,  Laboratory  of  Molecular  Microbiology 

National  Institute  of  Allergy 

and  Infectious  Diseases 
National  Institutes  of  Health 

Carolyn  W.  Slayman,  Ph.D. 
Professor  and  Chairman 
Department  of  Genetics 
Professor,  Department  of  Cellular 

and  Molecular  Physiology 
Yale  University  School  of  Medicine 

Harold  E.  Varmus,  M.D. 
American  Cancer  Society  Professor 

of  Molecular  Virology 
Department  of  Microbiology  and 

Immunology 
University  of  California,  San  Francisco, 

School  of  Medicine 

Immunology 

Joseph  M.  Davie,  M.D. 

(through  June  30,  1992) 
President 

G.D.  Searle  and  Company 

Matthew  D.  Scharff,  M.D. 
Professor  of  Cell  Biology 
Albert  Einstein  College  of  Medicine 

Ursula  Storb,  M.D. 

( effective  January  1,  1993 ) 
Professor 

Department  of  Molecular  Genetics 

and  Cell  Biology 
The  University  of  Chicago 

Emil  R.  Unanue,  M.D. 

Professor  and  Chairman 

Department  of  Pathology 

Washington  University  School  of  Medicine 


xxiv 


Scientific  Review  Board 


Ellen  S.  Vitetta,  Ph.D. 

Professor  of  Microbiology  and  Director, 

Cancer  Immunobiology  Center 
University  of  Texas  Southwestern  Medical 

Center  at  Dallas 

Neuroscience 

Floyd  E.  Bloom,  M.D. 
Chairman,  Department  of 

Neuropharmacology 
Member,  The  Scripps  Research  Institute 

Arthur  M.  Brown,  M.D.,  Ph.D. 
Professor  and  Chairman 
Department  of  Molecular  Physiology 

and  Biophysics 
Baylor  College  of  Medicine 

Zach  W.  Hall,  Ph.D. 
Professor  and  Chairman 
Department  of  Physiology 
University  of  California,  San  Francisco, 
School  of  Medicine 

Stephen  F.  Heinemann,  Ph.D. 

( effective  January  1,  1995 ) 
Faculty  Chairman 

Director  of  Molecular  Neurobiology 

Laboratory 
The  Salk  Institute 

CarlaJ.  Schatz,  Ph.D. 

{effective  July  1,  1993) 
Professor  of  Neurobiology 
Department  of  Molecular  and  Cell  Biology 
University  of  California,  Berkeley 


Melvin  I.  Simon,  Ph.D. 

(through  December  31,  1992) 
Biaggini  Professor  of  Biology 
California  Institute  of  Technology 

Structural  Biology 

David  R.  Davies,  Ph.D. 

Chief,  Section  on  Molecular  Structure 

Laboratory  of  Molecular  Biology 

National  Institute  of  Diabetes  and  Digestive 

and  Kidney  Diseases 
National  Institutes  of  Health 

Michael  N.  G.  James,  D.Phil. 
Professor  of  Biochemistry 
University  of  Alberta 

Dinshaw  Patel,  Ph.D. 

(through  December  31,  1992) 
Abby  Rockefeller  Mauze  Chair  in 

Experimental  Therapeutics 
Member,  Cellular  Biochemistry 

and  Biophysics  Program 
Memorial  Sloan-Kettering  Cancer  Center 

Michael  G.  Rossmann,  Ph.D. 

Hanley  Professor  of  Biological  Sciences 

Purdue  University 

Peter  E.  Wright,  Ph.D. 

(effective  January  1,  1993) 
Member  and  Chairman 
Department  of  Molecular  Biology 
The  Scripps  Research  Institute 


XXV 


Locations  of  Howard  Hughes  Medical  Institute 
Laboratories 


Alabama 
California 


Colorado 

Connecticut 

Georgia 

Illinois 

Indiana 

Iowa 
Maryland 

Massachusetts 


Michigan 
Missouri 
New  Jersey 
New  York 


University  of  Alabama  at  Birmingham  and  associated  hospitals 

California  Institute  of  Technology  and  associated  hospitals,  Pasadena 
The  Salk  Institute  for  Biological  Studies,  San  Diego 
Stanford  University  and  the  Stanford  University  Hospital,  Palo  Alto 
University  of  California,  Berkeley,  and  associated  hospitals 
University  of  California,  Los  Angeles,  and  associated  hospitals 
University  of  California,  San  Diego,  and  the  UCSD  Medical  Center 
University  of  California,  San  Francisco,  and  associated  hospitals 
University  of  Southern  California,  Los  Angeles,  and  associated  hospitals 

National  Jewish  Center  for  Immunology  and  Respiratory  Medicine,  Denver 
University  of  Colorado  at  Boulder  and  the  University's  Health  Sciences  Center 
University  of  Colorado  Health  Sciences  Center,  Denver,  and  associated  hospitals 

Yale  University  and  associated  hospitals,  New  Haven 

Emory  University  School  of  Medicine,  Atlanta,  and  associated  hospitals 

Northwestern  University  and  associated  hospitals,  Evanston 

The  University  of  Chicago  and  The  University  of  Chicago  Hospitals 

Indiana  University,  Bloomington,  and  associated  hospitals 

Indiana  University  School  of  Medicine,  Indianapolis,  and  associated  hospitals 

The  University  of  Iowa  and  associated  hospitals,  Iowa  City 

The  Carnegie  Institution  of  Washington,  Baltimore,  and  The  Johns 

Hopkins  Hospital 
The  Johns  Hopkins  University  and  Hospital,  Baltimore 

Brandeis  University,  Waltham,  and  associated  hospitals 

Brigham  and  Women's  Hospital,  Boston 

The  Children's  Hospital,  Boston 

Harvard  College,  Arts  and  Sciences,  Cambridge 

Harvard  Medical  School,  Boston 

Massachusetts  General  Hospital,  Boston 

Massachusetts  Institute  of  Technology  and  associated  hospitals,  Cambridge 
Tufts  University  School  of  Medicine  and  associated  hospitals,  Boston 
University  of  Massachusetts,  Worcester,  and  associated  hospitals 

University  of  Michigan  and  associated  hospitals,  Ann  Arbor 

Washington  University  and  associated  hospitals,  St.  Louis 

Princeton  University  and  associated  medical  centers,  Princeton 

Albert  Einstein  College  of  Medicine  of  Yeshiva  University,  Bronx,  and 

associated  hospitals 
Cold  Spring  Harbor  Laboratory  and  associated  hospitals.  Cold  Spring  Harbor 
Columbia  University  and  associated  hospitals.  New  York  City 
Cornell  University  Medical  College,  New  York  City 
Memorial  Sloan-Kettering  Cancer  Center,  New  York  City 
New  York  University  (Medical  Center  and  Washington  Square)  and  associated 

hospitals.  New  York  City 
The  Rockefeller  University  and  Rockefeller  University  Hospital,  New  York  City 
State  University  of  New  York  at  Stony  Brook  and  University  Hospital  at 

Stony  Brook 


North  Carolina       Duke  University,  including  Duke  University  Medical  Center,  Durham 


xxvi 


Locations  of  Howard  Hughes  Medical  Institute  Laboratories 


Oklahoma 

Oregon 

Pennsylvania 

Tennessee 

Texas 


Utah 

Washington 


Oklahoma  Medical  Research  Foundation  and  associated  hospitals, 
Oklahoma  City 

University  of  Oregon  and  associated  hospitals,  Eugene 

University  of  Pennsylvania  and  associated  hospitals,  Philadelphia 

St.  Jude  Children's  Research  Hospital,  Memphis 

Vanderbilt  University,  including  Vanderbilt  University  Hospital,  Nashville 

Baylor  College  of  Medicine  and  associated  hospitals,  Houston 
Rice  University  and  associated  hospitals,  Houston 
University  of  Texas  Southwestern  Medical  Center  at  Dallas  and 
associated  hospitals 

University  of  Utah,  including  University  of  Utah  Medical  Center,  Salt  Lake  City 

Fred  Hutchinson  Cancer  Research  Center,  Seattle 
University  of  Washington  and  associated  hospitals,  Seattle 


Wisconsin 


University  of  Wisconsin-Madison,  and  associated  hospitals 


Other  Institute  Facilities 

Maryland  HHMI-NIH  Cloister  at  the  Mary  Woodard  Lasker  Center  for  Science  and 

Education  on  the  NIH  campus,  Bethesda 

New  York  Synchrotron  Beam  Lines,  Brookhaven  National  Laboratory  (under  construction) 


xxvii 


I 


I 


( 

I 

( 

I 


I 


I 
I 

I 


1 


1 

! 

I 


Introduction 


THE  annual  publication  of  Research 
in  Progress  has  become  one  of  the 
highlights  of  the  Howard  Hughes  Medi- 
cal Institute's  research  activities.  By  pro- 
viding a  synoptic  overview  of  the  re- 
search being  conducted  by  the  Institute's 
investigators,  in  essentially  nontechnical 
language,  it  has  come  to  be  greatly  ap- 
preciated not  only  by  those  interested  in 
the  Institute's  activities  as  a  Medical  Re- 
search Organization,  but  by  many  others 
who  view  it  as  an  accessible  introduction 
to  current  biomedical  research. 
The  present  volume  in  this  series  follows  fairly 
closely  the  pattern  that  has  evolved  over  the  past 
three  or  four  years:  it  provides  a  snapshot  of  the 
status  of  the  Institute's  research  in  early  1992.  As 
in  the  previous  volume,  this  issue  includes  a  se- 
ries of  introductory  essays  that  are  intended  to 
assist  the  reader  who  has  had  little  or  no  back- 
ground in  biology  or  medicine.  These  essays  are 
not  intended  to  be  exhaustive  nor  simply  to  repli- 
cate material  readily  available  in  most  standard 
biology  or  medical  texts;  rather  they  are  intended 
to  serve  as  an  expanded  glossary  of  terms,  de- 
fining in  a  general  way  many  of  the  terms  used  in 
the  individual  reports.  To  emphasize  this  feature 
of  the  essays,  these  technical  terms  are  shown 
in  bold  type.  The  essays  are  based  on  an  initial 
set  of  drafts  prepared  for  each  of  the  five  pro- 
grammatic areas  in  which  the  Institute  conducts 
research:  cell  biology  and  regulation;  genetics; 
immunology;  neuroscience;  and  structural  biol- 
ogy. The  original  drafts  were  kindly  prepared  by 
Drs.  Richard  O.  Hynes,  Philip  Leder,  Charles  A. 
Janeway,  Eric  R.  Kandel,  and  Stephen  C.  Harrison, 
respectively.  We  are  grateful  to  them  and  to  a 
number  of  their  colleagues  within  the  Institute 
who  commented  on  the  essays  or  provided  addi- 
tional information  and,  in  some  cases,  clarified 
ambiguities  or  corrected  errors  in  the  original 
text.  As  in  last  year's  volume  the  text  of  the  essays 
has  been  supplemented  by  a  number  of  illustra- 


tions, derived  for  the  most  part  from  general  texts 
or  scientific  papers  that  are  identified  in  the  ac- 
companying legends.  We  hope  that  these  dia- 
grams and  photographs  will  make  the  essays  even 
more  useful. 

For  readers  who  wish  to  know  about  specific 
research  being  conducted  by  HHMI  investigators, 
we  have  provided  once  again  a  detailed  index. 
The  index  lists  almost  every  topic  or  term,  from 
abd-A  (a  gene  that  directs  the  segmental  develop- 
ment of  the  abdomen — first  identified  in  the  fruit 
fly  Drosophila  melanogaster)  to  the  zif268 
gene  and,  in  between,  the  less  arcane  subjects  of 
AIDS,  cancer,  cystic  fibrosis,  diabetes,  hemo- 
philia, muscular  dystrophy,  and  obesity.  Several 
readers  have  remarked  how  useful  the  addition  of 
an  index  has  been,  and  we  are  grateful  once  more 
to  Diana  Witt  for  preparation  of  the  index. 

The  greater  part  of  the  volume  consists  of  a 
series  of  short  reports  by  the  investigators  asso- 
ciated with  the  Institute  during  1992.  This  por- 
tion of  the  volume  is  again  larger  than  its  prede- 
cessor, reflecting  the  increased  number  of 
investigators  whose  work  is  represented.  In  addi- 
tion, reports  prepared  by  the  Institute's  Interna- 
tional Research  Scholars  are  included  in  a  section 
following  the  investigators'  reports.  These  24 
scientists  are  the  first  recipients  of  research 
grants  awarded  under  the  Institute's  new  interna- 
tional initiative.  The  reports  submitted  by  each 
investigator  and  International  Research  Scholar 
have  been  collated  and  edited  by  Dr.  Claire  H. 
Winestock,  Senior  Scientific  Officer;  Elizabeth 
Cowley,  Copy  Editor;  William  T.  Carrigan,  Edi- 
tor/Writer; Gail  Markley,  Manager  of  Publica- 
tions; and  Kimberly  A.  Cornejo,  Permissions  Edi- 
tor. Laura  North  also  assisted  with  the  editing  of 
manuscripts.  We  are  grateful  to  them  and  to  the 
many  investigators  who  have  provided  us  with 
suitable  illustrations  taken  from  their  ongoing  re- 
search. Not  only  do  the  illustrations  help  to  clar- 
ify the  written  text,  but  they  also  greatly  enhance 
the  aesthetic  appeal  of  the  volume. 


Cell  Biology  and  Regulation  Program 

The  Cell  Biology  and  Regulation  Program  is  the 
oldest  of  the  Institute's  research  programs.  Origi- 
nally referred  to  as  Metabolic  Regulation,  which 
reflected  the  program's  roots  in  clinical  studies 
of  metabolic  and  endocrine  disorders,  the  title  of 
this  program  was  changed  six  years  ago  to  reflect 
more  accurately  its  principal  theme — the  biol- 


ogy of  cells,  the  factors  that  regulate  their  normal 
growth  and  distinctive  functions,  and  the  ways  in 
which  cells  interact  with  each  other.  In  this  sense 
the  program  has  come  to  occupy  a  critical  posi- 
tion in  the  Institute's  research  endeavors,  and  it 
lies  firmly  within  the  mainstream  of  contempo- 
rary biological  research. 


Introduction 


The  human  body  contains  several  trilHon  (i.e., 
milHon  million)  cells  of  a  thousand  or  more  dis- 
tinct types.  Research  in  the  field  of  cell  biology 
seeks  to  understand  how  these  various  cells  are 
constructed  and  organized,  how  they  differ  from 
one  another,  how  they  sense  and  respond  to  out- 
side influences,  how  they  interact  with  their 
neighbors  to  form  more  complex  tissues  and  or- 
gans, and,  in  general,  how  the  cells  of  the  body 
are  integrated  to  produce  an  appropriately  func- 
tioning organism.  Equally  important,  research  in 
this  area  is  aimed  at  understanding  how  these  cel- 
lular functions  are  perturbed  by  disease.  To  this 
extent  the  problems  addressed  by  investigators  in 
the  Cell  Biology  and  Regulation  Program  inevita- 
bly impinge  on  related  work  in  genetics,  develop- 
ment, neuroscience,  and  immunology.  For  a 
Medical  Research  Organization  it  is  especially 
gratifying  to  see  how  many  of  the  insights  gleaned 
from  these  studies  are  already  beginning  to  throw 
light  on  such  medically  important  problems  as 
diabetes,  heart  disease,  cancer,  muscular  dys- 
trophy, cystic  fibrosis,  and  a  number  of  other  ge- 
netic disorders. 

The  strikingly  rapid  advances  that  have  oc- 
curred in  cell  biology  in  recent  years  have  been 
due,  in  large  part,  to  earlier  progress  in  biochem- 
istry and  cellular  physiology,  but  especially  to 
the  dramatic  developments  that  have  occurred  in 
molecular  biology  since  the  early  1960s.  The 
techniques  developed  in  these  fields  have  been 
invaluable  to  cell  biology,  which  has  always  been 
quick  to  apply  different  and  newly  emerging  ap- 
proaches to  the  solution  of  the  many  problems  of 
cell  structure  and  function.  To  understand  the 
types  of  research  being  conducted  in  contempo- 
rary cell  biology,  it  may  be  helpful  to  begin  with  a 
general  account  of  a  "typical"  animal  cell  (Fig- 
ure 1). 

Near  the  center  of  each  cell  is  the  nucleus, 
which  contains  the  genes  (Figure  2)  that  are 
made  of  DNA  and  encode  the  information  neces- 
sary to  construct  an  entire  organism  and  maintain 
its  day-to-day  activities.  The  entire  complement 
of  genes  is  the  genome,  which  comprises  a  set  of 
instructions  encoded  in  the  sequences  of  the  DNA 
molecules  (as  described  more  fully  in  the  section 
on  genetics).  The  human  genome  consists  of  46 
chromosomes — 22  pairs  of  autosomes  and  2  sex 
chromosomes.  The  two  copies  of  each  autosome 
are  inherited  from  the  mother  and  the  father,  re- 
spectively; in  females  there  are  two  X  chromo- 
somes (one  from  each  parent) ,  while  in  males  the 
Y  chromosome  is  always  inherited  from  the  father 


and  the  X  chromosome  (Figure  3)  is  always  in- 
herited from  the  mother.  The  46  chromosomes 
comprise  a  total  of  about  3  billion  pairs  of  nu- 
cleotides. Estimates  vary,  but  it  is  thought  that 
there  may  be  as  many  as  100,000  genes  in  the 
human  genome.  These  genes  vary  in  length  from 
around  1,000  to  about  2  million  nucleotides. 
Each  gene  encodes  the  information  for  a  particu- 
lar cellular  structure  or  function.  This  informa- 
tion, which  can  be  likened  to  a  computer  lan- 
guage, is  first  read  (transcribed)  into  RNA,  and 
the  message  contained  within  the  nucleotide  se- 
quence of  the  RNA  molecule  is  then  decoded 
(translated)  by  the  machinery  of  the  cell  into  a 
different  language,  or  chemical  structure.  While 
the  RNA  transcript  mirrors  exactly  the  DNA  se- 
quence of  the  gene,  the  messenger  RNA  (mRNA) 
that  is  translated  into  the  amino  acid  sequence  of 
the  encoded  protein  is  a  highly  edited  message 
(Figure  4).  Generally  several  intervening  (or 
noncoding)  sequences  called  introns  are  selec- 
tively removed  from  the  transcript.  Introns  ac- 
count for  a  considerable  proportion  of  the  DNA  in 
all  higher  organisms  and,  together  with  several 
other  noncoding  stretches  of  DNA  (spacer  DNA, 
satellite  DNA,  and  other  repetitive  DNA  se- 
quences) ,  are  sometimes  referred  to  as  selfish  or 
junk  DNA.  In  humans  it  is  estimated  that  more 
than  90  percent  of  the  DNA  in  the  genome  is  of 
this  kind.  The  possible  functions  (if  any)  of  this 
noncoding  DNA  are  not  known,  but  the  mecha- 
nisms whereby  the  coding  sequences  or  exons 
are  spliced  out  and  joined  together  (sometimes  in 
different  order — a  process  known  as  alternative 
splicing)  is  a  subject  of  considerable  interest  at 
present.  The  chemical  language  of  the  cell  has  20 
different  characters  or  units,  known  as  amino 
acids,  which  are  linked  together,  again  in  linear 
arrays,  to  make  proteins. 

Whereas  DNA  is  the  blueprint  directing  the 
cell's  development  and  function,  proteins  are  the 
molecules  from  which  cells  are  built  and  which 
carry  out  most  cellular  functions.  Most  genes  en- 
code proteins,  and  each  cell  contains  about 
10,000  different  types  of  protein.  That  is,  each 
cell  uses  only  about  10  percent  of  the  total  set  of 
genes  at  any  one  time.  This  raises  two  of  the  cen- 
tral questions  in  cell  biology  today:  1)  How  are 
genes  turned  on  and  off  so  that  each  cell  type 
expresses  only  its  appropriate  set  of  genes  and 
contains  only  its  correct  complement  of  proteins? 
2)  How  are  the  genes  in  a  given  cell  regulated,  so 
that  the  cell  can  respond  appropriately  to  outside 
influences  by  changing  either  the  pattern  of 


Introduction 


Figure  1.  A  small  section  of  a  typical  mammalian  cell  as  seen  in  the  electron  microscope. 
Part  of  the  nucleus  (N)  with  its  surrounding  membrane  and  nucleolus  (Nu )  are  shown  in 
the  upper  right;  the  cytoplasm  that  occupies  the  rest  of  the  electron  micrograph  contains 
several  different  organelles,  including  several  mitochondria  (M),  parts  of  the  Golgi  com- 
plex (GC),  and  the  rough  endoplasmic  reticulum  (RER).  Magnification  approximately 
50,000  X. 

Micrograph  provided  by  David  D.  Sabatini. 


Introduction 


Figure  2.  From  genes  and  chromosomes  to  cells,  organs,  and  entire  bodies. 

Adapted  from  an  illustration  by  Warren  Isensee  for  The  Chronicle  of  Higher  Educa- 
tion, September  3,  1986,  with  permission  from  the  publisher. 


Introduction 


X 


Figure  3-  A  schematic  representation  of  the  X  chro- 
mosome showing  the  location  of  the  characteristic 
banding  patterns  and  the  distribution  of  several  of 
the  genes  that  have  been  localized  to  this 
chromosome. 

Excerpt  from  Stephens,  J.C.,  Mador,  M.L.,  Cava- 
naugh,  M.L.,  Gradie,  M.I.,  andKidd,  K.K.  The  Human 
Genome  Map.  Science  250:  October  12,  1990.  Copy- 
right 1990  by  the  AAAS. 


INTRON 


EXON  INTKON 
(CODING  (NONCODING 
REGION)  REGION) 
,  1  , 


PNA- 


1 


■  2B    3    ■   4   ■  5 


rUANSC:RII'TION 


CAP 


PRIMARY 
niRNAl 
TRANSt:HIIT 


I'OLY  A 


INTRONS  ARE  CUT  OUT  AND 
COOING  REGIONS  ARE 
SI'LIGED  HX;ETHtR 


MATURE  inRNA  TRANSCRIH  1  |fl  TT 


Figure  4.  The  primary  RNA  transcript  of  a  gene  is  very  much  larger  than  the  message  that  is 
actually  translated  to  form  a  protein.  A  variable  number  of  noncoding  sequences  called  introns 
are  edited  out,  and  the  remaining  exons  are  spliced  together  by  a  complex  mechanism  shown  in 
the  drawings  to  the  left. 

From  Dugaiczyk,  A.,  Woo,  S.L.C.,  Colbert,  D.A.,  Lai,  B.C.,  Mace,  M.L.,  Jr.,  and  O'Malley,  B. 
1979.  Proc  Natl  Acad  Sci  USA  76:2253-2257. 


OCOCOCl I  % 


Introduction 


DNA 


mRNA 


RNA  POLYMERASE 


FORMING 
PROTEIN  CHAIN 


FORMING 
PROTEIN 


Figure  5.  Transcription  of  a  gene  by  the  enzyme  RNA  polymerase  and  the  subsequent  translation  of  this  messenger  RNA  ( mRNA ) 
by  ribosomes  to  form  a  protein. 

From  Raven,  P.H.,  and  Johnson,  G.B.  1988.  Understanding  Biology.  St.  Louis,  MO:  Times  Mirror /Mosby  College  Publishing, 
p.  267. 


EXTRACELLULAR 
SPACE 


apical  plasma 
membrane 


transport 
vesicle 


Golgi 
apparatus 


transport 
vesicle 


membrane-bound 
polyribosomes 
in  ER 


Figure  6.  The  synthesis  and  intracellular 
trafficking  of  proteins  from  the  endoplas- 
mic reticulum  through  the  Golgi  apparatus 
to  the  cell  membrane. 

From  Alberts,  B.,  Bray,  D.,  Lewis,  J.,  Raff, 
M.,  Roberts,  K.,  and  Watson,  J. D.  1989.  Mo- 
lecular Biology  of  the  Cell,  2nd  edition.  New 
York:  Garland,  p.  455. 


basal  plasma  membrane 


Introduction 


genes  it  uses  (and  thus  the  kinds  of  proteins  it 
produces)  or  the  amounts  of  each  protein  it 
makes  (Figure  5)?  In  the  next  section  we  describe 
the  considerable  progress  that  has  been  made 
recently  in  deciphering  the  DNA  sequence  ele- 
ments that  determine  whether  a  gene  is  tran- 
scribed. Proteins,  commonly  known  as  tran- 
scriptional factors,  bind  to  these  DNA 
sequence  elements  and  determine  when,  and 
how  frequently,  a  gene  is  transcribed.  Since  this 
often  influences  how  much  of  the  encoded  pro- 
tein is  produced  by  a  cell,  such  information  pro- 
vides one  level  of  insight  about  how  cells  differ 
from  one  another. 

Another  large  issue  concerns  the  way  in  which 
proteins  are  deployed  in  the  cell.  A  cell  is  not  just 
a  bag  of  randomly  distributed  molecules;  it  has  a 
highly  organized  internal  structure.  The  DNA  that 
makes  up  the  genes  and  the  machinery  for  gene 
transcription  are  packaged  in  the  nucleus,  which 
is  surrounded  by  a  membrane  with  distinctive 
pores  that  separates  it  from  the  cytoplasm, 
which  comprises  the  rest  of  the  cell.  Nearly  all 
cellular  functions  are  compartmentalized  in 
other  cellular  structures  collectively  referred  to 
as  organelles.  Among  the  more  prominent  or- 
ganelles in  animal  cells  are  the  mitochondria, 
which  are  the  cells'  principal  energy  source;  ly- 
sosomes,  which  are  concerned  largely  with  the 
degradation  of  foreign  materials  and  of  cellular 
proteins  whose  functions  have  been  fulfilled;  the 
rough  endoplasmic  reticulum,  a  complex,  ri- 
bosome-studded  system  of  membranes  responsi- 
ble for  the  synthesis  of  proteins  secreted  by  the 
cells;  and  the  Golgi  apparatus,  which  both  mod- 
ifies proteins  (by  adding  other  chemical  groups 
such  as  sugars)  and  also  packages  them  for  trans- 
port to  their  appropriate  locations,  such  as  the 
cell  membrane.  Most  of  these  organelles  are 
themselves  surrounded  by  membranes  that  sepa- 
rate their  functions  from  those  of  the  rest  of  the 
cell.  It  is  easy  to  see  how  such  a  compartmental- 
ized structure  allows  the  cell  to  organize  its  dif- 
ferent processes  efficiently,  but  it  poses  an  organi- 
zational problem  that  is  of  considerable  current 
interest  in  cell  biology:  How  are  particular  pro- 
teins routed  to  the  correct  organelles?  As  we  be- 
gan to  learn  about  the  structures  of  individual 
proteins,  it  was  discovered  that  there  are  specific 
"signals"  built  into  proteins  that  target  them  to 
particular  organelles  or  particular  locations 
within  the  cell  and  that  there  are  distinctive  cel- 
lular machineries  that  "detect"  these  signals  and 
steer  the  proteins  in  particular  directions  (Figure 


6).  Thus  certain  proteins  are  directed  to  the  nu- 
cleus, while  others  are  targeted  for  insertion  into 
the  surface  membrane  of  the  cell,  and  yet  others 
are  destined  for  export  out  of  the  cell  as  secretory 
products. 

At  another  level  of  organization,  it  has  become 
evident  that  the  organelles  within  a  cell  are  also 
not  distributed  randomly.  In  many  cells  one  can 
identify  a  distinct  "top"  and  "bottom."  Other 
cells,  while  not  polarized  in  this  manner,  have 
asymmetric  structures  arranged  in  such  a  way  that 
given  organelles  are  distributed  in  different,  but 
quite  reproducible,  patterns.  Still  other  cells,  es- 
pecially nerve  cells,  have  unusual  extensions  or 
processes  that  may  be  many  hundred  times  as 
long  as  the  body  of  the  cell.  In  each  case  organ- 
elles have  to  be  transported  to  particular  loca- 
tions and  maintained  there;  they  do  not  drift 
about  haphazardly  inside  the  cell. 

The  asymmetric  shapes  of  cells  and  the  loca- 
tions of  their  organelles  both  rely  on  cellular 
structures  known  collectively  as  the  cytoskele- 
ton.  The  cytoskeleton  consists  of  several  types  of 
elongated  threads  or  filaments  (microfilaments, 
microtubules,  intermediate  filaments),  each 
made  of  specific  proteins  that  are  so  designed  as 
to  assemble  spontaneously  into  filaments.  These 
cytoskeletal  elements  serve  as  a  form  of  internal 
scaffolding  to  maintain  the  shape  of  the  cell,  and 
as  a  system  of  tracks  along  which  organelles  can 
be  transported.  Recent  research  has  disclosed  a 
variety  of  proteins  that  function  as  molecular  mo- 
tors that  can  move  proteins  and  organelles  that 
attach  to  them  along  particular  cytoskeletal  fila- 
ments to  various  locations  within  the  cell.  We 
also  know  that  the  appropriate  organization  of 
cytoskeletal  filaments  and  motor  proteins  can,  in 
some  cases,  contribute  to  cell  motility,  that  is, 
the  movement  of  the  entire  cell  from  one  loca- 
tion to  another.  Such  cellular  movements  are  es- 
pecially important  in  development  but  continue 
to  play  an  integral  role  in  the  life  of  certain  cells 
even  in  adult  life. 

All  these  processes — gene  transcription,  pro- 
tein targeting,  organelle  movement,  and  cell  mo- 
tility— must  be  carefully  regulated  so  that  cells 
respond  appropriately  to  different  situations.  The 
same  is  true  of  many  other  cellular  processes.  For 
example,  the  proliferation  of  cells  that  takes 
place  by  cell  division  involves  copying  or  repli- 
cating the  genes,  the  breakdown  of  the  nuclear 
membrane,  the  separation  of  the  duplicated 
chromosomes  into  two  equivalent  sets,  divi- 
sion of  the  cell  into  two  daughter  cells,  the  re- 


Introduction 


formation  of  a  nucleus  in  each  of  the  daughter 
cells,  and  finally  the  resumption  of  normal  func- 
tion in  both  cells.  This  whole  process,  which  is 
known  as  the  cell  cycle,  takes  place  whenever 
cells  divide  and  remains  an  important  part  of  the 
life  of  all  but  a  few  cell  types.  Many  types  of  cells 
^for  example,  the  cells  in  the  blood  and  skin — 
are  continually  being  formed  and  replaced. 
Other  cells  proliferate  rarely,  and  some  divide 
only  during  early  development;  the  nerve  cells 
that  make  up  the  brain,  for  example,  proliferate 
rapidly  during  development,  but  no  further  cell 
division  occurs  before  death  of  the  individual 
some  70  or  more  years  later.  Obviously,  cell  pro- 
liferation must  be  tightly  controlled.  Recent  ad- 
vances have  shown  that  the  cell  cycle  is  con- 
trolled by  a  set  of  proteins  whose  role  is  to  modify 
other  proteins  selectively  and  thus  regulate  their 
functions.  One  common  way  in  which  proteins 
are  modified  (but  certainly  not  the  only  one)  is  to 
attach  a  small  chemical  group,  such  as  a  phos- 
phate group,  to  a  protein.  Proteins  that  attach 
phosphate  groups  to  other  proteins  are  called 
protein  kinases.  Control  of  many  aspects  of  the 
cell  cycle  and,  indeed,  of  many  other  cellular 
functions  relies  on  complex  control  networks  of 
protein  kinases  acting  on  key  proteins  at  pivotal 
stages  in  the  life  of  the  cell. 

Thus  far  we  have  considered  only  processes  oc- 
curring within  a  cell.  An  important  related  set  of 
issues  concerns  how  cells  interact  with  each 
other  and  how  they  respond  to  the  external  envi- 
ronment. Each  cell  is  surrounded  by  a  surface 
(or  plasma)  membrane,  which  serves  as  a  se- 
lective barrier  separating  the  inside  of  the  cell 
from  the  world  outside  itself.  Embedded  in  this 
membrane  are  several  types  of  proteins.  One  es- 
sential class  are  transporters,  specialized  for 
the  ordered  movement  in  and  out  of  the  cell  of 
nutrients,  ions  and  other  small  molecules  that  are 
essential  for  normal  cell  function. 

A  second  group  of  cell  surface  proteins  are  re- 
ceptors, which  bind  other  types  of  molecules 
that  interact  with  the  cell.  As  the  name  suggests, 
receptors  serve  to  receive  input  from  the  cell's 
external  environment.  They  are  of  many  different 
types.  The  largest  group  binds  peptide  hormones 
or  diffusible  factors  produced  locally  or  at  a  dis- 
tance by  other  cells,  but  another  important  group 
serves  to  transport  materials  like  cholesterol  from 
outside  to  the  interior  of  the  cell.  Typically  these 
receptors  have  three  parts:  an  external  part  or 
ligand-binding  domain  that  can  bind  the  hor- 
mone or  diffusible  factor,  a  transmembrane  do- 


main that  spans  the  cell  membrane,  and  an  intra- 
cellular part  that  can  interact  with  internal 
components  of  the  cell.  The  binding  of  a  hor- 
mone or  other  diffusible  factor  to  such  a  receptor 
triggers  in  some  way,  as  yet  undetermined,  a  sig- 
nal inside  the  cell.  These  signals  are  of  many 
types.  Some  receptors  are  protein  kinases  that  are 
selectively  activated  by  binding  the  appropriate 
external  factor;  others,  when  activated,  lead  to 
the  release  of  diffusible,  small  molecules,  such  as 
calcium  ions  or  cyclic  nucleotides.  These  diffusi- 
ble second  messenger  molecules  in  turn  acti- 
vate other  control  mechanisms  inside  the  cell, 
including  protein  kinases  and  other  regulatory 
molecules.  In  this  way  the  triggering  of  a  recep- 
tor from  outside  cells  can  result  in  a  cascade  of 
events  that  ultimately  controls  the  various  intra- 
cellular processes  discussed  earlier.  One  of  the 
current  "hot  topics"  in  cell  biology  research 
concerns  the  nature  and  mechanisms  of  cell  sur- 
face receptor  signaling  and  the  control  circuits 
inside  cells  that  link  receptor  activity  to  other 
control  mechanisms,  including  those  that  regu- 
late gene  function  and  the  control  of  the  cell 
cycle. 

A  special  subset  of  this  class  of  receptors  are 
those  that  respond  to  the  release  of  chemical  sig- 
nals (transmitters)  at  the  specialized  endings  of 
nerve  cell  processes  (Figure  7) .  The  released  neu- 
rotransmitters bind  to  the  external  part  of  the  cell 
surface  receptor  and  in  doing  so  may  open  an  ion 
channel  or  trigger  the  activation  of  a  second  in- 
tracellular message.  Since  the  majority  of  nerve 
signals  are  transmitted  from  cell  to  cell  in  this 
way,  the  analysis  of  this  class  of  receptors  is  (as 
we  shall  see  in  the  section  on  neuroscience) 
one  of  the  central  issues  in  contemporary 
neuroscience. 

Another  class  of  cell  surface  receptors  is  in- 
volved in  the  adhesion  of  cells,  either  to  their 
neighbors  or  to  the  extracellular  matrix,  a 
complex  group  of  secreted  proteins  and  polysac- 
charides that  assemble  into  an  organized  mesh- 
work  on  the  cell  surface.  Depending  upon  the 
cell  type  and  environment,  the  extracellular  ma- 
trix performs  various  functions  (Figure  8).  In  a 
petri  dish,  for  example,  the  extracellular  matrix 
provides  a  cushion  on  which  the  cell  sits.  In  the 
epidermis,  the  extracellular  matrix  helps  to  form 
the  basement  membrane,  which  anchors  the  epi- 
dermis to  the  rest  of  the  skin.  In  connective 
tissues,  the  extracellular  matrix  completely 
surrounds  most  cells  and  is  often  more  extensive 
in  its  distribution  than  the  cells  themselves.  In 


OCOCOC  l^l 


Introduction 


postsynapik:  ce)l 

Action  potential 
triggers  entry  of 
calcium  ions  (Ca^*) 
into  the  presynaptic 
terminal. 


Synaptic  vesicles 
fuse  withi  tfie  presynaptic 
membrane,  releasing 
transmitter. 


Tfie  transmitter  binds  to  proteins 
in  tfie  postsynaptic  membrane, 
changing  their  conformation  and 
either  opening  an  ion  channel  or 
activating  a  second  messenger 
system  v^/ifhin  the  postsynaptic  cell. 


The  transmitter  is  removed 
from  the  synaptic  cleft,  and 
the  postsynaptic  receptors 
revert  to  their  original 
conformation. 


Figure  7.  A  summary  of  the  essential  events  at  a  chemical  synapse  following  the  arrival  of  an  action  potential  in  the  axon 
terminal. 

Adapted  from  Alberts,  B.,  Bray,  D.,  Lewis,  J.,  Raff,  M.,  Roberts,  K.,  and  Watson,  J. D.  1989-  Molecular  Biology  of  the  Cell,  2nd 
edition.  New  York:  Garland,  p.  1077. 


Figure  8.  A:  A  section  of  human  skin  stained  with  an  anti 
body  against  the  protein  keratin.  Note  how  the  cells  are 
packed  together  to  form  the  distinctive  structure  of  skin.  The 
dark  structures  within  each  cell  are  the  nuclei  that  do  not 
contain  keratin  and  so  are  not  labeled. 

B:  A  fluorescence  micrograph  showing  the  presence  of  kera- 
tin filaments  in  cultured  skin  cells.  The  keratin  filaments 
have  been  revealed  by  a  fluorescently  labeled  antibody 
against  the  keratin  protein. 

Courtesy  of  Elaine  V.  Fuchs. 


xxxvii 


Introduction 


this  case,  the  extracellular  matrix  helps  to  pro- 
vide the  body's  architectural  framework. 

Cellular  adhesion,  which  plays  a  crucial  role 
in  cell,  tissue,  and  organ  structure  and  in  cell 
movements,  depends  on  specialized  cell  adhe- 
sion receptors  that  are  connected  to  the  intracel- 
lular cytoskeleton.  It  is  also  likely  that  cells  can 
signal  to  one  another  via  cell  adhesion  receptors. 
Decisions  as  to  whether  a  cell  remains  stationary, 
or  where  and  when  it  should  move,  and  what 
shape  it  should  take  up,  are  all  affected  by  cell 
adhesion  events.  And  these,  in  turn,  are  largely 
dependent  on  the  pattern  and  functions  of  cell 
adhesion  receptors  deployed  on  the  surfaces  of 
the  cells. 

Elucidating  the  normal  functions  of  cells  is  an 
important  first  step  in  understanding  how  these 
processes  go  awry  in  a  number  of  human  diseases . 
For  example,  it  is  now  known  that  alterations  in 
certain  normal  genes  (called  oncogenes)  can 
contribute  to  cancer.  It  is  also  clear  that  many 
oncogenes  encode  proteins  that  are  involved  in 
the  regulation  of  specific  cellular  functions: 
some  oncogene-encoded  proteins  are  growth  fac- 
tors; others  are  cell  surface  receptors;  yet  others 
are  signaling  molecules,  protein  kinases,  and 


transcription  factors.  Many  other  human  diseases 
are  known  to  be  due  to  defects  of  one  or  another 
of  the  cellular  processes  reviewed  above.  Thus 
disturbances  in  insulin  production  lead  to  dia- 
betes mellitus,  defects  in  the  extracellular  matrix 
can  cause  osteogenesis  imperfecta,  and  abnormal- 
ities in  cell  adhesion  receptors  may  result  in 
various  bleeding  disorders.  Indeed,  one  of  the 
major  insights  in  pathology  and  medicine  is  that 
all  disease  processes  are  ultimately  attributable 
to  the  breakdown  of  one  or  more  cellular  func- 
tions. In  the  twenty-first  century,  we  can  be  fairly 
certain  that  medicine  will  be  concerned  largely 
with  the  identification  and  treatment  of  specific 
disordered  cell  functions.  We  already  know  of 
many  disorders  that  can  be  attributed  to  distur- 
bances in  particular  organelles.  What  is  particu- 
larly encouraging  to  researchers  in  cell  biology 
today  is  that  new  avenues  are  beginning  to  be 
perceived  for  therapy,  as  the  molecular  bases  of 
various  disordered  functions  become  known.  In- 
deed, one  of  the  especially  appealing  aspects  of 
modern  cell  biological  research  is  the  imme- 
diacy with  which  fundamental  research  advances 
are  having  an  impact  on  medically  important 
problems. 


Investigators  in  the  Cell  Biology  and  Regulation  Program 


Alexander-Bridges,  Maria  C,  M.D.,  Ph.D. 

Beach,  David  H.,  Ph.D. 

Bennett,  G.  Vann,  M.D.,  Ph.D. 

Beutler,  Bruce  A.,  M.D. 

Bevilacqua,  Michael  P.,  M.D.,  Ph.D. 

Blackshear,  Perry  J.,  M.D.,  D.Phil. 

Blobel,  Gunter,  M.D.,  Ph.D. 

Bonadio,  Jeffrey  F.,  M.D. 

Brugge,  Joan  S.,  Ph.D. 

Campbell,  Kevin  P.,  Ph.D. 

Caron,  Marc  G.,  Ph.D.' 

Carroll,  Sean  B.,  Ph.D. 

Chin,  William  W.,  M.D. 

Crab  tree,  Gerald  R.,  M.D. 

Craig,  Nancy  L.,  Ph.D. 

Cunningham,  James  M.,  M.D. 

Davis,  Laura  I.,  Ph.D. 

Davis,  Roger  J.,  M.D. 

Dreyfuss,  Gideon,  Ph.D. 

Emr,  Scott  D.,  Ph.D. 

Esmon,  Charles  T.,  Ph.D. 

Exton,  John  H.,  M.D.,  Ph.D. 

Fuchs,  Elaine,  Ph.D. 

Ganem,  Donald  E.,  M.D. 

Garbers,  David  L.,  Ph.D. 

Gething,  Mary-Jane  H.,  Ph.D. 

Glomset,  John  A.,  M.D. 


Gomer,  Richard  H.,  Ph.D. 
Grosschedl,  Rudolf,  Ph.D. 
Habener,  Joel  F.,  M.D. 
Heintz,  Nathaniel,  Ph.D. 
Hynes,  Richard  O.,  Ph.D. 
Isberg,  Ralph  R.,  Ph.D. 
Kaback,  H.  Ronald,  M.D. 
Kim,  Peter  S.,  Ph.D. 
Kirkegaard,  Karla  A.,  Ph.D. 
Kobilka,  Brian  K,  M.D. 
Lai,  Michael  M.-C,  M.D.,  Ph.D. 
Lamb,  Robert  A.,  Ph.D.,  Sc.D. 
Lefkowitz,  Robert /.,  M.D. 
Lehmann,  Ruth,  Ph.D. 
Maas,  Richard  L.,  M.D.,  Ph.D. 
Mailer,  James  L.,  Ph.D. 
Massague,  Joan,  Ph.D. 
McKnigbt,  Steven  Lanier,  Ph.D. 
Nusse,  Roel,  Ph.D. 
O'Donnell,  Michael  E.,  Ph.D. 
Parker,  Keith  L.,  M.D.,  Ph.D. 
Pike,  Linda  J.,  Ph.D. 
Sadler,  J.  Evan,  M.D.,  Ph.D. 
Schekman,  Randy  W.,  Ph.D. 
Schoolnik,  Gary  K.,  M.D. 
Shenk,  Thomas  E.,  Ph.D. 
Sherr,  Charles  J.,  M.D.,  Ph.D. 


xxxviii 


Introduction 


Simpson,  Larry,  Ph.D. 
Spradling,  Allan  C,  Ph.D. 
Steiner,  Donald  P.,  M.D. 
Welsh,  Michael  J.,  M.D. 
Williams,  Lewis  T.,  M.D.,  Ph.D. 
Wilson,  James  M.,  M.D.,  Ph.D. 

Genetics  Program 

The  emergence  of  what  is  sometimes  referred 
to  as  the  "new  genetics"  has  contributed  more  to 
our  fundamental  understanding  of  biology  and 
medicine  in  the  past  two  decades  than  any  other 
advance  in  biomedical  science.  Not  surprisingly, 
this  development  has  come  to  assume  a  central 
place  in  all  biological  research,  making  it  possi- 
ble to  examine  biological  processes  at  a  level  of 
resolution  that  was  considered  quite  impossible 
just  20  years  ago.  "Gene  cloning,"  "recombinant 
DNA  technology,"  "genetic  engineering,"  and 
the  "Human  Genome  Initiative"  are  phrases  that 
have  entered  everyday  language,  but  the  possibili- 
ties they  offer  for  major  advances  in  biology  and 
medicine  have  yet  to  be  fully  appreciated.  Given 
the  central  role  of  genetics  in  modern  biology 
and  the  current  sense  of  excitement  that  the  new 
genetics  has  generated,  it  is  appropriate  that  the 
Genetics  Program  should  be  the  largest  research 
program  within  HHMI . 

Historically,  one  of  the  first  applications  of 
Mendel's  classic  laws  of  inheritance  was  to  the 
analysis  of  certain  human  diseases.  In  the  early 
years  of  this  century,  Archibald  Garrod,  an  En- 
glish physician,  noted  that  a  number  of  relatively 
rare  diseases  tended  to  occur  in  families,  often  in 
families  with  consanguineous  marriages.  The  pat- 
tern of  occurrence  of  these  rare  diseases  followed 
Mendel's  laws,  discovered  almost  50  years  ear- 
lier. Mendel's  work  on  inherited  characteristics 
in  plants  was  neglected  for  many  years,  but  after  it 
was  rediscovered,  around  the  turn  of  the  century, 
it  was  quickly  established  that  the  laws  of  genet- 
ics are  universal  and  govern  inheritance  in  organ- 
isms as  disparate  as  peas  and  worms,  mice  and 
fruit  flies,  bacteria  and  human  beings.  Beginning 
with  Garrod,  these  genetic  laws  were  applied  to  a 
host  of  inherited  diseases,  as  it  was  realized  that 
the  genetic  makeup  of  an  individual  can  have  a 
profound  effect  on  his  or  her  health  and  well- 
being.  However,  an  understanding  of  what  genes 
are  and  how  they  function  had  to  await  the  discov- 
ery, in  1944,  that  the  genetic  material  is  DNA, 
and,  in  1953  and  1961,  of  the  double  helix  and 
the  genetic  code,  respectively. 

Much  of  our  early  understanding  of  the  action 


Wolin,  Sandra  L.,  M.D.,  Ph.D. 
Yanagisawa,  Masashi,  M.D.,  Ph.D. 

*  This  investigator  was  appointed  after  manu- 
scripts were  submitted  for  publication.  His  re- 
search will  be  described  in  the  next  volume. 


of  genes  came  from  experiments  that  took  advan- 
tage of  the  universality  of  gene  action  by  using 
simple  organisms,  especially  bacteria  and  their 
viruses,  as  model  systems.  The  cardinal  discovery 
was  the  identification  of  deoxyribonucleic  acid 
(DNA)  as  the  fundamental  chemical  in  which  ge- 
netic information  is  encoded.  But  the  finding  that 
DNA  has  a  double-stranded,  mirror  image  struc- 
ture provided  the  first  clear  insight  as  to  how  this 
information  could  be  replicated  and  passed  on 
from  one  generation  of  organisms  to  the  next  (Fig- 
ures 9  and  10).  Understanding  how  the  chemical 
language  of  DNA  could  be  used  to  direct  the  syn- 
thesis of  other  cellular  constituents,  especially 
proteins,  came  with  the  discovery  of  the  nature  of 
the  genetic  code.  These  great  advances  will  al- 
ways be  viewed  as  the  high  watermark  of  the  early 
molecular  stage  of  modern  genetics. 

Notwithstanding  these  dramatic  developments 
— arguably  the  most  important  in  biology  since 
the  publication  of  Darwin's  essay  On  the  Origin 
of  Species  in  1859 — the  molecular  details  of  the 
genes  of  higher  organisms  remained  hidden  from 
view  by  the  enormous  complexity  of  their  ge- 
nomes (Figure  11).  Fortunately,  the  genes  of  sim- 
ple organisms  were  accessible,  because  they  are 
relatively  few  in  number  (involving  in  many 
cases  an  assemblage  of  as  few  as  3,000  base 
pairs,  as  the  building  blocks  of  DNA  are  called) 
compared  to  the  human  genome,  which  probably 
contains  about  3  billion  base  pairs. 

The  problem  of  genetic  complexity  has  been 
finally  overcome  in  the  past  1 0  years  by  the  devel- 
opment of  the  powerful  new  genetic  methods 
known  collectively  as  recombinant  DNA  tech- 
nology. This  technology  allows  researchers  to 
isolate  specific  genes  from  complex  mixtures, 
to  prepare  them  in  sufficiently  large  amounts 
that  their  entire  molecular  structure  can  be  de- 
termined, and  to  move  them  from  one  group  of 
cells  or  from  one  organism  to  another,  so  that 
their  functional  properties  can  be  identified  and 
their  products  produced  in  abundance.  In  a  num- 
ber of  cases,  medically  valuable  products  such 
as  human  insulin,  growth  hormone,  antihemo- 
philic factor,  erythropoietin,  and  TPA  (tissue 


rV^  4  fV* 
»Ar  fcA'  »vV 


Introduction 


sugar-phosphate 
backbone 


Figure  9-  The  famous  DNA  dou- 
ble helix. 

Prom  Alberts,  B.,  Bray,  D., 
Lewis,  J.,  Raff,  M.,  Roberts,  K., 
and  Watson,  J.D.  1989.  Molecu- 
lar Biology  of  the  Cell,  2nd  edi- 
tion. New  York:  Garland,  p.  99. 


Figure  10.  An  illustration  of  how  DNA  is  repli- 
cated so  as  to  form  two  new  strands  that  are 
exactly  complementary  to  the  original  DNA 
sequences  from  which  they  derive  their 
sequences. 

From  Raven,  P.H.,  and  Johnson,  G.B. 
1988.  Understanding  Biology.  St.  Louis,  MO: 
Times  Mirror /Mosby  College  Publishing, 
p.  277. 


xl 


chromosome  of  1 50,000,000  nucleotide  pairs,  containing  about  3000  genes 


0.5%  of  chromosome,  containing  15  genes 


one  gene  of  100,000  nucleotide  pairs 


1^ 


regulatory  region 


intron 


exon 

DNA  TRANSCRIPTION 


primary  RNA  transcript 


RNA  SPLICING 
3' 


mRNA 

Figure  11.  The  organization  of  genes  on  a  typical  vertebrate  chromosome.  Proteins 
that  bind  to  the  DNA  in  regulatory  regions  determine  whether  a  gene  is  transcribed; 
although  often  located  on  the  5'  side  of  a  gene,  as  shown  here,  regulatory  regions  can 
also  be  located  in  introns,  in  exons,  or  on  the  3'  side  of  a  gene.  The  intron  sequences 
are  removed  from  the  primary  RNA  transcripts  that  encode  protein  molecules  to 
produce  a  messenger  RNA  (mRNA )  molecule.  The  figure  given  here  for  the  number  of 
genes  per  chromosome  is  only  a  minimal  estimate. 

From  Alberts,  B.,  Bray,  D.,  Lewis,  J.,  Raff,  M.,  Roberts,  K.,  and  Watson,  J.D.  1989. 
Molecular  Biology  of  the  Cell,  2nd  edition.  New  York:  Garland,  p.  487. 


Introduction 


Normal  hemoglobin  beta  chain 


Vol 


ine 


Histidi 


me 


Leucine 


Th 


reonme 


Prol 


ine 


Sickl  e-cell  anemia  hemoglobin  beta  chain 


Vali 


ne 


Histidi 


ne 


Leucine 


Th 


reonme 


Proli 


ne 


Glutamic 
acid 


Vol 


ine 


Glutamic 
acid 


Glutamic 
acid 


Figure  12.  A  drawing  of  the  hemoglo- 
bin molecule  and  the  single-amino 
acid  change  in  the  P-chain  of  the  mole- 
cule that  causes  sickle  cell  anemia. 

From  Raven,  P.H.,  and  Johnson, 
G.B.  1988.  Understanding  Biology.  St. 
Louis,  MO:  Times  Mirror/Mosby  Col- 
lege Publishing,  p.  25 5. 


Figure  13.  We  can  determine  where  particular  genes 
are  being  expressed  by  using  a  radioactively  labeled 
probe  that  selectively  binds  to  the  UNA  transcript  of 
the  gene  and  examining  the  distribution  of  the  radio- 
activity in  a  tissue  autograph.  This  technique,  known 
as  in  vitro  hybridization,  was  used  in  this  case  to  show 
the  expression  of  one  of  the  keratin  genes  in  a  hair 
follicle.  The  micrograph  on  the  left  is  viewed  under 
darkfield  optics  ( the  exposed  silver  grains  in  the  auto- 
radiograph  appear  as  white  dots );  that  on  the  right  is 
seen  with  conventional  optics,  and  the  radioactivity  is 
marked  by  black  dots. 

Courtesy  of  Elaine  V.  Fuchs. 


xlii 


Introduction 


plasminogen  activator)  have  been  commercially 
manufactured  in  this  way  and  are  now  being  used 
therapeutically. 

Most  of  these  accomplishments  depend  on  the 
technique  commonly  referred  to  as  gene  clon- 
ing. In  this  process  a  gene  (for  example,  the  hu- 
man insulin  gene,  which  is  a  few  thousand  base 
pairs  in  length)  is  isolated  from  a  complex  mix- 
ture of  human  DNA  fragments  and  transferred 
into  the  genetic  apparatus  of  a  much  simpler  or- 
ganism such  as  a  bacterium.  Since  bacteria  multi- 
ply very  rapidly,  the  inserted  human  insulin  gene 
is  amplified  (cloned)  along  with  the  genes  of  its 
simple  host.  It  is  then  a  relatively  straightforward 
process  to  purify  the  cloned  gene  and  to  deter- 
mine the  sequence  of  nucleotides  that  encodes 
the  relevant  genetic  instructions. 

One  of  the  most  important  developments  to 
emerge  from  gene  cloning  and  sequencing  is 
that  they  permit  geneticists  to  determine  exactly 
how  a  gene  may  have  been  altered  (genetic  alter- 
ations of  this  kind  are  called  mutations)  to  pro- 
duce an  inherited  disease.  While  a  majority  of  the 
investigators  in  the  HHMI  Genetics  Program  are 
directing  their  efforts  toward  understanding  the 
principles  that  govern  the  action  of  genes,  a  large 
and  growing  number  are  trying  to  identify  the 
mutations  responsible  for  some  of  the  estimated 
4,000  human  genetic  defects.  Among  the  genes 
being  studied  are  those  responsible  for  mus- 
cular dystrophy,  cystic  fibrosis,  several  forms 
of  hemoglobinopathy,  chronic  granulomatous 
disease,  phenylketonuria,  polyposis  coli,  neuro- 
fibromatosis, osteogenesis  imperfecta,  and  hemo- 
philia. Most  of  the  genes  responsible  for  these 
diseases  have  already  been  cloned  and  their 
structures  determined;  in  this  way  the  precise  mo- 
lecular effects  of  the  mutations  that  cause  the  dis- 
orders are  now  being  determined.  Information 
that  has  been  derived  in  this  way  is  being  used  in 
several  ways.  For  example,  in  some  cases  it  is  be- 
ing used  to  counsel  affected  families  about  the 
risks  they  face  in  having  additional  children;  in 
others  it  is  being  used  to  develop  tests  that  are 
critical  for  prenatal  diagnosis,  and,  in  the  case  of 
hemophilia  and  chronic  granulomatous  disease, 
it  is  already  being  used  to  provide  appropriate 
therapy.  Information  about  some  of  these  genes 
and  their  protein  products  has  also  told  us  a  great 
deal  about  the  role  of  the  relevant  proteins  in 
development  (e.g.,  the  protein  dystrophin, 
which  is  normally  found  in  muscle  but  is  missing 
in  most  cases  of  muscular  dystrophy)  or  about  the 
mechanisms  required  for  normal  function  (e.g.. 


in  the  lungs  and  pancreas  in  cystic  fibrosis,  or  for 
the  mechanisms  of  blood  clotting  in  hemo- 
philia). It  is  the  continuous  interplay  between 
the  development  of  new  basic  knowledge  and  its 
application  to  the  understanding  of  human  dis- 
ease that  is  so  dramatically  informing  modern 
medicine. 

The  execution  of  a  genetic  program  in  a  cell 
obviously  cannot  be  left  to  chance.  To  bring 
about  an  ordered  series  of  changes  in  a  develop- 
mental or  metabolic  process,  each  genetic  in- 
struction must  be  activated  at  a  specific  time,  and 
the  product  of  the  gene  must  be  produced  in  an 
amount  commensurate  with  the  needs  of  the  or- 
ganism. As  was  pointed  out  in  the  section  on  cell 
biology,  different  sets  of  genes  are  expressed  in 
each  cell  type  so,  for  example,  many  of  those  ex- 
pressed in  muscle  cells  are  likely  to  be  quite  dif- 
ferent from  those  expressed  in  skin  cells  or  in 
bone.  Since  with  few  exceptions  (one  of  which 
will  be  discussed  in  the  section  on  immunology) 
each  cell  contains  the  same  genetic  information, 
there  must  be  some  mechanism  or  mechanisms 
that  insure  that  the  appropriate  genes  are  being 
expressed  in  each  cell  type.  The  processes  that 
govern  these  crucial  steps  are  generally  referred 
to  as  gene  regulation. 

Once  again,  the  study  of  inherited  human  dis- 
eases has  revealed  mutations  that  can  disrupt 
those  regulatory  programs.  For  example,  it  is  now 
known  that  certain  inherited  anemias  are  brought 
about  by  specific  mutations  that  affect  the  regula- 
tory apparatus  of  the  genes  that  encode  the  red 
blood  cell  protein,  hemoglobin  (Figure  12).  An 
even  larger  number  of  examples  could  be  cited 
from  work  on  simpler  organisms  to  show  how 
powerful  this  mutational  analysis  has  been  for 
our  understanding  of  genetic  regulation.  How- 
ever, it  will  suffice  to  say  here  that  the  work  of  a 
considerable  number  of  HHMI  investigators  has 
this  as  its  primary  theme. 

As  discussed  in  the  section  on  cell  biology  and 
regulation,  genetic  information  is  usually  con- 
veyed through  a  series  of  steps  from  the  nucleus 
of  the  cell  to  the  surrounding  cytoplasm,  where  it 
is  decoded  or  translated  to  form  the  protein  prod- 
ucts that  execute  the  relevant  genetic  program. 
This  process  involves  making  many  copies  of  the 
gene,  in  the  closely  related  chemical,  RNA.  The 
process  of  copying  a  gene  from  DNA  into  RNA  is 
called  transcription.  This  is  one  of  the  key 
points  at  which  the  flow  of  genetic  information 
can  be  regulated.  Genes  that  are  not  scheduled 
for  expression  in  a  particular  cell  type  are  not 


xliii 


Introduction 


transcribed:  while  still  present,  they  remain  si- 
lent and  ineffective. 

Since  it  plays  such  a  central  role  in  regulating 
the  expression  of  genetic  information,  it  is  not 
surprising  that  the  study  of  transcription  is  one  of 
the  most  active  areas  in  modern  genetic  research 
(Figure  13).  Investigators  have  taken  two  success- 
ful approaches  in  an  effort  to  understand  this  pro- 
cess. One  involves  the  study  of  mutations  that 
disrupt  regulation  in  a  variety  of  organisms.  The 
other  is  biochemical  and  involves  isolating  spe- 
cific proteins  that  are  parts  of  the  transcription 
machinery  and  then  determining  their  mode  of 
action  at  a  molecular  level.  The  ultimate  goals  of 
both  approaches  are  to  provide  a  complete  ac- 
count, in  chemical  terms,  of  the  processing  of 
genetic  information  and  to  provide  a  sound  basis 
for  our  understanding  of  the  types  of  disturbances 
in  this  process  that  can  lead  to  various  genetic 
defects  and  many  diseases  in  adult  life. 

It  is  perhaps  worth  commenting  on  the  broad 
spectrum  of  organisms  that  are  currently  used  as 
models  in  genetic  investigations.  Yeast,  worms, 
fruit  flies,  bacteria,  viruses,  mice,  and  human  be- 
ings all  provide  instructive  paradigms.  For  exam- 
ple, because  the  structure  of  the  nervous  system 
is  relatively  well  understood  in  the  primitive 
worm,  Caenorhabditis  elegans,  it  has  proved  to 
be  particularly  useful  for  elucidating  the  genetic 
program  that  directs  the  formation  of  the  nervous 
system.  The  fruit  fly  Drosophila  tnelanogaster, 
which  has  an  uncommonly  rich  background  of 
genetic  variation,  has  proved  to  be  the  most  valu- 
able organism  for  studying  the  genes  involved  in 
early  embryonic  development.  Similarly,  the 
mouse,  with  a  generation  time  of  just  a  few 
months,  is  a  convenient  stand-in  for  genetic  ex- 
periments that  cannot  be  contemplated  in  most 
mammals  or  human  beings.  Thus  it  has  become 
an  everyday  technique  to  introduce  new  genes 
into  the  genetic  makeup  of  mice.  Many  of  the 
resulting  animals,  called  transgenic  mice,  have 
proved  to  be  powerful  models  for  certain  human 
diseases.  Transgenic  mice  that  are  genetically 
cancer-prone  or  that  have  sickle  cell  anemia  have 
been  generated  recently  and  are  proving  useful 
not  only  for  understanding  the  molecular  bases  of 
these  disorders  but  also  for  exploring  possible 
means  for  their  treatment.  Complementing  this 
approach  has  been  the  development  of  tech- 
niques that  allow  one  to  knock  out  specific  genes 
selectively  and  to  generate  mice  of  essentially  any 
specified  genotype.  The  power  of  this  new  meth- 
odology (that  involves  homologous  recombi- 


nation) for  understanding  mammalian  develop- 
ment, for  studying  the  function  of  the  nervous 
system,  and  for  modeling  known  human  diseases 
has  already  captured  the  attention  of  many  genet- 
icists, including  several  in  the  HHMI  Genetics 
Program.  At  the  other  end  of  the  spectrum,  the 
simple  baker's  yeast,  Saccharomyces  cerevisiae, 
is  proving  to  be  especially  useful  for  cloning 
large  segments  of  mammalian  chromosomes.  Spe- 
cial carrier  elements  called  YACs  (yeast  artificial 
chromosomes)  have  been  developed  that  can  ac- 
commodate up  to  500,000  base  pairs  of  DNA. 
Since  this  is  more  than  20  times  as  large  as  the 
fragments  that  have  traditionally  been  cloned  in 
bacteria  and  viruses,  this  approach  and  the  asso- 
ciated method  for  separating  large  DNA  fragments 
are  beginning  to  play  an  important  part  in  the 
international  effort  to  map  the  human  genome. 

The  development  of  a  complete  map  of  the 
human  genome  is  one  of  the  great  challenges 
engaging  the  attention  of  geneticists  worldwide, 
including  several  HHMI  investigators.  Such  a  map 
will  permit  the  identification  of  genes  that  are 
close  to,  or  responsible  for,  a  large  number  of  as 
yet  uncharacterized  genetic  diseases.  Further- 
more, knowledge  of  the  map  will  allow  the  devel- 
opment of  easily  identifiable  genetic  markers  for 
specific  diseases.  These  genetic  markers,  called 
restriction  fragment  length  polymorphisms 
(RFLPs),  can  be  used  to  detect  carriers  of  many 
genetic  disorders,  to  determine  paternity,  and  to 
identify  individuals  for  forensic  purposes  (a  pro- 
cess commonly  referred  to  in  the  press  as  DNA 
fingerprinting) . 

Four  or  five  years  ago  an  extremely  sensitive 
technique  for  detecting  these  DNA  marker  frag- 
ments was  developed.  This  technique  (the  poly- 
merase chain  reaction  [PGR])  greatly  amplifies 
the  genetic  signal  and  has  made  these  studies 
both  simpler  and  faster.  With  this  and  other 
emerging  technologies,  the  structures  of  the  ge- 
nomes of  several  simple  bacteria  should  be 
known  in  the  near  future,  and  the  way  is  already 
clear  to  begin  the  systematic  structural  analysis  of 
the  genomes  of  a  number  of  more  complex  organ- 
isms, such  as  the  nematode  worm  C.  elegans  and 
the  fruit  fly  Drosophila.  It  is  confidently  pre- 
dicted that  with  further  improvements  in  the 
technology  for  sequencing  genes,  the  complete 
structure  of  the  human  genome  should  be  known 
within  12  or  15  years. 

While  the  new  genetics  has  opened  the  door  to 
an  understanding  of  an  important  range  of  funda- 
mental problems  such  as  the  mechanism  of  ch«-o- 


xliv 


Introduction 


mosome  replication,  the  genetic  basis  of  embry- 
onic development,  and  the  control  of  gene 
regulation,  it  also  holds  promise  for  an  assault  on 
the  even  more  complex  problems  that  confront 
those  interested  in  human  disease.  Cancer  has  a 
clear  genetic  basis.  Specific  genes,  generally  those 
concerned  with  cell  growth  and  gene  regulation, 
have  been  shown  to  be  involved  in  many  different 
malignancies.  Other  disorders,  such  as  heart  dis- 
ease and  hypertension,  have  clear  genetic  compo- 
nents. Even  distressing  behavioral  disorders  like 
manic  depression  and  schizophrenia  have  a  genetic 
basis  that  may  offer  important  clues  to  their  nature 
and  perhaps,  in  time,  to  their  treatment. 

The  real  power  of  the  new  genetics  is  that  it 

Investigators  in  the  Genetics  Program 

Barsh,  Gregory  S.,  M.D.,  Ph.D. 
Beachy,  Philip  A.,  Ph.D. 
Beaudet,  Arthur  L.,  M.D. 
Bell,  Graeme  I.,  Ph.D. 
Bellen,  Hugo  J.,  D.V.M.,  Ph.D. 
Belmont,  John  W.,  M.D.,  Ph.D. 
Brown,  Patrick  O.,  M.D.,  Ph.D. 
Capecchi,  Mario  R.,  Ph.D. 
Caskey,  C.  Thomas,  M.D. 
Cech,  Thomas  R.,  Ph.D. 
Church,  George  M.,  Ph.D. 
Cohen,  Stephen  M.,  Ph.D. 
Collins,  Francis  S.,  M.D.,  Ph.D. 
Corden,  Jeffry  L.,  Ph.D. 
Cullen,  Bryan  R.,  Ph.D. 
Desiderio,  Stephen  V.,  M.D.,  Ph.D. 
Desplan,  Claude,  Ph.D. 
Donelson,  John  E.,  Ph.D. 
Duyk,  Geoffrey  M.,  M.D.,  Ph.D. 
Feinberg,  Andrew  P.,  M.D.,  M.P.H. 
Francke,  Uta,  M.D. 
Friedman,  Jeffrey  M.,  M.D.,  Ph.D. 
Geliebter,  Jan,  Ph.D. 
Gesteland,  Raymond  F.,  Ph.D. 
Ginsburg,  David,  M.D. 
Gitschier,  Jane  M.,  Ph.D. 
Henikoff,  Steven,  Ph.D. 
Norwich,  Arthur  L.,  M.D. 
Kadesch,  Thomas  R.,  Ph.D. 
Kan,  Yuet  Wai,  M.D.,  D.Sc. 
Kaufman,  Thomas  C,  Ph.D. 
Kunkel,  Louis  M.,  Ph.D. 
Kurnit,  David  M  ,  M.D  ,  Ph.D. 
Laimins,  Laimonis  A.,  Ph.D. 
Lalouel,  Jean-Marc,  M.D.,  D.Sc. 
Leder,  Philip,  M.D. 
Ledley,  Fred  D.,  M.D. 
Liebhaber,  Stephen  A.,  M.D. 
Lindquist,  Susan  L.,  Ph.D. 


allows  investigators  to  approach  these  complex 
problems  using  a  reductionist  approach.  If,  for 
instance,  a  gene  contributes  to  the  development 
of  a  behavioral  disorder,  it  is  reasonable  to  as- 
sume that  it  also  plays  an  important  role  in  pro- 
gramming normal  behavior.  By  identifying  such 
genes  and  understanding  the  chemistry  of  their 
protein  products,  we  should  obtain  important  in- 
sights about  the  biological  basis  of  behavior. 
Thus  the  ultimate  promise  of  the  new  genetics  is 
that  it  will  help  us  to  understand  diseases  that  are 
not  commonly  thought  of  as  being  primarily  ge- 
netic and,  in  doing  so,  it  will  tell  us  much  about 
those  biological  processes  that  lie  at  the  very  core 
of  our  humanity. 


Lowe,  John  B.,  M.D. 

Malim,  Michael  H.,  Ph.D.* 

Nabel,  Gary  J.,  M.D.  Ph.D. 

Nadal-Ginard,  Bernardo,  M.D.,  Ph.D. 

Nathans,  Daniel,  M.D. 

Nathans,  Jeremy,  M.D.,  Ph.D. 

Nevins,  Joseph  R.,  Ph.D. 

Nussbaum,  Robert  L.,  M.D. 

Olson,  Maynard  V.,  Ph.D. 

Or  kin,  Stuart  H.,  M.D. 

Overbeek,  Paul  A.,  Ph.D. 

Page,  David  C,  M.D. 

Palmiter,  Richard  D.,  Ph.D. 

Perrimon,  Norbert,  Ph.D. 

Reeders,  Stephen  T.,  M.D. 

Rosbash,  Michael,  Ph.D. 

Sakonju,  Shigeru,  Ph.D. 

Seidman,  Jonathan  G.,  Ph.D. 

Singh,  Harinder,  Ph.D. 

Soriano,  Philippe  M.,  Ph.D.,  D.Sc. 

Steitz,  Joan  A.,  Ph.D. 

Sternberg,  Paul  W.,  Ph.D. 

Sukhatme,  Vikas  P..  M.D.,  Ph.D. 

Taub,  Rebecca  A.,  M.D. 

Thummel,  Carl  S.,  Ph.D. 

Tilghman,  Shirley  M.,  Ph.D. 

Tjian,  Robert,  Ph.D. 

Valle,  David  L.,  M.D. 

Warren,  Stephen  T.,  Ph.D. 

Weintraub,  Harold  M.,  M.D.,  Ph.D. 

White,  Raymond  L.,  Ph.D. 

Williams,  David  A.,  M.D. 

Woo,  Savio  L.  C,  Ph.D. 

Young,  Michael  W.,  Ph.D. 

*  This  investigator  was  appointed  after  manu- 
scripts were  submitted  for  publication.  His  re- 
search will  be  described  in  the  next  volume. 


xlv 


Introduction 


Immunology  Program 

One  of  the  most  important  developments  that 
occurred  during  the  evolution  of  vertebrates  was 
the  emergence  of  protective  mechanisms  that  en- 
able animals  to  defend  themselves  against  inva- 
sion by  foreign  organisms,  especially  viruses,  bac- 
teria, and  a  number  of  disease-causing  parasites. 
These  defenses  employ  tw^o  distinct  but  related 
strategies:  the  first  detects,  v^^ith  exquisite  sensi- 
tivity, the  presence  of  foreign  organisms,  cells,  or 
proteins;  the  second  involves  a  series  of  mecha- 
nisms that  act  cooperatively  to  rid  the  body  of  the 
invading  organisms  or,  at  the  least,  to  neutralize 
their  potentially  harmful  effects.  The  task  of  ef- 
fecting both  strategies  falls  to  the  immune 
system. 

Recognizing  the  strategic  importance  of  the 
immune  system  in  both  health  and  disease,  the 
Institute  selected  Immunology  to  be  one  of  its 
earliest  research  programs.  The  wisdom  of  that 
decision  has  been  amply  borne  out  by  the  truly 
remarkable  progress  that  has  been  made  in  immu- 
nology in  the  past  two  decades.  With  the  notable 
exception  of  molecular  genetics,  no  field  of  bio- 
medical research  has  witnessed  such  an  astonish- 
ing series  of  successes  at  almost  every  level,  from 
understanding  the  immune  system's  unique  rec- 
ognition mechanisms  to  the  elucidation  of  the 
cellular  and  chemical  means  used  to  destroy  or 
neutralize  invading  organisms. 

The  body's  initial  line  of  defense  against  inva- 
sion by  foreign  organisms  is  the  continuously  pa- 
trolling system  of  macrophages  and  other  types 
of  blood-borne  phagocytic  cells  that  act  both  as 
an  early  warning  system  and  as  a  "first-strike"  de- 
fense. These  cells  respond  by  ingesting  and 
breaking  up  the  invading  organisms  and  by 
releasing  soluble  signaling  molecules  like 
interleukin-1  that  serve,  among  other  things,  to 
mobilize  the  next  line  of  defense,  the  immune 
response  (Figure  14).  This  response  involves 
two  classes  of  lymphocytes,  called  T  and  B  ceils, 
reflecting  their  origin  from  the  thymus  and  bone 
marrow,  respectively. 

The  first  step  jn  the  immune  response  is  the 
activation  of  a  special  subclass  of  T  lympho- 
cytes called  helper  T  cells.  Macrophages  pre- 
sent fragments  of  foreign  proteins,  or  antigens, 
on  their  surfaces.  Recognition  of  these  antigens 
by  specialized  receptors  found  on  helper  T  cells 
then  initiates  the  two  responses:  a  cell-mediated 
immune  response  and  a  humoral  immune  re- 
sponse. The  cell-mediated  response  involves 
principally  the  stimulation  of  another  subclass  of 


T  lymphocytes  called  cytotoxic  T  cells  that  rec- 
ognize and  destroy  infected  cells.  The  humoral 
response,  on  the  other  hand,  involves  the  activa- 
tion of  the  second  major  class  of  lymphocytes,  the 
B  cells,  to  produce  circulating  antibodies.  Anti- 
bodies recognize  and  neutralize  soluble  antigens 
and  mark  cells  or  viruses  bearing  antigens  for  de- 
struction by  phagocytic  cells. 

One  of  the  central  problems  in  immunology 
concerns  the  way  in  which  lymphocytes  recog- 
nize antigens.  The  complexity  of  this  problem 
may  be  gauged  from  the  observation  that  humans 
and  other  higher  vertebrates  are  capable  of  form- 
ing antibodies  against  virtually  any  molecule  or 
part  of  a  molecule  (epitope),  including  even 
those  that  do  not  occur  naturally  but  are  chemi- 
cally synthesized  in  a  laboratory.  How  does  this 
occur?  And  how  does  the  immune  system  distin- 
guish foreign  molecules  from  those  produced  by 
its  own  cells?  In  a  word,  how  do  lymphocytes 
distinguish  self  from  non-self? 

The  key  to  the  first  issue,  as  we  now  know,  is  to 
be  found  in  the  almost  unlimited  variety  of  re- 
ceptors on  the  surfaces  of  lymphocytes.  The  dis- 
covery of  how  just  a  few  hundred  genes  are  capa- 
ble of  producing  such  extraordinary  receptor 
diversity  is  one  of  the  great  success  stories  of  mod- 
ern immunology.  The  essential  features  of  the  im- 
mune system's  capacity  for  generating  molecular 
diversity  can  be  summarized  briefly  by  stating 
that  lymphocyte  receptors  are  formed  by  pairs 
of  protein  chains  that  are  chemically  linked  to 
form  a  complex  receptor  structure.  Each  chain  of 
the  pair  has  a  constant  domain  and  a  variable  do- 
main. The  variable  domain  of  the  two  chains  is 
responsible  for  antigen  recognition  and  the  dis- 
crimination between  self  and  non-self.  The  con- 
stant (invariant)  domain  is  physically  linked  to 
other  membrane  proteins  of  the  receptor  com- 
plex that  activates  the  lymphocyte's  internal  sig- 
naling and  effector  mechanisms.  T  and  B  cells 
triggered  via  their  antigen  receptors  respond  to 
auxiliary  signaling  molecules  by  proliferating 
and  differentiating  to  a  mature  effector  stage.  In 
the  case  of  B  cells,  the  maturation  process  ulti- 
mately results  in  the  generation  of  plasma  cells 
that  produce  large  amounts  of  antibody  for  secre- 
tion into  bodily  fluids,  chiefly  the  bloodstream. 

The  complex  structure  of  the  variable  parts 
of  the  receptors  is  due  to  several  processes. 
First,  and  most  important,  the  genes  responsi- 
ble for  this  portion  of  the  receptor  are  assembled 
from  a  large  number  of  different  gene  segments 


xlvi 


Introduction 


Pathogens  Humoral  immunity 


Lymphokines  Lysis 


Figure  14.  When  a  pathogen  invades  the  body,  the  immune  system  responds  with  three  types  of  reaction.  The  cells  of  the 
humoral  immune  system  (B  cells )  secrete  antibodies  that  can  bind  to  the  pathogen.  Cells  of  the  cellular  system  (T  cells )  carry 
out  two  major  types  of  functions.  One  type  of  T  cell  (the  cytotoxic  T  cells,  CTL )  develops  the  ability  to  kill  pathogen-infected 
cells.  Helper  T  cells,  on  the  other  hand,  secrete  factors  (lymphokines)  that  stimulate  the  body's  overall  response. 

From  Molecular  Cell  Biology,  2nd  edition.  James  Darnell,  Harvey  Lodish,  and  David  Baltimore,  p.  1005.  Copyright  ®  1990 
by  Scientific  American  Books,  Inc.  Reprinted  with  permission  by  W.H.  Freeman  and  Company. 


xlvii 


Introduction 


Vh  segments  (hundreds) 


D  segments  (-20) 


Jh  segments  (4) 


Germ-line 
organization 


iVH 


First  rearrangement 


1  Iv.i    ioi  iol..! 

|jh 

N 


Second  rearrangement 


Vh 


Jh  IJh 


E 


N  N 

Figure  15.  The  specificity  of  antibodies  and  the  receptors  on  T  cells  is  brought  about  by  a  series  of  complex 
rearrangements  of  various  gene  segments  like  those  shown  in  this  figure. 

From  Molecular  Cell  Biology,  2nd  edition.  James  Darnell,  Harvey  Lodish,  and  David  Baltimore,  p.  1027. 
Copyright  ®  1990  by  Scientific  American  Books,  Inc.  Reprinted  with  permission  by  W.H.  Freeman  and 
Company. 


Figure  16.  A:  A  molecular  model  of  an  antibody  mol- 
ecule. Each  amino  acid  is  represented  by  a  small 
sphere.  The  heavy  chains  are  colored  blue,  the  light 
chains  red.  The  four  chains  wind  about  one  another 
to  form  a  Y  shape,  with  two  identical  antigen-bind- 
ing sites  at  the  arms  of  the  Y  and  a  tail  region  that 
serves  to  direct  the  antibody  to  a  particular  portion 
of  the  immune  response. 

B:  A  schematic  drawing  of  an  antibody  molecule. 
Each  molecule  is  composed  of  two  identical  light  (L) 
chains  and  two  identical  heavy  (H)  chains.  Carbo- 
hydrate is  sometimes  associated  with  the  H  chain. 
While  the  antigen- binding  sites  are  formed  by  a  com- 
plex of  both  H  and  L  chains,  the  tail  region  is  formed 
by  H  chains  alone. 

From  Raven,  P.H.,  and  Johnson,  G.B.  1988.  Under- 
standing Biology.  St.  Louis,  MO:  Times  Mirror/Mosby 
College  Publishing,  p.  692. 


xlviii 


Introduction 


THYMUS 


Precursors 


Hemopoietic 
Stem  Cell 


CD3/TCR/S 

o 

/ 


Epithelial 

Stromal  CD3/TCRa/3 
Cells 


CDS/TCR/S 

^(~\-  ySTCell 


— ^rnn  + 


Cytotoxic  T  Cell 


\       CD3/TCRa/3  X^-^CD8- 
(J) (^^^^^f^-=^  ^  CD3/TCR  0/3 

CD4,8+   Helper  T  Cell 


CD4' 
^~.CD8± 


IgM  +  lgD  igM 

a— o^(s>^ 


Natural  Killer  Cell 
Antibodies 


Stromal  Cell 


sIgM IgM+lgD+IgG,  2  30,4  '9Gi,2.3or4 
cllis^       ^         \\lgm+lgD+lgA,<„2     IgA,  or 


Fetal  Liver 
Bone  Marrow 


igM 


igG, 


2.3  or  4 


IgM+lgD+lgE 


IgA,  or  2 


Mature  B  Cells 


Plasma  Cells 


Figure  17.  Contemporary  model  of  immune  system  development  in  mammals.  T  and  B  cells,  which  form  the  major 
recognition  and  effector  limbs  of  the  immune  system,  are  derived  from  multipotent  stem  cells  that  also  give  rise  to  the 
other  types  of  blood  cells.  Definition  of  the  T  cell  receptors  and  accessory  cell  surface  molecules  has  allowed  the 
identification  of  two  major  sublineages  of  the  thymus-derived  T  cells,  each  of  which  expresses  a  different  type  of  T  cell 
receptor  and  exhibits  specialized  function.  The  B  lineage  cells,  generated  in  hemopoietic  tissues,  initially  express  one 
class  of  antibody  receptors  called  immunoglobulin  M  ( IgM ),  but  may  then  switch  to  the  expression  of  other  antibody 
classes,  each  of  which  has  special  biological  advantages.  Note  the  third  lineage  of  lymphocytes,  the  natural  killer 
cells.  They  are  the  most  recently  recognized  members  of  the  lymphocyte  family,  but  may  well  prove  to  be  the  most 
phylogenetically  ancient  elements  in  this  constellation  of  cells  involved  in  host  defense. 
Courtesy  of  Max  D.  Cooper. 


Introduction 


Figure  18.  A  schematic  representation  of 
how  the  thymus  gland  selects  T cells  that  are 
able  to  recognize  foreign  but  not  self 
proteins. 

From  Molecular  Cell  Biology,  2nd  edition. 
James  Darnell,  Harvey  Lodish,  and  David 
Baltimore,  p.  1037.  Copyright  ®  1990  by 
Scientific  American  Books,  Inc.  Reprinted 
with  permission  by  W.H.  Freeman  and 
Company. 


Bone-marrow  cells 


Rearrange  gene  segments  to  make 
a  unique  T-cell  receptor  (TCR) 
for  the  cell  surface 


TCR 


Thymus 
gland 


Into  the 
selection 
system 


Rejected:  TCR  binds  to 
self-MHC  plus  self-peptides 
(negative  selection) 


Rejected:  TCR  does  not  bind 
tightly  enough  to  self-MHC 
(positive  selection) 


Accepted  (-5%  of  cells):  TCR 
appropriate  to  recognize  foreign 
peptides  bound  to  self-MHC 


j  I  To  peripheral  lymphoid 

y  J  system 


Introduction 


(Figure  15).  Each  gene  segment  exists  in  several 
— and  in  some  cases  hundreds — of  different  cop- 
ies. These  segments  randomly  recombine  to  form 
new  genes  that  encode  the  virtually  limitless  re- 
pertoire of  recognition  elements.  To  take  just  one 
example,  T  cells  form  their  receptors  by  combin- 
ing a  number  of  different  gene  sequences:  V 
(variable),  D  (diversity),  and  J  (joining)  seg- 
ments. From  this  array  any  given  T  cell  derives  1 
from  about  100  possible  V  segments,  1  from 
about  6  D  segments,  and  1  from  about  50  J  seg- 
ments to  form  its  so-called  a-  or  heavy  chain, 
and  about  1  in  20  V,  1  in  2  D,  and  1  in  12  J 
segments  to  form  its  light  chain.  The  random 
recombination  of  V,  D,  and  J  segments  in  the  two 
chains  can  thus  code  for  literally  millions  of  dif- 
ferent possible  receptor  structures.  The  receptors 
on  B  cells  are  formed  in  basically  the  same  way, 
although  the  numbers  of  V,  D,  and  J  segments 
available  for  selection  and  recombination  differ 
somewhat.  Antibodies  (which  are  secreted  by 
plasma  cells)  are  generated  in  much  the  same 
way  as  their  receptors,  and  have  the  same  almost 
unlimited  capacity  for  diversity  (Figure  1 6) . 

At  one  time  it  was  thought  that  antigens  were 
capable  of  shaping  the  structure  of  lymphocyte 
receptors  and  antibodies  so  that  the  binding  sites 
of  receptors  and  antibodies  would  mold  them- 
selves in  some  way  to  fit  the  shape  of  the  antigen, 
much  as  a  rubber  glove  molds  itself  to  fit  one's 
hand.  We  now  know  that  this  "instructional"  hy- 
pothesis is  wrong.  Rather,  as  indicated  above,  the 
immune  system  produces  very  large  numbers  of 
different  types  of  receptors  and  antibodies,  and 
collectively  these  can  "fit"  essentially  every  pos- 
sible antigen  (Figure  1 7) .  Each  T  or  B  cell  bears 
only  one  type  of  receptor  on  its  surface  (although 
there  are  thousands  of  receptor  molecules  of  that 
given  structure  on  each  cell).  In  the  same  way, 
each  B  cell  secretes  antibodies  of  only  a  single, 
defined  structure.  Thus  the  capacity  of  the  body 
to  respond  to  an  enormous  variety  of  different 
antigens  is  due  to  the  existence  of  an  enormous 
number  of  different  T  or  B  cells,  each  able  to  rec- 
ognize a  single  antigen  (or  more  commonly,  a 
part  of  a  complex  antigen  known  as  an  antigenic 
determinant).  And  when  the  cell  recognizes 
and  binds  to  an  antigen,  it  responds  by  proliferat- 
ing to  form  a  large  number  of  cells  of  the  same 
type.  Such  a  population  of  cells,  all  derived  from 
a  single  progenitor,  is  known  as  a  clone,  and  the 
hypothesis  put  forward  to  account  for  the  selec- 
tive proliferation  of  lymphocytes  of  particular  re- 
ceptor type  in  response  to  a  specific  antigen  is 


known  as  the  clonal  selection  hypothesis.  This 
theory,  first  advanced  by  Sir  F.  Macfarlane  Burnet, 
has  withstood  every  test  and  is  rightly  viewed  as 
one  of  the  cornerstones  of  modern  immunology. 

The  intriguing  question  as  to  how  the  cells  of 
the  immune  system  distinguish  foreign  mole- 
cules from  those  on  the  surface  of  the  cells  of 
their  own  host  was  also  addressed  by  Burnet.  He 
suggested — and  there  is  now  a  large  body  of  evi- 
dence to  support  this  view — that  lymphocytes 
that  recognize  the  body's  own  tissues  (so-called 
self  antigens)  are  selectively  eliminated  during 
early  development — a  process  known  as  clonal 
deletion.  The  mechanisms  responsible  for  clonal 
deletion  of  T  and  B  cells  are  still  under  investiga- 
tion, but  at  least  for  the  T  cells  it  appears  that 
during  development  the  thymus  may  actively  se- 
lect, for  export  to  the  rest  of  the  immune  system, 
only  those  T  cells  that  are  capable  of  functioning 
in  the  host  (Figure  18).  These  "useful"  T  cells 
are  allowed  to  survive  and  mature,  while  the  po- 
tentially harmful  cells  die  and  are  removed.  This 
process  results  in  the  death  of  about  90  percent 
or  more  of  the  T  cells  that  are  initially  formed. 
The  bone  marrow  selection  of  B  cells  for  survival 
may  be  equally  stringent.  Although  this  seems  an 
astonishingly  wasteful  process,  comparable  cell 
deaths  are  known  to  be  a  rather  common  feature 
in  the  development  of  virtually  all  organs  and  of 
all  multicellular  organisms. 

For  reasons  that  remain  to  be  elucidated,  in 
some  conditions — commonly  referred  to  as  au- 
toimmune disorders — the  immune  system  may 
mistakenly  mount  an  attack  on  components  of  the 
host  organism's  own  cells.  For  example,  the  neu- 
rological condition  myasthenia  gravis  involves 
the  production  of  circulating  antibodies  directed 
against  the  receptor  molecules  on  the  surfaces  of 
muscle  cells  that  normally  enable  them  to  re- 
spond to  the  release  of  the  neurotransmitter  ace- 
tylcholine from  the  motor  nerves.  When  the  re- 
ceptor molecules  are  damaged  or  destroyed, 
there  is  a  progressive  loss  of  neuromuscular  con- 
trol, and  if  the  respiratory  muscles  are  involved 
the  condition  may  be  fatal.  Similarly,  type  I  or 
juvenile-onset  diabetes  is  now  known  to  be  due 
to  the  combined  attack  of  T  cells  and  antibodies 
directed  against  the  /S-cells  of  the  pancreas  that 
normally  produce  insulin,  the  hormone  that  regu- 
lates sugar  metabolism. 

Another  topic  of  considerable  current  interest 
in  immunology  concerns  antigen  presenta- 
tion. We  now  know  that  this  involves  a  complex 
set  of  genes  called  the  major  histocompatibil- 


li 


Introduction 


ity  complex  (MHC).  The  membrane  proteins 
encoded  by  these  genes  (of  which  there  are  two 
types  called  class  I  and  class  II)  are  able  to  selec- 
tively bind  short  segments  of  partially  digested 
protein  antigens,  termed  peptides.  These  pep- 
tides, arising  from  protein  breakdown  inside  the 
cell,  reach  the  surface  of  the  cell  together  with 
the  relevant  MHC  molecule.  Recent  x-ray  crystal- 
lographic  studies  indicate  that  the  peptide  anti- 
gen is  lodged  within  a  distinctive  groove  on  the 
outer  surface  of  the  MHC  molecule,  where  it  can 
be  detected  by  a  lymphocyte  bearing  the  appro- 
priate receptor.  The  receptors  on  T  cells  are  spe- 
cialized to  recognize  antigens  only  in  the  form  of 
such  a  peptide: MHC  complex.  There  are  acces- 
sory molecules  on  the  surfaces  of  T  cells,  called 
CD4  and  CDS,  that  are  selectively  expressed  on 
cells  that  recognize  antigens  presented  by  MHC  II 
and  MHC  I  molecules,  respectively.  Functionally 
these  accessory  molecules  form  part  of  the  T  cell 
receptor  for  peptide:MHC  complexes  by  binding 
to  both  the  MHC  molecule  and  the  T  cell  recep- 
tor. For  this  reason  CD4  and  CDS  are  sometimes 
called  co-receptors. 

Like  the  antigen  receptors  on  B  and  T  cells, 
MHC  molecules  show  considerable  diversity. 
However,  this  diversity  is  not  due  to  the  recombi- 
nation of  different  gene  segments  but  rather  to 
genetic  polymorphism.  There  may  be  as  many  as 
100  different  genetic  sequences  (alleles)  at  a 
single  MHC  locus,  and  T  cells  are  selected  during 
development  only  if  they  can  recognize  peptides 
presented  by  self  MHC  molecules.  How  this  oc- 
curs is  unknown,  but  its  role  in  T  cell  selection  is 
obviously  fundamental. 

The  CD4-bearing  T  cells  (also  called  T4  cells, 
helper  lymphocytes,  or  CD4^  cells)  have  become 
widely  known  because  of  their  role  in  the  devel- 
opment of  AIDS  (acquired  immune  defi- 
ciency syndrome) .  The  virus  that  causes  AIDS 
— the  human  immunodeficiency  virus  (or 
HIV) — selectively  invades  these  cells,  because 
the  CD4^  molecule  fortuitously  serves  also  as  a 
specific  receptor  for  the  virus  (Figure  19).  On 
entering  the  CD4^  T  cells,  the  genetic  material  of 
the  virus  (which  is  formed  of  RNA)  is  reverse 
transcribed  into  DNA,  and  this,  in  turn,  becomes 
integrated  into  the  T  cell's  own  genome.  In  this 
way  the  virus  subverts  the  cell's  genetic  machin- 
ery and,  when  activated,  the  cell  produces  more 
and  more  virus,  until  ultimately  the  cell  is  killed. 
When  the  cell  dies  it  releases  virus  into  the  bodily 
fluids,  where  it  is  free  to  invade  other  CD4^  T 
cells,  and  the  whole  process  may  be  repeated  un- 


til the  entire  T  cell  population  is  effectively  de- 
pleted. Since,  as  we  have  seen,  T  helper  cells  are 
essential  for  mounting  both  cell-mediated  and 
humoral  immune  responses,  patients  with  AIDS 
become  progressively  more  vulnerable  to  all 
forms  of  infection  and  commonly  succumb  to  op- 
portunistic infections  that  would  normally  be  eas- 
ily overcome. 

A  second  important  component  of  the  immune 
system  is  the  complement  system,  which  con- 
sists of  a  complex  series  of  proteins  in  the  serum 
and  in  cell  membranes  (Figure  20).  These  pro- 
teins perform  essential  roles  in  the  immune  re- 
sponse to  foreign  organisms  such  as  bacteria  and 
viruses,  and  in  the  response  to  tumors.  Deficien- 
cies of  any  of  the  complement  proteins  may 
lead  to  diseases,  including  those  that  involve 
infection,  hemolysis  of  red  blood  cells,  or 
autoimmune  diseases  such  as  systemic  lupus 
erythematosus. 

The  complement  system  is  activated  by  two 
general  mechanisms.  First,  antibodies  (Ab)  can 
activate  complement  when  they  bind  their  anti- 
gen (Ag).  In  addition  to  this  so-called  classical 
pathway  there  is  an  alternative  pathway  that  is 
continuously  active  at  a  low  level  marking  for- 
eign organisms  for  which  there  are  no  preformed 
antibodies  available. 

In  addition  to  these  roles,  complement  pro- 
teins help  to  regulate  the  immune  system  by  an- 
other mechanism.  This  involves  the  interaction  of 
specific  activated  complement  protein  fragments 
with  receptors,  or  binding  proteins,  that  are  on 
the  surface  of  immune  system  cells.  These  recep- 
tors allow  for  communication  with  the  interior  of 
the  cell,  and  their  activation  leads  to  a  change  in 
the  function  or  fate  of  the  cell. 

Overall,  the  complement  system  plays  a  funda- 
mental role  in  normal  or  abnormal  immune  re- 
sponses. Current  study  in  this  area  is  directed 
toward  understanding  not  only  the  molecular 
mechanisms  of  complement  activation  and  regu- 
lation but  also  the  general  effects  on  the  immune 
response  of  experimentally  altering  complement 
function. 

The  devastating  consequences  of  AIDS,  con- 
genital immunodeficiency  disorders,  and  the 
frequent  rejection  of  transplanted  organs  have 
made  the  public  increasingly  aware  of  the  im- 
portance of  the  immune  system  in  medical  prac- 
tice. The  development  of  immunosuppressive 
drugs  has  gone  a  long  way  toward  overcoming 
the  problem  of  tissue  rejection,  and  there  is  now 
considerable  interest  in  the  possible  develop- 


lii 


I 


Introduction 


Figure  19-  Stereoview  of  a  region  on  the  macromolecule  CD4,  the  T  cell  receptor  for  HIV.  CD4 
contains  four  tandem  immunoglobulin-like  domains,  D1-D4.  The  view  represents  Dl's  major 
binding  region  for  the  virus  coat  glycoprotein  gpl20.  Atoms  of  residues  41-59  are  shown  in  red. 
They  are  enveloped  by  the  surface  in  contact  with  a  water  molecule  probe.  This  figure  was  produced 
with  the  display  program  QUANTA. 

Reprinted  by  permission  from  Ryu,  S.-E.,  Kwong,  P.D.,  Truneh,  A.,  Porter,  T.G.,  Arthos,/.,  Rosen- 
berg, M.,  Dai,  X.,  Xuong,  N.-h.,  Axel,  R.,  Sweet,  R.W.,  and Hendrickson,  W.A.  Nature  348:419-426. 
Copyright  ®  1990  Macmillan  Magazines  Ltd. 

Biologic 

Activation  Regulation  Effects 


Ag  clearance 
Tumor  killing 
Cellular  activation 
Hemolysis 


fragments 

Figure  20.  The  complement  system.  Ag,  antigen;  Ab,  antibodies. 
Courtesy  of  V.  Michael  Holers. 


liii 


Introduction 


ment  of  vaccines  to  limit  the  spread  of  HIV. 
Many  of  us  remember  how  some  30  years  ago 
poliomyelitis  was  to  all  intents  and  purposes 
eliminated  in  this  country  by  the  development 
of  the  Salk  and  Sabin  vaccines,  and  we  are  all 
conscious  of  the  fact  that  many  illnesses  such  as 
measles,  rubella,  whooping  cough,  and  even 
smallpox  have  been  brought  under  control.  But 
it  is  still  not  widely  appreciated  that  the  immune 
system  is  itself  subject  to  a  number  of  serious 
disorders  such  as  lymphoma  and  leukemia.  Our 

Investigators  in  the  Immunology  Program 

Alt,  Frederick  W.,  Ph.D. 

Atkinson,  John  P.,  M.D. 

Bevan,  Michael  J.,  Ph.D. 

Bjorkman,  Pamela  J.,  Ph.D. 

Bloom,  Barry  R.,  Ph.D. 

Bottomly,  H.  Kim,  Ph.D. 

Chaplin,  David  D.,  M.D.,  Ph.D. 

Cooper,  Max  D.,  M.D. 

Cress  well,  Peter,  Ph.D. 

Davis,  Mark  M.,  Ph.D. 

Fischer  Lindahl,  Kirsten,  Ph.D. 

Flavell,  Richard  A.,  Ph.D. 

Ghosh,  Sankar,  Ph.D. 

Goodnow,  Christopher  C,  B.V.Sc,  Ph.D. 

Holers,  V.  Michael,  M.D. 

Jacobs,  William  R.,  Jr.,  Ph.D. 

Janeway,  Charles  A.,  Jr.,  M.D. 

Kappler,  John  W.,  Ph.D. 

Neuroscience  Program 

Among  the  most  challenging  problems  in  bio- 
medical research  are  those  posed  by  the  human 
brain.  How  do  we  perceive  the  world  around  us? 
How  do  we  learn  from  past  experiences?  How  do 
we  store  and  recall  information  derived  from 
those  experiences?  How  do  we  determine  when 
to  act  and  what  actions  to  carry  out?  What  is 
thought?  And  what  are  the  neural  mechanisms 
that  underlie  language?  In  a  word,  how  are  ail 
those  aspects  of  our  lives  that  most  specifically 
define  our  humanity  instantiated  in  the  function- 
ing of  our  brains?  The  answers  to  these  questions 
still  lie  far  in  the  future,  but  in  the  past  two  de- 
cades considerable  progress  has  been  made  in  our 
understanding  of  some  of  the  cellular  and  molec- 
ular mechanisms  involved  in  brain  function.  Rec- 
ognizing this,  in  1983  the  Institute  initiated  its 
Neuroscience  Program.  Until  recently,  the  Neu- 
roscience Program  has  been  largely  focused  on 
the  ways  in  which  nerve  cells  conduct  signals 


ability  to  deal  with  these  malignant  conditions  is 
still  very  limited,  but  we  are  beginning  to  under- 
stand what  may  cause  them.  While  these  dis- 
orders present  the  most  urgent  challenges  to 
clinical  immunology,  even  relatively  minor  al- 
lergic disorders  continue  to  pose  problems  both 
for  the  practicing  physician  and  for  the  patients 
who  suffer  from  them.  Many  of  the  reports  in  this 
volume  indicate  how  these  and  other  problems 
associated  with  the  immune  system  are  currently 
being  addressed. 


Korsmeyer,  Stanley  J.,  M.D. 
Leiden,  Jeffrey  M.,  M.D.,  Ph.D. 
Littman,  Dan  R.,  M.D.,  Ph.D. 
Loh,  Dennis  Y.-D.,  M.D. 
Marrack,  Philippa,  Ph.D. 
Nussenzweig,  Michel  C,  M.D.,  Ph.D. 
Pay  an,  Donald  G.,  M.D. 
Perlmutter,  Roger  M.,  M.D.,  Ph.D. 
Peterlin,  B.  Matija,  M.D. 
Schatz,  David  G.,  Ph.D. 
Smale,  Stephen  T.,  Ph.D. 
Thomas,  Matthew  L.,  Ph.D. 
Thompson,  Craig  B.,  M.D. 
Tonegawa,  Susumu,  Ph.D. 
Weiss,  Arthur,  M.D.,  Ph.D. 
Weissman,  Irving  L.,  M.D. 
Witte,  Owen  N.,  M.D. 


and  communicate  with  each  other  and  with  the 
effector  tissues  of  the  body  (such  as  muscle  and 
gland  cells)  and  on  the  cellular  mechanisms  in- 
volved in  the  development  of  the  nervous  system. 

Modern  neuroscience  is  founded  on  two  funda- 
mental concepts  that  derive  from  the  late  nine- 
teenth and  the  early  years  of  the  twentieth  cen- 
tury. The  first  of  these,  commonly  referred  to  as 
the  neuron  doctrine,  is  that  the  fundamental 
functioning  units  of  the  nervous  system  are  nerve 
cells,  or  neurons.  Among  the  cells  of  the  body, 
neurons  are  distinguished  anatomically  by  the 
fact  that  they  all  extend  processes  (some  of  con- 
siderable length)  that  are  of  two  general  types: 
shorter  tapering  processes  (dendrites)  that 
mainly  serve  to  receive  information  from  other 
cells,  and  longer  processes  (axons),  of  more  un- 
iform diameter,  that  serve  to  transmit  information 
to  other  parts  of  the  nervous  system  or  to  the  body 
at  large.  The  second  basic  concept  is  that  infor- 


liv 


Introduction 


mation  in  the  nervous  system  is  principally  en- 
coded in  a  series  of  signals  called  nerve  im- 
pulses, or  action  potentials.  These  are  brief, 
usually  all-or-nothing  electrical  changes  in  the 
nerve  cell  membrane  that  are  propagated  along 
the  axons  at  rates  between  about  3  and  400  feet 
per  second.  A  necessary  corollary  of  this  concept 
is  the  notion  that  nerve  cells  communicate  this 
encoded  information  to  each  other  at  specific 
sites  called  synapses,  where  the  axon  of  one  cell 
functionally  interacts  with  the  dendrites  or  the 
bodies  of  other  neurons. 

The  essential  morphological  features  of  neu- 
rons were  established  in  the  1870s  and  1880s, 
with  the  aid  of  a  number  of  selective  staining  pro- 
cedures, notably  the  metallic  impregnation  tech- 
nique developed  by  the  Italian  microscopist  Ca- 
millo  Golgi.  And  the  fundamental  principle  of 
the  neuron  doctrine,  namely,  that  nerve  cells  are 
anatomically  and  functionally  discrete  entities, 
was  convincingly  demonstrated  around  the  turn 
of  the  century  by  the  great  Spanish  neurohistolo- 
gist,  Santiago  Ramon  y  Cajal.  The  biophysical 
mechanisms  responsible  for  the  nerve  impulse 
and  for  synaptic  transmission  were  established  in 
the  early  1950s,  principally  through  the  work  of 
Hodgkin,  Huxley,  Katz,  and  Eccles  (Figures  21, 
22,  and  23).  In  brief,  activation  of  a  nerve  cell 
results  in  the  successive  opening  of  pores  or  ion 
channels  along  the  length  of  the  axon  that  result 
in  the  temporary  reversal  of  the  voltage  between 
the  inside  and  the  outside  of  the  axon  (this  tran- 
sient change  in  potential  is  the  action  potential; 
see  Figure  7) .  When  the  action  potential  reaches 
the  ends  of  the  axon  it  causes  the  release  of  a 
neurotransmitter  that  diffuses  across  the  micro- 
scopic gap  between  the  axon  terminal  and  the 
postsynaptic  cell.  The  binding  of  the  neurotrans- 
mitter to  specialized  receptors  in  the  membrane 
of  the  postsynaptic  cell  in  turn  triggers  a  response 
in  that  cell  which  may  either  be  the  opening  of  an 
ion  channel  or  the  activation  of  a  second  intracel- 
lular messenger  in  the  cell.  In  either  case  the 
binding  of  the  transmitter  to  the  receptor  is  re- 
flected in  the  generation  of  a  graded  voltage 
change  across  the  membrane  of  the  postsynaptic 
cell,  called  a  synaptic  potential.  Depending  on 
the  nature  of  the  transmitter  receptor,  the  re- 
sponse may  be  either  excitatory  or  inhibitory; 
i.e.,  the  postsynaptic  cell  may  either  be  activated 
or  rendered  less  likely  to  discharge  an  impulse. 
Finally,  the  released  neurotransmitter  is  either 
broken  down  by  a  specific  enzyme  within  the 
synaptic  cleft,  or  taken  up  by  selective  transport 


mechanisms  into  the  axon  terminal  (where  it  can 
be  reutilized)  or  into  the  surrounding  nonneural 
(glial)  cells. 

In  the  past  10  years  we  have  learned  a  good 
deal  about  the  molecular  mechanisms  involved 
in  both  impulse  conduction  and  synaptic  trans- 
mission, largely  as  the  result  of  the  successful 
cloning  of  the  genes  for  a  number  of  the  ion  chan- 
nels involved  (e.g.,  for  Na"^,  K"*",  and  Ca'^"'")  and  for 
many  neurotransmitter  receptors  like  those  for 
acetylcholine,  glutamate,  7-aminobutyric  acid 
(GABA),  serotonin,  norepinephrine,  dopamine, 
and  various  neuropeptides.  From  the  nucleotide 
sequence  of  these  genes  it  has  been  possible  not 
only  to  deduce  the  primary  amino  acid  sequence 
of  the  channel  or  transmitter  proteins  (and  from 
this  to  infer  the  probable  arrangement  of  the  rele- 
vant protein  in  the  membrane)  but  also  to  gener- 
ate hybridization  probes  to  identify  other  related 
channels  or  receptors.  And  using  some  of  the  es- 
tablished techniques  of  genetic  engineering,  like 
site-directed  mutagenesis,  it  has  been  possible  in 
some  cases  to  establish  the  regions  within  the 
channel  molecules  that  are  sensitive  to  changes 
in  voltage  (Figure  24),  or  the  ligand-binding  and 
second  messenger-activating  domains  of  recep- 
tors. One  example  will  suffice  to  demonstrate  the 
importance  of  this  approach  to  our  understand- 
ing of  these  fundamental  processes. 

It  has  been  known  for  almost  50  years  that  the 
relatively  simple  molecule  acetylcholine  is  the 
transmitter  at  the  junctional  region  between  mo- 
tor nerve  fibers  and  muscle  cells  and  also  at  cer- 
tain synapses  in  the  brain  and  spinal  cord.  With 
the  discovery  in  the  1970s  that  the  clinical  con- 
dition myasthenia  gravis  (previously  discussed 
in  the  immunology  section)  is  caused  by  circulat- 
ing antibodies  directed  against  the  receptor  for 
acetylcholine  in  the  muscle  membrane,  a  major 
effort  was  mounted  to  purify  and  biochemically 
characterize  the  acetylcholine  receptor  (AChR). 
This  work  served  to  establish  that  the  AChR  con- 
sists of  five  subunits:  two  designated  a,  and  one 
each  called  /3,  7,  and  b. 

In  the  early  1980s  Heinemann,  Patrick,  and 
their  colleagues  succeeded  in  cloning  the  genes 
for  the  a-subunit,  and  in  1983  Numa  and  his  co- 
workers presented  the  complete  nucleotide  se- 
quences encoding  all  four  kinds  of  subunits. 
From  these  sequences  we  gained  several  impor- 
tant insights.  First,  the  four  subunits  showed  a 
high  degree  of  homology,  which  suggested  that 
their  genes  were  probably  derived — by  duplica- 
tion and  divergence — from  a  single  ancestral 


Iv 


Introduction 


cell  body 


dendrites 


axon  (less  than  1/20  inch  to 
more  than  three  feet  in  length) 


terminal  branches  of  axon 


Figure  21.  A  schematic  diagram  of  a  typi- 
cal neuron.  The  arrows  indicate  the  di- 
rection in  which  nerve  signals  are  con- 
veyed. The  largest  axons  in  the  human 
brain  and  spinal  cord  extend  for  about 
three  feet  and  have  a  diameter  of  less 
than  1/1, 000th  of  an  inch.  Many  axons 
are  covered  by  an  insulating  layer 
known  as  the  myelin  sheath.  The  myelin 
sheath  is  interrupted  at  intervals  known 
as  nodes  of  Ranvier. 

From  Alberts,  B.,  Bray,  D.,  Lewis,  J., 
Raff,  M.,  Roberts,  K.,  and  Watson,  J.D. 
1989.  Molecular  Biology  of  the  Cell,  2nd 
edition.  New  York:  Garland,  p.  1061. 


1 


Figure  22.  A  few  of  the  many  types  of  neu- 
rons in  the  vertebrate  nervous  system. 
(S.  Ramon  y  Cajal,  Histologic  du  Systeme 
Nerveux  de  I'Homme  et  des  Vertebres. 
Paris:  Maloine,  1901- 191 1;  reprinted, 
Madrid:  C.S.I.C.,  1972.) 

From  Alberts,  B.,  Bray,  D.,  Lewis,  J., 
Raff,  M.,  Roberts,  K.,  and  Watson,  J.D. 
1989.  Molecular  Biology  of  the  Cell,  2nd 
edition.  New  York:  Garland,  p.  1061. 


Ivi 


Introduction 


Figure  23-  A:  The  anatomical  arrangement  of  a  typical  reflex,  in  this  case  the  knee  jerk.  Each  cell  actually  represents  a  population 
of  many  neurons.  Information  about  stretch  of  the  quadriceps  femoris  muscle  is  conveyed  by  afferent  neurons  to  several  loci 
within  the  central  nervous  system.  In  the  spinal  cord,  afferent  neurons  act  directly  on  the  motor  neurons  to  the  quadriceps  and, 
by  means  of  inhibitory  interneurons,  indirectly  on  the  motor  neurons  to  the  antagonistic  muscle,  the  biceps.  Both  of  these 
actions  combine  to  produce  the  coordinated  expression  of  the  reflex  behavior.  In  addition,  information  is  conveyed  to  higher 
regions  of  brain  to  update  them  about  the  information  coming  into  the  nervous  system  and  about  the  behavior  that  is  being 
generated.  These  higher  centers,  in  turn,  can  act  to  modify  the  reflex 
behavior. 

B:  The  sequences  of  signaling  changes  that  produce  the  reflex  action. 
Graded  stretch  of  the  muscle  produces  a  graded  receptor  potential  in  the 
muscle  spindle  of  the  afferent  neuron  that  propagates  passively  to  the  trig- 
ger zone  at  the  first  node  of  Ranvier.  If  the  potential  is  sufficiently  large,  it 
will  trigger  an  action  potential  that  will  propagate  actively  along  the  axon 
to  the  terminal  region.  At  the  terminal  the  change  in  membrane  potential, 
produced  by  the  action  potential,  gives  rise  to  a  secretory  potential  that 
leads  to  the  release  of  transmitter  substance.  The  transmitter  diffuses 
across  the  synaptic  cleft  and  interacts  with  receptor  molecules  on  the  mem- 
brane of  the  postsynaptic  motor  cell  to  initiate  a  synaptic  potential.  The 
synaptic  potential  then  propagates  passively  to  the  initial  segment  of  the 
axon,  where  it,  in  turn,  initiates  an  action  potential  that  propagates  to  the 
terminals  of  the  motor  neuron.  This  action  potential  leads  ultimately  to  a 
synaptic  potential  in  the  muscle,  which  initiates  an  action  potential  that 
causes  the  contraction  of  the  muscle. 

Reprinted  by  permission  of  the  publisher  from  Kandel,  E.R.,  and 
Schwartz,  J.H.  Principles  of  Neural  Science,  2nd  edition,  p.  20.  Copyright 
1985  by  Elsevier  Science  Publishing  Co.,  Inc. 

Cell  body  in 
dorsal  root  ganglion^ 

/  i^fii/j  II  I'  ^,''1 1 II! (film  III  1 1  /li  I  I  ^"^^ 

Quadriceps 
(extensor) 


Patella 


la  afferent  fiber 


Pyramidal 
tract 


To  contralateral 
spinal  cord 


Inhibitory 
Interneuron 


B 


Graded 

receptor 

potential 


Action 
potential 


Action 
potential 


J 


Secretory 
potential 


Synaptic 
potential 


Action 
potential 

Synaptic 
potential 


Action 
potential 


1  1 


Muscle  spindle  Myelin 


Dorsal  root 
ganglion  cell 


JL 

1  1 


Receptor  —  Trigger 
zone 


Input 


Stretch 


-3  E 


Axon 


Integrative  Conductile 


Synapse  —  Trigger 
zone 


Integrative 


Output  Input 


Axon 


Conductile 


Action 
potential 


Synaptic 
potential 


Synapse 


Muscle 


Output  Input 


Ivii 


Introduction 


CLOSED 


Figure  24.  A  local  reduction  of  the  voltage  differential  may 
induce  a  sodium  or  potassium  channel  to  change  confor- 
mation from  one  allosteric  form  to  another,  thus  opening 
the  channel  and  leading  to  the  free  passage  of  ions.  This 
change  is  presumably  caused  by  key  electrically  charged 
amino  acids  in  the  channel  protein,  which  shift  their  orien- 
tation in  response  to  the  changed  electrical  field. 

From  Discovering  Enzymes,  by  David  Dressier  and  Hun- 
tington Potter,  p.  235-  Copyright  ®  1991  by  David  Dressier. 
Reprinted  with  permission  by  W.H.  Freeman  and  Company. 


Iviii 


i 


Introduction 


gene.  Second,  the  similarities  in  the  predicted 
amino  acid  sequences  suggested  that  the  subunits 
are  probably  arranged  to  form  a  central  pore  or 
channel  between  them.  Third,  the  presence  in 
each  subunit  of  four  hydrophobic  regions,  each 
about  20  amino  acids  long,  immediately  sug- 
gested the  probable  disposition  of  the  subunits, 
with  four  transmembrane  domains  (Ml,  M2,  M3, 
and  M4)  and  intervening  intra-  and  extracellular 
linking  segments.  More  recent  work  has  identi- 
fied a  fifth  subunit  type  €  and  has  established  the 
precise  location  of  the  acetylcholine-binding  site 
and  the  existence  of  a  family  of  nicotinic  AChRs 
in  the  central  nervous  system;  much  has  also  been 
learned  about  the  regulation  of  the  receptor  dur- 
ing muscle  development  and  after  denervation, 
and  about  the  process  of  receptor  desensitization. 

The  nicotinic  AChR  belongs  to  a  large  class  of 
neurotransmitter  receptors  that  operate  by  selec- 
tively opening  ion  channels.  Another,  somewhat 
larger  class  of  receptors  acts  through  second  mes- 
sengers. For  example,  the  adrenergic  receptors, 
which  are  responsible  for  controlling  a  number 
of  vital  functions  such  as  heart  rate  and  blood 
pressure,  act  through  the  intermediary  of  a  class 
of  so-called  G  proteins  to  activate  the  enzyme 
adenylate  cyclase  and  increase  the  intracellular 
level  of  the  important  second  messenger,  cAMP. 
The  second  messenger,  in  turn,  usually  acts  by 
stimulating  protein  kinases  that  modify  (by  add- 
ing phosphate  groups)  other  proteins,  including 
ion  channels  and  proteins  that  regulate  gene  ex- 
pression in  the  responding  cell. 

The  regulation  of  gene  expression  by  synapti- 
cally  mediated  second  messenger  systems  has  be- 
come one  of  the  most  active  areas  for  research  in 
molecular  neuroscience.  Whereas  neurotransmit- 
ters usually  result  in  changes  that  have  a  time 
course  measured  in  the  millisecond  to  second 
range,  many  of  the  most  intriguing  phenomena  in 
neuroscience  are  those  that  occur  over  periods  of 
hours,  days,  and  even  months  or  years.  Recent 
work  has  demonstrated  that  in  addition  to  their 
more  or  less  immediate  and  short-lasting  effects, 
under  appropriate  conditions  (such  as  those  we 
commonly  associate  with  learning  and  memory) 
neurotransmitters  may,  through  second  messen- 
gers, activate  a  number  of  transcriptional  regula- 
tory proteins  that  "turn  on"  various  classes  of 
genes.  These,  in  turn,  may  regulate  the  expres- 
sion of  yet  other  genes  and  thus  unleash  a  com- 
plex cascade  of  events  within  the  responding 
nerve  cell,  modifying  its  growth  and  altering  its 


responsiveness  to  later  neurotransmitter  activa- 
tion over  long  periods  of  time. 

One  of  the  major  beneficiaries  of  the  applica- 
tion of  the  new  genetics  to  the  nervous  system  has 
been  the  field  of  developmental  neuroscience. 
Indeed  it  is  no  exaggeration  to  say  that  since  the 
late  1970s  this  field  has  been  transformed  from 
an  essentially  descriptive  science  into  one  in 
which,  for  the  first  time,  mechanistic  explana- 
tions are  emerging  to  account  for  the  growTih  of 
nerve  cells  and  their  processes,  for  the  deploy- 
ment of  cells  into  peripheral  ganglia  and  within 
cortical  layers  or  nuclear  groups  in  the  central 
nervous  system,  for  the  formation  of  specific  pat- 
terns of  connections,  and  for  the  elimination  of 
redundant  cells  and  inappropriate  connections. 

Because  of  the  complexity  and  inaccessibility 
of  the  mammalian  central  nervous  system,  until 
recently  much  of  the  most  definitive  work  on 
neural  development  has  been  carried  out  in 
simpler  forms  such  as  the  nematode  C.  elegans 
and  the  fruit  fly  Drosophila.  It  is  difficult  to  sum- 
marize the  broad  sweep  of  this  work,  except  to 
say  that  it  has  served  to  clarify  the  genetic  mecha- 
nisms that  determine  the  distinct  front-to-back 
and  top-to-bottom  organization  of  all  developing 
organisms,  that  determine  not  only  which  cells 
will  become  neurons  but  also  how  many  neurons 
will  be  generated  and  what  type  they  will  be 
(e.g.,  sensory  cells,  interneurons,  or  motor 
cells),  and  that  determine  finally  whether  the 
neurons  that  are  initially  formed  will  survive.  In 
some  instances  it  is  clear  that  the  character  or 
phenotype  of  the  nerve  cells  is  determined  by 
their  lineage;  in  other  cases  cell-cell  interactions 
are  more  important,  and  the  nature  of  the  signals 
that  developing  cells  transmit  to  their  neighbors 
is  currently  being  elucidated.  Of  special  impor- 
tance are  the  molecules  on  the  surfaces  of  cells 
that  enable  them  to  recognize  and  aggregate  with 
other  cells  of  like  kind  or  enable  them  to  migrate 
along  other  cells  or  across  territories  filled  with 
extracellular  matrix  materials.  While  much  re- 
mains to  be  discovered,  the  first  fruits  of  this  har- 
vest hold  great  promise  for  future  progress  in  this 
important  field. 

Finally,  no  account  of  progress  in  molecular 
neuroscience  would  be  complete  without  refer- 
ence to  the  striking  developments  in  our  under- 
standing of  the  basis  of  some  of  the  major  genetic 
disorders  that  affect  the  nervous  and  related  mus- 
cular systems.  Perhaps  the  most  striking  of  these 
developments  has  been  the  cloning  of  the  gene 


lix 


Introdtiction 


for  Duchenne  and  Becker  muscular  dystrophy. 
These  are  X-linked  recessive  disorders  that,  in  the 
more  severe  (Duchenne)  form  lead  inexorably 
from  muscular  weakness  to  muscular  atrophy  and 
finally  death.  The  extreme  size  of  this  gene  (it 
comprises  about  1  percent  of  the  X  chromosome 
and  almost  0.1  percent  of  the  total  human  ge- 
nome) renders  it  especially  vulnerable  to  muta- 
tion, and  in  many  of  the  identified  mutations,  the 
protein  encoded  by  the  gene,  dystrophin 
(which  appears  to  be  critical  for  coupling  muscle 
excitation  and  contraction),  is  either  absent  or 
markedly  deficient. 

Some  years  ago  the  general  location  of  the  gene 
responsible  for  the  severe  neurological  disorder 
known  as  Huntington's  disease  (HD)  was  deter- 
mined using  RFLPs,  as  discussed  in  the  section  on 
genetics.  Although  the  HD  gene  itself  has  so  far 
eluded  us,  there  is  every  reason  to  be  optimistic 
that  within  a  year  or  two  it  will  be  identified  and 
cloned.  In  the  meantime,  the  relevant  RFLP  has 
provided  a  useful  marker  for  identifying  carriers 
of  the  disordered  gene.  There  is  similarly  reason 
for  optimism  that  in  the  near  future  the  genetic 
basis  for  the  two  major  affective  disorders,  manic 
depression  and  schizophrenia,  will  be  eluci- 
dated. Careful  studies  of  family  histories  and  of 
identical  twins  raised  apart  have  clearly  estab- 
lished that  both  illnesses  have  an  important  ge- 
netic component,  and  while  neither  is  probably 
due  to  a  single  genetic  mutation,  RFLP  analysis 
and  other  genetic  approaches  should  reveal  the 
genes  involved.  That  such  complex  behavioral 

Investigators  in  the  Neuroscience  Program 

Adams,  Paul  R.,  Ph.D. 

Aldrich,  Richard  W.,  Ph.D. 

Anderson,  David  J.,  Ph.D. 

Artavanis-Tsakonas,  Spyridon,  Ph.D. 

Axel,  Richard,  M.D. 

Corey,  David  P.,  Ph.D. 

De  Camilli,  Pietro,  M.D. 

Evans,  Ronald  M.,  Ph.D. 

Goodman,  Corey  S.,  Ph.D. 

Horvitz,  H.  Robert,  Ph.D. 

Huganir,  Richard  L.,  Ph.D. 

Hurley,  James  B.,  Ph.D. 

Jahn,  Reinhard,  Ph.D. 

Jan,  Lily  Y.,  Ph.D. 

Jan,  Yuh  Nung,  Ph.D. 

Jessell,  Thomas  M.,  Ph.D. 

Kandel,  Eric  R.,  M.D. 

Lerner,  Michael  R.,  M.D.,  Ph.D. 

Miller,  Christopher,  Ph.D. 

Movshon,  J.  Anthony,  Ph.D. 


disorders  might  yield  to  this  type  of  approach  was 
unthinkable  only  a  decade  ago;  as  noted  in  the 
section  on  genetics,  nothing  serves  to  emphasize 
more  dramatically  the  power  of  the  new  genetics 
or  the  exciting  possibilities  it  portends. 

The  dramatic  advances  in  cellular  and  molecu- 
lar neuroscience  should  not  obscure  the  fact  that 
the  distinctive  role  of  the  nervous  system  in  the 
economy  of  an  organism  is  its  capacity  to  inte- 
grate sensory  information  that  is  received  (both 
from  within  and  from  outside  the  body)  and  to 
organize  it  into  patterns  of  behavior  that  allow 
the  organism  to  respond  to  changes  in  its  environ- 
ment in  appropriate  ways.  And  for  human  beings, 
it  is  through  the  nervous  system  that  we  learn 
both  from  personal  experience  and  the  accumu- 
lated wisdom  of  previous  generations  not  only 
how  to  survive  but  how  to  enjoy  and  profit  from 
the  richness  of  mental  experience  and  meaning- 
ful social  interactions.  To  understand  all  this  we 
will  need  to  learn  a  great  deal  more  about  the 
activities  of  large  populations  of  nerve  cells, 
about  the  computational  capacity  of  complex 
neural  networks,  and  about  the  extraordinary 
ways  in  which  the  human  brain,  with  its  100  bil- 
lion or  more  neurons  and  its  more  than  1  trillion 
synapses,  receives  and  processes  information.  We 
are  at  the  threshold  of  being  able  to  understand 
the  logic  of  the  simplest  organisms  at  both  the 
molecular  and  systems  levels;  the  exploration  of 
human  behavior  in  these  same  terms  stands  as 
perhaps  the  greatest  challenge  to  modern 
science. 


Reed,  Randall  R.,  Ph.D. 
Reichardt,  Louis  P.,  Ph.D. 
Rosenfeld,  Michael  G.,  M.D. 
Rubin,  Gerald  M.,  Ph.D. 
Sakmar,  Thomas  P.,  M.D. 
Scheller,  Richard  H.,  Ph.D. 
Sejnowski,  Terrence J.,  Ph.D. 
Siegelbaum,  Steven  A.,  Ph.D. 
Steller,  Hermann,  Ph.D. 
Stevens,  Charles  F.,  M.D.,  Ph.D. 
Struhl,  Gary,  Ph.D. 
SUdhof,  Thomas  C,  M.D. 
Tanabe,  Tsutomu,  Ph.D. 
Tsien,  Roger  ¥.,  Ph.D. 
Yau,  King- Wat,  Ph.D. 
Yellen,  Gary,  Ph.D. 
Ziff,  Edward  B.,  Ph.D. 
Zipursky,  S.  Lawrence,  Ph.D. 
Zuker,  Charles  S.,  Ph.D. 


Ix 


Introduction 


Structural  Biology  Program 

The  primary  goal  of  structural  biology  is  to  un- 
derstand, in  atomic  detail,  the  three-dimensional 
architecture  of  proteins,  protein  assemblies,  and 
the  complexes  formed  by  proteins  that  interact 
with  RNA  and  DNA.  Underlying  this  approach  is 
the  belief  that  fundamental  insights  into  the  func- 
tional role  of  biologically  interesting  molecules 
can  best  come  from  understanding  the  forms  of 
the  molecules  themselves.  As  Francis  Crick,  the 
co-discoverer  of  the  double-helical  structure  of 
DNA  (see  Figure  9),  remarked:  "to  understand 
function  it  is  essential  to  study  structure."  It  was 
with  this  in  mind  that  in  1985  the  Institute  made 
a  substantial  commitment  to  develop  a  new  Pro- 
gram in  Structural  Biology. 

At  present,  x-ray  crystallography  is  the  most 
powerful  approach  for  visualizing  the  three- 
dimensional  structures  of  large  molecules  (com- 
monly called  macromolecules) .  An  essential  pre- 
requisite for  x-ray  analysis  is  the  availability  of 
crystals  of  the  molecule  or  molecular  complex 
that  are  suitable  for  recording  the  diffraction  of 
x-rays.  The  production  of  crystals,  in  turn,  re- 
quires chemically  homogeneous  preparations. 
Moreover,  molecules  that  are  not  spatially  uni- 
form (too  "floppy")  must  be  broken  down  or  mo- 
lecularly  dissected  into  defined  and  rigid  com- 
ponents. For  example,  to  crystallize  antibody 
molecules,  it  was  important  first  to  cleave  them 
into  their  principal  fragments,  Fab  and  Fc,  because 
these  pieces  are  normally  connected  by  a  flexible 
hinge.  And  because  of  antibody  diversity  (de- 
scribed in  the  section  on  immunology) ,  it  became 
necessary  to  study  Fab  fragments  from  monoclonal 
immunoglobulins.  A  continuing  challenge  to 
structural  biologists  is  the  development  of  strate- 
gies for  crystallizing  membrane  proteins — by  solu- 
bilizing  them  with  detergents,  dissecting  them 
into  pieces,  or  altering  them  by  mutation. 

Genetic  engineering  has  transformed  struc- 
tural biology.  This  approach,  which  makes  it  pos- 
sible to  produce  large  quantities  of  pure  proteins, 
also  allows  an  investigator  either  to  choose  a  suit- 
able fragment  for  study  or  to  modify  genetically 
the  molecule  to  be  crystallized.  Other  method- 
ological advances  in  crystallography  itself  are 
transforming  the  field  by  extending  the  range  of 
problems  that  can  be  tackled  routinely. 

There  are  essentially  four  stages  in  determining 
a  structure  by  x-ray  diffraction  analysis  (Figures 
25  and  26):  1)  diffraction  experiments  (data  col- 
lection); 2)  complex  computations  that  pro- 
duce, in  effect,  an  image  of  the  molecule(s)  in 


the  crystal;  3)  interpretation  of  the  computed 
image  in  terms  of  a  molecular  model;  and  4)  re- 
finement of  the  model  by  further  computation. 
Synchrotron  x-ray  sources,  which  are  a  thou- 
sand or  more  times  stronger  than  conventional 
laboratory  x-ray  generators,  are  making  it  possi- 
ble to  study  structures  that  could  not  previously 
be  solved.  (HHMI  is  currently  developing  a 
synchrotron  resource  for  use  by  the  biological 
community  at  the  National  Synchrotron  Light 
Source  at  Brookhaven  National  Laboratory  on 
Long  Island.)  Recent  examples  from  HHMI  labo- 
ratories are  the  human  class  I  major  histocompati- 
bility antigen  and  the  DNA  virus  SV40.  At  the 
same  time,  position-sensitive  x-ray  detectors  have 
greatly  extended  the  applications  of  conven- 
tional radiation  sources.  Lastly,  novel  computa- 
tional methods  have  made  the  production  of  a 
molecular  image  (phase  determination)  less 
dependent  on  extensive  ancillary  data  from 
heavy-atom  modified  crystals  and  have  made  re- 
finement of  models  less  cumbersome  and  more 
objective. 

In  the  1950s  and  1960s,  x-ray  crystallography 
revealed  the  structures  of  the  first  biologically 
important  molecules,  including  DNA,  hemoglo- 
bin, and  insulin.  In  the  1970s  it  revolutionized 
the  field  of  enzymology  by  making  it  possible  to 
visualize  directly  the  active  sites  of  enzymes.  In 
the  1980s  it  made  comparably  far-reaching  con- 
tributions to  virology,  immunology,  and  mem- 
brane biology  by  revealing  the  structures  of  vi- 
ruses, antibodies,  and  a  photosynthetic  reaction 
center.  What  can  we  expect  in  the  1990s?  It 
seems  reasonable  to  predict  the  following: 

1.  Structures  of  different  classes  of  proteins 
or  protein/nucleic-acid  complexes.  Three  de- 
cades of  biological  crystallography  have  left  sev- 
eral major  areas  unexplored.  For  example,  we 
have  yet  to  know  what  any  of  the  major  proteins 
of  the  cytoskeleton  and  of  cellular  motility  look 
like  (actin,  myosin,  tubulin,  and  so  forth).  We 
have  yet  to  visualize  any  of  the  membrane  recep- 
tors referred  to  above,  and  we  have  yet  to  see  an 
ion  channel,  a  ribosome,  an  RNA  polymerase,  or  a 
ribozyme;  and,  with  the  exception  of  transfer 
RNAs  (tRNAs),  little  is  known  of  the  three- 
dimensional  structure  of  most  RNAs  and  RNA- 
protein  complexes.  Progress  toward  some  of 
these  goals  is  reported  in  this  volume;  others  will 
no  doubt  be  achieved  before  long,  as  more  and 
more  workers  are  drawn  into  the  field  and  as  new 
techniques  are  developed. 


Ixi 


Introduction 


Single 
crystiil 


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M.iihemalical 

analysis  of 
.callered  X  raw 


Diffraction  pattern 


Electron-density 
maps  of  sections 
through  the  molecule 


Electron-density  map 


Figure  25.  The  use  of  x-rays  to  determine  the  structure  of  enzymes.  When  an  enzyme  is 
in  a  crystalline  form,  its  molecules  are  arranged  in  regularly  repeating  arrays.  A  beam 
of  x-rays  passed  through  the  enzyme  is  diffracted  to  give  a  pattern  of  spots  on  a  detec- 
tor screen  behind  the  sample.  Each  spot  contains  information  about  the  electron  den- 
sity ( and  hence  the  types  of  atoms:  carbon,  oxygen,  nitrogen,  etc.)  in  various  parts  of 
the  crystal.  This  information  can  be  used  to  construct  electron  density  maps  whose 
contours  indicate  the  type  of  atoms  present.  The  contour  map  shown  at  the  right 
represents  a  short  segment  of  polypeptide  backbone  with  a  tyrosine  side  chain. 
Adapted  from  Geoffrey  Zubay,  Biochemistry,  Addison-Wesley,  1985. 


rosine  side  chain 


Backbone 


Electron-density  map 
with  inferred  polypeptide 
backbone  and  tyrosine 
side  chain  fitted  in 


-  -  -<i 


B 


Figure  26.  A:  The  x-ray  diffraction  pattern  given  by  the  enzyme  chymotrypsin. 
Courtesy  of  Thomas  A.  Steitz. 

B:  A  representation  of  the  three-dimensional  structure  of  chymotrypsin.  Carbon  atoms  are  shown  in 
black,  nitrogen  in  blue,  oxygen  in  red,  hydrogen  in  white,  and  sulfur  in  yellow.  The  diameter  of  the  enzyme 
is  about  45  A  (somewhat  less  than  a  millionth  of  an  inch).  The  hydrophilic  side  chain  of  arginine- 145  is 
clearly  visible  projecting  outward  from  the  right  side  of  the  molecule.  The  ridges  and  grooves  on  the  surface 
of  the  chymotrypsin  molecule  are  as  unique  as  the  mountains  and  craters  of  the  moon,  and  herein  lies  the 
fulfillment  of  the  lock-and-key  mechanism  hypothesized  by  Emil  Fischer  at  the  turn  of  the  century. 

Courtesy  of  Molecular  Simulations,  Waltham,  MA. 


Ixii 


Introduction 


2.  Time-resolved  images  of  events  at  the  ac- 
tive site  of  an  enzyme.  New  ways  of  using 
synchrotron  x-ray  radiation  permit  very  rapid 
measurements  of  diffraction  data,  so  that  in  prin- 
ciple it  should  be  possible  to  follow  the  struc- 
tural changes  that  occur  during  an  enzymatic  re- 
action. If  we  understood  these  changes,  it  might 
be  possible  to  develop  enzymes  with  usefully  al- 
tered properties  and  to  synthesize  enzyme  inhibi- 
tors with  enhanced  specificity. 

3.  The  role  of  molecular  recognition  in  the 
regulation  of  cellular  activity.  How  do  proteins 
that  control  transcription  recognize  specific  DNA 
sequences?  How  do  cell  surface  proteins  in  the 
immune  system  recognize  and  present  antigens? 
The  answers  to  these  questions  are  beginning 
to  emerge  from  crystal  structures  of  molecular 
complexes,  such  as  the  complexes  formed  by  reg- 
ulatory proteins  with  DNA,  those  formed  by  bind- 
ing proteins  with  their  appropriate  ligands,  of  an- 
tibodies with  antigens,  and  of  MHC  molecules 
with  peptides.  These  current  efforts  give  consid- 
erable promise  for  understanding  how  hormones 
or  neurotransmitters  trigger  a  cascade  of  events 
that  involves  the  formation  and  dissociation  of 
protein  assemblies  inside  cells.  A  large  and  medi- 
cally significant  class  of  regulatory  interactions 
involves  the  protein  products  of  oncogenes  or 
proto-oncogenes . 

As  our  knowledge  of  important  proteins  rap- 
idly increases,  we  can  only  hope  that  our  capacity 
to  anticipate  aspects  of  structures  not  yet  deter- 
mined will  keep  pace.  The  goal  of  accurately  pre- 
dicting the  three-dimensional  structure  of  any 
protein  from  its  amino  acid  sequence  is  still  a 
long  way  off.  But  recent  advances  in  computa- 
tional chemistry  make  it  possible  to  predict  the 
effects  of  small  perturbations,  such  as  point  mu- 
tations, on  the  folding  of  a  protein  and  to  calcu- 
late differences  in  binding  free  energies  for  re- 

Investigators  in  the  Structural  Biology  Program 

Agard,  David  A.,  Ph.D. 
Briinger,  Axel  T.,  Ph.D. 
Burley,  Stephen  K.,  M.D.,  D.Phil. 
Deisenhofer,  fohann,  Ph.D. 
Fox,  Robert  O.,  Ph.D. 
Harrison,  Stephen  C,  Ph.D. 
Hendrickson,  Wayne  A.,  Ph.D. 
Kuriyan,  John,  Ph.D. 


lated  ligands.  And  systematic  approaches  to 
designing  drugs,  such  as  antagonists  or  inhibitors 
of  enzymes,  are  beginning  to  emerge  now  that  we 
can  carry  out  meaningful  calculations  on  known 
structures. 

Nuclear  magnetic  resonance  (NMR)  meth- 
ods offer  an  alternative  route  to  determining  the 
three-dimensional  structures  of  peptides  and 
small  proteins.  The  DNA-binding  domains  of  tran- 
scriptional activators  and  repressors  are  good 
candidates  for  this  type  of  analysis.  The  past  two 
years  have  seen  the  determination  by  NMR  of  the 
structure  of  a  developmentally  important  DNA 
sequence  known  as  a  homeodomain  and  of  a 
DNA-binding  structure  known  as  a  zinc  finger. 
NMR  has  the  great  advantage  that  it  circumvents 
the  need  to  crystallize  the  protein  to  be  studied. 
At  the  other  end  of  the  size  scale,  imaginative 
combinations  of  light  and  electron  microscopy 
have  begun  to  reveal  important  patterns  and  re- 
gularities in  very  large  structures,  such  as  chro- 
mosomes, viruses,  and  receptors.  New  methods 
for  recording  images  and  enhancing  contrast  in 
light  microscopy  make  it  possible  to  record  in 
real  time  the  events  of  intracellular  transport  or 
the  process  of  chromosome  condensation.  As  the 
molecules  that  generate  these  large-scale  intra- 
cellular motions  are  characterized,  it  should  be- 
come possible  to  relate  such  changes  to  the  spe- 
cific molecular  recognition  events  that  control 
them.  It  is  fortunate,  but  not  coincidental,  that  as 
biologists  have  become  increasingly  aware  of  the 
need  to  know  the  precise  structure  of  the  mole- 
cules that  mediate  the  phenomena  they  are  inter- 
ested in,  a  whole  range  of  new  experimental 
methods  has  been  developed,  and  a  new  genera- 
tion of  structural  biologists  has  emerged  to  assist 
them  and  to  advance  their  understanding  of  the 
complex  relationships  between  structure  and 
function. 


Matthews,  Brian  W.,  Ph.D.,  D.Sc. 
Pabo,  Carl  O.,  Ph.D. 
Quiocho,  Florante  A.,  Ph.D. 
Sedat,John  W.,  Ph.D. 
Sigler,  Paul  B.,  M.D.,  Ph.D. 
Sprang,  Stephen  R.,  Ph.D. 
Steitz,  Thomas  A.,  Ph.D. 
Wiley,  Don  C,  Ph.D. 


W.  Maxwell  Cowan,  M.D.,  Ph.D. 

Vice  President  and  Chief  Scientific  Officer 


Ixiii 


I 


I 


I 


I 


■i 

I 


I 

i 


Electrical  Activity  of  Nerve  Cells 


Paul  R.  Adams,  Ph.D. — Investigator 

Dr.  Adams  is  also  Professor  of  Neurobiology  and  Behavior  and  of  Pharmacological  Sciences  at  the  State 
University  of  New  York  at  Stony  Brook.  He  received  his  B.A.  degree  in  physiology  and  pharmacology  from 
Cambridge  University  and  his  Ph.D.  degree  in  pharmacology  from  the  University  of  London.  His 
postdoctoral  work  was  done  with  Bert  Sakmann  at  the  Max  Planck  Institute,  Gottingen,  and  with 
Philippe  Ascher  at  the  Ecole  Normale,  Paris.  Dr.  Adams  is  currently  a  MacArthur  Fellow.  He  was  elected 
Fellow  of  the  Royal  Society. 


NERVE  cells  are  specialized  to  generate,  trans- 
mit, and  receive  rapid  electrical  messages. 
Electrical  impulses,  called  action  potentials,  last 
about  1,000th  of  a  second  and  can  travel  along 
specialized  nerve  cell  extensions  at  speeds  over 
100  mph.  Chemical  transmitter  substances  re- 
leased onto  the  nerve  cell  by  other  nerve  cells 
control  the  precise  timing  of  these  electrical 
pulses.  We  are  trying  to  understand  how  these 
pulses  are  generated  and  how  transmitters  im- 
pinging on  the  cell  control  them. 

Cell  membranes  are  normally  effective  barriers 
to  the  movement  of  ions  (electrically  charged 
atoms)  between  the  cell  environment  and  the 
cell  interior.  This  insulating  property  allows  the 
inside  of  a  nerve  cell  to  have  a  different  electrical 
voltage  from  the  outside  or  from  a  neighboring 
cell.  The  electrical  activities  described  above  are 
regulated  by  special  protein  molecules,  called 
ion  channels,  which  are  embedded  in  the  cell 
membrane.  There  are  many  types  of  ion  channel. 
Each  type  has  a  specific  role,  but  all  have  in  com- 
mon a  unique  feature  that  allows  certain  ions  to 
travel  easily  through  them.  The  protein  chains  that 
make  up  an  ion  channel  molecule  are  arranged  to 
create  a  minute  tunnel,  through  which  certain 
types  of  ions — for  example,  sodium,  potassium,  or 
calcium  ions — can  quickly  move. 

The  direction  that  the  ion  moves  is  not  con- 
trolled by  the  tunnel  but  by  the  ion  concentra- 
tions and  the  transmembrane  voltage.  The  tunnel 
does,  however,  control  the  type  of  ion  that 
moves.  Thus  the  sodium  channel  only  allows  so- 
dium ions  to  pass.  Because  sodium  ions  are  abun- 
dant outside,  but  not  inside,  nerve  cells,  the  exis- 
tence of  open  sodium  channels  leads  to  an  inward 
stream  of  sodium  ions,  making  the  cell  interior 
positive.  On  the  other  hand,  when  potassium 
channels  open,  potassium  streams  out  of  the  cell, 
making  it  negative.  Because  these  tunnels  are  not 
always  open  (indeed  are  closed  most  of  the 
time) ,  it  is  supposed  that  the  channel  must  have 
some  sort  of  gate. 

There  are  many  types  of  potassium  channel, 
differing  according  to  their  molecular  structure 
(see  the  articles  in  this  volume  by  Richard 


Aldrich  and  Lily  Jan),  speed  of  opening  and  clos- 
ing, and  the  way  in  which  their  gates  are  con- 
trolled. We  are  studying  potassium  channels  in 
bullfrog  sympathetic  ganglion  cells  to  under- 
stand better  how  they  are  controlled  and  how 
they  contribute  to  the  electrical  activity  of  nerve 
cells.  We  have  been  particularly  intrigued  by  a 
channel  we  call  the  M  channel.  This  channel  is 
turned  off  as  a  result  of  the  binding  of  certain 
neurotransmitters  (chemicals  released  from 
nerve  endings)  to  receptors  on  the  cell  surface. 
As  a  result,  less  potassium  leaves  the  cell,  which 
is  therefore  less  negative  and  more  able  to  fire 
electrical  pulses.  Tumofif  of  this  channel  occurs 
via  activation  of  a  G  protein  (see  the  article  by 
John  Exton) .  However,  after  the  neurotransmitter 
has  been  removed,  M  channels  turn  back  on  and 
transiently  become  more  numerous  than  initially 
observed.  We  have  shown  that  calcium  and  ara- 
chidonic  acid  are  involved  in  this  overshooting 
response.  Various  levels  of  calcium  were  per- 
fused into  nerve  cells  while  they  were  visualized 
with  a  special  calcium-detecting  microscope. 
Neurotransmitter  stimulation  of  receptors  pro- 
duces a  small  calcium  signal  that  is  sufficient  to 
increase  the  activity  of  M  channels.  However,  this 
is  not  seen  until  the  concomitant  G  protein- 
mediated  suppression  of  the  channels  is  termi- 
nated by  removing  the  transmitter. 

M  channels  work  in  concert  with  many  other 
types  of  channels,  which  we  have  also  character- 
ized. This  information  can  then  be  combined 
with  studies  of  calcium  diffusion  and  membrane 
geometry  to  predict  completely  the  cell's  electri- 
cal output.  We  are  making  detailed  quantitative 
morphological  measurements  of  cells  that  have 
been  previously  characterized  electrophysiologi- 
cally  in  both  bullfrog  ganglia  and  mammalian 
hippocampus  and  lateral  geniculate.  This  is 
achieved  by  automatic  three-dimensional  recon- 
struction of  dye-filled  cells,  which  are  optically 
sectioned  using  confocal  microscopy.  Our  voxel- 
based  reconstructions,  developed  in  collaboration 
with  the  computer  science  department  at  Stony 
Brook,  can  then  be  used  as  a  platform  for  Monte 
Carlo  simulations  of  the  movements  of  single  ions 
in  small  cell  structures,  such  as  dendritic  spines. 


Three-dimensional  computer  reconstructions  of  nerve  cells.  The  upper  image  shows 
a  sympathetic  ganglion  cell  of  a  bullfrog;  the  lower  image,  a  hippocampal  pyramidal 
cell  of  a  rat. 

Research  of  Rick  Avila,  Barry  Burbach,  Jim  Monckton,  Ted  Carnevale,  Arie  Kauf- 
man, and  Paul  Adams. 


2 


Three-Dimensional  Macromolecular 
and  Cellular  Structure 


David  A.  Agard,  Ph.D. — Associate  Investigator 

Dr.  Agard  is  also  Associate  Professor  of  Biochemistry  and  Biophysics  at  the  University  of  California,  San 
Francisco.  He  did  his  undergraduate  work  at  Yale  University  with  Frederic  Richards,  Hal  Wyckoff,  and 
Thomas  Steitz.  He  received  his  Ph.D.  degree  in  chemical  biology  from  the  California  Institute  of 
Technology,  where  he  studied  with  Robert  Stroud  and  began  a  continuing  collaboration  with  John  Sedat. 
His  postdoctoral  work  was  done  on  high-resolution  electron  microscopic  crystallography  at  the  MRC 
Laboratory  of  Molecular  Biology  in  Cambridge,  England,  with  Richard  Henderson.  There  he  also  began 
the  cloning  of  the  a-lytic  protease  gene  with  Sydney  Brenner. 


WE  study  chromosome  structure  in  close  col- 
laboration with  John  Sedat  (HHMI,  Univer- 
sity of  California,  San  Francisco);  hence  only  a 
subset  of  these  studies  will  be  discussed  here. 
Our  primary  aim  in  this  area  is  to  provide  a  physi- 
cal basis  for  understanding  chromosome  behav- 
ior and  function  by  directly  determining  the 
three-dimensional  structure  of  chromosomes  as  a 
function  of  both  transcriptional  state  and  the  cell 
cycle  stage.  Current  efforts  are  aimed  at  deter- 
mining how  fibers  of  nucleosomes  are  folded  into 
higher-order  structures  within  the  chromosome 
and  what  role  specific  chromosomal  proteins 
play  in  determining  these  structures. 

We  are  using  intermediate  voltage  electron  mi- 
croscope (IVEM)  tomography  to  examine  higher- 
order  chromosome  structure.  In  the  past  year  sig- 
nificant improvements  in  the  quality  of  the 
three-dimensional  reconstructions  have  led  to 
the  first  new  insights  into  the  structure  of  the  30- 
nm  fiber  (in  collaboration  with  Chris  Woodcock, 
University  of  Massachusetts) .  We  are  now  begin- 
ning to  be  able  to  trace  the  paths  of  the  30-nm 
chromatin  fibers  within  telophase  chromosomes. 
It  is  clear  that  the  existing  models  of  chromatin 
structure  are  seriously  flawed.  In  addition,  we 
have  made  dramatic  steps  toward  our  goal  of  fully 
automating  the  complex  task  of  collecting  three- 
dimensional  IVEM  tomography  data.  This  will 
simplify  the  arduous  task  of  collecting  100-150 
images  and  should  dramatically  reduce  radiation 
exposure  (from  3  hours  to  about  5  minutes) .  Sig- 
nificant progress  has  also  been  made  on  under- 
standing the  mechanism  of  image  formation  for 
thick  specimens  and  on  applying  a  powerful  new 
approach  to  the  problem  of  electron  microscopic 
(EM)  reconstruction.  Together  with  a  new  stain- 
ing approach  and  cryopreparation  methods, 
these  improvements  should  allow  us  to  trace  the 
paths  of  the  30-nm  fibers  throughout  the  chro- 
mosome and  will  finally  make  this  exciting  struc- 
tural approach  available  to  the  general  cell 
biologist. 

Structural  Basis  of  Enzyme  Specificity 

By  combining  solution  kinetic  analysis,  x-ray 


crystallographic  structural  analysis,  and  site- 
directed  mutagenesis,  we  have  been  able  to 
probe  the  structural  basis  for  substrate  specificity 
in  unprecedented  detail,  using  a-lytic  protease  as 
a  model  system.  The  availability  of  peptide  bor- 
onic  acid  inhibitors,  which  act  as  excellent 
mimics  of  the  reaction  transition  state,  or  nearby 
intermediates,  has  allowed  us  to  use  x-ray  crystal- 
lography to  examine  the  complex  set  of  interac- 
tions between  enzyme  and  substrate  that  mediate 
specificity. 

By  mutation,  we  have  been  able  to  alter  dramat- 
ically the  pattern  of  substrate  specificity  while 
maintaining  or  even  increasing  enzyme  activity. 
Approximately  40  high-resolution,  extremely 
well-refined  crystal  structures  have  now  been  de- 
termined and  analyzed.  Detailed  structural  analy- 
ses of  three  mutants  as  free  enzymes  and  as  com- 
plexes have  provided  surprising  insights  into  the 
mechanism  of  specificity  and  have  indicated  the 
crucial  role  that  protein  flexibility  plays  in  selec- 
tivity. During  the  past  year  we  have  made  numer- 
ous other  mutations  and  examined  their  kinetic 
and  structural  propenies.  Of  particular  note  is  a 
mutation  that  alters  specificity  indirectly  by 
changing  active-site  flexibility.  This  remarkable 
finding  is  the  first  demonstration  that  residues 
beyond  those  that  directly  contact  the  substrate 
can  play  a  significant  role  in  determining  the  pat- 
terns of  specificity. 

Not  long  ago  we  began  a  collaboration  with 
Vladimir  Basus  (University  of  California,  San  Fran- 
cisco) to  perform  a  complete  analysis  of  a-lytic 
protease  structure  in  solution  by  nuclear  mag- 
netic resonance  (NMR)  methods.  We  have 
now  made  '^N,''C  doubly  labeled  enzyme  and 
have  collected  sufficient  high-resolution  three- 
dimensional  NMR  data  sets  to  permit  complete 
backbone  assignment — a  significant  task  for  such 
a  large  protein.  Currently  we  are  working  on  the 
assignments  and  on  the  collection  of  data  for  the 
complete  side  chain  assignments.  We  anticipate 
that  the  NMR  data  will  provide  insights  into 
correlated  motions  within  the  enzyme  and  be 
crucial  for  the  folding  studies  described  below. 


3 


Three-Dimensional  Macromolecular  and  Cellular  Structure 


Last  year  we  had  developed  a  new  method  for 
predicting  the  energetics  of  protein-substrate  in- 
teractions. This  approach  (based  on  Ponder- 
Richards  rotamers  combined  with  energetics  and 
solvation  terms)  can  predict  k^^JK^  with  stun- 
ning accuracy.  We  have  used  this  method  to  de- 
sign a  new  enzyme  with  particular  properties, 
and  so  far  the  results  have  been  remarkable.  This 
year  we  have  been  working  to  extend  these  re- 
sults to  other  systems  and  to  improve  their  accu- 
racy. We  have  been  able  to  predict  quantitatively 
100  values  of  k^^JK^  for  subtilisin  with  equiva- 
lent accuracy.  This  approach  is  being  expanded 
to  allow  its  use  in  drug  design  situations  and  for 
modeling  proteins  based  on  the  structure  of  a  ho- 
mologous protein. 

Structural  and  Biochemical  Probes 
of  Folding  of  a-Lytic  Protease 

a-Lytic  protease  is  synthesized  as  a  prepro- 
enzyme.  Experiments  in  Escherichia  coli  have 
demonstrated  that  the  166-amino  acid  precursor 
domain  is  required  for  the  proper  folding  of  the 
198-amino  acid  protease  domain.  Furthermore, 
we  have  shown  that  the  pro  region  does  not  have 
to  be  attached  covalently  in  order  to  function. 
The  development  of  the  in  vitro  folding  system 
has  led  to  the  unprecedented  ability  to  trap  and 
purify  a  stable  folding  intermediate  under  non- 
denaturing  conditions.  Although  the  interme- 
diate is  unable  to  fold  by  itself,  it  is  rapidly  con- 
verted to  active  enzyme  upon  addition  of  the  pro 
region.  Analysis  of  the  folding  kinetics  proves 
that  the  pro  region  acts  essentially  as  a  "foldase" 
to  stabilize  the  transition  state  for  folding, 
thereby  speeding  up  folding  by  more  than  10^. 

Current  efforts  are  focused  on  characterizing 
the  folding  intermediate  and  analyzing  the  fold- 
ing reaction.  We  plan  to  use  a  combination  of 


approaches — including  NMR  and  crystallogra- 
phy— to  probe  the  structure  of  the  intermediate 
and  the  role  of  the  pro  region  in  the  final  stage  of 
folding. 

Receptor-Ligand  Targeting  and  Human 
Cholesterol  Metabolism 

Apolipoprotein  E  is  an  important  protein  in 
cholesterol  metabolism  in  mammals.  Responsi- 
ble for  targeting  chylomicrons,  very  low  density 
lipoprotein  (VLDL),  and  high-density  lipoprotein 
(HDL)  particles  to  the  low-density  lipoprotein 
(LDL)  receptor,  apolipoprotein  E  has  a  major  role 
in  triglyceride  and  cholesterol  metabolism.  The 
protein  itself  has  two  distinct  structural  and  func- 
tional domains:  the  amino-terminal  22-kDa  do- 
main contains  the  receptor-binding  functional- 
ity; lipid  binding  resides  primarily  with  the 
10-kDa  carboxyl-terminal  domain.  In  collabora- 
tion with  the  Mahley  group  at  the  Gladstone 
Foundation  Laboratories  for  Cardiovascular  Dis- 
ease, we  obtained  crystals  of  the  22-kDa  recep- 
tor-binding domain.  Last  year  we  reported  that 
the  protein  is  an  unusually  elongated  four-helix 
bundle.  Although  the  surface  is  exceptionally 
charged,  there  is  a  precise  balance  between 
groups  with  a  positive  charge  and  groups  with  a 
negative  charge,  except  in  what  we  believe  is  the 
receptor-binding  region.  We  have  recently  fin- 
ished the  analysis  of  a  naturally  occurring  human 
mutant  that  significantly  reduces  receptor  bind- 
ing and  can  lead  to  premature  atherosclerosis. 
The  structure  reveals  that  the  mutant  disrupts  the 
complex  set  of  surface  salt  bridges,  which  then 
causes  a  key  arginine  required  for  binding  to  be 
recruited  into  a  salt  bridge.  Current  efforts  are 
aimed  at  generating  a  soluble  fragment  of  the  LDL 
receptor-binding  domain  for  crystallographic 
study. 


4 


Molecular  Mechanisms  of  Ion  Channel  Function 


Richard  W.  Aldrich,  Ph.D. — Associate  Investigator 

Dr.  Aldrich  is  also  Associate  Professor  of  Molecular  and  Cellular  Physiology  at  Stanford  University.  He 
received  his  B.S.  degree  in  biology  from  the  University  of  Arizona  and  his  Ph.D.  degree  in  neuroscience 
from  Stanford.  He  carried  out  postdoctoral  research  at  Yale  University  with  Knox  Chandler  and  Charles 
Stevens.  Before  returning  to  Stanford,  he  was  Assistant  Professor  of  Molecular  Neurobiology  at  Yale. 
Among  his  awards  are  a  Searle  Scholars  Award,  the  Young  Investigator  Award  of  the  Society 
for  Neuroscience,  and  the  Young  Investigator  Award  of  the  Biophysical  Society. 


ION  channels  are  the  molecular  units  of  electri- 
cal signaling  in  cells.  They  are  proteins  that 
regulate  the  movement  of  ions,  such  as  sodium, 
calcium,  and  potassium,  into  and  out  of  cells. 
They  are  responsible  for  the  conversion  of  exter- 
nal sensory  signals  to  the  electrical  language  of 
the  nervous  system  and  for  the  integration  of 
these  signals  to  generate  appropriate  behavior.  In 
addition,  ion  channels  are  important  for  the  gen- 
eration and  regulation  of  the  heartbeat,  for  con- 
traction of  muscles,  and  for  the  release  of  hor- 
mones in  the  bloodstream.  A  very  large  variety  of 
ion  channel  types  are  found  in  the  body.  They  are 
specialized  to  select  for  certain  species  of  ions 
and  to  open  and  close  in  response  to  a  number  of 
different  stimuli,  such  as  the  binding  of  a  neuro- 
transmitter molecule  or  a  change  in  the  voltage 
that  exists  across  a  cell's  membrane.  Our  labora- 
tory is  interested  in  the  molecular  mechanisms  of 
ion  channel  function.  One  of  our  major  goals  is  to 
understand  the  conformational  changes  that  oc- 
cur as  the  channels  open  and  close  in  response  to 
appropriate  stimuli. 

Voltage-gated  ion  channels  are  an  important 
functional  class.  As  their  name  implies,  they  can 
open  in  response  to  changes  in  the  electrical  po- 
tential across  the  cell  membrane,  a  property  cru- 
cial for  the  generation  of  electrical  signals  and  for 
the  transmission  of  information  throughout  the 
body.  These  molecules  have  a  way  to  measure  the 
electrical  potential  and  open  accordingly.  In  ad- 
dition, a  number  of  these  channels  inactivate  or 
become  unavailable  for  opening  after  use.  In  re- 
cent years  we  have  studied  the  molecular  mecha- 
nisms of  inactivation  of  a  class  of  potassium  chan- 
nels that  were  cloned  in  Drosophila.  These 
channels  are  products  of  the  Shaker  gene.  They 
exhibit  the  fastest  inactivation  of  any  potassium 
channels  yet  cloned.  William  Zagotta  and  I  began 
by  using  single-channel  recording  methods  to 
study  the  gating  properties  of  wild-type  Shaker 
channels  in  their  native  tissue.  Such  methods  al- 
low us  to  record  the  behavior  of  a  single-channel 
molecule  as  it  opens  and  closes  on  a  millisecond 
time  scale. 

We    determined    that    the  conformational 


changes  associated  with  opening  the  channel  de- 
pended strongly  on  the  membrane  voltage  and 
therefore  involved  a  substantial  rearrangement  of 
an  electrically  charged  part  of  the  channel  in  the 
membrane.  On  the  other  hand,  the  inactivation 
process  did  not  involve  significant  charge  rear- 
rangement. This  result,  combined  with  our  abil- 
ity to  alter  inactivation  by  internal  enzymes,  led 
us  to  the  hypothesis  that  inactivation  involved  a 
conformational  change  on  the  inside  of  the  mem- 
brane that  blocked  the  flow  of  potassium  ions 
through  the  channel. 

Dr.  Zagotta,  Toshinori  Hoshi,  and  I  further  stud- 
ied this  hypothesis  by  making  altered  channels 
with  recombinant  DNA  methods  and  expressing 
the  normal  and  altered  channels  in  frog  oocytes. 
Our  results  are  strikingly  consistent  with  the 
"ball  and  chain"  model  of  inactivation  originally 
proposed  for  the  voltage-dependent  sodium 
channel  by  Armstrong  and  Bezanilla  in  1977.  The 
following  model  of  the  molecular  mechanism  of 
inactivation  emerges  from  our  results.  The  amino 
terminus  of  the  Shaker  channel  acts  as  an  inacti- 
vation particle  or  internal  plug  for  the  channel. 
When  the  inactivation  particle  is  bound  to  the 
receptor,  the  channel  closes.  We  tested  this 
model  further  by  applying  a  solution  containing 
free  synthetic  inactivation  particle  to  the  inside 
face  of  mutant  channels  that  did  not  inactivate.  In 
the  presence  of  the  synthetic  inactivation  parti- 
cle, the  mutant  channels  regained  inactivation, 
consistent  with  the  ball  and  chain  mechanism. 

Ruth  Murrell-Lagnado  and  I  have  used  syn- 
thetic peptide  inactivation  particles  with  altered 
amino  acid  composition  to  determine  the  impor- 
tant features  that  influence  an  ability  to  bind  to 
the  internal  mouth  of  the  channel.  The  naturally 
occurring  amino  acid  sequence  can  be  divided 
into  an  uncharged  region  and  a  highly  charged 
region.  The  charged  amino  acids  interact  with 
negative  charges  at  or  near  the  mouth  of  the  chan- 
nel to  influence  the  rate  of  binding.  The  more 
positive  charges  in  the  charged  region,  the  faster 
the  binding  rate.  Negative  charges  in  the  charged 
region  inhibit  binding.  A  surprising  result  is  that 
the  net  charge  in  this  region  seems  to  be  the  im- 
portant factor  for  the  binding  rate,  regardless  of 


5 


Molecular  Mechanisms  of  Ion  Channel  Function 


which  of  the  amino  acids  are  charged.  Con- 
versely, the  uncharged  region  is  important  for  de- 
termining the  unbinding  rate.  This  work  was  sup- 
ported in  part  by  the  National  Institutes  of  Health 
and  the  National  Institute  of  Mental  Health. 

Shaker  potassium  channels  also  exhibit  a 
slower  inactivation  process,  which  can  be  seen 
both  in  wild-type  channels  and  after  the  faster 
inactivation  process  has  been  removed  by  muta- 
genesis. The  slow  inactivation  does  not  require 
intact  fast  inactivation.  Although  it  also  does  not 
involve  rearrangement  of  charge  in  the  mem- 
brane, this  slower  inactivation  seems  to  involve  a 
mechanism  different  from  the  fast  process.  In  col- 
laboration with  Kathleen  Choi  and  Gary  Yellen, 
we  have  found  that  the  slow  inactivation  is  af- 
fected by  external  agents,  suggesting  that  the 
conformational  changes  for  this  process  involve 
external  structures.  Dr.  Hoshi,  Jose  Lopez  Barneo, 
and  I  have  found  that  the  rate  of  this  type  of  inac- 
tivation is  profoundly  affected  by  external  ions. 
Normally  sodium  is  the  predominant  positively 
charged  ion  outside  the  cell.  When  a  small 
amount  of  potassium  is  added  to  the  external  so- 


lution, as  could  occur  during  epileptic  activity  in 
the  brain  or  other  pathological  states,  the  inacti- 
vation is  significantly  slowed.  We  have  studied 
the  dependence  of  the  inactivation  rate  on  the 
type  of  external  ion  present  and  have  determined 
that  the  ability  of  an  ion  species  to  slow  inactiva- 
tion correlates  with  its  permeability  in  the  chan- 
nel. Ions  that  get  into  the  channel  better  from  the 
outside  inhibit  inactivation  more  effectively. 
These  results  are  consistent  with  a  hypothesis 
whereby  a  channel  that  is  occupied  by  an  ion  can- 
not undergo  slow  inactivation. 

The  slow  inactivation  process  occurs  by  greatly 
different  rates  in  variants  of  the  Shaker  channel 
with  differences  in  structure  at  the  carboxyl  end 
of  the  protein.  We  have  made  mutations  in  both 
of  these  variants  and  have  localized  the  differ- 
ence responsible  for  the  differences  in  slow  inac- 
tivation to  a  single  hydrophobic  amino  acid  in  a 
membrane-spanning  region  of  the  channel  mole- 
cule. Other  amino  acid  substitutions  at  this  posi- 
tion have  dramatic  effects  on  gating,  with  larger 
hydrophobic  amino  acids  leading  to  slower 
inactivation. 


6 


Divergent  Members  of  the  SRY  Family 
of  Transcriptional  Regulators  Bind  an 
Insulin-Responsive  Element,  IRE-A 


Maria  C.  Alexander-Bridges,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Alexander- Bridges  is  also  Assistant  Professor  of  Medicine  at  Harvard  Medical  School  and  Assistant  in 
Medicine  at  Massachusetts  General  Hospital,  Boston.  She  received  her  M.D.  and  Ph.D.  degrees  from 
Harvard  University  Medical  School,  where  she  was  a  member  of  the  Harvard-MIT  Health  Sciences  and 
Technology  program,  which  is  geared  toward  students  interested  in  academic  medicine.  She  developed  an 


/  abiding  interest  in  hormonal  regulation  of  cellular  metabolism  and,  as  a  graduate  student  in  physiology, 

L investigated  the  mechanism  of  insulin- stimulated  phosphorylation  of  cellular  proteins.  Dr.  Alexander- 
Bridges  then  served  as  an  intern  and  resident  at  the  Johns  Hopkins  University.  After  subspecialty  training 
in  endocrinology  at  Massachusetts  General  Hospital,  she  was  a  postdoctoral  fellow  with  Howard  Goodman. 


THE  main  function  of  insulin  is  to  allow  a 
starved  animal  to  adapt  to  a  glucose  load.  In- 
sulin does  this  by  activating  enzymes  that  pro- 
mote energy  storage  and  inactivating  enzymes 
that  break  down  energy  stores.  Over  the  long 
term,  insulin  alters  the  expression  of  these  en- 
zymes by  regulating  the  transcription  of  their 
genes.  The  work  in  our  laboratory  is  directed  to- 
ward understanding  the  mechanism  of  insulin  ac- 
tion on  expression  of  metabolically  active  genes 
in  tissues  that  modulate  glucose  utilization.  We 
use  the  glyceraldehyde- 3 -phosphate  dehydroge- 
nase (GAPDH)  gene  as  a  model  gene  for  the  ana- 
bolic effects  of  insulin,  because  it  is  highly  regu- 
lated by  insulin  1)  in  cultured  3T3  adipocytes,  2) 
during  nutritional  manipulations  such  as  fasting 
and  refeeding,  and  3)  during  the  induction  and 
treatment  of  diabetes. 

Transgenic  animals  that  express  GAPDH- 
growth  hormone  fusion  genes  have  been  made  in 
collaboration  with  Jeung  Yun  and  Tom  Wagner 
(Ohio  State  University).  Using  these  animals,  we 
have  confirmed  that  GAPDH  gene  transcription  is 
regulated  in  vivo  by  nutritional  manipulations 
that  lead  to  hyperinsulinemia.  For  example,  fast- 
ing a  rat  and  refeeding  it  a  high-carbohydrate, 
low-fat  diet  increases  circulating  glucose  and  in- 
sulin levels,  resulting  in  the  induction  of  glyco- 
lytic and  lipogenic  enzymes  and  the  repression  of 
gluconeogenic  and  lipolytic  enzymes. 

We  have  identified  a  cis-acting  sequence, 
insulin-responsive  element  A  (IRE-A),  in  the  up- 
stream region  of  the  GAPDH  gene  that  interacts 
with  an  insulin-responsive  DNA-binding  protein 
(IRP-A) .  Activation  of  GAPDH  gene  expression  in 
insulin-responsive  tissues  correlates  with  the 
presence  of  IRP-A.  Within  one  hour  of  exposure 
of  3T3  adipocytes  or  H35  hepatoma  cells  to  insu- 
lin, the  activity  of  this  protein  is  increased  two-  to 
fourfold.  The  activity  of  IRP-A  is  induced  four-  to 
eightfold  in  liver  and  fat  during  the  process  of 
refeeding  a  fasted  rat  a  high-carbohydrate,  low-fat 
diet.  These  observations  support  the  importance 
of  GAPDH  gene  regulation.  In  muscle,  where 


GAPDH  activity  is  not  rate  limiting  and  is  not  reg- 
ulated by  insulin,  IRP-A  binding  is  not  detectable. 

The  wild-type  IRE-A  motif  was  used  to  isolate  a 
clone  from  a  rat  adipocyte  library,  using  the 
Singh-Sharp  Southwestern  screening  approach. 
The  cloned  cDNA  encodes  a  protein  (IRE-ABP) 
that  binds  IRE-A  DNA  with  sequence  specificity 
that  overlaps  that  of  the  adipocyte  IRP-A  nuclear 
extract  protein.  IRE-ABP  is  expressed  in  liver  and 
fat  but  not  in  muscle,  which  provides  an  explana- 
tion for  the  tissue-specific  regulation  of  GAPDH 
gene  expression.  Expression  of  IRP-A  mRNA  is  in- 
duced during  the  process  of  fasting  and  refeed- 
ing. In  contrast,  one  hour  of  insulin  exposure  of 
cells  does  not  appear  to  alter  expression  of  the 
IRP-A  mRNA.  IRE-ABP  footprints  the  upstream  re- 
gion of  genes  that  are  inhibited  and  genes  that  are 
stimulated  by  insulin.  We  presume  that  IRE-ABP 
will  regulate  the  transcription  of  metabolic  genes 
with  diverse  functions  to  change  the  phenotype 
of  the  fat  and  liver  cell  during  the  switch  from  the 
fasted  to  the  refed  state  that  is  initiated  by  glucose 
and  insulin.  Studies  are  in  progress  to  determine 
whether  IRE-ABP  mediates  the  effect  of  insulin 
alone  or  in  association  with  another  protein. 

IRE-A  DNA-binding  Protein  Shares  Binding 
Specificity  with  the  Testis-determining 
Factor 

Surprisingly,  the  HMG  (high-mobility  group) 
box  domain  of  IRE-ABP  is  68  percent  identical  to 
SRY,  the  testis-determining  factor,  and  is  98  per- 
cent identical  to  an  autosomal  gene  that  was  iso- 
lated during  the  process  of  screening  a  whole 
mouse  embryo  cDNA  library  for  5i?F-related  se- 
quences. Furthermore,  IRE-ABP  and  SRY  share 
DNA-binding  specificity  for  IRE-A.  Although  IRE- 
ABP  shows  markedly  higher  affinity  for  the  IRE-A 
motif,  the  nucleotides  protected  by  these  two  di- 
vergent family  members  are  essentially  identical. 
The  sequence  in  IRE-A  that  is  contacted  by  IRE- 
ABP  and  SRY  is  highly  conserved:  the  sequence 
5'-Py-ctttg(a/t)-3',  previously  defined  by  Kather- 
ine  Jones  and  her  colleagues  as  a  consensus  motif, 


7 


Divergent  Members  of  the  SKY  Family  of  Transcriptional  Regulators  Bind  an 
Insulin-Responsive  Element,  IRE-A 


is  contained  in  several  T  cell-specific  genes  that 
are  bound  with  high  affinity  by  TCF-la  (T  cell 
factor  la).  Thus  diverse  members  of  the  HMG 
family  of  proteins  may  modulate  transcription 
through  a  similar  spectrum  of  sequences  that 
contain  a  core  motif.  Identification  of  such  a  mo- 
tif may  provide  a  clue  to  the  identity  of  important 
physiological  targets  of  the  IRE-ABP  and  SRY-like 
family  of  transcriptional  regulators. 

The  laboratories  of  Peter  Goodfellow  and  Ro- 
bin Lovell-Badge  have  identified  5/?Fas  the  testis- 
determining  region  on  the  basis  of  genetic  evi- 
dence. Several  patients  with  an  XY  genotype  who 
failed  to  differentiate  to  the  male  phenotype  have 
mutations  in  the  HMG  box  domain  of  SRY.  The 
SRY  locus  is  widely  presumed  to  encode  a  se- 
quence-specific DNA-binding  protein  because  it 
contains  an  HMG  domain  within  its  open  reading 
frame.  The  DNA-binding  properties  and  mecha- 
nism by  which  this  protein  regulates  transcrip- 
tion have  not  been  defined.  Certain  of  the  muta- 
tions that  were  found  in  the  sex-reversed  patients 
were  not  de  novo  mutations,  and  thus  it  was  not 
possible  to  deduce  whether  these  mutations  were 
completely  unrelated  to  the  sex-reversed  pheno- 
type or  contributed  to  it.  Because  IRE-ABP  was 
isolated  on  the  basis  of  its  ability  to  bind  the  IRE-A 
motif,  it  was  possible  to  examine  the  effect  of 
these  mutations  on  the  binding  affinity  of  IRE- 
ABP  and  SRY  derivatives.  Derivatives  that  con- 
tained the  mutations  associated  with  sex  reversal 
at  positions  3  and  7  in  the  HMG  box  domain 
showed  marked  impairment  in  their  ability  to 
protect  the  IRE-A  motif  from  DNase  I  digestion. 
Furthermore,  derivatives  of  IRE-ABP  and  5.^y  that 
contain  a  switch  between  IRE-ABP  and  SRYat  po- 
sition 3  in  the  HMG  box  domain  show  altered 
binding  affinity  for  the  IRE-A  motif.  Thus  posi- 
tions 3  and  7  appear  to  be  important  determi- 
nants of  binding  affinity  for  this  family. 

Future  Directions 

Identification  of  the  IRE-A  binding  protein  as  a 


member  of  the  5J?Ffamily  suggests  many  avenues 
of  investigation.  Studies  are  under  way  to  define 
the  binding  specificity  of  IRE-ABP-  and  SRY-like 
family  members.  Definition  of  the  preferred  bind- 
ing site  for  these  related  proteins  will  facilitate 
the  identification  of  other  insulin-sensitive  genes 
that  are  regulated  by  IRE-ABP  and  potential  tar- 
gets of  SRY.  For  example,  insulin  simultaneously 
activates  and  inhibits  diverse  metabolic  pro- 
cesses to  alter  the  flux  of  metabolites  into  glyco- 
gen and  fat.  We  have  located  the  proposed  con- 
sensus sequence  in  the  upstream  region  of  genes 
that  are  regulated  in  a  positive  and  negative  direc- 
tion by  insulin,  and  we  can  now  establish 
whether  IRE-ABP  plays  a  role  in  regulating  these 
diverse  metabolic  processes. 

The  observation  that  the  SRY  protein  and 
IRE-ABP  share  binding  specificity  for  a  se- 
quence located  in  a  glycolytic  gene  implies  that 
these  proteins  carry  out  similar  functions  in  the 
specific  tissues  in  which  they  are  expressed.  It 
is  clear,  for  instance,  that  spermatogenesis  in 
the  adult  testis  requires  high  lactate  produc- 
tion; thus  both  gene  products  may  be  involved 
in  regulating  glycolysis/gluconeogenesis  in 
their  respective  target  tissues.  Conversely,  IRE- 
ABP-like  genes  may  play  a  role  in  regulating 
processes  that  show  sexual  dimorphism  in 
adult  tissues  or  promoting  differentiation  of  its 
target  tissues  during  embryogenesis. 

Studies  on  the  regulation  of  IRP-A  gene  expres- 
sion have  led  to  an  understanding  of  the  mecha- 
nism by  which  insulin  modulates  the  expression 
of  specific  genes  in  specific  tissues  involved  in 
the  maintenance  of  normal  glucose  and  lipid  me- 
tabolism. Ultimately  we  expect  these  studies  will 
lead  to  an  understanding  of  the  signal  transduc- 
tion process  by  which  insulin  modulates  the  ex- 
pression of  these  genes.  Understanding  the  hor- 
monal control  of  lipid  metabolism  at  a  molecular 
level  will  provide  insights  into  two  disease  states 
of  major  importance,  obesity  and  diabetes. 


8 


Genetic  Mechanisms  Involved  in  the  Generation 
of  the  Antibody  Repertoire 


Frederick  W.  Alt,  Ph.D. — Investigator 

Dr.  Alt  is  also  Professor  of  Genetics  and  Pediatrics  at  the  Children 's  Hospital,  Boston,  and  Harvard  Medical 
School.  He  obtained  his  undergraduate  degree  in  biology  from  Brandeis  University  and  his  Ph.D.  degree 
in  biological  sciences  from  Stanford  University,  where  he  worked  with  Robert  Schimke.  He  did  postdoctoral 
work  with  David  Baltimore  at  the  Massachusetts  Institute  of  Technology,  after  which  he  was  Professor 
of  Biochemistry  and  Microbiology  at  Columbia  University  College  of  Physicians  and  Surgeons. 
His  honors  include  the  Irma  T.  Hirschl  Career  Scientist  Award,  the  Searle  Scholars  Award, 
and  the  Mallinckrodt  Scholar  Award. 


WE  are  interested  in  the  molecular  mecha- 
nisms that  underlie  the  generation  of  a  spe- 
cific immune  response.  The  mammalian  immune 
system  functions  through  complex  interactions 
between  various  cells  and  their  products.  Cells 
that  effect  specific  immunological  responses  fall 
into  two  general  categories:  B  lymphocytes  that 
mediate  humoral  immunity  (i.e.,  production  of 
antibodies  against  foreign  antigens)  and  T  lym- 
phocytes that  mediate  cellular  immunity  (e.g., 
foreign  graft  rejection).  During  the  earlier  stages 
of  lymphocyte  development,  stem  cells  proceed 
through  a  developmental  program  that  ultimately 
leads  to  the  generation  of  a  multitude  of  individ- 
ual B  or  T  lymphocyte  clones  (each  clone  is  an 
essentially  identical  set  of  cells  derived  from  a 
common  parent).  Each  set  of  clonal  cells  ex- 
presses a  novel  receptor  on  its  surface  that  will 
recognize  a  unique  set  of  antigens. 

The  ability  of  the  immune  system  to  respond 
specifically  to  a  vast  array  of  antigens  results  in 
substantial  part  from  the  unique  organization  of 
the  genes  that  encode  antigen  receptor  proteins. 
Unlike  most  genes,  antigen  receptor  genes  are  not 
inherited  intact  from  our  parents.  Instead,  these 
genes  are  encoded  in  cassettes  (gene  segments) 
in  the  germline  and  are  assembled  into  complete 
genes  only  during  the  somatic  differentiation  of 
lymphocytes.  This  lymphocyte-specific  gene  as- 
sembly process  is  referred  to  as  VDJ  recombina- 
tion. Because  there  are  many  individual  cassettes 
that  encode  various  portions  of  antibodies  and 
because  these  can  be  put  together  in  various  com- 
binations or  in  various  ways,  the  body  can  ran- 
domly assemble  a  vast  array  of  different  antibody 
genes  from  a  limited  amount  of  genetic  material. 

Much  of  our  work  is  aimed  at  determining  the 
genetic  mechanisms  by  which  antibody  genes  are 
assembled  from  gene  segments,  the  role  of  the 
gene  assembly  process  in  the  generation  of  anti- 
body diversity,  and  the  mechanisms  that  regulate 
this  gene  assembly  process  and  ensure  that  it  oc- 
curs only  in  appropriate  cell  types.  We  are  also 
working  on  the  elucidation  of  molecular  signals 
that  control  the  various  steps  of  B  lymphocyte 
differentiation. 


The  enzymatic  system  involved  in  the  assembly 
of  antigen  receptor  genes  involves  a  variety  of 
different  activities.  Some  of  these  activities  are 
expressed  specifically  in  developing  lympho- 
cytes and  are  likely  involved  in  the  early  pro- 
cesses of  gene  assembly,  including  recognition  of 
the  specific  gene  segments  that  will  be  joined  and 
cutting  them  away  from  the  surrounding  genetic 
material .  Other  activities  employed  in  VDJ  recom- 
bination are  likely  expressed  in  many  cell  types 
where  they  may  be  involved  in  other  processes, 
such  as  replication  of  genetic  material  or  repair 
of  lesions  in  genetic  material  (DNA  repair) .  The 
more  widely  expressed  activities  are  probably  re- 
cruited by  the  lymphocyte-specific  components 
of  the  system  to  carry  out  certain  aspects  of  the 
joining  event,  such  as  pasting  together  the  "cut" 
gene  segments. 

The  lymphocyte-specific  components  of  the 
VDJ  recombination  system  are  likely  encoded  by 
two  genes  (recently  isolated  by  David  Baltimore 
and  David  Schatz)  referred  to  as  recombination- 
activating  genes  1  and  2  {RAG-1  and  -2).  Simulta- 
neous expression  of  these  genes  was  found  to  oc- 
cur only  in  developing  lymphocytes;  artificially 
induced  expression  of  the  two  genes  (simulta- 
neously) in  nonlymphoid  cells  confers  them  with 
the  ability  to  undergo  VDJ  recombination.  How- 
ever, these  genes  have  been  found  to  be  ex- 
pressed in  other  tissues,  including  the  central 
nervous  system,  fueling  speculation  that  VDJ  re- 
combination activity  or  the  activity  of  one  or  the 
other  of  the  two  RAG  gene  products  might  be 
involved  in  developmental  processes  in  tissues 
other  than  the  immune  system. 

To  test  unequivocally  the  function  of  the  RAG- 
2  gene,  we  have  used  gene-targeting  technology 
to  eliminate  a  copy  of  this  gene  in  mouse  embry- 
onic stem  cell  lines.  These  cells  were  then  intro- 
duced into  developing  mouse  embryos,  where 
they  were  incorporated  into  the  germ  cells  of  the 
resulting  chimeric  mouse.  Mice  that  carried  a  sin- 
gle copy  of  the  targeted  mutation  in  their  germ- 
line  were  interbred  to  generate  animals  that  com- 
pletely lack  the  gene.  Mice  that  lack  the  RAG-2 


9 


Genetic  Mechanisms  Involved  in  the  Generation  of  the  Antibody  Repertoire 


gene  are  viable  and  appear  normal  at  birth.  How- 
ever, they  routinely  develop  severe  infections  in 
the  first  months  of  life  due  to  the  total  absence  of 
mature  T  or  B  lymphocytes — a  severe  combined 
immune  deficiency.  These  animals  do  have  large 
populations  of  very  immature  B  and  T  lympho- 
cytes in  their  primary  lymphocyte  differentiation 
organs;  however,  these  cells  are  unable  to  initiate 
the  VDJ  recombination  process.  Because  the 
.^G-2-deficient  mice  have  no  defects  in  any  cell 
type  besides  lymphocytes,  we  have  concluded 
that  VDJ  recombination  activity  and  RAG- 2  gene 
function  are  required  only  for  lymphocyte  devel- 
opment and  not  for  any  other  developmental 
process. 

Our  group  has  also  helped  characterize  the 
basis  of  a  naturally  occurring  murine  mutation 
that  when  homozygous  leads  to  severe  combined 
immune  deficiency  (the  scid  mutation).  Like 
iL4G-2-deficient  mice,  animals  homozygous  for 
the  scid  mutation  lack  functional  B  or  T  lympho- 
cytes due  to  inability  to  assemble  antigen  recep- 
tor gene  segments  correctly.  However,  mice  ho- 
mozygous for  the  scid  mutation  produce  normal 
RAG-1  and  -2  gene  products  and  can  efficiently 
initiate  the  VDJ  recombination  process.  Comple- 
tion of  the  joining  process  is  blocked  in  scid  mu- 
tant cells.  Moreover,  both  lymphoid  and  nonlym- 
phoid  cells  of  mice  homozygous  for  the  scid 
defect  are  unable  to  repair  certain  types  of  lesions 
in  their  genetic  material.  Thus  the  product  of 
the  gene  affected  by  the  scid  mutation  appears  to 
represent  an  example  of  a  component  of  the 
VDJ  recombination  system  that  is  used  both  in 
VDJ  recombination  and  in  more  general  cellular 
processes. 

To  define  further  generally  expressed  genes 
potentially  involved  in  VDJ  recombination,  we 


provided  the  lymphocyte-specific  components  of 
this  system  (RAG-l  and  -2)  to  mutant  Chinese 
hamster  ovary  cell  lines  that  are  defective  in  abil- 
ity to  repair  breaks  in  their  DNA.  By  introducing 
antigen  receptor  gene  segments  into  these  lines, 
we  could  ask  whether  the  cells  were  capable  of 
performing  correct  VDJ  recombination.  By  this 
approach,  we  have  now  derived  a  series  of  inde- 
pendent genetic  mutations  that  affect  both  DNA 
repair  and  VDJ  recombination.  Introduction  of 
specific  human  chromosomes  into  the  mutant 
cell  lines  restored  both  ability  to  repair  DNA  and 
to  undergo  VDJ  recombination.  We  are  now  in  the 
process  of  isolating  the  defective  genes  in  these 
cells.  Elucidation  of  these  genes  will  provide  in- 
sight into  the  VDJ  recombination  process  and  may 
also  provide  information  relevant  to  understand- 
ing the  basis  for  several  human  diseases  that  affect 
both  ability  to  repair  genetic  lesions  and  to  gener- 
ate a  normal  immune  system. 

We  are  also  analyzing  several  other  novel 
mouse  models  generated  to  study  various  factors 
involved  in  lymphocyte  differentiation  and  the 
generation  of  the  immune  response.  One  such 
model  is  a  mutant  mouse  line  that  cannot  pro- 
duce endogenous  antibody  molecules  because  it 
has  been  genetically  engineered  to  lack  germline 
gene  segments  necessary  for  forming  functional 
antibody  genes.  We  are  using  these  animals  to 
study  the  role  of  antibody  gene  products  in  regu- 
lating antibody  gene  assembly  and  lymphocyte 
development.  A  potential  practical  use  of  such 
mice  may  be  achieved  by  breeding  them  with 
transgenic  mouse  lines  that  have  been  engineered 
to  contain  functional  human  antibody  gene  cas- 
settes. The  hope  is  that  the  hybrid  animal  will 
rely  on  the  human  antibody  genes  for  its  immune 
system,  providing  a  more  effective  method  of 
generating  tailor-made  human  antibodies. 


10 


Control  of  Cell  Fate  During  Vertebrate 
Neuronal  Development 


David  J.  Anderson,  Ph.D. — Assistant  Investigator 

Dr.  Anderson  is  also  Associate  Professor  of  Biology  at  the  California  Institute  of  Technology  and  Adjunct 
Assistant  Professor  of  Anatomy  and  Cell  Biology  at  the  University  of  Southern  California  School  of 
Medicine.  He  received  his  A.B.  degree  in  biochemical  sciences  from  Harvard  College  and  a  Ph.D.  degree  in 
cell  biology  from  the  Rockefeller  University.  He  then  did  postdoctoral  research  in  molecular  neurobiology 
at  Columbia  University.  He  is  the  recipient  of  an  NSF  Presidential  Young  Investigator  Award,  a  Sloan 
Foundation  Fellowship  in  Neuroscience,  and  the  1989  Charles  Judson  Herrick  Award  in  Neurobiology 
from  the  American  Association  of  Anatomy. 


WE  are  interested  in  how  different  types  of 
nerve  cells  are  generated  during  the  devel- 
opment of  the  vertebrate  nervous  system.  We 
have  chosen  to  work  on  the  peripheral  auto- 
nomic nervous  system,  which  is  simpler  and 
more  accessible  than  the  brain.  Our  studies  have 
focused  on  the  development  of  two  specific  cell 
types:  the  sympathetic  neurons  that  lie  in  a  chain 
of  ganglia  along  the  spinal  cord  and  the  chromaf- 
fin cells  of  the  adrenal  medulla.  These  two  cells 
are  closely  related,  yet  distinct  in  major  respects. 
The  former  are  true  neurons,  with  long  branching 
axons  and  dendrites  that  send  and  receive  electri- 
cal signals.  The  latter  are  small,  round  secretory 
cells  that  release  epinephrine  (adrenaline)  into 
the  bloodstream  during  fright  or  excitement. 

Studies  in  a  number  of  laboratories,  including 
our  own,  have  established  that  these  two  cell 
types  derive  from  a  common  progenitor  cell.  This 
cell  arises  on  top  of  the  neural  tube  (the  develop- 
ing spinal  cord) ,  as  part  of  a  transient  structure 
called  the  neural  crest.  Like  parachutists,  the 
neural  crest  cells  peel  off  the  neural  tube  in  a 
wave  and  migrate  downward  through  the  em- 
bryo. Some  of  them  arrest  their  migration  in  a 
chain  of  small  clumps  along  a  blood  vessel, 
where  they  eventually  become  sympathetic  neu- 
rons. Others  continue  their  migration  downward 
to  invade  the  developing  adrenal  gland,  where 
they  become  chromaffin  cells. 

Using  a  sophisticated  fluorescence-activated 
cell  sorter  and  specific  monoclonal  antibodies  to 
tag  the  cells,  we  have  succeeded  in  isolating 
chromaffin  cell  precursors  from  the  fetal  adrenal 
gland  of  the  rat.  By  manipulating  the  cell  culture 
environment,  we  have  shown  that  these  precur- 
sors have  two  possible  developmental  fates:  if 
glucocorticoid  hormones  are  added  to  the 
growth  medium,  mimicking  the  environment  of 
the  adrenal  gland,  then  the  precursors  develop 
into  chromaffin  cells.  If,  on  the  other  hand,  fibro- 
blast growth  factor  (FGF)  and  nerve  growth  fac- 
tor (NGF)  are  added  to  the  medium,  the  precur- 
sors develop  into  sympathetic  neurons.  This 
suggests  that  the  fate  of  these  cells  is  determined 


in  large  part  by  signals  in  the  environments  to 
which  they  migrate.  However,  these  precursors 
seem  to  have  lost  the  ability  to  give  rise  to  other 
derivatives  of  the  neural  crest,  such  as  glial  cells. 
Therefore,  while  these  precursor  cells  have  a 
choice  of  fate,  it  is  a  restricted  one. 

Control  of  Neuronal  Survival 
by  Neurotrophic  Factors 

One  problem  in  studying  the  chromaffin-neu- 
ron  precursor  cell  at  the  molecular  level  is  the 
small  number  of  cells  that  can  be  recovered  after 
isolation  from  rat  fetuses.  To  circumvent  this 
problem,  wc  have  applied  recently  developed 
techniques  to  immortalize  the  cells.  Using  a  de- 
fective retrovirus  as  a  "disposable  molecular  sy- 
ringe," we  have  injected  the  cells  with  a  gene, 
v-myc,  that  allows  them  to  divide  forever  in  the 
culture  dish.  In  this  way  we  can  generate  an  end- 
less supply  of  cells  that  can  be  used  for  experi- 
ments at  any  time,  without  the  need  to  perform 
long  hours  of  dissection.  Fortunately,  these  im- 
mortalized precursor  cell  lines  still  appear  capa- 
ble of  undergoing  differentiation  into  sympa- 
thetic neurons  when  exposed  to  FGF  and  NGF. 

Sympathetic  neurons,  like  other  neurons,  de- 
pend on  specific  neurotrophic  factors  for  their 
survival.  Neurotrophic  factors  are  proteins  se- 
creted by  the  tissues  innervated  by  the  neurons; 
these  proteins  nourish  the  neurons  and  keep 
them  alive.  Different  types  of  neurons  use  differ- 
ent neurotrophic  factors:  NGF  is  the  factor  for 
sympathetic  neurons.  The  specificity  of  NGF  ac- 
tion is  due  to  a  specific  receptor  protein  present 
in  the  membrane  of  the  nerve  cell.  This  receptor 
binds  NGF  and  sends  a  signal  to  the  cell,  keeping 
it  alive.  We  are  using  the  immortalized  precursor 
cell  line  to  study  how  developing  neurons  ac- 
quire NGF  receptors  during  development.  The 
NGF  receptor  has  two  components  (subunits); 
our  data  suggest  that  different  factors  induce  the 
expresision  of  these  two  subunits.  One  factor  that 
seems  to  be  important  for  the  induction  of  func- 
tional NGF  receptors  is  electrical  activity.  This 
suggests  that  the  stimulation  of  the  developing 


Control  of  Cell  Fate  During  Vertebrate  Neuronal  Development 


neuron  by  its  presynaptic  partner  may  endow  it 
with  the  machinery  to  respond  to  key  survival 
factors  secreted  by  its  postsynaptic  target.  In  this 
way,  the  estabHshment  of  functional  connections 
between  a  neuron,  its  input,  and  its  output  target 
could  be  coordinated. 

Control  of  Neuron-Specific  Gene  Expression 

As  it  differentiates,  the  chromaffin-neuron  pre- 
cursor expresses  genes  that  are  not  expressed  in 
other  cell  types.  How  such  specific  expression  is 
achieved  is  a  basic  question  in  modern  develop- 
mental biology.  Studies  in  blood  and  lymphoid 
cells  have  revealed  that  genes  such  as  that  encod- 
ing hemoglobin  are  specifically  turned  on  in  the 
appropriate  cells  by  activator  proteins,  present 
only  in  those  cells.  We  have  studied  a  neuron- 
specific  gene,  SCGIO,  and  found  that  a  different 
mechanism  restricts  its  expression  to  developing 
neurons.  SCO  1 0  appears  to  be  specifically  turned 
off  in  all  tissues  except  neurons;  this  repression  is 
somehow  relieved  in  neurons  and  not  in  other 
cells.  We  have  made  advances  in  identifying  a 
protein  involved  in  the  repression  of  SCGIO.  This 
"silencer"  protein  is  present  in  nonneuronal 
cells  and  tissues  but  not  in  neuronal  cells,  consis- 
tent with  the  idea  that  it  shuts  off  SCGIO  every- 
where except  in  the  nervous  system.  This  same 
silencer  protein  also  appears  to  be  involved  in 
shutting  off  other  neuron-specific  genes.  This 
suggests  that  specific  de-repression  may  be  a 
common  mechanism  for  controlling  the  expres- 
sion of  neuron-specific  genes  and  that  a  common 
repressor  protein  may  silence  the  expression  of 
several  such  genes.  Future  efforts  will  be  directed 


at  cloning  the  gene  for  this  repressor  protein  and 
understanding  how  it  in  turn  is  controlled. 

Neural  Development  in  Mammals 
and  Drosophila  Uses  Similar 
Regulatory  Molecules 

We  have  used  the  chromaffin-neuron  precursor 
cell  lines  to  isolate  new  genes  that  may  be  candi- 
dates for  controlling  the  development  of  these 
cells.  Our  approach  is  based  on  the  idea  that  Dro- 
sophila genes  controlling  neural  development 
might  also  be  conserved  in  mammals.  One  impor- 
tant set  of  genes  that  control  neuronal  develop- 
ment in  Drosophila  are  those  of  the  achaete- 
scute  complex  (AS-C) .  These  scute  genes  encode 
a  group  of  related  proteins  that  act  by  binding  to 
DNA,  thereby  controlling  the  activity  of  other 
genes.  We  succeeded  in  isolating  two  scute- 
related  genes  from  the  rat  chromaffin-neuron 
precursor  cell  line.  The  structures  of  these  genes 
are  remarkably  similar  to  those  of  their  fruit  fly 
counterpans.  Moreover,  the  rat  scute-veliAed 
genes  appear  to  be  expressed  specifically  in  neu- 
ronal precursor  cells,  like  their  counterparts  in 
the  fly.  These  data  indicate  that  there  has  been  a 
striking  parallel  conservation  of  gene  structure 
and  cell  type  specificity  during  evolution.  They 
further  suggest  that  the  scwfe-related  genes  may 
control  the  development  of  mammalian  neurons. 
These  exciting  findings  suggest  that  the  molecu- 
lar mechanisms  controlling  nerve  cell  develop- 
ment in  vertebrate  and  invertebrate  organisms 
may  be  fundamentally  similar.  We  are  now  in  the 
process  of  testing  this  hypothesis  by  making  tar- 
geted mutations  in  these  genes  in  mice. 


12 


Cell  Fate  Choices  in  the  Nervous  System 
and  Elsewhere 


Spyridon  Artavanis-Tsakonas,  Ph.D. — Investigator 

Dr.  Artavanis-Tsakonas  is  also  Professor  of  Cell  Biology  and  Biology  at  Yale  University.  He  received 
his  M.A.  degree  in  chemistry  from  the  Federal  Institute  of  Technology  (Eidgenoessische  Technische 
Hochschule)  in  Zurich  and  his  Ph.D.  degree  in  molecular  biology  from  the  University  of  Cambridge, 
England,  for  work  done  at  the  MRC  Laboratory  of  Molecular  Biology.  His  postdoctoral  work  was  done 
at  the  Biozentrum  of  the  University  of  Basel  with  Walter  Gehring  and  at  Stanford  University 
with  David  Hogness. 


A fundamental  issue  in  the  biology  of  multi- 
cellular organisms  is  how  a  cell  acquires  its 
specific  developmental  fate.  The  molecular  rules 
by  which  neighboring  cells  choose  developmen- 
tally  distinct  paths  are  unknown.  We  are  particu- 
larly interested  in  how  these  rules  apply  to  the 
nervous  system  and  thus  have  been  exploring  the 
molecular  biology  of  early  neurogenesis  in  the 
fruit  fly. 

The  central  nervous  system  in  Drosophila  de- 
rives from  a  set  of  precursor  cells  (neuroblasts) 
that  segregate  from  the  epidermal  precursors 
(dermoblasts)  in  the  very  early  ectoderm.  Embry- 
ological,  genetic,  and  molecular  studies  have 
demonstrated  that  neuroblast  segregation  de- 
pends on  interactions  between  neighboring  cells. 
Several  genes  capable  of  interfering  with  this  pro- 
cess have  been  identified,  and  the  Notch  locus  is 
central.  As  we  have  shown,  Notch  codes  for  a 
transmembrane  protein  with  homology  to  the 
mammalian  epidermal  growth  factor  (EGF),  im- 
plying involvement  in  cell-surface  events. 

For  several  years  we  have  been  studying  Notch 
and  other  genetic  elements  in  the  cell  inter- 
actions underlying  neuroblast  differentiation. 
Through  these  studies  it  became  clear  that  the 
mechanisms  governing  the  segregation  of  the  neu- 
roblasts from  the  epidermal  precursors  in  the 
neural  ectoderm  are  used  not  only  in  neurogene- 
sis but  also  in  a  broad  spectrum  of  tissues  and  cell 
fate  choices. 

In  an  attempt  to  examine  the  complexity  of  the 
genetic  circuitry  in  which  Notch  is  integrated 
and  to  identify  genes  whose  products  may  inter- 
act directly  with  the  Notch  protein,  we  have  been 
using  genetic  screens  designed  to  identify  sup- 
pressors or  enhancers  of  specific  Notch  muta- 
tions. Through  this  analysis,  five  genes,  which  we 
operationally  refer  to  as  the  '"Notch  group,"  have 
been  identified,  and  extensive  interactions 
among  them  documented.  Besides  Notch,  the  set 
includes  four  members  that  code  for  cell-surface, 
cytoplasmic,  and  nuclear  elements.  Delta  and 
Serrate  both  code  for  transmembrane  proteins 
with  EGF  homologous  extracellular  domains;  En- 
hancer of  split  and  mastermind  code  for  nuclear 
proteins;  and  deltex  appears  to  be  cytoplasmic. 


Among  the  various  genetic  interactions  within 
the  Notch  group,  those  occurring  between  Notch 
and  Delta  most  clearly  suggest  that  they  interact 
on  the  molecular  level.  To  investigate  this  possi- 
bility, we  examined  the  effects  of  Notch  and 
Delta  expression  in  Drosophila  S2  cells.  We 
found  that  Notch-  and  De/to-expressing  cells 
formed  mixed  aggregates  and  that  this  occurs  via 
their  extracellular  EGF  homologous  domains.  In- 
terestingly, A'^o^c^-expressing  cells  exhibit  vesic- 
ular structures  containing  both  Delta  and  Notch 
proteins,  implying  that  Delta  is  internalized  via 
Notch  as  a  receptor.  This  relationship  is  currently 
being  analyzed  by  electron  microscopy. 

Deletion  mutagenesis  of  the  extracellular  do- 
main of  Notch  determined  that  only  two  of  the  36 
EGF  repeats  are  sufficient  and  necessary  to  medi- 
ate interactions  with  Delta.  Thus  the  extracellu- 
lar domain  of  Notch  seems  surprisingly  modular 
and  could  potentially  serve  to  bind  a  number  of 
other  proteins.  Some  of  these  may  bind  to  subsets 
of  the  EGF  repeats  or  to  parts  of  the  extracellular 
domain  of  Notch,  while  others  may  compete  for 
the  same  binding  sites.  Indeed,  Serrate,  which 
displays  homologies  to  the  Delta  protein,  inter- 
acts with  Notch  in  a  Delta-like  fashion  and,  since 
it  binds  to  the  same  EGF  repeats,  may  compete 
with  Delta  for  binding  to  Notch. 

On  the  basis  of  these  results,  it  was  proposed 
that  Notch  may  function  as  a  multifunctional  re- 
ceptor containing  a  series  of  ligand-binding  sites, 
each  of  which  may  interact  with  more  than  one 
specific  ligand.  Such  a  model  provides  a  plausi- 
ble explanation  for  the  pleiotropic  action  of 
Notch  and  suggests  a  general  function  for  the 
gene  throughout  development. 

If  Notch  acts  as  a  receptor,  how  are  Notch- 
mediated  extracellular  signals  transmitted  to  the 
nucleus?  This  question  is  central  to  our  current 
work  and  is  being  addressed  at  several  levels. 

Cloning  and  sequencing  of  deltex  (which  dis- 
plays allele-specific  interactions  with  Notch, 
Delta,  and  mastermind)  revealed  the  primary 
structure  of  its  product,  a  700-amino  acid  pro- 
tein with  no  known  homologues.  Preliminary 
results  indicate  that  the  deltex  protein  is  cyto- 


13 


Cell  Fate  Choices  in  the  Nervous  System  and  Elsewhere 


plasmic  and  appears  to  be  influenced  in  its  sub- 
cellular localization  by  the  expression  of  Notch. 
While  these  observations  are  compatible  with  the 
notion  that  Notch  interacts  with  a  cytoplasmic 
protein,  recent  findings  regarding  both  En- 
hancer of  split  and  the  intracellular  domain  of 
th&^Notch  protein  raise  the  possibility  that  Notch 
could  directly  interact  with  nuclear  components. 

Most  of  the  evidence  supporting  this  hypothe- 
sis stems  from  our  analysis  of  human  Notch  group 
homologues.  In  our  attempts  to  dissect  the  mo- 
lecular mechanisms  involving  Notch  group 
members,  we  have  been  exploring  their  involve- 
ment in  vertebrate  development.  While  helping 
to  establish  the  degree  to  which  the  Drosophila 
paradigm  is  valid  for  vertebrate  species,  this  work 
also  permits  us  to  make  use  of  the  vertebrate 
model's  experimental  advantages,  such  as  better- 
defined  cultured  cell  lines. 

The  Drosophila  Enhancer  of  split  locus  was 
shown  to  be  defined  genetically  by  several  tran- 
scripts. With  one  exception,  the  genes  contain 
the  helix-loop-helix  (HLH)  motif  found  in  a  vari- 
ety of  transcriptional  regulators,  and  they  are  par- 
tially redundant  functionally.  The  single  gene 
that  does  not  contain  the  HLH  motif  displays  ho- 
mology to  the  |S-subunit  of  the  signal-transducing 
G  proteins.  Cloning  of  human  homologues, 
which  were  named  TLEs  (for  transducin-like  En- 
hancer of  split),  revealed  an  extraordinary  con- 
servation between  fly  and  human  sequences.  In 
addition,  there  seems  to  be  a  family  of  TLE  genes 
in  the  human,  though  not  the  Drosophila,  ge- 
nome. Similarly,  we  found  that  the  human  ge- 
nome contains  at  least  two  Notch  homologous 
sequences. 

Comparisons  among  the  four  isolated  human 
TLE  genes  and  the  Drosophila  counterpart 


showed  that  they  harbor  a  structural  motif  impli- 
cated in  nuclear-cytoplasmic  transport.  Referred 
to  as  the  "CcN  motif,"  it  consists  of  a  closely 
spaced  combination  of  a  nuclear  localization  se- 
quence and  potential  phosphorylation  sites  for 
both  casein  kinase  II  and  cdc2  kinase.  Such  a  se- 
quence is  consistent  with  the  notion  that  TLEs  act 
as  nuclear  effector  molecules.  Indeed,  immuno- 
cytochemical  localizations  show  that  both  hu- 
man and  Drosophila  molecules  are  predomi- 
nantly nuclear  but  can  also  be  detected  in  the 
cytoplasm. 

Surprisingly,  comparisons  of  the  known  Notch 
homologues,  including  the  two  human  se- 
quences, reveal  the  existence  of  a  CcN  domain  in 
the  intracellular  part  of  Notch.  We  do  not  yet 
have  any  evidence  regarding  the  functional  signif- 
icance of  this  motif,  which  raises  the  provocative 
possibility  that  Notch  may  possess  some  nuclear 
functions. 

In  conclusion,  work  in  the  past  year  extended 
our  knowledge  regarding  the  molecular  rules  un- 
derlying the  action  of  the  Notch  group  genes. 
Our  studies — including  analyses  of  expression 
patterns,  genetic  interactions,  and  mutant  pheno- 
types — reinforce  the  notion  that  at  least  some  of 
these  genes  code  for  elements  of  a  pleiotropic 
cell  interaction  mechanism  that  is  involved  in 
cell  fate  choices  in  many  different  tissues,  in- 
cluding the  nervous  system.  Within  those  tissues, 
however,  cell  fate  choices  can  also  depend  on 
tissue-specific  interaction  events.  Our  current 
working  hypothesis  is  that  regulative  events  con- 
trolling cell  fate  choices  throughout  develop- 
ment rely  on  the  parallel  action  of  tissue-specific 
mechanisms  and  on  A^ofc^-mediated  interactions. 
A^o/^c^  activity,  while  apparently  general,  can  nev- 
ertheless be  modulated  by  different  ligands  such 
as  Delta  or  Serrate. 


Expression  of  Notch  protein  in  the  early  stages 
of  wild-type  Drosophila  egg  chambers  (from 
dissected  ovaries )  as  shown  by  confocal  micros- 
copy. Notch  is  highly  expressed  at  the  apical 
surface  of  the  follicle  cells  that  surround  the 
developing  egg  yolk. 

From  Xu,  T..  Caron,  L.A.,  Fehon,  R.G.,  and 
Artavanis-Tsakonas,  S.  1992.  Development 
115:913-922. 


14 


The  Complement  System 


John  P.  Atkinson,  M.D. — Investigator 

Dr.  Atkinson  is  also  Professor  of  Medicine  and  of  Molecular  Microbiology  at  Washington  University  School 
of  Medicine  and  Physician  at  Barnes  Hospital,  St.  Louis.  He  received  his  B.A.  degree  in  zoology  and  M.D. 
degree  from  the  University  of  Kansas.  His  training  in  internal  medicine  was  at  Massachusetts  General 
Hospital,  Boston,  and  at  NIH.  He  conducted  postdoctoral  research  at  NIH  and  Washington  University. 


MANY  poorly  understood  but  relatively  com- 
mon and  serious  human  diseases  involve 
aberrations  of  the  immune  system.  Such  condi- 
tions range  from  mild  forms  of  arthritis  to  life- 
threatening  autoimmune  illnesses  such  as  sys- 
temic lupus  erythematosus. 

The  immune  system  provides  a  powerful  ar- 
senal of  proteins  to  attack  and  eliminate  infecting 
microorganisms.  A  major  means  of  accomplishing 
this  task  is  through  the  special  biologic  partner- 
ship of  antibodies  and  complement.  Proteins 
from  these  two  systems  circulate  in  blood  and  are 
either  formed  in  response  to  the  organism  (anti- 
bodies) or  present  at  all  times  (complement). 

The  work  in  this  laboratory  centers  on  the  com- 
plement system.  Its  name  is  derived  from  its  func- 
tion of  "assisting"  antibody  in  immune  reactions. 
However,  we  now  know  that  the  complement 
system  also  serves  as  an  independent  immune  sys- 
tem capable  of  attacking  foreign  elements  by  it- 
self. This  first-line  defense  appears  to  have  oc- 
curred early  in  evolution,  preceding  antibody. 
The  complement  system  consists  of  at  least  30 
proteins  that  interact  with  each  other  in  reactions 
resembling  a  cascade  or  waterfall.  The  activated 
molecules  that  result  from  this  scheme  destroy 
the  invading  microbe  and  promote  the  inflamma- 
tory response.  The  complement  system  is  a  pow- 
erful, swift,  and  highly  effective  means  to  fight 
infectious  organisms.  As  might  be  anticipated,  an 
inherited  deficiency  of  a  complement  compo- 
nent predisposes  an  individual  to  infections. 

The  production  of  antibodies  is  triggered  when 
foreign  substances  invade  the  body.  Although 
complement  independently  attacks  the  patho- 
gen, the  binding  of  antibodies  to  a  target  such  as  a 
bacterium  initiates  and  potentiates  a  series  of  re- 
actions in  which  complement  proteins  swarm 
onto  the  surface  of  the  microbe.  Such  compo- 
nents serve  as  ligands  for  complement  receptors 
on  blood  cells.  A  foreign  particle  so  coated 
quickly  becomes  adherent  to  and  is  ingested  by 
these  blood  cells.  During  complement  activa- 
tion, small  fragments  (peptides)  that  promote  the 
inflammatory  response  are  liberated  from  the 
complement  proteins.  These  molecules  dilate 


blood  vessels  and  summon  scavenger  cells,  called 
phagocytes,  from  the  bloodstream.  The  phago- 
cytic white  blood  cells,  upon  arrival  at  the  site, 
find  organisms  that  are  already  prepared  for  in- 
gestion; i.e.,  they  are  coated  with  antibody  and 
complement.  This  phenomenon  is  known  as  op- 
sonization (from  the  Greek  word  opsonein,  to 
prepare  for  the  table).  As  a  result,  the  infection  is 
sequestered  and  eliminated  via  phagocytosis. 

Sometimes,  however,  the  immune  system,  in- 
stead of  synthesizing  antibodies  to  foreign  mate- 
rials, seemingly  makes  a  mistake  and  produces 
antibodies  that  react  with  its  own  cells.  For  exam- 
ple, in  certain  immune  disorders,  individuals 
make  antibodies  to  their  own  red  blood  cells.  An- 
tibody and  complement  then  attach  to  the  red 
blood  cells  and  destroy  them.  Such  conditions  are 
known  as  autoimmune  diseases,  in  this  case  au- 
toimmune hemolytic  anemia. 

In  other  pathologic  conditions,  the  immune 
system  does  not  efficiently  eliminate  the  in- 
fectious particles,  and  excessive  quantities  of  im- 
mune complexes  form.  Immune  complexes  con- 
sist of  antibodies,  complement  proteins,  and  the 
foreign  particle.  These  immune  complexes  are 
proinflammatory  and  cause  tissue  damage  if  de- 
posited in  undesirable  locations  such  as  joints, 
skin,  and  kidney.  This  leads  to  arthritis,  dermati- 
tis, and  glomerulonephritis,  respectively. 

Thus  there  are  two  general  ways  in  which  the 
humoral  immune  system  can  damage  its  own  tis- 
sue. The  first  is  to  produce  antibodies  to  self- 
components.  The  second  is  to  form  excessive 
amounts  of  immune  complexes. 

Our  goal  is  to  understand  how  immune  com- 
plexes form  and  are  processed.  We  are  studying  a 
biologic  mechanism  that  evolved  to  remove  im- 
mune complexes  from  the  circulation.  This  pro- 
cess helps  prevent  the  pathologic  accumulation 
of  these  complexes  in  tissue.  This  system  can  be 
likened  to  an  "inner  space  shuttle."  The  physio- 
logic vehicle  for  this  journey  is  the  red  blood 
cell.  The  red  blood  cell  participates  in  this  reac- 
tion through  a  complement  receptor  protein  on 
its  surface.  These  receptor  proteins  latch  onto 
complement-coated  foreign  particles,  such  as  vi- 


15 


The  Complement  System 


ruses  and  bacteria.  As  the  circulating  red  blood 
cell  passes  through  the  liver  or  spleen,  its  im- 
mune cargo  is  released  and  metabolized.  The  red 
blood  cell  then  returns  to  the  circulation  ready  to 
ferry  another  load. 

Our  laboratory  is  studying  the  complement  re- 
ceptor involved  in  this  process.  We  are  also  exam- 
ining the  complement  proteins  that  swarm  onto 
the  foreign  particles.  Furthermore,  we  are  inves- 
tigating certain  "housekeeping"  proteins  of  the 
complement  system.  Because  of  the  powerful  de- 
structive capabilities  of  the  complement  system, 
it  is  perhaps  no  surprise  that  the  body  must  keep 
this  system  tightly  regulated.  Special  proteins  are 
synthesized  to  protect  the  body's  own  cells  from 
damage  by  complement  factors.  Our  laboratory 
discovered  a  new  family  of  genes  that  encode  for 
at  least  six  complement  receptor  and  regulatory 
proteins.  Two  of  these  regulatory  proteins  occur 
on  almost  all  cells  of  the  body.  These  are  termed 
decay-accelerating  factor  and  membrane  cofactor 
protein.  Recently  these  regulatory  proteins  have 
been  demonstrated  to  be  expressed  in  relatively 
high  concentration  on  reproductive  tissue,  in- 
cluding placental  tissue  and  sperm.  A  new  direc- 
tion for  the  laboratory  concerns  the  role  of  these 
proteins  in  reproduction.  Modulating  the  func- 
tion of  these  regulatory  proteins  may  also  be  im- 


portant in  improving  the  destruction  of  tumor 
cells. 

A  further  potential  benefit  from  our  research 
involves  the  field  of  transplantation.  Presently 
the  supply  of  donor  organs  is  far  less  than  the 
number  of  patients.  Many  people  worldwide  die 
while  waiting  for  a  suitable  organ.  Use  of  animal 
organs  has  not  been  possible,  partly  because  of 
the  complement  system's  attack  on  these  trans- 
plants. However,  this  problem  could  be  sur- 
mounted by  using  animal  organs  genetically  engi- 
neered with  complement  regulatory  proteins 
designed  to  prevent  attack  by  the  host's  com- 
plement system.  We  are  investigating  these 
possibilities. 

Finally,  because  infectious,  autoimmune,  and 
immune  complex-mediated  illnesses  result  from 
aberrations  of  the  complement  system,  our  re- 
search efforts  are  helping  to  define  the  pathophys- 
iologic basis  of  such  diseases.  In  many  autoim- 
mune and  immune  complex-mediated  diseases, 
there  is  an  inherited  defect  in  the  handling  of 
immune  complexes  or  in  the  activation  or  regula- 
tion of  the  complement  system.  Variations  in  the 
structure,  function,  and  expression  of  comple- 
ment proteins  are  important  aspects  of  autoim- 
mune diseases.  The  information  gleaned  from 
such  research  efforts  could  provide  a  basis  for 
new  treatments. 


16 


The  Molecular  Biology  of  Smell 


Richard  Axel,  M.D. — Investigator 

Dr.  Axel  is  also  Higgins  Professor  of  Biochemistry  and  Molecular  Biophysics  and  of  Pathology  at  Columbia 
University  College  of  Physicians  and  Surgeons.  He  received  his  undergraduate  degree  from  Columbia 
College  and  his  M.D.  degree  from  the  Johns  Hopkins  University  School  of  Medicine.  He  then  came  to 
Columbia  University  as  a  resident  in  pathology  at  the  College  of  Physicians  and  Surgeons.  He  held 
fellowships  in  Columbia's  Institute  of  Cancer  Research  (in  Sol  Spiegelman's  laboratory)  and  the 
Department  of  Pathology.  Dr.  Axel  is  a  member  of  the  National  Academy  of  Sciences.  Among  his  many 
honors  are  the  Eli  lilly  Award  in  Biological  Chemistry  and  the  Richard  iounsbery  Award  from  the 
National  Academy  of  Sciences. 


PERIPHERAL  neurons  in  vertebrate  sensory 
systems  respond  to  environmental  stimuli 
and  transmit  these  signals  to  higher  sensory 
centers  in  the  brain,  where  they  are  processed  to 
allow  the  discrimination  of  complex  sensory  in- 
formation. Mammals  possess  an  olfactory  sensory 
system  of  enormous  discriminatory  power.  Hu- 
mans, for  example,  are  thought  to  be  capable  of 
distinguishing  among  thousands  of  distinct 
odors.  Even  subtle  alterations  in  molecular  struc- 
ture of  an  odorant  can  lead  to  profound  changes 
in  perceived  odor  quality.  How  is  this  diversity 
and  specificity  accomplished?  The  detection  of 
chemically  distinct  odors  presumably  results 
from  the  association  of  odorants  with  specific  re- 
ceptors on  olfactory  neurons  that  reside  in  spe- 
cialized epithelium  in  the  nose.  The  brain  must 
distinguish  which  receptors  or  which  neurons 
have  been  activated  to  allow  the  discrimination 
among  different  odorant  stimuli.  What  mecha- 
nisms have  vertebrates  evolved  to  allow  the  recog- 
nition of  this  huge  array  of  odorant  molecules? 
How  does  the  brain  know  what  the  nose  is  smell- 
ing? Insight  into  these  problems  is  likely  to  de- 
pend on  the  isolation  and  characterization  of  the 
odorant  receptors  expressed  in  the  nose. 

We  have  recently  identified  an  extremely  large 
multigene  family  whose  members  are  likely  to 
encode  a  diverse  family  of  odorant  receptors.  We 
have  determined  the  sequence  of  30  genes  and 
have  deduced  the  protein  sequence  of  30  differ- 
ent putative  olfactory  receptors.  The  olfactory 
proteins  are  clearly  members  of  a  superfamily  of 
receptors  that  traverse  the  membrane  seven 
times.  Analysis  of  the  proteins  reveals  structural 
features  that  may  render  this  family  particularly 
well  suited  for  the  detection  of  a  diverse  array  of 
structurally  distinct  odorants. 

These  olfactory  proteins  can  be  divided  into 
several  different  subfamilies  that  exhibit  signifi- 
cant divergence  in  the  region  of  the  receptor  mol- 
ecule thought  to  interact  with  the  odorous  li- 
gand.  These  observations  suggest  a  model  in 
which  each  of  the  individual  subfamilies  encodes 


receptors  that  bind  distinct  structural  classes 
of  odorants.  Within  a  given  subclass  of  odorous 
ligands,  the  members  would  recognize  more  sub- 
tle variations  among  odor  molecules  of  a  given 
structural  class.  As  such,  this  superfamily  of  mole- 
cules would  be  uniquely  suited  to  its  putative 
role  in  the  fine  discrimination  of  odor  molecules 
of  subtly  different  structures. 

The  isolation  of  this  large  family  of  genes  en- 
coding the  receptor  molecules  immediately  pro- 
vides one  solution  to  the  problem  of  olfactory 
perception.  How  do  we  recognize  so  diverse  an 
array  of  odors?  At  one  extreme,  we  would  argue 
that  the  recognition  of  diverse  odorants  could  be 
accomplished  by  a  small  number  of  promiscuous 
receptors,  each  capable  of  interacting  with  sev- 
eral structurally  distinct  odor  molecules.  Alterna- 
tively, olfactory  perception  would  result  from 
the  presence  of  a  large  number  of  different  recep- 
tor molecules,  each  capable  of  interacting  with 
one  or  a  small  number  of  specific  odorants.  The 
size  of  the  gene  family  we  have  characterized 
suggests  that  there  are  indeed  a  very  large  number 
of  odorant  receptors,  each  capable  of  interacting 
with  a  small  number  of  odorous  ligands.  These 
observations  are  in  sharp  contrast  to  other  sensory 
systems,  such  as  vision  or  touch,  where  discrimi- 
nation of  sensory  information  is  accomplished  by 
a  rather  small  number  of  receptor  modalities. 

How  then  does  the  brain  distinguish  which  re- 
ceptors or  which  neurons  have  been  activated  to 
allow  the  discrimination  between  different  odor- 
ant stimuli?  In  other  sensory  systems,  such  as  vi- 
sion and  touch,  neurons  in  the  brain  are  orga- 
nized in  a  topographic  map  that  identifies  the 
position  of  a  sensory  stimulus.  Thus  the  position 
of  a  given  neuron  in  the  brain  is  used  to  define  the 
location  of  a  sensory  stimulus  within  the  external 
environment.  Olfactory  processing  does  not  ex- 
tract spatial  features  of  the  odorant  stimulus.  Re- 
lieved of  the  necessity  to  encode  information 
about  the  spatial  localization  of  a  sensory  stimu- 
lus, the  olfactory  system  may  use  spatial  segrega- 
tion of  sensory  input  solely  to  encode  the  identity 
of  the  stimulus  itself.  Recent  data  utilizing  the 


17 


The  Molecular  Biology  of  Smell 


receptor  genes  as  molecular  probes  suggest  a 
model  of  olfactory  processing  in  which  sensory 
neurons  expressing  distinct  odorant  receptors  all 
project  to  a  restricted  region  within  the  olfactory 
bulb,  the  first  relay  station  in  the  brain.  In  this 
instance  the  discrimination  of  odors  would  be  a 
consequence  of  the  position  of  second-order  neu- 


rons in  the  olfactory  bulb,  such  that  spatially  lo- 
calized groups  of  neurons  would  preferentially 
respond  to  different  odorants.  The  molecular 
identification  of  the  genes  encoding  a  large  fam- 
ily of  olfactory  receptors  has  provided  initial  in- 
sight into  the  logic  of  olfactory  processing  in  the 
mammalian  brain. 


18 


Mammalian  Developmental  Genetics 


Gregory  S.  Barsh,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Barsh  is  also  Assistant  Professor  of  Pediatrics  at  Stanford  University.  He  received  his  M.D.  and  Ph.D. 
degrees  from  the  University  of  Washington,  where  he  studied  inherited  diseases  of  collagen  biosynthesis 
in  the  laboratory  of  Peter  Byers.  Dr.  Barsh's  postgraduate  training  was  in  internal  medicine  and  medical 
genetics  at  Harbor- University  of  California  Hospital,  Los  Angeles,  and  at  the  University  of  California, 
San  Francisco.  His  research  in  the  laboratory  of  Charles  Epstein  focused  on  a  molecular  and  genetic 
characterization  of  recessive  lethal  mutations  at  the  mouse  agouti  locus. 


VERY  little  is  known  about  the  genetic  control 
of  mammalian  development.  But  embryo- 
genesis  of  all  mammals  follows  a  similar  plan,  and 
the  basic  rules  discovered  in  one  species  are 
likely  to  apply  to  others.  By  studying  the  mouse,  a 
species  in  which  the  early  embryo  can  be  ob- 
served and  manipulated,  we  will  better  under- 
stand how  genes  control  human  development 
and  how  disruption  of  these  processes  may  lead 
to  such  abnormalities  as  miscarriages  and  birth 
defects. 

In  organisms  traditionally  subject  to  experi- 
mental genetic  analysis,  such  as  fruit  flies  and 
nematodes,  mutations  in  a  particular  develop- 
mental pathway  can  be  selected  in  a  comprehen- 
sive screening  experiment.  In  mice,  however, 
this  approach  has  been  hampered  by  the  inability 
to  study  and  recover  conditional  mutations  and 
by  the  inefficiency  of  generating  new  mutations 
through  the  insertion  of  mobile  genetic  ele- 
ments. As  a  result,  much  of  our  insight  into  mam- 
malian developmental  genetics  comes  from  the 
study  of  preexisting  mutations.  We  are  examining 
a  group  of  previously  identified  genes  that  affect 
development  around  the  time  of  implantation.  In 
addition,  we  are  developing  a  system  to  allow  the 
conditional  disruption  of  genes  with  recessive 
phenotypes  in  cell  culture  and  in  transgenic 
mice. 

Characterization  of  the  Mouse  agouti 
and  kreisler  Genes 

Located  within  a  small  region  of  mouse  chro- 
mosome 2  are  a  group  of  genes  required  for  fun- 
damental aspects  of  peri-implantation  develop- 
ment. The  mousc^agouti  {A)  locus,  originally 
described  as  a  coat  color  gene,  controls  the  tim- 
ing and  type  of  pigment  deposition  in  developing 
hair  follicles.  Several  A  mutations,  including  A^, 
a'^",  and  are  lethal  when  homozygous,  and  at 
least  three  genes  required  for  embryonic  develop- 
ment have  been  defined  at  or  near  the  A  locus. 
Closely  linked  to  A  is  the  segmentation  gene 
kreisler  (kr),  which  affects  formation  of  the  in- 
ner ear  by  interfering  with  the  number  and  pat- 


tern of  metameric  units,  the  so-called  rhombo- 
meres,  in  the  developing  hindbrain. 

Toward  the  eventual  goal  of  isolating  these 
genes,  we  have  constructed  high-resolution  ge- 
netic and  physical  maps  of  mouse  chromosome  2, 
using  classical  and  somatic  cell  genetic  ap- 
proaches. These  maps  are  based  on  a  variety  of 
molecular  markers  in  the  area,  including  the  in- 
sertion sites  of  two  endogenous  retroviruses, 
Emv-15  and  Xmv-10,  which  have  been  closely 
associated  with  the  A^  and  a  mutations,  respec- 
tively. Our  results  have  shown  that  neither  Emv- 
15  nor  Xmv-10  has  caused  an  A  mutation.  We 
have  also  established  the  order  and  relative  dis- 
tances between  many  closely  linked  molecular 
markers  in  the  region  and  are  beginning  to  lead 
toward  candidate  cDNAs  affected  by  the  A  and  kr 
mutations. 

We  have  further  characterized  the  relation- 
ships among  rhombomere  formation,  segmenta- 
tion, and  the  kr  mutation,  based  on  a  detailed 
analysis  of  branchial  arch  derivatives  in  kr/kr 
adults.  In  collaboration  with  Michael  Frohman 
and  Gail  Martin,  we  have  studied  the  expression 
of  rhombomere-specific  genes  in  fer/^r  embryos. 
Two  of  the  rhombomeres  affected  by  the  kr  muta- 
tion contribute  to  the  second  and  third  branchial 
arches,  which  ultimately  form,  among  other 
structures,  the  hyoid  bone.  In  kr/kr  adults,  the 
lower  part  of  the  hyoid,  normally  derived  from 
the  third  branchial  arch,  exhibits  some  morpho- 
logic characteristics  of  the  upper  part  of  the 
bone,  normally  derived  from  the  second  bran- 
chial arch. 

In  kr/kr  embryos,  the  expression  domains  of 
Krox-20,  Hox-2.6,  Hox-2.  7,  and  Hox-2.9,  which 
probably  code  for  transcription  factors,  and  Int- 
2,  which  codes  for  a  growth  factor  and  may  play  a 
role  in  morphogenesis  of  the  inner  ear,  are  all 
shifted  rostrally  toward  the  head.  Taken  together, 
these  results  suggest  that  fer  affects  the  location  of 
position-specific  gene  expression  along  the  ros- 
trocaudal  axis  and  that  the  kr*  gene  product  plays 
a  key  role  in  the  acquisition  of  positional  iden- 
tity. By  isolating  the  kr  gene  and  further  charac- 
terizing the  fer/fer  phenotype,  we  hope  to  clarify 


19 


Mammalian  Developmental  Genetics 


the  molecular  pathways  of  mammalian  segmenta- 
tion and  the  role  of  Hox-2  genes  in  rhombomere 
morphogenesis. 

Insertional  Mutagenesis 

An  important  development  in  mammalian  ex- 
perimental embryology  has  been  the  ability  to 
isolate  embryonic  stem  cells  from  preimplanta- 
tion  mouse  embry^os,  which  can  be  modified  in 
cell  culture  and  then  used  to  reconstitute  an  in- 
tact animal.  Like  the  whole  organism,  these  cells 
are  diploid  and  contain  two  copies  of  every  auto- 
somal gene.  When  embryonic  stem  cells  are 
mixed  with  a  fragment  of  "reporter"  DNA,  such 
as  the  coding  regions  of  the  neomycin  resistance 
gene  or  the  /3-galactosidase  gene,  insertion  of  the 
exogenous  DNA  provides  a  "gene  trap, ' '  in  which 
expression  of  the  reporter  sequences  is  con- 
trolled by  regulatory^  elements  of  an  endogenous 
gene. 

Insertion  of  the  reporter  DNA  is  likely  to 
disrupt  expression  of  the  endogenous  gene,  but 
in  most  cases,  expression  from  the  uninterrupted 
homologue  will  be  sufficient  to  prevent  pheno- 
typic  eff^ects.  To  block  this  expression  in  a  single 
step,  and  in  a  conditional  fashion,  we  have  con- 
structed a  series  of  gene  trap  vectors  that  contain 
an  inducible  promoter  on  the  strand  opposite 
the  reporter  sequences.  "Captured"  cell  clones 
are  first  selected  by  antibiotic  resistance  or  /3- 
galactosidase  staining.  After  removal  of  selective 
pressure  and  activation  of  the  inducible  pro- 
moter, an  antisense  transcript  is  generated  against 
coding  sequences  of  the  endogenous  gene.  In 
certain  cases,  this  transcript  will  function  in 
trans,  thereby  inhibiting  expression  of  the  endog- 
enous gene  from  the  uninterrupted  homologue. 

Control  experiments  suggest  that  these  DNA 
vectors  are  capable  of  trapping  endogenous 
genes  and  that  basal  expression  of  the  inducible 
promoter  does  not  reduce  appreciably  the  fre- 
quency of  trapping.  We  are  currently  isolating  a 
panel  of  captured  cell  clones.  The  efficiency  of 
these  antisense  promoters  in  cis  will  be  analyzed 


by  measuring  expression  of  the  reporter  se- 
quences before  and  after  antisense  induction.  Po- 
tential eff'ects  of  trans  inhibition  will  then  be 
tested  by  examining  the  phenotypes  of  chimeric 
mice  that  contain  the  mutant  embryonic  stem 
cells.  This  approach  will  allow  the  phenotypic 
effects  of  a  recessive  mutation  to  be  studied  in  a 
diploid  organism  by  altering  only  one  of  the  two 
copies  and  may  be  applicable  to  many  organisms 
and  developmental  systems. 

Ultimately  we  plan  to  place  the  gene  trap  vec- 
tors in  the  context  of  retroviral  packaging  ele- 
ments to  allow  their  introduction  into  the  mouse 
germline  by  infection  of  developing  gametes. 
Theoretical  advantages  of  using  retrotransposons 
as  insertional  mutagens  include  an  enzymatic 
mechanism  of  integration  that  conserves  host 
DNA  sequences,  accessibility  to  most  if  not  all 
segments  of  the  genome,  and  the  efficient  detec- 
tion and  recovery  of  new  integration  sites.  These 
advantages,  however,  have  been  difficult  to 
achieve  in  the  mouse,  in  part  because  of  the  low 
efficiency  of  retrotransposition  in  the  germline. 

In  the  case  of  endogenous  ecotropic  proviruses 
and  exogenous  Moloney  murine  leukemia  virus 
(MoMuLV) ,  a  block  to  retroviral  infection  of  de- 
veloping gametes  seems  likely  to  be  due  to  a  com- 
bination of  factors,  including  decreased  accessi- 
bility of  germline  cells  to  viral  particles  with  very 
short  infectious  half-lives  and  a  transcriptional 
block  to  expression  from  the  retroviral  long  ter- 
minal repeat  (LTR).  As  a  first  step  toward  over- 
coming these  hurdles,  we  have  constructed,  in 
collaboration  with  Patrick  Brown  (HHMI,  Stan- 
ford University) ,  transgenic  mice  that  contain  the 
transcriptional  control  sequences  for  the  mouse 
protamine  1  gene  fused  to  the  coding  sequences 
for  MoMuLV.  Because  the  mouse  protamine  1 
gene  is  highly  expressed  in  postmeiotic  sperma- 
tids, the  transgene  should  provide  a  rich  source 
of  native  retroviral  particles  in  the  vicinity  of  de- 
veloping male  gametes.  Our  preliminary  results 
show  that  the  chimeric  protamine-MoMuLV 
transgene  is  functional  and  can  lead  to  retroviral 
infection  of  somatic  and  germline  tissues. 


20 


Cell  Cycle  Control 


David  H.  Beach,  Ph.D. — Investigator 

Dr.  Beach  is  also  Senior  Staff  Scientist  at  Cold  Spring  Harbor  Laboratory  and  Adjunct  Associate  Professor 
in  the  Department  of  Microbiology  at  the  State  University  of  New  York  at  Stony  Brook.  He  received 
his  undergraduate  degree  at  the  University  of  Cambridge,  followed  by  a  Ph.D.  degree  at  the  University  of 
Miami,  where  he  worked  with  Marcus  Jacobson.  His  postdoctoral  studies  were  done  with  Sydney  Shall 
at  the  University  of  Susex  and  with  Amar  Klar  at  Cold  Spring  Harbor  Laboratory. 


THE  cell  division  cycle  interests  an  increasing 
number  of  scientists  and  continues  to  be  the 
central  focus  of  our  research.  The  two  most  criti- 
cal events  of  the  cycle  are  the  replication  of  DNA 
that  occurs  during  the  S  (synthetic)  phase  and  the 
segregation  of  chromosomes  into  daughter  cells 
that  occurs  in  the  M  (mitotic)  phase.  DNA  replica- 
tion and  mitosis  are  profoundly  different  cellular 
events,  both  molecularly  and  in  terms  of  cellular 
mechanics,  but  both  are  regulated  by  closely  re- 
lated enzymes,  members  of  a  family  known  as  the 
cyclin  kinases. 

Each  of  these  kinases  has  a  catalytic  subunit,  of 
which  cdc2  is  the  prototype,  and  a  regulatory  sub- 
unit  that  is  a  cyclin.  Cyclins  derive  their  name 
simply  from  the  property  by  which  they  were  first 
recognized.  Their  abundance  oscillates  in  the  di- 
vision cycle,  with  the  same  periodicity  as  the  cy- 
cle itself.  Many  cyclins  have  been  discovered,  all 
related  in  their  amino  acid  sequence.  In  humans 
there  are  at  least  five  groups  of  such  proteins, 
called  cyclins  A,  B,  C,  D,  and  E.  Within  each 
group  there  may  be  further  members,  such  as  Dl , 
D2,  D3  or  Bl,  B2.  Cyclins  of  different  classes  ap- 
pear to  act  at  different  stages  of  the  cell  cycle — 
B-type  cyclins  at  mitosis,  A  type  at  S  phase,  and 
so  on. 

Also,  in  the  case  of  the  D-type  cyclins,  each 
member  of  the  family  displays  a  specific  tissue 
distribution.  D-type  cyclins  have  attracted  partic- 
ular interest  because  they  appear  to  act  early  in 
the  cell  division  pathway  and  can  also  mutate  to 
give  rise  to  oncogenic  forms.  With  cyclin  Dl ,  the 
gene  is  amplified  or  rearranged  in  a  wide  range  of 
human  tumors,  including  cancers  of  the  breast, 
esophagus,  bladder,  and  lymphatic  system.  It  is 
not  yet  known  how  overexpression  or  activation 
of  cyclin  Dl  contributes  to  the  pathology  of 
cancer,  but  a  clear  connection  has  been 
established. 

A  major  activity  in  the  Beach  laboratory  has 
centered  on  identification  of  the  regulatory  mole- 
cules controlling  cyclin  kinases,  particularly  the 
cyclin  B/cdc2  enzyme  that  functions  in  mitosis. 
This  enzyme  is  influenced  by  at  least  three  phos- 
phorylation events,  some  activating  and  others  in- 


hibiting. Tyrosine  phosphorylation  of  cdc2  is  the 
best-characterized  inhibitory  mechanism,  and 
the  relevant  tyrosine  kinases  and  phosphatases 
have  been  identified  both  in  yeast  and  human 
cells. 

The  tyrosine  kinases  are  encoded  by  the  mikl'^ 
and  weel^  genes  of  yeast,  and  the  tyrosine  phos- 
phatases by  the  cdc25^  gene.  Kinases  and  phos- 
phatases act  antagonistically.  Thus,  in  the  ab- 
sence of  the  tyrosine  kinase,  cells  enter  mitosis 
prematurely  before  the  completion  of  DNA  repli- 
cation. Lack  of  the  tyrosine  phosphatase  causes 
arrest  in  the  G2  phase  of  the  division  cycle. 

A  further  class  of  negative  regulators  of  mitosis 
has  recently  been  discovered.  The  piml^  (pre- 
mature initiation  of  mitosis)  gene  of  yeast  en- 
codes a  homologue  of  a  human  protein  called 
RCCl.  In  the  absence  of  piml/^CCl  function, 
cells  also  enter  mitosis  before  the  completion  of 
DNA  replication.  The  gene  product  pirn  1  /RCCl 
is  an  integral  nucleosomal  protein  (present  at 
one  copy  per  nucleosome)  and  is  responsible  for 
signaling  the  state  of  the  chromatin  to  the  cdc2/ 
cyclin  B  enzyme.  Only  when  the  chromatin  is 
fully  replicated  can  the  cell  proceed  to  division 
by  activation  of  the  cyclin  B/cdc2  enzyme.  The 
mechanism  by  which  the  pirn  1 /RCCl  nucleoso- 
mal protein  communicates  with  the  cyclin  ki- 
nases is  an  area  of  major  current  interest. 

Many  substrates  of  the  cyclin  kinases  have  been 
discovered.  They  include  major  structural  pro- 
teins such  as  lamins  and  vimentin,  which  play  a 
vital  role  in  the  structural  reorganization  of  the 
cell  that  occurs  during  mitosis.  Other  substrates 
include  transcription  factors  and  also  negative 
growth  regulators  such  as  the  p53  and  rb 
proteins. 

It  is  unclear  why  such  different  cellular  activi- 
ties as  DNA  replication  and  cell  division  are  regu- 
lated by  a  family  of  cyclin  kinases  that  are  clearly 
related  evolutionarily.  The  evolutionary  similar- 
ity implies  that  there  was  once  only  one  cyclin 
kinase  and  thus  that  this  enzyme  regulated  only 
one  cell  cycle  event  in  a  primitive  cell.  Was  that 
event  DNA  replication  or  cell  division?  It  is  very 
difficult  to  imagine  the  primitive  cell,  because  all 


21 


Cell  Cycle  Control 


known  cells  are  exquisitely  interdependent  enti- 
ties. For  example,  the  replication  of  DNA  is  de- 
pendent on  proteins,  but  the  proteins  are  en- 
coded by  DNA,  and  so  on.  It  is  clear,  however. 


that  the  primitive  cyclin  kinase  has  evolved  in 
present-day  cells  into  a  very  complicated  family 
of  enzymes  that  regulate  all  stages  of  the  cell  divi- 
sion cycle. 


22 


Genetic  Control  of  Morphogenesis  in  Drosophila 


Philip  A.  Beachy,  Ph.D. — Assistant  Investigator 

Dr.  Beachy  is  also  Assistant  Professor  in  the  Department  of  Molecular  Biology  and  Genetics  at  the  Johns 
Hopkins  University  School  of  Medicine.  He  did  his  graduate  work  in  the  Department  of  Biochemistry  at 
Stanford  University  School  of  Medicine  with  David  Hogness.  Before  joining  the  Hughes  Institute  at  Johns 
Hopkins,  he  spent  two  years  as  Staff  Associate  at  the  Carnegie  Institution's  Department  of  Embryology. 
He  has  been  the  recipient  of  several  fellowships,  including  a  Sloan  Foundation  Fellowship  in  Neuroscience. 


TWO  conceptually  distinct  processes  operate 
during  Drosophila  embryogenesis.  In  the 
first  of  these,  a  detailed  system  of  spatial  informa- 
tion is  generated  by  a  cascade  of  interactions 
among  several  groups  of  genes  and  their  prod- 
ucts. During  morphogenesis,  the  second  phase, 
interpretation  of  the  blueprint  results  in  the  com- 
plex arrangement  of  structures  seen  in  the  larva 
and  adult. 

The  first  process  is  initiated  during  oogenesis 
by  maternal  input  of  broad  spatial  cues.  The  in- 
teracting genes  that  receive  and  refine  this  infor- 
mation in  the  embryo  have  been  extensively 
characterized,  in  most  cases  at  the  molecular 
level.  Much  less  is  known  about  how  morphogen- 
esis is  directed,  though  it  is  generally  believed  to 
occur  through  regulation  of  target  genes  by  the 
localized  proteins  generated  during  construction 
of  the  blueprint.  This  laboratory's  current  inter- 
ests center  upon  two  groups  of  genes  that  operate 
at  the  interface  between  these  processes,  the  seg- 
ment polarity  genes  and  the  homeotic  genes. 


The  hedgehog  Segment  Polarity  Gene 

The  segment  polarity  group  is  the  latest  acting 
of  the  segmentation  gene  groups.  It  functions  in 
the  refinement  of  positional  information  at  the 
level  of  individual  cells  within  embryonic  seg- 
ments. During  the  earlier,  syncytial  period  of  Dro- 
sophila embryogenesis,  the  absence  of  cell  mem- 
branes permits  diff'usion  between  nuclei,  which 
can  account  for  many  of  the  interactions  between 
early  acting  segmentation  genes  and  their  prod- 
ucts. Some  segment  polarity  genes,  however,  are 
expressed  and  function  long  beyond  the  syncytial 
stages.  Communication  between  cells  for  refine- 
ment of  positional  information  requires  signaling 
across  cell  membranes.  Consistent  with  a  role  in 
signaling,  the  products  of  many  segment  polarity 
genes  are  secreted  or  localized  to  nonnuclear 
compartments. 

During  the  past  year  our  laboratory  isolated  and 
characterized  the  segment  polarity  gene  hedge- 
hog. The  hedgehog  locus  was  identified  in  the 
exhaustive  genetic  screens  carried  out  by  Chris- 


tiane  Niisslein-Volhard,  Eric  Wieschaus,  and  their 
colleagues,  and  was  named  for  the  bristle  pattern 
observed  in  mutant  larvae.  The  phenotype  resem- 
bles that  of  a  number  of  other  segment  polarity 
genes,  including  wingless. 

The  wingless  gene  is  expressed  within  each 
segment  in  a  stripe  of  cells  adjacent  to  that  in 
which  engrailed,  another  segment  polarity  gene, 
is  expressed.  The  normal  function  of  each  gene  is 
required  within  its  own  stripe  for  maintenance  of 
the  other  gene's  expression  within  the  neighbor- 
ing (but  not  overlapping)  stripe.  The  mutual  re- 
quirement of  each  gene  for  the  other's  continued 
expression  in  a  neighboring  cell  implies  a  mecha- 
nism for  communication  between  these  cells. 
Since  wingless  encodes  a  secreted  peptide  factor 
and  engrailed  a  homeodomain-containing  tran- 
scription factor,  additional  components  must  be 
involved  in  the  signaling  mechanism. 

Interest  in  this  pathway  has  been  stimulated  by 
recent  results  elsewhere  indicating  that  targeted 
inactivation  of  the  murine  homologues  of  wing- 
less and  engrailed  produce  specific  developmen- 
tal defects  of  the  midbrain  and  cerebellum.  Other 
genes  that  may  be  involved  in  this  pathway  are 
hedgehog  and  patched,  which  from  genetic  evi- 
dence appear  to  interact.  While  patched  encodes 
a  protein  with  apparent  multiple  membrane- 
spanning  domains,  the  product  of  the  hedgehog 
gene  had  not  previously  been  characterized.  We 
gained  entry  into  the  hedgehog  locus  with  the  aid 
of  an  "enhancer  trap"  P-transposable  element  in- 
serted near  the  gene.  Analysis  revealed  a  se- 
quence capable  of  encoding  a  protein  of  471 
amino  acid  residues.  This  includes  a  region  near 
the  amino  terminus  suggestive  of  targeting  to  the 
secretory  pathway,  either  as  a  secreted  polypep- 
tide or  as  an  integral  membrane  protein. 

Either  possibility  would  permit  interaction  of 
the  hedgehog  protein  with  the  membrane-span- 
ning patched  protein,  in  accordance  with  the  ge- 
netic prediction.  Also  consistent  with  such  an  in- 
teraction, the  hedgehog  gene  is  expressed 
coincidently  with  engrailed,  while  patched  is 
expressed  coincidently  with  wingless  in  adjacent 
cells.  The  physical  nature  of  the  interaction  re- 


23 


Genetic  Control  of  Morphogenesis  in  Drosophila 


mains  to  be  characterized,  along  with  the  intra- 
cellular consequences  of  this  signaling  event. 

Regulatory  Functions  of  Homeotic  Genes 

Homeotic  genes  play  a  primary  role  in  conver- 
sion of  the  spatial  information  generated  by  the 
earlier-acting  segmentation  genes  into  morpho- 
logical structure.  The  products  of  homeotic 
genes  direct  morphogenesis  in  a  manner  that 
produces  the  diversity  of  structures  distinguish- 
ing the  segments.  This  task  is  presumably  ac- 
complished by  differential  regulation  of  target 
genes,  though  the  identities  of  these  targets 
and  the  mechanisms  by  which  homeotic  genes 
achieve  their  biological  specificity  remain 
largely  unknown. 

We  have  studied  in  detail  the  DNA  sequence- 
recognition  properties  of  homeodomains  from 


the  homeotic  genes  Deformed  and  Ultrabitho- 
rax.  While  these  domains  are  very  closely  re- 
lated, we  have  demonstrated  clear  differences  in 
DNA  sequence  preferences  that  lead  to  moderate 
differences  in  affinity  for  individual  binding 
sites.  Our  recent  work  demonstrates  that  these 
differences  in  sequence  preference  map  to  the 
two  DNA-contacting  portions  of  the  homeodo- 
main.  Even  small  differences  in  binding  to  indi- 
vidual sites  can  lead  to  large  differences  in  regula- 
tory effect  when  multiple  sites  are  present.  This  is 
due  to  cooperative  binding  of  homeodomain  pro- 
teins to  multiple  sites,  a  property  we  have  studied 
extensively  for  the  Ultrabithorax  protein.  Dif- 
ferences in  DNA  sequence  preference  by  homeo- 
tic gene  products  may  thus  account  for  a  substan- 
tial part  of  the  biological  specificity  of  homeotic 
gene  function. 


24 


Molecular  Studies  of  Human  Genetic  Disease 


Arthur  L.  Beaudet,  M.D. — Investigator 

Dr.  Beaudet  is  also  Professor  in  the  Institute  for  Molecular  Genetics  and  the  Departments  of  Pediatrics  and 
Cell  Biology  at  Baylor  College  of  Medicine.  He  received  his  B.S.  degree  in  biology  from  Holy  Cross  College 
and  his  M.D.  degree  from  Yale  University.  After  completing  his  pediatric  residency  at  the  Johns  Hopkins 
Hospital,  he  performed  postdoctoral  research  at  NIH. 


EXTRAORDINARY  progress  in  the  understand- 
ing and  diagnosis  of  human  genetic  diseases 
has  occurred  over  the  past  decade.  Much  of  this 
progress  was  made  possible  by  the  application  of 
recombinant  DNA  techniques  to  the  analysis  of 
those  diseases  caused  by  an  alteration  in  a  single 
gene.  There  are  thousands  of  such  disorders,  in- 
cluding well-known  conditions  such  as  cystic  fi- 
brosis, Duchenne  muscular  dystrophy,  hemo- 
philia A  and  B,  sickle  cell  anemia,  and  Tay  Sachs 
disease.  Although  this  progress  in  the  diagnosis  of 
single-gene  disorders  continues  unabated,  the 
ability  to  treat  genetic  disease  virtually  stands 
still  by  comparison.  There  is  hope  that  at  least 
some  of  these  diseases  will  yield  to  somatic  gene 
therapy. 

The  extraordinary  diagnostic  ability  that  allows 
anticipation  of  genetic  symptoms  in  individuals 
and  their  offspring  raises  important  societal  op- 
portunities and  concerns.  These  include  preven- 
tion of  genetic  disease  through  reproductive 
planning,  treatment  prior  to  development  of 
symptoms,  and  possible  discrimination  (as  for  in- 
surance or  employment)  on  the  basis  of  one's  ge- 
netic predispositions. 

Abnormalities  of  a  single  gene,  however,  are 
not  responsible  for  most  of  the  common  medical 
problems  of  adult  life.  Rather,  these  are  caused 
by  differences  in  a  number  of  genes  in  combina- 
tion with  environment  and  life-style.  Problems  of 
this  type  include  atherosclerosis,  hypertension, 
diabetes  mellitus,  and  autoimmune  disease. 
Work  in  our  laboratory  involves  various  aspects 
of  precise  genetic  diagnosis,  efforts  to  develop 
somatic  gene  therapy,  and  attempts  to  understand 
the  complex  genetic  basis  of  some  of  these  multi- 
factorial disorders. 

Cystic  Fibrosis 

Cystic  fibrosis  (CF)  is  a  common  genetic  dis- 
ease alfecting  approximately  1  in  2,500  Cauca- 
sians. Most  CF  patients  die  of  progressive  lung 
disease  during  childhood  or  young  adult  life. 
About  1  in  25  Caucasians  carry  an  abnormal  CF 
gene,  and  about  1  in  625  couples  are  at  high  risk 
of  having  an  affected  child.  Our  laboratory  has 


been  actively  involved  in  developing  and  imple- 
menting DNA  testing  for  purposes  of  prenatal 
diagnosis  and  carrier  detection  of  CF.  Prenatal 
diagnosis  for  high-risk  couples  who  have  a  l-in-4 
chance  that  a  pregnancy  will  be  affected  with  CF 
is  now  a  routine  matter,  and  such  diagnosis  is  per- 
formed at  many  laboratories  throughout  the 
world.  Similarly,  carrier  detection  for  close  rela- 
tives of  CF  patients  is  routine. 

Much  more  controversial  is  whether  carrier 
testing  for  CF  should  be  offered  to  most  or  all 
couples  prior  to  reproduction.  Although  a  single 
common  defect  is  present  in  the  majority  of  CF 
chromosomes,  the  remaining  fraction  of  abnor- 
mal chromosomes  contains  dozens  of  different 
mutations.  One  goal  of  efforts  in  our  laboratory  is 
to  develop  efficient  methods  for  detection  of 
such  multiple  abnormalities.  Currently  carrier 
testing  identifies  80-90  percent  of  carriers  and 
would  detect  up  to  80  percent  of  couples  at  risk 
for  having  a  CF  child.  Although  it  is  unclear 
whether  CF  carrier  testing  should  be  offered  to  all 
couples  immediately  or  in  the  very  near  future,  it 
is  our  view  that  carrier  testing  will  be  the  starting 
point  for  multiphasic  DNA  testing  to  provide  in- 
formation regarding  reproductive  and  health 
risks. 

Precise  genetic  diagnosis  for  CF,  and  for  other 
genetic  disorders,  does  not  guarantee  an  effective 
treatment.  It  would  be  desirable  to  have  an  ani- 
mal model  for  easier  analysis  and  therapeutic 
trials.  As  one  step  in  the  process  of  developing  an 
animal  model  in  mice,  we  have  characterized  the 
sequence  of  the  mouse  CF  gene.  We  have  pre- 
pared DNA  clones  that  are  suitable  for  disrupting 
the  normal  mouse  gene  (which  is  similar  to  the 
human  gene)  in  cultured  embryonic  stem  (ES) 
cells.  These  altered  ES  cells  can  be  used  to  gener- 
ate mice  affected  with  CF.  The  work  to  obtain  a 
CF  mutant  mouse  is  supported  in  part  by  a  grant 
from  the  Cystic  Fibrosis  Foundation. 

Somatic  gene  therapy  for  CF  focuses  on  the 
lung,  since  it  is  involvement  of  the  lung  that 
proves  fatal  for  the  vast  majority  of  CF  patients. 
Treatment  of  the  lung  presents  a  unique  circum- 
stance, for  therapeutic  agents  can  be  delivered  by 


25 


Molecular  Studies  of  Human  Genetic  Disease 


inhalation  rather  than  injection.  We  are  preparing 
to  evaluate  various  methods  for  pulmonary  gene 
therapy.  Liposomes  containing  DNA,  retroviral 
vectors,  and  adenoviral  vectors  will  be  used  for 
direct  instillation  into  the  lungs  or  for  various 
aerosol  deliveries,  beginning  with  normal  ro- 
dents and  perhaps  progressing  to  primates. 

Leukocyte  and  Endothelial  Cell 
Adhesion  Molecules 

Leukocytes  (white  blood  cells)  are  involved  in 
defense  against  infection  and  in  most  inflamma- 
tory responses.  Through  complex  mechanisms, 
the  various  forms  of  leukocytes  roll  along  the 
blood  vessel  walls  and  eventually  migrate 
through  the  endothelial  layer.  This  ability  is  me- 
diated to  a  large  extent  by  carefully  regulated  ad- 
hesion molecules  on  the  surface  of  leukocytes 
and  endothelial  cells. 

We  are  attempting  to  understand  the  role  of 
these  proteins  in  normal  biology  and  in  multifac- 
torial genetic  diseases.  This  involves  preparing 
mutant  mice  and  analyzing  naturally  occurring 
genetic  variation  in  humans.  It  is  quite  possible 
that  variation  in  these  genes  could  be  an  impor- 
tant factor  in  the  predisposition  to  multifactorial 
disorders  such  as  atherosclerosis,  autoimmune 
disease,  and  a  wide  range  of  inflammatory  pro- 
cesses, including  arthritis  and  diabetes  mellitus. 

We  are  focusing  initially  on  CD  18,  which  is  a 
subunit  of  leukocyte  integrin,  and  on  intracellu- 
lar adhesion  molecule- 1  (ICAM-1),  a  member  of 
the  immunoglobulin  gene  family.  CD  18  is  found 
on  the  surface  on  many  leukocytes,  and  its  adhe- 
siveness can  be  sharply  increased  through  an  acti- 
vation process.  ICAM-1  is  found  on  many  cells, 
particularly  endothelial  cells,  where  it  can  bind 
the  CD18-containing  integrins  and  mediate  firm 
cell  attachment  and  migration  of  leukocytes  out 
of  the  bloodstream. 

Individual  genes  can  be  altered  in  mice  by  the 
technique  of  homologous  recombination  (ex- 
change of  DNA  between  related  molecules) .  Us- 
ing this  methodology  in  ES  cells,  a  mutation  was 
introduced  into  the  mouse  CD  18  gene,  and  the 
alteration  was  transmitted  through  the  germline 
so  that  the  altered  animals  could  be  bred.  The 
mutant  animals  have  a  partial  rather  than  com- 
plete impairment  of  the  CD  18  gene's  function. 
Leukocyte  function  shows  significant  alteration 
in  these  animals,  and  they  will  be  studied  for  any 
differences  in  inflammatory  responses  or  suscep- 
tibility to  atherosclerosis. 


A  mutation  has  also  been  introduced  into  the 
ICAM-1  gene,  and  mice  carrying  this  mutation  in 
the  majority  of  their  cells  are  being  bred  to  obtain 
transmission  of  the  defect  to  the  germline.  Eff^orts 
have  also  been  initiated  to  prepare  a  mutation  in 
the  P-selectin  gene,  whose  product  is  expressed 
on  platelets  and  endothelial  cells.  Adhesion  pro- 
teins of  the  selectin  family  bind  to  sugar  mole- 
cules, rather  than  to  proteins,  on  other  cells.  The 
work  to  obtain  mouse  mutations  in  leukocyte  and 
endothelial  cell  adhesion  molecules  is  supported 
in  part  by  a  grant  from  the  National  Institutes  of 
Health. 

A  defect  in  CD  18  causes  a  rare  human  genetic 
disease.  In  its  severe  form,  this  condition,  known 
as  leukocyte  adhesion  deficiency  (LAD),  leads  to 
fatal  susceptibility  to  infections.  LAD  is  an  attrac- 
tive model  for  development  of  somatic  gene  ther- 
apy for  bone  marrow-derived  cells.  A  recombi- 
nant retrovirus  encoding  the  human  CD  18 
protein  has  been  prepared  and  has  been  used  to 
transfer  the  gene  into  mouse  and  human  cells. 
Bone  marrow  transplantation  was  performed  in 
mice,  and  the  infected  cells  express  human  CD  18 
when  reimplanted  into  the  mouse.  Cultured  cells 
from  LAD  patients  were  infected  with  retrovirus, 
and  the  adhesion  properties  of  the  cells  were  re- 
stored to  normal.  Bone  marrow  cells  from  af- 
fected patients  are  being  cultured  in  the  labora- 
tory and  infected  with  the  retrovirus  in 
preparation  for  performing  human  gene  therapy. 

Genetic  variation  in  the  adhesion  molecules  of 
human  leukocytes  and  endothelial  cells  is  being 
identified.  In  the  ICAM-1  gene,  for  example, 
there  is  an  amino  acid  difference  that  is  prevalent 
in  the  population.  It  is  of  interest  to  determine 
whether  different  forms  of  the  ICAM-1  gene  in 
the  human  population  are  associated  with  differ- 
ent susceptibilities  to  inflammatory  diseases  and 
atherosclerosis.  Some  of  the  long-term  goals  of 
this  project  are  to  identify  naturally  occurring  ge- 
netic variations  that  alter  susceptibility  to  com- 
mon disease  processes.  In  addition,  the  mutant 
mice  can  be  used  to  determine  if  decreased  func- 
tion of  some  of  these  cell  adhesion  molecules  re- 
duces chronic  inflammatory  disease  processes 
(e.g.,  autoimmune  disease,  arthritis,  multiple 
sclerosis,  diabetes  mellitus).  If  reduced  gene 
function  were  to  lessen  the  disease  processes  in 
mouse  models,  this  would  provide  evidence  that 
the  function  of  these  proteins  might  be  blocked 
by  drugs  as  a  means  of  slowing  the  analogous  pro- 
cesses in  humans. 


26 


Molecular  Genetics  of  Diabetes  Mellitus 


Graeme  I.  Bell,  Ph.D. — Investigator 

Dr.  Bell  is  also  Professor  of  Biochemistry  and  Molecular  Biology,  Medicine,  and  Genetics  at  the  University 
of  Chicago.  He  received  his  B.S.  degree  in  zoology  and  M.S.  degree  in  biology  from  the  University  of 
Calgary,  Canada.  He  earned  his  Ph.D.  degree  in  biochemistry  and  biophysics  from  the  University  of 
California,  San  Francisco,  where  he  also  did  his  postdoctoral  research.  Before  moving  to  the  University  of 
Chicago,  Dr.  Bell  served  as  Senior  Scientist  at  the  Chiron  Corporation.  Dr.  Bell  has  received  a  number  of 
awards  for  his  work,  including  the  Outstanding  Scientific  Achievement  Award  from  the  American  Diabetes 
Association,  the  Rolf  Luft  Award  from  the  Swedish  Medical  Society,  and  the  Distinguished  Alumni  Award 
from  the  University  of  Calgary. 


DIABETES  mellitus  is  a  disorder  of  carbohy- 
drate metabolism  characterized  by  elevated 
blood  glucose  levels.  In  the  United  States,  an  esti- 
mated 6  million  persons  are  known  to  have  dia- 
betes, and  there  are  probably  an  equal  number 
with  unrecognized  disease.  About  10  percent  of 
persons  over  age  65  have  diabetes,  and  the  com- 
plications of  the  cardiovascular,  kidney,  visual, 
and  nervous  systems  are  major  causes  of  morbid- 
ity and  mortality. 

Clinically  diabetes  is  a  heterogeneous  dis- 
order. One  form,  insulin-dependent  diabetes 
mellitus,  primarily  affects  children  and  adoles- 
cents. It  results  from  immunological  destruction 
of  the  insulin-producing  cells  of  the  pancreas; 
because  of  the  absolute  deficiency  of  insulin,  pa- 
tients require  insulin  therapy  for  survival.  The 
more  common  form  of  diabetes,  non-insulin- 
dependent  diabetes  mellitus  (NIDDM),  includes 
about  90  percent  of  diabetic  patients.  This  form 
results  from  reduced  insulin  levels  in  some  pa- 
tients and  a  relative  deficiency  in  others  and  is 
due  to  abnormal  functioning  of  the  insulin- 
producing  cells  or  decreased  responsiveness  of 
tissues  to  insulin.  In  these  patients  the  blood  glu- 
cose levels  can  usually  be  controlled  by  diet  or  by 
drugs  that  can  be  taken  orally  to  improve  insulin 
secretion  and  action. 

As  with  other  common  diseases  of  middle  age 
such  as  cardiovascular  disease  and  hypertension, 
genetic  factors  contribute  to  the  development  of 
NIDDM.  Our  goal  is  to  identify  the  genes  that  in- 
crease the  risk  of  developing  diabetes  and  to  de- 
termine how,  together  with  environmental  and 
life-style  factors,  they  result  in  the  elevated  blood 
glucose  levels  that  define  this  disorder. 

We  have  taken  an  approach  that  combines  ge- 
netics and  molecular  biology.  Our  working  hy- 
pothesis is  that  a  relatively  small  number  of  po- 
tentially identifiable  major  genes  increase  the 
risk  of  developing  diabetes  and  that  the  individ- 
ual's overall  genetic  background,  together  with 
environmental  and  life-style  factors,  influences 
the  phenotypic  expression  of  the  major  suscepti- 
bility genes. 


In  our  genetic  studies,  we  are  studying  diabetes- 
prone  families  in  which  NIDDM  has  an  early  age- 
at-onset  and  a  clear  autosomal  dominant  mode  of 
inheritance.  In  one  such  family  we  have  identi- 
fied a  DNA  marker  on  the  long  arm  of  human 
chromosome  20  that  is  tightly  linked  to  the 
diabetes-susceptibility  gene.  We  are  presently 
trying  to  isolate  this  gene  and  to  identify  the  mu- 
tation that  impairs  its  normal  function  and  leads 
to  failure  of  the  insulin-secreting  pancreatic  ^- 
cells.  The  identification  of  this  gene  may  suggest 
others  whose  mutation  could  also  cause  NIDDM. 

Drawing  on  our  understanding  of  the  patho- 
physiology of  NIDDM,  we  are  also  cloning  and 
characterizing  genes  that  might  reasonably  con- 
tribute to  diabetes  susceptibility.  Since  the  pan- 
creatic /3-cell  plays  an  important  role  in  the  patho- 
genesis of  all  forms  of  diabetes  mellitus,  we  are 
particularly  interested  in  studying  the  genes  that 
are  responsible  for  its  unique  physiological  char- 
acteristics. These  include  genes  encoding  hor- 
mones such  as  insulin  and  islet  amyloid  polypep- 
tide (a  newly  discovered  hormone-like  peptide 
of  uncertain  function),  ion  channels,  and  recep- 
tors for  hormones  and  other  agents  that  regulate 
insulin  secretion.  In  addition  to  cloning  and 
characterizing  genes  expressed  in  /3-cells,  we  are 
also  identifying  DNA  polymorphisms  in  these 
genes  to  facilitate  genetic  studies  of  their  contri- 
bution to  development  of  NIDDM.  Furthermore, 
we  are  studying  the  effects  of  hyperglycemia  and 
other  altered  metabolic  states  on  their  expression 
in  order  to  determine  if  changes  in  the  levels  of 
the  proteins  encoded  by  these  genes  can  provide 
a  molecular  explanation  for  the  failure  of  the 
cell  to  secrete  appropriate  amounts  of  insulin  in 
response  to  glucose  in  patients  with  NIDDM. 

Recent  studies  of  the  receptor  for  the  polypep- 
tide hormone  somatostatin  are  beginning  to  shed 
new  light  into  the  mechanism  by  which  this  hor- 
mone inhibits  insulin  secretion.  In  addition,  they 
are  leading  us  into  the  area  of  neurobiology  and 
hormonal  regulation  of  neuronal  function.  So- 
matostatin is  a  cyclic  1 4-amino  acid  polypeptide 


27 


Molecular  Genetics  of  Diabetes  Mellitus 


found  throughout  the  gastrointestinal  tract,  in- 
cluding the  pancreas,  and  nervous  systems.  It  has 
many  diverse  functions,  including  inhibiting  the 
secretion  of  insulin  and  glucagon  from  the  cells 
of  the  pancreatic  islets  and  of  growth  hormone 
from  the  pituitary.  In  the  central  nervous  system, 
somatostatin  acts  as  a  neurotransmitter  to  regu- 
late neuronal  activity  and  to  facilitate  the  release 
of  other  neurotransmitters.  It  may  also  play  a  role 
in  centrally  mediated  behaviors  such  as  move- 
ment and  cognition.  We  have  recently  cloned 
the  genes  encoding  three  somatostatin  receptors. 
All  are  members  of  the  seven-transmembrane- 
spanning,  GTP-binding  protein  family  of  recep- 
tors. One  of  these,  SSTR2,  is  expressed  in  the  pan- 
creatic /S-cell  and  is  likely  to  be  responsible  for 
mediating  the  inhibition  of  insulin  release  by  so- 
matostatin. The  three  somatostatin  receptor  iso- 
forms  that  we  have  characterized  have  unique 
pharmacological  properties  and  couple  to  differ- 
ent intracellular  effector  systems,  thus  providing 
at  least  a  partial  explanation  for  the  diverse  physi- 
ological effects  of  somatostatin.  We  are  continu- 
ing our  studies  of  this  family  of  receptors  to  gain  a 
better  understanding  of  how  somatostatin  bind- 
ing results  in  inhibition  of  insulin  secretion. 

In  addition  to  genes  expressed  in  the  pancre- 
atic /3-cell,  we  have  also  characterized  genes  for 
membrane  proteins  involved  in  the  transport  of 
glucose  across  the  plasma  membrane.  Our  stud- 
ies have  revealed  unexpected  functional  com- 


plexity that  could  have  important  implications 
for  the  treatment  of  diabetes.  These  studies  indi- 
cate that  facilitative  glucose  transport  is  not  the 
property  of  a  single  protein  but  rather  involves  a 
family  of  at  least  five  structurally  related  pro- 
teins. These  proteins  have  distinct  patterns  of  tis- 
sue distribution  and  different  kinetic  properties 
and  are  independently  regulated.  These  features 
allow  the  precise  disposal  of  dietary  glucose 
under  varying  physiological  conditions.  Recent 
studies  have  shown  that  one  of  the  five  proteins 
we  have  identified  is  the  major  glucose  trans- 
porter of  neuronal  cells  and  that  the  major  func- 
tion of  another  is  to  transport  fructose  across  the 
plasma  membrane.  This  fructose  transporter  is 
expressed  on  the  luminal  surface  of  the  absorp- 
tive epithelial  cells  of  the  small  intestine  and  kid- 
ney tubules  and  is  also  present  in  early  and  late 
spermatids  in  the  testes.  Sperm  require  a  fructose 
transporter,  because  they  utilize  fructose  in  semi- 
nal fluid  as  an  energy  source. 

Our  studies  are  leading  to  a  better  understand- 
ing of  the  causes  of  diabetes  mellitus.  In  addition, 
they  are  providing  new  insight  into  the  function 
of  somatostatin  in  the  regulation  of  neuronal 
function.  The  identification  of  the  fructose  trans- 
porter in  sperm  may  also  have  implications  in  re- 
productive physiology.  Our  results  also  illustrate 
how  studies  in  one  area  can  impact  those  in  an- 
other, because  of  the  exquisite  manner  in  which 
the  organism  is  able  to  utilize  similar  proteins  for 
different  physiological  functions. 


28 


Development  of  the  Drosophila  Peripheral 
Nervotis  System 


Hugo  J.  Bellen,  D.V.M.,  Ph.D. — Assistant  Investigator 

Dr.  Bellen  is  also  Assistant  Professor  in  the  Institute  for  Molecular  Genetics,  the  Division  of  Neuroscience, 
the  Department  of  Cell  Biology,  and  the  Program  in  Developmental  Biology  at  Baylor  College  of  Medicine. 
Educated  in  Belgium,  he  received  a  degree  in  commercial  engineering  and  began  research  in  sociometry, 
but  decided  to  pursue  a  career  in  medical  science.  He  obtained  a  D.V.M.  degree  from  the  University  of 
Ghent  and  a  Ph.D.  degree  in  genetics  from  the  University  of  California,  Davis,  where,  in  John  Kiger's 
laboratory,  he  studied  mutations  that  affect  behavior  in  the  fruit  fly.  Later,  in  Basel,  Switzerland,  as 
a  postdoctoral  fellow  with  Walter  Gehring,  he  helped  to  develop  the  enhancer  detection  system. 


OUR  research  is  centered  on  the  development 
of  the  peripheral  nervous  system  (PNS)  of 
the  fruit  fly  Drosophila  melanogaster.  The  re- 
sults will  help  us  analyze  the  development  of  the 
nervous  system  of  many  eukaryotic  species.  We 
have  focused  on  the  PNS  of  Drosophila  because 
its  cells  are  relatively  easy  to  study  and  because 
sophisticated  genetics  can  be  applied  to  this 
model  organism. 

We  are  presently  studying  the  couch  potato 
(cpo)  and  neuromusculin  (nrm)  genes,  which 
are  expressed  at  the  onset  of  embryonic  nervous 
system  development  and  later  in  embryogenesis 
in  most  cells  of  the  PNS.  These  genes  were  identi- 
fied in  enhancer  detector  screens  in  which  regula- 
tory sequences  of  genes  are  identified  by  means 
of  a  ;8-galactosidase  reporter.  Here  we  describe 
the  preliminary  characterization  of  these  essen- 
tial genes  and  the  development  of  a  novel  tech- 
nique to  ablate  nervous  system  cells. 

The  cpo  gene  was  so  named  because  homozy- 
gous mutant  flies  are  viable  but  have  poor  jump 
responses,  poor  flight  abilities,  slow  recovery 
after  ether  anesthesia,  and  are  generally  hypoac- 
tive.  More  than  10  insertional  cpo  mutations 
were  recovered  from  several  enhancer  detector 
screens;  none  of  the  insertions  caused  a  complete 
loss  of  function  of  the  gene.  Some  insertional  al- 
leles cause  recessive  embryonic  lethality,  but  no 
defects  were  observed  in  the  PNS  of  the  mutant 
embryos. 

A  molecular  analysis  of  cpo  was  initiated  with 
the  isolation  of  cpo  cDNAs.  In  situ  hybridizations 
to  whole-mount  embryos  showed  that  the  gene  is 
expressed  in  the  precursor  cells  of  the  PNS  as 
well  as  glia  of  the  PNS  and  CNS  (central  nervous 
system).  Sequencing  data  show  that  the  gene  en- 
codes at  least  three  different  proteins  that  contain 
many  stretches  of  the  same  amino  acids.  Two  of 
these  proteins  contain  a  domain  that  may  allow 
binding  to  RNA  molecules.  This  binding  domain 
is  found  in  many  proteins  that  are  involved  in 
processing  immature  RNA  in  a  form  that  can  be 
readily  translated  into  a  protein.  The  CPO  protein 
has  most  significant  homology  with  two  other 


RNA-binding  proteins  that  are  found  in  nervous 
tissue:  HuD,  a  human  brain  paraneoplastic  en- 
cephalomyelitis antigen,  and  elav,  the  protein 
from  a  Drosophila  nervous  system-specific  gene 
{embryonic  lethal  abnormal  vision). 

The  cpo  gene  sequence  was  used  to  produce  a 
polyclonal  antibody  to  CPO.  The  antibody  allows 
the  localization  of  this  nuclear  protein  in  the  de- 
veloping embryo,  in  essentially  the  same  cells  as 
the  transcript.  In  addition,  the  antibody  recog- 
nizes a  protein  that  is  associated  with  polytene 
chromosomal  bands  isolated  from  the  salivary 
gland  of  third  instar  larvae.  This  association  may 
be  mediated  through  the  binding  of  CPO  to  na- 
scent RNA  that  is  being  synthesized  from  the  DNA 
of  the  giant  polytene  chromosomes.  Thus  it  is  pos- 
sible that  CPO  acts  as  an  essential  PNS  diff^erentia- 
tion  factor  by  controlling  some  aspects  of  RNA 
maturation  specific  to  the  cells  in  which  CPO  is 
expressed. 

In  addition  to  its  biological  functions,  the  cpo 
gene  is  also  a  hot  spot  for  P-element  enhancer 
detector  insertions.  Ten  insertions  that  cause  a 
variety  of  phenotypes  were  mapped  at  the  nu- 
cleotide level,  and  molecular  defects  in  cpo  mu- 
tant strains  that  carry  an  enhancer  detector  were 
determined.  All  insertions  recovered  so  far  are 
clustered  in  a  200-bp  genomic  fragment  that  con- 
tains key  regulatory  regions  of  the  cpo  gene.  Most 
insertions  are  integrated  upstream  of  the  first  nu- 
cleotide of  the  longest  cDNA  but  downstream  of 
the  consensus  binding  sites  of  three  known  Dro- 
5op^«7a  transcription  factors.  Thus  they  are  proba- 
bly located  between  the  enhancer-binding  sites 
and  the  promoter  of  cpo.  Five  insertions  causing 
different  phenotypes  have  inserted  at  exactly  the 
same  location.  There  is  a  perfect  correlation  be- 
tween the  observed  defects  (or  their  lack)  in  em- 
bryos or  adults  and  the  size  and  the  orientation  of 
these  insertions — e.g.,  large  insertions  are  less 
detrimental  than  smaller  insertions.  These  data 
suggest  that  the  distance  between  the  binding 
sites  of  the  transcription  factors  and  the  transcrip- 
tion initiation  site  is  critical  for  proper  cpo  regu- 
lation. In  addition,  insertions  with  the  /«cZ gene 


29 


Development  of  the  Drosophila  Peripheral  Nervous  System 


in  the  opposite  orientation  of  cpo  transcription 
cause  much  less  severe  defects  than  insertions  in 
the  same  orientation.  These  observations  provide 
a  unique  opportunity  to  study  the  interactions  be- 
tween enhancers  and  promoters  in  vivo.  The 
study  of  cpo  is  also  supported  by  the  Muscular 
Dystrophy  Association. 

The  nrm  gene  is  expressed  early  in  the  develop- 
ment of  the  PNS  and  muscles.  It  maps  at  cytologi- 
cal  band  80A  and  was  also  identified  in  an  en- 
hancer detector  screen.  To  our  knowledge  only  a 
single  insertion  has  been  identified  in  nrm,  and 
the  jS-galactosidase  expression  pattern  in  em- 
bryos of  this  strain  is  virtually  identical  to  that  of 
cpo  during  early  development.  Flies  homozygous 
for  this  particular  insertion  are  homozygous  via- 
ble. A  mutagenesis  experiment  allowed  us  to  re- 
cover 10  recessive  lethal  mutations  that  fail  to 
complement  each  other.  Although  many  subtle 
defects  have  been  observed  in  different  mutant 
strains,  no  consistent  morphological  defects  have 
been  observed. 

The  cloned  nrm  gene  encodes  a  single  4.6-kb 
transcript  that  is  expressed  transiently  in  the  sen- 
sory mother  cells  and  developing  neurons  and 
support  cells  of  the  PNS  (5-10  h).  Later  in  embry- 
onic development  (14-17  h)  the  transcript  is 
also  observed  in  most  embryonic  muscles.  The 
earliest  expression  of  nrm  in  muscles  coincides 
with  the  formation  of  the  neuromuscular  junc- 


tion. Sequencing  data  show  that  the  nrm  gene 
encodes  a  novel  protein  that  contains  a  signal 
peptide  and  eight  immunoglobulin  domains.  The 
protein  is  possibly  anchored  in  the  membrane  of 
the  cells  by  a  secondary  modification  of  the  pro- 
tein. On  the  basis  of  these  molecular  data  and  the 
expression  pattern  of  nrm,  we  propose  that  nrm 
is  a  novel  neural  cell  adhesion  molecule  that  may 
play  an  important  role  in  growth  cone  guidance 
in  the  PNS  and  may  affect  the  formation  of  the 
neuromuscular  junction.  These  hypotheses  are 
presently  being  tested. 

To  study  the  role  of  glial  cells  in  the  develop- 
ment of  the  PNS,  we  are  developing  a  method  that 
allows  temporally  induced  arrest  of  most  func- 
tions in  specific  cells.  These  arrests  are  induced 
by  temperature  shifts  of  flies  that  express  temper- 
ature-sensitive (ts)  forms  of  diphtheria  toxin 
(DT-A'*)  under  the  control  of  cell-  or  tissue- 
specific  regulatory  sequences.  DT-A'*  genes  were 
isolated  in  a  mutagenesis  screen  using  the  yeast 
Saccharomyces  cerevisiae  and  subsequently 
tested  in  PNS  neurons,  namely  the  R1-R6  photo- 
receptor cells  of  transgenic  fruit  flies.  Four  DT-A'* 
have  been  partially  characterized  in  yeast  cells, 
and  three  have  been  tested  in  Drosophila.  These 
toxins  show  similar  temperature  dependence, 
suggesting  that  they  may  be  useful  in  a  wide  range 
of  species.  We  are  presently  trying  to  ablate  glial 
cells  in  the  PNS  and  CNS,  in  an  attempt  to  exam- 
ine the  role  of  glia  in  the  living  organism. 


Expression  pattern  of  the  couch  potato  protein  in  an  almost  fully  developed  Drosophila 
embryo.  This  protein  is  expressed  in  all  cells  of  the  peripheral  nervous  system  and  in  a 
subset  of  cells  of  the  central  nervous  system.  Anterior,  left,  dorsal,  up. 

Research  and  photograph  by  Sandra  Kooyer  and  Diana  D  'Evelyn  in  the  laboratory  of 
Hugo  Bellen. 


30 


Genetic  Manipulation  of  Hematopoietic 
Stem  Cells 


John  W.  Belmont,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Belmont  is  also  Assistant  Professor  of  Molecular  Genetics,  Pediatrics,  and  Microbiology  and 
Immunology  at  Baylor  College  of  Medicine.  He  received  his  undergraduate  degree  from  the  University  of 
Texas,  Austin,  and  his  M.D.  and  Ph.D.  degrees  from  Baylor  College  of  Medicine,  where  he  worked  with 
Robert  Rich.  After  internship  and  residency  training  in  pediatrics  at  Children  's  Hospital,  Washington, 
D.  C,  he  completed  a  fellowship  in  medical  genetics  at  Baylor  College  of  Medicine. 


PLURIPOTENT  hematopoietic  stem  cells  are 
the  "seed"  for  the  development  of  all  blood 
cells.  These  cells,  which  normally  reside  in  the 
bone  marrow,  arise  in  early  fetal  development 
and  persist  throughout  adult  life.  They  can  be  re- 
moved from  the  bone  marrow  and  transplanted 
into  a  prepared  recipient;  they  will  then  stably 
reconstitute  the  entire  blood  and  immune  sys- 
tems. We  have  developed  techniques  for  the  effi- 
cient transfer  of  genes  into  mouse  and  human 
stem  cells,  using  retroviral  vectors.  The  long- 
term  goal  of  these  studies  is  to  perfect  methods 
that  could  be  used  for  the  treatment  of  various 
genetic  and  acquired  diseases. 

A  viral  vector  system  based  on  the  Moloney  mu- 
rine leukemia  virus  (MoMuLV)  has  been  chosen 
because  of  its  potential  for  very  high  gene 
transfer  efficiency.  The  unique  life  cycle  of  this 
retrovirus  makes  it  attractive  for  adaptation  as  a 
vector,  since  the  foreign  genetic  material  is  stably 
integrated  into  the  host  cell  genome.  MoMuLV 
vector  particles  are  able  to  carry  their  genetic  ma- 
terial to  the  target  cells  but  are  incapable  of  repli- 
cating and  spreading  as  a  live  infectious  agent. 

We  have  used  two  model  systems  to  investigate 
properties  of  the  stem  cells.  In  one  model  the 
bacterial  antibiotic  resistance  gene  neo  intro- 
duces distinct  genetic  tags  into  individual  stem 
cells.  The  second  model  uses  the  human  enzyme 
adenosine  deaminase  (ADA)  as  the  molecular 
marker  for  gene  transfer.  This  system  is  particu- 
larly suitable  for  studies  of  expression  of  genes  by 
the  retroviral  vectors.  In  addition,  the  genetic  de- 
ficiency of  ADA  causes  a  form  of  severe  combined 
immune  deficiency,  so  that  successful  laboratory 
experiments  with  this  gene  may  in  time  facilitate 
the  clinical  application  of  the  gene  transfer 
procedures. 

Our  earlier  work  demonstrated  that  genes 
could  be  introduced  into  hematopoietic  stem 
cells  but  that  the  process  was  much  less  efficient 
than  in  the  more  mature  cells  of  the  marrow.  In 
mouse  transplant  experiments,  only  about  50 
percent  of  the  animals  retained  expression  of  the 
human  ADA  enzyme  in  their  blood  for  more  than 
six  months.  This  has  led  to  an  investigation  of  the 


conditions  in  cell  culture  that  would  optimally 
support  the  survival  or  proliferation  of  the  stem 
cells. 

In  collaboration  with  Doug  Williams  (Im- 
munex,  Seattle),  we  have  evaluated  the  efi'ects  of 
several  recombinant  hematopoietic  growth  fac- 
tors on  retroviral  vector-mediated  gene  transfer 
into  stem  cells.  These  factors  included  interleu- 
kin-3,  -6,  and  -7;  granulocyte  colony-stimulating 
factor  (G-CSF);  and  leukemia  inhibitory  factor 
(LIF).  LIP  has  been  of  special  interest  because 
among  its  many  biological  functions,  it  appears  to 
prevent  the  difi'erentiation  of  mouse  embryonic 
stem  cells.  If  it  had  a  similar  action  on  hematopoi- 
etic stem  cells,  it  might  allow  the  preservation  of 
their  developmental  capacity  in  culture. 

A  novel  assay  using  inbred  transgenic  mice  was 
used  to  test  the  activity  of  LIF  on  stem  cells.  These 
experiments  indicate  that  LIF  preserves  the  stem 
cells  during  the  culture  period  required  for  gene 
transfer.  Inclusion  of  LIF  in  the  bone  marrow  cul- 
tures allows  about  70  percent  of  the  stem  cells  to 
be  infected  with  the  retrovirus.  Subsequently  all 
the  mice  transplanted  with  such  cells  maintain 
high-level  expression  of  human  ADA  in  all  their 
blood  and  immune  system  organs  for  at  least  six 
months.  This  finding  has  recently  been  supported 
in  collaborative  experiments  with  Savio  Woo 
(HHMI,  Baylor  College  of  Medicine)  using  a  re- 
troviral vector  that  encodes  the  human  a j -anti- 
trypsin gene. 

We  suspect  that  LIF  acts  in  concert  with  one  or 
more  other  growth  factors  in  our  experimental 
model.  The  recently  isolated  ligand  for  the  c-kit 
cell  surface  receptor  is  a  promising  candidate  for 
a  factor  that  causes  stem  cells  to  replicate.  In  an 
effort  to  survey  critical  growth  factor/ligand  sys- 
tems expressed  by  stem  cells,  we  have  tried  a 
cloning  method  based  on  the  conservation  of 
gene  sequence  to  identify  new  candidate  recep- 
tors. Messenger  RNA  extracted  from  stem  cell- 
enriched  fractions  of  bone  marrow  has  been  used 
as  a  template  for  amplification  of  conserved  re- 
ceptor tyrosine  kinase  sequences.  Among  the 
clones  are  three  that  had  not  previously  been  de- 
scribed. We  are  currently  sequencing  these  genes 


31 


Genetic  Manipulation  of  Hematopoietic  Stem  Cells 


to  determine  if  they  are  novel  receptors  involved 
in  the  regulation  of  hematopoiesis. 

To  analyze  better  the  behavior  of  stem  cells  and 
their  progeny  in  culture  and  after  transplant,  we 
are  testing  a  new  method  for  introducing  unique 
genetic  identifiers  into  individual  stem  cells. 
This  method  allows  identification  of  the  stem 
cells  by  the  polymerase  chain  reaction  (PGR), 
which  is  especially  suited  for  analysis  of  very 
small  numbers  of  cells.  A  family  of  vectors  carry- 
ing the  bacterial  gene  neo  have  been  constructed 
that  are  identical  except  for  small  variations  in 
size.  This  size  variation  is  conveniently  distin- 
guished by  PGR.  When  mixed  together  these  vec- 
tors provide  an  array  of  potential  markers  that  can 
integrate  into  each  stem  cell.  Because  each  stem 
cell  receives  one  to  four  vectors,  the  integration 
of  a  particular  subset  of  the  vectors  provides  a 
genetic  fingerprint  unique  to  each  stem  cell  fam- 
ily. Preliminary  results  with  these  vectors  indi- 


cate that  we  can  mark  the  most  primitive  hemato- 
poietic precursors  very  efficiently.  We  plan  to 
use  this  method  to  compare  different  culture 
conditions  for  stem  cells. 

Retroviral  vector  genetic  marking  may  also 
prove  to  be  very  informative  in  clinical  bone 
marrow  transplantation.  GoUaborative  studies 
with  Albert  Deisseroth  (M.D.  Anderson  Hospital, 
Houston)  investigated  whether  the  techniques 
that  have  been  used  to  optimize  mouse  bone 
marrow  stem  cell  gene  transfer  could  be  used  for 
human  cells.  These  experiments  demonstrate  the 
importance  of  supporting  cells  in  the  culture 
(called  stromal  cells)  for  efficient  gene  transfer. 
The  work  was  performed  using  an  assay  of  human 
stem  cells  that  depends  on  their  persistence  in 
culture.  We  hope  that  many  valuable  lessons  will 
be  learned  from  these  studies  that  will  contribute 
to  the  long-term  goal  of  treating  disease  by  the 
use  of  therapeutic  vectors. 


Human  bone  marrow  colony  called  an  erythroid  burst  or  BFU-E.  The  colony  grows  from  a 
single  cell  to  form  3-9  clusters  of  red  blood  cells.  Measurement  of  gene  transfer  into  such 
colonies  has  helped  to  improve  gene  therapy  protocols. 
Research  and  photograph  by  Kateri  Moore  in  the  laboratory  offohn  Belmont. 


32 


Proteins  of  the  Spectrin-based  Membrane  Skeleton 


G.  Vann  Bennett,  M.D.,  Ph.D. — Investigator 

Dr.  Bennett  is  also  Professor  of  Biochemistry  at  Duke  University  Medical  Center.  He  received  his  M.D.  and 
Ph.D.  degrees  from  the  Johns  Hopkins  University  Medical  School.  He  completed  postdoctoral  training  at 
Harvard  University  in  membrane  protein  biochemistry  with  Daniel  Branton.  Before  joining  Duke 
University,  he  was  on  the  faculty  in  the  Department  of  Cell  Biology  and  Anatomy  at  Johns  Hopkins. 


STRUCTURAL  proteins  in  the  cytoplasm  and 
membranes  of  cells  provide  the  basis  for  spa- 
tial organization  of  the  diverse  components  of  eu- 
karyotic  cells.  These  proteins  thus  are  principal 
participants  in  fundamental  cellular  activities 
such  as  cell  motility  and  cell-cell  interactions. 
Our  work  over  the  past  1 0  years  has  focused  on 
plasma  membranes.  We  initiated  these  studies  in 
the  human  erythrocyte.  This  relatively  simple 
cell  has  provided  an  experimentally  accessible 
model  system  for  detailed  dissection  of  protein- 
protein  interactions  that  are  responsible  for  the 
structure  and  organization  of  the  plasma 
membrane. 

The  principal  structural  protein  in  the  eryth- 
rocyte membrane  is  the  flexible  rod-shaped 
molecule  spectrin,  which  is  organized  in  a  two- 
dimensional  network  attached  to  the  cytoplasmic 
surface  of  the  plasma  membrane.  Spectrin  mole- 
cules are  attached  at  their  ends  to  form  a  series  of 
hexagons  and  pentagons  that  closely  resembles  a 
geodesic  dome.  The  binding  of  spectrin  to  the 
protein  ankyrin  attaches  the  spectrin  network  to 
the  plasma  membrane.  Ankyrin  also  interacts 
with  high  affinity  with  the  cytoplasmic  domain  of 
an  integral  membrane  protein  (a  protein  that  tra- 
verses the  membrane  and  actually  has  portions 
exposed  on  both  the  inner  and  outer  membrane 
surfaces) .  The  spectrin-based  membrane  network 
or  skeleton  is  required  for  normal  stability  of 
erythrocytes  in  the  circulation.  Abnormalities  in 
amounts  or  function  of  spectrin  and  associated 
proteins  result  in  hemolytic  anemias  and  are  the 
basis  for  diseases  such  as  hereditary  spherocytosis 
and  hereditary  elliptocytosis. 

Proteins  closely  related  to  spectrin  are  present 
in  many  vertebrate  cells  and  are  associated  in 
most  cases  with  the  plasma  membrane.  Spectrin 
is  present  in  especially  high  amounts  in  brain, 
where  it  comprises  3  percent  of  the  total  mem- 
brane protein.  The  spectrin-based  membrane 
skeleton  in  brain  and  other  tissues  is  likely  to  play 
an  important  role  in  providing  organization  of 
integral  membrane  proteins  in  the  plasma  mem- 
brane and  for  coupling  membrane  proteins  to  ele- 
ments of  the  cytoskeleton.  Potential  physiologi- 


cal consequences  of  these  activities  include 
stabilization  of  the  lipid  bilayer  and  organization 
of  membrane  proteins  in  specialized  regions  on 
the  cell  surface  in  polarized  cells. 

Specific  aims  of  this  laboratory  are  to  elucidate 
the  proteins  in  erythrocytes  and  other  cells  that 
mediate  interaction  of  spectrin  with  membranes, 
determine  how  these  protein  interactions  are  reg- 
ulated, and  understand  the  cellular  functions  of 
the  spectrin  skeleton. 

Ankyrins  in  the  Nervous  System 

Ankyrin  appears  to  function  as  an  adapter  be- 
tween certain  membrane  proteins  and  the  spec- 
trin skeleton.  We  have  discovered  that  brain  con- 
tains multiple  forms  of  ankyrin,  with  diversity 
due  to  distinct  genes  as  well  as  alternative  splic- 
ing of  RNAs  encoded  by  the  same  gene.  We  have 
determined  the  complete  amino  acid  sequence  of 
the  major  form  of  ankyrin  in  human  brain  and 
have  discovered  an  unusual  alternative  form  of 
this  protein  that  contains  a  large  inserted  se- 
quence. The  large  form  of  brain  ankyrin  is  the 
first  ankyrin  detected  during  brain  development 
and  is  targeted  to  neuronal  processes  including 
unmyelinated  axons. 

The  same  gene  that  encodes  ankyrin  in  erythro- 
cytes also  is  expressed  in  brain.  This  form  of  an- 
kyrin is  localized  in  the  plasma  membranes  of 
certain  neurons  and  is  abundant  in  cerebellum, 
brain  stem,  and  spinal  cord.  The  erythrocyte  form 
of  ankyrin  is  missing  in  a  strain  of  mutant  mice 
developed  at  the  Jackson  Laboratory.  Ankyrin- 
deficient  mice  experience  degeneration  of  Pur- 
kinje  cells,  a  major  type  of  neuron  in  the  cerebel- 
lum, and  develop  a  stagger  and  difficulty  in 
walking.  Neurological  problems  of  ankyrin-defi- 
cient  mice  may  have  counterparts  in  humans  with 
slowly  progressive  diseases  due  to  death  of  nerve 
cells. 

Another  isoform  of  ankyrin  is  highly  concen- 
trated along  with  the  voltage-dependent  sodium 
channel  at  the  nodes  of  Ranvier  of  nerve  axons. 
Nodes  of  Ranvier  are  specialized  regions  on  the 
axons  of  nerves  where  the  myelin  or  insulation  of 
the  axon  is  interrupted  and  where  ions  can  enter 


33 


Proteins  of  the  Spectrin- based  Membrane  Skeleton 


or  leave  the  axon  through  ion  channels.  Localiza- 
tion of  the  voltage-dependent  sodium  channel  at 
nodes  of  Ranvier  is  important  for  normal  conduc- 
tion of  nerve  impulses.  We  hope  to  identify  the 
gene  encoding  the  form  of  ankyrin  at  the  node  of 
Ranvier  and  eventually  to  understand  the  role  of 
nodal  ankyrin  in  organization  of  this  specialized 
membrane  domain.  These  studies  will  have  rele- 
vance to  diseases  of  neurons  such  as  multiple  scle- 
rosis, where  the  myelin  coating  of  axons  is  lost 
and  sodium  channels  are  no  longer  restricted  to 
the  nodes  of  Ranvier. 

Diversity  of  ank)'rins  suggests  that  this  family  of 
proteins  may  interact  with  many  membrane  pro- 
teins. Characterized  membrane  proteins  in  brain 
that  associate  with  ankyrin  in  in  vitro  assays  in- 
clude the  voltage-dependent  sodium  channel  and 
sodium/potassium  ATPase.  We  have  used  the 
membrane-binding  domain  of  the  major  form  of 
brain  ankyrin  to  isolate  ankyrin-binding  proteins 
and  have  identified  a  family  of  ankyTin-binding 
proteins  found  in  plasma  membranes  of  neurons 
and  glial  cells.  These  ankyrin-binding  proteins 
represent  0.3  percent  of  adult  brain  membrane 
protein  and  appear  late  in  postnatal  develop- 
ment. An  important  goal  for  future  work  will  be 
to  isolate  the  cDNAs  encoding  these  proteins  and 
determine  their  function  in  adult  brain. 

Ankyrin-Independent  Membrane 
Attachment  Sites  for  Spectrin 

Brain  spectrin  can  also  associate  directly 
with  membrane  proteins  through  an  interaction 
that  is  independent  of  ankyrin.  We  have  discov- 
ered that  calcium,  in  concert  with  three  differ- 
ent calcium-regulated  proteins  (calmodulin,  a 
calcium-activated  protease,  and  protein  kinase 
C),  inhibits  the  direct  spectrin-membrane  link- 
age but  has  no  effect  on  spectrin-ankyrin  interac- 
tions. These  results  suggest  that  the  spectrin  skel- 
eton includes  both  stable,  ankyrin-mediated 
linkages  and  dynamic  calcium-sensitive  associa- 
tions that  are  subject  to  metabolic  control.  Iden- 
tification of  the  spectrin  "receptor"  is  the  first 
step  in  understanding  the  role  of  this  type  of 
spectrin-membrane  interaction  in  cells. 

Ankyrin  Structure 

Ankyrin  contains  three  independently  folded 


domains:  one  that  interacts  with  certain  mem- 
brane proteins,  another  that  associates  with  spec- 
trin, and  a  third  that  regulates  associations  of  the 
binding  domains.  Surprisingly,  the  membrane- 
binding  domain  of  ankyrin  includes  an  amino 
acid  sequence  that  is  homologous  to  regions  of 
sequence  in  a  group  of  apparently  unrelated  pro- 
teins from  flies,  yeast,  and  even  viruses.  We  have 
recently  discovered  that  this  conserved  portion 
of  the  ankyrin  sequence  is  responsible  for  the  in- 
teraction of  ankyrin  with  at  least  one  membrane 
protein.  Ankyrin  thus  contains  a  highly  conserved 
and  ancient  structural  motif  that  may  have  a  gen- 
eral role  in  molecular  recognition.  We  hope  to 
determine  the  three-dimensional  structure  of  this 
portion  of  ankyrin,  with  the  expectation  that  this 
structure  will  help  us  understand  interactions  of 
ankyrin  and  other  proteins  with  related  sequences. 

Adducin 

The  protein  adducin  is  a  candidate  to  play  a 
role  in  assembly  of  the  spectrin  skeleton  in  eryth- 
rocytes, brain,  and  certain  epithelial  tissues.  We 
have  found  that  adducin  is  localized  at  sites  of 
cell-cell  contact  in  epithelial  tissues.  Adducin 
and  spectrin  are  colocalized  at  cell  contact  sites 
and  may  be  arranged  in  a  structure  analogous  to 
the  spectrin  network  of  erythrocytes.  The  associa- 
tion of  adducin  with  cell-cell  contact  sites  occurs 
before  assembly  of  other  types  of  specialized  cell 
junctions  such  as  desmosomes. 

Our  working  hypothesis  is  that  adducin  pro- 
motes assembly  of  a  stable  spectrin  network  at 
sites  of  cell-cell  contact.  A  further  hypothesis  is 
that  the  spectrin  network  is  an  essential  precon- 
dition for  assembly  of  specialized  cell  junctions. 
Formation  of  appropriate  cell-cell  contacts  and 
cell  junctions  is  an  essential  event  in  embryogen- 
esis  and  is  one  of  the  processes  that  is  disturbed  in 
diseases  such  as  cancer.  We  are  excited  by  the 
possibility  that  adducin  and  spectrin  may  partici- 
pate in  such  a  fundamental  activity  of  cells.  We 
have  determined  the  complete  protein  sequence 
of  both  adducin  subunits  and  are  in  the  process  of 
determining  a  physical  model  for  organization  of 
domains  and  subunits  in  the  adducin  molecule. 
Future  experiments  will  evaluate  the  role  of  ad- 
ducin and  spectrin  in  formation  of  junctions  be- 
tween cells. 


34 


TNF  and  the  Molecular  Pathogenesis  of  Shock 


Bruce  A.  Beutler,  M.D. — Associate  Investigator 

Dr.  Beutler  is  also  Associate  Professor  of  Internal  Medicine  at  the  University  of  Texas  Southwestern  Medical 
Center  at  Dallas.  After  receiving  his  M.D.  degree  from  the  University  of  Chicago  (Pritzker  School  of 
Medicine),  he  served  as  an  intern  and  resident  at  the  Southwestern  Medical  Center.  His  postdoctoral 
fellowship  with  Anthony  Cerami  was  completed  at  the  Rockefeller  University,  which  he  left  as  an  assistant 
professor  to  assume  a  position  at  the  Southwestern  Medical  Center. 


THIS  laboratory  studies  basic  mechanisms  that 
lead  to  septic  shock,  a  serious  condition  aris- 
ing as  a  result  of  many  types  of  infection.  We  have 
learned  that  the  final  common  pathway  leading  to 
shock  involves  the  production  of  certain  cyto- 
kines, particularly  tumor  necrosis  factor  (TNF), 
by  host  cells  known  as  macrophages  and  by  other 
cell  types  as  well. 

Once  TNF  has  been  produced,  it  alters  the  me- 
tabolism of  cells  throughout  the  body,  triggering 
a  breakdown  of  protein  and  fat  stores.  If  TNF  is 
chronically  secreted  at  low  levels,  a  state  of  wast- 
ing called  cachexia  will  develop.  This  condition 
is  seen  in  cancer  and  many  other  forms  of  chronic 
illness.  On  the  other  hand,  if  massive  quantities 
of  TNF  are  released  over  a  short  time,  as  in  wide- 
spread injury,  the  protein  activates  neutrophils 
and  endothelial  cells  in  such  a  way  that  shock 
occurs. 

Because  TNF  is  a  critically  important  molecule 
in  various  human  disease  processes,  we  have 
sought  to  understand  how  its  biosynthesis  is  con- 
trolled. Probably  the  most  potent  stimulus  for 
TNF  release  is  a  molecule  known  as  lipopolysac- 
charide,  or  endotoxin.  This  molecule  is  pro- 
duced by  gram-negative  bacteria,  which  have  a 
remarkable  tendency  to  cause  shock.  In  the 
course  of  a  gram-negative  infection,  endotoxin  is 
released  into  the  bloodstream.  It  is  harmless  to 
most  cells  but  is  a  powerful  activator  of  mono- 
cytes and  macrophages,  triggering  their  release  of 
TNF  with  all  of  its  attendant  consequences. 

By  studying  different  portions  of  the  TNF  gene, 
we  have  shown  that  endotoxin  causes  two  sepa- 
rate responses  within  the  macrophage:  1)  It 
causes  increased  transcription  of  the  TNF  gene, 
leading  to  a  marked  accumulation  of  TNF  mRNA 
within  macrophages.  2)  It  causes  far  more  effi- 
cient translation  of  the  mRNA — i.e.,  increases  the 
speed  with  which  the  mRNA  is  read  to  produce 
TNF  protein.  Acting  in  concert,  these  two  effects 
are  responsible  for  a  10,000-fold  increase  in  the 
rate  of  TNF  biosynthesis  and  thus  a  massive  net 
effect. 

For  a  number  of  technical  reasons,  it  has  been 
very  difficult  to  demonstrate  the  major  sources  of 


TNF  in  living  animals.  It  is  not  clear,  for  example, 
whether  it  is  made  by  normal  tissues  in  healthy 
animals  or  whether  such  "baseline"  production 
is  important  for  maintenance  of  physiological  or 
immunological  processes.  Similarly,  it  is  not 
clear  whether  the  TNF  that  arises  in  cancer  is  de- 
rived from  cells  of  the  tumor  or  from  host  cells 
that  act  in  response  to  the  tumor.  TNF  is  believed 
to  be  made  in  a  variety  of  autoimmune  or  allergic 
diseases,  but  again,  the  principal  source  of  the 
protein  remains  uncertain. 

To  address  these  questions,  our  laboratory  has 
produced  transgenic  mice  that  express  a  reporter 
construct  in  which  an  easily  measurable  enzyme 
(chloramphenicol  acetyltransferase,  or  CAT)  is 
employed  as  a  marker  for  TNF.  In  other  words, 
CAT  synthesis  also  occurs  in  cells  that  produce 
TNF.  CAT  remains  confined,  however,  to  the  cell 
of  origin,  whereas  TNF  is  secreted  and  becomes 
widely  dispersed  in  the  organism.  Using  these  an- 
imals, we  have  found  that  during  normal  develop- 
ment TNF  is  made  by  cells  of  the  thymus.  Other 
investigators  have  further  reported  that  thymic 
production  of  TNF  is  essential  for  normal  devel- 
opment. Although  the  protein  does  not  appear  to 
be  produced  elsewhere  in  healthy  animals,  it  is 
readily  induced  by  administration  of  lipopolysac- 
charide  or  by  various  authentic  infections. 

A  second  tissue  in  which  CAT  synthesis  marks 
TNF  production  is  the  placenta,  the  organ  that  is 
formed  in  pregnant  mammals  to  nourish  the  de- 
veloping fetus.  The  placenta  contains  cells  of  two 
separate  origins:  part  of  the  placenta,  the  deci- 
dua,  is  derived  from  the  mother.  The  remainder 
of  the  placenta,  the  trophoblast,  is  derived  from 
the  fertilized  egg.  Studies  utilizing  the  reporter 
construct  have  effectively  proved  that  the  TNF 
gene  is  constitutively  expressed  by  the  tropho- 
blast. TNF  may  also  be  constitutively  expressed 
by  trophoblastic  tumors.  Its  function  within  the 
placenta  remains  to  be  established;  however,  it  is 
interesting  to  consider  that  the  protein  may  play 
an  important  role  in  normal  pregnancy. 

Our  laboratory  has  also  made  progress  in  un- 
derstanding the  mechanism  of  action  of  drugs 
that  inhibit  TNF  biosynthesis  and  in  devising  mol- 
ecules that  block  the  action  of  TNF  once  it  has 


35 


TNF  and  the  Molecular  Pathogenesis  of  Shock 


been  released.  These  studies  might  lead  to  better 
therapies  for  shock  and  other  disorders.  Gluco- 
corticoid hormones  (e.g.,  prednisone,  dexameth- 
asone,  and  Cortisol)  have  long  been  used  as  anti- 
inflammatory drugs.  One  of  their  principal 
effects  appears  to  be  a  blockade  of  TNF  biosynthe- 
sis.,, which  depends  upon  inhibition  of  both  TNF 
gene  transcription  and  mRNA  translation.  Other 
drugs  of  a  class  known  as  phosphodiesterase  in- 
hibitors (e.g.,  theophylline,  caffeine,  and  pen- 
toxifylline) also  block  TNF  biosynthesis,  achiev- 
ing their  effect  by  preventing  TNF  mRNA 
accumulation.  They  appear  to  function  at  a  dif- 
ferent locus  than  do  glucocorticoids.  We  have 
shown  that  the  two  classes  of  drugs  when  com- 
bined exert  a  synergistic  effect. 


Recently  the  cell-surface  receptor  for  TNF  was 
cloned  in  a  number  of  laboratories.  We  have  engi- 
neered a  recombinant  molecule  in  which  the  TNF 
receptor  is  attached  to  a  portion  of  a  normal  anti- 
body, yielding  a  new  protein  molecule  in  which 
two  TNF-binding  sites  are  expressed.  This  biva- 
lent TNF-binding  protein  strongly  inhibits  the  bi- 
ological effects  of  TNF,  is  highly  stable  in  vivo, 
and  may  be  produced  in  large  quantities  by  re- 
combinant techniques.  We  anticipate  that  this 
type  of  molecule  will  allow  a  thorough  investiga- 
tion of  the  many  effects  of  TNF  in  health  and  dis- 
ease and  may  also  be  useful  as  a  therapeutic  tool. 
Of  particular  interest  will  be  its  use  in  studies  of 
TNF  production  and  action  in  the  thymus  and  in 
the  placenta. 


36 


Cytotoxic  T  Lymphocyte  Recognition 


Michael  J.  Sevan,  Ph.D. — Investigator 

Dr.  Bevan  is  also  Professor  of  Immunology  at  the  University  of  Washington,  Seattle.  He  received  his  Ph.D. 
degree  for  work  performed  at  the  National  Institute  for  Medical  Research,  Mill  Hill,  london.  He  did  his 
postdoctoral  work  at  the  Salk  Institute  in  the  laboratory  of  Melvin  Cohn,  after  which  he  was  a  faculty 
member  in  the  Center  for  Cancer  Research  and  the  Department  of  Biology  at  the  Massachusetts  Institute 
of  Technology.  He  later  conducted  research  in  immunology  at  the  Scripps  Research  Institute  before  moving 
to  the  University  of  Washington.  Dr.  Bevan  was  elected  Fellow  of  the  Royal  Society  of  london. 


TWO  functionally  different  types  of  T  lympho- 
cytes mature  in  the  thymus  and  populate  the 
peripheral  lymphoid  organs.  Helper  T  lympho- 
cytes, the  first  type,  respond  to  antigen  by  releas- 
ing lymphokines,  which  activate  macrophages  or 
augment  the  response  of  antibody-producing  B 
cells.  Cytotoxic  T  lymphocytes,  on  the  other 
hand,  when  induced  with  antigen,  specifically 
lyse  target  cells  expressing  the  antigen  and  re- 
lease interferon-7.  Their  function  is  thought  to  be 
crucial  in  the  response  to  intracellular  patho- 
gens, such  as  viruses  and  bacteria,  and  they  may 
eliminate  some  tumor  cells. 

T  lymphocytes  can  only  recognize  antigen  pre- 
sented in  association  with  cell  surface  glycopro- 
teins encoded  by  the  major  histocompatibility 
complex  (MHC) .  Recent  work  has  shown  that  cy- 
totoxic T  cells  recognize  short  (usually  nona- 
meric)  antigenic  peptides  presented  in  the 
groove  of  class  I  MHC  molecules. 

Since  class  I  molecules  are  expressed  on  vir- 
tually all  tissues,  any  cell  type  can  be  a  target  for 
cytotoxic  cells.  The  peptides  presented  by  these 
MHC  molecules  derive  from  degraded  intracellu- 
lar proteins,  so  all,  or  most,  of  a  cell's  normal 
components  can  provide  class  I-binding  pep- 
tides. Normally,  however,  the  T  cells  tolerate 
these  self-peptide/self-MHC  complexes.  Viral  in- 
fection, on  the  other  hand,  leads  to  the  produc- 
tion of  new  proteins  in  the  cell.  These  may  pro- 
vide nonamer  peptides  that  can  combine  with 
class  I  and  be  presented  on  the  surface.  If  cyto- 
toxic T  lymphocyte  surveillance  works  well, 
these  foreign-peptide/self-MHC  complexes  will 
be  recognized  and  the  virus-infected  cell 
destroyed. 

The  class  I  MHC  proteins  expressed  on  the  cell 
surface  have  three  components:  class  I  heavy 
chain,  (82microglobulin,  and  a  tightly  bound 
peptide.  This  trimolecular  complex  is  assembled 
shortly  after  synthesis  of  the  heavy  and  light 
chains  in  a  pre-Golgi  compartment. 

Many  of  the  peptides  that  are  bound  to  class  I 
actually  derive  from  cytosolic  and  nuclear  pro- 
teins. It  seems  likely  that  cytosolic  proteins  are 
degraded  in  the  cytosol  by  complex  structures 


called  proteosomes  and  that  the  peptide  degrada- 
tion products  pass  into  the  endoplasmic  reticu- 
lum (ER).  Some  of  the  components  of  proteo- 
somes are  encoded  within  the  MHC  in  close 
proximity  to  a  pair  of  genes  that  belong  in  the 
superfamily  of  ATP-dependent  transport  pro- 
teins. These  gene  products  may  mediate  the 
translocation  of  peptide  antigen  from  the  cyto- 
plasm into  the  ER  lumen. 

Antigen  Presentation-Defective  Variants 

A  number  of  mouse  and  human  cell  lines  have 
been  described  that  synthesize  normal  class  I 
heavy  and  /32-microglobulin  chains  but  neither  as- 
semble these  chains  for  surface  expression  nor 
present  endogenous  antigens  to  cytotoxic  T  cells. 
We  provided  evidence  that  the  defect  in  the 
mouse  cell  line  RMA-S  maps  to  chromosome  17, 
the  chromosome  encoding  the  MHC.  When  this 
cell  line  was  fused  with  a  wild-type  partner,  ex- 
pression of  MHC  antigens  was  restored.  When  the 
wild-type  chromosome  17  was  selected  against, 
expression  of  the  MHC  antigens  was  lost.  John 
Monaco  (Virginia  Commonwealth  University, 
Richmond)  had  previously  identified  and  cloned 
Ham  l  and  Ham-2,  two  ABC  transporter  genes 
from  the  mouse  MHC.  In  collaboration  with  his 
group  and  with  James  Forman  and  Kirsten  Fischer 
Lindahl  (HHMI,  University  of  Texas  Southwest- 
ern Medical  Center  at  Dallas),  we  were  able  to 
show  that  transfection  of  the  Ham-2  gene  into 
RMA-S  restored  surface  expression  of  class  I  MHC 
antigens,  as  well  as  the  ability  to  present  five  en- 
dogenous antigens  on  the  surface  for  recognition 
by  cytotoxic  T  lymphocytes.  Thus  a  defect  in  the 
Ham-2  gene  leads  to  loss  of  surface  class  I  ex- 
pression. In  conjunction  with  previous  experi- 
ments with  the  human  defective  cell  lines,  this 
suggests  that  both  Ham- 1  and  Ham-2  may  be  es- 
sential for  class  1  surface  expression,  and  in  fact 
they  may  operate  as  heterodimers. 

In  other  experiments  we  have  shown  that  the 
RMA-S  cell  line  expresses  a  subset  of  endogenous 
peptides  on  the  surface  with  class  1.  For  example, 
the  octameric  peptide  derived  from  the  vesicular 
stomatitis  virus  nucleoprotein  is  presented  to  cy- 


37 


Cytotoxic  T  Lymphocyte  Recognition 


totoxic  T  lymphocytes  by  RMA-S.  After  viral  infec- 
tion or  transfection  of  the  nucleoprotein  gene 
alone,  this  epitope  appears  on  the  cell  surface.  By 
moving  this  sequence  of  eight  residues  around, 
we  hope  to  be  able  to  discover  what  makes  these 
escape  peptides  special — is  it  the  peptides  them- 
selves or  surrounding  sequences?  The  leakiness 
in  this  cell  line  also  suggests  that  a  Ham- 1  homo- 
dimer  may  function  inefficiently  in  this  mutant 
cell  line. 

Cytotoxic  Responses 
to  Intracellular  Bacteria 

Listeria  monocytogenes  is  a  frequent  food- 
borne  pathogen  that  causes  severe  disease  in 
immunocompromised  individuals.  It  is  a  gram- 
positive  facultative  anaerobe.  Following  uptake 
by  macrophages.  Listeria  escapes  from  the  hos- 
tile environment  of  the  phagolysosome  by  secret- 
ing a  hemolysin,  listeriolysin,  which  ruptures  the 
phagosome  membrane.  Once  into  the  cytoplasm, 
the  bacterium  replicates  and  can  push  itself  into  a 
neighboring  cell  by  polymerizing  actin.  Previous 
workers  had  demonstrated  a  cytotoxic  T  cell  re- 
sponse in  mice  to  Listeria  infection.  Our  main 
priority  was  to  determine  which  of  the  approxi- 
mately 4,000  proteins  made  by  Listeria  were 
providing  class  I-associated  peptide  epitopes  for 
cytotoxic  T  lymphocytes. 

We  determined  that  the  secreted  listeriolysin 
molecule  itself  provided  a  strong  class  I-re- 
stricted  epitope  in  the  mice  we  studied.  To  pin- 
point the  presumed  nonamer  within  the  600 
amino  acid  residues  of  the  listeriolysin  molecule, 
we  used  class  I  peptide-binding  motifs  that  had 
predicted  tyrosine  at  position  2  and  leucine  at 
position  9.  The  listeriolysin  contained  three  non- 
amers  that  fit  this  criteria.  These  and  others  were 


made  as  synthetic  peptides,  one  of  which  tar- 
geted our  cytotoxic  T  lymphocytes  at  picomolar 
concentration.  Thus  we  have  used  these  motifs 
for  the  first  time  and  shown  that  they  are  valid  in 
pinpointing  cytotoxic  T  lymphocyte  epitopes. 

In  more  recent  studies  we  have  been  able  to 
show  that  cytotoxic  T  lymphocyte  immunity  to 
this  short  9-residue  peptide  presented  by  a  class  I 
molecule  is  sufficient  to  confer  adoptive  immu- 
nity to  the  whole  bacterium.  Lines  of  cytotoxic  T 
lymphocytes  with  specificity  only  to  this  epi- 
tope, when  transferred  into  syngeneic  mice, 
confer  protection  against  lethal  doses  of  Listeria 
monocytogenes. 

The  listeriolysin  epitope  of  Listeria  that  in- 
duces conventional  cytotoxic  T  cell  immunity  is 
restricted  by  a  classical  class  I  molecule.  "Classi- 
cal" refers  here  to  the  highly  polymorphic  H-2K, 
D,  or  L  loci  in  mice  and  their  equivalent  HLA-A,  B, 
and  C  loci  in  humans.  We  have  evidence  that 
other  peptides  derived  from  this  bacterium  are 
presented  by  nonclassical,  nonpolymorphic  class 
I  molecules.  We  know  that  class  I  is  involved  in 
presenting  these  peptides,  because  cell  lines  that 
lack  j82"r"icrogIobulin  are  unable  to  present  the 
peptides.  Furthermore,  we  know  that  the  class  I 
molecule  maps  just  outside  the  MHC,  as  revealed 
by  experiments  with  congenic  mice  differing 
only  in  this  region. 

But  unlike  the  classical  class  I  molecules,  this 
I'estricting  element  is  rather  nonpolymorphic: 
i.e.,  cells  from  most  mouse  strains  are  able  to 
bind  and  present  the  peptide.  We  have  yet  to 
identify  the  peptide  derived  from  Listeria  or  the 
class  I  molecule  involved.  It  will  be  interesting  in 
the  future  to  determine  whether  cytotoxic  T  lym- 
phocytes directed  to  these  peptides,  seen  in  the 
context  of  nonpolymorphic  MHC  molecules, 
confer  protective  immunity. 


38 


Vascular  Endothelium  in  Inflammation 
and  Metastasis 


Michael  P.  Bevilacqua,  M.D.,  Ph.D.  — Associate  Investigator 

Dr.  Bevilacqua  is  also  Associate  Professor  of  Pathology  and  a  member  of  the  division  of  Cellular  and 
Molecular  Medicine  at  the  University  of  California,  San  Diego.  He  received  his  undergraduate  degree  in 
biology  from  the  University  of  Pennsylvania  and  his  M.D.  and  Ph.D.  degrees  in  pathology  from  the  State 
University  of  New  York  at  Brooklyn.  As  a  postdoctoral  research  fellow  with  Michael  Gimbrone,  Jr.,  and 
Ramzi  Cotran  at  Brigham  and  Women 's  Hospital  and  Harvard  Medical  School,  he  initiated  work  on 
endothelial  leukocyte  adhesion  molecules.  He  continued  these  studies  as  a  faculty  member  at  Harvard 
Medical  School  before  assuming  his  present  position.  Dr.  Bevilacqua  is  a  Pew  Scholar  in  the  Program  in 
the  Biomedical  Sciences. 


VASCULAR  endothelial  cells  form  a  cobble- 
stone-like lining  of  blood  vessels  throughout 
the  body.  Despite  accurate  predictions  by  certain 
investigators  in  the  18th  century,  the  highly  re- 
sponsive and  changeable  nature  of  vascular  endo- 
thelium was  not  recognized  until  the  early 
1980s.  Endothelium  was  widely  regarded  as  a 
"nonstick"  surface  that  prevented  blood-clotting 
and  resisted  the  adhesion  of  circulating  leuko- 
cytes. It  is  now  apparent  that  the  simple  struc- 
tural features  of  vascular  endothelium  belie  a 
complex  functional  nature. 

Our  research  has  focused  on  the  mechanisms 
by  which  the  vascular  endothelium  can  regulate 
inflammation,  immunity,  thrombosis,  and  tumor 
metastasis.  It  was  demonstrated  that  soluble  pro- 
tein mediators  known  as  cytokines  could  act  di- 
rectly on  endothelial  cells  to  increase  the  expres- 
sion of  prothrombotic  activities  and  promote  the 
adhesion  of  blood  leukocytes.  Investigation  into 
the  mechanisms  of  leukocyte-endothelial  adhe- 
sion led  to  the  identification  and  characterization 
of  two  endothelial  cell-surface  glycoproteins  that 
were  designated  endothelial  leukocyte  adhesion 
molecule- 1  (ELAM-1)  and  inducible  cell  adhe- 
sion molecule- 110  (INCAM-110).  This  discus- 
sion focuses  on  endothelial  adhesion  molecules 
and  their  potential  roles  in  human  disease 
processes. 

Endothelial  Adhesion  Molecules 
in  Inflammation 

The  study  of  inflammation  has  one  of  the 
richest  histories  in  biomedical  research.  Particu- 
larly noteworthy  is  the  work  of  Julius  Conheim 
(1839-1884),  which  provided  one  of  the  first 
(and  best)  microscopic  descriptions  of  the  pro- 
cess. He  carefully  observed  blood  vessels  after  a 
local  injury  in  transparent  membranes  such  as  the 
tongue  of  a  frog  and  described  the  vasodilation, 
edema,  and  leukocyte  emigration  with  great  accu- 
racy. He  also  suggested  that  the  blood  vessel  lin- 
ing in  the  area  of  inflammation  became  sticky  for 
leukocytes.  This  concept  was  largely  lost  during 
the  next  century,  in  which  a  theory  of  leukocyte 


response  to  soluble  mediators  (chemotactic  fac- 
tors) dominated. 

Until  the  early  1980s  endothelial  cells  were 
thought  to  move  politely  aside  or  be  destroyed  as 
leukocytes  exited  blood  vessels  to  fight  bacterial 
infections  or  contain  foreign  substances.  By  1985 
the  view  of  vascular  endothelium  had  begun  to 
change.  Recombinant  cytokines  were  found  to 
act  directly  on  endothelial  cells  to  increase  dra- 
matically the  adhesion  of  blood  leukocytes,  and  it 
became  apparent  that  the  endothelium  produces 
a  "sticky"  surface.  By  1987  the  nature  of  that 
surface  was  being  elucidated,  with  the  identifica- 
tion of  two  cytokine-inducible  endothelial  adhe- 
sion molecules  designated  ELAM-1  and  ICAM-1 
(intracellular  adhesion  molecule-1).  Studies  on 
human  tissues  demonstrated  that  these  mole- 
cules are  expressed  on  endothelium  at  sites  of 
inflammation. 

A  solid  working  hypothesis  emerged:  foreign 
substances  such  as  bacterial  products  introduced 
into  tissues  would  stimulate  local  cells  such  as 
macrophages  to  synthesize  and  secrete  cytokines. 
The  cytokines  would  then  act  on  vascular  endo- 
thelium to  promote  the  expression  of  adhesion 
molecules  that  would  bind  passing  leukocytes. 
Leukocytes  could  then  exit  the  blood  vessel  and 
migrate  toward  the  foreign  substances  following 
chemotactic  gradients.  By  1990,  through  the  ef- 
forts of  multiple  laboratories,  five  endothelial 
cell-surface  molecules  that  participate  in  leuko- 
cyte adhesion  and  inflammation  had  been  identi- 
fied, cloned,  and  characterized.  These  molecules 
fall  into  two  families  based  on  their  structures. 

A  newly  described  family,  the  vascular  selec- 
tins,  contains  three  related  molecules,  two  of 
which  can  be  found  on  endothelial  cells:  E- 
selectin  (a  new  name  for  ELAM-1 )  and  P-selectin 
(previously  known  as  GMP-140  or  PADGEM). 
The  second  group  of  endothelial  leukocyte  adhe- 
sion molecules  are  part  of  a  much  larger  family 
known  as  the  immunoglobulin  superfamily. 

The  pattern  of  expression  of  endothelial  adhe- 
sion molecules  as  well  as  the  specificity  of  their 
binding  interactions  with  leukocytes  contributes 

39 


Vascular  Endothelium  in  Inflammation  and  Metastasis 


importantly  to  the  control  of  inflammatory  pro- 
cesses. Acute  inflammation  is  of  relatively  short 
duration,  lasting  as  little  as  a  few  minutes  or  as 
long  as  1  -  2  days.  It  is  characterized  by  the  exuda- 
tion of  plasma  proteins  and  the  emigration  of  leu- 
kocytes, especially  neutrophils.  Chronic  inflam- 
mation is  of  longer  duration  and  is  associated 
with  the  extravasation  of  blood  lymphocytes  and 
macrophages  and  with  the  morphological  changes 
in  blood  vessels  and  connective  tissues. 

In  most  cases  the  pattern  of  E-selectin  expres- 
sion correlates  well  with  acute  inflammation. 
This  observation  is  consistent  with  the  demonstra- 
tion that  E-selectin  supports  the  adhesion  of  es- 
sentially all  neutrophils,  but  only  a  small  portion 
of  lymphocytes.  In  contrast,  INCAM-1 10  (known 
also  as  VCAM-1)  supports  the  adhesion  of  lym- 
phocytes but  not  neutrophils,  and  its  pattern  of 
expression  is  consistent  with  its  primary  role  in 
chronic  inflammatory  processes. 

The  inflammatory  process  is  essential  for  host 
defense.  In  certain  settings,  however,  it  can  also 
contribute  to  debilitating  and  life-threatening 
diseases.  Examples  of  human  diseases  with  signifi- 
cant inflammatory/immunological  components 
are  adult  respiratory  distress  syndrome  (ARDS), 
myocardial  reperfusion  injury,  rheumatoid  ar- 
thritis, and  various  autoimmune  diseases.  Un- 
derstanding the  mechanisms  of  endothelial- 
leukocyte  adhesion  may  allow  us  to  enhance  our 
defense  mechanisms,  or  dampen  their  responses 
when  appropriate. 

Vascular  Selectins  and  Their 
Carbohydrate  Ligands 

Each  of  the  three  selectins  is  a  cell-surface  gly- 
coprotein with  a  mosaic  structure.  The  portion  of 
the  molecule  that  is  external  to  the  cell,  and 
hence  reaches  out  into  the  bloodstream,  contains 
a  lectin  domain.  Lectins  are  protein  molecules 
that  bind  to  carbohydrates  (sugars) .  Our  labora- 
tory is  utilizing  the  recombinant  E-  and  P-selectin 
proteins  and  soluble  synthetic  carbohydrates  to 
determine  the  structures  involved  in  recognition. 

Early  studies  had  suggested  that  E-selectin  me- 
diates the  adhesion  of  blood  leukocytes  through 
the  binding  of  a  carbohydrate  known  as  sialyl- 
Lewis  X.  This  structure  contains  a  terminal 
sialic  acid  as  well  as  a  fucose  group  bound  to 
a  carbohydrate  backbone  of  galactose  and  A'^ 
acetylglucosamine.  Surprisingly,  we  demon- 
strated that  a  soluble  form  of  sialyl-Lewis  X  was 
only  a  modest  inhibitor  of  E-selectin-dependent 
adhesion,  whereas  a  related  carbohydrate  called 


sialyl-Lewis  A,  difi'ering  only  in  the  linkage  of  the 
backbone  sugars,  was  much  more  effective.  Sialyl- 
Lewis  A  is  not  found  on  blood  neutrophils  but  is 
expressed  by  a  variety  of  cancers,  including  those 
of  the  colon.  The  relative  blocking  activities  of 
sialyl-Lewis  X  and  sialyl-Lewis  A  were  reversed 
for  P-selectin. 

In  addition,  a  third  carbohydrate  compound 
designated  CD65,  which  is  found  on  leukocytes 
and  some  cancer  cells,  was  shown  to  be  an  efl'ec- 
tive  blocker  of  adhesion  to  both  E-  and  P-selectin. 
Subsequent  studies  using  modified  carbohydrates 
have  demonstrated  that  the  presence  and  precise 
arrangement  of  sialic  acid  and  fucose  are  impor- 
tant in  the  recognition  by  the  two  selectins.  Fur- 
thermore, minor  modifications  in  the  carbohy- 
drate structures  can  result  in  greatly  increased 
activity  in  blocking  leukocyte  adhesion. 

A  Possible  Role  in  Cancer  Metastasis 

If  cancers  grew  and  invaded  tissues  locally  but 
failed  to  metastasize,  most  of  them  could  be 
cured  by  surgery.  Indeed,  the  lethal  effects  of  a 
cancer  are  generally  ascribed  to  its  metastatic 
abilities.  Hematogenous  metastasis  involves  re- 
lease of  cells  from  a  primary  tumor  into  blood 
vessels  and  transport  to  new  tissues.  Substantial 
evidence  indicates,  however,  that  the  blood  is 
hostile  to  cancer  cells  and  generally  destroys 
them.  Thus  most  cancer  cells  must  exit  the 
bloodstream  in  order  to  survive.  Our  laboratory 
has  focused  on  the  mechanisms  by  which  tumor 
cells  bind  the  vessel  wall  and  extravasate.  We 
now  appreciate  that  many  tumor  cells  can  inter- 
act with  the  vascular  endothelium  through  the 
same  molecules  blood  leukocytes  use. 

As  noted  above,  colon  cancers  can  express  car- 
bohydrate ligands  for  E-selectin.  In  a  parallel  line 
of  investigation,  we  have  demonstrated  that  hu- 
man melanomas  can  express  the  integrin  a4l3l, 
which  is  a  receptor  for  the  endothelial  cell- 
surface  molecule  INCAM-1 10  or  VCAM-1.  These 
observations  suggest  the  possibility  of  novel  ther- 
apeutic approaches  to  block  the  metastatic 
spread  of  cancer. 

In  summary,  vascular  endothelium  can  exist  in 
different  functional  states.  These  states  are  deter- 
mined by  stimuli  such  as  cytokines  and  bacterial 
products.  Activated  endothelial  cells  express  ad- 
hesion molecules  for  circulating  leukocytes  and 
can  thereby  regulate  inflammatory  processes.  In 
addition,  it  appears  that  cancer  cells  can  utilize 
these  same  adhesion  molecules  during  the  pro- 
cess of  hematogenous  metastasis. 


40 


Structural  Studies  of  Molecules  Involved 
in  the  Immune  Recognition  of  Infected  Cells 


Pamela  J.  Bjorkman,  Ph.D. — Assistant  Investigator 

Dr.  Bjorkman  is  also  Assistant  Professor  in  the  Division  of  Biology  at  the  California  Institute  of 
Technology  and  Adjunct  Professor  of  Biochemistry  at  the  University  of  Southern  California  School  of 
Medicine,  Los  Angeles.  She  received  a  B.A.  degree  in  chemistry  from  the  University  of  Oregon  and  a  Ph.D. 
degree  in  biochemistry  and  molecular  biology  from  Harvard  University,  where  her  thesis  advisor  was  Don 
Wiley.  She  completed  a  low-resolution  structure  of  a  human  histocompatibility  molecule  for  her  thesis 
and  then  stayed  on  to  finish  the  work.  She  continued  her  postdoctoral  training  at  Stanford  University  in 
the  laboratory  of  Mark  Davis  before  joining  the  staff  at  Caltech.  Dr.  Bjorkman  received  the  William  B. 
Coley  Award  for  Distinguished  Research  in  Fundamental  Immunology  from  the  Cancer  Research  Institute. 


THE  technique  of  x-ray  crystallography  allows 
visualization  of  the  three-dimensional  struc- 
tures of  proteins  in  atomic  detail.  In  other  words, 
we  get  a  picture  of  the  protein  that  shows  the 
location  of  all  the  atoms  and  how  they  interact. 
The  shape  of  a  protein  and  the  location  of  individ- 
ual atoms  with  respect  to  one  another  are  impor- 
tant for  determining  how  the  protein  functions. 
With  such  knowledge  it  is  often  possible  to  de- 
sign compounds  that  modify  the  protein  for  medi- 
cal intervention. 

The  proteins  that  we  are  studying  structurally 
are  those  that  mediate  the  immune  response 
against  viruses  and  other  pathogens.  The  immune 
system  has  evolved  so  that  highly  specific  mole- 
cules on  the  surfaces  of  lymphocytes  can  recog- 
nize a  virally  infected  cell.  In  the  infected  cell, 
pieces  of  viral  proteins  are  fragmented  and  bound 
to  a  cellular  protein  called  a  histocompatibility 
molecule.  If  the  complex  formed  between  the 
histocompatibility  molecule  and  the  viral  frag- 
ment is  recognized  by  a  protein  on  the  lympho- 
cyte, the  infected  cell  is  destroyed.  The  lympho- 
cytes that  bear  the  recognizing  proteins  are  T 
cells,  and  the  proteins  on  their  surface  are  T  cell 
receptors. 

The  three-dimensional  structure  of  a  histocom- 
patibility protein  gives  us  a  picture  of  how  and 
where  viral  molecules  bind  and  how  T  cell  recep- 
tors might  bind  to  the  complex  formed  between 
the  viral  and  histocompatibility  molecules.  My 
laboratory  now  seeks  to  determine  a  three- 
dimensional  structure  for  a  T  cell  receptor,  in 
order  to  understand  the  atomic  details  of  its  inter- 
action with  the  surface  of  an  infected  cell.  (This 
work  is  being  done  in  collaboration  with  Mark 
Davis,  HHMI,  Stanford  University.)  We  have  crys- 
tallized a  T  cell  receptor  and  now  seek  to  make 
crystals  of  a  complex  between  a  T  cell  receptor 
and  histocompatibility  molecule.  An  understand- 
ing of  how  T  cell  receptors  interact  with  histo- 
compatibility molecules  complexed  to  viral  frag- 
ments should  increase  our  understanding  of  how 
the  immune  system  distinguishes  normal,  healthy 


cells  from  virally  infected  cells  that  need  to  be 
destroyed. 

Our  laboratory  is  also  using  protein  expression 
systems  in  mammalian  and  bacterial  cells  to  pro- 
duce the  large  quantities  of  proteins  necessary  for 
crystallization  and  structural  studies.  Using  mo- 
lecular biological  techniques,  it  is  possible  to 
transfect  a  protein-encoding  gene  into  a  cell  in 
which  it  is  not  normally  found,  thus  persuading 
that  cell  to  manufacture  that  protein.  Many  pro- 
teins normally  occur  in  small  quantities;  the  use 
of  such  protein  expression  systems  allows  the 
isolation  of  much  more  of  the  protein  than  would 
be  otherwise  possible.  We  have  expressed  histo- 
compatibility proteins  in  bacteria  and  formed 
complexes  between  these  proteins  and  antigenic 
peptides. 

We  have  also  used  a  similar  system  to  make  an 
Fc  receptor,  a  protein  that  binds  to  the  Fc  portion 
of  antibody  molecules.  This  particular  Fc  recep- 
tor is  found  in  the  intestine  of  newborn  mammals 
and  binds  immunoglobulin  found  in  mother's 
milk,  thus  transferring  partial  immunity  from 
mother  to  infant.  The  amino  acid  sequence  and 
structural  organization  of  this  molecule  show  sim- 
ilarities to  histocompatibility  molecules.  We 
have  made  a  soluble  (non-membrane-bound) 
version  of  this  molecule,  which  is  still  capable  of 
binding  to  Fc  molecules.  We  have  recently  puri- 
fied and  crystallized  this  Fc  receptor  and  initiated 
a  three-dimensional  structure  determination.  We 
have  also  crystallized  a  complex  between  the  re- 
ceptor and  the  Fc  portion  of  an  antibody;  this  will 
ultimately  allow  us  to  obtain  a  picture  of  how  the 
two  proteins  interact.  A  comparison  of  the  Fc  re- 
ceptor structure  and  its  interaction  with  antibod- 
ies should  increase  our  understanding  of  the 
structurally  related  histocompatibility  proteins 
and  perhaps  reveal  reasons  for  the  evolution  of 
this  type  of  structure  in  the  immune  system.  We 
also  have  crystals  of  a  structurally  unrelated  Fc 
receptor  and  are  interested  in  comparing  how  the 
two  types  of  receptors  bind  to  their  common 
ligand. 


41 


Mechanisms  of  Insulin  Action 


Perry  J.  Blackshear,  M.D.,  D.Phil. — Investigator 

Dr.  Blackshear  is  also  Professor  of  Medicine  and  Assistant  Professor  of  Biochemistry  at  Duke  University 
Medical  Center.  He  received  his  D.Phil,  degree  in  biochemistry  from  Trinity  College,  Oxford  University, 
and  his  M.D.  degree  from  Harvard  Medical  School.  Before  moving  to  Duke  University,  he  was  Assistant 
Professor  of  Medicine  at  Harvard  Medical  School.  Dr.  Blackshear  has  received  the  Young  Investigator 
Award  for  Clinical  Research  from  the  American  Federation  for  Clinical  Research. 


OUR  laboratory  is  mainly  interested  in  the  mo- 
lecular mechanisms  of  action  of  insulin  and 
polypeptide  growth  factors.  The  studies  involv- 
ing insulin  action  are  particularly  relevant  to 
common  clinical  disorders  of  insulin  resistance, 
such  as  type  II  (adult-onset)  diabetes  and  obesity, 
in  which  the  locus  of  the  resistance  is  thought  to 
be  at  a  "postreceptor"  step  within  the  cells  of 
muscle,  liver,  and  adipose  tissue.  Our  work  is 
aimed  at  understanding  the  biochemical  steps  in- 
volved in  insulin  action  in  these  tissues,  with  the 
ultimate  hope  of  identifying  the  abnormal  steps 
in  these  insulin-resistant  states  and  possibly  using 
this  knowledge  to  develop  novel  drugs  aimed  at 
correcting  the  abnormalities. 

Another  major  area  of  interest  is  the  molecular 
steps  involved  in  mediating  the  effects  of  a  wide 
variety  of  hormones,  neurotransmitters,  and 
drugs  on  their  target  cells.  The  common  denomi- 
nator of  these  agents  is  that  the  mechanism  of 
action  involves  the  stimulated  breakdown  of  cer- 
tain membrane  lipid  compounds,  leading  to  the 
generation  of  intracellular  lipid  mediators  known 
as  diacylglycerols.  These  in  turn  can  activate  an 
important  cellular  enzyme,  protein  kinase  C  (PKC). 

Over  the  past  several  years,  a  large  amount  of 
information  has  accumulated  about  the  way  in 
which  hormones  stimulate  the  breakdown  of 
these  membrane  lipids  and  about  the  molecular 
biology  and  biochemistry  of  the  PKC  family  of 
enzymes.  However,  almost  nothing  is  yet  known 
about  the  proteins  that  this  kinase  phosphorylates 
in  cells  and  tissues,  or  about  the  involvement  of 
these  phosphorylated  proteins  in  mediating  a  vari- 
ety of  cellular  effects.  To  understand  the  role  of 
these  PKC  substrates  in  the  cell  is  a  major  goal  of 
our  laboratory.  Within  the  past  year  we  have 
made  several  advances  in  each  of  these  areas. 

With  regard  to  signal  transduction,  we  are  in- 
vestigating the  molecular  nature  of  a  family  of 
PKC  substrates  known  by  the  acronym  MARCKS 
(myristoylated  alanine-rich  C-kinase  substrates). 
These  are  widely  distributed  cellular  proteins 
that  are  phosphorylated  within  seconds  of  PKC 
activation  in  intact  cells,  and  it  is  presumed  that 
this  phosphorylation  serves  some  function  in  me- 


diating the  effects  of  the  activated  kinase.  Our 
group  has  established  that  these  proteins  are  ex- 
cellent substrates  for  PKC,  and  that  phosphoryla- 
tion leads  to  changes  in  their  properties  that  may 
influence  their  behavior  in  the  cell.  For  example, 
phosphorylation  of  the  protein  disrupts  its  associ- 
ation with  model  cellular  membranes,  perhaps 
allowing  for  phosphorylation-dependent  changes 
in  the  protein's  intracellular  location. 

Similarly,  phosphorylation  of  the  protein 
disrupts  its  ability  to  form  a  tight  complex  with 
calmodulin,  the  ubiquitous  calcium-dependent 
regulator  of  a  wide  variety  of  cellular  enzymes. 
We  postulate  that  such  disruption  leads  to  an  in- 
crease in  the  cellular  content  of  free  calmodulin, 
which  might  then  lead  to  concomitant  activation 
of  calmodulin-sensitive  enzymes.  In  this  way 
phosphorylation  of  the  MARCKS  protein  and  its 
relatives  by  PKC  might  lead  to  increases  in  the 
activity  of  calcium/calmodulin-dependent  pro- 
cesses, a  synergistic  relationship  that  has  long 
been  postulated  but  not  proved. 

The  overall  goal  of  these  studies  is  to  try  to 
determine  the  role  that  the  MARCKS  protein  fam- 
ily plays,  if  any,  in  mediating  the  actions  of  PKC 
in  various  tissues.  One  important  series  of  studies 
now  under  way  involves  attempts  to  render  cells 
and  intact  animals  deficient  in  these  proteins,  us- 
ing the  techniques  of  antibody  microinjection, 
antisense  RNA  expression,  and  ultimately  gene 
disruption.  It  is  hoped  that  within  the  next  year 
or  two,  such  deficient  cells  and  animals  will  be 
available  to  study  the  potential  role  of  this  family 
of  proteins  in  development,  particularly  of  the 
nervous  system,  in  which  the  MARCKS  proteins 
and  their  relatives  are  highly  expressed. 

Considerable  progress  has  also  been  made  in 
the  studies  of  the  molecular  mechanisms  of  insu- 
lin action.  In  one  group  of  studies  involving  two 
genes  whose  transcription  is  rapidly  stimulated 
by  insulin,  we  have  made  progress  in  identifying 
regions  of  the  DNA  that  are  implicated  in  the  in- 
sulin reaction.  We  have  also  identified  proteins 
that  bind  to  these  regions  and  have  demonstrated 
that  they  are  modified  in  some  way  by  rapid  insu- 
lin exposure  of  the  cells.  Current  studies  in  the 
laboratory  are  attempting  to  clone  and  sequence 


43 


Mechanisms  of  Insulin  Action 


these  binding  proteins  and  to  determine  the  na- 
ture of  the  insulin-stimulated  modification. 

Another  group  of  studies  involves  the  rapid 
insulin-stimulated  translation  of  messenger  RNA, 
using  the  enzyme  ornithine  decarboxylase  in  a 
rnodel  system.  Within  the  past  year,  we  have  de- 
termined that  insulin  can  rapidly  stimulate  the 
translation  of  certain  types  of  mRNA  that  contain 
extremely  extensive  and  convoluted  secondary 
structures  at  their  5'-untranslated  ends.  We  have 
proposed  that  insulin  does  this  by  means  of  a  spe- 
cific ability  to  activate  translation  initiation  fac- 
tors that  can  unwind  or  "melt"  the  RNA  second- 
ary structure,  and  have  determined  that  insulin 
can  rapidly  stimulate  the  phosphorylation  and 


presumably  the  activation  of  these  initiation  fac- 
tors in  intact  cells. 

Current  studies  are  focusing  on  the  protein  ki- 
nases and  phosphatases  involved  in  this  initiation 
factor  phosphorylation,  as  well  as  on  other  rap- 
idly stimulated,  insulin-activated  protein  kinases 
that  could  play  a  role  in  the  phosphorylation  of 
the  transcription  factors  alluded  to  above.  In  all 
of  these  cases,  the  ultimate  goal  is  the  elucidation 
of  the  biochemical  steps  between  insulin  binding 
to  its  receptor  and  its  ultimate  intracellular  ef- 
fect, such  as  gene  transcription,  with  the  hope 
that  steps  in  this  pathway  that  are  abnormal  in 
states  of  insulin  resistance  can  be  identified,  lead- 
ing to  the  development  of  specific  therapies. 


44 


Intracellular  Protein  Traffic 
and  Nuclear  Organelles 


Gunter  Blobel,  M.D.,  Ph.D. — Investigator 

Dr.  Blobel  is  also  Professor  of  Cell  Biology  at  the  Rockefeller  University.  He  received  bis  M.D.  degree  from 
the  University  of  Tiibingen  and  his  Ph.D.  degree  with  Van  Potter  in  oncology  from  the  McArdle  Laboratory 
at  the  University  of  Wisconsin-Madison.  Thereafter,  he  did  postdoctoral  work  with  George  Palade  at 
Rockefeller.  Dr.  Blobel  is  a  member  of  the  National  Academy  of  Sciences  and  of  several  other  distinguished 
societies.  He  has  received  many  honors,  including  the  Gairdner  Foundation  Award. 


NUMEROUS  structurally  and  functionally  di- 
verse proteins  can  be  translocated  across  a 
few  distinct  cellular  membranes.  It  is  now  estab- 
lished that  targeting  to  these  membranes  and 
translocation  across  them  is  specified  by  a  mem- 
brane-specific "signal"  sequence  that  is  a  tran- 
sient (or  permanent)  part  of  the  protein  to  be 
translocated.  The  primary  structure  of  numerous 
representatives  for  such  a  sequence  has  been  es- 
tablished in  our  laboratory  and  by  others.  Present 
work  focuses  on  the  identification  and  character- 
ization of  machinery  involved  in  the  recognition 
of  a  signal  sequence,  in  its  targeting  to  the  proper 
membrane,  and  in  protein  translocation.  Collec- 
tively, these  components  comprise  what  has  been 
termed  a  protein  translocon. 

Our  laboratory  is  working  on  translocons  for 
four  distinct  cellular  entities:  1 )  the  endoplasmic 
reticulum  (ER)  of  animal  (and  yeast)  cells, 
which  is  able  to  translocate  proteins  from  the  cy- 
tosol  to  the  ER  lumen;  2)  bacterial  plasma  mem- 
branes (gram-negative  bacteria),  able  to  translo- 
cate proteins  from  the  cytoplasm  to  the 
periplasmic  space;  3)  yeast  mitochondria,  able  to 
translocate  protein  from  the  cytoplasm  to  the  mi- 
tochondrial interior  ("matrix")  across  outer  and 
inner  mitochondrial  membranes;  and  4)  plant 
cell  (pea  and  spinach)  chloroplasts,  able  to  trans- 
locate proteins  from  the  cytoplasm  to  the  chloro- 
plast  interior  ("stroma")  across  outer  and  inner 
chloroplast  membranes. 

From  studies  on  these  four  translocons  so  far, 
but  especially  from  studies  on  the  ER  translocon, 
it  is  likely  that  a  translocon  is  composed  of  at  least 
four  entities:  1)  a  soluble  signal-recognition 
factor  (SRF),  2)  a  homing  receptor,  3)  a  signal 
sequence-gated  protein-conducting  channel, 
and  4)  a  signal-removing  peptidase.  The  SRF  has 
two  functions:  recognition  of  the  signal  sequence 
and  targeting  to  the  homing  receptor,  which  is 
restricted  in'  its  localization  to  a  translocon- 
specific  membrane.  Interaction  of  the  signal  se- 
quence-SRF  complex  with  the  homing  receptor 
leads  to  dissociation  of  the  signal  sequence  and 
its  presentation  to  a  signal  sequence  receptor, 
which  might  be  a  subunit  of  the  protein- 
conducting  channel.  This  channel  would  close 


immediately  following  completion  of  transloca- 
tion, only  to  open  again  after  presentation  of  an- 
other signal  sequence.  The  signal  peptidase 
would,  in  most  cases,  remove  the  signal  sequence 
either  during  or  shortly  after  translocation. 

In  the  case  of  the  ER,  the  SRF  was  isolated  and 
shown  to  be  a  ribonucleoprotein  particle.  Re- 
ferred to  as  a  signal-recognition  particle  (SRP), 
this  consists  of  one  7S  RNA  molecule  and  six  dif- 
ferent proteins.  Likewise,  a  homing  receptor  (re- 
ferred to  as  the  SRP  receptor)  and  a  signal -remov- 
ing peptidase,  a  complex  of  five  proteins,  were 
isolated.  More  recently  we  were  able  to  demon- 
strate the  existence  of  a  protein-conducting 
channel,  using  electrophysiological  methods. 
The  protein  constituents  of  this  channel  remain 
to  be  identified.  We  have  been  able  to  solubilize 
the  ER  membranes  by  detergent  and  to  reconsti- 
tute translocation-competent  vesicles.  Using  this 
method,  it  should  be  possible  to  identify  the 
channel  proteins. 

We  recently  identified  components  of  other 
translocons.  An  SRF  was  isolated  for  signal  se- 
quence targeted  to  the  bacterial  plasma  mem- 
brane. Moreover,  we  identified  signal  sequence- 
binding  subunits  for  the  mitochondrial  and 
chloroplast  translocons.  These  integral  mem- 
brane proteins  are  located  in  contact  sites  be- 
tween outer  and  inner  organelle  membranes  and 
are  candidates  for  subunits  of  a  protein-conduct- 
ing channel  in  the  outer  organelle  membrane 
linked  to  a  protein-conducting  channel  in  the  in- 
ner membrane. 

Our  other  major  research  effort  focuses  on  the 
organelles  associated  with  the  cell's  nuclear  en- 
velope membranes.  These  organelles  are  thought 
to  organize  the  large  amount  of  information  in 
the  linear  structure  of  the  DNA  into  numerous 
structurally  and  functionally  distinct  three- 
dimensional  superstructures,  allowing  only  a  lim- 
ited amount  of  that  information  to  be  expressed. 
Characterization  of  these  organelles  should  ad- 
vance understanding  of  such  fundamental  pro- 
cesses as  differential  gene  expression,  cell  differ- 
entiation, and  development. 

Our  efforts  focus  on  the  structural  and  func- 
tional characterization  of  two  morphologically 


45 


Intracellular  Protein  Traffic  and  Nuclear  Organelles 


distinct  structures.  One,  the  nuclear  pore  com- 
plex (NPC),  is  located  in  the  nuclear  envelope. 
We  speculate  that  this  organelle  is  involved  in 
"gene  gating";  i.e.,  each  pore  complex  is  at- 
tached to  a  set  of  actively  transcribed  genes.  We 
have  identified  and  isolated  several  proteins  of 
the  NPC  of  mammalian  cells.  So  far  we  have  es- 
tablished the  primary  structure  of  3  of  the  more 
than  100  proteins  of  this  large  organelle.  Re- 
cently we  succeeded  in  isolating  NPCs  from 
yeast.  These  genetically  amenable  cells  should  fa- 
cilitate the  structural  and  functional  analysis  of 
the  many  NPC  proteins.  We  have  also  established 
the  primary  structure  of  two  membrane  proteins 
that  are  specifically  located  in  the  pore  mem- 
brane domain  of  the  nuclear  envelope  mem- 
brane. We  are  using  cell-free  systems  for  protein 
uptake  into  the  nucleus  to  isolate  the  hypotheti- 
cal SRF,  the  homing  receptor,  and  the  so-called 
transporter  of  the  pore  complex. 

The  other  morphologically  distinct  structure 
associated  with  the  nuclear  envelope  that  we  are 
studying  is  the  nuclear  lamina,  a  fibrous  mesh- 
work  associated  with  the  nuclear  side  of  the  inner 
nuclear  envelope  membrane.  The  lamina  consists 
of  three  proteins,  which  we  have  termed  lamins 
A,  B,  and  C.  We  have  speculated  that  the  nuclear 
lamina  is  involved  in  the  three-dimensional  orga- 
nization of  nontranscribed  chromatin. 


By  molecular  cloning  and  cDNA  sequencing  of 
the  three  lamins,  we  and  others  showed  recently 
that  these  proteins  are  members  of  the  interme- 
diate filament  protein  family.  We  demonstrated 
that  lamin  B  binds  to  the  carboxyl-terminal  por- 
tion of  cytoplasmic  intermediate  filament  pro- 
teins and  that  ankyrin,  a  protein  associated 
with  the  plasma  membrane,  binds  to  the  amino- 
terminal  portion  of  cytoplasmic  intermediate  fila- 
ment proteins.  Recently  we  have  investigated  the 
interaction  of  such  a  protein  with  nuclear  lamin  B 
in  more  detail  and  have  localized  the  interacting 
regions  to  the  near  carboxyl-terminal  segment  of 
these  proteins.  Interestingly,  a  synthetic  peptide, 
representing  this  lamin  B-binding  site  of  the  cy- 
toplasmic intermediate  filament  protein,  when 
microinjected  into  cells,  led  to  the  detachment  of 
intermediate  filaments  from  the  nucleus. 

These  data  indicate  a  direct  connection  be- 
tween the  plasma  membrane  skeleton  (ankyrin) , 
the  cytoskeleton  (intermediate  filaments),  and 
the  peripheral  nucleoskeleton  (lamina)  through 
the  NPC.  Moreover,  we  recently  identified  a  la- 
min B  receptor  in  the  inner  nuclear  membrane. 
Its  primary  structure  has  been  determined  by  mo- 
lecular cloning  and  cDNA  sequencing.  This  re- 
ceptor has  a  number  of  interesting  sites,  suggest- 
ing that  it  may  not  only  interact  with  lamin  B  but 
also  with  DNA. 


46 


Immunity  and  Pathogenesis  of  Third  World 
Diseases:  Leprosy  and  Tuberculosis 


Barry  R.  Bloom,  Ph.D. — Investigator 

Dr.  Bloom  is  also  Weinstock  Professor  of  Microbiology  and  Immunology  at  Albert  Einstein  College  of 
Medicine.  He  received  his  B.A.  degree  and  an  honorary  Sc.D.  degree  from  Amherst  College  and  his  Ph.D. 
degree  from  the  Rockefeller  University.  He  is  active  as  an  advisor  to  the  World  Health  Organization  in  the 
areas  of  tropical  diseases  and  vaccine  development.  Dr.  Bloom  also  serves  on  the  Board  of  Science 
and  Technology  in  Development  of  the  U.S.  National  Research  Council  and  the  National  Vaccine 
Advisory  Committee.  He  is  a  member  of  the  National  Academy  of  Sciences,  the  Institute  of  Medicine, 
and  the  American  Academy  of  Arts  and  Sciences. 


THE  commitment  of  our  laboratory  is  to  inves- 
tigate basic  scientific  problems  that  have  par- 
ticular relevance  for  health  in  the  Third  World. 
Three-quarters  of  the  world's  population  lives  in 
the  Third  World,  and  one-fourth  of  that  popula- 
tion suffers  from  malnutrition  and  disease.  The 
premises  of  our  research  are  that  the  advances  in 
molecular  biology  and  immunology  have  a  great 
deal  to  offer  for  understanding  infectious  diseases 
afflicting  people  in  developing  countries  and,  re- 
ciprocally, that  the  study  of  some  of  those  dis- 
eases can  provide  insights  into  fundamental  im- 
munological and  pathogenetic  mechanisms  of 
relevance  to  people  in  the  industrialized  world. 

The  Importance  of  Mycobacterial  Diseases 

Tuberculosis  and  leprosy  are  both  caused  by 
mycobacteria.  Leprosy  afflicts  4-6  million  peo- 
ple in  the  world  and  produces  deformity  in  30 
percent  if  untreated.  Throughout  time  and  in  all 
cultures,  leprosy  has  engendered  a  unique  fear 
and  stigma.  Although  Mycobacterium  /eprae  was 
the  first  major  human  bacterial  pathogen  de- 
scribed, it  remains  one  of  the  few  that  has  never 
been  cultivated  in  the  test  tube.  However,  its  an- 
tigens can  be  produced  and  studied  vicariously  in 
Escherichia  coli  by  means  of  recombinant  DNA 
technology. 

Tuberculosis  is  the  major  cause  of  death  from  a 
single  infectious  disease  in  the  world  today.  Each 
year  there  are  8  million  new  cases  of  tuberculosis 
and  3  million  deaths,  afflicting  primarily  the 
most  productive  element  of  society — young 
adults.  Infection  with  HIV  (human  immunodefi- 
ciency virus)  causes  a  breakdown  of  resistance  to 
tuberculosis;  this  has  produced  a  grave  increase 
in  the  disease,  in  both  the  developing  and  the 
industrialized  countries.  In  1985,  following  a 
32-year  decline  in  the  number  of  cases,  the  inci- 
dence of  tuberculosis  in  the  Unites  States  began 
to  increase,  reaching  25,701  cases  reported  in 
1990. 

Resistance  to  M.  tuberculosis 

The  devastatingly  heightened  susceptibility  of 


HIV-infected  individuals  to  a  galloping  course  of 
tuberculosis  is  compelling  evidence  that  there 
are  powerful  mechanisms  of  immunity  in  im- 
munocompetent hosts.  The  cellular  and  molecu- 
lar mechanisms  mediating  that  immunity  remain 
enigmatic.  We  have,  however,  established  that 
the  most  common  microbicidal  product  of  acti- 
vated macrophages,  oxygen  radicals,  is  ineffec- 
tive at  killing  virulent  human  tubercle  bacilli, 
but  reactive  nitrogen  intermediates  (particularly 
nitric  oxide)  can  inhibit  growth  and  kill  M.  tu- 
berculosis in  vitro. 

Immunologic  Unresponsiveness 
and  Leprosy 

One  fundamental  issue  in  immunology  is  the 
nature  of  immunological  tolerance,  i.e.,  the 
mechanisms  by  which  cells  in  the  immune  sys- 
tem discriminate  between  foreign  antigens  and 
self-antigens  and  prevent  responses  to  self.  A 
breakdown  of  tolerance  to  self-antigens  leads  to 
autoimmune  diseases,  such  as  rheumatoid  arthri- 
tis, juvenile  diabetes,  and  perhaps  multiple  scle- 
rosis. The  principal  mechanism  for  developing 
tolerance  is  thought  to  be  deletion  of  clones  of 
potentially  autoreactive  T  cells  in  the  thymus 
during  neonatal  life.  Clearly,  however,  not  all 
such  clones  can  be  deleted  in  the  thymus;  there 
must  be  additional  mechanisms  by  which  self- 
reactive  cells  can  be  rendered  unresponsive  after 
birth. 

Leprosy  provides  a  unique  model  with  which 
to  study  immunoregulation  and  unresponsive- 
ness. The  disease  comprises  a  spectrum  of  clini- 
cal entities.  In  the  tuberculoid  form,  strong  cell- 
mediated  immunity  kills  the  organism  but 
damages  nerves  in  the  process.  In  the  leproma- 
tous  form,  at  the  other  end  of  the  spectrum,  pa- 
tients are  unable  to  respond  immunologically  to 
M.  leprae  antigens.  Because  infection  occurs 
after  birth,  there  is  little  evidence  of  clonal  dele- 
tion of  T  cells  capable  of  reacting  to  this  organ- 
ism. Therefore  understanding  the  mechanisms  of 
that  unresponsiveness  is  relevant  to  preventing 


47 


Immunity  and  Pathogenesis  of  Third  World  Diseases:  Leprosy  and  Tuberculosis 


autoimmune  disease  and  increasing  transplant 
survival. 

We  have  learned  that  the  unresponsiveness  in 
leprosy  is  antigen-specific-,  lepromatous  leprosy 
patients  unable  to  respond  to  antigens  of  M.  le- 
prae usually  respond  to  those  of  M.  tuberculosis, 
which  is  a  closely  related  mycobacterium.  How  is 
it  possible  for  T  cells  to  recognize  antigens  in  the 
tubercle  bacillus  and  yet  be  unable  to  recognize 
the  same  or  closely  related  antigens  associated 
with  the  leprosy  bacillus?  We  proposed  that  there 
might  be  one  or  a  few  unique  antigens  associated 
with  M.  leprae  that  induce  active  T  cell  suppres- 
sion of  potentially  reactive  T  cell  clones.  Sup- 
pressor cells  in  immunology  have  been  a  contro- 
versial subject,  but  the  idea  that  some  T  cells  can 
down-regulate  immune  responses,  particularly 
self-destructive  ones,  is  compelling.  About  85 
percent  of  patients  with  lepromatous  leprosy 
have  a  subset  of  T  cells  capable  of  being  triggered 
specifically  by  leprosy  antigens  to  suppress  re- 
sponses of  immune  T  cells.  Although  they  repre- 
sent a  minor  subset  of  T  cells  in  the  blood,  they 
are  the  major  lymphocyte  subset  in  lepromatous 
lesions.  By  establishing  long-term  T  cell  clones 
directly  from  the  lesions  and  blood,  we  found 
that  the  suppressor  cells  have  a  pattern  of  antigen 
recognition  different  from  other  cytotoxic  or 
lymphokine-producing  T  cells.  They  carry  the 
surface  marker  CDS  and  recognize  foreign  anti- 
gens in  association  with  the  HLA-DQ  region  of  the 
human  major  histocompatibility  complex  (MHC) 
class  II.  We  speculate  that  presentation  of  anti- 
gens by  this  MHC  subset  predisposes  the  immune 
responses  toward  negative  rather  than  positive 
responses.  Our  studies  suggest  that  functionally 
distinct  subsets  of  T  cells  are  characterized  by 
distinct  patterns  of  lymphokines  produced  upon 
antigenic  stimulation. 

New  Vaccines  from  Old — Recombinant  BCG 
as  a  Multivaccine  Vehicle 

Vaccines  represent  the  most  cost-effective  med- 
ical intervention.  Yet  three  general  problems 
limit  the  use  of  many  current  vaccines:  1)  they 
require  multiple  booster  shots  to  be  effective;  2) 
they  cannot  be  given  for  6-12  months  after  birth, 
because  of  transferred  maternal  antibodies  that 


inactivate  them;  and  3)  the  cost.  BCG  (bacille 
Calmette-Guerin) ,  the  most  widely  used  vaccine 
in  the  world,  is  a  live,  attenuated  bovine  tuber- 
cle bacillus  given  to  protect  children  against 
tuberculosis. 

BCG  has  been  given  to  more  than  2.5  billion 
people  and  has  a  very  low  incidence  of  serious 
side  effects.  It  is  one  of  only  two  childhood  vac- 
cines that  can  be  given  at  birth  or  any  time  there- 
after. It  is  a  single-shot  vaccine  that  engenders 
long-lasting  cellular  immunity  and  costs  only 
$0.10  a  dose. 

The  unique  attributes  of  BCG  suggested  to  us 
that,  if  it  could  be  genetically  engineered  to  ex- 
press a  variety  of  foreign  antigens  protective  for 
different  pathogens,  a  single  immunization  might 
be  capable  of  engendering  protective  responses 
to  multiple  pathogens  simultaneously.  One  prob- 
lem, however,  was  the  paucity  of  molecular  ge- 
netic information  about  the  Mycobacteria.  In  col- 
laboration with  William  Jacobs  (HHMI,  Albert 
Einstein  College  of  Medicine),  we  developed  ge- 
netic systems  for  introducing  and  expressing  for- 
eign genes  in  mycobacteria,  particularly  BCG 
strains.  We  developed  a  shuttle  strategy  in  which 
mycobacterial  DNA  could  be  genetically  cloned 
and  manipulated  in  E.  coli  and  then  transferred 
into  mycobacteria.  Our  first  approach  was  to  use 
mycobacteriophages  (viruses  that  infect  bacte- 
ria) as  vectors  to  target  foreign  genes  to  a  specific 
site  in  the  bacterial  chromosome.  This  enabled  us 
to  introduce  foreign  DNA  into  BCG  for  the  first 
time.  Recently  we  have  developed  shuttle  plas- 
mid  vectors  that  are  able  to  produce  many  copies 
of  foreign  genes  in  BCG. 

With  several  collaborators  at  Medlmmune, 
Inc.,  and  the  University  of  Pittsburgh,  we  have 
developed  the  first  experimental  recombinant 
BCG  vaccines.  These  express  protective  antigens 
from  M.  leprae,  schistosomes,  malaria,  measles 
virus,  leishmania,  and  HIV.  Initial  experiments  in 
mice  indicate  that  three  major  types  of  protective 
immune  responses  can  be  generated  in  vivo — 
namely  immunoglobulin  antibodies,  T  cell  lym- 
phokines, and  cytotoxic  T  lymphocytes.  Continu- 
ing efforts  will  be  made  to  define  antigens  that 
will  engender,  through  recombinant  BCG,  pro- 
tective immunity  against  a  variety  of  viral,  bacte- 
rial, and  parasitic  pathogens. 


48 


Molecular  Biology  of  the  Extracellular  Matrix 


Jeffrey  p.  Bonadio,  M.D. — Assistant  Investigator 

Dr.  Bonadio  is  also  Assistant  Professor  of  Pathology  and  Member  of  the  Program  in  Bioengineering  at 
the  University  of  Michigan  Medical  School.  He  received  his  bachelor's  degree  in  biology  from  Marquette 
University  and  his  M.D.  degree  from  the  Medical  College  of  Wisconsin,  Milwaukee.  He  studied  anatomical 
pathology  with  Bruce  Beckwith  and  medical  molecular  genetics  with  Peter  Byers  at  the  University 
of  Washington,  Seattle. 


THE  long-term  goal  of  our  research  is  to  under- 
stand how  extracellular  matrix  proteins  con- 
tribute to  skeletal  structure  and  function.  Quanti- 
tative and  qualitative  changes  in  these  proteins 
occur  during  morphogenesis  and  as  part  of  the 
wound  healing  process.  These  observations  sug- 
gest that  both  the  organization  and  protein  com- 
position of  the  matrix  are  precisely  regulated.  It 
is  clear  that  this  regulation  occurs  in  part  at  the 
level  of  gene  expression  and  in  part  at  the  level  of 
the  assembly  of  proteins  into  a  matrix-like 
configuration. 

I  have  chosen  to  focus  for  the  most  part  on  the 
matrix  molecule  type  I  collagen.  This  collagen  is 
a  polymer  of  two  related  proteins  whose  se- 
quence has  been  determined.  Moreover,  the  mul- 
tidomain  structure  of  the  molecule  and  a  general 
outline  of  collagen  biosynthesis  are  known,  and 
the  molecule  is  recognized  to  be  distributed 
widely  within  tissues  such  as  bone,  tendon,  liga- 
ment, tooth,  dermis,  and  sclera.  Previous  studies 
have  implied  that  type  I  collagen  makes  an  im- 
portant contribution  to  the  structure,  integrity, 
and  normal  homeostasis  of  these  tissues.  Over  the 
past  year  we  have  continued  our  work  to  establish 
model  systems  that  would  allow  us  to  study  this 
contribution  at  the  molecular  level. 

One  system  is  designed  to  investigate  the  intra- 
cellular assembly  of  the  collagen  molecule.  In 
general,  this  work  involves  site-specific  mutagen- 
esis and  assays  that  quantify  the  effects  of  muta- 
tion on  the  assembly  process.  These  effects  are 
studied  at  two  levels.  First,  we  have  established 
conditions  that  allow  synthetic  peptides  to  fold 
into  a  collagen-like  triple  helix.  Peptide  folding 
is  slow  enough  that  the  process  can  be  character- 
ized by  methods  such  as  circular  dichroism.  In 
addition,  the  triple  helix  formed  in  vitro  is  stable 
enough  that  its  structure  can  be  characterized  by 
nuclear  magnetic  resonance  (NMR)  techniques. 
Therefore  the  effect  of  a  given  mutation  can  be 
quantified  by  directly  comparing  the  behavior  of 
a  normal  peptide  with  that  of  mutant  peptide. 
Second,  cellular  transfection  methods  have  been 
developed  to  express  and  assemble  collagen  mol- 
ecules in  vitro.  Again,  the  effect  of  mutation  on 


the  assembly  process  can  be  quantified  by  di- 
rectly comparing  the  behavior  of  normal  and  mu- 
tant molecules. 

In  our  initial  mutagenesis  experiments,  we 
characterized  a  highly  conserved  region  of  the 
triple-helical  domain  and  demonstrated  that  it 
made  an  important  contribution  to  the  assembly 
of  collagen  molecules  into  a  thermodynamically 
stable  conformation.  We  speculate  that  this  re- 
gion was  conserved  during  evolution  because  it 
plays  an  important  role  in  collagen  biosynthesis, 
i.e.,  in  folding  the  collagen  molecule  into  its 
correct  conformation.  In  the  future,  we  hope  to 
use  this  model  system  to  define  further  the  nor- 
mal contribution  made  by  other  collagen  do- 
mains to  the  assembly  process.  In  addition,  we 
are  interested  in  characterizing  those  regions  of 
the  molecule  that  mediate  interactions  between 
collagen  and  other  matrix  molecules  such  as 
fibronectin,  heparin  sulfate  proteoglycan,  and  in- 
tegrins.  These  interactions  are  important  because 
they  represent  a  molecular  basis  for  the  assembly 
of  collagen  within  the  matrix. 

A  second  system  is  designed  to  investigate  the 
function  of  type  I  collagen  at  the  level  of  connec- 
tive tissue.  Our  initial  set  of  experiments  utilized 
a  transgenic  mouse  strain  that  expressed  only  half 
the  normal  amount  of  type  I  collagen.  We  demon- 
strated that  the  mutation  adversely  affected  the 
connective  tissue  of  bone  and  skin  dermis.  In  ad- 
dition, the  mutant  mice  were  profoundly  deaf. 
We  utilized  biomechanical  tests  to  quantify  the 
effect  of  the  collagen  deficiency  at  the  tissue 
level,  and  these  studies  demonstrated  that  the 
major  role  of  type  I  collagen  is  to  provide  con- 
nective tissue  with  a  high  degree  of  resiliency. 

More  recently,  we  also  demonstrated  that  the 
skeleton  of  these  transgenic  mice  is  able  to  adapt 
to  the  inherited  collagen  deficiency.  This  adapta- 
tion involves  a  thickening  of  cortical  bone  and 
results  from  the  synthesis  of  new  bone  matrix. 
Particularly  intriguing  was  our  observation  that 
the  adaptation  was  associated  with  a  significant 
improvement  in  bone  strength.  This  result  is  im- 
portant because  it  suggests  the  basis  for  a  strategy 
to  strengthen  the  fragile  skeleton. 


49 


Functional  Heterogeneity  in  CD4-bearing 
T  Lymphocytes 


H.  Kim  Bottomly,  Ph.D. — Associate  Investigator 

Dr.  Bottomly  is  also  Associate  Professor  in  the  Section  of  Immunobiology  and  the  Department  of  Biology 
at  Yale  University  School  of  Medicine.  She  received  her  Ph.D.  degree  from  the  University  of  Washington, 
Department  of  Biological  Structure,  where  she  studied  with  Roy  Schwartz.  Her  postdoctoral  training 
was  received  in  the  field  of  immunology  with  Don  Mosier  at  NIH. 


LYMPHOCYTE  interactions  during  an  immune 
response  are  necessary  for  the  induction  of 
antigen-specific  lymphocytes.  The  resulting  ef- 
fector phase  of  an  immune  response  is  described 
as  either  humoral  or  cell  mediated:  each  of  these 
phases  combats  different  types  of  microorgan- 
isms. Although  these  effector  mechanisms  are 
well  characterized,  the  precise  mechanism  by 
which  the  response  to  a  given  antigen  or  in- 
fectious agent  is  directed  into  the  humoral  or 
cell-mediated  mode  is  not  known.  What  is  clear, 
however,  is  that  both  types  of  immunity  depend 
on  the  activation  of  CD4-bearing  T  lymphocytes, 
which  in  turn  induce  other  cell  types  to  respond 
to  the  foreign  antigen.  These  responses  include 
activation  of  B  cells  to  proliferate  and  to  secrete 
antibody,  induction  of  delayed-type  hypersensi- 
tivity reactions,  activation  of  CDS  cytolytic  T 
cells,  and  activation  of  macrophages. 

Two  questions  were  then  asked.  1 )  Could  the 
same  CD4  T  cell  activate  all  these  target  cells, 
therefore  mediating  both  humoral  and  cell- 
mediated  responses?  2)  Is  the  CD4  T  cell  popula- 
tion functionally  heterogeneous;  i.e.,  do  some 
CD4-bearing  T  cells  activate  B  cells  and  play  a 
primary  role  in  the  induction  of  humoral  immu- 
nity, and  do  some  activate  macrophages  and  CD8- 
bearing  T  cells  and  play  a  primary  role  in  cell- 
mediated  immunity? 

For  several  years  we  have  focused  on  the  hetero- 
geneity in  CD4  T  cell  function  and  the  activation 
conditions  that  lead  to  it.  These  studies  have  suc- 
cessfully shown  that  monoclonal  CD4  T  lympho- 
cytes, obtained  by  T  cell  cloning  and  expansion 
in  tissue  culture,  belong  to  two  distinct  subsets  of 
T  cell.  One  subset  can  help  B  cell  antibody  secre- 
tion but  cannot  activate  macrophages;  the  other 
set  activates  macrophages  but  is  a  poor  B  cell  acti- 
vator. To  reflect  their  main  functions,  these  two 
subsets  are  called  helper  T  cells  (Th2)  and  inflam- 
matory T  cells  (Th  1 ) .  The  distinct  functional  abil- 
ities of  Th2  and  Thl  subsets  are  reflected  in  their 
release,  upon  activation,  of  distinct  panels  of  cy- 
tokines. Several  cytokines  are  produced  selec- 
tively by  one  or  the  other  subset.  In  particular, 
interIeukin-2,  interferon-7,  and  lymphotoxin  are 


produced  by  the  Thl  but  not  Th2  cell  subset. 
Interleukin-4  is  produced  by  the  Th2  but  not  Th  1 
cell  subset. 

Thus  there  is  a  correlation  between  cytokine 
production  and  function.  Interleukin-4  is  a  po- 
tent B  cell  activator  involved  in  B  cell  prolifera- 
tion and  secretion  of  immunoglobulins  IgGl  and 
IgE.  By  contrast,  interferon-7,  lymphotoxin,  and 
interleukin-2  are  associated  with  responses  in- 
volving the  activation  of  macrophages,  lysis  of 
target  cells,  and  induction  of  cytolytic  T  cells, 
which  is  consistent  with  the  known  function  of 
these  cytokines.  One  can  conclude  from  these 
studies  that  T  cells  are  committed  to  the  release 
of  a  distinct  panel  of  lymphokines  when  acti- 
vated, with  the  released  effector  molecules  de- 
termining their  effector  function. 

Recent  studies  in  this  laboratory  have  focused 
on  determining  whether  functionally  distinct 
subsets  of  CD4  T  cell  exist  in  vivo  and  whether 
the  selective  activation  of  one  subset  or  the  other 
has  the  expected  functional  consequences.  Nu- 
merous clinical  studies  have  suggested  that 
various  immunization  schemes  induce  primarily 
a  humoral  or  cell-mediated  immune  response.  It 
is  critically  important  to  determine  if  such  major 
differences  in  protective  immunity  reflect  differ- 
ences in  the  proportion  of  CD4  T  cell  subsets 
activated.  If  this  is  true,  one  might  propose  that 
the  form  of  the  antigen  or  the  antigen  presenta- 
tion must  somehow  direct  which  CD4  T  cell  will 
be  preferentially  activated.  To  test  this  possibil- 
ity, several  questions  about  normal  CD4  T  cells 
have  been  asked. 

First,  is  there  a  separation  of  resting  CD4  T  cells 
into  subsets?  To  answer  this,  analysis  of  the  re- 
sponses to  antigens  that  give  rise  to  primarily  hu- 
moral or  cell-mediated  immunity  has  been  per- 
formed. When  humoral  immunity  dominates, 
Th2-like  CD4  T  cells  are  activated.  By  contrast, 
when  cell-mediated  immunity  dominates,  Thl- 
like  CD4  T  cells  are  activated.  Thus  during  the 
course  of  an  immune  response,  CD4  T  cells  may 
become  specialized  in  their  functional  capabili- 
ties, and  these  T  cells  in  vivo  resemble,  in  their 
activities,  Thl  and  Th2  cloned  lines. 


51 


Functional  Heterogeneity  in  CD4-bearing  T  Lymphocytes 


Second,  when  during  development  or  activa- 
tion do  subsets  of  CD4 -bearing  T  cells  arise?  A 
particularly  interesting  question  is  whether  the 
commitment  of  CD4 -bearing  T  cells  to  the  Thl 
and  Th2  subsets  is  a  consequence  of  antigen 
priming.  Analysis  of  memory  and  virgin  CD4- 
bearing  T  cells  indicates  that  the  functional  spe- 
cialization characteristic  of  Thl  and  Th2  subsets 
resides  mainly  in  the  memory  population.  This 
suggests  that  contact  with  foreign  antigen  initi- 
ates a  commitment  of  CD4-bearing  T  cells  to  a 
particular  effector  response. 

The  form  an  immune  response  takes  must  be 
appropriate  to  the  microorganism  that  is  causing 
the  disease.  This  is  seen  clearly  in  leprosy,  where 
one  response  produces  abundant  antibody  and,  at 


the  same  time,  overwhelming  growth  of  the  lep- 
rosy bacterium  in  macrophages.  This  is  probably 
an  example  of  selective  activation  of  the  helper 
(Th2),  rather  than  the  inflammatory  (Thl),  sub- 
set of  CD4  T  cells.  What  controls  the  form  such  a 
response  will  take?  Model  systems  to  explore 
these  questions  are  needed,  so  that  an  inappropri- 
ate immune  response  can  be  redirected  to  be- 
come curative.  Future  research  in  this  laboratory 
will  focus  on  this  question  of  the  control  of  CD4 
T  cell  subset  activation  and  regulation. 

Studies  analyzing  those  factors  important  in  the 
generation  of  memory  and  effector  CDA^  T  lym- 
phocytes and  those  analyzing  the  selective  activa- 
tion of  CD4  T  cell  subsets  are  supported  by  grants 
from  the  National  Institutes  of  Health. 


52 


Retroviral  Replication 


Patrick  O.  Broum,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Brown  is  also  Assistant  Professor  of  Pediatrics  and  of  Biochemistry  at  Stanford  University  School  of 
Medicine.  He  received  his  B.A.  degree  in  chemistry  from  the  University  of  Chicago.  His  graduate  work  with 
Nicholas  Cozzarelli  at  the  University  of  Chicago  was  focused  on  the  mechanisms  of  DNA  topoisomerases. 
He  received  his  Ph.D.  and  M.D.  degrees  from  the  University  of  Chicago,  completed  a  pediatrics  residency 
at  Children's  Memorial  Hospital  in  Chicago,  and  then  joined  Michael  Bishop's  laboratory  at  the  University 
of  California,  San  Francisco.  There  he  began  to  investigate  the  mechanism  of  retroviral  integration, 
which  has  continued  to  be  the  major  focus  of  his  research. 


RETROVIRUSES  are  an  important  cause  of  dis- 
ease in  most  vertebrate  species.  In  humans, 
retroviral  infections  can  lead  to  AIDS  (acquired 
immune  deficiency  syndrome),  leukemia,  lym- 
phoma, and  degenerative  diseases  of  the  central 
nervous  system.  Millions  of  people  are  infected 
with  the  human  immunodeficiency  virus,  HIV, 
and  will  likely  succumb  to  AIDS  unless  an  effec- 
tive treatment  is  developed. 

The  retroviral  genes  are  carried  in  the  virus 
particle  as  RNA  molecules.  When  the  virus  infects 
a  cell,  it  transcribes  these  molecules,  its  RNA  ge- 
nome, into  a  double-stranded  DNA  molecule  and 
inserts  this  into  a  chromosome  in  the  nucleus  of 
the  host  cell.  Thus  the  viral  genome,  then  called  a 
provirus,  becomes  an  integral  part  of  the  cell's 
genome.  Integration  of  a  provirus  into  its  host 
cell's  DNA  is  essential  for  retroviral  reproduc- 
tion. This  distinctive  feature  of  the  retroviral  life 
cycle  accounts  for  many  of  the  characteristics  as- 
sociated with  retroviral  infection,  including  in- 
sertional  mutagenesis,  induction  of  tumors,  and 
the  latent  and  persistent  nature  of  many  retroviral 
infections.  Moreover,  the  fact  that  retroviruses 
are  designed  to  introduce  foreign  genes  into  cel- 
lular DNA  makes  them  exceptionally  useful  as 
tools  for  genetic  engineering. 

How  does  a  retrovirus  get  its  DNA  into  a  cell's 
nucleus  and  integrate  it  into  the  cell's  DNA,  and 
how  does  the  cell  regulate  these  processes? 

To  investigate  the  molecular  mechanism  by 
which  a  retrovirus  inserts  its  DNA  into  that  of  the 
infected  cell,  we  have  developed  a  variety  of 
methods  for  studying  the  retroviral  integration  re- 
action in  a  test  tube.  We  have  used  this  approach 
to  define  several  discrete  steps  in  the  joining  of 
viral  to  cellular  DNA  and  to  determine  the  re- 
quirements for  the  reaction.  The  enzymatic  ma- 
chinery that  carries  out  integration  can  be  iso- 
lated from  infected  cells  in  a  stable  complex  with 
the  unintegrated  viral  DNA  molecule.  We  are 
currently  using  electron  microscopy  as  well  as 
biochemical  methods  to  define  the  structure  of 
this  viral  replication  intermediate. 

To  study  the  enzymology  of  integrase,  the  viral 


protein  that  actually  catalyzes  integration,  we 
have  constructed  genetically  engineered  bacte- 
rial strains  that  produce  abundant  quantities  of 
the  integrase  proteins  from  HIV  and  murine  leu- 
kemia virus  and  have  developed  simple  purifica- 
tions of  these  proteins.  Using  small  synthetic  DNA 
molecules  as  model  substrates,  we  can  now 
readily  study  their  catalytic  activities,  which  in- 
clude the  sequence-specific  processing  of  the 
ends  of  the  viral  DNA  and  the  joining  of  these 
ends  to  a  target  DNA  molecule.  We  have  made 
progress  in  the  past  year  toward  understanding 
the  organization  of  integrase  and  how  it  binds  the 
viral  and  target  DNA  substrates.  For  example,  we 
have  identified  DNA  substrates  that  function  pref- 
erentially either  as  donors  or  as  targets  in  a  DNA 
joining  assay.  By  studying  the  competition  of 
these  DNAs  for  binding  to  the  enzyme,  we  have 
identified  two  distinct  sites  that  bind  viral  and 
target  DNA,  respectively.  By  analyzing  the  rever- 
sal of  the  usual  DNA  joining  reaction,  we  have 
discovered  that  integrase  has  a  previously  unrec- 
ognized DNA  splicing  activity.  We  are  investigat- 
ing the  possibility  that  this  new  activity  may  play 
a  role  in  integration  and  in  the  high-frequency 
recombination  that  occurs  between  viral  ge- 
nomes during  replication. 

The  structure  of  integrase  is  clearly  of  central 
importance  to  understanding  integration.  Be- 
cause efforts  to  obtain  conventional  crystals  of 
integrase  have  been  unrewarding,  we  are  prepar- 
ing to  use  electron  diffraction  methods  to  deter- 
mine a  structure  from  two-dimensional  crystals. 
By  constructing  specific  mutants  and  defining 
their  biochemical  defects,  we  have  begun  to 
identify  the  functions  of  specific  protein  regions. 
To  extend  this  approach,  we  are  developing  a 
new  genetic  system  that  we  hope  will  enable  us 
to  screen  for  functional  defects  among  millions  of 
mutant  integrases,  based  on  their  ability  to  carry 
out  recombination  in  bacteria.  This  system 
should  also  facilitate  our  efforts  to  develop  genet- 
ically altered  integrases  with  properties  more  fa- 
vorable for  therapeutic  applications.  For  exam- 
ple, we  would  like  to  develop  an  integrase  that 


53 


Retroviral  Replication 


can  selectively  integrate  the  viral  DNA  into  pre- 
determined sites  in  the  target  DNA. 

The  ultimate  goal  of  our  work  on  integration  is 
to  understand  in  molecular  detail  how  the  pro- 
teins of  the  integration  machinery  recognize  the 
viral  DNA,  assemble  into  an  active  complex,  rec- 
ognize the  target  DNA,  and  finally  catalyze  the 
DNA  breakage  and  joining  reactions  that  lead  to 
integration  of  the  provirus.  It  is  hoped  that  this 
understanding  will  lead  to  the  development  of 
new  agents  for  inhibiting  the  replication  of  patho- 
genic retroviruses  and  to  improved  systems  for 
the  therapeutic  introduction  of  genes  into  mam- 
malian cells. 

It  has  been  recognized  for  many  years  that  es- 
tablishment of  a  retroviral  provirus  proceeds 
much  more  readily  in  actively  dividing  cells  than 
in  their  resting  counterparts.  However,  the  basis 
for  this  phenomenon  remains  obscure.  To  bring 
this  observation  into  clearer  focus,  we  have  in- 
vestigated the  dependence  of  specific  steps  in  the 
life  cycle  of  the  murine  leukemia  virus  on  the 
host  cell's  stage  in  its  own  division  cycle.  Using 
drugs  or  a  mutation  in  the  cell-cycle  control  gene 
cdc2  to  regulate  the  cell  cycle  of  the  host  cells, 
we  have  found  that  integration  in  vivo  depends 
on  mitosis.  Yet  synthesis  of  viral  DNA  and  accu- 
mulation of  integration-competent  intermediates 
occur  normally  in  cells  blocked  from  entering 
mitosis.  The  intermediates  accumulate  in  the  cy- 
toplasm and  remain  stable  for  hours,  awaiting  mi- 
tosis. Current  investigations  are  aimed  at  under- 
standing why  mitosis  is  required  for  integration. 
Preliminary  evidence  suggests  that  disassembly 
of  the  nuclear  envelope  at  mitosis  may  provide  a 
route  of  entry  for  viral  replication  intermediates. 


Understanding  how  cellular  functions  can  deter- 
mine the  fate  of  an  infecting  retrovirus  may  lead 
to  new  approaches  to  antiviral  therapy  and  to  im- 
provements in  the  use  of  retroviruses  as  vectors 
for  gene  therapy. 

Our  work  on  retroviral  integration  is  supported 
by  a  grant  from  the  National  Institutes  of  Health. 

New  Methods  for  Linkage  Mapping 
in  Complex  Genomes 

A  major  impediment  to  defining  and  character- 
izing the  genes  that  influence  complex  human 
traits  has  been  the  difficulty  of  collecting  suitable 
large  families  in  which  the  trait  segregates.  Such 
families  are  generally  needed  to  find  genes  by 
conventional  linkage  mapping.  The  same  genes 
could  in  principle  be  mapped  more  easily  by  an 
alternative  strategy  that  involves  collecting  and 
analyzing  pairs  of  relatives  that  share  a  trait  of 
interest.  However,  linkage  mapping  with  small 
sets  of  relatives  generally  requires  analysis  of  a 
large  number  of  closely  spaced  and  highly  poly- 
morphic genetic  markers,  which  makes  this  strat- 
egy impractical  with  current  technology. 

We  are  developing  a  new  set  of  genetic  tools 
that  will  allow  widespread  application  of  these 
highly  efficient  linkage  mapping  methods.  Ex- 
periments are  in  progress  to  test  these  new  meth- 
ods in  model  systems.  In  parallel  with  our  efforts 
to  develop  new  biochemical  methods,  we  are 
working  to  find  optimal  statistical  methods,  using 
high-resolution  maps  of  genetic  identity  between 
pairs  of  relatives.  Our  aim  is  to  apply  this  technol- 
ogy to  map  genes  for  complex  human  traits. 

This  work  is  supported  by  a  grant  from  the  Na- 
tional Institutes  of  Health. 


54 


Regulation  of  Cellular  Processes 

by  Protein-Tyrosine  Phosphorylation 


Joan  S.  Brugge,  Ph.D. — Investigator 

Dr.  Brugge  is  also  Professor  of  Microbiology  at  the  University  of  Pennsylvania  School  of  Medicine.  She 
received  her  B.A.  degree  in  biology  from  Northwestern  University  and  her  Ph.D.  degree  in  virology  from 
Baylor  College  of  Medicine.  Her  postdoctoral  research  was  done  with  Raymond  Erikson  at  the  University 
of  Colorado  School  of  Medicine,  Denver.  Before  moving  to  the  University  of  Pennsylvania,  Dr.  Brugge 
was  a  member  of  the  Department  of  Microbiology  at  the  State  University  of  New  York  at  Stony  Brook. 


THE  modification  of  intracellular  proteins  by 
the  reversible  addition  of  phosphate  groups 
(phosphorylation/dephosphorylation)  is  the  most 
common  mechanism  for  regulating  their  activity. 
Enzymes  that  specifically  transfer  phosphate  to 
tyrosine  residues  of  proteins  (protein-tyrosine  ki- 
nases) play  critical  roles  in  intracellular  signal 
transduction  events — biochemical  processes 
that  transform  interactions  at  the  surface  of  the 
cell  into  an  intracellular  response.  Our  labora- 
tory is  interested  in  investigating  the  role  of  tyro- 
sine phosphorylation  in  these  processes.  We  have 
chosen  for  these  studies  two  model  systems  that 
offer  unique  advantages:  platelets,  which  are  ide- 
ally suited  for  studies  of  cell  adhesion,  secretion, 
and  cytoskeletal  rearrangements,  and  PCI  2  cells, 
which  allow  dissection  of  events  that  are  in- 
volved in  the  differentiation  of  neuronal  cells 
after  treatment  with  nerve  growth  factors. 

Tyrosine  Phosphorylation  in  Platelets 

Platelets  are  small,  anucleate  peripheral  blood 
cells  that  contain  many  intracellular  vesicles 
whose  components  are  released  upon  activation 
by  cellular  hormones.  Platelet  aggregation  and 
the  released  products  from  platelets  are  responsi- 
ble for  the  formation  of  blood  clots  and  wound 
healing.  We  have  found  that  hormones  that  acti- 
vate platelet  aggregation  and  secretion  cause 
rapid  changes  in  the  phosphorylation  of  multiple 
proteins  on  tyrosine.  In  collaboration  with  San- 
ford  Shattil  (University  of  Pennsylvania  School  of 
Medicine) ,  we  have  shown  that  the  phosphoryla- 
tion of  several  of  these  proteins  requires  platelet 
aggregation. 

This  process  is  mediated  by  interactions  be- 
tween the  serum  adhesion  protein  fibrinogen  and 
its  receptor,  GP  Ilb-IIIa,  on  the  platelet  surface. 
GP  Ilb-IIIa  is  a  member  of  the  integrin  family  of 
receptors  that  bind  to  extracellular  matrix  and 
adhesion  proteins.  These  receptors  are  believed 
to  be  important  in  causing  changes  in  cell  behav- 
ior that  are  mediated  by  cell  adhesion.  Our  stud- 
ies demonstrating  that  inhibition  of  GP  Ilb-IIIa- 
induced  cell  aggregation  prevents  tyrosine 
phosphorylation  of  cellular  proteins  suggest  that 


this  receptor  may  regulate  the  activation  of  tyro- 
sine kinases,  which  play  a  role  in  aggregation- 
dependent  events. 

We  have  recently  found  that  a  cytoplasmic  tyro- 
sine protein  kinase,  pi  25-FAK,  is  phosphorylated 
on  tyrosine  and  activated  following  platelet  ag- 
gregation. This  kinase  was  first  identified  by  Tom 
Parsons  (University  of  Virginia  School  of  Medi- 
cine) as  a  substrate  phosphorylated  in  Rous  sar- 
coma virus-transformed  cells.  The  enzyme  is  lo- 
calized in  focal  adhesion  plaques — sites  where 
integrin  receptors  couple  with  extracellular  ma- 
trix proteins  and  intracellular  microfilaments.  It 
is  not  phosphorylated  on  tyrosine  or  activated  in 
platelets  from  patients  with  Glanzmann's  throm- 
bocytopenia, a  disorder  caused  by  the  absence  of 
a  functional  GP  Ilb-IIIa.  These  results  suggest 
that  pi  25-FAK  may  be  activated  by  events  follow- 
ing GP  Ilb-IIIa-dependent  platelet  aggregation. 
It  is  also  phosphorylated  on  tyrosine  in  fibro- 
blasts following  activation  of  other  integrin  re- 
ceptors and  could  play  a  critical  role  in  integrin- 
mediated  activation  of  intracellular  signal 
transduction  events  mediated  by  cell-cell  and 
cell-matrix  interactions. 

GP  Ilb-IIIa  may  also  be  important  for  the  assem- 
bly of  intracellular  cytoskeletal  complexes  that 
follow  thrombin-induced  platelet  aggregation. 
Together  with  Joan  Fox  (Gladstone  Foundation), 
we  have  found  that  the  assembly  of  these  cytoskel- 
etal proteins  with  enzymes  involved  in  signal 
transduction  is  dependent  on  GP  Ilb-IIIa  and 
platelet  aggregation.  We  have  found  that  four 
members  of  the  Src  family  of  protein-tyrosine  ki- 
nases— Src,  Fyn,  Lyn,  and  Yes — are  part  of  these 
assemblies,  as  are  the  cytoskeletal  proteins  actin, 
vinculin,  talin,  and  GPIV.  In  platelets  from 
Glanzmann's  thrombocytopenia  patients,  these 
proteins  do  not  associate  with  the  detergent- 
insoluble  cell  fraction,  indicating  that  GP  Ilb-IIIa 
is  critical  for  these  cytoskeletal  rearrangements. 
Thus  platelet  aggregation  mediated  by  fibrinogen 
binding  to  GP  Ilb-IIIa  appears  to  play  an  impor- 
tant role  in  nucleating  the  assembly  of  cytoskele- 
tal signaling  complexes  that  may  be  important  for 
activation  of  intracellular  processes,  and  these 


55 


Regulation  of  Cellular  Processes  by  Protein- Tyrosine  Phosphorylation 


studies  raise  the  possibility  that  integrin  recep- 
tors in  other  cell  types  play  similar  roles  in  cou- 
pling with  cytoplasmic  tyrosine  kinases. 

We  have  also  found  that  another  platelet  mem- 
brane receptor,  GPIV,  or  CD36,  is  tightly  cou- 
pled with  three  other  protein-tyrosine  kinases. 
These  kinases,  Fyn,  Lyn,  and  Yes,  are  members  of 
the  Src  family  of  protein-tyrosine  kinases,  which 
have  been  shown  to  be  linked  with  transmem- 
brane receptors  in  other  cell  types.  The  ligand 
that  binds  to  GPIV/CD36  has  not  been  identified; 
however,  the  evidence  that  this  receptor  is  asso- 
ciated with  protein-tyrosine  kinases  strongly  im- 
plicates it  is  an  intracellular  signal  transducer. 

The  Fc  receptors  for  the  complement-binding 
domain  of  antibody  molecules  are  also  found  on 
platelets.  Activation  of  these  receptors  causes 
platelet  aggregation  and  secretion,  and  the  in- 
duction of  tyrosine  phosphorylation  of  the  same 
proteins  that  are  phosphorylated  in  thrombin- 
treated  platelets.  However,  unlike  other  platelet 
receptors,  the  FC-7RII  receptor  is  itself  phos- 
phorylated on  tyrosine.  The  site(s)  of  tyrosine 
phosphorylation  of  this  receptor  lies  within  a 
peptide  motif  that  is  shared  with  several  other 
lymphocyte  receptors  in  T  cells,  B  cells,  natural 
killer  cells,  mast  cells,  and  basophils.  This  motif 
appears  to  be  involved  in  coupling  these  recep- 
tors with  protein-tyrosine  kinases.  Tyrosine  phos- 
phorylation of  the  conserved  tyrosine  residues  in 
this  motif  may  be  important  for  these  coupling 
interactions. 

Tyrosine  Phosphorylation  in  Neuronal 
PCI 2  Cells 

We  are  interested  in  defining  the  role  of  tyro- 
sine phosphorylation  in  mediating  neuronal  dif- 
ferentiation. PCI 2  cells,  derived  from  a  rat 
pheochromocytoma,  provide  a  useful  model  sys- 
tem, since  these  cells  differentiate  into  cells  re- 
sembling sympathetic  neurons  after  treatment 


with  nerve  growth  factor  (NGF)  or  fibroblast 
growth  factor  (FGF) .  Both  of  these  receptors  are 
transmembrane  proteins  whose  cytoplasmic  do- 
mains contain  protein-tyrosine  kinase  activity. 
Growth  factor  binding  to  these  cells  causes  a 
burst  of  tyrosine  phosphorylation  of  multiple 
cellular  proteins. 

Recently,  in  collaboration  with  Simon  Ha- 
legoua,  we  have  found  that  tyrosine  phosphoryla- 
tion of  two  serine/threonine  protein  kinases, 
p42MAPK       p44MAPK  jg  dependent  on  the  small 

GTP-binding  protein  Ras.  Expression  of  a  mutant 
of  Ras  that  interferes  with  the  activity  of  the  en- 
dogenous Ras  protein  blocks  tyrosine  phosphory- 
lation of  these  proteins,  and  expression  of  a  con- 
stitutively  activated  form  of  Ras  leads  to  their 
tyrosine  phosphorylation.  These  mutant  forms  of 
Ras  have  no  effect  on  the  activity  of  the  NGF  re- 
ceptor itself  or  on  the  phosphorylation  of  pro- 
teins that  are  direct  substrates  of  this  kinase. 

The  p42  and  p44  MAP  kinases  are  referred  to  as 
"switch  kinases."  They  are  activated  by  phos- 
phorylation on  tyrosine  but  phosphorylate  other 
proteins  on  serine  and  threonine.  MAP  kinases  are 
activated  by  a  variety  of  mitogens  and  growth  fac- 
tors, and  are  believed  to  serve  as  critical  compo- 
nents of  intracellular  signaling  pathways  by  inte- 
grating signals  from  a  diverse  array  of  receptors. 
Our  studies  suggest  that  tyrosine  phosphoryla- 
tion of  MAP  kinase  is  dependent  on  pathways  reg- 
ulated by  the  Ras  GTP-binding  protein,  and  that 
this  event  can  thus  be  distinguished  from  phos- 
phorylation events  that  are  directly  mediated  by 
the  NGF  receptor  itself.  Since  Ras  activity  is  es- 
sential for  NGF-  and  FGF-induced  neuronal  dif- 
ferentiation of  PC  12  cells,  these  results  raise  the 
possibility  that  MAP  kinases  are  also  essential 
components  of  this  process. 

Dr.  Brugge  is  now  Scientific  Director  at  Ariad 
Pharmaceuticals,  Cambridge,  Massachusetts. 


56 


Computational  Structural  Biology 


Axel  T.  Brunger,  Ph.D. — Assistant  Investigator 

Dr.  Brunger  is  also  Associate  Professor  of  Molecular  Biophysics  and  Biochemistry  at  Yale  University.  He 
was  born  in  Leipzig,  Germany.  He  received  his  diploma  in  physics  at  the  University  of  Hamburg  and  his 
Ph.D.  degree  from  the  Technical  University  of  Munich.  He  held  a  NATO  postdoctoral  fellowship  and 
subsequently  became  a  research  associate  with  Martin  Karplus  in  the  Department  of  Chemistry  at  Harvard 
University  before  joining  the  faculty  at  Yale.  His  research  has  focused  on  molecular  dynamics  studies 
of  protein  structure  and  function  and  on  methods  in  protein  crystallography  and  nuclear  magnetic 
resonance  spectroscopy. 


OUR  research  lies  at  the  interface  between 
theory  and  experiment  in  the  area  of  struc- 
tural biophysics.  The  research  tools  are  simula- 
tion methods  of  computational  chemistry  adapted 
to  the  requirements  of  macromolecular  systems. 
Macromolecular  simulations  are  an  important  ad- 
dition to  the  arsenal  of  methods  available  to  struc- 
tural biologists  working  with  x-ray  crystallo- 
graphic  or  nuclear  magnetic  resonance  (NMR) 
spectroscopic  data.  In  one  class  of  projects,  we 
are  trying  to  understand  the  detailed  microscopic 
interactions  that  govern  stability  and  recognition 
in  biological  systems  and  to  test  the  reliability  of 
the  theoretical  methods  as  tools  for  this  purpose. 
In  another  class  of  projects,  we  are  directly  com- 
bining macromolecular  simulation  with  experi- 
mental data  in  order  to  make  data  analysis  possi- 
ble or  more  efficient. 

Accuracy  of  Crystal  and  Solution 
NMR  Structures 

As  methods  for  determining  macromolecular 
three-dimensional  structure  continue  to  become 
more  powerful  and  are  being  applied  to  many 
biologically  interesting  systems,  concern  has 
been  raised  about  the  verification  of  final  atomic 
models.  A  common  problem  arises  when  models 
are  fitted  against  preliminary  experimental  data 
of  mediocre  quality.  The  recent  revision  of  a  num- 
ber of  published  structures,  both  x-ray  and  solu- 
tion NMR,  illustrates  the  need  to  develop  im- 
proved tools  for  checking  the  accuracy  of  the 
final  atomic  models. 

Structure  determination  of  macromolecules  by 
crystallography  involves  fitting  atomic  models  to 
the  observed  diff'raction  data.  The  traditional 
measure  of  the  quality  of  this  fit,  and  presumably 
the  accuracy  of  the  model,  is  the  R  value.  Despite 
stereochemical  restraints,  it  is  possible  to  overfit 
or  "misfit"  the  diffraction  data:  an  incorrect 
model  can  be  refined  to  fairly  good  R  values,  as 
several  recent  examples  have  shown.  We  recently 
proposed  a  reliable  and  unbiased  indicator  of  the 
accuracy  of  such  models. 

In  analogy  to  testing  statistical  models  by  cross- 
validation,  we  defined  a  statistical  quantity,  Rjee. 


that  measures  the  agreement  between  observed 
and  computed  structure  factor  amplitudes  for  a 
"test"  set  of  reflections  that  is  omitted  in  the  mod- 
eling and  refinement  process.  As  examples  show, 
there  is  a  high  correlation  between  Rj^e  and  the 
accuracy  of  the  atomic  model  phases.  This  is  use- 
ful, since  experimental  phase  information  is 
usually  inaccurate,  incomplete,  or  unavailable. 

The  enhanced  sensitivity  of  11^,^^  with  respect  to 
model  errors  was  illustrated  for  the  crystal  struc- 
ture of  the  plant  ribulose-1 ,5-bisphosphate  car- 
boxylase/oxygenase (RuBisCO)  (David  Eisen- 
berg.  University  of  California,  Los  Angeles).  A 
partially  incorrect  model  for  RuBisCO,  which  es- 
sentially had  the  small  subunit  traced  backward, 
showed  only  a  4  percent  conventional  R  value 
difference  from  the  correct  model.  On  the  other 
hand,  RX^g  showed  a  13  percent  difference,  sug- 
gesting that  the  incorrect  model  had  been  overfit. 

We  concluded  that  Rj^g  represents  a  reliable 
and  unbiased  parameter  by  which  to  evaluate  the 
information  content  of  a  model  produced  by  x- 
ray  crystallography.  It  is  not  restricted  to  high- 
resolution  diffraction  data.  The  observation  that 
Rjee  cai^  distinguish  between  a  random  distribu- 
tion of  scatterers  and  a  distribution  close  to  the 
protein  suggests  applications  to  ab  initio  phas- 
ing. Presently  we  shall  apply  this  method  to  as- 
sess models  of  thermal  motion  and  disorder, 
time-averaging,  and  bulk  solvent  in  protein 
crystals. 

A  similar  approach  might  be  useful  for  the 
three-dimensional  structure  determination  by  so- 
lution NMR.  Using  molecular  dynamics  refine- 
ment, we  have  just  succeeded  in  implementing 
the  free  R  approach.  At  present  we  are  testing  it 
for  a  number  of  model  systems.  The  question  of 
accuracy  is  an  even  more  fundamental  problem 
for  solution  NMR  structures  because  of  the  ad- 
verse observable-to-parameter  ratio. 

This  work  is  also  supported  by  the  National 
Science  Foundation. 

Predictions  of  Helix-Helix  Association 
and  Stability 

Prediction  of  the  three-dimensional  structure 

^  7 


Computational  Structural  Biology 


of  proteins  based  on  their  sequence  remains  im- 
possible. This  fundamental  problem  of  structural 
biology  (the  "folding  problem")  is  still  un- 
solved, despite  improvements  in  computational 
techniques  for  macromolecular  simulation  and 
CQjnputer  hardware.  Nevertheless,  macromolecu- 
lar simulation  has  been  successful  in  predicting 
localized  conformations  if  sufficient  experimen- 
tal constraints  or  restraints  (e.g.,  in  the  form  of  an 
x-ray  structure)  are  available.  It  is  therefore  con- 
ceivable that  other  more  global  predictions  are 
possible  if  appropriate  experimental  information 
is  available.  We  have  embarked  on  trying  to  pre- 
dict the  association  and  stability  of  helices  that 
form  coiled  coils.  Conformational  search  strate- 
gies are  being  employed  with  empirical  energy 
functions,  using  molecular  dynamics  and  energy 
minimization. 

Presently  we  shall  apply  this  approach  to  the 
family  of  leucine  zipper  proteins,  which  are  se- 
quence-specific DNA-binding  proteins  that  regu- 
late gene  expression  in  certain  mammalian  cells. 
We  have  successfully  predicted  the  structure  of 
the  dimerization  domain  of  GCN4,  for  which  a 
high-resolution  x-ray  has  become  available 
(Thomas  Alber,  University  of  Utah). 


Macromolecular  Simulation  of  Free-Energy 
Differences 

We  are  involved  in  a  number  of  projects 
aimed  at  simulating  free-energy  differences  be- 
tween two  states  of  a  biological  system,  using 
the  so-called  free-energy  perturbation  tech- 
nique. The  goal  is  to  investigate  microscopi- 
cally the  structure  and  stability  of  protein  sec- 
ondary structural  elements  and  protein-peptide 
complexes.  Furthermore,  we  would  like  to  eval- 
uate the  reliability  of  free-energy  calculations 
and  molecular  dynamics  simulations  as  tools 
for  this  purpose. 

One  project  concerns  the  complexes  of  bovine 
pancreatic  ribonuclease  S  and  a  number  of  mu- 
tants of  the  S-peptide  for  which  x-ray  crystal 
structures,  binding  free  energies,  and  enthalpies 
have  been  obtained  by  Frederic  Richards  and  Ju- 
lian Sturtevant  (Yale  University) .  Another  project 
involves  the  study  of  a  number  of  site-directed 
mutants  of  |8-turns  in  staphylococcal  nuclease  in  a 
joint  project  with  Robert  Fox  (HHMI ,  Yale  Univer- 
sity) for  which  x-ray  crystal  structures  and  infor- 
mation about  the  cis  to  trans  equilibria  of  a  pro- 
line side  chain  in  the  turn  have  been  obtained  in 
Dr.  Fox's  laboratory. 


Illustration  of  hulk  solvent  regions  in  protein  crystal  struc- 
tures. The  regions  are  indicated  by  blue  dots  in  this  unit  cell  of  a 
crystal  of penicillopepsin  from  Penicillium  janthinellum,  whose 
structure  was  solved  in  Michael  fames'  laboratory  (Alberta, 
Canada)  at  1.8- A  resolution.  The  protein  backbone  atoms  ap- 
pear as  yellow  lines;  ordered  water  molecules,  as  pink  dots. 
Bulk  solvent  typically  constitutes  40-60 percent  of  the  unit  cell 
contents,  yet  its  physical  characteristics  and  biological  func- 
tion are  poorly  understood. 

Research  and  photograph  by  Axel  Brunger,  using  a  graphics 
interface  developed  by  Warren  Delano. 


58 


Biophysical  Studies  of  Eukaryotic  Gene  Regulation 
and  Molecular  Recognition 


Stephen  K.  Burley,  M.D.,  D.Phil. — Assistant  Investigator 

Dr.  Burley  is  also  Assistant  Professor  and  Co-Head  of  the  Laboratory  of  Molecular  Biophysics  at  the 
Rockefeller  University.  He  received  a  B.Sc.  degree  in  physics  from  the  University  of  Western  Ontario,  a 
D.Phil,  degree  in  molecular  biophysics  from  Oxford  University,  and  an  M.D.  degree  from  Harvard  Medical 
School  in  the  Harvard  MIT  Joint  Program  in  Health  Sciences  and  Technology.  While  a  medical  student, 
he  carried  out  research  in  protein  crystallography  with  Gregory  Petsko.  During  his  clinical  training 
at  Brigham  and  Women 's  Hospital,  he  also  conducted  postdoctoral  research  in  protein  crystallography 
with  William  Lipscomb  at  Harvard  University,  where  he  solved  the  three-dimensional  structure 
of  leucine  aminopeptidase. 


WE  are  interested  in  developing  a  detailed 
understanding  of  the  physical  principles 
that  govern  the  general  problem  of  molecular  rec- 
ognition in  biological  systems.  Our  approach  is 
to  use  x-ray  crystallography  and  complementary 
biophysical  methods  to  determine  and  character- 
ize the  three-dimensional  structure  and  function 
of  biological  macromolecules  and  their  com- 
plexes with  DNA,  proteins,  or  smaller  ligands. 
These  structures  contain  a  wealth  of  atomic  detail 
that  we  can  analyze  with  biochemical,  molecular 
genetic,  and  theoretical  methods  to  provide  a 
functional  description  of  the  intra-  and  intermo- 
lecular  interactions  responsible  for  stabilizing 
macromolecular  complexes. 

In  the  long  term,  we  hope  that  our  biophysical 
studies  and  analyses  will  allow  us  to  exploit  the 
powerful  formalism  of  physics  to  classify  system- 
atically the  interactions  between  individual 
atoms  that  are  responsible  for  molecular  recogni- 
tion in  biological  systems.  We  believe  that  such  a 
quantitative  understanding  will  ultimately  per- 
mit us  to  harness  the  machinery  of  molecular  rec- 
ognition and,  thereby,  make  defined  interven- 
tions into  important  biochemical  processes  such 
as  disease  states. 

Eukaryotic  Gene  Regulation 

We  are  examining  the  problem  of  eukaryotic 
gene  regulation,  with  the  goal  of  improving  our 
understanding  of  the  structural  and  physical 
bases  of  transcriptional  control  of  genes.  Three 
distinct  classes  of  proteins  are  active  in  transcrip- 
tion, and  we  are  studying  representative  mem- 
bers of  each  class.  First,  we  are  collaborating  with 
Robert  Roeder  (Rockefeller  University)  on  x-ray 
crystallographic  and  complementary  biophysical 
studies  of  transcription  factor  IID  (TFIID)  and 
other  components  of  the  basic  transcription  ma- 
chinery. These  proteins  form  a  stable  multipro- 
tein,  or  preinitiation,  complex  with  DNA  se- 
quences found  immediately  upstream  of  the 
transcription  start  site,  where  they  mediate  tran- 
scription by  RNA  polymerase  II.  We  have  pu- 
rified, characterized,  and  crystallized  TFIID, 


which  begins  preinitiation  complex  formation  by 
binding  to  the  TATA  consensus  sequence.  In  ad- 
dition, we  have  started  work  on  TFIIB,  the  second 
protein  to  be  recruited  to  the  preinitiation 
complex. 

Second,  we  are  also  collaborating  with  Dr. 
Roeder  on  studies  of  upstream  stimulatory  factor 
(USF),  a  member  of  the  c-mjr-related  family  of 
DNA-binding  proteins  that  contains  both  a  helix- 
loop-helix  motif  and  a  leucine  repeat.  We  have 
used  various  biophysical  methods  to  purify  and 
extensively  characterize  USF  and  its  mechanisms 
of  action.  Moreover,  we  were  recently  able  to 
grow  small  cocrystals  of  USF  and  DNA.  During 
transcription,  TFIID,  the  other  basic  factors,  and 
USF  bind  to  DNA  in  close  proximity  and  interact 
with  one  another  to  enhance  both  DNA  binding 
and  transcription.  After  determining  the  three- 
dimensional  structures  of  each  of  these  proteins 
and  their  complexes  with  their  respective  pro- 
moter DNA  elements,  we  hope  to  determine  the 
structures  of  some  biologically  relevant  multi- 
protein-DNA  complexes. 

Third,  we  are  collaborating  with  Eseng  Lai  (Me- 
morial Sloan-Kettering  Cancer  Center)  on  struc- 
tural studies  of  human  hepatocyte  nuclear  factor 
3.  This  transcriptionally  active  protein  belongs  to 
a  gene  family  in  mammals  that  is  homologous  to 
the  Drosophila  homeotic  gene  fork  head.  These 
diverse  proteins  share  a  highly  conserved  DNA- 
binding  region  and  influence  transcription  by 
binding  to  DNA  elements,  known  as  enhancer  se- 
quences, that  are  located  far  from  the  transcrip- 
tion start  site.  During  the  past  year,  we  have  co- 
crystallized  a  member  of  this  family  with  DNA 
and  are  proceeding  with  a  three-dimensional 
structure  determination.  Detailed  structural  and 
biophysical  studies  of  these  three  distinct  classes 
of  participants  in  eukaryotic  transcription  should 
provide  insights  into  the  precise  role  molecular 
recognition  plays  in  gene  regulation. 

Molecular  Recognition 

During  the  past  year,  we  have  also  begun 
to  study  other  biological  systems  that  function 


59 


Biophysical  Studies  of  Eukaryotic  Gene  Regulation  and  Molecular  Recognition 


via  molecular  recognition,  including  steroid-  minations  of  these  proteins  will  be  pursued  once 
binding  proteins,  polypeptide-binding  proteins,  they  are  fully  characterized  and  suitable  crystals 
and  enzymes.  Three-dimensional  structure  deter-    and  cocrystals  become  available. 


F-Actin 

Proposed  model  of  the  dystrophin-glycoprotein  complex.  Dystrophin  dimers  link  the  cyto- 
skeleton  with  the  membrane  glycoprotein  complex  by  binding  to  actin  at  the  amino  ter- 
minus (N)  and  to  the  50-,  43-,  and  35-kDa  transmembrane  proteins  at  the  carboxyl  ter- 
minus (C). 

From  Ervasti,  J.M.,  and  Campbell,  K.P.  1991.  Ceil  66:1121-1131-  Copyright©  1991  by 
Cell  Press. 


60 


Molecular  Studies  of  Calcium  Channels 
and  the  Dystrophin-Glycoprotein  Complex 


Kevin  p.  Campbell,  Ph.D. — Investigator 

Dr.  Campbell  is  also  Professor  of  Physiology  and  Biophysics  at  the  University  of  Iowa,  Iowa  City.  He 
received  his  B.S.  degree  in  physics  from  Manhattan  College,  his  master's  degree  from  the  University  of 
Rochester  School  of  Medicine  and  Dentistry,  and  his  Ph.D.  degree  from  the  Department  of  Radiation 
Biology  and  Biophysics  at  the  University  of  Rochester.  He  did  postdoctoral  studies  in  the  laboratory  of 
David  MacLennan  at  the  Banting  and  Best  Department  of  Medical  Research,  University  of  Toronto, 
before  coming  to  Iowa. 


CALCIUM  ion  (Ca^^)  functions  as  a  ubiquitous 
intracellular  messenger  regulating  a  wide  va- 
riety of  cellular  responses,  including  contrac- 
tion, secretion,  and  cell  proliferation.  Two  com- 
mon pathways  by  which  intracellular  Ca^"^ 
transients  can  be  triggered  have  been  identified. 
The  first  involves  the  influx  of  Ca^"^  into  the  cyto- 
plasm from  the  extracellular  medium  through 
specific  plasma  membrane  channels,  and  the  sec- 
ond involves  the  release  of  Ca^"^  into  the  cyto- 
plasm from  intracellular  stores  through  specific 
release  channels. 

Over  the  past  10  years,  we  have  investigated 
the  structure  and  function  of  the  membrane 
components  involved  in  Ca^"*^  fluxes  across  mem- 
branes. In  particular,  we  have  focused  on  identi- 
fying, purifying,  and  characterizing  the  mem- 
brane proteins  that  function  as  surface  Ca^^ 
channels  and  intracellular  Ca^"^  release  channels 
in  excitable  cells. 

Neuronal  Calcium  Release  Channels 

A  major  area  of  research  in  my  laboratory  con- 
cerns the  structure  and  function  of  intracellular 
Ca^^  release  channels.  In  skeletal  muscle,  Ca^"^ 
release  from  the  sarcoplasmic  reticulum  initi- 
ates contraction.  We  previously  purified  a  high- 
affinity  ryanodine  receptor  from  skeletal  muscle 
sarcoplasmic  reticulum  and  showed  it  to  be  iden- 
tical to  the  sarcoplasmic  reticulum  Ca^^  release 
channel. 

In  neurons,  inositol  1 ,4,5-trisphosphate  (IP3) 
is  an  important  second  messenger  involved  in 
Ca^"^  release.  However,  physiological  and  pharma- 
cological evidence  has  indicated  the  presence  of 
non-IPj-gated  Ca^^  pools  in  neurons.  Recently 
my  laboratory  also  identified  and  purified  a  neu- 
ronal ryanodine  receptor.  The  purified  receptor 
was  shown  to  be  a  homotetramer  composed  of 
protein  subunits  of  approximately  500  kDa.  Re- 
constitution  of  the  purified  ryanodine  receptor 
into  lipid  bilayers  has  demonstrated  that  it  func- 
tions as  a  caffeine-  and  ryanodine-sensitive  Ca^^ 
release  channel  that  is  distinct  from  the  brain  IP3 
receptor.  Immunoblotting  experiments  indicate 
that  the  brain  receptor  is  more  like  the  cardiac 


receptor  than  the  skeletal  receptor.  Thus  we  be- 
lieve that  the  brain  ryanodine  receptor  may  oper- 
ate as  a  Ca^^-induced  release  channel  for  intracel- 
lular Ca^^  pools  in  neurons.  In  the  upcoming  year 
further  studies  of  this  receptor  should  provide 
insights  into  its  role  in  neuronal  Ca^^  homeostasis 
and  its  distribution  within  the  central  nervous 
system. 

Voltage-gated  Calcium  Channels 

A  second  major  area  of  research  concerns  volt- 
age-gated Ca^^  channels  in  excitable  cells.  We  have 
studied  in  skeletal  muscle  the  dihydropyridine- 
sensitive  Ca^^  channel,  which  is  essential  to  exci- 
tation-contraction coupling.  The  skeletal  muscle 
dihydropyridine  receptor  has  been  purified  and 
consists  of  four  subunits  (aj,  and  7). 

In  neurons,  voltage-gated  Ca^^  channels  exist 
as  several  types  (L,  N,  T,  and  P)  with  different 
kinetic  and  pharmacological  properties.  Dihy- 
dropyridines  bind  specifically  to  L-type  Ca^"*" 
channels  and  alter  their  channel  activity.  For  N- 
type  channels,  which  are  likely  responsible  for 
triggering  neurotransmitter  release  at  synapses, 
oj-conotoxin  is  largely  specific.  We  are  now  using 
antibodies  and  cDNA  probes  to  the  various  sub- 
units  of  the  dihydropyridine-sensitive  channels 
to  study  oj-conotoxin-sensitive  Ca^^  channels. 

We  have  demonstrated  that  the  brain 
conotoxin-sensitive  Ca^^  channel  contains  a 
component  homologous  to  the  18-subunit  of  the 
dihydropyridine-sensitive  Ca'^'^  channel  of  skele- 
tal muscle.  We  have  also  isolated  a  cDNA  clone 
from  a  brain  cDNA  library  encoding  a  protein 
with  high  homology  to  the  /8-subunit  of  the  skele- 
tal muscle  dihydropyridine-sensitive  Ca^^  chan- 
nel. This  brain  /3-subunit  cDNA  encodes  numer- 
ous consensus  phosphorylation  sites,  suggesting 
a  role  in  Ca^^  channel  regulation. 

We  are  now  purifying  the  w-conotoxin-sensi- 
tive  Ca^^  channel,  using  affinity  chromatography, 
in  order  to  analyze  its  subunit  composition  and  to 
demonstrate  that  it  is  identical  to  the  N-type  Ca^^ 
channel.  Experiments  are  also  in  progress  to 
coexpress  the  brain  /3-subunit  with  the  aj-subunit 


61 


Molecular  Studies  of  Calcium  Channels  and  the  Dystrophin-Glycoprotein  Complex 


in  order  to  study  how  the  ;8-subunit  regulates 
Ca^"""  channel  activity. 

Dystrophin-Glycoprotein  Complex 

A  third  major  project  in  my  laboratory  is  aimed 
at-understanding  the  function  of  dystrophin  in 
normal  muscle  and  determining  how  the  absence 
of  dystrophin  leads  to  Duchenne  muscular  dys- 
trophy (DMD).  Dystrophin  is  localized  to  the  in- 
ner surface  of  the  sarcolemma  in  normal  muscle 
but  is  absent  in  skeletal  muscle  of  DMD  patients 
and  mdx  mice.  We  previously  showed  that  dys- 
trophin is  tightly  linked  to  a  large  oligomeric 
complex  of  sarcolemmal  glycoproteins.  We  have 
isolated  a  dystrophin-glycoprotein  complex  and 
have  shown  that  it  consists  of  cytoskeletal,  trans- 
membrane, and  extracellular  components.  These 
data  have  allowed  us  to  propose  a  model  for  the 
organization  of  the  dystrophin-glycoprotein 
complex  (see  figure).  The  membrane  organiza- 
tion of  the  dystrophin-glycoprotein  complex  and 
the  high  density  of  dystrophin  in  the  sarcolemma 
membrane  suggest  that  this  complex  could  have 
an  important  structural  role  in  skeletal  muscle. 

In  the  past  year  we  have  investigated  the  rela- 
tive abundance  of  each  of  the  components  of  the 
dystrophin-glycoprotein  complex  in  skeletal 
muscle  from  normal  and  mdx  mice.  Our  results 
demonstrate  that  all  of  the  dystrophin-associated 
glycoproteins  (DAGs)  are  significantly  reduced 
in  mdx  skeletal  muscle  and  suggest  that  the  loss 
of  DAGs  is  due  to  the  absence  of  dystrophin  and 
not  to  secondary  effects  of  muscle  fiber  degrada- 
tion. Furthermore,  we  recently  showed  that  the 
absence  of  dystrophin  in  skeletal  muscle  from 


DMD  patients  leads  to  a  dramatic  loss  of  all  the 
components  of  the  dystrophin-glycoprotein  com- 
plex. Thus  the  abnormal  expression  of  the  DAGs 
may  play  a  crucial  role  in  molecular  pathogenesis 
in  DMD. 

In  order  to  identify  the  normal  function  of 
the  DAGs,  we  recently  established,  by  cDNA 
cloning,  the  primary  sequence  of  two  compo- 
nents of  the  dystrophin-glycoprotein  complex. 
The  43-  and  1 56-kDa  DAGs  are  encoded  by  the 
same  mRNA,  and  post-translational  modifica- 
tion of  a  97-kDa  precursor  protein  results  in 
two  mature  proteins:  the  transmembrane  43- 
kDa  DAG  and  the  extracellular  1 56-kDa  DAG. 
In  addition,  we  have  shown  that  the  1 56-kDa 
DAG  binds  laminin,  a  well-characterized  com- 
ponent of  the  extracellular  matrix.  Our  results 
demonstrate  that  the  43/1 56-kDa  DAG  (named 
dystroglycan)  is  a  novel  laminin-binding  glyco- 
protein and  suggest  that  the  function  of  the 
dystrophin-glycoprotein  complex  is  to  provide 
a  linkage  between  the  subsarcolemma  cytoskel- 
eton  and  the  extracellular  matrix.  Our  findings 
strongly  support  the  hypothesis  that  in  DMD  a 
dramatic  reduction  in  a  1  56-kDa  DAG  leads  to 
the  loss  of  a  linkage  between  the  sarcolemma 
and  extracellular  matrix.  This  may  render  mus- 
cle fibers  more  susceptible  to  necrosis  or  may 
disrupt  the  integrity  of  muscle. 

Our  goal  for  the  next  year  is  to  clone  the  other 
DAGs  in  order  to  express  the  entire  complex  in 
nonmuscle  cells  to  study  its  function.  We  also 
plan  to  examine  the  possible  involvement  of  the 
DAGs  in  other  muscular  dystrophies. 

This  work  was  also  supported  by  the  Muscular 
Dystrophy  Association. 


62 


Gene  Targeting 


Mario  R.  Capecchi,  Ph.D. — Investigator 

Dr.  Capecchi  is  also  Professor  of  Human  Genetics  at  the  University  of  Utah  School  of  Medicine  and 
Professor  of  Biology  at  the  University  of  Utah.  He  received  his  B.S.  degree  in  chemistry  and  physics  from 
Antioch  College  and  his  Ph.D.  degree  in  biophysics  from  Harvard  University,  where  he  worked  with  James 
Watson.  Dr.  Capecchi  remained  at  Harvard  as  a  Junior  Fellow  of  the  Society  of  Fellows  and  then  joined 
the  Harvard  faculty.  Before  moving  to  the  University  of  Utah,  he  was  Associate  Professor  of  Biochemistry 
at  Harvard  School  of  Medicine.  Dr.  Capecchi  is  a  member  of  the  National  Academy  of  Sciences. 


GENE  targeting — homologous  recombination 
between  DNA  sequences  in  the  chromo- 
somes of  mouse  embyro-derived  stem  (ES)  cells 
and  newly  introduced,  exogenous  DNA  se- 
quences— has  been  used  to  create  mice  with  null 
(ablating)  mutations  in  members  of  two  families 
of  genes.  The  first  set  of  genes  are  members  of  the 
int  proto-oncogene  family  (int  l,  int-2,  etc.). 
This  set  of  genes  is  believed  to  be  involved  in 
localized  developmental  decisions  mediated 
through  cell-cell  signaling.  Their  protein  prod- 
ucts resemble  growth  factors,  and  int-2  is  a 
member  of  the  fibroblast  growth  factor  family. 
These  genes  were  initially  identified  through 
their  pathological  role  in  the  genesis  of  mouse 
mammary  carcinomas.  We  are  addressing  the  ques- 
tion of  their  normal  role  during  embryogenesis. 

Targeted  disruption  of  int-1  (wnt-l)  resulted 
in  mice  with  a  range  of  phenotypes  from  death  at 
birth  to  survival  to  adulthood.  Those  mice  surviv- 
ing exhibited  severe  ataxia  (loss  of  balance  and 
coordinated  movement).  Those  int- 1~ / int- 1~ 
mice  that  died  at  or  near  birth  showed  severe  ab- 
normalities in  the  formation  of  the  entire  cerebel- 
lum and  the  major  portion  of  the  midbrain, 
whereas  the  defect  in  the  survivors  was  restricted 
to  the  formation  of  the  anterior  region  of  the  cere- 
bellum. We  have  shown  that  a  pre-existing  mu- 
tant mouse,  identified  by  its  ataxic  behavior,  con- 
tains a  frameshift  mutation  in  the  int-1  {wnt-1) 
gene.  This  allele  also  appears  to  be  a  null  muta- 
tion and  exhibits  the  same  range  of  variation  in 
expressivity  as  our  targeted  null  allele. 

We  have  created  mice  with  null  mutations  in 
int-2.  These  mice  also  exhibit  a  variable  pheno- 
type  from  death  at  birth  to  survival  to  adulthood. 
The  survivors  are  even  fertile.  The  int-2'' /int- 2~ 
mice  have  defects  in  the  formation  of  the  vesti- 
bule and  cochlea,  resulting  in  loss  of  balance  and 
deafness.  However,  the  degree  of  malformation 
of  these  inner  ear  compartments  varies. 

The  second  set  of  genes  we  are  analyzing  con- 
stitute part  of  the  developmental  program  that 
specifies  positional  information  along  the  antero- 
posterior axis  of  the  early  embryo.  These  genes, 
collectively  known  as  the  Hox  genes,  code  for 
transcription  factors  in  both  the  human  and 


mouse.  The  38  Hox  members  are  present  on  four 
linkage  groups  of  four  separate  chromosomes. 
The  four  linkage  groups  are  believed  to  have 
arisen  early  in  chordate  evolution,  as  a  result  of 
quadruplication  of  a  single  ancestral  group  com- 
mon to  both  vertebrates  and  invertebrates.  Ex- 
pansion of  this  gene  complex  may  have  had  a 
critical  role  in  the  progression  from  invertebrates 
to  vertebrates,  by  supplying  the  necessary  com- 
plexity to  this  network  of  genes  to  accommodate 
the  development  of  our  complex  body  plan. 

In  Drosophila  these  genes  act  as  master 
switches  directing  the  course  of  morphogenic  de- 
velopment of  each  parasegment.  A  mutation  in 
one  gene  can  result  in  dramatic  homeotic  trans- 
formations of  one  body  part  into  another,  such  as 
the  conversion  of  antennae  to  legs.  Determining 
the  function  of  the  corresponding  genes  in  mam- 
mals is  just  beginning. 

We  have  initiated  a  systematic  genetic  analysis 
of  the  Hox  genes  in  mice.  First,  we  are  creating 
mice  with  null  mutations  in  each  of  these  genes 
to  define  their  individual  functions.  From  such  an 
analysis,  patterns  should  emerge  that  define  the 
zones  governed  by  these  genes.  Second,  through 
epistasis  and  molecular  analysis  of  combinations 
of  hox  mutations,  we  hope  to  define  how  this  set 
of  genes  functions  as  a  network  to  specify  posi- 
tional information  along  the  body  axis  of  the  em- 
bryo. To  date  we  have  analyzed  mice  containing 
targeted  disruptions  in  the  two  closely  linked 
Hox  genes,  hox- 1.5  and  hox- 1.6. 

Disruption  of  hox- 1.5  or  -7.6  resulted  in  mice 
with  complex  but  regionally  restricted  develop- 
mental defects.  Interestingly,  the  sets  of  defects 
associated  with  disrupting  these  two  genes  are 
distinct  and  non-overlapping.  The  hox-1.5~/ 
hox- 1.5'^  mice  are  athymic,  aparathyroid,  and 
have  reduced  thyroid  and  submaxillary  tissue. 
They  also  exhibit  a  wide  spectrum  of  throat  abnor- 
malities (including  shortened  necks,  abnormal 
larynx,  truncated  soft  palate,  and  poor  organiza- 
tion of  throat  musculature)  and  defects  of  the 
heart  and  major  arteries.  This  collection  of  defi- 
ciencies is  remarkably  similar  to  those  afflicting 
humans  with  the  congenital  DiGeorge  syndrome. 

On  the  other  hand,  hox- 1 .6~ /hox- 1 .6"  mice 


63 


Gene  Targeting 


executing  each  of  those  pathways.  The  non- 
overlap  of  phenotypes  associated  with  disrupting 
these  two  genes  argues  for  their  independent 
character.  For  example,  if  a  nonredundant  com- 
binatorial code  of  box  genes  is  used  to  specify 
each  cell  type  in  a  given  region,  then  the  com- 
bined presence  of  both  of  these  gene  products 
does  not  specify  any  tissue. 


Disruption  of  the  hox-1.6  gene  results  in  regionally  restricted  developmental  defects,  including 
formation  of  the  ear,  cranial  nerves,  and  ganglia,  as  well  as  the  brain  stem.  This  is  illustrated  by 
these  embryos  of  El  0.5  mice  immunoreacted  with  a  monoclonal  antibody  against  the  155-kDa 
neurofilament  protein,  a)  cowfro/ hox-1 .6"^/hox-l  .6^  embryo;  b,  c)  two  mutant  hox-l  .6~/hox- 
1.6"  embryos. 

Reprinted  by  permission  from  Chisaka,  O.,  Musci,  T.S.,  and  Capecchi,  M.R.  1992.  Nature 
355:516-520.  Copyright©  1992  Macmillan  Magazines  Limited. 


exhibit  profound  defects  in  the  formation  of  the 
ear,  hindbrain  nuclei,  and  cranial  nerves  and  gan- 
glia. The  glossopharyngeal  and  vagus  nerves  are 
poorly  developed,  and  the  preganglionic  connec- 
tions with  the  brain  stem  are  not  formed. 

In  both  hox-l. 5  and  -7. 6 mutants,  the  affected 
tissues  are  formed  by  very  different  embryonic 
pathways,  yet  the  two  hox  genes  are  involved  in 


64 


Genetic  Control  of  Pattern  Formation 
in  Drosophila 


Sean  B.  Carroll,  Ph.D. — Assistant  Investigator 

Dr.  Carroll  is  also  Associate  Professor  of  Molecular  Biology,  Genetics,  and  Medical  Genetics  at  the 
University  of  Wisconsin-Madison.  He  obtained  his  B.A.  degree  in  biology  from  Washington  University  in 
St.  Louis  and  his  Ph.D.  degree  in  immunology  from  Tufts  University  School  of  Medicine  in  Boston.  He 
received  postdoctoral  training  in  developmental  genetics  working  with  Matthew  Scott  at  the  University 
of  Colorado.  In  addition  to  his  central  work  on  pattern  formation  in  Drosophila,  Dr.  Carroll  has 
also  conducted  basic  research  on  new  types  of  snake  antivenoms,  which  are  now  under  evaluation 
as  potential  pharmaceuticals.  His  honors  include  the  NSF  Presidential  Young  Investigator  Award. 


INSECTS  are  the  dominant  group  of  animals  on 
earth  today.  Nearly  1  million  species  have  been 
classified  among  an  estimated  20  million  species 
extant.  The  innumerable  sizes,  shapes,  and  colors 
of  insects  have  evolved  from  a  basic  segmented 
body  plan  consisting  of  three  broad  divisions — 
head,  thorax,  and  abdomen — with  three  pairs  of 
ambulatory  legs  and,  in  some  orders,  wings. 

One  insect,  the  fruit  fly  Drosophila  melano- 
gaster,  has  emerged  as  a  key  model  for  investiga- 
tions into  the  genetics  and  molecular  biology  of 
animal  development.  An  enormous  amount  of  in- 
formation has  been  gained  about  the  organization 
of  the  Drosophila  egg,  the  dynamic  regulatory 
mechanisms  guiding  embryonic  development, 
and  the  cellular  and  molecular  processes  in- 
volved in  tissue  differentiation  and  organogene- 
sis. By  addressing  the  intricacies  of  Drosophila 
development  at  the  genetic,  cellular,  and  molecu- 
lar levels,  one  of  the  central  puzzles  of  biology  is 
being  deciphered:  How  do  complex  animals 
form  from  a  simple  egg? 

In  addition,  the  stage  is  being  set  for  compara- 
tive studies  that  will  integrate  the  growing  knowl- 
edge of  Drosophila  development  with  contempo- 
rary views  of  animal  evolution.  Efforts  in  our 
laboratory  are  aimed  at  both  a  detailed  under- 
standing of  Drosophila  embryology  as  a  model 
for  animal  development  and  comparative  studies 
of  other  insects  (e.g.,  beetles  and  butterflies)  as 
models  for  evolution. 

Pattern  Formation  in  Drosophila  Embryos 

The  genetic  control  of  pattern  formation  can  be 
broken  down  conceptually  into  at  least  three 
phases.  The  first  consists  of  a  molecular  prepat- 
tern,  revealed  as  chemical  changes  that  take  place 
in  different  regions  of  the  animal  and  foreshadow 
the  cellular  events  to  follow.  For  example,  cer- 
tain key  regulatory  proteins  in  Drosophila  come 
to  be  expressed  in  stripes  of  cells  encircling  the 
embryo — stripes  representing  future  segmental 
divisions.  The  second  phase  involves  the  specifi- 
cation of  groups  of  precursor  cells  that  populate 
the  different  tissues  of  the  animal.  For  example. 


genes  such  as  those  of  the  achaete- scute  complex 
(AS-C)  are  activated  in  clusters  of  cells  that  give 
rise  to  the  central  and  peripheral  nervous  sys- 
tems. Finally,  in  the  third  phase,  these  precursor 
cells  divide  and  differentiate,  giving  rise  to  the 
full  complement  of  specialized  cells  that  make 
up  different  tissues  and  organs  and  express  dis- 
tinct structural  genes  to  carry  out  their  individual 
tasks. 

From  molecular  prepattern  to  the  formation  of 
stem  cells  to  the  differentiation  of  their  progeny, 
there  is  a  flow  of  genetic  information.  The  pre- 
pattern specifies  the  spatial  domains  of  genes  that 
are  activated  in  stem  cells,  and  these  genes  in  turn 
regulate  cell  type-specific  gene  expression.  Our 
laboratory  is  interested  in  the  genetic  basis  of  this 
information  flow  and  focuses  on  three  aspects  of 
pattern  formation. 

First,  we  are  studying  the  molecular  regulation 
and  function  of  the  pair-rule  genes,  the  first  genes 
expressed  in  a  segmentally  repeating  prepattern. 
Second,  we  are  studying  how  the  early  prepattern 
genes  regulate  the  expression  of  the  stem  cell- 
specific  proneural  genes  of  the  AS-C.  Third,  we 
are  trying  to  identify  some  of  the  molecular  pre- 
patterns  that  govern  pattern  formation  in  imagi- 
nal  discs — the  distinct  pouches  of  cells  that  are 
set  aside  during  embryogenesis,  proliferate  exten- 
sively during  larval  development,  and  give  rise  to 
adult  structures  such  as  the  wing,  leg,  and  eye. 

In  addition,  we  have  recently  initiated  studies 
of  other  insects  (guided  by  our  knowledge  of  Dro- 
sophila genes)  to  compare  the  molecular  aspects 
of  segmentation  in  species  that  exhibit  different 
styles  of  oogenesis  and  early  development  and  to 
explore  the  genetic  basis  of  wing  development 
and  evolution  among  the  flying  insects. 

The  Pair-Rule  Segmentation  Genes 

During  early  Drosophila  development,  small 
batteries  of  genes  are  expressed  in  rapid  succes- 
sion to  establish  the  segmental  prepattern.  The 
first  genes  expressed  in  a  periodic  pattern  are  the 
pair-rule  genes,  deployed  in  one  stripe  every  two 
segments.  The  striped  patterns  of  certain  pair- 


65 


Genetic  Control  of  Pattern  Formation  in  Drosophila 


rule  genes  are  generated  by  the  aperiodic  pat- 
terns of  gap  proteins,  a  handful  of  transcription 
factors  expressed  in  broad,  partially  overlapping 
regions  of  the  early  embryo. 

Three  aspects  of  pair-rule  gene  regulation  and 
function  are  under  study.  First,  we  wish  to  deter- 
mine the  molecular  mechanisms  of  gap  gene  reg- 
ulation of  the  pair-rule  hairy  gene.  Second,  we 
want  to  know  how  the  pair-rule  genes  regulate 
the  spatial  expression  of  proneural  genes  during 
early  formation  of  the  nervous  system.  And  third, 
we  are  investigating  whether  the  same  sort  of  seg- 
mentation genes  function  in  insects  with  differ- 
ent styles  of  early  development  (e.g.,  beetles  and 
butterflies) .  To  this  end,  we  are  now  isolating  gap 
and  pair-rule  genes  from  other  insects  and  arthro- 
pods to  study  their  embryonic  function. 

Proneural  Genes  and  Early  Neurogenesis 

Once  the  embryo  is  subdivided  by  the  segmen- 
tation genes,  the  formation  of  different  tissues  be- 
gins. One  of  the  earliest  events  in  embryogenesis 
is  the  segregation  of  the  central  nervous  system 
precursor  cells  (neuroblasts)  from  the  overlying 
ectoderm.  The  proneural  genes,  which  are 
named  for  their  role  in  promoting  the  neural  over 
the  epidermal  pathway  in  the  insect  embryonic 
ectoderm,  are  expressed  in  segmentally  repeat- 
ing clusters  of  4-6  cells.  A  single  proneural- 
expressing  neuroblast  will  segregate  from  each 
cluster. 

We  have  recently  determined  that  the  estab- 
lishment of  proneural  gene  expression  in  these 
clusters  is  almost  entirely  regulated  by  up  to 
eight  pair-rule  genes  (all  of  which  appear  to  be 
nuclear  regulatory  proteins)  and  an  unknown 
number  of  genes  acting  along  the  dorsoventral 
axis.  This  demonstrates  that  the  pair-rule  genes 
are  not  simply  involved  in  regulating  the  expres- 
sion of  other  segmentation  genes,  but  also  direct 


the  expression  of  genes  that  govern  the  behavior 
of  small  groups  of  cells.  They  serve  to  integrate 
the  global  regulatory  system  governing  segmenta- 
tion with  the  local  regulatory  system  that  speci- 
fies tissue  architecture. 

The  pair-rule  genes  are  not  sufficient  to  specify 
individual  cell  fates,  only  the  position  of  pro- 
neural cell  clusters.  Interactions  between  cells 
within  each  cluster  determine  their  neural  or  epi- 
dermal fate.  We  have  shown  that  the  so-called 
neurogenic  genes — a  handful  of  loci  that  encode 
a  variety  of  different  proteins  involved  in  cell  sur- 
face interactions,  signal  transduction,  and  gene 
regulation — are  required  to  single  out  just  one 
neuroblast  from  the  proneural  cluster  and  pre- 
vent the  other  cluster  cells  from  entering  the 
neural  pathway. 

Development  and  Evolution  of  Insect 
Appendages 

One  of  the  best  known  but  least  understood 
aspects  of  Drosophila  development  concerns  the 
morphogenesis  of  adult  structures  from  the  larval 
imaginal  tissues.  The  eyes,  wings,  legs,  antennae, 
and  other  structures  are  derived  from  sacs  of  cells 
that  undergo  extensive  growth  and  morphogene- 
sis during  the  larval  and  pupal  stages.  We  are 
most  interested  in  the  development  of  the  wing. 
We  have  recently  shown  that  the  product  of  the 
vestigial  gene  is  precisely  expressed  in  those 
cells  of  the  imaginal  wing  disc  that  will  form  the 
actual  flight  appendage  and  is  not  expressed  in 
the  other  disc  cells  that  will  form  structural  com- 
ponents of  the  thorax.  In  the  absence  of  vestigial 
gene  function,  fruit  flies  are  wingless,  suggesting 
that  vestigial  plays  a  special  role  in  the  develop- 
ment of  wings  from  imaginal  tissues.  Further  stud- 
ies into  the  regulation  of  vestigial  expression  in 
Drosophila  may  provide  some  interesting  clues 
to  the  evolution  and  function  of  the  vestigial 
gene  in  winged  and  wingless  insects. 


66 


The  WMc/ear  vestigial  gene  product  controls  wing  and  haltere  development  in  the  Drosophila  em- 
bryo. Vestigial  protein  is  specifically  expressed  in  cells  of  the  imaginal  discs  that  will  form  the 
flight  appendages — wing  (top  left)  and  haltere  (bottom  left).  The  nonexpressing  cells  of  these 
discs  will  form  structural  parts  of  the  thorax.  During  morphogenesis  the  vestigial-expression 
region  everts  (top  right)  and  forms  the  wing  blade  (bottom  right).  In  mutants  lacking  yestigidA 
protein,  no  wing  or  halteres  form. 

Research  and  photography  by  Jim  Williams  and  Steve  Paddock  in  the  laboratory  of  Sean 
Carroll.  From  Williams,  J.,  and  Carroll,  S.B.  1991.  Genes  Dev  5:2481-2495. 


67 


Human  Disease  Gene  Identification 
and  Correction 


C.  Thomas  Caskey,  M.D. — Investigator 

Dr.  Caskey  is  also  Professor  of  Molecular  Genetics,  Biochemistry,  Medicine,  and  Cell  Biology  at  Baylor 
College  of  Medicine.  He  received  his  M.D.  degree  at  Duke  University.  His  internship  and  residency  training 
were  in  internal  medicine,  also  at  Duke;  his  postdoctoral  training  was  at  NIH,  under  the  supervision 
of  Marshall  Nirenberg.  Dr.  Caskey  is  a  past  president  of  the  American  Society  for  Human  Genetics 
and  was  named  Distinguished  Service  Professor  by  the  Board  of  Trustees  of  Baylor. 


MOLECULAR  genetics  offers  unprecedented 
opportunities  for  the  discovery  of  disease 
genes,  the  development  of  simple  DNA-based 
diagnostics,  and  correction  of  single-gene  de- 
fects. Recent  isolation  of  the  gene  responsible  for 
the  fragile  X  syndrome  has  led  to  improved  diag- 
nostic procedures  and  discovery  of  a  novel  mech- 
anism for  the  occurrence  of  genetic  disease.  Fur- 
ther knowledge  regarding  the  genetic  mechanism 
and  pathology  of  two  other  X  chromosome  dis- 
orders has  also  been  gained.  In  addition,  our  labo- 
ratory has  made  significant  progress  in  the  devel- 
opment of  gene  replacement  therapies  for  three 
diseases. 

Disease  Gene  Identification  and  Diagnosis 

The  gene  that  contains  the  breakage  point  in 
fragile  X  syndrome  has  been  isolated  as  the  result 
of  a  collaboration  with  Stephen  Warren  (HHMI, 
Emory  University)  and  Ben  Oostra  (Erasmus  Uni- 
versity). This  gene  contains  a  three-nucleotide 
sequence  repeated  in  tandem,  and  the  repeat  re- 
gion forms  the  fragile  site  itself.  The  number  of 
repeats  is  variable  and  correlates  with  occurrence 
of  the  fragile  X  disorder.  Normal  individuals  have 
approximately  6-50  repeats;  affected  individ- 
uals, over  200  repeats;  and  those  with  an  inter- 
mediate number  appear  to  harbor  a  "premuta- 
tion" that  is  very  likely  to  give  rise  to  the  full 
mutation  by  inheritance  through  females.  This 
phenomenon  of  genetic  disease  caused  by  se- 
quence amplification  was  previously  unknown, 
but  may  prove  to  play  a  part  in  other  inherited 
disorders. 

Since  the  length  of  this  particular  region  of 
DNA  is  an  indicator  of  the  affected  or  carrier  sta- 
tus of  the  individual,  DNA  diagnosis  of  fragile  X 
syndrome  can  be  performed  by  Southern  analysis, 
and  more  recently  by  the  polymerase  chain  reac- 
tion. These  DNA-based  methods  improve  on  cyto- 
genetic diagnosis,  particularly  in  the  detection  of 
female  carriers  and  unaffected  males  who  never- 
theless can  transmit  the  disorder. 

Study  of  Genetic  Disorders 

The  central  region  of  the  dystrophin  gene  is  a 


"hot  spot"  for  the  deletion  end-points  causing 
Duchenne  and  Becker  muscular  dystrophies.  This 
region  was  studied  in  greater  detail  to  determine 
possible  mechanisms  that  could  explain  why  de- 
letions were  so  frequent.  In  two  independent  pa- 
tients with  Duchenne  muscular  dystrophy,  the 
ends  of  deletion  lay  within  a  transposable  ele- 
ment (a  portion  of  DNA  that  can  move  around  the 
genome)  in  the  dystrophin  gene.  It  is  likely  that 
such  transposable  elements  are  involved  in  other 
deletions  in  this  and  other  genetic  disorders. 

Lesch-Nyhan  syndrome  in  humans  results  from 
lack  of  the  enzyme  hypoxanthine  guanine  phos- 
phoribosyltransferase  (HPRT).  The  neurological 
aspects  of  this  incurable  disorder  are  poorly  un- 
derstood. Study  of  the  syndrome  and  develop- 
ment of  therapeutic  measures  would  greatly  ben- 
efit from  the  generation  of  an  animal  model. 
Transgenic  mice  have  been  engineered  to  lack 
the  uricase  enzyme  (which  is  absent  in  humans), 
and  these  are  being  bred  with  mice  lacking  the 
HPRT  enzyme.  This  strategy  has  potential  for  gen- 
erating a  mouse  model  of  the  human  Lesch-Nyhan 
syndrome. 

Genetic  Correction  of  Inlierited  Disease 

Human  genes  can  now  be  cloned,  placed  into 
defective  (safe)  viral  vectors,  and  transferred  into 
other  cultured  cells,  embryonic  cells,  and  ani- 
mals. These  encouraging  developments  increase 
the  likelihood  of  successful  gene  replacement 
therapy.  Our  laboratory  is  developing  technology 
toward  that  objective  for  three  heritable  diseases. 
Each  disease  offers  different  technical  and  strate- 
gic challenges. 

Adenosine  deaminase  (ADA)  deficiency  is  an 
inherited  autosomal  recessive  disease.  Bone 
marrow  transplantation  is  curative,  but  carries 
the  risk  of  a  graft  rejection .  Administration  of  PEG 
ADA  (the  enzyme  ADA  attached  to  polyethylene 
glycol)  on  a  continuing  basis  has  provided  im- 
provement in  the  immunologic  function  of  pa- 
tients, but  is  not  a  cure. 

The  goal  of  this  laboratory  is  the  development 
of  a  clinical  gene  therapy  protocol  for  ADA  defi- 
ciency. This  will  require  the  treatment  of  human 


69 


Human  Disease  Gene  Identification  and  Correction 


cells  with  supernatant  containing  vector  viruses 
(and  no  cells) ,  rather  than  with  virus-producing 
cells,  because  the  former  method  is  considered  to 
be  safer.  Supernatant  viral  infection  of  bone 
marrow  cells  resulting  in  the  acquisition  of  ADA 
activity  can  be  achieved,  but  high  efficiency  re- 
quires the  presence  of  other  supporting  cells, 
which  is  impractical  in  the  context  of  an  autolo- 
gous bone  marrow  transplant.  Attempts  are  under 
way  to  purify  bone  marrow  stem  cells,  which  are 
responsible  for  the  regeneration  of  the  entire  he- 
mopoietic system,  since  targeting  this  group  of 
cells  would  provide  a  more  efficient  approach  to 
the  permanent  repopulation  of  bone  marrow 
with  genetically  corrected  cells. 

Ornithine  transcarbamylase  (OTC)  deficiency, 
the  most  common  urea  cycle  defect  in  humans,  is 
inherited  in  an  X-linked  recessive  manner.  Coma, 
seizures,  and  retardation  are  the  result  of  hyper- 
ammonemia secondary  to  the  enzyme  deficiency. 
Liver  transplantation  provides  a  once-per-lifetime 
cure,  although  again  with  the  potential  compli- 
cations of  a  graft-versus-host  reaction.  This  condi- 
tion is  poorly  managed  through  dietary  protein 
restriction  and  medical  therapy,  making  the  dis- 
order another  excellent  candidate  for  gene 
therapy. 

Two  mouse  models  of  the  human  disorder  are 
available  for  developing  gene  therapy.  In  this  lab- 
oratory, the  OTC  deficiency  in  one  of  the  strains 


of  mice  has  been  corrected  by  retroviral  delivery 
of  the  human  OTC  gene  to  the  small  intestine.  In 
current  studies  toward  the  development  of  hu- 
man gene  therapy  for  OTC  deficiency,  retrovi- 
ruses and  defective  (safe)  adenovirus  vectors  are 
used  to  transfer  the  human  gene  to  cultured  liver 
cells. 

Duchenne  muscular  dystrophy  (DMD)  is  a  se- 
vere disorder,  also  inherited  in  an  X-linked  reces- 
sive manner.  Deficiency  of  the  protein  dystro- 
phin leads  to  multiple  muscle  abnormalities  and 
eventually  death.  The  gene  is  large  and  complex. 
Several  mouse  models  of  DMD  are  available  for 
study,  and  the  dystrophin  deficiency  in  one  of 
these  strains  has  been  corrected  by  introduction 
of  a  construct  expressing  mouse  dystrophin. 

In  collaboration  with  Helen  Blau  (Stanford 
University),  myoblasts  (precursor  muscle  fiber 
cells)  are  being  isolated  from  very  young  patients 
with  DMD.  Different  constructs  will  be  tested  for 
the  transfer  of  the  dystrophin  gene  and  for  creat- 
ing gene-corrected  myoblasts  that  can  be  re- 
turned to  the  patient.  The  gene  is  so  large  that 
currently  used  viral  vectors  will  not  accommo- 
date it.  Approaches  being  used  to  overcome  this 
problem  include  modifying  the  vectors,  truncat- 
ing the  gene,  and  using  physical  methods  (that  do 
not  present  a  size  limitation)  for  transferring  the 
gene,  such  as  electroporation  or  ballistic  gene 
transfer  (using  the  Du  Pont  "gene  gun"). 


70 


Enzymatic  RNA  Molecules  and  the  Structure 
of  Chromosome  Ends 


Thomas  R.  Cech,  Ph.D. — Investigator 

Dr.  Cech  is  also  American  Cancer  Society  Professor  at  the  University  of  Colorado  at  Boulder  and  Professor 
of  Biochemistry,  Biophysics,  and  Genetics  at  the  University  of  Colorado  Health  Sciences  Center,  Denver. 
He  received  his  B.A.  degree  in  chemistry  from  Grinnell  College  and  his  Ph.D.  degree  in  chemistry  from  the 
University  of  California,  Berkeley.  His  postdoctoral  work  in  biology  was  conducted  in  the  laboratory  of 
Mary  Lou  Pardue  at  the  Massachusetts  Institute  of  Technology.  Dr.  Cech  is  a  member  of  the  National 
Academy  of  Sciences.  Among  his  many  honors  are  the  Lasker  Award  and  the  1989  Nobel  Prize  in  chemistry. 


THE  nucleic  acids,  DNA  and  RNA,  are  best 
known  as  the  information-carrying  molecules 
of  a  living  cell.  For  example,  a  molecule  of  DNA 
or  RNA  might  carry  the  instructions  to  build  myo- 
sin, a  protein  involved  in  muscle  movement, 
or  pepsin,  a  protein  enzyme  that  helps  digest 
food.  Our  laboratory  is  investigating  the  non- 
informational  roles  of  nucleic  acids,  situations  in 
which  a  nucleic  acid  molecule  has  an  important 
cellular  function  other  than  encoding  a  protein. 
In  the  area  of  RNA  catalysis,  we  wish  to  under- 
stand how  a  folded  RNA  structure  can  have  enzy- 
matic activity.  In  the  area  of  chromosome  func- 
tion, we  are  characterizing  the  DNA  and 
associated  protein  necessary  for  proper  mainte- 
nance of  a  chromosome  end.  In  both  projects, 
chemical  and  biological  approaches  are  com- 
bined for  fuller  analysis  of  a  biochemical 
problem. 

RNA  Catalysis 

A  cell  must  orchestrate  thousands  of  chemical 
reactions  in  order  to  live,  grow,  and  respond  to  its 
environment.  These  chemical  reactions,  rarely 
spontaneous,  are  usually  catalyzed  by  macromole- 
cules  called  enzymes.  It  was  long  thought  that  all 
enzymes  were  proteins.  More  recently  we  and 
others  have  found  that  a  nucleic  acid,  RNA,  can  in 
some  cases  act  as  an  enzyme. 

The  finding  of  RNA  catalysis  has  several  impli- 
cations. First,  it  means  that  RNA  is  not  restricted 
to  being  a  passive  carrier  of  genetic  information 
but  can  participate  actively  in  directing  cellular 
biochemistry.  In  particular,  many  RNA-process- 
ing  reactions  are  at  least  partly  catalyzed  by  RNA. 
Second,  the  study  of  RNA  enzymes,  called  ribo- 
zymes,  may  reveal  hitherto  unknown  mecha- 
nisms of  biologic  catalysis.  Third,  ribozymes  can 
be  used  as  sequence-specific  RNA  cleavage  agents 
in  vitro,  providing  useful  tools  for  study  of  RNA 
biochemically.  Finally,  on  a  more  speculative 
note,  RNA  catalysis  has  the  potential  of  providing 
new  therapeutic  agents.  For  example,  ribozymes 
efficiently  cleave  and  thereby  destroy  viral  RNAs 
under  controlled  laboratory  conditions,  suggest- 
ing that  they  might  be  able  to  inactivate  viruses  in 
a  living  organism. 


Many  of  our  studies  of  RNA  catalysis  concern 
the  Tetrahymena  ribozyme,  named  for  the  sin- 
gle-celled animal  from  which  it  was  originally 
isolated.  This  RNA  enzyme  is  capable  of  cleaving 
other  RNA  molecules  (substrates)  in  a  sequence- 
specific  manner.  One  of  our  objectives  is  to  un- 
derstand the  mechanisms  by  which  this  RNA  mol- 
ecule acts  as  a  catalyst.  A  second  goal,  in  the  area 
of  structural  biology,  is  to  obtain  a  detailed  pic- 
ture of  the  active  site  of  this  ribozyme. 

Last  year  we  demonstrated  that  this  ribozyme 
uses  a  novel  mode  of  RNA  recognition  to  bind  its 
RNA  substrate.  In  addition  to  the  well-established 
mode  of  binding  by  formation  of  base  pairs  (as  in 
the  "ladder"  of  the  DNA  double  helix),  the  ribo- 
zyme also  binds  two  of  the  sugar  groups  that  form 
the  "backbone"  of  the  RNA  substrate  chain.  We 
have  now  located  the  other  partner  in  this  inter- 
action, a  specific  adenine  base  within  the  RNA's 
catalytic  core.  In  addition  to  support  from  HHMI, 
a  grant  from  the  National  Institutes  of  Health 
supported  a  graduate  student  working  on  this 
project. 

In  a  separate  study,  nucleic  acid  chemistry  was 
used  to  change  individual  oxygen  atoms  to  sulfur 
atoms  near  the  cleavage  site  in  the  RNA  substrate. 
With  one  of  the  sulfur-substituted  RNAs,  we  ob- 
served a  change  in  metal-ion  specificity  in  the 
cleavage  reaction.  Thus  we  believe  we  have  lo- 
cated one  of  the  long-postulated  active-site  metal 
ions  and  have  established  that  this  RNA,  like  some 
protein  enzymes,  is  a  metalloenzyme. 

In  the  structural  area,  we  have  developed  a 
new  technique  for  identifying  parts  of  the  folded 
RNA  molecule  that  are  in  proximity  to  some 
known  site.  We  attach  a  controllable  agent  of  de- 
struction to  a  small  molecule  known  to  dock  in 
the  ribozyme's  active  site,  allow  it  to  bind,  initi- 
ate the  chemical  reaction,  and  map  the  sites  of 
damage.  The  method  has  revealed  convincing  in- 
formation about  the  higher-order  folding  of  this 
RNA  catalyst. 

Telomere  Structure 

Unlike  the  circular  chromosomes  of  bacteria, 
the  chromosomes  found  in  the  nuclei  of  higher 


71 


Enzymatic  RNA  Molecules  and  the  Structure  of  Chromosome  Ends 


organisms  are  linear  DNA  molecules.  The  ends  of 
linear  chromosomes,  called  telomeres,  must  be 
protected  from  degradation,  and  special  features 
are  required  to  ensure  their  replication.  We  are 
studying  telomere  structure  and  function,  with 
special  emphasis  on  the  protein  that  caps  off  the 
ends  of  each  chromosome. 

Most  cells  have  only  a  few  dozen  chromosomes 
and  therefore  not  many  telomeres.  We  chose  to 
work  with  a  ciliated  protozoan,  Oxytricha  nova, 
because  it  has  26  million  miniature  chromo- 
somes per  cell.  This  facilitated  our  earlier  purifi- 
cation of  the  telomeric  protein  and  our  cloning 
and  sequencing  of  the  genes  that  encode  it.  Simi- 
larities in  DNA  sequences  in  the  telomeres  of  Oxy- 
tricha and  those  of  higher  cells,  including  human 


cells,  give  us  reason  to  believe  that  our  findings 
in  Oxytricha  will  be  of  some  generality. 

Last  year  we  used  genetic  engineering  methods 
to  produce  the  two  subunits  of  the  telomere- 
binding  protein  in  bacteria.  This  has  now  allowed 
us  to  assess  the  individual  contributions  of  the 
two  subunits  to  the  protein-DNA  complex  (work 
supported  mainly  by  a  grant  from  the  National 
Institutes  of  Health).  It  has  also  allowed  the  puri- 
fication of  large  amounts  of  the  protein,  enabling 
attempts  to  crystallize  this  complex,  in  collabora- 
tion with  Steve  Schultz's  research  group  (Univer- 
sity of  Colorado).  If  successful,  crystallization 
could  lead  to  structure  determination  via  x-ray 
diffraction,  providing  an  atomic-resolution  pic- 
ture of  the  end  of  a  chromosome. 


72 


Molecular  and  Cellular  Physiology  of  Acute 
Inflammatory  Cytokines 


David  D.  Chaplin,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Chaplin  is  also  Associate  Professor  of  Medicine,  Genetics,  and  Molecular  Microbiology  at  Washington 
University  School  of  Medicine  and  Assistant  Physician  at  Barnes  Hospital,  St.  Louis.  He  received  his  A.B. 
degree  in  biochemistry  from  Harvard  University  and  his  M.D.  and  Ph.D.  degrees  in  cellular  and 
developmental  biology  from  Washington  University.  Following  a  residency  in  internal  medicine  at 
Parkland  Memorial  Hospital,  Dallas,  he  received  postdoctoral  training  in  genetics  at  Harvard  Medical 
School  with  Jonathan  Seidman.  He  then  returned  to  Washington  University  as  a  faculty  member. 


PROPER  regulation  of  immune  defenses  re- 
quires the  coordinate  action  of  an  array  of 
different  host  responses.  Many  of  these  responses 
depend  for  their  regulation  on  the  interactions  of 
several  different  types  of  immune  cells.  Some  of 
these  interactions  are  mediated  by  physical  con- 
tact between  the  participating  cells.  Others,  how- 
ever, are  mediated  by  soluble  proteins  that  are 
released  from  one  cell  to  act  by  binding  to  spe- 
cific receptors  on  other  cells.  These  soluble  medi- 
ators have  been  designated  cytokines. 

Three  cytokines  appear  particularly  important 
for  the  initiation  of  acute  inflammatory  and  im- 
mune responses:  interleukin-1  (IL-1),  tumor  ne- 
crosis factor  (TNF),  and  interleukin-6  (IL-6). 
IL-1,  TNF,  and  IL-6  are  pleiotropic  cytokines. 
They  are  produced  by  a  wide  variety  of  cells,  and 
they  act  on  many  tissues,  showing  a  broad  array  of 
activities.  In  general,  these  activities  are  proin- 
flammatory, favoring  first  the  recruitment  of 
white  blood  cells  to  sites  of  inflammation,  then 
activation  of  these  cells  to  express  their  full  ef- 
fector potential.  This  recruitment  and  activation 
is  absolutely  necessary  for  host  defense  against 
invading  microbes;  however,  when  it  occurs  inap- 
propriately, it  can  lead  to  acute  and  chronic  dam- 
age to  self  tissues. 

Although  a  great  deal  of  information  has  been 
accumulated  concerning  the  activities  of  these 
cytokines  when  added  to  cultured  cells  or  when 
injected  into  live  animals,  little  is  known  about 
their  true  physiological  function  in  normal  host 
responses.  In  the  case  of  IL-1,  this  uncertainty  is 
underscored  by  studies  of  the  cytokine's  struc- 
ture. First,  biochemical  and  molecular  genetic 
analysis  of  IL- 1  has  established  that  there  are  actu- 
ally two  forms  of  IL- 1  (IL- 1  a  and  IL- 1  /?) ,  each  the 
product  of  a  discrete  gene.  The  relative  produc- 
tion of  each  form  is  variable,  with  some  types  of 
cells  producing  primarily  IL- 1  a  and  some  primar- 
ily IL-1/3.  Both  forms  are  synthesized  in  the  IL-1- 
producing  cell  as  a  larger  precursor  molecule 
(pro-IL-la  and  pro-IL-1/3),  and  both  are  cleaved 
to  a  smaller  mature  form  that  is  found  outside  the 
cell.  In  vitro,  mature  IL-1  a  and  -1|8  show  appar- 
ently identical  activities.  Surprisingly,  the  two 


cytokines  share  only  25  percent  amino  acid  se- 
quence identity.  Early  studies  from  our  labora- 
tory showed  that  the  genes  encoding  the  two  IL- 1 
proteins  were  generated  by  duplication  of  an  an- 
cestral IL-1 -like  gene,  with  subsequent  extensive 
mutation  to  produce  the  existing  IL-1  a  and  -1|8 
genes.  We  interpret  the  separate  lineages  of  the 
two  genes  to  indicate  that  there  has  been  selec- 
tive pressure  for  the  independent  evolution  of 
each  gene.  Although  this  pressure  probably  acts 
at  the  level  of  unique  functions  for  each  of  the 
two  IL-1  molecules,  in  vitro  studies  have  not  re- 
vealed any  functional  diff^erences  between  the 
two  cytokines.  This  indicates  to  us  that  existing 
in  vitro  models  do  not  adequately  represent  the 
normal  physiology  of  IL-1  and  that  new  models 
must  be  explored  to  disclose  the  functional  dif- 
ferences that  must  exist  in  vivo. 

Just  as  there  are  fundamental  questions  con- 
cerning the  unique  functions  of  each  of  the  IL- 1 
proteins,  there  are  basic  questions  concerning 
the  mechanism  by  which  these  proteins  exit  the 
cell  to  perform  their  extracellular  functions. 
Normally  proteins  that  are  destined  to  act  outside 
the  cell  contain  a  characteristic  signal  peptide,  a 
short  amino  acid  sequence  that  directs  them  into 
the  cellular  secretory  pathway.  Neither  IL- 1  pre- 
cursor protein  contains  a  secretion  signal  pep- 
tide. Additionally,  unlike  conventionally  se- 
creted proteins,  pro-IL-la  and  -1/5  reside  in  the 
cellular  cytoplasm,  not  associated  with  any  mem- 
branous cellular  compartment.  Consequently  it 
has  been  suggested  that  the  IL- 1  proteins  are  re- 
leased via  a  novel  secretory  pathway. 

Recent  studies  from  our  laboratory  have 
yielded  new  insights  into  the  mechanism  of  lL-1 
release  and  maturation.  In  these  studies  we  have 
shown  that  release  of  IL-1  a  and  -1/8  from  acti- 
vated cells  does  not  involve  a  unique  secretory 
mechanism  but  occurs  as  a  consequence  of  injury 
to  the  IL-1 -producing  cell.  This  conclusion  is 
based  on  the  observation  that  IL-1  release  is  ac- 
companied by  parallel  release  of  other  cytoplas- 
mic proteins,  such  as  the  enzyme  lactate  dehydro- 
genase (LDH).  Agents  that  enhance  IL-1  release 
either  induce  increased  IL- 1  synthesis  or  increase 


73 


Molecular  and  Cellular  Physiology  of  Acute  Inflammatory  Cytokines 


the  degree  of  cellular  injury  and  cause  greater 
release  of  LDH.  Conversely,  agents  that  reduce 
IL-1  release  either  reduce  IL-1  synthesis  or  en- 
hance cell  viability. 

Although  the  release  of  IL- 1  from  IL- 1  -produc- 
ing cells  is  not  via  a  specific  release  pathway,  the 
outcome  of  release  can  be  modulated.  This  is 
demonstrated  by  studies  in  which  IL-1 -produc- 
ing cells  are  deliberately  injured.  In  the  most  gen- 
eral terms,  there  are  two  types  of  cellular  injury. 
One  type  occurs  when  vital  cellular  functions  are 
interrupted,  leading  to  cellular  necrosis.  This  oc- 
curs as  a  consequence  of  many  stress  injuries,  in- 
cluding hypoxia,  alterations  in  local  pH,  ex- 
tremes of  temperature,  and  physical  attack  by 
infectious  lytic  viruses.  Morphologically,  ne- 
crotic cells  exhibit  disruptions  in  their  plasma 
membrane  and  disintegration  of  the  intracellular 
organelles.  The  consequence  of  necrosis  is  the 
release  into  the  extracellular  space  of  all  of  the 
soluble  cellular  components.  As  expected,  when 
an  IL-1 -producing  cell  sustains  a  necrotic  injury, 
the  IL- 1  precursors  are  released  along  with  all  of 
the  other  cellular  components;  however,  there  is 
failure  of  processing  of  the  IL-1|8  precursor  to  its 
mature  form.  This  failure  is  important,  because 
maturation  is  required  for  the  IL-l/?  precursor  to 
acquire  receptor-binding  activity  (and,  conse- 
quently, for  the  acquisition  of  all  of  the  known 
IL-1  functional  activities). 

In  contrast  to  cellular  necrosis,  cell  death  can 
occur  in  a  regulated  fashion.  Normal  develop- 
ment and  tissue  remodeling  frequently  require 
removal  of  otherwise  viable  cells.  This  occurs  by 
activating  the  pathway  of  programmed  cell  death 


leading  to  apoptosis.  Morphologically,  apoptosis 
is  very  different  from  necrosis,  with  apoptotic 
cells  showing  retention  of  the  integrity  of  their 
plasma  membranes  and  maintenance  of  the  struc- 
ture of  intracellular  organelles.  Portions  of  the 
cell  are  released  as  membrane-coated  blebs  and 
are  subsequently  ingested  by  phagocytic  cells 
and  digested.  Apoptosis  permits  the  termination 
of  the  life  of  a  cell  with  minimal  release  of  cellu- 
lar debris  into  the  extracellular  environment. 
This  pathway  is  activated  during  many  stages  of 
embryological  development  and  to  initiate  nor- 
mal cell  turnover.  In  the  immune  system,  re- 
moval of  self-reactive  T  lymphocytes  in  the  thy- 
mus occurs  by  apoptosis,  as  does  the  death  of 
target  cells  attacked  by  cytotoxic  lymphocytes. 
When  this  pathway  of  programmed  cell  death  is 
activated  in  macrophages  expressing  the  IL-1 
precursors,  there  is  striking,  rapid,  and  complete 
maturation  and  release  of  both  forms  of  IL- 1 . 
Thus,  in  cells  expressing  only  IL-l/?  (such  as 
blood  monocytes  and  certain  cells  in  the  spleen 
and  thymus),  apoptosis — in  contrast  to  necrosis 
— affords  an  efficient  method  for  generating 
bioactive  IL-1 . 

These  results  have  given  us  a  new  appreciation 
of  IL-1  as  a  physiological  signal  of  cell  and  tissue 
injury.  IL- 1  does  not  appear  to  be  released  from 
the  cell  that  produces  it  unless  a  significant  cell 
injury  occurs.  If  the  injury  is  necrotic  in  nature, 
then  IL-1/3  is  released  in  only  its  inactive  precur- 
sor form.  If,  on  the  other  hand,  the  injury  leads  to 
apoptosis,  then  mature  bioactive  IL-1/3  is  released 
in  a  form  ready  to  activate  the  systemic  responses 
that  lead  to  tissue  repair. 


74 


Hormonal  Regulation  of  Gene  Expression 


William  W.  Chin,  M.D. — Investigator 

Dr.  Chin  is  also  Associate  Professor  of  Medicine  at  Harvard  Medical  School  and  Senior  Physician  at 
Brigham  and  Women's  Hospital,  Boston.  He  obtained  his  undergraduate  degree  in  chemistry  from 
Columbia  College  and  his  M.D.  degree  from  Harvard  Medical  School.  His  postdoctoral  work  was  performed 
with  Jacob  Maizel  and  Philip  Leder  at  NIH  and  with  Joel  Habener  at  Massachusetts  General  Hospital, 
Boston.  His  awards  include  the  Bowditch  Lectureship  Award  of  the  American  Physiological  Association, 
the  Van  Meter- USV  Award  of  the  American  Thyroid  Association,  and  the  Outstanding  Investigator's  Award 
from  the  American  Federation  for  Clinical  Research. 


HORMONES,  key  players  in  the  endocrine  and 
nervous  systems,  are  produced  by  special- 
ized tissues  in  the  body,  effect  extracellular  com- 
munication, and  regulate  cellular  function.  Our 
recent  studies  have  considered  the  molecular 
mechanisms  of  gene  regulation  by  a  specific  hor- 
mone, thyroid  hormone. 

Thyroid  Hormone  Action 

Our  early  work  has  focused  on  the  regulation 
of  the  thyrotropin  (thyroid-stimulating  hormone 
[TSH])  subunit  genes  by  thyroid  hormones.  TSH  is 
a  polypeptide  hormone  that  is  produced  and  se- 
creted by  a  single  cell  type  in  the  anterior  pitu- 
itary gland.  It  comprises  two  different  sugar- 
containing  polypeptide  subunits,  a  and  /?,  which 
are  encoded  by  genes  located  on  different  chro- 
mosomes. Importantly,  TSH  stimulates  the  thy- 
roid gland  to  produce  the  thyroid  hormones,  T3 
and  T4.  These  modified  amino  acids  regulate  me- 
tabolism and  gene  expression  in  almost  every  cell 
of  the  body.  To  maintain  a  constant  level  of  T3  and 
T4  in  the  bloodstream,  these  hormones  act  back 
on  the  pituitary  to  decrease  TSH  production,  in- 
cluding subunit  gene  expression,  and  secretion. 
Hence  TSH  and  thyroid  hormones  are  involved  in 
a  classic  negative-feedback  regulatory  system. 

To  understand  further  the  molecular  mecha- 
nisms involved  in  the  negative  regulation  of  TSH 
synthesis  by  thyroid  hormones  at  the  transcrip- 
tion level,  we  have  isolated  and  analyzed  the 
genes  encoding  the  a-  and  /5-subunits  of  TSH  in 
the  rat  and  identified  putative  thyroid  hormone- 
responsive  elements  (TREs).  These  TREs,  which 
mediate  a  negative  regulation  by  the  thyroid  hor- 
mones and  thyroid  hormone  receptors  (TRs), 
contain  several  consensus  TR-binding  half-sites 
in  a  direct  repeat  orientation  and  are  located  im- 
mediately upstream  of  the  TATA  box  (a-subunit) 
or  near  the  start  of  transcription  (TSH;3  subunit) . 

Multiple  Thyroid  Hormone  Receptors 
and  Other  Nuclear  Factors 

Thyroid  hormones,  in  general,  act  at  the  cellu- 
lar level  by  entering  the  cell  either  as  Tj  or  T4.  T4 


may  be  converted  to  T3  in  certain  tissues,  such  as 
the  pituitary.  T3  then  enters  the  nucleus,  where  it 
interacts  with  the  TR,  a  protein  encoded  by  the 
proto-oncogene  c-erbA.  The  T3-TR  complex  then 
binds  directly  to  cis-DNA  elements  within  thyroid 
hormone-responsive  genes  to  activate  the  appro- 
priate responses. 

The  TRs  are  encoded  by  two  genes,  a  and  /3, 
each  expressing  at  least  two  related  molecules 
obtained  by  alternative  promoter  choice  and 
splicing.  The  rat  TRa  gene  encodes  a  bona  fide 
TR,  TRal ,  and  a  related  but  non-Tj-binding  form, 
c-erhAa2.  The  rat  TRjS  gene  encodes  two  func- 
tional TRs,  TRjSl  and  TR/32,  which  are  identical 
except  for  different  regions  amino  terminal  to  the 
DNA-binding  domain.  Remarkably,  TR/32  was 
most  highly  expressed  in  the  pituitary  gland.  This 
observation  has  potential  physiological  relevance 
because  of  the  major  role  of  thyroid  hormones  in 
regulating  important  hormone  genes  in  the  pitu- 
itary, including  TSH.  The  finding  also  stands  in 
contrast  to  the  general  tissue  distribution  of  the 
other  receptor  forms. 

We  recently  demonstrated  the  existence  of  an- 
other nuclear  protein(s)  that  can  augment  the 
binding  of  TRs  to  various  TREs.  This  ubiquitous 
60-  to  65-kDa  protein  (TRAP;  TR  auxiliary  pro- 
tein) interacts  with  TR  to  form  a  heterodimer  and 
also  binds  specific  sequences  within  the  TRE.  Sev- 
eral regions  of  the  carboxyl-terminal  or  ligand- 
binding  domain  of  the  TR  are  important  for  this 
heterodimerization.  Preliminary  results  show 
that  the  TR-TRAP  interaction  may  be  critical  in 
thyroid  hormone-mediated  transactivation.  Fur- 
thermore, the  recent  identification  of  the  reti- 
noid X  receptor  as  a  potential  TRAP  by  several 
groups  fuels  additional  excitement  in  this  area. 

In  summary,  the  thyroid  hormone  receptor  fam- 
ily is  complex.  At  least  three  biologically  active 
forms  are  expressed  in  a  tissue-specific  fashion, 
and  another  form  may  have  an  important  role  in 
modulating  the  effects  of  the  others.  Knowledge 
of  the  functions  of  these  receptor  isoforms  and 
their  interactions  with  other  nuclear  factors  will 
be  critical  for  our  full  understanding  of  thyroid 
hormone  action. 


75 


Hormonal  Regulation  of  Gene  Expression 


Thyroid  Hormone  Action 

at  the  Post-transcriptional  Level 

In  addition  to  the  well-known  transcriptional 
effect  of  thyroid  hormone  on  TSH  synthesis,  we 
have  recently  examined  the  effect  of  thyroid  hor- 
mone on  the  half-lives  of  TSH  subunit  mRNAs  in 
vitro  and  in  vivo.  The  turnover  rate  of  the  TSH|8 
mRNA  was  greater  in  the  presence  of  thyroid  hor- 
mone. This  phenomenon  was  associated  with  a 
shortening  of  the  poly(A)  tail  of  the  mRNA.  These 
events  occurred  in  a  dose-  and  time-dependent 
fashion.  Thus  thyroid  hormone  reduces  the  half- 
life  of  the  TSH/3  mRNA  and  causes  a  concomitant 
decrease  in  its  poly  (A)  size.  These  data  indicate 
that  thyroid  hormones  may  also  exert  their  effects 


on  gene  expression  at  the  post-transcriptional 
level. 

Summary 

Our  work  has  focused  on  the  molecular  mecha- 
nisms of  thyroid  hormone  action,  with  a  focus  on 
the  regulation  of  TSH  gene  expression  by  thyroid 
hormones,  including  the  cis-elements  and  trans- 
factors  that  are  involved  in  these  processes.  The 
effects  of  these  hormones  are  manifest  at  both  the 
transcriptional  and  post-transcriptional  levels. 
We  hope  that  our  efforts  will  provide  insight  into 
the  hormonal  regulation  of  gene  expression  and 
cellular  function  in  normal  and  pathological  en- 
docrine states  and  in  cancer. 


76 


Technologies  for  Genome-sequencing  Projects 


George  M.  Church,  Ph.D. — Assistant  Investigator 

Dr.  Church  is  also  Assistant  Professor  of  Genetics  at  Harvard  Medical  School.  He  received  his  B.A.  degree  in 
zoology  and  chemistry  from  Duke  University  and  his  Ph.D.  degree  in  biochemistry  and  molecular  biology 
from  Harvard  University.  Before  moving  to  Harvard  Medical  School,  Dr.  Church  was  a  scientist  at  Biogen 
Research  Corporation  and  a  Life  Sciences  Research  Foundation  Fellow  in  the  Department  of  Anatomy 
at  the  University  of  California,  San  Francisco. 


THE  study  of  the  linear  sequence  of  bases  in 
genomic  DNA  and  messenger  RNA  is  steadily 
gaining  recognition,  in  part  as  a  result  of  the  in- 
creasing ease  and  advantage  of  using  shared  com- 
puter databases  to  find  connections  among  dis- 
tant concepts  and  distant  biological  systems.  For 
example,  connections  have  been  found  between 
human  oncogenes  and  yeast  transcription  factors, 
between  differentiation  antigens  and  bacterial 
chaperone  proteins,  and  between  developmental 
regulatory  genes  and  bacterial  DNA-binding 
proteins. 

Unfortunately,  these  database  searches  are  fre- 
quently unsuccessful,  because  not  all  classes  of 
genetic  elements  are  represented.  The  complete 
sequence  of  a  few  small  genomes  should  rectify 
this.  Sequencing  projects  have  begun  for  ge- 
nomes of  various  bacteria  {Mycoplasma,  Myco- 
bacteria, Escherichia,  and  Thermococcus) ,  a 
yeast  (Saccharomyces) ,  a  plant  (Arabidopsis) , 
and  a  worm  (Caenorhabditis} ,  chosen  for  their 
well-studied  genetics,  their  small  genome  sizes, 
and  their  representation  of  all  major  branches  of 
the  evolutionary  tree.  The  genome  closest  to 
completion  is  that  of  Escherichia,  with  about  38 
percent  of  its  4.7  million  base  pairs  already  in  the 
database,  through  the  effort  of  2,000  researchers. 

To  improve  the  accuracy  and  efficiency  of 
these  projects,  we  have  developed  new  sequenc- 
ing technologies.  One,  called  multiplex  se- 
quencing, is  a  way  of  keeping  a  large  set  of  DNA 
fragments  as  a  precise  mixture  throughout  most 
of  the  steps  of  sequencing.  Because  each  mixture 
can  be  handled  with  the  same  effort  as  a  single 
sample  in  previous  methods,  more  fragments  can 
be  handled. 

The  mix  is  deciphered  by  strategically  tagging 
the  fragments  at  the  beginning  with  unique  bits 
of  DNA  and  then,  at  the  end,  hybridizing  to  the 
sequencing  reactions  complementary  bits  of  DNA 
that  have  been  spread  out  by  size  and  immobi- 
lized on  large  membranes.  This  method  also  im- 
proves the  accuracy,  since  the  mixtures  contain 


internal  standards  of  known  sequence  that  help 
in  the  computer  analysis  of  the  film  data. 

The  number  of  probings  obtainable  per  mem- 
brane represents  the  increased  efficiency  factor 
of  this  method.  This  number  exceeds  50  now 
(the  higher  the  better)  and  is  likely  to  increase. 
We  have  designed  and  tested  devices  to  facilitate 
most  of  the  steps  in  multiplex  sequencing,  in- 
cluding DNA  preparation,  sequencing  reactions, 
gel  loading,  hybridization,  film  exposure,  and 
film  reading.  All  of  these  devices  have  been  ap- 
plied to  collect  over  1  million  bases  of  raw  data 
and  are  undergoing  further  development.  Multi- 
plexing has  also  allowed  chemiluminescent  de- 
tection to  replace  the  radioactivity  normally  used 
in  DNA  sequencing,  reducing  exposure  times  1 0- 
fold.  To  fill  in  specific  gaps  in  the  sequences,  we 
have  devised  multiplex  oligonucleotide  synthe- 
sis for  use  in  multiplex  DNA  sequence  walking 
strategies. 

Toward  the  goal  of  modeling  cell  structure  and 
gene  expression,  we  have  searched  for  abundant 
cellular  proteins  that  have  nonetheless  eluded 
the  extensive  biochemical  and  genetics  studies  of 
Escherichia  coli.  This  has  been  done  by  systemati- 
cally correlating  amino-terminal  protein  se- 
quence data  obtained  from  two-dimensional 
gel  spots  with  the  DNA  sequence  and  two- 
dimensional  gel  databases.  Of  300  sequences  de- 
termined so  far,  over  50  are  candidates  for  such 
major  novel  proteins. 

We  have  extended  our  methods  for  detecting 
in  vivo  molecular  interactions  by  analyzing  the 
protection  of  individual  DNA  bases  from  enzy- 
matic methylation.  DNA  protein  interactions  in- 
volved in  cAMP  and  pyrimidine  feedback  regula- 
tion have  been  studied  in  this  way. 

In  the  future,  with  new  sequencing  technolo- 
gies such  as  automated  multiplex  sequencing, 
with  examples  of  most  of  the  basic  genetic  mod- 
ules, and  with  an  eye  to  sequence  elements  con- 
served among  species,  the  analysis  and  modeling 
by  investigators  worldwide  of  human  sequences 
and  genetics  should  become  more  manageable. 


77 


Molecular  Genetics  of  Limb  Development 
in  Drosophila 


Stephen  M.  Cohen,  Ph.D. — Assistant  Investigator 

Dr.  Cohen  is  also  Assistant  Professor  in  the  Department  of  Cell  Biology  and  in  the  Institute  for  Molecular 
Genetics  at  Baylor  College  of  Medicine.  He  obtained  his  Ph.D.  degree  at  Princeton  University,  working 
with  Malcolm  Steinberg.  He  did  postdoctoral  work  with  Harvey  Lodish  at  the  Whitehead  Institute 
and  with  Herbert  Jackie  at  the  Max  Planck  Institute  for  Developmental  Biology,  Tiibingen, 
and  at  the  University  of  Munich. 


MY  laboratory  is  interested  in  the  processes 
by  which  genetic  information  can  be  used 
to  organize  the  three-dimensional  pattern  of  the 
body  during  embryonic  development.  The  partic- 
ular problem  on  which  we  have  focused  involves 
the  organization  of  the  limbs  in  the  fruit  fly  Dro- 
sophila. We  would  like  to  understand  the  genetic 
and  molecular  mechanisms  that  the  fly  embryo 
uses  to  determine  where  the  limbs  will  develop 
with  respect  to  the  rest  of  the  body  pattern.  We 
are  investigating  how  the  appropriate  embryonic 
cells  become  committed  to  develop  as  limb  pre- 
cursors and  subsequently  how  these  cells  cooper- 
atively organize  the  spatial  pattern  of  the  leg. 

Specification  of  Limb  Cell  Identity 

Using  classical  genetics,  we  have  identified  the 
gene  Distal-less,  which  seems  to  function  as  a 
critical  genetic  switch  that  initiates  limb  develop- 
ment in  the  embryo.  Embryos  that  lack  Distal- less 
gene  function  do  not  develop  larval  or  adult 
limbs.  These  observations  indicate  that  Distal- 
less  activity  is  critically  required  but  do  not  tell 
us  what  the  gene  is  actually  doing.  Using  molecu- 
lar probes,  we  can  visualize  the  RNA  product  of 
the  Distal-less  gene  in  the  embryo  and  in  the  de- 
veloping adult  limbs.  All  of  the  cells  that  will  go 
on  to  form  limb  structures  express  Distal-less.  An 
additional  genetic  experiment  indicates  that 
these  cells  need  the  Distal- less  gene  product  to 
be  limb  cells.  If  the  gene  is  removed  from  a  group 
(or  clone)  of  cells  that  were  already  committed 
to  develop  into  limb  structures,  these  cells  in- 
stead become  body  wall  cells.  Distal-less  there- 
fore specifies  their  identity  as  limb  cells.  Distal- 
less  is  a  member  of  a  family  of  important 
regulatory  genes  that  encode  a  sequence-specific 
DNA-binding  motif  called  the  homeodomain. 
Distal-less  therefore  can  be  presumed  to  define 
cell  identity  by  regulating  the  expression  of  other 
genes.  We  wish  to  identify  the  transcriptional  tar- 
gets through  which  Distal-less  acts. 

How  Does  the  Embryo  Position  Its  Limbs? 

The  critical  first  event  in  limb  development  is 


determining  the  position  at  which  the  legs  will 
develop.  We  are  using  a  combination  of  genetic 
and  molecular  methods  to  identify  the  source  of 
the  instructions  specifying  the  identity  of  the  leg 
cells  and  the  nature  of  the  response  that  this  sig- 
nal elicits.  In  the  Drosophila  embryo,  the  legs 
begin  as  clusters  of  cells.  These  clusters  are  estab- 
lished at  a  well-defined  location  within  each  tho- 
racic segment,  with  respect  to  both  anterior- 
posterior  and  dorsal-ventral  position.  One  way  to 
mark  a  unique  point  is  to  draw  two  intersecting 
lines.  This  rather  simple  solution  appears  to  de- 
scribe what  the  embryo  does  to  identify  the  cell 
clusters.  Thus  the  spatial  cues  that  the  embryo 
uses  to  position  its  legs  appear  to  be  stripes  of 
cells  that  act  as  sources  of  secreted  intercellular 
signals.  The  earliest  detectable  consequence  of 
this  signaling  process  is  that  presumptive  leg 
cells  express  the  Distal-less  gene. 

Our  previous  work  identified  the  segmentally 
repeated  stripes  of  cells  expressing  the  wingless 
gene  as  one  of  the  signaling  centers  responsible 
for  initiating  leg  development.  Stripes  of  cells  ex- 
pressing wingless  bisect  the  nascent  leg  primor- 
dia.  The  wingless  gene  encodes  a  secreted  inter- 
cellular signaling  molecule  related  to  the 
vertebrate  oncogene  INT- 1 .  The  secreted  wing- 
less protein  is  required  to  signal  nearby  cells  to 
express  Distal- less  but  is  not  sufficient  to  do  so. 
An  intersecting  stripe  of  information  is  required 
to  specify  the  cluster.  The  decapentaplegic  gene 
is  a  good  candidate  to  encode  this  second  signal. 
A  stripe  of  cells  expressing  decapentaplegic  in- 
tersects the  wingless  stripe  at  precisely  the  posi- 
tion where  the  leg  is  formed.  The  decapentaple- 
gic gene  encodes  a  product  homologous  to  the 
transforming  growth  factor-/3  (TGF-|5)  class  of  se- 
creted signaling  molecules.  A  group  of  cells  that 
lies  near  the  intersection  of  these  two  stripes 
should  receive  both  signals.  Although  we  are  not 
yet  certain  whether  decapentaplegic  plays  a  di- 
rect role  in  this  process,  the  simultaneous  receipt 
of  the  wingless  and  TGF-/3  signals  should,  in  prin- 
ciple, be  sufficient  to  specify  the  identity  of  the 
leg  precursor  cells. 


79 


Molecular  Genetics  of  Limb  Development  in  Drosophila 


why  Do  Insects  Have  Six  Legs? 

Insects  develop  legs  only  in  their  thoracic  seg- 
ments, while  more  primitive  arthropods  may  de- 
velop abdominal  legs  as  well.  A  class  of  genes 
known  collectively  as  homeotic  genes  distin- 
guish segments  from  one  another.  The  homeotic 
genes  of  the  Bithorax  complex  act  as  negative  reg- 
ulators of  thoracic  segment  identity.  The  Bithorax 
genes  repress  Distal- less  expression  and  block 
leg  development  in  abdominal  segments.  Accord- 
ing to  our  model,  the  intercellular  signal  re- 
sulting from  the  intersection  of  wingless-  and 
decapentaplegic-express'mg  cells  should  in  prin- 
ciple be  capable  of  eliciting  limb  development  in 
the  abdomen.  The  signaling  sources  are  ex- 
pressed in  precisely  the  same  pattern  in  thoracic 
and  abdominal  segments.  We  have  found  that  this 
response  is  blocked  by  the  Bithorax  genes.  Re- 
moving Bithorax  genes  permits  Distal-less  ex- 
pression in  the  abdominal  segments  and  leads  to 
ectopic  limb  development  in  the  abdomen. 
Therefore,  the  signal  to  specify  leg  primordia  in 
the  abdominal  segments  exists,  but  the  conse- 
quences of  signaling  are  overridden. 

Molecular  analysis  of  the  control  elements  that 
regulate  Distal-less  expression  in  the  embryo 
provides  compelling  evidence  that  the  intercel- 
lular signal  is  actually  received  and  correctly  in- 
terpreted by  cells  in  the  abdominal  segments,  just 
as  in  the  thorax.  We  have  identified  a  small  piece 
of  DNA,  known  as  an  enhancer,  that  acts  as  the 
target  of  this  signal.  By  functional  dissection  of 
this  enhancer  we  have  identified  a  regulatory  ele- 
ment that  mediates  the  repression  of  limb  devel- 
opment in  the  abdominal  segments.  Removing  a 
small  piece  of  DNA  renders  the  enhancer  element 
insensitive  to  the  repressor  genes  of  the  Bithorax 
complex  but  has  no  effect  on  its  response  to  the 
activation  signal.  Gene  expression  driven  by  this 
mutant  version  of  the  enhancer  element  is  there- 
fore constitutive ly  "de-repressed"  in  the  abdo- 
men. These  observations  demonstrate  that  the 
wingless-  and  decapentaplegic-dependent  acti- 
vation signal  is  both  received  and  correctly  inter- 
preted by  the  would-be  abdominal  leg  cells.  Re- 
pression of  leg  development  must  therefore  lie  in 
the  prevention  of  Distal- less  expression  by  these 
cells.  These  observations  strongly  suggest  that 


turning  on  the  Distal-less  gene  in  these  cells  is 
the  only  signal  necessary  to  specify  their  identity 
as  leg  cells. 

Proximal-Distal  Pattern  Formation 
in  the  Leg 

As  mentioned  above,  flies  that  lack  Distal- less 
gene  activity  do  not  develop  any  leg  structures. 
Flies  in  which  Distal-less  gene  activity  is  im- 
paired, but  not  eliminated,  develop  abnormal 
legs.  Distal-less  mutant  legs  are  foreshortened 
along  the  proximal-distal  axis.  The  characteris- 
tics of  different  Distal-less  mutations  tell  us  that 
the  amount  of  activity  of  the  gene  is  important  in 
controlling  the  range  of  structures  that  the  leg 
can  develop.  Distal  parts  of  the  leg  require  more 
Distal-less  gene  activity  than  do  proximal  parts  to 
develop  normally.  These  observations  suggest 
that  Distal- less  may  play  an  important  role  in  or- 
ganizing the  proximal-distal  axis  of  the  leg. 

Early  in  development,  presumptive  leg  cells 
express  Distal-less,  whereas  presumptive  body 
wall  cells  do  not.  As  the  leg  matures,  this  simple 
pattern  transforms  into  a  graded  distribution  of 
Distal-less  RNA  across  the  developing  limb.  The 
distal-most  region  of  the  limb  expresses  a  high 
level  of  the  gene  product,  intermediate  levels  ex- 
press lower  levels  of  Distal- less  RNA,  and  proxi- 
mal regions  express  little  or  no  Distal- less  RNA. 
These  observations  are  particularly  intriguing  in 
view  of  the  regional  differences  in  the  require- 
ment for  the  activity  of  the  gene  along  the  leg.  We 
are  interested  in  understanding  the  transition 
from  the  initial  simple,  two-state  system  of  the 
embryo  to  the  complex,  graded  distribution  of 
the  gene  product  that  we  see  at  later  stages. 

Understanding  pattern  formation  in  the  devel- 
oping fly  leg  will  teach  us  about  fundamental 
mechanisms  important  for  the  development  of 
vertebrate  embryos.  We  are  studying  a  system  in 
which  intercellular  signaling  molecules  are  used 
to  assign  cells  to  functional  developmental  units. 
Although  the  particular  details  of  the  systems 
vary,  the  fundamental  principles  and  molecular 
mechanisms  will  prove  to  be  of  wider  applicabil- 
ity. In  this  context  we  are  intrigued  by  the  role 
that  Distal-less  plays  as  a  pattern  organizer  at  the 
genetic  level  and  its  prospective  role  as  a  regula- 
tor of  gene  expression  at  the  molecular  level. 


80 


Tracking  Genes  That  Cause  Human  Disease 


Francis  S.  Collins,  M.D.,  Ph.D. — Investigator 

Dr.  Collins  is  also  Professor  of  Internal  Medicine  and  Human  Genetics  at  the  University  of  Michigan 
Medical  School.  He  received  his  Ph.D.  degree  in  physical  chemistry  from  Yale  University  and  his  M.D. 
degree  from  the  University  of  North  Carolina.  After  completing  his  internship  and  residency  in  internal 
medicine  at  the  North  Carolina  Memorial  Hospital,  he  went  on  to  a  fellowship  in  human  genetics  at  Yale. 
He  recently  received  the  Young  Investigator  Award  of  the  American  Federation  of  Clinical  Research 
and  the  National  Medical  Research  Award  of  the  National  Health  Council  and  was  elected  to  the  Institute 
of  Medicine  and  the  American  Association  of  Physicians. 


THE  theme  of  our  laboratory  is  the  study  of 
human  genetic  disease  at  the  molecular 
level.  Our  goal  is  to  identify  genes  involved  in 
specific  genetic  disorders,  to  define  their  struc- 
ture and  function,  to  understand  the  control  of 
their  expression,  and  to  use  this  information  to 
develop  potential  new  therapies. 

Over  4,000  genetic  disorders  are  listed  in  the 
most  recent  edition  of  Mendelian  Inheritance  in 
Man.  For  the  majority  of  these,  the  normal  func- 
tion of  the  gene  involved  is  not  known.  The  iden- 
tification of  disease-causing  genes  without  knowl- 
edge of  their  protein  product  or  its  normal  role, 
now  referred  to  as  positional  cloning,  is  a  major 
endeavor  in  our  research. 

Only  recently  has  it  become  possible  to  iden- 
tify such  genes,  and  the  process  is  still  laborious. 
First  the  gene  must  be  mapped  to  a  specific  hu- 
man chromosome,  using  a  process  known  as  link- 
age analysis.  This  involves  identification  of  fami- 
lies with  the  disorder  and  analysis  of  DNA  from 
these  families  with  a  panel  of  probes  from  all 
parts  of  the  human  genome.  The  probes  are  used 
to  establish  the  chromosomal  location  of  a  DNA 
sequence  that  may  have  been  inherited  in  associa- 
tion with  a  disease  gene  that  will  probably  lie 
close  to  it.  Such  sequences,  or  "markers,"  can 
pinpoint  the  chromosome  on  which  the  disease 
gene  resides.  Additional  probes  from  that  chro- 
mosome can  then  be  tested  to  identify  markers 
that  are  even  closer.  It  is  often  possible  to  narrow 
the  responsible  region  to  about  1  percent  of  a 
particular  chromosome.  Although  this  is  a  major 
achievement,  such  a  region  may  yet  contain  30  to 
50  genes,  only  one  of  which  is  responsible  for  the 
disease.  Thus  additional  refinements  must  be 
made  before  candidates  for  the  responsible  gene 
can  be  identified. 

In  1989,  as  part  of  a  collaborative  effort  with 
investigators  at  the  Hospital  for  Sick  Children  in 
Toronto,  we  were  successful  in  identifying  the 
gene  for  cystic  fibrosis  (CF),  a  common  severe 
genetic  disease  characterized  by  lung  infections, 
pancreatic  insufficiency,  and  elevations  in  sweat 
chloride  concentration. 


Although  one  mutation  (AF508)  is  responsible 
for  about  75  percent  of  CF  chromosomes  in  the 
United  States,  numerous  other  mutations  have 
now  been  found  in  other  individuals  with  the  dis- 
ease. This  year  we  identified  one  mutation  that 
gives  rise  to  such  a  mild  form  of  the  disorder  that 
it  was  not  recognized  as  being  caused  by  this 
same  gene,  because  the  affected  individuals  had 
normal  sweat  chloride.  This  observation  indi- 
cates that  mutations  in  the  CF  gene  may  have  a 
broader  clinical  spectrum  than  previously 
suspected. 

Another  important  observation  in  CF  this  year 
has  been  the  demonstration  by  our  group  that  the 
AF508  mutation  does  not  completely  inactivate 
the  gene.  In  fact,  using  a  frog  oocyte  system,  we 
were  able  to  show  that  the  AF508  protein  can  be 
activated  to  almost  normal  levels  by  using  drugs 
that  raise  levels  of  cAMP,  an  intracellular  second 
messenger.  This  suggests  that  drug  therapy  aimed 
at  this  second  messenger  might  benefit  patients 
with  the  disease  by  activating  their  defective  CF 
protein. 

Another  major  project  in  our  laboratory  is  an 
investigation  of  von  Recklinghausen  neurofibro- 
matosis (NFl),  a  common  genetic  disorder  some- 
times incorrectly  referred  to  as  the  Elephant  Man 
disease.  Having  identified  this  gene  in  1990,  we 
are  now  intensively  attempting  to  identify  muta- 
tions that  cause  the  disease  and  to  determine  the 
normal  function  of  the  protein  product  of  this 
gene.  Previous  studies  have  demonstrated  that 
this  protein  interacts  with  another  class  of  pro- 
teins (encoded  by  the  ras  genes)  involved  in  the 
regulation  of  cell  growth.  Recent  data  from  our 
group  have  shown  that  the  NFl  protein  also  inter- 
acts with  the  cytoskeleton  and  can  be  seen  to  co- 
localize  with  microtubules  using  immunofluores- 
cence techniques.  This  unexpected  observation 
suggests  that  the  protein  product  of  the  NFl  gene 
may  play  a  crucial  role  in  the  regulation  of  cell 
division.  In  the  longer  term,  it  is  hoped  that  this 
improved  understanding  of  the  biology  of  the  dis- 
ease will  lead  to  improved  therapies. 

The  Huntington  disease  (HD)  gene  has  been 


81 


Tracking  Genes  That  Cause  Human  Disease 


localized  by  a  collaborative  group,  which  in- 
cludes our  laboratory.  It  is  now  known  to  lie  in  a 
region  near  the  tip  of  the  short  arm  of  chromo- 
some 4,  within  an  interval  of  approximately  2 
million  base  pairs.  Using  yeast  artificial  chromo- 
some cloning,  we  have  generated  an  overlapping 
set  of  genomic  clones  from  this  region  and  are 
utilizing  a  variety  of  novel  technologies  to  search 
for  candidate  genes. 

A  new  project  in  our  laboratory  this  year  is  an 
intense  effort  to  identify  the  gene  responsible  for 
early  onset  breast  cancer,  which  has  recently 
been  mapped  by  Mary-Claire  King  and  her  col- 


leagues  (University  of  California,  Berkeley)  to 
chromosome  17.  Using  genetic  linkage  analysis 
in  families,  as  well  as  the  analysis  of  tumors,  we 
are  narrowing  down  the  responsible  interval.  Sig- 
nificant portions  of  the  candidate  interval  have 
now  been  cloned  in  yeast  artificial  chromosomes. 
Utilizing  the  relatively  new  technology  of  physi- 
cal microdissection  of  chromosomes,  we  have 
been  able  to  produce  many  new  markers  in  this 
region  of  the  long  arm  of  chromosome  17;  thus 
the  opportunity  to  identify  a  major  gene  contrib- 
uting to  this  common  and  devastating  disease  in 
Western  society  is  now  quite  real. 


•■.»• 


Colocalization  of  neurofibromin  (the  normal  protein  product  of  the  neurofibromatosis  gene) 
and  microtubules.  The  same  rodent  cell  has  been  double-labeled  and  visualized  by  fluorescence 
microscopy.  Red  shows  staining  with  an  antibody  directed  against  tubulin,  the  major  component 
of  cytoplasmic  microtubules.  Green  shows  staining  of  neurofibromin  that  appears  to  decorate  the 
microtubules. 

Research  of  Paula  Gregory,  David  Gutmann,  and  Francis  Collins. 


82 


Development  of  the  Immune  System 


Max  D.  Cooper,  M.D. — Investigator 

Dr.  Cooper  is  also  Professor  of  Medicine,  Pediatrics,  and  Microbiology  at  the  University  of  Alabama  at 
Birmingham.  He  received  his  M.D.  degree  and  specialty  training  in  pediatrics  at  Tulane  Medical  School 
and  his  postdoctoral  training  in  immunology  at  the  University  of  Minnesota. 


INFORMATION  obtained  from  studies  of  im- 
mune system  development  in  a  variety  of  verte- 
brate species  is  used  to  investigate  diseases  of  the 
immune  system  in  humans.  We  are  particularly 
interested  in  the  pathogenesis  of  immunodefi- 
ciency diseases  and  lymphoid  malignancies. 

Comparative  studies  in  birds  and  mammals  ini- 
tially revealed  the  separate  developmental  path- 
ways of  T  and  B  lymphocytes,  the  two  major  types 
of  immunocompetent  cells.  T  cells  provide  help 
for  antibody-producing  B  cells  and  are  primarily 
responsible  for  immunity  against  viruses  and 
fungi.  We  wish  to  know  whether  the  thymus  is 
the  only  source  of  T  cells  in  vertebrates  or 
whether  they  may  also  arise  in  other  tissues. 

The  central  tissues  may  vary  for  production  of  B 
cells,  the  source  of  antibodies.  In  birds,  immuno- 
globulin (Ig) -bearing  B  cells  are  derived  from  the 
hindgut  bursa  of  Fabricius,  whereas  in  mammals 
such  cells  are  generated  in  blood-forming  organs, 
mainly  the  fetal  liver  and  adult  bone  marrow. 
Multipotent  stem  cells  in  these  tissues  use  an  elab- 
orate gene  program  to  generate  millions  of  T  and 
B  lymphocyte  clones,  each  expressing  a  T  cell 
receptor  (TCR)  or  Ig  receptor  of  different  antigen 
specificity.  These  are  seeded  via  the  bloodstream 
to  the  peripheral  tissues,  where  they  execute  im- 
mune surveillance  of  foreign  and  self  antigens. 

Inherited  or  acquired  gene  defects  may  specifi- 
cally alter  growth  or  maturation  of  these  cell 
lines  to  cause  immune  system  dysfunction  or 
malignancy. 

Comparative  Analysis  of  T  Cell  Development 

We  have  embarked  on  a  comparative  analysis  of 
T  cell  development  in  representative  mamma- 
lian, avian,  and  amphibian  species.  Our  studies  in 
birds  reveal  remarkable  evolutionary  conserva- 
tion of  the  pattern  of  T  cell  development  found  in 
mammals,  including  the  sequential  development 
of  cells  bearing  either  the  yh  or  a/3  TCR. 

Cells  expressing  the  yb  TCR  are  generated  first 
during  ontogeny.  They  may  not  undergo  clonal 
selection  during  their  intrathymic  development. 
They  migrate  preferentially  to  splenic  red  pulp 
regions  and  the  intestinal  epithelium.  Unlike  afi 


T  cells,  the  yb  T  cells  cannot  recognize  class  II 
molecules  of  the  major  histocompatibility 
(MHC)  gene  complex  to  initiate  a  graft-versus- 
host  (GVH)  attack.  Analysis  of  their  physiological 
role  is  facilitated  by  their  relative  abundance  in 
birds  (25-50  percent  of  T  cells),  where  we  are 
examining  their  development  and  functional 
capabilities. 

In  collaboration  with  Craig  Thompson  (HHMI, 
University  of  Michigan) ,  we  have  used  the  TCR2 
and  TCR3  antibodies  to  identify  two  discrete  sub- 
lineages  of  a/3  cells  that  use  different  variable- 
region  gene  families:  V/Sl  and  V|82.  The  avian  V/Jl 
and  V/32  genes  contain  highly  conserved  se- 
quences that  distinguish  the  two  major  sub- 
groups of  mammalian  V/3  genes.  Birds  thus  pro- 
vide a  simple  model  system  for  study  of  the 
functional  significance  of  these  prototypic  gene 
families. 

In  birds,  as  in  mammals,  TCR/?  diversity  is  cre- 
ated largely  by  nucleotide  sequence  variations  in 
the  joints  between  rearranged  V/3,  D  (diversity), 
and  J  (joining)  genes.  Since  V/51  and  V|82  genes 
both  combine  with  the  same  D  and  J  genes,  the 
V|S  1  ^  and  V/32^  T  cells  should  be  capable  of  recog- 
nizing the  same  spectrum  of  antigenic  peptides. 
However,  the  V/31  and  Vj82  genes  have  little  se- 
quence homology  (less  than  30  percent),  so  their 
protein  products  may  interact  differently  with 
peptide-presenting  MHC  molecules. 

Indeed,  although  both  V/31+  and  V/32+  T  cells 
can  cause  GVH  disease  in  recipients  with  the 
same  MHC  class  II  genes,  they  do  so  with  differ- 
ent efficiency.  Selective  inhibition  of  VjSl  T  cell 
development  revealed  that  only  the  V;3l^  T  cells 
can  help  B  cells  produce  protective  IgA  antibod- 
ies along  the  mucous  surfaces  of  the  body. 
Current  studies  explore  in  more  detail  the  func- 
tional capabilities  of  the  VjSl  and  V(82  subpopula- 
tions  of  avian  afi  T  cells. 

To  analyze  T  cell  development  in  an  amphib- 
ian, we  have  made  monoclonal  antibodies  that 
identify  7^  and  a/3  TCR  candidates  expressed  by 
separate  lymphocyte  subpopulations  in  Xenopus 
laevis.  Frog  T  cells  bearing  the  putative  yb  and 
TCR  homologues  exhibit  the  same  tissue  distri- 


83 


Development  of  the  Immune  System 


bution  patterns  shown  previously  for  their  avian 
and  mammalian  counterparts.  Cloning  of  the  frog 
TCR  genes  is  one  current  focus,  and  the  thymus 
dependence  and  functional  capabilities  of  the 
two  sublineages  are  being  explored.  The  goals  of 
th^se  studies  are  to  understand  the  evolutionary 
strategy  for  generating  T  cells  that  can  discrimi- 
nate between  self  and  nonself  and  to  gain  fresh 
clues  for  some  of  the  unresolved  mysteries  of  the 
human  immune  system. 

B  Cell  Development 

Bone  marrow  stromal  cells  can  influence  the 
growth  and  development  of  B  cell  precursors,  ei- 
ther by  direct  cell  contact  or  via  soluble  products 
like  interleukin-7  (IL-7).  We  are  examining  cell 
surface  molecules  through  which  pre-B  cells 
may  receive  these  environmental  cues.  One  such 
candidate  molecule  is  an  ectoenzyme  named 
aminopeptidase  A  (APA),  the  increased  expres- 
sion of  which  is  induced  by  exposure  to  IL-7.  We 
are  testing  the  hypothesis  that  IL-7  is  either  a  li- 
gand  or  a  catalytic  substrate  for  APA.  We  are  using 
a  mouse  gene  probe  to  identify  the  human  gene 
for  APA  for  sequence  analysis,  with  the  goal  of 
understanding  its  expression  as  a  function  of  nor- 
mal and  leukemic  pre-B  cell  differentiation. 

Before  pre-B  cells  rearrange  their  light-chain 
genes  to  become  B  cells,  they  express  a  complex 


receptor  that  is  composed  of  surrogate  light- 
chain  proteins  (VpreB  and  X5),  m  heavy  chains,  and 
two  small  transmembrane  proteins  called  a  and 
/3.  We  have  produced  monoclonal  antibodies 
against  exposed  configurations  of  these  protein 
receptor  units  in  order  to  explore  the  nature  of 
the  receptor  ligand(s)  and  the  signals  transduced 
to  the  pre-B  cell.  One  of  these  antibodies,  which 
is  specific  for  the  /3-chain  of  the  Ig  receptor  unit, 
may  prove  to  be  a  universal  B  cell  suppressant 
because  it  down-modulates  the  antibody  recep- 
tors on  all  B  cells. 

Immunodeficiency  Diseases 

The  pathogenesis  of  three  primary  immunodefi- 
ciency diseases  is  being  investigated.  X-Iinked 
agammaglobulinemia  features  an  arrest  in  pre-B 
cell  differentiation,  the  precise  nature  of  which  is 
under  study  in  affected  boys.  Other  studies  ad- 
dress the  hypothesis  that  selective  IgA  deficiency 
(IgA-D)  and  common  variable  immunodeficiency 
(CVID)  may  represent  polar  ends  of  a  clinical 
spectrum  reflecting  a  single  underlying  gene  de- 
fect. The  cellular  defect  in  both  deficiencies  con- 
sists of  arrested  B  cell  differentiation.  Both  dis- 
orders frequently  occur  in  members  of  the  same 
family,  and  the  same  MHC  class  III  haplotypes  are 
associated  with  IgA-D  and  CVID.  Current  studies 
focus  on  the  C4A  complement  gene. 


84 


Structure  and  Function  of  RNA  Polymerase  II 


Jeffry  L.  Corden,  Ph.D. — Associate  Investigator 

Dr.  Corden  is  also  Associate  Professor  of  Molecular  Biology  and  Genetics  at  the  Johns  Hopkins  University 
School  of  Medicine.  He  received  his  B.S.  and  Ph.D.  degrees  in  biochemistry  and  biophysics  from  Oregon 
State  University.  His  postdoctoral  work  was  done  with  Pierre  Chambon  in  the  Laboratoire  de  Genetique 
Moleculaire  des  Eucaryotes  at  the  Faculte  de  Medecine,  Strasbourg,  France. 


THE  first  step  in  the  expression  of  genetic  in- 
formation is  carried  out  by  RNA  polymer- 
ase II.  How  this  enzyme  determines  which  genes 
are  to  be  expressed  in  which  cells  and  under 
which  physiological  conditions  remains  largely 
unknown. 

RNA  polymerase  II  contains  more  than  10  dif- 
ferent proteins  whose  precise  functions  are  only 
now  beginning  to  be  addressed.  The  goal  of  my 
laboratory  is  to  understand  the  structure  and 
function  of  the  subunits  of  RNA  polymerase  II. 
Several  years  ago  we  began  to  analyze  the  largest 
subunit  of  the  mouse  RNA  polymerase  II  com- 
plex. This  subunit  comprises  one-half  of  the  mass 
of  the  enzyme  and,  through  the  work  of  many 
laboratories,  is  known  to  be  involved  in  the  enzy- 
matic synthesis  of  RNA.  We  have  cloned  and  se- 
quenced the  mouse  gene  encoding  this  largest 
subunit  and  have  also  isolated  and  characterized 
several  mutations  in  this  gene.  These  mutant 
genes  are  being  used  to  study  the  function  of  dif- 
ferent domains  of  the  largest  subunit.  The  aim  of 
these  experiments  is  to  understand  how  RNA 
polymerase  II  orchestrates  the  ordered  expres- 
sion of  100,000  genes  during  the  vertebrate  life 
cycle. 

The  gene  encoding  the  largest  subunit  of  RNA 
polymerase  II  comprises  28  segments  (exons) 
that  cover  30,000  bases  of  mouse  chromosomal 
DNA  near  the  center  of  chromosome  1 1 .  The 
amino  acid  sequence  deduced  from  the  DNA  se- 
quence has  revealed  two  interesting  properties  of 
the  subunit.  The  major  portion  of  the  protein  is 
similar  in  sequence  to  a  bacterial  RNA  polymer- 
ase subunit  (from  Escherichia  coif)  that  carries 
out  an  equivalent  function.  This  evolutionary 
conservation  is  much  stronger  than  had  been  ex- 
pected and  has  allowed  us  to  predict  that  the 
mouse  subunit  is  involved  in  the  transcription 
elongation  process. 

Although  the  major  part  of  the  largest  subunit  is 
related  to  the  bacterial  enzyme,  our  DNA  se- 
quence analysis  has  also  revealed  a  domain  that  is 
unique  to  RNA  polymerase  II.  This  domain  is  lo- 
cated at  one  end  of  the  molecule  and  constitutes  a 
52-fold  repeat  of  a  seven-amino  acid  sequence. 


This  unusual  sequence,  although  absent  in  bacte- 
ria, is  found  in  other  large  subunits  of  virtually 
every  RNA  polymerase  II,  including  those  of  ani- 
mals, plants,  insects,  and  protists.  We  are 
currently  focusing  our  efforts  on  understanding 
the  role  of  this  domain  in  the  process  of 
transcription. 

Our  genetic  approach  to  the  function  of  this 
carboxyl-terminal  domain  (CTD)  has  grown  from 
analysis  of  mutations  in  the  largest  subunit  gene. 
We  first  isolated  mutant  mouse  tissue-culture  cell 
lines  that  are  resistant  to  the  mushroom  toxin  a- 
amanitin.  The  largest  subunit  genes  from  several 
of  these  cell  lines  have  been  cloned  and,  by  re- 
introduction  into  amanitin-sensitive  cells,  have 
been  shown  to  confer  resistance  to  a-amanitin. 
We  have  used  this  gene  transfer  technique  to 
map  the  mutations  responsible  for  amanitin 
resistance. 

The  availability  of  a  well-defined,  selectable  ge- 
netic marker  in  the  largest  subunit  gene  has 
proved  useful  in  the  analysis  of  the  role  of  the 
CTD.  Deletion,  insertion,  and  substitution  muta- 
tions have  been  created  in  the  CTD  of  an  amani- 
tin-resistance  gene.  The  effect  of  these  mutations 
has  been  tested  by  introducing  the  altered  resis- 
tance genes  into  cells  and  scoring  for  amanitin 
resistance.  Removing  the  entire  CTD  eliminates 
the  ability  to  transfer  amanitin  resistance,  demon- 
strating that  the  CTD  plays  an  essential  role  in 
transcription.  We  have  also  shown  that  up  to  20 
of  the  52  repeats  are  dispensable  for  growth  in 
tissue  culture,  indicating  either  that  the  CTD  is 
functionally  redundant  or  that  dispensable  re- 
peats are  only  necessary  in  some  cell  types.  Para- 
doxically, we  have  recently  shown  that  the  CTD 
is  highly  conserved  among  all  mammals.  We 
are  now  designing  experiments  to  assess  the  role 
of  dispensable  heptapeptide  repeats  during 
development. 

We  have  also  been  examining  postsynthetic 
modifications  of  the  CTD.  This  domain  is  rich 
in  amino  acids  (such  as  serine,  threonine,  and 
tyrosine)  that  can  be  modified  by  addition  of 
phosphate  groups.  RNA  polymerase  II  is  a  phos- 
phorylated  enzyme,  but  no  function  for  phos- 
phorylation is  known.  We  have  used  synthetic 


85 


Structure  and  Function  of  RNA  Polymerase  II 


CTD  peptide  repeats  as  substrates  to  identify  en- 
zymes (CTD  kinases)  that  carry  out  this  phos- 
phorylation. Two  CTD  kinases  have  been  iso- 
lated, each  containing  protein  kinase  catalytic 
subunits  previously  identified  in  yeast  as  cell- 
division  control  proteins.  Further  cloning  studies 
have  revealed  several  more  related  mouse  kinases 
whose  role  in  CTD  phosphorylation  is  currently 
under  investigation.  This  investigation  is  sup- 
ported by  a  grant  from  the  National  Institutes  of 
Health. 

Using  the  purified  CTD  kinases,  we  have 
mapped  the  sites  of  phosphorylation  on  the  CTD. 
These  sites,  serines  that  precede  proline  residues, 
correspond  to  sites  in  other  proteins  that  are  mod- 
ified in  a  cell  cycle-dependent  fashion.  Current 
studies  are  directed  toward  examining  the  level 
of  CTD  phosphorylation  at  different  times  in  the 
cell  cycle.  One  consequence  of  CTD  phosphory- 


lation is  that  the  CTD  becomes  greatly  extended. 
The  basis  of  this  conformational  change  is  also  an 
area  of  investigation  in  our  laboratory. 

To  define  more  precisely  the  amino  acid  se- 
quences required  for  CTD  function,  we  have  de- 
vised a  strategy  to  clone  synthetic  CTD  repeats. 
This  work  is  being  done  in  the  yeast  Saccharo- 
myces  cerevesiae,  where  as  few  as  1 0  of  the  re- 
peats are  sufficient  for  viability.  We  have  con- 
structed mutant  CTDs  in  which  the  residues 
identified  as  phosphorylation  sites  have  been 
changed.  In  most  cases  these  mutations  are  lethal, 
indicating  that  phosphorylation  is  essential  for 
RNA  polymerase  II  function.  We  are  currently  us- 
ing yeast  genetics  to  isolate  suppressors  of  these 
mutations  in  an  effort  to  identify  proteins  that 
interact  with  the  CTD.  Through  characterization 
of  such  proteins  we  hope  to  determine  the  func- 
tion of  the  CTD  in  RNA  polymerase  II  function. 


Hair  cells  from  the  bullfrog  inner  ear.  A  tuft  of  cilia,  which  sense  mechanical 
stimuli,  protrude  from  the  apical  surface  of  each  cell.  Deflections  of  the  bundle 
toward  the  longer  cilia  are  excitatory. 
Research  of  David  Corey  and  fohn  Assad. 


86 


Mechanically  Activated  Ion  Channels 


David  p.  Corey,  Ph.D. — Associate  Investigator 

Dr.  Corey  is  also  Associate  Professor  of  Neuroscience  at  Harvard  Medical  School  and  Assistant  Physiologist 
at  Massachusetts  General  Hospital,  Boston.  He  studied  physics  as  an  undergraduate  at  Amherst  College, 
conducted  research  for  a  year  at  Harvard  Medical  School,  and  then  entered  the  neurobiology  program  at 
the  California  Institute  of  Technology.  His  thesis  work,  with  James  Hudspeth,  focused  on  mechanical 
transduction  in  auditory  receptor  cells.  His  postdoctoral  work  with  Charles  Stevens  at  Yale  Medical  School 
was  on  voltage- sensitive  ion  channels. 


OUR  laboratory  is  interested  in  how  protein 
channels  in  cell  membranes  mediate  the 
electrical  activity  of  the  brain.  Such  channels, 
which  open  and  close  to  regulate  the  flow  of  min- 
ute amounts  of  electrical  current  into  a  cell,  are 
intimately  involved  in  the  brain's  information 
processing.  They  are  important  in  detecting  sen- 
sory signals  such  as  light  and  sound,  in  the  trans- 
mission of  this  information  from  the  sense  organ 
to  the  brain,  and  in  communication  from  one 
brain  cell  to  another.  We  are  focusing  primarily 
on  ion  channels  in  the  sensory  receptor  cells  of 
the  inner  ear,  especially  on  the  mechanism  of  ac- 
tivation of  those  channels  that  detect  sound. 

The  sound-activated  channels  occur  in  a  spe- 
cial type  of  inner-ear  cell  called  a  hair  cell.  These 
cells  are  named  for  a  bundle  of  cilia  that  extends 
from  their  top  surface  and  that  tilts  back  and  forth 
with  each  cycle  of  a  sound  wave.  Moving  the  cilia 
opens  and  closes  the  channels.  Over  10  years  ago, 
it  was  found  that  the  ion  channels  could  open 
extremely  quickly — in  just  a  few  millionths  of  a 
second — when  the  bundle  was  deflected.  That 
speed  rules  out  the  kind  of  biochemical  chain 
used  by  receptor  cells  of  the  eye  and  nose.  We 
proposed  instead  that  mechanical  forces  on  the 
cilia  could  pull  the  channels  open  directly. 
Others  have  shown  that  this  was  true,  and  it  was 
possible  to  measure  the  opening  movement  of 
channels  when  force  was  applied  to  them.  But 
where  are  the  channels,  and  what  pulls  on  them? 

Other  workers,  having  explored  around  the 
bundle  with  a  fine  electrode,  indicated  that  the 
channels  were  near  the  tips  of  the  cilia.  This  was  a 
surprising  result,  since  a  lever  action  of  the  cilia 
might  focus  forces  near  the  bases.  We  have  re- 
cently confirmed  this,  however,  by  putting  a  fluo- 
rescent dye  inside  the  cell  that  reports  the  entry 
of  calcium  through  the  channels.  When  calcium 
is  allowed  to  enter  the  cell,  the  fluorescent  signal 
appears  first  at  the  tips,  showing  that  the  channels 
are  there. 

With  attention  focused  on  the  tips  of  the  cilia,  a 
group  in  England  discovered  that  the  tips  are 
linked  by  extremely  tiny  filaments,  just  1 50  nano- 
meters long,  which  they  called  tip  links.  To- 


gether these  clues  led  to  a  beautifully  simple 
theory  for  the  operation  of  hair  cells.  Deflecting 
the  bundle  in  one  direction  would  stretch  the  tip 
links,  and  they  would  pull  directly  on  the  ion 
channels  to  open  them  and  let  ions  into  the  cell. 
Moving  in  the  other  direction  would  relax  the 
links  and  allow  channels  to  close.  However,  no 
way  to  test  the  theory  could  be  found. 

This  past  year,  we  have  been  able  to  confirm  it. 
We  found  that  removing  calcium  from  the  fluid 
around  the  cilia,  for  just  a  few  seconds,  com- 
pletely eliminated  the  tip  links,  as  observed  with 
either  transmission  electron  microscopy  or  scan- 
ning electron  microscopy.  When  we  tested  the 
mechanical  sensitivity  of  the  hair  cells,  by  mov- 
ing a  single  bundle  and  measuring  the  electrical 
response  of  the  cells,  we  found  that  the  same 
treatment  eliminated  the  electrical  response  in  a 
few  tenths  of  a  second.  A  further  test  indicated 
that  the  electrical  response  was  lost  specifically 
because  the  mechanical  tension  on  the  channels 
was  gone.  Thus  tip  links  do  convey  the  stimulus 
tension  to  the  ion  channels. 

In  addition  to  this  transduction  mechanism  in 
these  cells,  there  is  an  adaptation  mechanism  that 
enables  them  to  be  sensitive  to  extremely  small 
displacements  while  retaining  an  ability  to  re- 
spond over  a  large  range  of  stimuli.  Hair  cells 
have  a  very  narrow  range  of  sensitivity.  A  deflec- 
tion of  about  a  third  of  a  micrometer — the  diame- 
ter of  one  cilium — is  sufficient  to  open  all  chan- 
nels. We  found  some  years  ago,  however,  that  a 
steady  deflection  that  opens  all  channels  is  rap- 
idly followed  by  the  spontaneous  closure  of 
channels.  To  reconcile  this  with  the  context  of 
the  tip-links  model,  one  must  suppose  that  clo- 
sure is  a  consequence  of  relaxing  the  tension  on 
the  channels.  A  further  deflection  to  stretch  the 
tip  links  does,  in  fact,  reopen  channels.  Thus 
there  seems  to  be  a  continuous  adjustment  sys- 
tem, acting  to  set  the  tension  on  the  ion  channels. 
Indeed,  another  group  found  that  the  stiffness  of 
the  bundle  relaxed  with  the  same  time  course  as 
adaptation,  in  keeping  with  a  tension  adjustment 
scheme. 

In  contrast,  experiments  from  a  different  group 


87 


Mechanically  Activated  Ion  Channels 


suggested  that  adaptation  is  not  a  mechanical  ad- 
justment, but  comes  about  through  calcium  entry 
through  the  transduction  channels.  Calcium  in- 
side the  cell  would  close  channels,  to  create  a 
similar  kind  of  negative  feedback.  It  was  clearly 
important  to  distinguish  these  models. 

In  the  past  two  years,  we  have  obtained  good 
evidence  for  the  mechanical  adjustment  hypoth- 
esis. A  key  feature  of  this  hypothesis  is  that  the 
putative  adjustment  of  tension  in  the  tip  links 
would  also  be  felt  by  the  cilia  themselves  and 
would  act  to  move  the  bundle.  We  found,  first, 
that  we  could  change  the  rate  of  adaptation  by 
changing  the  voltage  across  the  cell  membrane. 
Because  voltage  affected  the  relaxing  more  than 
the  tightening,  we  could  then  predict  that  posi- 
tive voltage  would  increase  the  tension,  and  how 
much  and  how  quickly.  Judging  from  the  activa- 
tion of  transduction  channels,  these  predictions 
were  borne  out.  Third,  we  could  then  predict 
how  much  the  tension  change  should  move  the 
bundles.  The  predicted  change  was  only  about 
100  nanometers — a  quarter  the  wavelength  of 
light — so  we  worked  out  a  video  microscopy  sys- 
tem to  measure  the  motion  with  a  resolution  of  5 
nanometers.  When  the  voltage  was  changed,  the 
bundles  did  move,  with  the  predicted  amplitude 
and  time  course.  Thus  a  quantitative  theory  that 
describes  the  adaptation  also  describes  the  move- 
ment, strongly  suggesting  that  adaptation  is 
mechanical. 

How  could  tension  on  the  tip  links  be  adjusted? 
Others  have  speculated  that  the  adaptation  comes 
about  by  a  movement  of  the  points  where  the  tip 
links  are  attached  to  the  structure  of  the  cilia. 
When  the  tip  links  are  stretched  to  open  chan- 
nels, the  attachment  points  might  slip  to  allow 
the  links  to  shorten.  Conversely,  if  the  tip  links 
are  relaxed,  the  attachment  points  move  up  to 
stretch  the  links  to  restore  the  resting  tension. 

This  past  year,  we  began  to  test  this  structural 
theory,  by  measuring  the  position  of  the  attach- 
ment points  before  and  after  deflections  that 
cause  adaptation.  Bundles  were  given  calibrated 
deflections  and  then  fixed  with  glutaraldehyde  in 
the  deflected  position.  Measurements  from  trans- 
mission electron  micrographs  suggest  that  the  at- 
tachment points  do  move  in  response  to  deflec- 
tion, although  we  still  need  to  test  whether  the 
movement  is  quantitatively  consistent  with  the 
adaptation. 

What  could  move  the  attachment  point?  Some 
circumstantial  evidence  suggests  that  the  "mo- 


tor" molecule  is  like  the  myosin  protein  that 
causes  the  contraction  of  muscle:  The  stiff  cores 
of  the  cilia  are  composed  largely  of  actin,  on 
which  myosin  moves  in  muscle;  the  motor  mole- 
cule in  these  cilia  moves  in  the  same  direction 
and  at  the  same  rate  as  myosin;  and  glass  beads 
coated  with  muscle  myosin  do  move  on  the  actin 
cores  of  these  cilia.  We  are  now  seeking  further 
evidence  as  to  whether  the  motor  is  a  form  of 
myosin. 

Our  ultimate  aim  is  to  describe  each  link  in  the 
mechanical  chain  from  cilia  to  channels,  in  terms 
of  the  protein  identity  of  the  links,  their  biophysi- 
cal properties,  and  their  relationship  to  each 
other.  Answers  to  these  specific  questions  will 
contribute  to  the  long-range  goal  of  a  compre- 
hensive theory  of  mechanically  activated  chan- 
nels, not  only  in  the  ear  but  in  the  many  other  cell 
types  that  display  a  mechanical  sensitivity. 

In  the  past  two  years,  we  have  started  to  use 
some  of  what  has  been  learned  about  ion  chan- 
nels to  understand  human  disease.  Hyperkalemic 
periodic  paralysis  is  a  dominantly  inherited  mus- 
cle disease  that  causes  sporadic  weakness  or  paral- 
ysis. Exercise  or  certain  foods  that  raise  the  level 
of  potassium  in  the  blood  can  bring  on  a  paralytic 
attack.  Colleagues  at  Massachusetts  General  Hos- 
pital had  found  that  the  voltage-sensitive  sodium 
channel  in  muscle  was  genetically  linked  to  this 
disease,  suggesting  that  a  defect  in  the  channel 
might  be  the  cause.  Last  year  we  were  able  to 
show  that  the  channels  are  in  fact  defective.  In 
the  presence  of  excess  potassium,  they  fail  to  in- 
activate normally,  which  allows  sodium  to  enter 
the  muscle  and  (indirectly)  causes  the  paralysis. 

A  puzzle,  however,  is  that  paralytic  episodes 
are  often  preceded  in  patients  by  myotonia — an 
excessive  contraction  of  the  muscle.  Could  the 
same  defect  cause  both  myotonia  and  paralysis? 
This  year  we  have  used  a  toxin  derived  from  sea 
anenomes,  which  mimics  the  genetic  defect  in 
sodium  channels  when  applied  to  normal  mus- 
cle. Toxin-treated  muscle  did,  in  fact,  show  clas- 
sic myotonia,  with  both  increased  electrical  activ- 
ity and  increased  tension.  Thus  a  single  genetic 
defect  can  have  a  graded  effect.  Induced  only 
slightly,  it  causes  myotonia;  induced  to  a  greater 
extent  by  potassium,  it  causes  paralysis.  A  com- 
puter model  of  electrical  activity  in  muscle 
shows  the  same  result  with  a  single  defect.  Ex- 
plaining the  pathology  at  the  molecular  level 
gives  hints  for  effective  drug  treatment  of  the 
disorder. 


88 


Genetic  Regulatory  Mechanisms 
in  Cellular  Differentiation 


Gerald  R.  Crahtree,  M.D. — Associate  Investigator 

Dr.  Crabtree  is  also  Associate  Professor  of  Pathology  at  Stanford  University  School  of  Medicine.  He  received 
his  B.S.  degree  from  West  Liberty  State  College,  West  Virginia,  and  his  M.D.  degree  from  Temple  University 
School  of  Medicine,  Philadelphia.  He  was  a  senior  investigator  at  NIH  before  coming  to  Stanford  University. 


CELLS  acquire  their  final  differentiated  func- 
tion by  a  complex  interplay  between  primary 
genetic  regulatory  events  in  the  nucleus  and  in- 
teractions at  the  cell  membrane.  Building  on  con- 
cepts largely  provided  from  studies  on  lower 
animals,  our  laboratory  has  been  exploring  regu- 
latory mechanisms  that  help  determine  how 
mammalian  cells  differentiate  to  assume  their 
normal  functions. 

Isolation  of  a  Trans-acting  Regulator 
of  Homeodomain  Protein  Function 

Several  years  ago,  we  identified  a  tissue- 
specific  transcription  factor,  HNF-la,  that  inter- 
acts with  essential  regions  of  the  promoters  of  a 
large  family  of  genes  expressed  in  endodermally 
derived  tissues.  After  purifying  the  protein  and 
cloning  its  gene,  we  found  that  HNF-1  contains  a 
homeodomain  similar  to  that  found  in  genes  de- 
termining body  form  in  insects.  Curiously,  the 
protein  dimerizes  through  an  amino  acid  se- 
quence unlike  that  found  in  other  homeodomain- 
containing  proteins.  This  led  us  to  look  for  a  pro- 
tein that  might  heterodimerize  with  it  and  hence 
diversify  its  regulatory  capabilities.  We  found 
such  a  protein  by  screening  a  hepatocyte  cDNA 
library  at  low  stringency.  This  protein,  HNF-1/8, 
which  is  expressed  in  a  partially  overlapping 
group  of  tissues  with  HNF-la,  contains  a  dimer- 
ization  and  homeodomain  similar  to  those  of 
HNF-la,  but  a  different  transcriptional  activation 
domain. 

Surprisingly,  HNF-la  was  only  able  to  activate 
transcription  in  certain  tissues,  suggesting  the  ex- 
istence of  a  second  tissue-specific  protein  that 
regulated  its  function.  Dirk  Mendel  began  a 
search  for  such  a  protein  by  purifying  HNF- 1  a  by 
means  that  do  not  disturb  hydrophobic  interac- 
tions. HNF-la  copurified  with  an  1 1-kDa  protein 
that  participated  in  the  formation  of  a  tetrameric 
complex.  The  1 1  -kDa  protein,  which  was  cloned, 
expressed,  and  found  to  enhance  the  affinity  of 
dimerization  between  HNF  molecules,  was 
named  DCoH  (dimerization  cofactor  for  HNF- 1 ) . 
In  both  cases  DCoH  required  the  amino-terminal 
32  amino  acids  that  constitute  the  dimerization 


domain  for  binding  to  the  complex  consisting  of 
two  molecules  of  HNF-la  and  two  molecules  of 
DCoH.  Developmentally,  the  DCoH  protein  is  ex- 
pressed early  (about  day  9  or  10  after  fertiliza- 
tion) in  mice  and  is  expressed  in  a  group  of  tis- 
sues that  do  not  always  express  HNF-la  or  -1/3, 
suggesting  that  other  tissues  such  as  the  brain 
contain  proteins  that  can  bind  to  DCoH.  When 
co-expressed  with  HNF-la,  DCoH  enhances  tran- 
scriptional activation  by  several  hundredfold;  we 
are  presently  investigating  the  mechanism  by 
which  this  occurs. 

T  Lymphocyte  Activation 
and  Differentiation 

T  lymphocytes  undergo  two  biologically  and 
medically  important  types  of  differentiation.  The 
first  occurs  in  the  thymus  and  generates  cells  ca- 
pable of  directing  an  immune  response  to  nearly 
any  antigen.  However,  the  cells  produced  by  the 
thymus  that  circulate  in  our  blood  are  immuno- 
logically nearly  inert.  They  acquire  immunologic 
function  as  a  result  of  a  second  process  of  cellular 
differentiation  that  takes  about  10-14  days  and 
produces  T  cells  that  coordinate  the  actions  of 
other  cells  involved  in  the  immune  response  by 
production  of  cytokines  and  cell-cell  interac- 
tions. This  differentiation  pathway  is  initiated  by 
a  complex  interaction  between  the  T  cell  and  an 
antigen-presenting  cell.  The  essential  require- 
ment for  a  commitment  to  specialized  function  is 
a  highly  specific  interaction  between  histocom- 
patibility molecules,  antigen,  and  the  antigen  re- 
ceptor. This  critical  interaction  is  only  effective 
when  stabilized  by  transient  nonspecific  interac- 
tions based  on  intracellular  adhesive  molecules. 
Finally,  lymphokines  such  as  interleukin-1  and  -6 
that  are  the  secreted  products  of  the  antigen- 
presenting  cell  are  accessory  signals  necessary  to 
initiate  differentiation.  These  three  requirements 
for  the  initiation  of  differentiation — a  highly  spe- 
cific cell-cell  interaction,  a  nonspecific  adhesive 
interaction,  and  cytokines — are  similar  to  the  re- 
quirements for  the  cellular  commitment  to  dif- 
ferentiate in  other  systems. 

Because  the  interaction  between  the  antigen- 


89 


Genetic  Regulatory  Mechanisms  in  Cellular  Differentiation 


presenting  cell  and  a  T  lymphocyte  is  transient, 
all  of  the  molecular  events  required  for  the  deci- 
sion to  proceed  down  this  1 0-  to  1 4-day  pathway 
of  cellular  differentiation  must  occur  in  the  short 
period  during  which  the  T  cell  and  the  antigen- 
presenting  cell  interact.  Our  laboratory  is  seeking 
an  understanding  of  the  molecular  basis  of  this 
cellular  decision. 

Our  approach  to  understanding  the  events  that 
initiate  this  pathway  was  to  begin  by  defining  the 
molecules  essential  for  the  activation  of  genes 
that  are  known  to  be  essential  for  T  cell  activation 
and  difl'erentiation.  This  approach  led  us  to  de- 
fine two  transcription  factors  that  specifically  re- 
ceive signals  from  the  antigen  receptor  and  are 
responsible  for  activation  of  early  genes  such  as 
interleukin-2.  One  of  these  proteins,  nuclear  fac- 
tor of  activated  T  cells  (NFAT),  is  expressed  al- 
most exclusively  in  T  lymphocytes,  and  its  tran- 
scriptional activity  is  under  rigorous  control  by 
the  antigen  receptor.  Furthermore,  its  transcrip- 
tional activity  is  induced  by  the  antigen  receptor 
immediately  before  the  activation  of  most  early 
genes.  We  thus  focused  our  attention  on  this  tran- 
scription factor  rather  than  others  that  are  acti- 
vated in  many  cell  types  and  under  many  biologic 
circumstances. 

Immunosuppressants  Cyclosporin  A 
and  FK-506  Block  Nuclear  Translocation 
of  the  Cytosolic  Component  of  NFAT 

Cyclosporin  A  and  FK-506  specifically  inhibit 
T  cell  activation,  a  characteristic  that  underlies 
their  clinical  use  as  immunosuppressants  to  pre- 
vent transplant  rejection.  Although  the  mecha- 
nism of  action  of  these  drugs  is  unknown,  they 
appear  to  work  early  during  the  commitment  pe- 
riod for  T  cells.  By  acting  early,  they  block  the 
late  functions  of  T  cells  and  many  of  the  functions 
of  B  lymphocytes  and  other  hematopoietic  cells 


that  are  directed  by  T  cells.  Other  groups  have 
found  that  these  drugs  bind  and  inhibit  the  func- 
tion of  cis-trans  prolyl  isomerases.  These  en- 
zymes accelerate  the  folding  of  newly  synthe- 
sized proteins.  In  studies  with  Stuart  Schreiber 
(Harvard  University) ,  we  have  shown  that  the  iso- 
merase  activity  is  not  involved  in  the  action  of  the 
cyclosporin  and  FK-506,  but  rather  that  an  inhibi- 
tory complex  formed  between  either  cyclosporin 
A  and  cyclophilin  or  between  FK-506  and  FKBP 
blocks  signal  transduction.  This  inhibitory  com- 
plex binds  and  blocks  the  activity  of  calcineurin, 
a  heteromeric  protein  phosphatase  also  known  as 
PP2B.  This  inhibition  is  likely  to  be  related  to  the 
mechanism  of  action  of  the  FK-506  and  cyclo- 
sporin A,  since  immunosuppressive  drugs  form 
complexes  that  block  the  activity  of  calcineurin 
but  inactive  ones  do  not.  We  have  found  that  the 
specific  transcriptional  activity  of  NFAT,  but  not 
its  DNA-binding  activity,  is  affected  by  cyclo- 
sporin A  and  FK-506.  Mike  Flanagan,  Peter  Kao, 
and  Blaise  Corthesy  found  that  NFAT  is  a  complex 
heterodimeric  protein,  one  subunit  of  which  is 
constitutive,  T  cell  specific,  and  located  in  the 
cytoplasm  of  resting  cells;  the  other  subunit  is 
located  in  the  nucleus  and  is  induced.  The  tran- 
scriptionally active  protein  forms  when  the  cyto- 
plasmic component  translocates  to  the  nucleus  in 
response  to  stimulation  through  the  antigen  re- 
ceptor of  T  cells.  Cyclosporin  A  and  FK-506  ap- 
pear to  function  by  inhibiting  the  translocation 
but  do  not  interfere  with  the  induction  of  the 
nuclear  component.  The  inhibition  of  nuclear 
import  is  specific  to  the  extent  that  the  drugs  do 
not  block  nuclear  import  of  NF-kB.  Recently, 
Peter  Kao  and  Blaise  Corthesy  have  purified  the 
proteins  and  isolated  the  genes  for  the  two  sub- 
units  of  NFAT.  This  should  allow  us  to  determine 
directly  if  calcineurin  is  involved  in  the  signaling 
pathway  initiating  T  cell  activation. 


90 


The  Mechanism  of  a  Bacterial  Transposition 
Reaction 


Nancy  L.  Craig,  Ph.D. — Associate  Investigator 

Dr.  Craig  is  also  Associate  Professor  of  Molecular  Biology  and  Genetics  at  the  Johns  Hopkins  University 
School  of  Medicine.  After  receiving  a  bachelor's  degree  in  biology  and  chemistry  from  Bryn  Mawr  College, 
she  did  graduate  work  on  bacterial  responses  to  DMA  damage  with  Jeffrey  Roberts  at  Cornell  University, 
leading  to  her  Ph.D.  degree  in  biochemistry.  She  did  postdoctoral  research  on  the  integration/excision 
cycle  of  the  bacteriophage  X  with  Howard  Nash  at  the  National  Institute  of  Mental  Health.  Before  joining 
HHMI,  she  was  Associate  Professor  of  Microbiology  and  Immunology  and  of  Biochemistry  and  Biophysics 
at  the  University  of  California,  San  Francisco. 


DESPITE  DNA's  essential  role  in  maintaining 
accurate  genetic  information,  this  molecule 
displays  a  surprising  degree  of  plasticity.  We  now 
know  that  DNA  rearrangements — i.e.,  the  reorga- 
nization of  DNA  sequences  through  breakage, 
translocation,  and  rejoining  reactions — mediate 
a  wide  variety  of  fundamental  cellular  processes. 
DNA  rearrangements  play  an  important  role  in  the 
control  of  gene  expression  during  development, 
the  acquisition  of  new  genetic  elements  such  as 
viruses,  the  repair  of  damaged  DNA,  and  the  cre- 
ation of  genetic  diversity.  We  are  interested  in 
understanding  at  the  molecular  level  how  DNA 
rearrangements  occur  and  are  controlled. 

We  are  particularly  interested  in  the  type  of 
recombination  called  transposition.  In  this  reac- 
tion, a  discrete  DNA  segment  moves  from  one  po- 
sition in  a  genome  into  another,  nonhomologous 
target  position.  This  translocation  may  occur  be- 
tween different  positions  on  the  same  chromo- 
some, between  chromosomes,  or  between  chro- 
mosomes and  extrachromosomal  elements  such 
as  plasmids.  Transposable  elements  have  been 
identified  in  a  wide  variety  of  organisms.  A  nota- 
ble consequence  of  transposition  is  the  promo- 
tion of  rapid  and  extensive  genetic  change.  Trans- 
poson  insertion  results  in  the  stable  linkage  of 
information  encoded  by  the  transposon  with  the 
target  DNA.  Transposon  insertion  into  a  gene 
will  likely  inactivate  the  gene,  and  insertion  into 
DNA  sequences  that  control  the  expression  of 
nearby  genes  may  inactivate  or  activate  those 
genes.  Probably  because  of  its  potential  for 
profound  influence,  transposition  is  highly 
regulated. 

Our  research  is  focused  on  understanding  the 
transposition  of  Tn7,  a  bacterial  transposon  with 
several  unusual  properties.  Of  particular  note  is 
Tn7's  unusual  target  selectivity.  Most  transpos- 
able elements  display  little  insertion-site  selectiv- 
ity, inserting  into  many  different  targets.  By  con- 
trast, Tn7  inserts  at  high  frequency  into  a  single, 
specific  site  in  the  chromosomes  of  many  bacte- 
ria. In  Escherichia  colt,  the  organism  in  which 


we  study  Tn7,  this  special  target  is  called  an  at- 
tachment site  and  designated  attTnl.  When 
attTn  7  is  unavailable,  Tn7  resembles  most  other 
transposable  elements,  inserting  at  low  fre- 
quency into  many  target  sites. 

Like  most  mobile  DNA  segments,  Tn7  encodes 
the  machinery  that  mediates  its  transposition 
from  place  to  place.  We  have  established  that  Tn7 
encodes  a  surprisingly  complex  array  of  transposi- 
tion proteins,  and  we  have  also  identified  the  par- 
ticular DNA  sequences  at  the  ends  of  Tn7  and  at 
its  insertion  sites  that  are  the  actual  substrates  for 
transposition.  The  high-frequency  insertion  of 
Tn7  into  attTnl  is  mediated  by  four  Tn7- 
encoded  genes,  tnsABC  +  tnsD\  Tn7  insertion 
into  other  random  target  sites,  a  low-frequency 
reaction,  is  mediated  by  a  distinct  set  of  Tn7-en- 
coded  genes,  tnsABC  +  tnsE.  Tn7  also  encodes 
resistance  to  the  antibiotics  trimethoprim  and 
streptomycin/spectinomycin.  The  ability  of  Tn7 
and  other  mobile  DNA  segments  that  encode  anti- 
biotic resistance  to  insert  into  and  thus  be  joined 
to  plasmids  that  can,  in  turn,  move  among  a  vari- 
ety of  bacterial  species  underlies  the  rapid  dis- 
semination of  antibiotic  resistance  among  bacte- 
rial populations. 

Our  overall  goals  are  to  understand  in  molecu- 
lar detail  how  Tn7  moves  from  place  to  place 
and  how  the  frequency  of  this  movement  is 
modulated.  We  expect  that  understanding  the 
macromolecular  interactions  that  underlie  Tn7 
transposition  will  contribute  not  only  to  the 
understanding  of  DNA  recombination  but  also 
to  the  understanding  of  other  complex  protein- 
nucleic  acid  transactions  such  as  DNA  replica- 
tion, transcription,  and  RNA  processing.  We  are 
using  a  variety  of  biochemical  and  genetic  ap- 
proaches to  dissect  Tn7  transposition. 

The  fundamental  steps  in  transposition  are  the 
DNA  cleavages  that  separate  the  transposon  from 
its  flanking  donor  DNA  and  the  subsequent  break- 
age of  the  target  DNA  and  the  joining  of  the  trans- 
poson to  the  target  DNA.  Little  is  known  in  molec- 
ular terms  about  how  such  reactions  occur.  Our 


91 


The  Mechanism  of  a  Bacterial  Transposition  Reaction 


HHMI-supported  effort  is  focused  on  dissecting 
the  mechanism  of  the  DNA  strand  breakage  and 
joining  reactions  during  Tn7  transposition. 

We  previously  developed  a  cell-free  system  for 
Tn7  transposition  to  attTnl.  Tn7  inserts  effi- 
ciently and  specifically  into  attTnl  in  vitro  in 
the  presence  of  four  purified  TnV-encoded  pro- 
teins: TnsA  +  TnsB  +  TnsC  +  TnsD.  Thus  these 
proteins  participate  directly  in  transposition.  We 
have  also  established  that  Tn7  strand  breakage 
and  joining  can  be  carried  out  by  a  subset  of  these 
proteins:  TnsA  +  TnsB  +  TnsC.  We  now  want  to 
know  which  transposition  protein  carries  out 
which  particular  step(s)  in  transposition  and  the 
chemical  basis  of  these  reactions.  We  are  probing 
these  questions  by  examining  the  ability  of  each 
transposition  protein  to  individually  execute  a 
subset  of  the  chemical  steps  that  underlie  the 
complete  transposition  reaction.  Our  strategies 
to  reveal  what  may  be  usually  suppressed  activi- 
ties include  manipulating  the  reaction  conditions 
and  using  DNA  substrates  derivatized  in  particu- 
lar ways  that  we  suspect  may  provoke  strand 
cleavage  and  joining. 

Another  biochemical  method  we  are  using  to 
identify  the  domains  of  the  recombination  pro- 
teins that  are  most  intimately  involved  in  DNA 
strand  breakage  and  joining  is  to  determine, 
through  protein-DNA  crosslinking  studies,  the 
segments  of  the  recombination  proteins  that  most 
closely  appose  the  positions  of  DNA  strand  break- 
age and  joining  during  transposition. 

In  a  complementary  genetic  approach,  we  are 
seeking  to  identify  "active  sites"  in  the  recombi- 
nation proteins  that  promote  DNA  strand  break- 
age and  joining,  by  isolating  and  characterizing 
mutant  transposition  proteins  altered  in  their  abil- 
ity to  promote  these  reactions.  We  suspect  that 


these  active  sites  actually  lie  in  TnsB,  which  binds 
specifically  to  the  ends  of  Tn7,  i.e.,  the  sites  of 
strand  breakage  and  joining  during  transposition. 
We  are  also  particularly  interested  in  TnsB  be- 
cause there  is  some  sequence  similarity  between 
TnsB  and  the  recombinases  of  retrotransposons, 
including  the  integrases  of  retroviruses.  Our 
long-term  goals  are  to  describe  in  detail  the  chem- 
ical steps  that  underlie  DNA  breakage  and  joining 
and  to  understand  how  the  recombination  pro- 
teins promote  these  reactions. 

We  are  also  interested  in  understanding  how 
transposition  is  controlled  and,  in  particular,  in 
understanding  the  interplay  between  Tn7  and  its 
bacterial  host. 

Much  of  our  biochemical  characterization  of 
Tn7  transposition  has  focused  on  dissecting  the 
high-frequency  insertion  of  Tn7  into  its  spe- 
cific insertion  site  attTn  7.  We  are  now  working 
to  extend  the  biochemical  analysis  of  Tn7  trans- 
position to  its  low-frequency  insertion  into  ran- 
dom target  sites.  We  are  developing  a  cell-free 
system  for  Tn7  insertion  into  random  target 
sites.  We  will  then  be  poised  to  dissect  the  dif- 
ferences between  high-  and  low-frequency  Tn7 
transposition  in  molecular  detail.  We  also  sus- 
pect that  characterization  of  Tn7  insertion  into 
random  target  sites  will  reveal  host  proteins 
that  likely  participate  in  this  recombination  re- 
action. We  are  also  examining  how  the  struc- 
ture of  the  E.  coli  chromosome  may  influence 
Tn7  transposition.  Another  strategy  we  are  us- 
ing to  probe  the  control  of  Tn7  transposition  is 
to  isolate  and  characterize  mutant  Tns  proteins 
that  display  altered  transposition  properties. 
The  above  work  on  the  control  of  Tn7  transposi- 
tion is  supported  by  a  grant  from  the  National 
Institutes  of  Health. 


92 


Mechanisms  of  Antigen  Processing 


Peter  Cresswell,  Ph.D. — Investigator 

Dr.  Cresswell  is  also  Professor  of  Immunobiology  at  Yale  University  School  of  Medicine.  He  was  born 
and  educated  in  the  United  Kingdom.  He  received  undergraduate  degrees  in  chemistry  and  microbiology 
from  the  University  of  Newcastle  upon  Tyne  and  his  Ph.D.  degree  in  biochemistry  and  immunology 
from  London  University.  Dr.  Cresswell  took  postdoctoral  training  at  Harvard  University  with 
Jack  Strominger.  Before  assuming  his  position  at  Yale,  he  was  Chief  of  the  Division  of  Immunology 
at  Duke  University  Medical  Center. 


FOREIGN  protein  antigens  must  be  proteo- 
lyzed  into  peptides  and  must  associate  with 
specialized  membrane  glycoproteins  before  they 
can  be  recognized  by  T  lymphocytes.  These 
membrane  glycoproteins  are  known  as  major  his- 
tocompatibility complex  (MHC)  molecules,  be- 
cause of  their  original  definition  as  the  critical 
antigens  responsible  for  organ  graft  rejection  be- 
tween members  of  the  same  species.  Two  types  of 
MHC  molecules  have  evolved,  apparently  to  deal 
with  two  different  categories  of  antigens.  Class  I 
MHC  molecules  bind  peptides  derived  from  cyto- 
solic  proteins  synthesized  by  the  cell  that  bears 
them,  and  are  critical  for  T  cell  recognition  of 
virally  infected  cells.  Class  H  MHC  molecules 
bind  peptides  derived  from  proteins  internalized 
by  a  number  of  class  Il-positive  cell  types,  such 
as  B  cells  or  macrophages,  collectively  known  as 
antigen-presenting  cells.  The  major  current  inter- 
est of  my  laboratory  is  in  the  molecular  mecha- 
nisms involved  in  generating  these  MHC-peptide 
complexes. 

Class  I  MHC-Peptide  Association 

The  association  of  cytosolic  protein-derived 
peptides  with  class  I  MHC  molecules  presents  a 
topological  problem,  in  that  the  peptides  or  their 
precursor  proteins  must  cross  an  intracellular 
membrane  for  binding  to  occur.  Evidence  from 
the  literature  suggests  that  peptide  binding  oc- 
curs early  in  the  intracellular  transport  of  class  I 
MHC  molecules,  perhaps  in  the  endoplasmic  re- 
ticulum (ER). 

A  mutant  cell  line,  T2,  derived  in  our  labora- 
tory from  a  similar  cell  line,  .174  (produced  by 
Robert  DeMars  at  the  University  of  Wisconsin- 
Madison)  ,  is  defective  in  one  or  more  of  the  steps 
involved  in  generating  class  I  MHC-peptide  com- 
plexes. Somatic  cell  genetic  evidence  suggested 
that  the  gene  (or  genes)  involved  is  localized  in  a 
region  of  the  MHC  that  is  deleted  in  T2.  Two  of 
these  genes  are  homologous  to  a  group  of  pro- 
teins with  multiple  membrane-spanning  domains 
known  as  the  ATP-binding  cassette  (ABC)  family 
of  membrane  transporters. 

These  molecules  are  generally  involved  in  the 


active  transport  of  small  molecules,  or  occasion- 
ally larger  proteins,  across  membranes.  It  has 
been  proposed  that  the  members  of  the  family 
encoded  in  the  MHC  are  responsible  for  the  trans- 
location of  cytosolic  peptides  into  the  ER,  where 
they  bind  to  class  I  MHC  molecules.  Data  from  my 
laboratory  have  shown  that  most  class  I  alleles 
expressed  in  T2  indeed  lack  peptides  when  they 
are  affinity  purified  and  associated  peptides  are 
analyzed  chromatographically. 

Human  class  I  MHC  (HLA)  molecules  generally 
fail  to  be  transported  to  the  cell  surface  when 
expressed  in  T2,  a  probable  consequence  of  the 
lack  of  associated  peptides.  An  exceptional  al- 
lele, HLA-A2,  is  significantly  surface-expressed  at 
20-50  percent  of  wild-type  levels.  HLA-A2  is  as- 
sociated with  a  limited  set  of  peptides  in  T2, 
three  of  which  have  been  isolated  and  se- 
quenced. Two  proved  to  be  derived  from  the  sig- 
nal sequence  of  an  interferon-inducible  protein 
known  as  IP- 30,  and  the  third  corresponded  to  no 
known  protein. 

The  two  identified  peptides,  a  nonamer  and 
1 1  -mer  respectively,  are  presumably  transpor- 
ted into  the  ER  by  the  conventional  signal- 
recognition  mechanism  involved  in  the  translo- 
cation of  secretory  and  transmembrane  proteins. 
This  would  explain  their  association  with  HIA-A2 
in  a  cell  line  defective  in  the  normal  mechanisms 
of  peptide  transport.  In  wild-rype  cells,  class  I- 
associated  peptides  are  generally  nonameric.  The 
existence  of  the  1 1-mer  peptide,  and  the  fact  that 
the  third  peptide  is  a  1 3-mer,  argues  that  the  gen- 
eration of  nonamers  in  wild-type  cells  may  be  a 
normal  facet  of  peptide  generation  and  transport. 

Curiously,  mouse  class  I  MHC  (H-2)  molecules 
in  general  are  well  surface-expressed  in  the  T2 
cell  line,  even  though  they  are  devoid  of  asso- 
ciated peptides.  This  has  led  us  to  hypothesize 
that  the  human  class  I  molecules  are  subject  to  a 
specific  retention  mechanism  that  prevents  them 
from  leaving  the  ER  unless  they  are  associated 
with  a  peptide.  Understanding  the  difi'erential 
transport  properties  of  class  I  H-2  and  HLA  mole- 
cules in  both  normal  and  mutant  cells  is  a  major 
area  of  emphasis  in  our  laboratory  and  should  un- 


93 


Mechanisms  of  Antigen  Processing 


cover  additional  details  of  the  mechanisms  in- 
volved in  the  in  vivo  generation  of  class  I  MHC- 
peptide  complexes. 

Class  II  MHC-Peptide  Association 

Class  II  MHC  molecules  associate  late  in  trans- 
port with  peptides  derived  from  internalized  pro- 
tein antigens.  It  is  believed  that  in  the  early  stages 
of  transport,  they  follow  the  same  pathway  as 
class  I  molecules,  namely  from  the  ER  through 
the  Golgi  complex.  This  leads  to  the  question. 
Why  do  class  II  molecules  nothind  peptides  from 
the  same  set  of  cytosolically  derived  peptides  that 
associate  with  class  I  MHC  molecules? 

At  least  a  partial  answer  lies  in  the  association 
of  class  II  molecules  with  an  additional  glycopro- 
tein, the  invariant  chain.  This  molecule  is  not  a 
product  of  the  MHC  and  is  not  structurally  related 
to  class  I  or  class  II  glycoproteins.  It  associates 
with  class  II  molecules  immediately  upon  synthe- 
sis in  the  ER,  forming  a  large  nine-chain  structure 
that  we  have  found  to  consist  of  a  core  trimer  of 
invariant  chains  associated  with  three  class  II  mol- 
ecules, each  of  which  is  a  two-subunit  hetero- 
dimer.  Class  II  molecules  in  this  structure  cannot 
bind  antigenic  peptides,  though  they  acquire  the 
capacity  to  do  so  if  released  by  mild  denaturation 
or  partial  proteolysis  of  the  invariant  chain.  Pre- 
sumably their  association  with  the  invariant  chain 
in  vivo  prevents  class  II  MHC  molecules 
from  binding  inappropriate  peptides  early  in 
transport. 

After  assembly,  the  nine-chain  class  II  MHC- 
invariant  chain  complex  is  transported  from  the 


ER  and  through  the  Golgi  apparatus.  We  have 
found  that  the  invariant  chain  then  targets  the 
complex  to  endosomal  structures,  where  it  en- 
counters internalized  foreign  antigens,  shown 
strikingly  when  influenza  virus  is  used  as  a  test 
antigen .  Evidence  obtained  with  proteinase  inhib- 
itors, such  as  leupeptin,  suggests  that  the  invari- 
ant chain  is  proteolytically  degraded  in  the  endo- 
some,  releasing  class  II  molecules  capable  of 
binding  peptides. 

The  simplest  model  for  the  generation  of  class 
II  MHC-peptide  complexes  suggests  that  internal- 
ized proteins  are  endosomally  degraded  into  pep- 
tides that  bind  to  the  newly  released  class  II 
molecules.  Recent  evidence,  however,  suggests 
that  this  model  is  too  simple.  We  have  found 
that  the  T2  cell  line,  referred  to  above  as  a  class  I 
MHC  antigen-processing  mutant,  is  also  a  class 
II  MHC  antigen-processing  mutant.  T2  itself  lacks 
class  II  molecules  as  a  result  of  the  homozygous 
deletion  of  the  MHC  region  encoding  class  II  mol- 
ecules. However,  if  mouse  class  II  molecules  (I- 
A'')  are  expressed  in  T2  by  transfection,  the  cell 
line  is  unable  to  process  internalized  protein  an- 
tigens into  class  II  MHC-associated  peptides  rec- 
ognizable by  T  cells.  To  date,  it  appears  that  both 
mouse  and  human  class  II  molecules  expressed  in 
T2  associate  normally  with  the  invariant  chain 
and  are  transported  to  an  endosomal  compart- 
ment where  the  invariant  chain  is  proteolytically 
cleaved.  The  missing  steps  in  generating  func- 
tional class  II  MHC-peptide  complexes  remain  to 
be  determined,  but  again  appear  to  be  the  proper- 
ties of  MHC-linked  genes. 


94 


Regulation  of  Human  Retroviral 
Gene  Expression 


Bryan  R.  Cullen,  Ph.D. — Associate  Investigator 

Dr.  Cullen  is  also  Associate  Professor  in  the  Section  of  Genetics  and  the  Department  of  Microbiology  and 
Immunology  and  Associate  Medical  Research  Professor  in  the  Department  of  Medicine  at  Duke  University 
Medical  Center.  He  received  his  master's  degree  in  virology  from  the  University  of  Birmingham,  England. 
After  emigrating  to  the  United  States,  Dr.  Cullen  worked  as  a  research  technician  for  several  years  before 
reentering  graduate  school  at  the  University  of  Medicine  and  Dentistry  of  New  Jersey,  where  he  received 
his  Ph.D.  degree  in  microbiology.  Before  accepting  his  current  position  at  Duke,  Dr.  Cullen  studied  gene 
regulation  in  higher  eukaryotes,  as  a  laboratory  head  in  the  Department  of  Molecular  Genetics 
at  Hoffmann- la  Roche. 


RETROVIRUSES  derive  their  name  from  their 
ability  to  reverse  the  normal  flow  of  genetic 
information  from  DNA  to  RNA.  They  possess  the 
remarkable  ability  to  synthesize  a  double- 
stranded  DNA  copy  of  their  single-stranded  RNA 
genome  and  then  to  integrate  this  DNA  copy  into 
the  genome  of  the  infected  host  cell.  Once  the 
genome  of  a  retrovirus  is  integrated  into  a  host 
chromosome,  it  is  indistinguishable  from  a  host 
gene  and  may  be  actively  transcribed  by  the  host's 
transcriptional  machinery. 

The  infection  of  an  animal  by  retroviruses  can 
result  in  a  number  of  disease  states,  of  which  the 
most  common  is  leukemia.  Indeed,  the  avian  leu- 
kemia virus  (ALV)  was  the  first  oncogenic  virus  to 
be  defined  experimentally.  ALV  and  the  some- 
what similar  murine  leukemia  viruses  continue  to 
be  studied  extensively  as  models  for  this  virus 
group.  This  research  has  not  only  helped  to  delin- 
eate the  retroviral  replication  cycle  but  has  also 
greatly  advanced  our  understanding  of  retroviral 
oncogenesis.  Most  importantly,  this  research 
has  allowed  the  definition  of  a  number  of  cellular 
genes,  the  oncogenes,  whose  inappropriate  ex- 
pression can  contribute  to  cellular  transformation. 

Although  animal  retroviruses  have  been  the 
subject  of  scientific  research  for  some  time,  the 
discovery  of  human  retroviruses  occurred  only 
within  the  last  decade.  Two  major  groups  of 
pathogenic  human  retroviruses  have  been  identi- 
fied thus  far.  Human  T  cell  leukemia  viruses 
(HTLV-I  and  HTLV-II)  are  known  to  be  causative 
agents  of  human  leukemias,  including  adult  T 
cell  leukemia,  and  are  significant  disease  agents 
in  several  parts  of  the  world,  including  Japan  and 
the  Caribbean  basin.  Of  even  more  concern  are 
the  human  immunodeficiency  viruses  (HIV-1  and 
HIV-2),  which  are  a  leading  cause  of  disease  and 
death  in  parts  of  Africa  and  in  the  United  States.  A 
third  group  of  retroviruses,  the  human  foamy  vi- 
ruses (HFV),  has  recently  been  detected  in  hu- 
man populations  in  both  Africa  and  Oceania  but 
has  not  yet  been  clearly  associated  with  any 
disease. 


A  striking  feature  of  all  three  classes  of  human 
retroviruses  is  that  they  each  encode  regulatory 
proteins  that  control  both  the  quantity  and  qual- 
ity of  viral  gene  expression.  This  regulatory  com- 
plexity is  not  observed  in  many  animal  retrovi- 
ruses, including  the  avian  and  murine  leukemia 
viruses,  and  may  strongly  influence  the  patho- 
genic potential  of  these  "complex  retroviruses." 
The  major  focus  of  this  laboratory  has  been  the 
determination  of  the  role  and  mechanism  of  ac- 
tion of  these  viral  trans-activators,  with  a  concen- 
tration particularly  on  the  Tat  and  Rev  regulatory 
proteins  of  HIV- 1 . 

In  the  past  we  demonstrated  that  the  Tat  pro- 
tein of  HIV-1  acts  on  sequences  located  within 
the  HIV-1  promoter  element  (the  long  terminal 
repeat  or  LTR)  to  increase  the  level  of  expression 
of  linked  genes.  This  increased  viral  gene  expres- 
sion occurs  via  a  bimodal  mechanism  that  in- 
volves an  increase  in  the  rate  of  transcription  of 
HIV-1  mRNAs  and  in  the  efficiency  of  transla- 
tional  utilization  of  those  RNAs.  The  target  se- 
quence for  Tat  is  a  59-nucleotide  RNA  stem-loop 
structure  located  at  the  very  5'  end  of  all  viral 
mRNA  molecules.  The  direct  interaction  of  Tat 
with  this  RNA  structure  leads  to  an  enhancement 
of  viral  transcription  initiation  and  elongation. 
This  mechanism,  which  may  be  unique  to  Tat, 
remains  poorly  understood  but  is  likely  to  in- 
volve the  interaction  of  Tat  with  cellular  proteins 
that  are  currently  poorly  defined.  The  identifica- 
tion and  characterization  of  these  cellular  pro- 
teins is  a  major  research  aim  of  this  laboratory. 

A  second  HIV- 1  protein,  Rev,  is  required  for  the 
expression  of  viral  structural  proteins  but  is  dis- 
pensable for  the  expression  of  viral  regulatory 
proteins.  Our  research  has  demonstrated  that  Rev 
acts  post-transcriptionally  to  induce  the  cytoplas- 
mic expression  of  the  unspliced  or  incompletely 
spliced  RNAs  that  encode  the  viral  structural  pro- 
teins Gag  and  Env,  while  simultaneously  repress- 
ing the  expression  of  the  fully  spliced  RNAs  that 
encode  the  viral  regulatory  proteins,  including 


95 


Regulation  of  Human  Retroviral  Gene  Expression 


Rev  itself.  The  Rev  protein  therefore  regulates  its 
own  expression  via  a  negative  feedback  mecha- 
nism. It  has  been  proposed  that  Rev  achieves  this 
effect  by  specifically  regulating  the  export  of 
viral  RNAs  from  the  cell  nucleus  to  the  cyto- 
plasm. This  specificity  is  conferred  by  a  cis-acting 
viral  RNA  target  sequence,  the  Rev  response  ele- 
ment (RRE),  which  has  been  shown  to  form  a 
complex  RNA  secondary  structure.  Recent  data 
demonstrate  that  Rev  specifically  recognizes,  and 
binds  to,  a  short,  approximately  13-nucleotide 
primary  sequence  within  the  context  of  the  larger 
RRE  structure.  Rev  function  also  appears  to  re- 
quire the  subsequent  binding  of  additional  Rev 
protein  monomers  to  secondary  target  sites 
within  the  RRE. 

Mutational  analysis  of  the  Rev  protein  has 
demonstrated  the  existence  of  two  functional 
domains.  The  first  is  a  sequence-specific  RNA- 
binding  domain  required  for  binding  to,  and  mul- 
timerization  on,  the  RRE,  while  the  second  is  be- 
lieved to  interact  with  a  currently  unidentified 


cellular  protein  that  may  form  part  of  the  cellular 
RNA  transport  machinery.  Mutations  of  this  latter 
domain,  the  Rev  activation  domain,  give  rise  to 
Rev  proteins  that  act  as  competitive  inhibitors  of 
the  wild-type  Rev  trans-activator.  Mutant  HIV-1 
proteins  of  this  type  (dominant  negative  mu- 
tants) may  have  future  application  in  the  gene 
therapy  of  HIV-1 -infected  individuals.  A  major 
focus  of  this  laboratory  is  the  development  of 
these  trans-dominant  Rev  mutants  and,  in  particu- 
lar, the  further  investigation  of  the  role  of  cellu- 
lar proteins  in  the  Rev  response. 

Finally,  we  have  begun  to  expand  our  research 
to  other  human  retroviruses,  including  HIV-2  and 
the  apparently  nonpathogenic  HFV,  as  well  as  to 
related  animal  retroviruses,  such  as  visna  virus. 
The  elucidation  of  similarities  and  differences  in 
the  regulation  of  gene  expression  among  these 
retroviruses  should  facilitate  the  identification 
and  understanding  of  the  cis-  and  trans-acting  ele- 
ments required  for  their  replication  and 
pathology. 


96 


Mechanism  of  Retrovirus  Infection 


James  M.  Cunningham,  M.D. — Assistant  Investigator 

Dr.  Cunningham  is  also  Assistant  Professor  of  Medicine  at  Brigham  and  Women's  Hospital  and  Harvard 

Medical  School.  He  received  a  B.S.  degree  in  chemistry  from  the  University  of  Michigan  and  an  M.D.  degree 

from  Stanford  University  School  of  Medicine.  After  clinical  training  in  internal  medicine  (Peter  Bent 

Brigham  Hospital)  and  oncology  (Dana  Farber  Cancer  Institute),  he  was  a  postdoctoral  fellow 

in  the  laboratory  of  Robert  Weinberg  at  the  Massachusetts  Institute  of  Technology.  Dr.  Cunningham  was 

an  HHMI  Associate  at  Brigham  and  Women 's  Hospital  and  Harvard  Medical  School 

before  assuming  his  current  appointment. 


VIRUSES  are  parasites.  They  cannot  produce 
progeny  on  their  own,  but  must  rely  on  the 
machinery  provided  by  the  host  cell  to  replicate 
the  viral  genome  and  assemble  new  virus  parti- 
cles. Infection  is  initiated  by  attachment  of  the 
virus  to  the  host  cell — the  first  step  in  a  complex 
reaction  that  results  in  transfer  of  the  viral  ge- 
nome through  the  cell  membrane  and  into  the 
cytoplasm.  This  attachment,  or  binding,  is  a  con- 
sequence of  the  interaction  between  proteins  ex- 
posed on  the  surface  of  the  virus  and  the  host  cell 
plasma  membrane. 

Cells  that  do  not  express  a  suitable  virus- 
binding  protein,  called  a  receptor,  are  not  suscep- 
tible to  infection  by  a  particular  virus.  Indeed, 
the  ability  of  many  pathogenic  viruses,  such  as 
human  immunodeficiency  virus,  poliovirus,  and 
certain  herpesviruses,  to  infect  specific  host  tis- 
sues has  been  closely  correlated  with  the  expres- 
sion of  specific  receptors. 

Our  laboratory  has  been  interested  in  the  mech- 
anism of  infection  utilized  by  Moloney  murine 
leukemia  virus  (MoMuLV) ,  a  member  of  a  group 
of  related  leukemogenic  retroviruses  found  in  vir- 
tually all  vertebrates.  We  have  isolated  a  molecu- 
lar clone,  MCAT,  which  confers  MoMuLV  infectiv- 
ity  upon  introduction  into  mammalian  cells  that 
are  not  normally  susceptible  to  infection.  Subse- 
quent experiments  have  demonstrated  that  MCAT 
encodes  for  a  membrane  protein  that  serves  as  the 
MoMuLV  receptor.  Our  current  research  is  ad- 
dressed toward  dissecting  the  molecular  details 
of  the  virus-receptor  interaction  that  mediates  in- 
fection and  understanding  the  function  of  the  re- 
ceptor in  normal  cell  metabolism. 

The  MoMuLV  receptor  is  not  present  in  mice 
for  the  convenience  of  the  virus,  but  rather  pro- 
vides a  portal  for  entry  of  lysine,  arginine,  and 
ornithine,  amino  acids  that  carry  a  net  positive 
charge.  There  is  a  similarity  between  MCAT  and 
two  membrane  proteins  in  yeast  that  are  also 
amino  acid  transporters.  This  suggests  conserva- 
tion of  a  single  mechanism  for  transport  of  these 
amino  acids  over  evolutionary  time  and  predicts 
that  proteins  similar  to  the  MoMuLV  receptor  are 


used  by  all  animals.  Inherited  disorders  of  cat- 
ionic  amino  acid  transport  have  been  described 
in  patients  that  may  be  explained  by  mutations  in 
MCAT  genes,  a  hypothesis  we  are  now  examining. 

A  protein  that  is  closely  related  to  the  MoMuLV 
receptor  has  been  identified  in  liver  tissue.  We 
have  demonstrated  that  this  protein  is  also  an 
amino  acid  transporter  with  properties  that  may 
help  explain  the  specific  requirements  for  argi- 
nine metabolism  by  the  liver:  the  capacity  of  he- 
patocytes  to  clear  the  portal  vein  of  the  high  con- 
centration of  amino  acids  present  after  a  big  meal 
and  the  regulation  of  the  urea  cycle,  the  meta- 
bolic pathway  used  to  eliminate  nitrogen  waste. 
Related  forms  of  this  protein  are  expressed  in  T 
and  B  lymphocytes  and  macrophages  that  are  ac- 
tivated as  part  of  the  host  response  to  bacterial 
and  parasitic  infection.  Within  the  past  few  years, 
arginine  has  been  identified  as  the  substrate  for 
nitric  oxide,  an  important  mediator  of  the  host 
defense  against  these  pathogens.  Currently  we 
are  investigating  how  the  family  of  related  amino 
acid  transporters  can  influence  nitric  oxide  pro- 
duction by  regulating  arginine  availability.  As 
part  of  these  studies,  we  have  identified  a  molecu- 
lar clone  that  encodes  nitric  oxide  synthase,  the 
enzyme  that  is  responsible  for  the  synthesis  of 
nitric  oxide  from  arginine.  Expression  of  the  gene 
encoding  this  enzyme  is  stimulated  in  macro- 
phages that  are  activated  in  response  to  infection. 

Our  laboratory  remains  interested  in  how  retro- 
viruses interact  with  MCAT  to  gain  entry  into  the 
cell.  Recently  we  have  prepared  an  antibody  that 
can  recognize  the  MCAT  protein  and  thereby  per- 
mits examination  of  its  synthesis  in  infected  and 
uninfected  cells.  These  studies  have  demon- 
strated that  MCAT  is  normally  modified  by  the 
addition  of  carbohydrate  during  its  transit  from 
the  ribosome  to  the  plasma  membrane.  In  cells 
that  have  been  infected  with  MoMuLV,  we  have 
identified  an  interaction  between  MCAT  and  the 
virus  envelope  protein  that  impairs  MCAT  matura- 
tion and  decreases  amino  acid  uptake.  This  de- 
crease may  result  from  failure  of  the  mature 
MCAT  protein  to  arrive  at  the  plasma  membrane. 
This  finding  has  important  implications  for  un- 


97 


Mechanism  of  Retrovirus  Infection 


derstanding  the  host  response  to  retrovirus  infec- 
tion and  also  for  understanding  virus  interference 
— the  production  by  cells  of  retroviruses  that  are 
resistant  to  additional  infection  by  the  same  virus. 
To  investigate  this  problem,  we  are  now  examin- 
ing the  behavior  of  MCAT  proteins  that  contain 


mutations  at  the  site  of  virus  attachment.  Our 
long-term  goal  is  to  understand  the  chemical 
basis  of  the  virus-transporter  interaction  in  suffi- 
cient molecular  detail  to  design  small  molecules 
that  can  block  virus  binding  and  prevent 
infection. 


98 


The  Nuclear  Pore  Complex 


Laura  I.  Davis,  Ph.D. — Assistant  Investigator 

Dr.  Davis  is  also  Assistant  Professor  in  the  Section  of  Genetics  and  the  Department  of  Cell  Biology  at  Duke 
University  Medical  Center.  She  received  her  undergraduate  degree  from  the  University  of  California,  San 
Diego,  and  her  Ph.D.  degree  from  the  Rockefeller  University,  where  she  studied  with  Giinter  Blobel. 
Her  postdoctoral  work  was  done  with  Gerald  Fink  at  the  Whitehead  Institute. 


ONE  of  the  hallmarks  of  eukaryotic  cells  is  the 
presence  of  membranous  barriers  that  di- 
vide the  cell  into  functional  compartments  called 
organelles.  How  these  compartments  are  gener- 
ated and  how  traffic  through  them  is  controlled 
are  matters  of  intense  interest  in  cell  biology.  Our 
focus  is  on  the  nuclear  envelope,  which  separates 
the  genomic  material  from  the  rest  of  the  cell. 
Both  RNAs  and  proteins  move  across  the  enve- 
lope, between  the  nucleus  and  cytoplasm,  and 
the  populations  found  in  the  two  compartments 
are  very  different. 

For  example,  newly  synthesized  mRNA  must  be 
extensively  edited  or  processed  before  leaving 
the  nucleus.  The  mechanism  that  restricts  its  ex- 
port until  processing  is  complete  remains  un- 
clear. Similarly,  only  certain  proteins  are  im- 
ported into  the  nucleus.  These  contain  specific 
signals  that  must  be  recognized  by  some  compo- 
nent of  the  import  apparatus.  Recently  it  has  be- 
come clear  that  the  cell  can  regulate  the  availabil- 
ity of  these  signals  by  changing  the  context  in 
which  they  are  found.  For  example,  transcrip- 
tional activators  that  control  the  growth  state  of 
the  cell  by  turning  on  gene  expression  can  be 
held  in  an  inactive  form  in  the  cytosol  if  their 
signals  are  hidden  from  the  import  apparatus. 
When  the  cell  is  stimulated  to  begin  growth,  the 
signals  are  uncovered,  allowing  the  transcrip- 
tional activators  to  enter  the  nucleus  and  exert 
their  function.  Thus  nucleocytoplasmic  transport 
has  an  important  regulatory  role  in  controlling 
gene  expression. 

It  is  thought  that  all  nucleocytoplasmic  trans- 
port proceeds  through  large  proteinaceous  chan- 
nels that  perforate  the  nuclear  envelope.  Called 
nuclear  pore  complexes  (NPCs),  these  structures 
are  about  30  times  as  large  as  an  ion  channel  and 
are  probably  composed  of  over  200  different 
polypeptides.  Among  the  few  known  compo- 
nents of  the  NPC  are  members  of  a  family  of  re- 
lated proteins  called  nucleoporins.  These  pro- 
teins have  been  localized  to  an  iris-like  structure 
in  the  middle  of  the  NPC,  called  the  central  trans- 
porter, and  are  thought  to  play  an  essential  role  in 
mediating  protein  (and  perhaps  RNA)  transport. 


They  are  required  for  binding  of  proteins  to  the 
NPC  prior  to  nuclear  import,  and  antibodies  that 
bind  to  the  nucleoporins  can  block  the  energy- 
dependent  movement  of  proteins  through  the 
NPC.  While  it  is  possible  that  the  nucleoporins 
themselves  recognize  the  signals  on  proteins  des- 
tined for  the  nucleus,  most  of  the  available  evi- 
dence suggests  that  they  are  more  likely  to  act  as 
docking  points  for  cytosolic  receptors  that  actu- 
ally recognize  the  signals. 

Our  goal  has  been  to  understand  more  about 
the  function  of  the  nucleoporins  and  to  identify 
other  components  of  the  transport  apparatus.  We 
use  the  budding  yeast  Saccharomyces  cerevi- 
siae,  since  it  is  amenable  to  both  genetic  and  bio- 
chemical analysis.  Using  antibodies  that  recog- 
nize the  mammalian  nucleoporins,  I  previously 
identified  homologues  of  these  proteins  in  yeast 
and  cloned  the  gene  encoding  one  of  them 
{NUPl).  The  genes  encoding  two  others  have 
now  been  isolated  (NUP2  and  NSPl) .  The  pro- 
tein encoded  by  NUPl  is  essential  for  viability, 
and  we  have  isolated  nupl  mutants  that  confer 
growth  at  one  temperature  but  not  another.  These 
conditional  mutants  have  been  used  to  assay  the 
phenotype  resulting  from  loss  of  NUPl  function. 
Using  immunofluorescence,  we  have  found  that 
protein  import  into  the  nucleus  is  severely  inhib- 
ited at  the  nonpermissive  temperature,  providing 
in  vivo  evidence  that  NUPlp  is  required  for  pro- 
tein transport. 

Conditional  mutants  will  also  be  useful  for 
identifying  proteins  that  functionally  interact 
with  NUPlp.  To  do  this,  we  are  using  genetic 
screens  to  find  mutations  in  new  genes  that  either 
enhance  or  suppress  the  conditional  phenotype 
of  the  nupl  mutants.  The  assumption  is  that  mu- 
tations in  proteins  that  functionally  interact  with 
NUPlp  will  either  exacerbate  the  defect  in  nupl 
mutants  and  lead  to  death  at  all  temperatures,  or 
will  overcome  the  defect  and  allow  grouth  at  the 
restrictive  temperature.  Using  this  approach,  we 
hope  to  identify  new  NPC  components,  as  well  as 
cytoplasmic  and  nuclear  proteins  that  may  inter- 
act transiently  with  the  nucleoporins  in  a  func- 
tional manner. 

We  have  also  taken  a  biochemical  approach  to 


99 


The  Nuclear  Pore  Complex 


identifying  proteins  that  interact  with  NUPlp. 
Using  a  specific  antibody,  we  isolated  the  NUPl 
protein  from  cell  extracts  and  have  found  it  to  be 
stably  associated  with  several  other  proteins. 
Some  of  these  appear  to  be  nucleoporins,  while 
others  are  as  yet  unidentified.  We  are  now  purify- 


ing the  individual  members  of  the  complex,  in 
order  to  obtain  protein  sequences  that  we  can  use 
to  isolate  the  genes  encoding  them.  We  expect 
some  of  these  proteins  to  correspond  with  those 
we  identify  through  genetic  interactions  with 
NUPl. 


100 


Molecular  Approaches  to  T  Lymphocyte 
Recognition  and  Differentiation 


Mark  M.  Davis,  Ph.D. — Investigator 

Dr.  Davis  is  also  Professor  of  Microbiology  and  Immunology  at  Stanford  University  School  of  Medicine.  He 
received  his  B.A.  degree  from  the  Johns  Hopkins  University  and  his  Ph.D.  degree  in  molecular  biology  from 
the  California  Institute  of  Technology.  He  held  positions  at  NIH  as  a  postdoctoral  fellow  and  Staff  Fellow 
before  joining  the  staff  at  Stanford.  He  is  the  recipient  of  the  Eli  Lilly  award  in  Microbiology 
and  Immunology  and  a  Gairdner  Foundation  award. 


WE  have  focused  on  several  major  areas  in 
immunology  that  generally  resolve  into 
two  questions:  How  do  T  cells  recognize  foreign 
entities?  How  is  lymphocyte  differentiation  con- 
trolled, both  in  the  thymus  and  in  the  periphery? 
An  additional  goal  is  to  refine  and  better  integrate 
recombinant  DNA  technology  with  other  power- 
ful techniques  in  immunology,  as  an  approach  to 
defining  the  function  of  unknown  genes  or 
poorly  understood  genes  and  their  products. 

Topology  of  T  Cell  Recognition 

The  work  of  many  investigators  over  the  years 
has  shown  that  T  cells,  through  their  antigen  re- 
ceptor molecules,  recognize  fragments  of  foreign 
proteins  (peptides)  embedded  in  major  histo- 
compatibility complex  (MHC)  molecules.  In 
contrast,  antibodies,  although  closely  related  to  T 
cell  antigen  receptors  (TCRs) ,  bind  intact  foreign 
particles  directly.  This  suggests  fundamental  dif- 
ferences in  the  rules  governing  T  cell  recognition 
versus  antibody-mediated  recognition  by  B  cells. 
Because  of  the  consistently  high  concentration  of 
sequence  diversity  in  the  V-J  junctional  region  of 
TCRs  (equivalent  to  the  third  complementation- 
determining  region  [CDR3]  of  immunoglobulins) 
as  well  as  structural  considerations,  we  have  pro- 
posed that  this  is  the  important  region  for  peptide 
recognition  and  that  other  V  region-encoded  resi- 
dues might  contact  the  surface  of  the  MHC 
molecule. 

To  test  this  hypothesis,  we  recently  developed 
an  immunological  version  of  the  classical  genetic 
technique  of  second-site  suppression.  In  these 
experiments  we  change  residues  that  are  impor- 
tant for  T  cell  recognition  (and  not  MHC  interac- 
tion) on  a  peptide,  immunize  mice,  and  then  ana- 
lyze the  responding  T  cells  that  emerge  with 
respect  to  their  TCR  sequences.  To  hold  part  of 
the  original  receptor  constant,  we  use  mice  that 
are  transgenic  for  either  chain  of  the  original 
TCR.  With  this  approach,  we  have  shown  that  one 
of  the  two  residues  on  the  peptide  that  are  impor- 
tant in  the  T  cell  response  is  governed  by  the 
CDR3  of  the  Va  polypeptide,  and  that  the  other 
(three  amino  acids  downstream  on  the  peptide 


sequence)  is  specified  by  the  CDR3  of  the  VjS 
polypeptide.  Thus  we  have  generated  significant 
support  for  the  original  hypothesis.  The  results 
indicate  that  T  cell  recognition  is  a  much  more 
stylized  event  than  antibody-antigen  interactions. 

Kinetics  of  T  Cell  Recognition 
and  Activation 

To  learn  more  about  the  dynamics  of  TCR- 
peptide-MHC  interactions,  we  have  developed 
expression  systems  that  allow  us  to  produce  ei- 
ther TCR  or  MHC  class  II  heterodimers  in  a  solu- 
ble form.  This  involves  replacing  the  normal 
membrane-spanning  sequences  of  these  polypep- 
tides with  a  signal  sequence  for  lipid  linkage, 
such  as  employed  normally  by  a  number  of  cell 
surface  proteins.  Molecules  expressed  in  this  fash- 
ion can  then  be  conveniently  cleaved  from  the 
surface  of  expressing  cells  with  the  enzyme  phos- 
phatidylinositol-specific  phospholipase  C.  By 
utilizing  high-density  mammalian  cell  culture 
machines,  we  are  able  to  make  milligram  quanti- 
ties of  a  soluble  TCR  and  its  cognate  MHC  mole- 
cule. This  has  provided  us  with  the  raw  material 
to  initiate  structural  studies  such  as  x-ray  crystal- 
lography and  nuclear  magnetic  resonance  analy- 
sis. We  have  also  used  the  soluble  MHC  protein  to 
show  greatly  enhanced  uptake  of  antigenic  pep- 
tides at  low  pH.  This  is  important  both  in  making 
significant  quantities  of  a  pure  antigen-MHC 
complex  and  in  understanding  the  biology  of  this 
type  of  MHC  molecule  (class  II)  that  recycles 
through  low-pH  endosomal  corripartments.  Our 
current  data  suggest  that  the  low  pH  triggers  a 
specific  conformational  change  in  the  MHC  mole- 
cule, which  allows  it  to  bind  new  peptides  more 
easily. 

Recently  we  have  studied  the  kinetics  of  TCR 
peptide-MHC  interactions,  using  soluble  MHC- 
peptide  complexes  that  competitively  inhibit 
binding  of  labeled  anti-TCR  antibody  fragments 
to  TCRs.  In  several  cases  studied,  we  derive 
values  for  this  interaction  of  5  X  10~^  M,  1,000- 
to  10,000-fold  weaker  than  antibodies  to  protein 
ligands  of  comparable  size.  These  values  are  con- 
sistent with  the  scanning  nature  of  T  cell  recogni- 


101 


Molecular  Approaches  to  T  Lymphocyte  Recognition  and  Differentiation 


tion  and  indicate  that  TCR  binding  to  peptide- 
MHC  ligands  is  so  weak  energetically  that  other, 
antigen-independent  receptor-Iigand  systems  must 
govern  the  initial  stages  of  T  cell  contact  with 
antigen-presenting  or  target  cells.  The  best  candi- 
dates for  such  regulators  of  T  cell  interaction  are 
adhesion  molecules,  such  as  LFA-1  (lymphocyte 
function  antigen  1)  or  CD2.  This  would  give  ad- 
hesion molecule-ligand  interactions  a  major  role 
in  orchestrating  which  T  cells  interact  with 
which  antigen-presenting  cells.  This  has  impor- 
tant implications  for  autoimmunity:  normally  T 
cells  would  be  expected  to  focus  on  appropriate 
antigen-presenting  cells  (such  as  B  cells  or  macro- 
phages) and  would  be  hindered  in  surveying 
most  other  cells  or  tissues  (which  would  lack  the 
appropriate  ligand  expression) . 

Generation  of  Memory  T  Cells  in  Vitro 

A  hallmark  of  the  vertebrate  immune  system  is 
its  ability  to  respond  much  more  strongly  and 
quickly  to  a  second  encounter  with  an  antigen. 
Although  some  of  this  effect  is  due  to  the  clonal 
expansion  of  B  and  T  cells,  a  significant,  and  per- 
haps the  major,  contribution  is  thought  to  involve 
the  induction  of  antigen-specific  memory  lym- 
phocytes. Evidence  suggests  that  this  differen- 
tiation step  is  crucial  to  mounting  a  successful 
immune  response,  both  in  appropriate  and  inap- 


propriate (e.g.,  autoimmune)  circumstances. 
Studies  of  B  or  T  lymphocyte  memory  have  been 
hampered  by  the  lack  of  well-defined  systems  in 
which  uniform  populations  of  primary  and  mem- 
ory cells  can  be  studied.  We  have  approached  this 
problem  in  T  lymphocytes  by  making  use  of  TCR 
a/3  transgenic  mice  that  have  an  essentially  mono- 
clonal T  cell  immune  system,  thereby  eliminating 
the  clonal  expansion  component  of  T  cell  mem- 
ory and  allowing  us  to  focus  on  the  physiological 
changes  that  characterize  this  transition. 

We  find  that  primary  T  cells  can  only  produce 
interleukin-2  (IL-2)  in  response  to  TCR  stimula- 
tion with  immobilized  peptide-MHC  complexes 
if  PMA  (phorbol  12-myristate  12-acetate)  is  in- 
cluded in  the  culture.  In  contrast,  secondary,  or 
memory,  T  cells,  derived  from  the  identical  start- 
ing population,  readily  produce  lymphokine  on 
this  substrate,  irrespective  of  PMA  addition.  Both 
T  cell  populations  are  inhibited  by  protein  kinase 
C  (PKC)  inhibitors  H7  and  staurosporine.  Thus 
secondary  T  cells  are  functionally  distinct  from 
primary  cells;  the  establishment  of  T  cell  mem- 
ory, which  may  include  alterations  in  PKC  signal- 
ing, followed  by  the  addition  of  exogenous  IL-2, 
is  sufficient  to  convert  primary  T  cells  to  second- 
ary cells  in  vitro.  This  provides  an  excellent  op- 
portunity to  characterize  and  manipulate  this  im- 
portant juncture  in  T  cell  differentiation. 


102 


Signal  Transduction  by  the  Epidermal  Growth 
Factor  Receptor 


Roger  J.  Davis,  Ph.D. — Assistant  Investigator 

Dr.  Davis  is  also  Associate  Professor  in  the  Program  in  Molecular  Medicine  and  the  Department 
of  Biochemistry  and  Molecular  Biology  at  the  University  of  Massachusetts  Medical  School.  He  received 
his  undergraduate  and  graduate  education  at  Cambridge  University  and  was  a  postdoctoral  fellow 
with  Michael  Czech  at  the  University  of  Massachusetts. 


CELLULAR  proliferation  is  a  highly  regulated 
process.  During  embryonic  development, 
rapid  cell  growth  is  required  to  form  the  tissues 
of  the  body.  In  contrast,  cellular  proliferation  in 
adults  is  slow,  primarily  serving  to  replace  senes- 
cent cells.  Adults,  however,  retain  a  limited  ca- 
pacity for  rapid  growth — for  example,  during 
wound  healing.  Regulation  of  this  proliferative 
capacity  is  critically  important.  Errors  in  growth 
control  can  result  in  a  variety  of  diseases,  includ- 
ing cancer. 

The  local  production  of  protein  growth  factors 
is  an  important  mechanism  that  can  account  for 
the  control  of  cellular  proliferation.  Our  research 
group  is  investigating  the  action  of  a  family  of 
peptides  that  includes  epidermal  growth  factor 
(EGF)  and  transforming  growth  factor-a  (TGF- 
a).  These  agents  are  synthesized  as  cell  surface 
glycoproteins  that  are  split  to  release  small  solu- 
ble peptides.  Both  the  membrane-bound  precur- 
sor and  the  diffusible  peptides  are  biologically 
active  and  bind  to  specific  receptor  molecules 
located  at  the  surface  of  responsive  cells.  Secre- 
tion of  these  peptide  growth  factors  contributes 
to  the  rapid  growth  of  some  tumors. 

The  long-term  goal  of  this  laboratory  is  to  un- 
derstand the  molecular  basis  for  the  control  of 
cellular  proliferation  by  the  EGF  receptor.  It  is 
known  that  the  binding  of  growth  factors  to  this 
receptor  at  the  cell  surface  triggers  a  complex 
series  of  chemical  reactions  that  lead  to  DNA  syn- 
thesis within  the  nucleus  and  to  cell  division. 
However,  the  molecular  details  of  the  signaling 
pathways  utilized  by  the  receptor  are  poorly 
understood. 

Regulation  of  EGF  Receptor  Function 

The  EGF  receptor  is  a  glycoprotein  consisting 
of  an  extracellular  domain  that  binds  growth  fac- 
tor, a  membrane-spanning  domain,  and  a  cyto- 
plasmic domain.  The  cytoplasmic  domain  is  an 
enzyme,  tyrosine  kinase,  that  causes  the  covalent 
attachment  of  phosphate  to  tyrosine  components 
of  substrate  proteins  (phosphorylation).  The 
binding  of  EGF  to  the  receptor's  extracellular  do- 
main causes  an  increase  in  the  tyrosine  kinase  ac- 


tivity of  the  cytoplasmic  domain.  EGF  also  causes 
the  receptor  to  aggregate  and  to  associate  tran- 
siently with  intracellular  regulatory  molecules  to 
form  a  signal  transduction  complex.  We  are  study- 
ing these  interactions  and  investigating  the  con- 
sequences of  the  phosphorylation  process. 

The  ability  of  EGF  to  increase  the  tyrosine  ki- 
nase activity  of  its  receptor  is  blocked  when  cells 
are  incubated  with  a  tumor  promoter  or  with 
other  growth  factors.  Under  these  conditions,  the 
EGF  receptor  is  itself  phosphorylated  at  multiple 
serine  and  threonine  residues.  We  are  investigat- 
ing the  significance  of  this  phosphorylation.  Our 
approach  is  to  construct  receptors  with  point 
mutations  at  the  sites  of  phosphorylation,  using 
recombinant  DNA  technology.  These  studies  have 
demonstrated  that  the  phosphorylation  of  a  single 
threonine  residue  blocks  the  ability  of  EGF  to 
stimulate  the  receptor's  tyrosine  kinase  activity. 
Phosphorylation  also  alters  the  internalization  of 
the  receptor.  We  are  investigating  the  structural 
basis  for  the  effects  of  phosphorylation  on  the 
regulation  of  EGF  receptor  function.  These  stud- 
ies are  supported  by  a  grant  from  the  National 
Institutes  of  Health. 

Signaling  by  the  EGF  Receptor 

A  principal  question  that  we  must  answer  to 
understand  the  mechanism  of  signal  transduction 
by  the  EGF  receptor  is  how  a  signal  that  is  initi- 
ated at  the  cell  surface  can  be  transmitted  to  the 
nucleus  to  cause  DNA  replication.  One  class  of 
regulatory  molecules  that  could  account  for  this 
process  is  the  protein  kinases.  We  are  focusing 
our  research  on  one  subclass  of  these  enzymes 
that  cause  the  phosphorylation  of  proline-rich 
target  sequences  in  substrate  proteins.  Examples 
are  represented  by  the  mitogen-activated  protein 
kinases  and  the  cyclin-dependent  protein  ki- 
nases. Each  of  these  types  of  protein  kinases  exists 
in  multiple  forms  as  part  of  an  extended  family  of 
enzymes  that  are  regulated  by  growth  factors  and 
by  the  cell  cycle.  We  are  investigating  the  struc- 
ture of  additional  members  of  this  family  by  mo- 
lecular cloning. 

Substrates  for  these  growth  factor-regulated 


103 


Signal  Transduction  by  the  Epidermal  Growth  Factor  Receptor 


protein  kinases  that  we  have  identified  include 
the  EGF  receptor  and  the  nuclear  transcription 
factors  expressed  by  the  proto-oncogenes  c-myc 
and  c-jun.  We  are  investigating  the  mechanism 
and  significance  of  the  phosphorylation  of  these 
proteins  in  EGF-treated  cells.  The  overall  goal  of 
these  studies  is  to  establish  the  molecular  details 
of  a  signal  transduction  pathway  that  is  initiated 
at  the  EGF  receptor  and  leads  to  the  regulation  of 
nuclear  function. 

Tissue  Specificity  of  Tumor  Induction 

The  gene  for  the  EGF  receptor  is  a  frequent  site 
of  integration  by  avian  leukosis  viruses.  Insertion 
of  the  virus  causes  the  expression  of  a  truncated 
EGF  receptor.  The  formation  of  a  virus  containing 
a  copy  of  the  truncated  receptor  gene  can  also 


occur.  This  truncated  gene  is  the  dominantly  ac- 
tive oncogene  erbB.  The  primary  disease  asso- 
ciated with  erbB  is  erythroblastosis.  However, 
mutations  in  the  carboxyl  terminus  of  erbB  that 
occur  during  viral  replication  cause  additional 
tumors — fibrosarcomas  and  angiosarcomas.  We 
are  studying  the  molecular  basis  for  the  change  in 
tissue  specificity  of  the  erbB  oncogene. 

The  approach  we  are  taking  is  to  construct  re- 
combinant viruses  containing  erbB.  The  advan- 
tage of  this  procedure  is  that  we  can  undertake  a 
systematic  analysis  of  the  effects  of  erbB  muta- 
tions on  the  tissue  specificity  of  tumor  formation. 
The  results  should  help  explain  why  erbB  causes 
tumors  in  one  tissue  but  not  in  others.  This  infor- 
mation will  be  useful  in  designing  strategies  for 
clinical  intervention  in  tumor  development. 


A  hippocampal  neuron  in  primary  culture,  showing  axosomatic  and  axo- 
dendritic synapses  at  the  surface.  The  perikaryon  and  the  dendrites  of  the 
neuron  are  visualized  by  immunostain  (green  fluorescence)  for  MAP2,  a 
protein  associated  with  microtubules  of  perikarya  and  dendrites.  Presynap- 
tic nerve  terminals  originating  from  neurons  not  visible  in  the  field  are 
visualized  by  immunostain  (orange-yellow  fluorescence)  for  the  synaptic 
vesicle  protein  synaptotagmin. 

From  the  work  of  Michela  Malleoli,  Gary  Banker,  Thomas  Siidhof  and 
Pietro  De  Camilli. 


104 


Traffic  of  Synaptic  Vesicle  Proteins  in  Neurons 
and  Endocrine  Cells 


Pietro  De  Camilli,  M.D. — Investigator 

Dr.  De  Camilli  is  also  Professor  of  Cell  Biology  at  Yale  University  School  of  Medicine.  He  received  his  M.D. 
degree  from  the  University  of  Milano,  Italy,  where  he  worked  with  Jacopo  Meldolesi.  He  did  his 
postdoctoral  studies  with  Paul  Greengard  at  Yale  University.  Prior  to  his  current  position,  he  held 
appointments  both  at  Yale  and  the  University  of  Milano.  Dr.  De  Camilli  is  a  recipient  of  a  McKnight 
Scholars  Award  and  a  member  of  the  European  Molecular  Biology  Organization  (EMBO ). 


WE  are  interested  in  the  mechanisms  of  sig- 
naling between  nerve  cells,  with  emphasis 
on  neuronal  secretion  of  neurotransmitter  mole- 
cules. Most  neurotransmitters  are  stored  in  vesi- 
cles within  nerve  terminals  and  are  secreted  in 
response  to  depolarization  of  the  terminal  by  fu- 
sion of  the  vesicle  with  the  plasmalemma,  a  pro- 
cess called  exocytosis.  Most,  and  possibly  all, 
nerve  terminals  contain  two  classes  of  secretory 
vesicles:  synaptic  vesicles  (SVs)  and  the  so-called 
large  dense-core  vesicles  (LDCVs),  which  differ 
in  a  variety  of  properties,  including  neurotrans- 
mitter content. 

SVs  are  small,  morphologically  homogeneous 
vesicles  (50-nm  diameter),  containing  nonpep- 
tide  neurotransmitters  only.  They  are  very  abun- 
dant in  nerve  terminals,  and  clusters  of  SVs  ap- 
posed to  the  plasmalemma  represent  a  typical 
structural  feature  of  axon  endings.  Their  exocyto- 
sis takes  place  selectively  at  the  presynaptic  plas- 
malemma, is  faithfully  linked  to  nerve  terminal 
depolarization,  and  plays  the  dominant  role  in 
the  fast,  point-to-point  intercellular  signaling  typ- 
ical of  the  nervous  system  (the  so-called  synap- 
tic transmission).  SV  membranes  are  rapidly  re- 
internalized  after  exocytosis  and  are  used  to 
re-form  SVs,  which  are  loaded  locally  with 
neurotransmitters . 

LDCVs  are  larger  vesicles  with  a  dense  protein- 
rich  core.  Their  contents  are  predominantly  pep- 
tide neurotransmitters,  though  they  may  also 
contain  amines.  Their  exocytosis  is  preferentially 
triggered  by  trains  of  action  potentials  and  is  in- 
volved primarily  in  a  modulatory  type  of  signal- 
ing. They  have  long  been  recognized  as  the  neuro- 
nal organelles  equivalent  to  secretory  granules  of 
endocrine  cells,  which  secrete  peptide  hormones 
and  amines.  In  contrast,  many  of  the  properties  of 
SVs  set  them  apart  from  other  secretory  organ- 
elles. The  elucidation  of  these  properties  repre- 
sents the  main  focus  of  our  laboratory.  We  are 
following  three  major  lines  of  research.  (Several 
aspects  of  this  research  have  previously  been  sup- 
ported by  the  National  Institutes  of  Health.) 

Biogenesis  and  Traffic  of  Synaptic  Vesicles 

First,  we  are  investigating  the  biogenesis  and 


traffic  of  SVs,  using  in  vivo  and  in  vitro  systems. 
The  large  body  of  information  recently  accumu- 
lated on  the  molecular  structure  of  SVs  has  made 
available  numerous  molecular  probes  that  can  be 
used  in  these  studies  (some  of  which  are  carried 
out  in  collaboration  with  Reinhard  Jahn  [HHMI, 
Yale  University]  and  Thomas  Siidhof  [HHMI,  Uni- 
versity of  Texas  Southwestern  Medical  Center  at 
Dallas]).  We  have  developed  an  immunocyto- 
chemical  assay  to  detect  SV  exocytosis.  Using  this 
assay  on  hippocampal  neurons  in  primary  cul- 
ture, we  have  shown  that  SVs  undergo  a  high  rate 
of  constitutive  exocytosis  and  recycling  in  devel- 
oping neurons  before  synaptic  contacts  are 
formed. 

These  findings  suggest  that  synapse  formation 
coincides  with  a  reorganization  of  the  exocytotic 
machinery  (rather  than  with  its  de  novo  assem- 
bly) and  that  signaling  via  SV  exocytosis  may  play 
an  important  role  in  nervous  system  development 
prior  to  synapse  formation.  We  are  working  at  de- 
veloping assays,  some  cell  free,  to  define  specific 
steps  of  the  exo-endocytotic  pathway  of  SVs.  Such 
assays  aid  greatly  in  elucidating  the  underlying 
molecular  mechanisms. 

Synapse-like  Microvesicles 
in  Endocrine  Cells 

Second,  we  are  exploring  the  relationship  of 
SVs  to  organelles  found  in  nonneuronal  cells.  Do 
SVs  represent  the  adaptation  of  recycling  vesicles 
to  organelles  found  in  other  cells?  How  is  the 
exo-endocytotic  recycling  of  SVs  related  to  the 
plasmalemma-endosome  recycling  that  operates 
in  all  cells  and  occurs,  for  example,  in  the  recy- 
cling of  receptors? 

We  and  others  have  found  that  a  variety  of  SV 
proteins  are  expressed  at  significant  levels  by 
peptide-secreting  endocrine  cells.  Investigating 
the  subcellular  localization  of  these  proteins,  we 
have  found  that  at  least  some  of  them  (synapto- 
physin,  protein  p29)  are  concentrated  on  a  popu- 
lation of  microvesicles  (synapse-like  microvesi- 
cles, SLMVs)  distinct  from  the  secretory  granules 
that  store  and  secrete  peptide  hormones. 

A  first  biochemical  characterization  of  these 


105 


Traffic  of  Synaptic  Vesicle  Proteins  in  Neurons  and  Endocrine  Cells 


microvesicles  has  demonstrated  that  they  are  simi- 
lar in  composition  to  bona  fide  neuronal  SVs. 
Consistent  with  these  findings,  SLMVs,  like  SVs, 
undergo  exocytosis,  endocytosis,  and  recycling. 
We  have  therefore  explored  the  possibility  that 
SLMVs  may  share  with  SV  the  property  to  store 
and  secrete  nonpeptide  neurotransmitters.  The 
model  endocrine  cell  we  have  used  for  these  stud- 
ies is  the  insulin-secreting  cell  of  the  endocrine 
pancreas,  also  called  pancreatic  |8-cell. 

Previous  work  had  suggested  that  the  nonpep- 
tide neurotransmitter  GABA,  the  major  inhibitory 
neurotransmitter  in  the  brain,  may  play  a  para- 
crine role  in  the  physiology  of  the  endocrine  pan- 
creas. GABA  was  reported  to  be  concentrated  in 
pancreatic  jS-cells  and  to  act  on  surrounding  pan- 
creatic endocrine  cells.  Work  we  have  carried  out 
so  far  has  suggested  that  SLMVs  of  /3-cells  may  in- 
deed store  GABA.  These  findings  support  the  hy- 
pothesis that  the  neuronal  secretory  pathway  in- 
volving SVs  represents  an  amplification  and  an 
adaptation  of  a  secretory  pathway  that  is  ex- 
pressed in  a  more  rudimentary  form  in  peptide- 
secreting  endocrine  cells. 

Such  results,  in  addition  to  having  important 
implications  for  the  field  of  endocrine  physiol- 
ogy, raise  the  possibility  of  using  endocrine  cells 
to  answer  questions  on  the  life  cycle  of  SVs  that 
would  not  be  feasible  to  address  in  neurons.  Us- 
ing endocrine  cell  lines  as  a  model  system,  we 
have  obtained  evidence  indicating  that  the  bio- 
genesis of  SLMVs  (and  presumably  of  SVs)  is  ef- 
fected by  protein  sorting  from  endosomal 
membranes. 

Molecular  Mechanisms  of  Exocytosis 

Third,  we  are  interested  in  elucidating  the  mo- 
lecular mechanisms  of  SV  exocytosis.  The  ap- 
proach we  are  currently  taking  is  based  on  the 
assumption  that  at  least  some  basic  feature  of  the 
exocytotic  process  may  have  been  conserved  in 
evolution.  We  are  exploring  the  potential  of  yeast 
genetics  to  identify  some  of  the  proteins  in- 
volved. A  variety  of  temperature-sensitive  yeast 
mutants  defective  in  vesicle  exocytosis  {sec  mu- 


tants), and  therefore  in  growth,  have  been  iso- 
lated in  recent  years.  We  are  currently  searching 
for  gene  products  that  rescue  sec  mutations  in  a 
functional  assay,  by  transfecting  yeast  sec  mutants 
with  mammalian  brain  cDNAs. 

Stiflf-Man  Syndrome  and  Diabetes 

The  presence  of  SV-like  organelles  in  endocrine 
cells  suggests  that  pathological  processes  affecting 
SV  proteins  (mutations,  autoimmunity)  might  af- 
fect both  the  nervous  and  endocrine  systems. 
These  considerations  led  us  to  identify  a  possible 
link  between  stiff-man  syndrome  and  insulin- 
dependent  diabetes  mellitus  (IDDM).  Stiff-man 
syndrome  is  a  rare  and  severe  disease  of  the  central 
nervous  system  characterized  by  a  progressive  ri- 
gidity of  the  body  musculature.  In  a  series  of  stud- 
ies that  involved  a  collaboration  with  the  Univer- 
sity of  Milano,  we  have  obtained  evidence  for  an 
autoimmune  pathogenesis  of  stiff-man  syndrome 
and  identified  the  GABA-synthesizing  enzyme, 
GAD,  as  the  major  autoantigen. 

GAD  is  concentrated  at  the  cytoplasmic  surface 
of  SVs  of  GABAergic  neurons  and  of  SLMVs  of  pan- 
creatic jS-cells.  IDDM,  which  results  from  an  au- 
toimmune destruction  of  (S-cells,  was  found  to 
occur  at  high  frequency  in  stiff-man  syndrome 
patients.  This  observation  then  led  us  to  hypothe- 
size an  identity  between  GAD  and  the  64-kDa 
autoantigen  of  IDDM  previously  identified  by 
Steinunn  Baekkeskov,  Ake  Lemmark,  and  their  co- 
workers (Hagedorn  Research  Laboratory,  Den- 
mark). A  collaboration  between  our  group  and 
that  of  Dr.  Baekkeskov  (now  at  the  University  of 
California,  San  Francisco)  confirmed  that  the  64- 
kDa  antigen  is  GAD. 

These  findings  may  lead  to  a  diagnostic  assay 
for  IDDM  and  for  preclinical  stages  of  the  disease. 
In  addition,  it  opens  the  possibility  of  investigat- 
ing molecular  mechanisms  of  autoimmunity  in 
IDDM. 

During  the  next  year  the  work  on  SV  exocytosis 
and  on  GAD  autoimmunity  in  stiff-man  syndrome 
will  be  partially  supported  by  the  National  Insti- 
tutes of  Health. 


106 


Three-Dimensional  Structures  of  Biological 
Macromolecules 


Johann  Deisenhofer,  Ph.D. — Investigator 

Dr.  Deisenhofer  is  also  Regental  Professor  and  Professor  of  Biochemistry,  and  he  holds  the  Virginia  and 
Edward  Linthicum  Distinguished  Chair  in  Biomolecular  Science  at  the  University  of  Texas  Southwestern 
Medical  Center  at  Dallas.  He  was  born  and  educated  in  Germany.  His  Ph.D.  research  in  protein 
crystallography  was  done  at  the  Max  Planck  Institute  for  Biochemistry,  Martinsried,  and  at  the  Technical 
University  of  Munich.  As  a  postdoctoral  fellow  and  as  a  staff  scientist  at  the  Max  Planck  Institute,  he 
continued  his  structural  analysis  of  biological  macromolecules  by  x-ray  crystallography.  He  has  received 
several  honors  for  his  structure  analysis  of  a  photosynthetic  reaction  center,  including  the  1988  Nobel 
Prize  in  chemistry,  which  he  shared  with  Hartmut  Michel  and  Robert  Huber. 


MY  colleagues  and  I  study  the  three- 
dimensional  structures  of  proteins  to  un- 
derstand their  folding,  structural  stability,  and 
function.  We  are  particularly  interested  in 
protein-pigment  complexes  catalyzing  photo- 
chemical energy  conversion,  energy  transfer,  and 
electron  transfer,  and  in  membrane-spanning  and 
membrane-associated  proteins. 

Cytochrome  b/ Cj  complex 

The  b/Cj  complexes  (also  called  ubiquinol- 
cytochrome  c  oxidoreductases)  are  integral 
membrane  proteins  that  play  crucial  roles  in  pho- 
tosynthesis and  cell  respiration.  Their  function  in 
these  fundamental  processes  is  to  oxidize  quinols 
to  quinones  and  to  transfer  electrons  and  protons 
through  the  membrane.  The  electrons  go  to  cy- 
tochrome c,  and  the  protons  build  up  an  electro- 
chemical gradient  across  the  membrane,  which, 
for  example,  drives  the  synthesis  of  ATP. 

Photosynthetic  purple  bacteria  have  the  sim- 
plest b/c,  complexes,  consisting  of  only  three  or 
four  different  protein  subunits  with  three  heme 
groups  and  an  iron-sulfur  cluster  as  cofactors. 
Photosynthetic  reaction  centers  and  b/c,  com- 
plexes cooperate  in  the  bacterial  inner  mem- 
brane; b/Ci  complexes  occur  at  a  concentration 
significantly  lower  than  that  of  reaction  centers 
and  are  therefore  more  difficult  to  isolate.  David 
Knafif's  research  group  (Texas  Technical  Univer- 
sity, Lubbock)  succeeded  in  purifying  the  b/Cj 
complex  from  Rhodospirillum  ruhrum.  In  col- 
laboration with  this  group  we  have  prepared  this 
complex  in  milligram  quantities  with  high  pu- 
rity. This  preparation  serves  as  the  starting  mate- 
rial for  crystallization  experiments. 

Mitochondria  of  higher  organisms  have  in  their 
inner  membranes  b/Cj  complexes  consisting  of  at 
least  10  different  polypeptide  chains,  3  of  which 
are  similar  to  those  in  the  b/Cj  complex  of  purple 
bacteria.  We  collaborate  with  Chang-An  Yu  and 
his  colleagues  (Oklahoma  State  University,  Still- 
water), who  produced  large  crystals  of  the  b/Cj 
complex  from  beef  heart  mitochondria.  Prelimi- 


nary diffraction  experiments  at  the  Cornell  High 
Energy  Synchrotron  Source  (CHESS)  were  encour- 
aging, and  we  hope  to  begin  a  structure  analysis  at 
medium  resolution  (approximately  5  A)  in  the 
near  future. 

DNA  Photolyase 

Light  energy  plays  an  important  role  in  reac- 
tions other  than  photosynthesis.  The  molecular 
machinery  that  enables  cells  to  repair  DNA  dam- 
aged by  ultraviolet  (UV)  light  includes  an  en- 
zyme that  uses  light  energy  to  drive  the  repair  of 
one  frequent  type  of  damage  caused  by  UV  irra- 
diation: the  crosslinking  of  two  neighboring  thy- 
mine bases  in  a  strand  of  DNA.  Most  of  these 
crosslinks  are  in  the  form  of  cyclobutane  rings 
connecting  four  carbon  atoms,  two  from  each 
thymine  base.  The  enzyme  DNA  photolyase  can 
locate  and  bind  to  such  defects,  and  upon  input 
of  light  of  suitable  wavelength  (300-500  nm) 
cleave  the  carbon-carbon  bonds  between  the 
bases,  thus  repairing  the  damage. 

DNA  photolyase  has  been  found  in  prokaryotes, 
eukaryotes,  and  archaebacteria.  Aziz  Sancar  and 
his  colleagues  (University  of  North  Carolina  at 
Chapel  Hill)  sequenced,  overexpressed,  and  puri- 
fied the  enzyme  from  Escherichia  coll.  It  consists 
of  a  single  polypeptide  chain  of  471  amino  acids 
and  two  cofactors — a  flavin-adenine  dinucleo- 
tide  (FAD)  and  5, 10-methenyltetrahydrofolate. 
The  FAD  cofactor  fully  reduced  to  FADH2  is  es- 
sential for  the  enzyme's  function;  the  folate  acts 
as  a  light-harvesting  antenna. 

Despite  significant  problems  with  the  en- 
zyme's tendency  to  denature,  we  were  able  to 
crystallize  DNA  photolyase  from  E.  coli  in  two 
crystal  forms;  both  forms  difi'ract  x-rays  to  at  least 
2.8-A  resolution.  We  collected  x-ray  diffraction 
intensity  data  from  one  of  these  crystal  forms;  to 
solve  the  phase  problem,  experiments  are  under 
way  to  bind  heavy-atom  compounds  to  the  pro- 
tein in  the  crystal.  We  are  also  trying  to  crystallize 
the  enzyme  in  complex  with  a  substrate,  a  five- 
nucleotide  piece  of  single-stranded  DNA  contain- 


107 


Three-Dimensional  Structures  of  Biological  Macromolecules 


ing  a  crosslinked  pair  of  thymine  bases.  These  ex- 
periments have  to  be  done  in  the  dark  or  under 
yellow  or  red  light,  to  prevent  the  enzyme  from 
repairing  and  releasing  the  substrate. 

Because  light  is  an  essential  ingredient  of  the 
enzymatic  reaction  of  DNA  photolyase,  the  en- 
zyme-substrate complex  will  be  a  suitable  system 
to  study  the  time  course  of  the  reaction,  using  the 
Laue  technique.  This  technique  employs  the 
broad  spectrum  of  x-ray  wavelengths  in  the 
synchrotron's  powerful  beam  to  record  within  a 
very  short  time  a  sufficient  fraction  of  a  crystal's 
dilfraction  pattern  for  structural  interpretation. 
In  such  an  experiment  the  crystal  is  irradiated 
with  a  light  pulse  suitable  to  trigger  the  enzy- 
matic reaction,  and  diffraction  patterns  are  re- 
corded at  different  times  after  the  pulse.  These 
data  can  provide  snapshots  of  the  structural  rear- 
rangements during  the  reaction  and  thus  contrib- 
ute to  an  understanding  of  the  enzyme's 
mechanism. 

Cytochrome  P-450  Enzymes 

P-450  enzymes  are  members  of  a  superfamily 
of  b-type  heme  proteins  that  catalyze  hydroxyla- 
tion  of  organic  substrates  using  electrons  from 
reduced  nicotinamide  adenine  dinucleotide 
phosphate  (NADPH)  and  molecular  oxygen. 
They  consist  of  a  polypeptide  chain  of  about  420 
amino  acids  and  a  heme  group  as  part  of  their 
active  site.  P-450s  occur  in  organisms  as  different 
as  bacteria  and  humans.  Some  of  them  play  spe- 
cific roles  in  biosynthetic  pathways,  such  as  for- 
mation of  glucocorticoids,  androgens,  and  estro- 
gens from  cholesterol;  others  are  involved  in 
detoxification  of  foreign  substances  by  making 
them  more  soluble  and  so  facilitating  their  excre- 
tion. However,  in  some  cases  hydroxylation  of 
foreign  substances  creates  highly  efficient  carcin- 
ogens; thus  the  effects  of  P-450s  are  not  always 
beneficial. 

P-450s  occur  in  two  classes,  distinguished  by 
the  way  in  which  electrons  from  NADPH  are 
transferred  to  the  heme  group.  Class  I  P-450s  re- 
ceive the  electrons  via  a  flavoprotein  and  an  iron- 
sulfur  protein;  class  H  enzymes  are  reduced  di- 
rectly by  a  protein  containing  the  cofactors  flavin 
mononucleotide  and  FAD.  The  three-dimensional 
structure  of  only  one  class  I  P-450,  the  camphor 
hydroxylase  P-450cam'  is  known  at  atomic 
resolution. 

To  learn  more  about  the  structural  variation 


within  the  cytochrome  P-450  family,  about  dif- 
ferences between  class  I  and  class  II  enzymes,  and 
about  the  spectrum  of  reactions  catalyzed  by 
P-450s,  we  collaborate  with  Julian  Peterson  and 
his  colleagues  (University  of  Texas  Southwestern 
Medical  Center  at  Dallas)  to  analyze  the  three- 
dimensional  structures  of  a  class  I  and  a  class  II 
enzyme.  The  class  I  P-450  was  isolated  from 
Pseudomonas;  it  hydroxylates  the  hydrocarbon 
a-terpineol  and  was  therefore  named  P-450,p^. 
The  class  II  P-450  is  the  cytochrome  part  of  an 
enzyme  from  Bacillus  megaterium  that  is 
unique  in  combining  a  P-450  domain  and  a  P-450 
reductase  domain  in  a  single  polypeptide  chain 
of  1,066  amino  acids.  The  cytochrome  domain, 
P-450bm-3.  consists  of  the  471  amino-terminal 
amino  acids;  it  was  cloned  and  expressed  at  high 
levels  in  E.  coli.  Its  functions  include  hydroxyla- 
tion and  epoxidation  of  long-chain  fatty  acids. 

Well-ordered  crystals  of  both  P-450s  have  been 
grown.  At  CHESS,  crystals  of  P-450t^rp  diffracted 
to  about  2.0 -A  resolution,  and  crystals  of 
P-450bm-3  diffracted  to  better  than  1.5 -A  resolu- 
tion. Molecular  replacement  calculations,  using 
the  P-450cam  model,  produced  a  plausible  solu- 
tion for  the  crystal  structure  of  P-450terp-  Multiple 
isomorphous  replacement  with  three  heavy-atom 
derivatives  allowed  the  calculation  of  an  electron 
density  map  at  3-A  resolution  for  P-450bm.3;  this 
map  is  currently  being  interpreted.  Model  build- 
ing and  crystallographic  refinement  will  result  in 
accurate  models  of  both  proteins  in  the  near  fu- 
ture. We  also  plan  to  study  substrate  binding  and 
structures  of  site-specific  mutants. 

Other  Projects 

In  addition  to  the  projects  described  above,  we 
also  work  on  the  determination  of  the  three- 
dimensional  structures  of  several  other  proteins, 
including  the  catalytic  domain  of  human  HMG- 
CoA  reductase,  a  key  enzyme  in  the  synthesis  of 
cholesterol  and  a  likely  target  for  drugs;  human 
synapsin  I,  a  protein  binding  to  synaptic  vesicles 
and  mediating  their  release;  the  small  GDP- 
binding  protein  smgp25A  from  bovine  brain;  the 
DNA-binding  protein  myogenin;  the  SecA  protein 
from  E.  coli,  one  of  the  key  parts  of  the  protein 
export  system;  mammalian  phosphofructokinase; 
and  ribonuclease  inhibitor  from  pig  liver. 

The  work  on  cytochrome  b/Cj  complexes  and 
on  DNA  photolyase  is  also  supported  by  a  grant 
from  the  Welch  Foundation. 


108 


Molecular  Mechanisms  of  Lymphocyte 
Differentiation 


Stephen  V.  Desiderio,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Desiderio  is  also  Associate  Professor  of  Molecular  Biology  and  Genetics  at  the  Johns  Hopkins  University 
School  of  Medicine.  He  received  his  undergraduate  degree  in  Biology  and  in  Russian  from  Haverford 
College  and  his  M.D.  and  Ph.D.  degrees  from  the  Johns  Hopkins  University  School  of  Medicine  in 
biochemistry,  and  cellular  and  molecular  biology.  After  a  postdoctoral  fellowship  at  the  Massachusetts 
Institute  of  Technology  with  David  Baltimore,  he  returned  to  Johns  Hopkins. 


ONE  remarkable  feature  of  the  immune  sys- 
tem is  its  ability  to  recognize  and  respond  to 
an  extraordinary  variety  of  foreign  molecules,  or 
antigens.  This  exquisite  specificity  is  achieved 
through  protein  receptors,  which  bind  tightly  to 
specific  antigens.  Such  receptors  are  found  on 
the  surfaces  of  two  types  of  immune  cells:  B  and  T 
cells. 

One  type  of  antigen  receptor  is  the  antibody 
molecule,  or  immunoglobulin.  The  site  on  the 
antibody  molecule  that  binds  to  a  specific  anti- 
gen is  genetically  encoded  by  multiple  short  seg- 
ments of  DNA.  At  the  onset  of  development  of  the 
immune  system,  these  DNA  segments  are  located 
at  separate  places  in  the  genome,  but  during  the 
maturation  of  antibody-producing  cells  (B  cells), 
segments  are  joined  to  form  intact  immunoglobu- 
lin genes. 

In  addition  to  antibody,  another  class  of  anti- 
gen receptor  is  found  on  the  surfaces  of  cells  that 
mediate  cellular  immunity  (T  cells).  The  T  cell 
receptor's  antigen-binding  site,  like  that  of  the 
antibody  molecule,  is  encoded  by  multiple  DNA 
segments  that  are  brought  together  during  T  cell 
maturation. 

After  their  genes  are  assembled,  the  antigen  re- 
ceptor molecules  are  expressed  at  the  surfaces  of 
B  and  T  cells.  Here,  several  interactions  combine 
to  trigger  cell  division  and  further  maturation. 
These  include  the  binding  of  antigens  and  special- 
ized hormones  called  lymphokines  with  their  re- 
spective receptors. 

Lymphocyte  Activation 

A  major  goal  of  our  laboratory  is  to  find  out  how 
specific  antigens  and  growth  factors  trigger  the 
activation  of  B  and  T  cells.  Biochemical  evidence 
has  long  suggested  that  enzymes  called  tyrosine 
kinases  might  be  intimately  involved.  These  en- 
zymes regulate  the  activities  of  other  proteins, 
and  of  each  other,  by  adding  regulatory  chemical 
groups,  phosphates,  to  specific  sites.  The  kinases 
involved  in  B  and  T  cell  activation  have,  for  the 
most  part,  proved  elusive.  We  therefore  set  out  to 
find  new  tyrosine  kinase  genes  that  are  expressed 
in  B  or  T  cells,  and  we  identified  seven.  Two  are 


expressed  only  in  immune  cells,  and  we  have 
concentrated  on  those. 

One  of  the  genes  resembles  a  growth- 
regulating  gene  called  c-src.  Unlike  c-src,  how- 
ever, which  is  turned  on  in  many  different  kinds 
of  cells,  this  new  gene  is  only  turned  on  in  B  cells 
and  their  developmental  precursors.  Accord- 
ingly, we  call  the  gene  blk,  for  B  lymphoid  ki- 
nase. The  blk  gene  is  activated  early  in  B  cell  de- 
velopment and  is  expressed  along  with  several 
proteins  that  are  known  to  transmit  signals  across 
the  B  cell  membrane,  including  the  antibody 
heavy  chain. 

When  B  cells  develop  into  antibody-secreting 
or  plasma  cells,  they  no  longer  express  these  sig- 
nal transmission  proteins  or  blk.  This  pattern  sug- 
gests that  the  product  of  the  blk  gene  interacts 
with  a  receptor  that  spans  the  B  cell  membrane 
and  that  senses  the  presence  of  antigen  or  a  spe- 
cific growth  signal.  In  the  spleen,  the  protein  en- 
coded by  blk  is  found  specifically  in  those  loca- 
tions where  resting  B  cells  encounter  antigen, 
reinforcing  the  idea  that  this  kinase  functions  in 
the  triggering  of  the  B  cell's  immune  response. 

How  might  extracellular  signals  activate  the 
blk  kinase?  The  kinase  is  inactivated  by  addition 
of  a  phosphate  group  to  a  specific  site,  and  is 
activated  when  the  phosphate  is  removed.  If  the 
target  site  on  the  blk  kinase  is  mutated  so  that  it 
cannot  accept  the  inactivating  chemical  group, 
the  enzyme  is  locked  into  the  "on"  state.  Cells 
that  contain  this  "on"  version  of  the  blk  kinase 
grow  in  an  unregulated  way.  Having  an  activated 
version  of  the  blk  kinase  has  made  it  easier  to 
identify  its  targets.  We  have  found  one  target  to 
be  an  enzyme  called  phospholipase  C-7  (PLC-7), 
a  key  component  of  the  B  cell  activation  pathway. 
Evidence  from  other  systems  suggests  that  PLC-7 
is  activated  by  tyrosine  kinases.  We  speculate  that 
activation  of  the  blk  kinase  in  B  cells  activates 
PLC-7,  initiating  subsequent  signaling  events. 

The  hormone  interleukin-2  (IL-2)  plays  a  cen- 
tral role  in  the  immune  response  by  inducing  the 
multiplication  of  T  cells.  The  binding  of  IL-2  to 
its  receptor  turns  on  a  tyrosine  kinase  of  unknown 
identity.  We  have  recently  identified  six  new  tyro- 
sine kinase  genes  that  are  expressed  in  cells  de- 


109 


Molecular  Mechanisms  of  Lymphocyte  Differentiation 


pendent  on  IL-2  for  growth.  One  of  these  genes, 
which  we  call  itk  (for  IL-2-inducible  T  cell  ki- 
nase), is  expressed  specifically  in  T  cells.  The  itk 
gene  encodes  a  tyrosine  kinase  that  resembles  src 
kinases,  but  from  its  sequence  we  predict  that  it 
will  differ  from  those  kinases  with  respect  to  its 
location  in  the  cell  and  the  regulation  of  its  activ- 
ity. IL-2  is  known  to  regulate  expression  of  a  num- 
ber of  genes,  including  a  gene  for  its  own  recep- 
tor. In  cells  that  have  been  withdrawn  from  IL-2, 
itk  is  expressed  at  low  levels.  Soon  after  addition 
of  IL-2,  itk  is  strongly  turned  on  in  parallel  with 
the  IL-2  receptor  gene,  suggesting  that  itk  func- 
tions in  the  IL-2  response. 

Antibody  Gene  Rearrangement 

The  total  number  of  immunoglobulin  or  T  cell 
receptor  gene  segments  is  large,  but  when  any 
particular  immunoglobulin  or  T  cell  receptor 
gene  is  assembled,  only  a  handful  of  segments  are 
selected  and  joined.  As  a  result,  many  different 
combinations  of  segments  are  possible.  It  is  this 
shuffling  of  small  bits  of  DNA  that  generates 
much  of  the  diversity  of  the  immune  response. 

By  using  artificial  DNA  molecules  that  rear- 
range after  they  are  introduced  into  immature  B 
cells,  we  have  been  able  to  outline  the  general 
features  of  antibody  gene  assembly,  but  the  mech- 
anism at  the  level  of  interactions  between  mole- 
cules is  not  known.  The  products  of  at  least  three 
genes  are  known  to  play  an  intimate  role  in  rear- 
rangement. Two  of  these  genes,  RAG-1  and  RAG- 
2,  have  been  shown  by  others  to  activate  some- 
how antibody  gene  rearrangement.  A  third  gene 
encodes  a  protein  that  all  cells  use  to  help  repair 
DNA  damage  and  that  also  functions  at  a  late  step 
in  antibody  gene  assembly.  In  addition  to  the 
products  of  these  genes,  our  laboratory  has  found 
a  protein — NBP — that  binds  specifically  to  a 
DNA  sequence  element  required  for  rearrange- 
ment, suggesting  that  it  may  be  a  part  of  the  re- 
combinational  machinery.  Members  of  our  labora- 
tory are  studying  the  products  of  the  RAG  genes 
and  NBP,  with  the  aim  of  understanding  their 
functions  and  interrelationships. 

Since  NBP  was  isolated  from  calf  thymus,  we 
cloned  the  RAG-1  and  RAG-2  genes  of  the  cow 
and  identified  regions  of  identity  among  the 
genes  from  cow,  mouse,  and  human.  Based  on  the 
predicted  protein  sequences  of  these  conserved 
regions,  antibodies  were  raised  that  specifically 
bind  to  the  products  of  the  RAG-1  and  RAG-2 
genes.  Using  these  antibodies,  RAG-1  and  RAG-2 
proteins  were  identified  in  extracts  of  calf  thy- 


mus and  isolated.  We  found  these  proteins  are 
distinct  from  NBP.  Interestingly,  the  sizes  of  the 
RAG-1  or  RAG-2  proteins  are  slightly  different 
from  those  predicted  by  their  gene  sequences, 
suggesting  that  they  are  modified  after  they  are 
made.  We  have  gone  on  to  find  that  both  RAG-1 
and  RAG-2  are  modified  by  addition  of  phosphate 
groups.  As  was  discussed  earlier,  this  type  of  mod- 
ification often  serves  to  regulate  protein  func- 
tion. We  are  intrigued  by  the  possibility  that  such 
is  the  case  for  RAG-1  and  RAG-2. 

Molecular  Mimicry  by  Antibody  Molecules 

As  a  result  of  antibody  gene  rearrangement,  the 
structures  of  potential  antigen-binding  sites  vary 
almost  endlessly.  It  has  been  a  long-standing  no- 
tion in  immunology  that  the  structure  of  an  anti- 
gen could  be  mimicked  by  a  special  type  of  anti- 
body— an  antibody  raised  against  another 
antibody  that  was  originally  elicited  by  the  anti- 
gen of  interest.  We  can  draw  an  analogy  for  this 
idea  from  the  art  of  casting.  A  sculptor  creates  a 
positive  image,  which  is  used  to  produce  a  nega- 
tive image,  the  mold,  which  in  turn  is  used  to 
reproduce  the  final  work.  An  essential  difference 
between  this  metaphor  and  reality  is  that  in  cast- 
ing, one  object  is  shaped  by  another,  while  in  the 
antibody  response,  the  binding  of  antigen  selects 
a  complementary  shape  from  a  universe  of  preex- 
isting ones. 

We  have  tested  this  idea  in  collaboration  with 
research  groups  in  Baltimore  and  Paris  (led  by 
Mario  Amzel  of  the  Johns  Hopkins  School  of  Medi- 
cine and  Pierre  Ronco,  INSERM,  Hopital  Tenon). 
Antibodies  (Abl)  were  produced  against  a  hor- 
mone, angiotensin  II.  Then  a  second  antibody 
(Ab2)  was  raised  against  the  anti-angiotensin  II 
antibody.  A  third  antibody  (Ab3)  was  in  turn 
raised  against  Ab2.  The  Ab3  antibodies  bound  to 
angiotensin  II  just  as  well  as  the  original  Abl  anti- 
bodies, suggesting  that  Ab2  could  mimic  the  hor- 
mone's structure. 

This  was  proved  by  analyzing  the  structures  of 
Abl  and  Ab3.  Remarkably,  we  found  that  the  se- 
quences of  their  antigen-binding  sites  were 
nearly  identical.  We  then  determined  the  three- 
dimensional  structure  of  the  antigen-binding  site 
of  Ab3  in  complex  with  angiotensin  II.  We  discov- 
ered that  the  atoms  in  critical  contact  with  angio- 
tensin II  in  this  complex  are  also  found  in  the 
sequence  of  Abl.  Thus  both  Abl  and  Ab3  were 
elicited  by  similar  structures,  one  represented  by 
the  original  antigen,  angiotensin  II,  and  the  other 
by  a  surface  feature  of  the  Ab2  antibody  that  mim- 
ics angiotensin  II. 


110 


1 


Expression  of  the  gene  BK27  in  a  Drosophila  embryo  with  an  extended  germ  band.  This  gene, 
which  encodes  a  homeodomain  and  a  Paired  domain,  exhibits  prominent  expression  in  the 
ventral  labial  lobe,  marking  the  progenitor  cells  for  the  salivary  gland  placode.  The  staining  seen 
in  the  head  is  localized  in  the  subantennal  region. 
Research  and  photograph  by  Susie  Jun  in  the  laboratory  of  Claude  Desplan. 


112 


Transcription  Control  During  Early 
Drosophila  Development 


Claude  Desplan,  Ph.D. — Associate  Investigator 

Dr.  Desplan  is  also  Associate  Professor  and  Head  of  the  Laboratory  of  Molecular  Genetics  at  the  Rockefeller 
University.  He  graduated  from  the  Ecole  Normale  Superieure  de  Saint  Cloud  in  France.  He  received 
his  D.Sc.  degree  from  the  University  of  Paris,  working  with  Baty  Moukhtar  and  Monique  Thomasset 
at  the  INSERM  on  calcium  regulation.  He  was  a  Maitre  de  Conference  at  the  Ecole  Normale  Superieure 
de  Fontenay  until  he  moved  to  the  University  of  California,  San  Francisco,  to  work  as  a  postdoctoral 
fellow  with  Pat  O'Farrell  on  the  functions  of  homeodomain  proteins  during  early  development. 


ALL  information  required  for  the  develop- 
ment of  a  fertilized  egg  into  a  complete  or- 
ganism is  contained  in  its  own  genetic  material, 
contributed  by  both  parents,  and  in  products  pro- 
vided by  the  mother  as  the  egg  is  formed.  Genetic 
studies  on  the  fruit  fly  Drosophila  have  identi- 
fied most  of  the  genes  involved  in  the  process  of 
pattern  formation.  Many  of  these  genes  appear  to 
encode  transcription  factors  and  to  share  some 
common  protein  motifs,  such  as  homeodomains 
or  zinc  finger  domains.  The  zygotic  genome  re- 
sponds to  maternal  organizing  factors  through  a 
network  of  transcriptional  regulators  to  set  up  the 
body  pattern  of  the  embryo.  Many  of  the  Drosoph- 
ila developmental  genes  have  mammalian  homo- 
logues  expressed  during  embryogenesis.  Thus  it 
is  likely  that  the  mechanisms  uncovered  in  flies 
are  of  general  significance  for  the  development  of 
multicellular  organisms. 

The  goal  of  our  laboratory  is  to  understand  the 
molecular  mechanisms  involved  in  the  regula- 
tory interactions  among  developmental  genes.  In 
light  of  the  complexity  of  the  system,  a  produc- 
tive approach  is,  first,  to  characterize  the  molecu- 
lar interactions  in  vitro  and,  second,  to  design  in 
vivo  systems  to  test  models  consistent  with  prop- 
erties uncovered  through  the  first  approach.  We 
are  investigating  the  events  leading  to  patterning 
in  two  major  systems.  One  involves  the  establish- 
ment of  the  early  anterior  pattern,  while  the  other 
is  a  structure-function  analysis  of  a  gene  that  con- 
trols later  steps  of  development.  Independent 
support  will  be  sought  for  the  research  described 
in  the  first  part  of  this  report. 

Interactions  Between  Maternal 
and  Zygotic  Genes 

The  maternal  homeodomain-encoding  gene  bi- 
coid  is  one  of  the  few  examples  of  a  morphogen. 
A  concentration  gradient  of  its  protein  product 
appears  to  control  the  establishment  of  the  ex- 
pression pattern  of  the  first  set  of  zygotic  genes, 
in  particular  the  anterior  gap  gene  hunchback 
{hb^.  Using  transgenic  flies  that  carry  artificial 
genes  containing  combinations  of  binding  sites 
for  the  products  of  bicoid  and  hb,  we  have  shown 
that  the  establishment  of  the  simple  hb  pattern 


requires  not  only  bicoid  but  also  the  synergistic 
participation  of  hb  itself.  This  hb  autoregulation 
may  help  explain  the  sharp,  precise  boundaries 
of  its  expression  domain.  It  also  has  evolutionary 
implications  for  the  development  of  other  organ- 
isms that  do  not  possess  bicoid. 

The  role  of  bicoid  is  not  only  to  control  hb 
expression  but  also  to  direct  the  expression  of  a 
set  of  newly  discovered  genes  required  for  head 
formation.  Using  the  same  approach  as  described 
above,  we  have  demonstrated  that  bicoid 
action  is  modulated  by  the  maternal  genes  from 
the  terminal  torso  group.  These  genes,  which 
encode  a  cascade  of  kinases,  appear  to  act  post- 
transcriptionally  on  the  bicoid  protein  (Bed)  to 
repress  its  action  in  the  terminal  region.  This 
leads  to  the  expression  of  the  head  gap  genes  as 
an  anterior  stripe,  responsible  for  development 
of  a  specific  head  region.  While  bicoid  is  setting 
up  their  posterior  margin,  the  torso  group,  pre- 
venting activation  by  bicoid,  sets  up  their  ante- 
rior margin. 

We  recently  identified  a  gene  that  has  such  an 
expression  pattern  and  encodes  a  homeodomain 
with  a  DNA-binding  specificity  related  to  that  of 
Bed.  We  are  investigating  the  control  of  the  new 
gene's  pattern  of  expression,  and  are  analyzing 
phenotypes  in  the  head  region  for  genetic  dele- 
tions of  the  locus. 

The  demonstration  of  a  direct  interaction  be- 
tween bicoid  and  the  maternal  torso  group  is  the 
first  clue  for  understanding  the  formation  of  head 
structure.  It  also  presents  us  with  a  unique  oppor- 
tunity to  use  a  combination  of  biochemical  and 
genetic  approaches  to  address  the  nature  and  reg- 
ulation of  the  post-transcriptional  modifications 
of  a  transcription  factor  in  the  context  of  a  devel- 
oping organism. 

Regulation  of  the  Segmentation  Gene  hb 

Molecular  dissection  of  the  hb  promoter  by  sev- 
eral groups  has  allowed  definition  of  the  sites  of 
action  of  bed.  Our  in  vivo  studies  have  uncov- 
ered a  more  complex  regulation  of  hb.  As  de- 
scribed above,  hb  appears  to  be  involved  in  the 
establishment  of  its  own  early  expression,  in  syn- 
ergy with  bed.  We  have  identified  in  vitro,  in  the 


113 


Transcription  Control  During  Early  Drosophila  Development 


region  of  the  hb  promoter,  an  Hb  protein-bind- 
ing site  potentially  required  for  full  anterior  acti- 
vation. Mutations  of  this  site  are  being  tested  in 
vivo  for  their  ability  to  respond  to  hb  function.  In 
another  region  of  the  hb  promoter,  we  have  iden- 
tified a  cluster  of  Hb-binding  sites  that  may  medi- 
ate the  other  instance  of  hb  autoregulation  at  a 
later  stage  in  development. 

Interestingly,  these  two  examples  of  positive 
hb  autoregulation  contrast  with  the  general  re- 
pressive nature  of  hb.  We  have  obtained  in  vivo 
evidence  that,  depending  on  the  promoter  con- 
text, hb  can  act  as  a  positive  or  a  negative  regula- 
tor of  gene  expression.  Finally,  the  hb  promoter 
is  also  regulated  by  the  neighboring  gap  gene 
Kriippel,  for  which  two  putative  binding  sites 
have  been  identified.  This  description  and  the  ex- 
periments presented  above  may  allow  us  to  re- 
construct a  functional  hb  promoter  made  exclu- 
sively of  minimal  response  elements  identified 
for  the  regulators  of  hb. 

DNA-binding  Specificity 
of  the  Homeodomain 

Most  developmental  processes  involve  genes 
that  encode  a  homeodomain  (HD).  The  HD  in- 
cludes a  domain  similar  to  the  helix-turn-helix 
motif  present  in  many  prokaryotic  DNA-binding 
transcriptional  regulators.  Our  analysis  of  the 
function  of  the  HD  has  led  us  to  propose  that  the 
specificity  changes  among  classes  of  HDs  are  due 
to  the  nature  of  a  single  amino  acid  at  position  9 
of  the  recognition  helix.  This  position  is  not  criti- 
cal in  the  prokaryotic  helix-turn-helix  proteins, 
and  indeed  structural  analysis  of  HD-DNA  com- 
plexes recently  showed  that  the  HD-DNA  interac- 
tion involves  a  new  mode  of  recognition.  In  this 
mode,  amino  acid  9  fits  in  the  major  groove  of 
DNA,  in  close  contact  with  the  base  pairs  being 
recognized. 

We  have  generalized  the  role  of  amino  acid  9  to 
several  classes  of  HDs.  Using  a  powerful  selection 
procedure  from  a  library  of  random  sequences, 
we  have  isolated  specific  sequences  recognized 
by  a  set  of  mutant  HD  proteins  carrying  different 
residues  at  position  9.  These  sequences  confirm 
our  previous  observations,  but  indicate  that  the 
different  proteins  interact  with  DNA  in  different 
modes. 

The  paired  Gene  Encodes  a  Multifunctional 
Transcription  Factor 

In  addition  to  an  HD,  the  paired  gene  product, 
the  Prd  protein,  contains  regions  that  are  also 


found  in  other  developmental  genes.  We  have 
shown  that  the  so-called  Paired  domain  is  a  sec- 
ond DNA-binding  domain  in  the  Prd  protein, 
which  makes  Prd  a  bifunctional  transcription  fac- 
tor. Although  both  the  Paired  domain  and  the  HD 
can  bind  to  DNA  independently,  they  can  also 
bind  cooperatively  to  adjacent  sites,  but  only 
when  both  domains  are  present  in  the  same  mole- 
cule. The  cooperation  between  the  two  motifs 
may  refine  the  functional  specificity  of  genes 
containing  highly  related  domains. 

We  have  undertaken  an  in  vivo  structure- 
function  analysis  of  the  product  of  prd.  In  this  we 
are  attempting  to  correlate  the  multiple  DNA- 
binding  functions  of  Prd  defined  in  vitro  with  the 
multiple  genetic  functions  of  the  prd  gene,  as  a 
regulator  of  segmentation  genes  and  of  genes  in 
the  nervous  system.  Transgenic  flies  that  carry  the 
prd  coding  sequence  driven  by  its  own  promoter 
rescue  the  lethal  phenotype  of  prd"  flies.  Other 
lines  carrying  versions  of  the  prd  gene  modified 
in  regions  encoding  each  of  the  subdomains  of 
the  Prd  protein  are  now  being  tested  for  their 
ability  to  rescue  some  of  the  differential  molecu- 
lar phenotypes  of  prd~ . 

From  Segmentation  to  Organogenesis 

We  recently  discovered  a  gene  that  appears  to 
be  involved  in  the  early  steps  of  organogenesis  of 
the  salivary  gland,  but  not  in  segmentation.  This 
gene,  highly  homologous  to  prd,  is  first  ex- 
pressed in  a  group  of  cells  that  represent  the  pro- 
genitor of  the  salivary  gland  placode,  at  a  stage 
when  no  tissue  differentiation  has  occurred.  This 
expression  is  controlled  by  positional  cues  from 
the  dorsoventral  and  anteroposterior  determi- 
nants. Later,  when  the  placode  invaginates,  other 
transcription  factor-encoding  genes  are  ex- 
pressed in  the  same  tissue.  Since  the  expression 
of  this  pr^Z-related  gene  precedes  morphological 
events,  it  may  provide  an  excellent  model  of  tran- 
scriptional commitment  to  a  particular  differen- 
tiation pathway.  We  are  now  generating  mutants 
to  analyze  the  early  expression  of  the  putative 
target  genes  and  the  resulting  morphological 
phenotype. 

Because  the  protein  encoded  by  this  gene  has  a 
Prd-like  organization  but  a  Prd  domain  from  a  di- 
vergent class,  this  molecule  will  also  be  very  use- 
ful in  dissecting  the  residues  that  determine  the 
specificity  of  the  Prd  domain.  In  collaboration 
with  John  Kuriyan's  group  (HHMI,  Rockefeller 
University) ,  we  have  undertaken  a  structural  anal- 
ysis of  the  Prd  domain  and  the  HD  present  in  Prd. 


114 


Immune  Evasion  by  Parasites  Causing 
Tropical  Diseases 


John  E.  Donelson,  Ph.D. — Investigator 

Dr.  Donelson  is  also  Distinguished  Professor  in  the  Department  of  Biochemistry  at  the  University  of  Iowa, 
Iowa  City.  He  obtained  his  bachelor's  degree  in  biophysics  from  Iowa  State  University,  served  as  a  Peace 
Corps  volunteer  for  two  years  in  Ghana,  West  Africa,  and  then  completed  a  Ph.D.  degree  in  biochemistry 
at  Cornell  University.  His  postdoctoral  research  was  conducted  at  the  MRC  laboratory  of  Molecular 
Biology  in  Cambridge,  England,  and  at  Stanford  University.  He  has  received  the  Iowa  Governor's 
Science  Medal  and  the  Burroughs  Wellcome  Award  in  Molecular  Parasitology. 


THE  risk  of  acquiring  parasitic  infections  is  a 
part  of  the  daily  lives  of  more  than  3  billion 
people  living  in  the  developing  countries  of  the 
tropics.  The  various  protozoan  and  helminthic 
parasites  that  are  responsible  for  these  infectious 
diseases  possess  a  variety  of  mechanisms  for  evad- 
ing the  immune  response  of  their  hosts.  Our  labo- 
ratory studies  the  interactions  between  several  of 
these  parasites  and  the  immune  system,  in  antici- 
pation that  a  more  detailed  understanding  of 
these  immune  evasion  mechanisms  will  suggest 
better  ways  to  combat  or  prevent  the  infections. 
The  parasites  that  cause  three  of  these  tropical 
diseases  are  described  here. 

Trypanosomiasis 

African  trypanosomes  are  protozoan  parasites 
that  cause  sleeping  sickness  or  trypanosomiasis 
throughout  equatorial  Africa.  They  are  transmit- 
ted from  tsetse  flies  to  the  mammalian  blood- 
stream, where  they  continually  confront  the  hu- 
moral and  cellular  immune  systems.  Each 
trypanosome  in  the  bloodstream  is  covered  by 
about  10  million  copies  of  a  single  protein,  the 
variant  surface  glycoprotein  (VSG).  The  trypano- 
some population  survives  the  continuous 
immune  assault  because  individual  parasites 
occasionally  switch  spontaneously  from  the  ex- 
pression of  one  VSG  to  another — a  process  called 
antigenic  variation.  A  new  immune  response 
must  be  mounted  against  the  VSG  of  the  switched 
parasite  and  its  descendants,  enabling  the  trypan- 
osome population  as  a  whole  to  stay  "one  step 
ahead"  of  the  host  immune  defenses.  We  are  ex- 
amining the  events  at  the  DNA  and  RNA  level  that 
are  responsible  for  this  antigenic  variation. 

We  know  that  the  trypanosome  genome  con- 
tains about  1,000  different  genes  encoding  anti- 
genically  distinct  VSGs.  Usually  one,  and  only 
one,  of  these  VSG  genes  is  expressed  at  a  time. 
Rearrangements  and  duplications  of  these  genes 
are  partly  responsible  for  the  selection  of  which 
VSG  gene  is  to  be  transcribed  and  for  the  switch 
event  itself. 

The  rearrangements  maneuver  specific  VSG 
genes  into  and  out  of  special  chromosomal  loca- 


tions, called  expression  sites,  where  transcrip- 
tion occurs.  These  expression  sites  are  always  lo- 
cated near  the  ends  of  the  chromosomes,  i.e., 
near  the  chromosomal  telomeres,  for  reasons  that 
are  not  well  understood.  The  expression  process 
is  complicated  by  the  fact  that  several,  and  per- 
haps many,  potential  telomere-linked  expression 
sites  exist  in  the  genome,  yet  only  one  is  normally 
activated  at  any  time.  Our  goal  is  to  understand 
this  activation  mechanism  at  the  molecular  level. 

In  one  project  we  have  identified  a  protein  that 
specifically  binds  to  a  region  upstream  of  a  VSG 
gene  in  an  expression  site  activated  during  the 
final  developmental  stage  of  the  parasite  in  the 
tsetse  fly,  i.e.,  the  metacyclic  stage.  We  think  that 
this  protein  may  contribute  to  the  developmental 
regulation  of  that  VSG  gene's  transcription.  In  an- 
other project  we  have  detected  a  high  rate  of  mu- 
tation in  the  telomere-linked  VSG  genes,  which 
may  increase  the  effectiveness  of  antigenic  varia- 
tion still  further.  A  third  project  involves  a  charac- 
terization of  a  group  of  very  small  chromosomes 
that  are  unique  to  African  trypanosomes  and  con- 
tain many  of  the  VSG  genes  destined  for  sequen- 
tial expression.  In  still  another  project  we  have 
introduced  into  trypanosomes  several  plasmids 
containing  combinations  of  sequences  upstream 
of  an  expressed  VSG  gene  and  an  easily  detected 
luciferase  gene  in  an  effort  to  identify  the  se- 
quences that  regulate  the  transcription  of  the 
VSG  gene. 

Leishmaniasis 

Leishmania  parasites  are  protozoan  pathogens 
that  cause  a  spectrum  of  diseases,  including  cuta- 
neous, mucocutaneous,  and  visceral  leishmania- 
sis, in  many  tropical  countries.  During  their  life 
cycle  these  parasites  reside  in  both  the  sandfly 
vector  and  a  mammalian  host.  Within  sandflies 
they  exist  as  uniflagellar  promastigotes  that  de- 
velop from  a  less-infectious  form  to  the  final 
highly  infectious  form.  This  developmental  pro- 
cess can  be  mimicked  during  cultivation  of  pro- 
mastigotes in  liquid  medium.  After  transmission 
from  the  sandfly  to  the  mammalian  host  blood- 
stream, the  promastigotes  penetrate  host  macro- 


115 


Immune  Evasion  by  Parasites  Causing  Tropical  Diseases 


phages,  where  they  reside  as  spherical  amasti- 
gotes  within  acidic  phagolysosomes.  Thus  the 
parasites  evade  the  immune  response  by  "hid- 
ing" inside  macrophages — one  of  several  cell 
types  of  the  immune  system  that  normally  help  to 
destroy  foreign  pathogens  and  substances.  When 
a  sandfly  ingests  amastigote-laden  macrophages 
during  a  blood  meal,  the  life  cycle  is  completed. 
With  a  grant  from  the  National  Institutes  of 
Health,  we  are  studying  how  these  organisms  can 
survive  in  hostile  environments  as  diverse  as  a 
sandfly  midgut  and  an  acidic  macrophage 
phagolysosome. 

A  major  glycoprotein  on  the  surface  of  both 
promastigotes  and  amastigotes  is  a  metal  lopro- 
tease  of  about  63  kDa  (gp63) .  This  enzyme  partic- 
ipates in  the  promastigotes'  uptake  by  macro- 
phages and  contributes  to  the  amastigotes' 
survival  within  them.  The  amount  of  gp63  on  the 
surface  of  promastigotes  increases  about  1 0-fold 
as  the  parasites  develop  into  the  highly  in- 
fectious, virulent  form  during  growth  in  culture. 
We  have  discovered  that  three  different  RNA  spe- 
cies encoding  gp63  occur  during  cultured  pro- 
mastigote  growth. 

One  RNA  species  occurs  in  the  promastigotes 
only  during  their  early,  logarithmic  growth  when 
they  are  less  infectious  and  have  a  small  amount 
of  gp63  on  their  surface.  This  gp63  RNA  is  re- 
placed by  another  gp63  RNA  species  as  the  pro- 
mastigotes enter  stationary  phase  and  become 
more  infectious.  The  presence  of  this  second  RNA 
correlates  with  the  increased  amount  of  gp63 
protein.  The  third  gp63  RNA  species  is  continu- 
ously present  at  a  low  level  throughout  cultured 
growth  and  encodes  an  altered,  transmembrane 
form  of  the  protein. 

The  three  RNA  species  are  derived  from  a  fam- 
ily of  about  1 5  different  genes,  some  of  which  are 
identical  and  all  of  which  are  continuously  tran- 
scribed independent  of  the  stage  of  cultured 
growth.  The  3'-untranslated  regions  of  the  three 


RNA  species  have  completely  different  se- 
quences, and  we  have  shown  that  these  se- 
quences contribute  to  the  different  steady-state 
amounts  of  the  RNAs  at  the  different  growth 
stages.  In  addition,  we  are  introducing  recombi- 
nant DNAs  into  leishmania  to  amplify,  alter,  or 
delete  individual  gp63  genes  so  that  we  may  de- 
termine the  functions  of  each  of  the  three  differ- 
ent gp63  proteases  during  the  promastigote  and 
amastigote  stages.  One  of  our  long-term  goals  is 
to  alter  genetically,  or  attenuate,  the  parasite  so 
that  it  can  be  readily  maintained  under  laboratory 
culture  conditions  but  cannot  survive  in  human 
macrophages.  Such  parasites  may  be  useful  in  the 
development  of  a  vaccine  against  leishmaniasis. 

Onchocerciasis 

Onchocerca  volvulus  is  a  filarial  nematode 
that  causes  onchocerciasis,  or  river  blindness,  in 
Africa  and  Latin  America.  Female  O.  volvulus 
worms  grow  to  50  cm  in  length  and  reside 
throughout  the  body  within  nodules.  In  the  nod- 
ules they  produce  thousands  of  microfilariae 
each  day  that  migrate  throughout  the  body  and 
enter  the  eyes,  where  they  produce  lesions  that 
can  lead  to  blindness.  It  is  not  known  how  any  of 
the  developmental  stages  of  the  parasite  evade 
the  immune  response  of  an  infected  person. 
The  parasites  are  difficult  to  study  in  the  labora- 
tory, because  there  is  no  good  experimental  ani- 
mal model;  they  infect  only  humans  and 
chimpanzees. 

We  have  constructed  cDNA  expression  librar- 
ies of  the  mRNAs  from  the  infective  L3  stage  lar- 
vae of  the  parasite,  and  we  are  using  specific 
cDNA  clones  to  overproduce  parasite  antigens 
from  this  larval  stage  that  are  recognized  by  anti- 
sera  from  onchocerciasis  patients.  This  approach 
has  revealed  several  proteins  that  are  unique  to 
the  parasite  and  may  be  valuable  for  improved 
diagnosis,  treatment,  and  prevention  of  the 
disease. 


116 


Post-transcriptional  Regulation 

of  Gene  Expression,  RNA-Protein  Complexes, 

and  Nuclear  Structures 


Gideon  Dreyfuss,  Ph.D. — Investigator 

Dr.  Dreyfuss  is  also  Professor  of  Biochemistry  and  Biophysics  at  the  University  of  Pennsylvania  School 
of  Medicine.  He  received  his  Ph.D.  degree  in  biological  chemistry  from  Harvard  University  and  his 
postdoctoral  research  training  as  a  Helen  Hay  Whitney  fellow  at  the  Massachusetts  Institute  of  Technology. 
Prior  to  his  present  appointment,  he  was  Professor  and  Established  Investigator  of  the  American  Heart 
Association  at  Northwestern  University. 


MESSENGER  RNAs  (mRNAs)  are  the  func- 
tional translatable  intermediates  in  the 
pathway  of  gene  expression  from  DNA  to  pro- 
teins. They  are  formed  in  the  nuclei  of  eukaryotic 
cells  by  extensive  and  tightly  regulated  post- 
transcriptional  processing  of  primary  RNA  poly- 
merase II  transcripts.  These  transcripts  are 
termed  heterogeneous  nuclear  RNAs  (hnRNAs) ,  a 
term  that  describes  their  size  heterogeneity  and 
cellular  localization.  The  terms  hnRNA  and  pre- 
mRNA  are  often  used  interchangeably,  though  it 
is  possible  that  only  a  subset  of  hnRNAs  are  actu- 
ally precursors  to  mRNA  and  that  the  rest  turn 
over  in  the  nucleus.  From  the  time  hnRNAs 
emerge  from  the  transcription  complex  and  as 
long  as  they  remain  in  the  nucleus,  they  are  asso- 
ciated with  proteins. 

The  collective  term  for  the  proteins  that  bind 
hnRNAs  (but  are  not  stable  components  of  other 
classes  of  ribonucleoprotein  complexes,  such  as 
snRNPs  [small  nuclear  ribonucleoproteins])  is 
hnRNPs.  The  significance  of  hnRNPs  is  that  they 
are  bound  to  the  hnRNAs  and  thus  influence  their 
structure  and  therefore  their  fate  and  processing 
into  mRNAs.  They  are  also  abundant  in  the  nu- 
cleus and  in  hnRNA-hnRNP  complexes  (hnRNP 
complexes)  and  of  interest  as  major  nuclear 
structures. 

Once  formed,  the  mRNAs  are  transported  to  the 
cytoplasm  via  nuclear  pores — a  process  that  ap- 
pears to  be  one  of  the  most  important  regulatory 
steps  in  the  post-transcriptional  pathway  of  gene 
expression  and  about  which  very  little  is  pres- 
ently known.  In  the  cytoplasm,  mRNAs  are  asso- 
ciated with  proteins — the  mRNPs — and  these  are 
likely  to  be  involved  in  the  regulation  of  the 
translation  and  stability  of  mRNAs  and  in  their 
cellular  localization.  Our  goal  is  to  understand, 
in  molecular  detail  and  cellular  architecture, 
how  the  post-transcriptional  portion  of  the  path- 
way of  gene  expression  operates  in  the  cell.  To 
that  end,  we  investigate  the  structure,  function, 
and  localization  of  the  hnRNPs  and  mRNPs  and 
the  RNP  complexes. 

We  have  used  photochemical  RNA-protein 
crosslinking  in  intact  cells  and  affinity  chromato- 


graphic methods  to  identify  and  purify  the 
hnRNPs  and  mRNPs  and  have  produced  monoclo- 
nal antibodies  to  many  of  them.  The  monoclonal 
antibodies  were  used  for  immunopurification  of 
hnRNP  complexes  from  vertebrate  and  Drosoph- 
ila  melanogaster  cells  and  for  the  characteriza- 
tion of  the  hnRNPs.  Immunopurified  hnRNP 
complexes  contain  large  RNA  of  up  to  10,000 
nucleotides  and  at  least  20  major  proteins,  desig- 
nated A-U,  in  the  range  of  34- 1 20  kDa.  There  are 
also  many  less  abundant  hnRNPs,  and  these  ap- 
pear to  bind  only  to  specific  subsets  of  hnRNAs. 
The  specific  arrangement  of  the  proteins  on  spe- 
cific hnRNAs  is  probably  important  in  determin- 
ing the  structure  of  the  hnRNA  and  is  one  of  the 
areas  on  which  we  concentrate  our  investigation. 

Related  to  this  issue,  we  found  that  several  of 
the  hnRNPs  have  RNA-binding  specificities.  Inter- 
estingly, some  of  the  specificities  of  the  major 
hnRNPs  are  for  sequences  important  in  pre- 
mRNA  processing  and  polyadenylation,  and  it  is 
likely  that  this  binding  specificity  is  directly  re- 
lated to  a  role  for  these  proteins  in  mRNA 
formation. 

The  molecular  cloning  and  sequencing  of 
cDNAs  for  several  RNPs  made  possible  the  discov- 
ery of  a  conserved  RNA-binding  domain  (RBD) 
and  a  ribonucleoprotein  consensus  sequence 
(RNP-CS).  The  RNP-CS,  Lys/Arg-Gly-Phe/Tyr- 
Gly/Ala-Phe-Val-X-Phe/Tyr,  is  the  most  highly 
conserved  segment  in  a  generally  conserved  do- 
main of  about  90  amino  acids  found  in  many 
RNA-binding  proteins  of  the  nucleus  and  cyto- 
plasm in  all  eukaryotes  examined.  Overall,  RNP- 
CS  proteins  have  a  modular  structure  reminiscent 
of  DNA-binding  transcription  factors.  They  fre- 
quently contain  several  similar  but  nonidentical 
RBDs,  and  all  contain  at  least  one  auxiliary 
domain  that  is  unique  to  each  type  of  protein 
(e.g.,  glycine-rich,  glutamine-rich,  or  acidic)  and 
that  most  likely  functions  in  protein-protein 
interactions. 

The  hnRNP  C1/C2  proteins  are  abundant,  avid 
hnRNA  binders.  Deletional  analysis  showed  that  a 
93-amino  acid  segment  of  the  human  hnRNP  C 
protein  that  contains  the  RNP-CS  is,  as  predicted, 


117 


Post-transcriptional  Regulation  of  Gene  Expression,  RNA-Protein  Complexes, 
and  Nuclear  Structures 


sufficient  for  RNA  binding.  This  RBD  was  pro- 
duced in  bacteria  and  purified  to  homogeneity  in 
active  form.  Nuclear  magnetic  resonance  (NMR) 
methods  (in  collaboration  with  Luciano  Mueller 
and  Michael  Wittekind,  Bristol-Myers  Squibb), 
including  '^C-  and  ^^N-edited  three-dimensional 
NMR,  were  used  to  determine  the  structure  of  the 
RBD  in  solution.  The  compact  folded  structure 
exhibits  a  four-stranded  antiparallel  |8-sheet  and 
two  well  defined  a-helices.  The  structure  of  this 
RBD  complexed  with  an  RNA  substrate  is  being 
determined. 

Experiments  on  mitotic  cells  unexpectedly 
provided  important  insights  into  the  assembly 
and  general  nature  of  hnRNP  complexes  and  into 
the  transport  of  proteins  to  the  nucleus.  In  mito- 
sis, as  the  nuclear  envelope  breaks  down,  hnRNPs 
disperse  throughout  the  cell  but  remain  asso- 
ciated in  complexes  with  RNA.  After  mitosis, 
once  the  nuclear  envelope  re-forms,  preexisting 
hnRNPs  return  to  the  nucleus.  We  observed,  how- 
ever, using  double-label  immunofluorescence 
microscopy  with  monoclonal  antibodies  to 
various  hnRNPs  on  postmitotic  cells,  that  at  the 
end  of  mitosis  the  hnRNP  complexes  dissociate  in 
the  cytoplasm  and  the  different  proteins  are  trans- 
ported to  the  nucleus  separately.  Some,  includ- 
ing CI,  C2,  and  U,  like  snRNPs  and  lamins,  are 
transported  immediately  (early  group),  while 
others,  including  Al,  A2,  Bl,  B2,  E,  G,  H,  and  L, 
are  transported  into  the  nucleus  later  (late 
group).  Thus,  immediately  following  reassembly 
of  the  nuclear  envelope  at  the  end  of  mitosis, 
pairs  of  cells  are  detected  in  which  some  hnRNPs 
are  in  the  nucleus,  and  others  are  in  the  cyto- 
plasm. These  observations  show  that  hnRNP  com- 
plexes are  dynamic  structures,  in  that  hnRNPs 
can  dissociate  from  the  complexes  and  return  to 
the  nucleus  separately. 

Surprisingly,  the  transport  of  the  late  group  re- 
quires transcription  by  RNA  polymerase  II:  inhibi- 
tors of  this  polymerase  cause  the  late  proteins  to 
remain  in  the  cytoplasm.  Thus  there  are  two  path- 
ways for  nuclear  transport  localization  of  pro- 


teins: a  transcription-independent  pathway  and  a 
novel  transcription-dependent  pathway.  The  dif- 
ferent hnRNPs  utilize  one  of  the  two  pathways, 
and  both  pathways  operate  throughout  the  cell 
cycle.  The  signals  in  the  proteins  that  spec- 
ify the  pathway  to  be  used,  the  mechanism  of 
transcription-dependent  transport  localization, 
and  the  relevance  to  mRNA  transport  are  being 
investigated. 

Immunofluorescence  microscopy  indicated 
that  the  hnRNP  proteins  A,  B,  C,  E,  I,  K,  L,  M,  and 
U  are  nucleoplasmic — that  is,  localized  to  the 
nucleus  but  excluded  from  nucleoli.  In  contrast, 
the  mRNPs  that  have  been  characterized  so  far  [in 
particular  the  poly  (A) -binding  protein]  are  con- 
fined to  the  cytoplasm.  It  was  therefore  con- 
cluded that  the  mRNA  must  exchange  most  if  not 
all  of  the  proteins  with  which  it  is  associated  in 
the  nucleus  as  it  is  transported  to  the  cytoplasm. 
The  dissociation  of  these  proteins  from  the  mRNA 
and  the  subsequent  binding  of  mRNPs  must  be  an 
important  aspect  of  nuclear-cytoplasmic  trans- 
port of  mRNA. 

Recently  we  found  that  several  of  the  abundant 
hnRNPs,  including  Al,  are  not  confined  to  the 
nucleus  but  rather  shuttle  continuously  between 
the  nucleus  and  the  cytoplasm.  Thus  hnRNPs  may 
have  cytoplasmic  functions  in  addition  to  their 
nuclear  roles  in  the  processing  of  pre-mRNA  to 
mRNA.  Furthermore,  Al  is  bound  to  mRNA  in  the 
cytoplasm  and  its  return  to  the  nucleus  requires 
RNA  polymerase  II  transcription.  It  is  therefore 
possible  that  the  novel  cytoplasmic  ribonucleo- 
protein  complex  of  mRNA  with  hnRNPs  is  the 
substrate  of  RNA  nuclear-cytoplasmic  transport. 

Thus  it  is  likely  that  hnRNPs  play  an  active  role 
in  mRNA  export  and  that  the  mRNA  is  transported 
to  the  cytoplasm  as  a  result  of  its  association  with 
the  shuttling  hnRNPs.  Understanding  the  traffick- 
ing of  hnRNPs  in  the  cell  and  the  mechanisms 
that  regulate  the  assembly  and  disassembly  of 
RNPs  with  hnRNAs  and  mRNAs  should  be  impor- 
tant for  elucidating  the  post-transcriptional  path- 
way of  gene  expression,  including  the  nuclear 
transport  process  of  mRNA. 


118 


Genetic  Basis  of  Hearing  Loss 


Geoffrey  M.  Duyk,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Duyk  is  also  Assistant  Professor  of  Genetics  at  Harvard  Medical  School  and  a  member  of  the  Eaton- 
Peabody  Laboratory  at  the  Massachusetts  Eye  and  Ear  Infirmary.  He  received  his  undergraduate  degree 
at  Wesleyan  University  and  his  M.D.  degree  and  a  Ph.D.  degree  in  biochemistry  at  Case  Western  Reserve 
University.  After  internship  and  residency  at  the  University  of  California,  San  Francisco,  he  held  a 
fellowship  in  medical  genetics  under  the  direction  of  Charles  Epstein.  His  postdoctoral  research  work  was 
also  at  UCSF,  with  David  Cox  and  Richard  Myers.  While  at  UCSF,  Dr.  Duyk  was  awarded  an  HHMI 
Physician  Research  Fellowship  and  was  a  Lucille  P.  Markey  Scholar  in  Biomedical  Science. 


HEARING  loss  is  the  commonest  form  of  sen- 
sory impairment.  In  the  United  States  the  in- 
cidence of  congenital  deafness  is  approximately 
1  per  1,000,  and  at  least  half  of  these  cases  are 
likely  to  be  determined  genetically.  It  is  esti- 
mated that  by  age  65  one  in  six  of  us  will  have  a 
clinically  significant  hearing  loss.  While  environ- 
mental factors  play  a  role  in  hearing  impairment, 
genetic  factors  often  determine  predisposition. 
Deafness  is  a  major  aspect  of  over  100  defined 
genetic  disorders. 

Our  laboratory  is  interested  in  sensorineural 
hearing  loss  in  which  the  abnormality  should  lie 
along  the  pathway  between  the  sensory  receptors 
of  the  inner  ear  and  the  auditory  centers  of  the 
brain.  Our  strategy  is  to  combine  genetic  map- 
ping and  positional  cloning  with  candidate  gene 
approaches  to  identify  genes  responsible  for 
hearing  loss  and  to  further  our  understanding  of 
the  molecular  mechanisms  of  hearing. 

Candidate  Genes 

Within  the  inner  ear  reside  the  sensory  organs 
for  hearing  and  balance.  Both  of  these  systems 
represent  mechanical  transduction.  In  each,  a 
specialized  "hair  cell"  serves  as  a  sensor  of  sound 
or  motion.  Hair  cells  are  highly  polarized,  with 
the  basal  surface  forming  synapses  with  afferent 
and  efferent  nerve  fibers.  Finger-like  projections 
referred  to  collectively  as  the  hair  cell  bundle  are 
distinctive  features  of  the  hair  cell's  apical  sur- 
face. Deflection  of  the  hair  cell  bundle  by  the 
sensory  input  activates  mechanically  sensitive 
ion  channels,  initiating  signal  transduction.  This 
process  is  distinct  from  other  sensory  systems 
such  as  taste,  olfaction,  and  vision,  where  a  ligand 
binds  to  a  specific  receptor,  which  in  turn  acti- 
vates a  second  messenger  system  to  initiate  sen- 
sory transduction.  The  molecular  components  of 
this  highly  specialized  mechanical  sensory  sys- 
tem are  logical  targets  for  specific  mutations  re- 
sulting in  hearing  loss. 

In  collaboration  with  David  Corey  (HHMI, 
Massachusetts  General  Hospital),  we  have  begun 
the  process  of  identifying  the  molecular  compo- 


nents of  the  hearing  apparatus  by  combining  mo- 
lecular biological  and  biophysical  approaches. 
Although  extensive  microanatomical  and  bio- 
physical characterization  of  this  apparatus  has 
been  undertaken,  little  biochemical  or  molecular 
information  is  available.  As  a  starting  point,  we 
are  constructing  cDNA  libraries  from  microdis- 
sected  inner  ear  material  highly  enriched  for  hair 
cells.  In  addition,  we  are  exploring  strategies  for 
analyzing  mRNA  populations  of  single  cells  with 
the  polymerase  chain  reaction  (PGR)  technique 
to  augment  the  traditional  approaches  for  re- 
covering and  studying  genes  from  specialized  tis- 
sue sources.  As  we  identify  components  of  the 
sensory  transduction  pathway,  human  and  mu- 
rine homologues  will  be  recovered  and  mapped 
to  specific  chromosomes,  and  closely  linked  DNA 
polymorphic  markers  will  be  identified.  The  de- 
rived information  will  be  an  important  resource 
as  we  begin  our  search  for  the  mutations  responsi- 
ble for  this  group  of  inherited  disorders. 

"Reverse  Genetics" 

The  genetic  analysis  of  hearing  loss  in  human 
populations  is  complicated  by  the  difficulty  of 
diff^erentiating  many  of  these  syndromes  (genetic 
heterogeneity)  and  the  fact  that  deaf  individuals 
often  intermarry  (nonassortive  mating).  As  a  con- 
sequence, most  of  the  families  suitable  for  study 
are  small,  increasing  the  problem  of  placing  the 
disease  locus  with  the  degree  of  precision  re- 
quired to  clone  the  gene.  The  availability  of 
mapped  candidate  genes  helps  to  bridge  the  gap 
between  genetic  and  physical  mapping.  Analysis 
of  these  small  families  will  be  enhanced  by  the 
availability  of  dense  genetic  maps.  Toward  that 
end,  we  have  developed  an  efficient  technique 
for  the  construction  of  genomic  libraries  aug- 
mented for  selected  classes  of  simple  sequence 
repeats  (SSRs).  SSRs  correspond  to  runs  of  di-, 
tri-,  or  tetranucleotide  sequences  (e.g.,  [CA]„  or 
[GGC]„)  that  often  demonstrate  length  polymor- 
phisms detectable  by  PGR  assay.  These  libraries 
will  aid  in  the  production  of  high-resolution  ge- 


119 


Genetic  Basis  of  Hearing  Loss 


netic  maps  composed  of  very  polymorphic,  ho- 
mogeneously distributed  PCR-based  markers. 

Genetic  mapping  can  localize  a  disease  gene  to 
a  1-  to  2-Mb  region  of  a  particular  chromosome. 
The  next  challenge  is  to  identify  the  genes  in  this 
region.  We  have  developed  a  strategy,  termed 
exon  trapping,  that  utilizes  splicing  signals  as 
primary  identifiers  of  coding  sequence  in  cloned 
genomic  DNA.  This  approach  is  also  of  great  util- 


ity for  cloning  the  human  homologue  of  a  gene  of 
interest  when  the  temporal  or  spatial  pattern 
of  gene  expression  limits  the  availability  of 
mRNA  for  analysis.  We  are  developing  second- 
generation  exon-trapping  strategies  based  on 
large-capacity  cloning  vectors  (e.g.,  cosmids, 
PI )  that  should  extend  the  range  for  gene  search- 
ing and  help  to  integrate  the  recovery  of  genes 
with  physical  mapping. 


120 


Molecular  Genetics  of  Intracellular 
Protein  Sorting 


Scott  D.  Emr,  Ph.D. — Associate  Investigator 

Dr.  Emr  is  also  Professor  of  Cellular  and  Molecular  Medicine  at  the  University  of  California,  San  Diego, 
School  of  Medicine.  He  did  his  graduate  work  in  microbiology  and  molecular  genetics  with  Thomas 
Silhavy  and  Jonathan  Beckwith  at  Harvard  Medical  School,  where  he  identified  and  characterized  the  first 
secretion- defective  signal  sequence  mutants  as  well  as  the  first  component  of  the  bacterial  protein  export 
apparatus.  Dr.  Emr  did  postdoctoral  work  on  protein  secretion  in  yeast  with  Randy  Schekman  at  the 
University  of  California,  Berkeley.  He  was  a  faculty  member  at  the  California  Institute  of  Technology 
before  moving  to  UCSD.  Dr.  Emr  counts  among  his  honors  a  Searle  Scholars  Award  and  an  NSF 
Presidential  Young  Investigator  Award. 


AN  essential  feature  of  all  eukaryotic  cells  is 
their  highly  compartmentalized  organiza- 
tion. Different,  often  competing  biochemical 
processes  are  segregated  into  distinct  compart- 
ments. The  identity,  stability,  and  function  of 
each  of  these  compartments,  or  organelles,  is 
conferred  in  large  part  by  the  unique  set  of  pro- 
teins that  reside  within  it.  These  proteins  must  be 
routed  from  their  common  site  of  synthesis  in  the 
cytoplasm  to  their  unique  site  of  function  in  the 
appropriate  intracellular  organelle.  Toward  a  de- 
tailed understanding  of  the  molecular  mecha- 
nisms that  direct  the  delivery  of  one  class  of  these 
proteins,  we  have  focused  our  attention  on  the 
transport  and  sorting  of  proteins  through  the 
Golgi  complex  to  the  lysosome. 

Lysosomal  Hydrolase  Sorting 

Our  laboratory  is  using  a  simple  unicellular  eu- 
karyote,  the  yeast  Saccharomyces  cerevisiae,  as  a 
model  genetic  system  to  study  protein  trafficking 
through  the  secretory  pathway  to  the  lysosome- 
like  vacuole.  The  fundamental  similarities  be- 
tween yeast  and  other  eukaryotic  cells  in  their 
pathways  and  mechanisms  for  protein  delivery 
have  clearly  established  yeast  as  an  important 
model  system  for  the  study  of  these  problems. 

The  importance  of  the  lysosomal  protein  sort- 
ing pathway  in  humans  is  revealed  when  the  ef- 
fects of  mislocalizing  such  enzymes  are  exam- 
ined. A  number  of  lysosomal  hydrolases  are 
secreted  from  naturally  occurring  tumor  cells 
(e.g.,  cathepsin  D  by  breast  cancer  cells).  It  has 
been  proposed  that  the  mislocalization  of  lyso- 
somal enzymes  enhances  cell  growth  and  in- 
creases the  metastatic  potential  of  tumor  cells  by 
contributing  to  the  hydrolysis  of  extracellular 
matrix  components  of  target  tissues.  The  impor- 
tance of  this  sorting  pathway  is  further  exempli- 
fied by  the  serious  inherited  disorders  (e.g.,  I  cell 
disease)  that  result  in  mislocalization  of  lyso- 
somal hydrolases. 

Proteins  destined  for  the  vacuole/lysosome  in 
yeast  and  mammalian  cells  transit  through  early 


stages  of  the  secretory  system.  The  transport  and 
processing  characteristics  of  several  vacuolar  hy- 
drolases in  yeast,  including  the  soluble  protease 
carboxypeptidase  Y  (CPY),  have  been  well  char- 
acterized. CPY  is  translocated  into  the  endoplas- 
mic reticulum  (ER),  where  it  is  modified  with 
four  core  oligosaccharides  to  generate  the  ER 
precursor  form  of  CPY  (pi CPY).  Subsequent 
transport  events  are  mediated  by  vesicular  car- 
riers. Delivery  from  the  ER  to  the  Golgi  complex 
and  passage  through  the  Golgi  are  accompanied 
by  elongation  of  the  core  oligosaccharides  on 
CPY,  resulting  in  formation  of  the  Golgi- 
modified  form  of  CPY  (p2CPY).  Sorting  of 
p2CPY  from  proteins  destined  for  secretion  ap- 
pears to  take  place  in  the  late  Golgi.  Upon  arrival 
in  the  vacuole,  an  amino-terminal  propeptide 
segment  on  p2CPY  is  proteolytically  removed, 
generating  the  active  mature  form  of  the  protease 
(mCPY). 

Sorting-Defective  Mutants 

We  have  focused  our  efforts  on  identifying  and 
characterizing  the  cellular  machinery  that  directs 
the  sorting  and  transport  of  vacuolar  hydrolases. 
A  gene  fusion-based  selection  scheme  we  de- 
signed enabled  us  to  isolate  more  than  600  yeast 
mutants  that  exhibit  severe  defects  in  vacuolar 
protein  sorting.  Each  of  the  mutants  missorts  and 
secretes  CPY  and  other  vacuolar  enzymes.  The 
recessive  mutations  define  more  than  33  comple- 
mentation groups  referred  to  as  vps  (vacuolar 
protein  sorting  defective) .  Extensive  genetic,  bio- 
chemical, and  morphological  characterization  of 
the  vps  mutants  has  allowed  us  to  organize  them 
into  phenotypically  related  groups.  Each  group 
appears  to  function  at  a  common  stage  of  the  pro- 
tein sorting  pathway. 

The  apparent  genetic  complexity  of  the  vps 
mutant  collection  presumably  reflects  the  bio- 
chemical complexity  of  the  protein  sorting  reac- 
tion. Specific  cellular  components  must  recog- 
nize vacuolar  proteins,  segregate  them  from 
other  secretory  proteins,  and  package  them  into 


121 


Molecular  Genetics  of  Intracellular  Protein  Sorting 


specific  transport  vesicles  that  ultimately  must 
recognize  and  fuse  with  the  appropriate  target 
organelle,  the  vacuole.  Therefore  the  list  of  po- 
tential activities  and  structures  required  for  the 
sorting  and  transport  of  lysosomal/vacuolar  pro- 
teins can  easily  accommodate  the  large  number 
of  gene  products  presently  implicated  by  the  ge- 
netic studies. 

Role  for  a  Protein  Kinase  Complex 

An  understanding  of  this  protein  sorting  path- 
way and  the  individual  activities  of  different  VPS 
gene  products  is  being  facilitated  by  the  molecu- 
lar isolation  and  characterization  of  these  genes. 
Thus  far,  we  have  cloned  and  sequenced  eight 
VPS  genes.  Comparison  of  the  yeast  VPS  gene  se- 
quences with  other  known  mammalian  genes  has 
revealed  several  informative  structural  and  func- 
tional similarities.  Two  of  the  genes,  VPS\ 5  and 
VPS54,  are  of  particular  interest.  The  sequence  of 
the  VPSl  5  gene  predicts  a  protein  with  the  fol- 
lowing features:  a  consensus  site  for  amino- 
terminal  myristoylation,  a  region  that  shares  sig- 
nificant sequence  similarity  with  the  family  of 
Ser/Thr  protein  kinases,  and  a  region  of  homol- 
ogy with  the  regulatory  subunit  of  protein  phos- 
phatase 2A  (PP2A).  The  sequence  similarity 
Vpsl  5  protein  (Vpsl  5p)  shares  with  Ser/Thr  pro- 
tein kinases  and  the  PP2A  regulatory  subunit 
raises  the  interesting  possibility  that  protein 
phosphorylation/dephosphorylation  may  play  an 
important  role  in  the  regulation  of  protein  sort- 
ing events. 

To  assess  the  functional  significance  of  these 
sequence  similarities,  we  used  site-directed  mu- 
tagenesis to  change  several  highly  conserved 
amino  acid  residues  in  the  putative  kinase  do- 
main of  the  Vpsl  5p.  Each  of  the  mutations  inacti- 
vates the  complementing  activity  of  the  VPS15 
gene.  The  mutant  cells  exhibit  extreme  vacuolar 
protein  sorting  defects;  amino  acid  changes  in 
less  highly  conserved  positions  gave  only  weak 
protein  sorting  defects. 

These  data  suggest  that  the  Vpsl  5p  is  an  active 
kinase  and  that  this  kinase  activity  is  required 
during  some  step  in  vacuolar  protein  sorting. 
Protein  phosphorylation  may  act  as  a  "molecular 
switch"  in  this  protein  sorting  pathway  by  ac- 
tively diverting  vacuolar  hydrolases  away  from 
the  default  secretion  path  and  toward  the  vacu- 
ole. One  can  imagine  several  points  in  the  path- 
way that  may  need  to  be  regulated  precisely,  such 


as  the  budding  and  transport  of  carrier  vesicles  or 
the  recognition  and  fusion  of  these  with  the 
correct  target  organelle. 

Biochemical  and  genetic  evidence  indicate 
that  the  Vpsl 5  protein  forms  a  complex  (on  the 
cytoplasmic  face  of  the  membrane)  with  another 
Vps  protein,  Vps34p.  Overexpression  of  Vps34p 
suppresses  the  vacuolar  protein  sorting  and 
growth  defects  caused  by  mutations  within  the 
kinase  domain  of  Vpsl  5p,  but  will  not  suppress  a 
null  allele  of  VPSl  5.  Therefore,  Vps34p  cannot 
bypass  the  cells'  requirement  for  Vpsl5p.  This 
genetic  interaction  between  VPSl  5  and  VPS54  is 
consistent  with  the  observation  that  mutations  in 
both  the  VPSl  5  and  VT534  genes  result  in  a  com- 
mon set  of  phenotypes. 

The  Vps34  protein  shares  sequence  similarity 
with  a  mammalian  gene  recently  identified  in 
Mike  Waterfield's  laboratory  that  codes  for  the 
catalytic  subunit  of  the  phosphatidylinositol 
3-kinase  (PI3-kinase).  P13-kinase  catalyzes  the 
formation  of  PI3-phosphate,  a  rare  membrane 
lipid  that  has  been  proposed  to  act  as  a  second 
messenger  in  cell  signaling.  The  enzyme  appears 
to  play  an  important  role  in  cell  proliferation  and 
transformation.  PI3-kinase  activity  has  been 
shown  to  be  associated  with  several  cell  surface 
protein-tyrosine  kinase  receptors  (e.g.,  the  PDGF 
[platelet-derived  growth  factor],  insulin,  and 
CSF-1  [colony-stimulating  factor  1]  receptors). 
The  role  of  this  lipid-modifying  enzyme  in  pro- 
tein trafficking  events  is  not  yet  clear.  However, 
we  are  in  the  process  of  mutating  several  con- 
served sequence  motifs  to  analyze  the  signifi- 
cance of  this  intriguing  sequence  similarity  in 
Vps34  protein  function. 

Among  its  functions,  PI3-phosphate  formation 
at  localized  sites  in  the  membrane  (adjacent  to 
membrane  receptor  proteins)  may  facilitate  vesi- 
cle formation/targeting  required  during  protein 
transport  to  the  lysosome  from  both  the  Golgi 
complex  (delivery  of  newly  synthesized  lyso- 
somal hydrolases)  and  the  cell  surface  (endocytic 
uptake  and  down-regulation  of  cell  surface  re- 
ceptors). The  Vps  15  kinase  may  regulate  the  ac- 
tivity of  Vps34p/PI3-kinase  by  phosphorylation 
of  Vps34p. 

We  recently  developed  an  in  vitro  assay  that 
reconstitutes  intercompartmental  protein  trans- 
port to  the  yeast  vacuole.  We  are  now  using  this 
assay  to  assign  the  Vpsl  5  and  Vps34  proteins  to  a 
specific  step(s)  in  the  reaction. 


122 


Mechanisms  Involved  in  Preventing  Unwanted 
Blood  Clots 


Charles  T.  Esmon,  Ph.D. — Investigator 

Dr.  Esmon  is  also  a  member  of  the  Oklahoma  Medical  Research  Foundation  and  Professor  of  Pathology 

and  Associate  Professor  of  Biochemistry  at  the  University  of  Oklahoma  Health  Sciences  Center,  Oklahoma 

City.  He  received  his  B.S.  degree  in  chemistry  from  the  University  of  Illinois  and  his  Ph.D.  degree 

in  biochemistry  from  Washington  University.  He  conducted  his  postdoctoral  research  at  the  University 

of  Wisconsin  before  joining  the  faculty  at  the  University  of  Oklahoma  Health  Sciences  Center. 

later  he  joined  the  Oklahoma  Medical  Research  Foundation. 


PROTEIN  C,  protein  S,  and  thrombomodulin 
constitute  one  of  the  natural  anticoagulant 
complexes  that  prevents  unwanted  blood  clots. 
We  have  focused  our  attention  on  this  system  be- 
cause we  have  been  able  to  identify  patients  with 
a  history  of  unwanted  blood  clots  who  have  ab- 
normal protein  C,  protein  S,  or  thrombomodulin. 
To  understand  how  the  system  functions,  it  is 
useful  to  review  the  function  of  the  components. 
Thrombomodulin  is  found  primarily  on  the  sur- 
face of  endothelial  cells,  the  cells  that  line  the 
blood  vessels.  Thrombin,  the  enzyme  that  causes 
blood  to  clot,  can  bind  to  thrombomodulin; 
when  thrombin  is  bound,  it  no  longer  clots  the 
blood  but  instead  converts  protein  C  into  the  ac- 
tive blood  clotting  inhibitor,  activated  protein  C. 
Activated  protein  C  then  binds  to  protein  S  on  the 
surface  of  platelets  (small  cells  in  the  blood)  or 
endothelial  cells,  where  it  functions  as  an  antico- 
agulant. The  activated  protein  C-protein  S  com- 
plex works  as  an  anticoagulant  by  cutting  up  and 
inactivating  two  of  the  clotting  proteins,  factor 
VIII  (the  protein  missing  in  hemophilia)  and  fac- 
tor V. 

This  broad  outline  of  how  the  system  functions 
fails  to  tell  us  much  about  where,  when,  or  how 
the  system  might  function  in  human  disease  pro- 
cesses. This  knowledge  is  important  both  in 
terms  of  understanding  the  basic  properties  of  the 
system  and  in  the  design  of  new  therapeutic  ap- 
proaches to  diagnosis  and  prevention  of  blood 
clots.  Of  particular  interest,  and  still  unex- 
plained, is  the  observation  that  administration  of 
activated  protein  C  at  levels  that  can  prevent  un- 
wanted blood  clots  does  not  increase  blood  loss 
at  surgical  sites.  This  contrasts  with  available  anti- 
coagulants, such  as  heparin,  which  block  un- 
wanted clot  formation  but  also  dramatically  in- 
crease blood  loss  at  surgical  sites.  One  of  our 
goals  is  to  understand  how  this  natural  anticoagu- 
lant can  accomplish  this  remarkable  specificity. 
New  therapeutic  agents  with  these  properties 
could  greatly  decrease  morbidity  and  mortality 
associated  with  thrombotic  complications. 

Most  healthy  individuals  have  adequate 
amounts  of  protein  C  and  the  other  components 


of  the  system  that  control  blood  clot  formation 
under  normal  circumstances.  When  people  be- 
come sick,  unwanted  clotting  is  often  a  problem. 
Studies  from  other  laboratories  have  shown  that 
protein  S  circulates  in  humans  both  free  and 
bound  to  an  inhibitor  of  the  complement  system 
(the  system  that  helps  protect  from  infection), 
called  C4b-binding  protein  (C4bBP). 

We  found  that  only  the  free  form  of  protein  S 
could  work  to  form  the  anticoagulant.  Patients 
with  clinical  conditions  known  to  cause  an  in- 
creased risk  of  blood  clots  also  had  reduced  lev- 
els of  free  protein  S  and  more  C4bBP-protein  S 
complex.  Families  with  inherited  thrombotic 
complications  were  identified  in  which  the  fam- 
ily members  who  developed  blood  clots  had  high 
levels  of  the  complex.  These  observations  sug- 
gested that  alteration  in  the  levels  of  free  protein 
S  might  contribute  to  the  clotting  complications 
observed  in  these  patients.  To  test  this  hypothe- 
sis, we  developed  some  animal  models  that  in- 
volved blood  clotting  as  a  complication. 

Clearly  thrombosis  is  caused  by  more  than  sim- 
ply an  alteration  of  C4bBP  levels.  Infection  and 
agents  that  cause  inflammation  are  known  to  trig- 
ger coagulation  by  a  variety  of  mechanisms. 
Three  very  different  responses  occur.  The  first  is 
related  to  bacterial  infection,  which  can  result  in 
septic  shock,  a  process  characterized  by  small 
blood  clots  in  the  circulation,  damage  to  organs, 
and  death.  Once  this  process  begins,  treatment  is 
very  difficult.  A  second  response  is  formation  of 
solid  blood  clots  in  the  small  vessels.  This  results 
in  death  of  the  affected  organs.  A  third  response  is 
occlusion  of  the  large  vessels.  Why  inflammation 
causes  these  three  dramatically  different  re- 
sponses is  unknown,  complicating  rational  ap- 
proaches for  both  early  diagnosis  and  effective 
therapy. 

Animal  models  for  these  three  processes  were 
developed  in  collaboration  with  Fletcher  Taylor. 
These  models  indicate  that  shifts  in  the  balance 
between  free  protein  S  and  protein  S  bound  to 
C4bBP  contribute  to  the  dift'crent  disease  pro- 
cesses and  can  be  involved  in  shifting  the  ob- 
served type  of  thrombosis.  Increased  levels  of 


123 


Mechanisms  Involved  in  Preventing  Unwanted  Blood  Clots 


C4bBP-protein  S  complex  cause  the  inflamma- 
tory response  to  low  levels  of  bacteria  to  convert 
from  a  mild  response  to  a  severe  response  with  all 
of  the  characteristics  of  toxic  shock.  Moreover, 
under  certain  conditions  the  shift  in  amounts  of 
G4bBP-protein  S  complex  can  change  the  re- 
sponse from  that  of  circulating  small  clots  to  oc- 
clusion of  small  vessels.  Most  data  suggest  that 
bacteria  cause  clotting  by  causing  the  formation 
of  inflammatory  mediators,  called  cytokines.  In- 
flammatory cytokines  do  not  lead  to  the  small 
clots  characteristic  of  bacterial  infection  but 
rather  to  blood  clots  in  the  major  vessels.  This 
response  occurs  only  when  the  C4bBP-protein  S 
levels  are  high.  In  every  case  tested  to  date,  the 
deleterious  effects  of  the  C4bBP  can  be  reversed 
by  adding  protein  S. 

Why  then  do  bacterial  infections  lead  to  the 
small  clots  in  the  circulation?  Possible  insights 
were  gained  from  the  observation  that  small  cell 
fragment-like  particles  can  convert  this  throm- 
botic response  back  into  the  small-clot  syndrome 
seen  in  septic  shock.  Complement  is  known  to 
cause  such  cell  fragments  to  be  formed.  These 
studies  may  provide  insights  into  how  different 
inflammatory  and  coagulation  components  work 
in  concert  to  generate  such  distinct — but 
related — thrombotic  complications. 

These  results  suggest  that  the  balance  between 
free  protein  S  and  that  bound  to  C4bBP  may  be 
critical  to  thrombotic  complications  as  a  result  of 


inflammation.  The  data  imply  that  increasing 
protein  S  would  prevent  these  thrombotic  com- 
plications, suggesting  new  therapeutic  ap- 
proaches to  thrombotic  diseases.  If  we  could 
block  the  decrease  in  free  protein  S,  the  risk  of 
thrombosis  might  be  reduced.  This  unique  ap- 
proach could  potentially  return  the  patient  to 
normal  status  without  significantly  increasing  the 
risk  of  bleeding. 

A  major  interest  in  our  laboratory  has  been  to 
understand  how  thrombomodulin  causes  throm- 
bin to  change  its  function  from  a  clotting  to  a 
clot-inhibiting  enzyme.  To  understand  this,  we 
have  examined  the  binding  of  thrombin  to  throm- 
bomodulin and  to  its  other  targets.  Of  particular 
interest  is  the  ability  of  thrombin  to  activate  cells 
and  platelets,  leading  to  platelet  plugs  and  clots, 
especially  in  the  arteries.  Thrombomodulin  can 
block  platelet  activation  by  thrombin,  but  how  it 
does  so  had  been  a  mystery.  In  collaboration  with 
Shaun  Coughlin's  group,  we  demonstrated  that 
the  thrombin  receptor  on  platelets  and  thrombo- 
modulin bind  thrombin  at  the  same  place, 
thereby  explaining  the  earlier  observations.  Both 
protein  C  and  the  thrombin  receptor  share  com- 
mon sequences  near  the  cleavage  sites,  and  both 
sequences  are  inherently  poor  sequences  for 
cleavage  by  thrombin.  Binding  of  thrombomodu- 
lin fragments  or  the  fragments  from  the  thrombin 
receptor  results  in  changing  how  thrombin 
cleaves  its  substrates.  Thus  both  receptors  have  a 
built-in  switch  that  allows  thrombin  to  function. 


124 


Molecular  Genetics  of  Steroid  and  Thyroid 
Hormone  Receptors 


Ronald  M.  Evans,  Ph.D. — Investigator 

Dr.  Evans  is  also  Professor  at  the  Gene  Expression  Laboratory  of  the  Salk  Institute  for  Biological  Studies 
and  Adjunct  Professor  in  the  Departments  of  Biology  and  of  Biomedical  Sciences  at  the  University  of 
California,  San  Diego.  He  received  his  Ph.D.  degree  in  microbiology  and  immunology  from  the  University 
of  California,  Los  Angeles,  School  of  Medicine.  After  postdoctoral  training  with  James  Darnell  at  the 
Rockefeller  University,  he  joined  the  faculty  of  the  Salk  Institute.  Dr.  Evans  is  a  member  of  the 
National  Academy  of  Sciences.  His  research  interests  are  in  developmental  and  inducible  regulation 
of  gene  expression. 


AN  understanding  of  the  mechanisms  by 
which  apparently  distinct  regulatory  sys- 
tems integrate  to  modulate  body  function  and  be- 
havior poses  one  of  the  most  important  chal- 
lenges of  modern  biology.  Hence  we  have 
focused  our  attention  on  the  action  of  steroid, 
retinoid,  and  thyroid  hormones  in  regulatory  cel- 
lular and  organ  physiology.  This  field  has  under- 
gone an  extraordinary  development  in  the  last 
several  years  as  a  consequence  of  the  cloning  and 
sequencing  of  the  genes  encoding  the  receptors 
for  these  hormones  in  target  cells. 

It  has  been  demonstrated  that  these  receptors 
are  all  structurally  related  and  constitute  a  super- 
family  of  nuclear  regulatory  proteins  that  are  ca- 
pable of  modulating  gene  expression  in  a  ligand- 
dependent  fashion.  One  challenge  is  to  define 
each  receptor's  molecular  properties  that  deter- 
mine its  interactions  with  the  transcription  ma- 
chinery regulating  gene  expression.  Another 
challenge  is  to  elucidate  the  contributions  of  indi- 
vidual regulatory  systems  to  the  integrated  and 
complex  processes  associated  with  cell  growth, 
differentiation,  and  organ  function. 

A  Novel  Retinoic  Acid  Response  Pathway 

The  retinoids,  a  group  of  compounds  that  in- 
clude retinoic  acid,  retinol  (vitamin  A),  and  a  se- 
ries of  natural  and  synthetic  derivatives,  exert 
profound  effects  on  development  and  differentia- 
tion in  a  wide  variety  of  systems.  (The  retinoic 
acid  receptors  are  collectively  designated  RARs.) 
Retinoic  acid  has  also  been  shown  to  induce  the 
transcription  of  several  genes,  suggesting  a  role 
analogous  to  those  of  steroid  and  thyroid  hor- 
mones. In  previous  studies  we  described  the 
cloning  and  characterization  of  a  retinoic  acid- 
dependent  transcription  factor  referred  to  as 
RARa.  Additional  RAR-related  genes  have  been 
isolated,  and  at  least  three  different  RAR  subtypes 
(a,  /3,  and  7)  are  now  known  in  mice  and  humans. 

Retinoic  acid  receptors  share  homology  with 
the  superfamily  of  steroid  and  thyroid  hormone 
receptors  and  have  been  shown  to  regulate 
specific  gene  expression  by  a  similar  ligand- 


dependent  mechanism.  Complicating  these  ob- 
servations is  our  recent  identification  of  a  group 
of  receptors  termed  RXRs  (retinoid  X  receptors) , 
which  are  only  distantly  related  to  the  RARs.  The 
discovery  of  this  second  retinoid  transduction 
pathway  led  us  to  investigate  its  functional  prop- 
erties and  determine  its  relationship  to  the  RARs. 
We  now  know  that  there  are  at  least  three  RXR- 
related  genes  (termed  a,  j3,  and  7)  located  at  ge- 
netically distinct  loci.  Northern  blot  analyses  of 
the  RXRs  indicate  that  each  isoform  has  a  unique 
pattern  of  expression  in  adult  tissue  and  is  tempo- 
rally and  spatially  expressed  in  the  embryo. 
These  studies  suggest  a  role  for  RXRs  in  adult 
physiology  and  embryonic  development. 

Binding  experiments  demonstrate  that  the  RXR 
protein  has  low  affinity  for  retinoic  acid  (RA) 
and,  taken  together  with  the  transactivation  stud- 
ies, indicate  that  the  RXR  ligand  may  be  a  metabo- 
lite of  RA.  Based  on  these  assumptions,  we  de- 
vised a  strategy  to  identify  the  putative 
metabolite  referred  to  as  retinoid  X.  The  implicit 
concept  of  the  strategy  was  that  all-trans  RA  may 
be  converted  to  retinoid  X  by  a  natural  cellular 
process.  Accordingly,  high  doses  of  all-trans  RA 
were  fed  to  recipient  tissue  culture  cells.  After 
allowing  for  metabolic  conversion,  material  ex- 
tracted from  these  cells  was  fractionated  to  re- 
solve the  various  retinoid  peaks,  and  each  peak 
was  assayed  for  its  ability  to  activate  the  RXRs  in  a 
transfection  assay.  A  specific  peak,  termed  reti- 
noid X,  was  identified  and  characterized  by  mass 
spectrometry. 

Based  on  this  process,  we  have  now  demon- 
strated that  9-cis  RA  is  the  high-affinity  ligand  for 
the  RXR.  While  not  previously  seen  in  living  or- 
ganisms, 9-cts  RA  is  apparently  a  new  and  widely 
used  vertebrate  hormone.  It  transactivates  RXRa 
up  to  40  times  more  efficiently  than  all-trans  RA 
and  binds  to  RXR  with  high  affinity.  We  also  con- 
firmed that  all-trans  RA  shows  no  detectable 
binding  affinity  for  the  RXR.  Furthermore,  each 
of  the  RXR  subtypes  (a,  /3,  and  7)  is  activated  by 
9-c«  RA  with  increased  potency  and  efficacy  rela- 
tive to  all-trans  RA. 


125 


Molecular  Genetics  of  Steroid  and  Thyroid  Hormone  Receptors 


A  point  of  potential  physiological  significance 
is  that  9-cis  RA  also  binds  to  and  transactivates 
both  RXRs  and  RARs  and  may  thus  serve  as  a  com- 
mon or  "bifunctional"  ligand.  Conversion  of  the 
all-trans  to  the  9-cis  isomer  could  provide  a 
novel  means  for  differential  cell-speciftc  regula- 
tion of  the  activity  of  these  retinoid  pathways. 
The  hypothesis  that  9-cis  RA  may  be  functionally 
distinct  from  its  all-trans  precursor  raises  the  in- 
triguing possibility  that  the  regulation  of  its  iso- 
merization  could  be  a  key  step  in  retinoid  physiol- 
ogy. It  is  unknown  whether  this  reaction  is 
catalyzed  by  an  enzyme. 

The  3-4-5  Rule 

Members  of  the  receptor  superfamily  modulate 
target  gene  expression  by  binding  as  either  homo- 
or  heterodimers  to  hormone  response  elements 
(HREs).  We  recently  described  the  properties  of 
direct  repeats  of  the  consensus  half-site  sequence 
AGGTCA  as  HREs  for  nuclear  receptors.  Receptor 
specificity  for  binding  and  activation  was  shown 
to  be  conferred  through  the  number  of  nucleo- 
tides separating  the  two  half-sites.  Spacers  of  3,  4, 
or  5  nucleotides  were  originally  shown  to  serve 
as  optimal  response  elements  for  the  vitamin  D 
receptor  (VDR),  thyroid  hormone  receptor  (TR), 
and  RAR,  respectively.  We  now  refer  to  this  physi- 
ological code  built  into  HREs  as  the  "3-4-5  rule." 

More  recently,  we  have  characterized  in  the  up- 
stream regulatory  region  of  the  cellular  retinol- 
binding  protein  type  II  (CRBPII)  gene  an  HRE 
that  confers  selective  responsiveness  to  the  RXRs. 
This  response  element  consists  of  tandem  repeats 


of  the  AGGTCA  sites  separated  by  a  single 
nucleotide. 

The  ability  of  the  VDR,  TR,  and  RAR  to  recog- 
nize their  cognate  response  elements  is  depen- 
dent upon  their  ability  to  form  a  heterodimeric 
complex  with  an  unknown  nuclear  factor.  Re- 
markably, the  search  for  this  factor  has  recently 
identified  RXR  as  a  common  heterodimeric 
partner  for  the  VDR,  TR,  and  RAR.  These  results 
emerged  from  our  initial  finding  of  a  functional 
interaction  between  the  RAR  and  RXR,  followed 
by  the  demonstration  that  these  two  receptors 
form  stable  heterodimers  in  solution.  Further- 
more, the  heterodimer  binds  target  DNA  with 
more  than  100-fold  increased  efficiency  over  ei- 
ther partner  alone. 

Similarly,  RXR  heterodimers  with  the  VDR  and 
TR  have  greatly  increased  affinity  for  the  respec- 
tive cognate  response  elements.  Apparently  RXR 
is  serving  as  a  type  of  master  receptor,  gating  the 
activities  of  vitamin  D,  thyroid  hormone,  and  reti- 
noic  acid.  As  noted  above,  however,  RXR  can  act 
independently  of  all  these  receptors  when  re- 
sponding to  its  novel  hormone  9-cis  RA.  Despite 
the  apparent  level  of  complexity  revealed  by 
these  interactions,  it  appears  that  the  physiologi- 
cal response  is  built  upon  a  series  of  simple  prin- 
ciples in  which  heterodimers,  each  consisting  of 
an  RXR  component,  interact  with  common  DNA 
sequences,  each  varying  by  a  single  nucleotide.  It 
is  through  this  simplicity  that  such  exquisite 
specificity  can  be  maintained  and  thus  permit  co- 
ordinate control  of  a  vast  gene  network  in  a 
highly  selective  and  orderly  fashion. 


Opposite:  Expression  pattern  of  the  genes  for  RXRa,  ^,  and  7  in  a  mouse  embryo.  RXRs  are 
receptor  proteins  that  mediate  the  actions  of  the  newly  described  hormone,  9-cys  retinoic  acid. 
Parasagittal  sections  from  the  embryo  taken  at  gestation  day  16.5  were  hybridized  in  situ  with 
radioactive  probes  that  mark  the  specific  areas  where  RXR  mRNA  is  expressed.  After  exposure  to 
x-ray  film,  the  resulting  image  is  digitally  scanned  into  a  computer  and  colorized.  Dark  red  to 
yellow  colors  represent  low  to  high  levels  of  RXR  expression.  Thus  the  figure  demonstrates  that 
RXRa  is  strongly  expressed  in  skin  and  metabolic  organs  such  as  intestine;  RXR^  is  expressed 
ubiquitously;  RXRy  shows  marked  expression  in  the  pituitary  and  corpus  striatum,  the  brain 
center  that  regulates  muscle  coordination  and  is  affected  in  parkinsonian  disorders. 

Reprinted  with  permission  from  Mangelsdorf  D.J.,  Borgmeyer,  U.,  Heyman,  R.A.,  Zhou,J.Y., 
Ong,  E.S.,  Oro,  A.E.,  Kakizuka,  A.,  and  Evans,  R.M.  1992.  Genes  Dev  6:329-344.  Computer 
photograph  by  Jamie  Simon,  the  Salk  Institute  for  Biological  Studies. 


126 


1  mtji  I        fji  mry 


DEVELOPMENT 


alpha 


gamma 


Spring  Harbor  Laboratory  Press 

in  association  with 


The  Genetical  Sticiety  of  Great  Britain 


127 


Molecular  Mechanisms  Involved  in  the  Actions 
of  Calcium-mediated  Hormones 


John  H.  Exton,  M.D.,  Ph.D. — Investigator 

Dr.  Exton  is  also  Professor  of  Molecular  Physiology  and  Biophysics  and  of  Pharmacology  at  the  Vanderbilt 
University  School  of  Medicine.  He  received  his  medical  degree  from  the  University  of  New  Zealand  and  his 
Ph.D.  degree  in  biochemistry  from  the  University  of  Otago,  New  Zealand.  His  postdoctoral  research  was 
done  in  the  Department  of  Physiology  at  Vanderbilt  University  School  of  Medicine  with  Charles  Park, 
where  he  has  remained  as  a  faculty  member.  His  honors  include  the  Lilly  Award  of  the  American  Diabetes 
Association  and  the  M.D.  degree  with  distinction  from  the  University  of  Otago. 


THE  major  objective  of  my  laboratory  is  to  elu- 
cidate the  mechanisms  of  action  of  hor- 
mones, neurotransmitters,  and  other  agents  that 
transmit  information  in  the  nervous  system  and 
other  organ  systems  by  altering  membrane  lipids 
and  increasing  the  concentration  of  calcium  ions 
in  their  target  cells.  Agents  that  act  this  way  in- 
clude regulators  of  heart  function  and  blood 
flow,  such  as  epinephrine,  norepinephrine,  ace- 
tylcholine, angiotensin,  and  vasopressin;  other 
neurotransmitters,  such  as  serotonin,  neuroten- 
sin, and  substance  P;  and  agents  that  control  cer- 
tain pituitary  and  pancreatic  secretions,  food  di- 
gestion, bladder  and  uterine  contraction,  platelet 
aggregation,  and  certain  responses  to  trauma  and 
infection. 

We  initially  established  that  many  hormones 
and  neurotransmitters  act  by  increasing  the  intra- 
cellular concentration  of  calcium  ions.  The  next 
phase  of  our  work  involved  the  demonstration 
that  the  increase  is  due  to  both  mobilization  of 
calcium  ions  from  internal  stores  and  stimulation 
of  their  inflow  across  the  cell  membrane.  We  also 
demonstrated  that  the  receptors  for  calcium-me- 
diated agents  are  located  on  the  outer  surface 
of  their  target  cells.  Thus  these  cells  must 
have  some  means  of  signaling  from  the  receptors 
to  the  internal  calcium  stores,  and  our  eff^orts 
were  directed  toward  elucidating  the  signaling 
mechanism. 

Initially  we  tested  the  hypothesis  that  the  sig- 
nal is  generated  by  the  breakdown  of  phosphati- 
dylinositol,  a  phospholipid  in  the  cell  mem- 
brane. However,  this  was  found  to  be  too  slow  to 
account  for  the  changes  in  calcium.  Attention  was 
then  focused  on  a  related  phospholipid,  phos- 
phatidylinositol  4,5-bisphosphate  (PIP2),  which 
breaks  down  more  rapidly  in  response  to  hor- 
mones. The  situation  became  clearer  when  inosi- 
tol 1,4,5-trisphosphate  (IP3)  was  identified  as 
the  signaling  molecule  for  intracellular  calcium 
release.  This  compound  is  generated  when  PIP2  is 
broken  down  by  the  enzyme  phospholipase  C.  The 
other  compound  produced  is  1 ,2-diacylglycerol 
(DAG),  which  is  also  a  signaling  molecule,  since 
it  activates  a  specific  protein-phosphorylating 
enzyme,  protein  kinase  C. 


The  present  activities  of  the  laboratory  encom- 
pass two  major  research  areas.  The  first  involves 
elucidating  how  calcium-mediated  agents  stimu- 
late the  breakdown  of  PIP2.  A  major  discovery  has 
been  the  finding  that  a  G  protein  (a  regulatory 
protein  that  binds  the  nucleotide  GTP)  is  in- 
volved in  coupling  the  receptors  for  these  agents 
to  the  phospholipase  C  enzyme  that  breaks  down 
PIP2.  Our  work  has  involved  purifying  and  char- 
acterizing the  relevant  G  proteins  from  liver  cell 
membranes  and  reconstituting  them  with  other 
components  of  the  signaling  system.  Reconstitu- 
tion  of  the  G  proteins  with  PIP2  phospholipase  C 
has  been  achieved,  and  the  system  has  been  used 
to  purify  the  G  proteins  to  homogeneity  in  both 
the  complete  form  {a^y  heterotrimers)  and  in 
the  form  of  free  a-subunits.  Two  a-subunits  (42 
and  43  kDa)  have  been  identified,  and  both  have 
been  shown  immunologically  to  be  members  of 
the  Gq  family  of  G  proteins.  Partial  sequencing  of 
tryptic  peptides  of  both  proteins  has  confirmed 
that  they  are  Gaq  and  Gq:,i. 

Purification  of  the  G  protein  activators  of  the 
phospholipase  C  in  the  heterotrimeric  form  has 
likewise  yielded  two  G  proteins  corresponding  to 
Gq  and  Gn.  Using  Gofq  and  Gofn  activated  by  a 
GTP  analogue,  we  have  shown  that  the  specific 
isozyme  form  of  the  PIP2  phospholipase  C  con- 
trolled by  these  G  proteins  in  both  liver  and  brain 
is  the  148-kDa  -isozyme.  Evidence  that  Gq 
and  G,]  are  coupled  to  receptors  for  calcium- 
mobilizing  agents  has  also  been  obtained.  Three 
calcium-mediated  agents  (vasopressin,  epineph- 
rine, and  angiotensin)  specifically  stimulate  the 
labeling  of  Gaq  and  Ga,,  by  a  radioactive,  light- 
reactive  analogue  of  GTP  in  liver  cell  mem- 
branes. Binding  of  a  GTP  analogue  is  enhanced  by 
an  acetylcholine  analogue  when  Gq  and  G,j  are 
reconstituted  with  the  M,  (calcium-mobilizing) 
muscarinic  receptor  but  not  with  the  M2  recep- 
tor. Co-reconstitution  of  the  purified  Mj  recep- 
tor, Gq/Gi,,  and  phospholipase  C-/3i  permits 
agonist-stimulated  hydrolysis  of  PIP2  in  a  GTP  ana- 
logue-dependent manner.  The  activation  of  the 
phospholipase  is  paralleled  by  activation  of  the  G 
proteins,  as  measured  by  their  binding  of  the 


129 


Molecular  Mechanisms  Involved  in  the  Actions 
of  Calcium-mediated  Hormones 


GTP  analogue.  These  findings  demonstrate  the 
complete  reconstruction  of  a  signaling  pathway 
for  a  calcium-mediated  agonist  from  purified 
components. 

The  second  major  research  area  arose  from  the 
observation  that  many  hormones,  neurotransmit- 
ters, and  growth  factors  break  down  another  cell 
membrane  phospholipid,  phosphatidylcholine 
(PC),  in  a  wide  variety  of  cells.  This  breakdown 
yields  DAG  and  phosphatidic  acid  (PA)  and  in- 
volves two  other  phospholipases  (C  and  D), 
which  are  being  purified  and  characterized.  Fur- 


ther work  has  shown  that  PC  breakdown  is  regu- 
lated by  mechanisms  involving  G  proteins,  cal- 
cium ions,  protein  kinase  C,  and  tyrosine  kinases. 
The  latter  two  mechanisms  are  currently  being 
explored  in  several  cell  types. 

In  a  related  area,  the  regulation  of  different  iso- 
zymes of  protein  kinase  C  by  certain  molecular 
species  of  DAG  and  PA  is  being  studied.  This  in- 
volves the  purification  of  a  protein  kinase  C  iso- 
zyme that  is  stimulated  by  PA  but  not  DAG.  These 
studies  should  elucidate  the  functions  and  mecha- 
nisms of  this  novel  signaling  system. 


130 


Tumor-Suppressor  Genes 


Andrew  p.  Feinberg,  M.D.,  M.P.H. — Associate  Investigator 

Dr.  Feinberg  is  also  Associate  Professor  of  Internal  Medicine  and  Human  Genetics  at  the  University 
of  Michigan  Medical  School.  He  received  his  B.A.,  M.D.,  and  M.P.H.  degrees  from  the  Johns  Hopkins 
University.  He  received  clinical  training  at  the  University  of  Pennsylvania  and  Johns  Hopkins  and  did 
postdoctoral  research  at  the  University  of  California,  San  Diego,  and  Johns  Hopkins.  Before  moving  to  the 
University  of  Michigan,  Dr.  Feinberg  was  Assistant  Professor  of  Oncology  and  Medicine  at  Johns  Hopkins. 


ONE  of  the  most  important  areas  of  cancer  ge- 
netics is  the  identification  and  characteriza- 
tion of  tumor-suppressor  genes,  whose  inactiva- 
tion  contributes  to  cancer.  Since  almost  all  genes 
are  present  in  two  copies  in  the  cell,  cancer 
would  develop  from  deletion  or  inactivation  of 
both  copies  of  these  suppressor  genes.  Inactiva- 
tion of  one  copy  could  be  transmitted  in  families 
from  parent  to  child.  Thus  individuals  inheriting 
one  nonfunctional  copy  of  the  gene  would  be  at 
increased  cancer  risk. 

Wilms'  tumor  (WT),  a  childhood  kidney 
cancer,  was  one  of  the  earliest  models  of  suppres- 
sor gene  action.  Strong  and  Knudson  showed  20 
years  ago  that  WT  apparently  resulted  from  two 
mutations,  based  on  two  peaks  in  the  age  of  onset. 
Furthermore,  it  was  discovered  in  the  1970s  that 
some  children  with  WT  lack  a  large  portion  of 
chromosome  1 1  (in  band  llpl3),  which  is  visi- 
ble microscopically.  Before  joining  HHMI,  I 
observed  (with  Eric  Fearon  and  Bert  Vogelstein) 
that  more  subtle  gene  deletions  can  be  detected 
indirectly  on  chromosome  1 1  in  WTs,  through 
use  of  restriction  fragment  length  polymor- 
phisms (RFLPs),  which  can  distinguish  the  ma- 
ternal and  paternal  copies  of  a  given  gene. 

In  collaboration  with  David  Schlessinger 
(Washington  University,  St.  Louis)  and  Bryan  Wil- 
liams (Hospital  for  Sick  Children,  Toronto),  our 
laboratory  has  now  cloned  the  1 1  p  1 3  WT  gene 
region  in  yeast  artificial  chromosomes  (YACs) .  In- 
terestingly, we  found  that  this  region  contains 
multiple  genes  turned  on  specifically  in  develop- 
ing kidney.  At  least  two  of  these  genes  showed 
reduced  or  absent  expression  in  approximately 
half  of  sporadically  occurring  WTs,  notably  those 
of  the  same  histologic  type  that  occur  in  children 
with  1  lpl3  deletions.  One  of  these  genes  codes 
for  a  DNA-binding  protein  that  is  mutated  in 
some  WTs. 

These  mutations,  however,  occur  infrequently, 
and  the  laboratory  is  investigating  whether  re- 
duced expression  of  the  gene  may  be  a  more 
common  mechanism  for  tumorigenesis  and 
whether  other  genes  from  this  complex  may  play 
a  role.  We  are  also  "retrofitting"  WT  YACs  with  a 
gene  that  allows  growth  in  mammalian  cells,  in 


order  to  demonstrate  directly  a  tumor-suppressor 
phenotype  of  the  1  lpl3  WT  gene(s)  and  to  de- 
termine by  deletion  experiments  the  functional 
role  of  the  multiple  genes  from  this  region. 

In  addition  to  the  known  gene  on  chromosome 
1 1  that  predisposes  to  WT,  we  have  discovered  a 
second  predisposing  gene  at  a  different  location 
on  the  chromosome  (band  11  p  1 5  )  .  This  gene  ap- 
pears to  be  involved  in  bladder,  breast,  and  lung 
cancer,  as  well  as  WT.  Thus  WT  causation  is  more 
complex  than  investigators  had  previously  be- 
lieved, and  this  second  WT  gene  may  turn  out  to 
be  important  in  common  cancers. 

Consistent  with  this  gene  being  a  tumor  sup- 
pressor, the  laboratory  has  applied  genetic 
linkage  analysis  to  map  to  the  same  region 
of  11  pi  5  a  cancer-predisposing  disorder,  Beckwith- 
Wiedemann  syndrome  (BWS).  Using  YACs,  the 
laboratory  has  now  isolated  several  DNA  break- 
points from  BWS  patients  with  germline  chromo- 
somal rearrangements.  This  should  enable  the  lab- 
oratory to  determine  whether  the  BWS  gene  is 
indeed  the  second  WT-suppressor  gene  on  the 
llpl5  band. 

Adding  to  the  complexity  of  WT  is  the  fact  that 
some  non-BWS  families  with  hereditary  predispo- 
sition to  WT  do  not  show  linkage  of  this  trait  to 
chromosome  1 1 .  Recently  the  laboratory  found, 
in  collaboration  with  former  sabbatical  member 
Anthony  Reeve  (University  of  Otago,  New  Zea- 
land), involvement  of  chromosome  16. 

The  laboratory  has  recently  developed  a  novel 
approach  to  isolating  tumor-suppressor  genes  di- 
rectly. Human  chromosomes  are  fragmented  into 
2-5  million  base  pair  "superfragments"  in  a  way 
that  allows  their  transfer  into  any  mammalian 
cell.  The  advantage  of  this  technique  is  that  it 
enables  one  to  transfer  the  giant  DNA  pieces  into 
a  recipient  cell  and  screen  directly  for  a  func- 
tional gene.  It  may  thus  have  immediate  ap- 
plication to  cloning  other  chromosome  1 1 
tumor-related  genes,  or  more  general  application 
to  cloning  a  variety  of  genes  on  other  chromo- 
somes— genes  for  which  one  can  currently 
screen  but  not  select,  such  as  those  for  cellular 
aging. 

For  example,  one  should  be  able  to  exploit  the 


131 


Tumor-Suppressor  Genes 


cancer-suppressing  property  of  tumor-suppressor 
genes  to  identify  the  key  chromosomal  region 
containing  the  gene.  In  a  test  of  this  method,  8  of 
nearly  100  chromosome  11  superfragments  that 
the  laboratory  isolated,  when  introduced  into  WT 
cells,  suppressed  their  neoplastic  growth.  Inter- 
estingly, the  tumor-suppressing  hybrids  contain  a 
small  portion  of  1  lpl5,  demonstrating  the  exis- 
tence of  a  suppressor  gene  on  this  band.  The  labo- 
ratory is  now  testing  whether  these  superfrag- 
ments also  suppress  important  common  cancers 
that  involve  lip,  such  as  lung  cancer.  This  novel 
technique  may  allow  investigators  to  bridge  a  gap 
in  cloning  methods  between  chromosome-size 
pieces  (averaging  100  million  nucleotides)  and 
YACs  (averaging  300,000  nucleotides),  and  it 
may  have  general  application  to  cloning  a  wide 
variety  of  genes. 

Another  long-standing  interest  of  the  labora- 
tory is  the  role  of  epigenetic  changes  (not  involv- 
ing DNA  sequence,  and  thus  potentially  re- 
versible) in  cancer.  The  laboratory  recently 


identified  several  CpG  islands  (DNA  sequences 
rich  in  cytosine-guanine  dinucleotides,  often 
found  near  actively  expressed  genes)  within  the 
1  Ipl  3  WT  gene  region.  Some  of  these  sequences 
were  methylated,  a  reversible  modification  of  the 
nucleotide  cytosine.  Previously  CpG  islands 
were  only  known  to  be  methylated  on  the  inac- 
tive X  chromosome.  The  surprising  finding  of 
methylated  autosomal  CpG  islands  suggests  that 
epigenetic  changes  may  play  a  role  in  Wilms'  tu- 
morigenesis.  We  are  now  testing  this  hypothesis 
by  examining  the  islands  directly. 

We  also  observed  that  balanced  BWS  chromo- 
somal translocations  on  llpl5  always  involve 
the  maternal  chromosome,  while  unbalanced 
BWS  duplications  always  involve  the  paternal 
chromosome,  suggesting  an  epigenetic  differ- 
ence (or  imprint)  between  the  two  alleles.  If 
such  epigenetic  changes  are  found  to  contribute 
to  inactivation  of  1  Ip  suppressor  genes,  this  will 
represent  an  exciting  convergence  of  our  two 
major  interests. 


132 


'  '  '      '  w 

Genetics,  Structure,  and  Function 
of  Histocompatibility  Antigens 


Kirsten  Fischer  Lindahl,  Ph.D. — Investigator 

Dr.  Fischer  Lindahl  is  also  Professor  of  Microbiology  and  Biochemistry  at  the  University  of  Texas 
Southwestern  Medical  Center  at  Dallas.  She  began  the  study  of  histocompatibility  with  Morten  Simonsen 
in  Copenhagen,  Denmark,  and  received  her  Ph.D.  degree  in  immunobiology  from  the  University  of 
Wisconsin-Madison.  She  was  a  postdoctoral  fellow  with  Darcy  Wilson  at  the  University  of  Pennsylvania, 
Philadelphia,  and  with  Klaus  Rajewsky  at  the  Institute  for  Genetics  in  Cologne,  West  Germany.  Before 
accepting  her  current  position,  she  was  a  member  of  the  Basel  Institute  of  Immunology  in  Switzerland. 


HISTOCOMPATIBILITY  (H)  antigens  are  cell 
surface  molecules  that,  when  foreign,  lead 
to  the  rejection  of  grafted  tissues  and  organs  by 
the  vertebrate  immune  system.  Because  they  form 
a  major  obstacle  to  clinical  transplantation,  H  an- 
tigens have  been  studied  for  over  50  years.  They 
are  complexes  of  a  small  peptide  ligand  and  an 
MHC  molecule  (encoded  by  genes  of  the  major 
histocompatibility  complex).  A  given  individual 
has  MHC  molecules  of  a  few  different  kinds,  each 
of  which  can  present  to  the  immune  system  a 
large  variety  of  peptides  on  the  surface  of  cells. 
These  peptides  might  be  derived  from  proteins 
produced  by  intracellular  parasites,  bacteria,  or 
viruses  or  by  the  body's  own  cells,  such  as  tumor- 
specific  antigens  or  minor  H  antigens.  The  amino 
acid  side  chains  that  line  the  peptide-binding 
groove  of  an  MHC  molecule  determine  which 
peptides  that  molecule  can  bind  and  therefore 
what  antigens  can  be  presented  to  induce  an  im- 
mune response  in  the  individual  with  this  MHC. 

The  immune  system  is  capable  of  recognizing  a 
difference  in  either  of  the  H  antigens'  two  parts.  A 
difference  in  the  MHC  molecule  itself  will  alter 
many  complexes  and  induce  a  strong  immune  re- 
sponse, hence  the  term  "major"  H  antigen.  By 
contrast,  a  difference  in  a  peptide  alters  only  one 
of  many  kinds  of  complexes  and  induces  a  weaker 
response,  hence  the  term  "minor"  H  antigen.  Un- 
like the  major  H  antigens,  human  minor  H  anti- 
gens remain  ill  defined.  In  the  mouse,  however, 
more  than  50  genes  that  encode  minor  H  antigens 
have  been  mapped.  Almost  every  chromosome, 
including  the  mitochondrial  genome,  carries  at 
least  one. 

The  Maternally  Transmitted  Antigen 

In  the  mouse  mitochondrial  protein  NDl,  the 
sixth  amino  acid  is  polymorphic.  When  cells  with 
one  form  are  transplanted  to  a  mouse  with  an- 
other form,  the  amino-terminal  peptide  of  NDl 
will  act  as  a  transplantation  antigen,  called  Mta. 
This  peptide  is  presented  on  the  cell  surface  by 
an  MHC  class  I  molecule  called  M3.  M3  binds  the 
NDl  peptide  only  when  the  methionine  at  the 


end  carries  a  formyl  group,  and  M3  can  also  bind 
other  peptides  with  a  formyl-methionine.  This 
characteristic  of  the  amino  terminus  of  mito- 
chondrial and  bacterial  proteins  distinguishes 
them  from  proteins  made  in  the  cytoplasm  of 
mammalian  cells.  We  are  collaborating  with 
Michael  Bevan  (HHMI,  University  of  Washington, 
Seattle) ,  who  is  studying  the  immune  response  of 
mice  to  the  bacterium  Listeria.  His  group  reports 
that  the  M3  molecules  present  a  Listeria  antigen 
on  the  cell  surface  to  cytotoxic  T  cells,  which  kill 
the  infected  cells. 

It  is  of  great  interest  to  understand  how  the 
formyl-methionine  peptides  are  bound  by  M3  and 
which  amino  acids  in  M3  are  important  for  this 
specificity.  We  cloned  the  gene  for  M3  from  mice 
and  rats.  The  rat  and  mouse  M3  genes  are  more 
similar  to  each  other  than  they  are  to  the  other 
MHC  class  I  genes  of  their  own  species.  This  is 
particularly  striking  in  the  rat,  where  the  class  I 
genes  are  otherwise  very  similar.  These  results 
suggest  that  the  specialized  function  of  M3 
evolved  long  ago  in  a  species  from  which  both 
rats  and  mice  are  descended,  and  that  it  has  been 
conserved  in  both  species  during  their  separate 
evolution,  presumably  because  it  is  useful  in  the 
immune  response  against  bacterial  peptides. 

We  can  express  M3  in  mouse  fibroblasts,  where 
Mta  can  be  detected  by  killer  T  lymphocytes.  This 
system  allows  us  to  change  single  amino  acids  in 
the  protein  by  introducing  mutations  at  specific 
sites  in  the  gene.  We  are  currently  looking  at 
amino  acids  34  and  171,  which  differ  in  M3  from 
the  consensus  of  MHC  class  1  molecules  and  are 
near  the  site  where  the  formyl-methionine  of  the 
peptide  is  expected  to  lodge.  In  sequencing  natu- 
rally occurring  variants  of  the  M3  gene  from  wild 
mice,  we  are  learning  more  about  which  amino 
acids  are  essential  for  the  ability  to  present  the 
NDl  peptide.  Amino  acid  95  points  straight  up  in 
the  peptide-binding  site,  probably  right  under 
the  variable  sixth  residue  of  the  peptide.  The  sin- 
gle change  of  95  from  leucine  to  glutamine  com- 
pletely abolishes  T  cell  recognition  of  Mta.  We 
are  collaborating  with  the  laboratory  of  Johann 
Deisenhofer  (HHMI,  University  of  Texas  South- 


133 


Genetics,  Structure,  and  Function  of  Histocompatibility  Antigens 


western  Medical  Center  at  Dallas)  to  produce  M3 
in  amounts  sufficient  for  a  structural  analysis. 

RMA-S  Mutant  Cells 

The  heavy  chains  of  MHC  class  I  molecules  are 
not  stable  in  the  properly  folded  conformation  at 
body  temperature  unless  they  bind  a  peptide  as 
well  as  the  182-microglobulin  light  chain.  RMA-S 
mutant  cells  make  MHC  class  I  heavy  chains  and 
j82-microglobulin,  but  do  not  display  them  on 
their  surface.  The  defect  can  be  circumvented  by 
adding  suitable  synthetic  peptides  to  the  mutant 
cells,  which  will  then  display  those  MHC  mole- 
cules that  bind  the  added  peptide.  Not  only  clas- 
sical MHC  class  I  molecules  and  M3,  which  were 
already  known  to  present  peptides,  are  reduced 
or  missing  from  these  cells,  but  also  other  MHC 
class  I  molecules,  such  as  Qa-1 .  These  molecules 
are  recognized  by  antibodies  or  killer  T  lympho- 
cytes, but  their  physiological  function  is  not  yet 
clear.  The  RMA-S  cell  line  shows  that  they  all  bind 
peptides. 

The  RMA-S  mutation  affects  one  of  two  MHC 
genes  whose  protein  products  together  make  a 
channel  to  transport  peptides  from  the  cytoplasm 
into  the  endoplasmic  reticulum,  where  MHC 
class  I  molecules  fold.  Surface  display  of  all  such 
molecules,  including  Mta  and  Qa-1,  is  restored 
when  a  normal  version  of  this  gene  is  expressed 
in  the  RMA-S  cell  line.  This  tells  us  that  the  mito- 
chondrial peptide  is  transported  from  the  cyto- 
plasm into  the  endoplasmic  reticulum  by  the 
same  mechanism  that  other  peptide  antigens  use. 
We  have  found  that  the  drug  oligomycin,  which 
stimulates  protein  degradation  in  mitochondria, 
increases  the  surface  display  of  both  Mta  (as  ex- 
pected) and  Qa-1  on  RMA-S  cells,  suggesting  that 
Qa-1  can  also  bind  a  mitochondrial  peptide.  We 
believe  that  the  increased  supply  of  these  pep- 


tides floods  the  inefi'ective  transport  system  and 
overcomes  the  defect. 

/?2-Microglobulin  Polymorphism 

Mice  are  the  only  mammals  in  which  allelic 
forms  of  /32-microglobulin  have  been  described. 
Because  of  its  intimate  interaction  with  the  pep- 
tide-binding  parts  of  MHC  class  I  heavy  chains,  an 
allelic  difi'erence  of  182-microglobulin  that  alters 
amino  acid  85  from  aspartic  acid  to  alanine  can 
subtly  aff'ect  the  structure  of  MHC  class  I  mole- 
cules and  their  ability  to  bind  particular  pep- 
tides. We  have  found  four  new  alleles  of  (82- 
microglobulin  among  wild  mice  of  the  species 
Mus  musculus.  These  differ  by  only  one  or  two 
amino  acids  relative  to  the  |82microglobulin  of 
inbred  mice.  Three  of  these  wild  alleles  have  va- 
line in  position  85  and  may  therefore  alter  pep- 
tide binding. 

We  also  looked  at  the  |82-niicroglobulin  gene 
from  the  species  Mus  spretus,  which  has  been 
separated  from  Mws  by  about  1  million 

years  of  evolution.  To  our  surprise,  this  (82- 
microglobulin  differed  from  that  of  inbred  mice 
by  12  amino  acids  and  had  only  a  single,  silent 
mutation,  which  afi'ected  the  DNA  sequence,  but 
not  the  protein.  For  comparison,  rat  and  mouse 
/32-microglobulins  differ  by  14  amino  acids.  We 
would  have  expected  at  least  an  equal  number  of 
silent  mutations  and  amino  acid  changes  for  the 
spretus  form. 

Such  drastic  divergence  contrasts  with  the  mod- 
est changes  seen  in  other  members  of  the  immu- 
noglobulin superfamily  and  suggests  that  the 
molecule  has  been  selected  for  its  differences. 
The  diversification  may  be  part  of  the  speciation 
process,  but  it  may  be  limited  to  molecules 
that  can  tolerate  change.  All  changes  in  the 
microglobulin  molecule  are  in  loops  or  on  the 
face  away  from  the  MHC  class  I  heavy  chain,  spar- 
ing the  face  that  interacts  with  the  conserved  do- 
main of  the  heavy  chains. 


134 


Borrelia  burgdorferi,  the  Lyme  disease  agent  in  the  gut  of  deer  ticks.  Ticks  that  feed  on  immunized 
mice  show  elimination  of  the  spirochetes  ( right),  whereas  ticks  that  feed  on  control  mice  contain 
numerous  spirochetes  (left). 

From  Fikrig,  E.,  Telford,  S.,  Barthold,  S.W.,  Kantor,  F.S.,  Spielman,  A.,  and  Flavell,  R.A.  1992. 
Proc  Natl  Acad  Sci  USA  89:5418-5421. 


136 


Genetic  Approaches  to  Immune  Function 
and  Tolerance 


Richard  A.  Flavell,  Ph.D. — Investigator 

Dr.  Flavell  is  also  Professor  of  Immunobiology  at  Yale  University  School  of  Medicine.  He  received  k>is  B.Sc. 
and  Ph.D.  degrees  in  biochemistry  from  the  University  of  Hull,  England,  and  performed  postdoctoral  work 
in  Amsterdam  and  Zurich.  Before  accepting  his  current  position,  Dr.  Flavell  was  first  Assistant  Professor 
at  the  University  of  Amsterdam,  then  Head  of  the  Laboratory  of  Gene  Structure  and  Expression  at  the 
National  Institute  for  Medical  Research,  Mill  Hill,  London,  and  subsequently  President  and  Chief  Scientific 
Officer  of  Bio  gen  Research  Corporation,  Cambridge,  Massachusetts.  Dr.  Flavell  is  a  Fellow  of  the  Royal 
Society  and  a  member  of  other  distinguished  societies. 


MY  laboratory  has  concerned  itself  for  many 
years  with  the  regulation  and  function  in 
the  immune  system  of  the  genes  of  the  murine 
MHC  (major  histocompatibility  complex).  In  the 
mouse  these  genes  are  encoded  on  chromosome 
17,  and  prior  work  has  shown  that  there  are  a 
large  number  of  linked  class  1-related  genes  and 
a  handful  of  class  II  genes.  Class  I  genes  encode  a 
protein  of  approximately  45,000  molecular 
weight  that  is  found  in  association  with  a  small 
subunit,  i82"r"icroglobulin.  Together  this  com- 
plex forms  a  symmetrical  molecule  consisting  of 
four  extracellular  globular  domains  anchored 
through  the  cell  membrane  with  the  transmem- 
brane segment  and  having  a  short  stretch  of 
amino  acids  that  extend  into  the  cytoplasm.  Class 
II  molecules  achieve  a  similar  symmetry,  but 
with  two  polypeptide  chains,  a  and  /?,  each  of 
which  has  two  extracellular  domains  and  a  trans- 
membrane and  cytoplasmic  segment. 

Both  class  I  and  class  II  gene  products  serve  as 
recognition  elements,  which  bind  antigenic  pro- 
tein fragments  and  present  them  to  T  cells.  In  the 
case  of  class  I  genes,  the  presentation  is  to  T  cells 
carrying  the  CDS  co-receptor  molecule.  These 
cells  are  usually  cytotoxic  T  cells,  whose  role  is 
to  destroy  cells  that  are  virally  infected.  In  the 
case  of  class  II  molecules,  it  is  commonly  intra- 
cellular pathogens  that  are  presented,  this  time  to 
helper  or  inflammatory  T  cells  that  carry  the  CD4 
co-receptor.  Both  types  of  T  cells  secrete  hor- 
mone-like molecules  called  lymphokines,  which 
in  turn  act  on  other  cell  types — for  example,  on  B 
cells,  which  are  stimulated  to  multiply  and  to 
make  antibody. 

Class  I  and  class  II  genes  are  both  regulated  in 
vivo  by  various  lymphokines.  Of  these,  the  inter- 
ferons are  important  and  are  currently  utilized  in 
the  therapy  of  several  human  diseases.  For  exam- 
ple, interferon-7  secreted  by  activated  T  cells 
stimulates  the  synthesis  of  MHC  class  1  and  II  mol- 
ecules and,  as  a  result,  presumably  renders  a  cell 
better  able  to  present  antigen  and  thus  to  poten- 
tiate an  immune  response.  We  have  taken  a  ge- 
netic approach  to  attempt  to  understand  how  in- 


terferon-7 activates  the  synthesis  of  these  MHC 
molecules.  A  new  strategy  was  used  to  isolate  a 
series  of  mutant  cell  lines  that  are  not  capable  of 
responding  to  interferon-7.  These  mutants  ap- 
pear to  have  a  series  of  different  defects.  In  some 
of  these  cell  lines,  the  mutations  result  in  a  com- 
plete loss  of  the  ability  of  all  genes  to  respond  to 
interferon-7  and  even,  surprisingly,  interferon-a 
and  which  were  previously  believed  to  use 
distinct  mechanisms.  Other  cell  lines  are  defec- 
tive only  in  the  response  of  the  class  II  and  related 
genes.  This  genetic  approach  should  help  us  un- 
derstand how  these  important  molecules  regulate 
gene  expression  by  elucidating  the  molecular 
steps  that  the  cell  utilizes  to  activate  genes 
through  interferons. 

One  important  issue  in  the  functioning  of  the 
immune  system  is  how  the  body  discriminates  its 
own  tissues  (self)  from  foreign  components  such 
as  pathogens.  The  process  that  protects  the  indi- 
vidual against  the  destruction  of  self  tissues  is 
known  as  immune  tolerance.  Tolerance  is  gener- 
ally believed  to  be  established  during  the  produc- 
tion of  new  T  cells  in  the  thymus  by  a  process 
called  negative  selection,  which  is  mediated  by 
clonal  deletion;  that  is,  self-reactive  T  cells  are 
destroyed  at  the  site  of  synthesis.  The  failure  of 
self-tolerance  leads  to  the  autoimmunity  that 
characterizes  human  diseases  such  as  autoim- 
mune diabetes  (insulin-dependent  diabetes  mel- 
litus  [IDDM])  and  rheumatoid  arthritis. 

In  the  past  few  years  we  have  been  interested  in 
determining  the  mechanisms  of  tolerance  to 
those  components  of  the  body  that  are  never 
found  in  the  thymus  and  therefore  pose  a  prob- 
lem for  tolerance  mechanisms  operating  in  the 
thymus.  Transgenic  mice  can  be  used  to  study 
this  process,  since  the  expression  of  a  given  gene 
— and  hence  the  protein  encoded  by  that  gene — 
can  be  directed  to  the  tissue  of  choice  by  linking 
the  gene  for  the  desired  protein  to  the  regulatory 
signals  that  function  in  that  specific  tissue.  We 
have  previously  performed  such  experiments  by 
directing  the  synthesis  of  MHC  class  II  proteins  to 
the  pancreatic  jS-cells  of  transgenic  mice.  In  these 


137 


Genetic  Approaches  to  Immune  Function  and  Tolerance 


experiments  the  mice  were  indeed  found  to  be 
tolerant  to  the  MHC  antigens.  T  cells  that  would 
normally  react  with  this  MHC  product  are  not 
eliminated,  which  is  what  would  be  found  if  the 
class  II  antigen  is  expressed  in  thymic  tissues.  In- 
stead, these  T  cells  are  present  but  have  been  in- 
activated in  some  way,  such  that  they  are  no 
longer  able  to  respond  to  the  MHC  antigen,  either 
in  the  animal  or  in  test-tube  experiments.  As  a 
result,  no  destruction  of  the  pancreatic  tissue 
occurs. 

To  determine  mechanisms  of  peripheral  toler- 
ance to  protein  antigens,  rather  than  MHC,  we 
made  transgenic  mice  that  express  the  T  cell  re- 
ceptor (TCR)  reactive  with  a  fragment  of  the  T 
antigen  of  SV40  (simian  virus  40).  We  have  ob- 
tained transgenic  mice  that  express  SV40  T  anti- 
gen in  various  peripheral  tissues  in  the  body,  in- 
cluding the  |8-cells  of  the  pancreas  and  the 
secretory  exocrine  cells  of  the  pancreas  that  pro- 
duce digestive  enzymes.  By  crossing  the  TCR 
transgenics  with  mice  expressing  the  antigen,  we 
can  determine  the  immune  status  to  SV40  T  anti- 
gen. Tolerance  is  found  to  the  SV40  T  antigen 
provided  that  expression  has  occurred  prior  to 
the  release  of  T  cells  from  the  thymus  in  the  new- 
born animal.  This  tolerance  results  in  partial  elim- 
ination of  antigen-specific  T  cells  and  clonal 
inactivation  of  the  remainder.  If,  however,  ex- 
pression of  antigen  is  delayed,  the  T  cells 
are  not  tolerant  but  instead  become  activated, 
with  resultant  autoimmune  destruction  of  the 
pancreas. 

Delayed  expression  of  antigen  therefore  seems 
to  place  an  individual  at  risk  for  autoimmunity. 
However,  even  in  individuals  genetically  predis- 
posed to  become  autoimmune,  nongenetic  fac- 
tors play  an  important  role.  One  model  suggests 
that  a  precipitating  event  for  autoimmunity  can 
be  the  onset  of  a  local  inflammatory  response  in 


the  tissue,  for  example,  as  a  consequence  of  in- 
fection. To  test  this  we  made  transgenic  mice  that 
express  the  inflammatory  cytokine  TNF  (tumor 
necrosis  factor)  on  the  pancreatic  islets,  which 
simulates  local  inflammation.  This  cytokine  ex- 
pression causes  a  massive  infiltration  of  T  and  B 
lymphocytes,  which  mimics  the  infiltrate  seen  in 
mouse  autoimmune  diabetes  and  causes  partial 
destruction  of  the  insulin-producing  j8-cells  of 
the  pancreas.  We  are  currently  determining 
whether  the  inflammatory  cells  present  in  the 
TNF  transgenic  mice  are  specific  for  pancreatic 
antigens,  as  is  the  case  in  true  IDDM. 

We  also  study  the  response  of  the  immune  sys- 
tem to  the  spirochete  Borrelia  burgdorferi, 
which  causes  the  notorious  inflammatory  dis- 
ease, Lyme  disease.  We  showed  last  year  that  mice 
vaccinated  against  an  outer  surface  protein  of 
Borrelia  (OspA)  are  protected  against  infection 
by  Borrelia  burgdorferi.  We  have  now  shown 
that  protection  can  also  be  mediated  by  a  second 
protein  (OspB)  but  not  by  the  flagellar  antigen, 
even  though  a  potent  antibody  response  is  ob- 
tained to  this  protein. 

For  such  a  vaccine  to  work  it  must  be  eff'ective 
against  most,  if  not  all,  strains  of  the  infectious 
agent  and  be  effective  against  a  natural  challenge. 
Since  the  natural  vector  is  the  deer  tick,  we  also 
infected  laboratory  mice  with  B.  burgdorferi  by 
placing  infected  ticks  on  these  mice.  Encourag- 
ingly, mice  vaccinated  with  either  OspA  or  OspB 
were  protected;  furthermore,  spirochetes  in  the 
gut  of  ticks  feeding  upon  vaccinated  mice  were 
eliminated,  whereas  they  were  unaffected  in  non- 
vaccinated  mice.  This  shows  an  additional  poten- 
tial benefit  of  the  vaccine,  which  is  to  eliminate 
spirochetes  from  the  vector.  We  are  now  con- 
sidering strategies  for  the  eradication  of  the  spiro- 
chetes in  the  wild  population  of  ticks  by  a  related 
approach. 


138 


Biophysical  Genetics  of  Protein  Structure 
and  Folding 


Robert  O.  Fox,  Ph.D. — Associate  Investigator 

Dr.  Fox  is  also  Associate  Professor  of  Molecular  Biophysics  and  Biochemistry  at  the  Yale  University  School 
of  Medicine.  He  received  his  B.S.  degree  in  biochemistry  from  the  University  of  Pittsburgh  and  his  M.Phil, 
and  Ph.D.  degrees  in  molecular  biophysics  and  biochemistry  from  Yale  University  while  working  with 
Frederic  Richards  in  the  area  of  x-ray  crystallography.  He  carried  out  postdoctoral  studies  at  Yale  University 
in  protein  engineering  with  Nigel  Grindley  and  studied  protein  folding  using  NMR  spectroscopy 
with  Christopher  Dobson  at  Oxford  University  as  a  Fellow  of  the  Jane  Coffin  Childs  Memorial  Fund 
for  Medical  Research.  Before  moving  to  Yale,  Dr.  Fox  was  Assistant  Professor  in  the  Department 
of  Cell  Biology  at  Stanford  University  Medical  School. 


ALTHOUGH  the  information  that  directs  the 
folding  of  a  protein  molecule  into  a  defined 
three-dimensional  structure  is  genetically  en- 
coded, the  mechanisms  and  pathways  of  the  fold- 
ing process  are  poorly  understood.  One  approach 
to  this  problem  is  an  analysis  of  partially  struc- 
tured folding  intermediates,  combined  with  a 
mutational  analysis.  We  use  nuclear  magnetic  res- 
onance (NMR)  spectroscopy  and  chemical  meth- 
ods to  probe  for  structural  and  kinetic  interme- 
diates in  the  folding  process. 

Many  polypeptide  sequences  adopt  a  common 
folded  motif,  but  they  frequently  differ  in  the  de- 
tailed arrangement  or  conformation  of  structural 
elements  in  ways  that  are  functionally  significant. 
Certain  loops  of  the  immunoglobulins  (antibod- 
ies) are  examples.  We  are  using  staphylococcal 
nuclease  as  a  model  protein  system  to  understand 
how  the  amino  acid  sequence  of  a  secondary 
structural  element  dictates  its  detailed  conforma- 
tion in  the  context  of  a  folded  protein  molecule. 
We  combine  a  number  of  methodologies  in  these 
studies,  including  x-ray  crystallography,  NMR 
spectroscopy,  and  molecular  biology. 

Mapping  Structure  in  the  Unfolded  State 
of  Proteins 

Protein  molecules  in  the  unfolded  and  molten 
globule  states  are  often  more  compact  than 
would  be  expected  for  a  true  random-coil  confor- 
mation. If  this  conformational  bias  is  toward  that 
of  the  folded  structure,  it  may  explain  the  rapid 
rate  at  which  proteins  fold.  We  have  developed  a 
chemical  approach  to  map  close  contacts  be- 
tween a  variable-reporter  residue  site  and  all 
other  residues  of  a  protein  chain  in  these  states. 
This  approach  is  being  used  to  investigate  staphy- 
lococcal nuclease  variants,  nuclease  fragments, 
and  the  molten  globule  state  of  myoglobin.  A  po- 
lar chelator  has  been  designed  and  synthesized 
that  can  be  specifically  attached  to  a  cysteine  resi- 
due engineered  into  the  protein  chain.  When  this 
chelator  is  loaded  with  iron  and  the  reaction  is 
initiated  with  a  reducing  agent,  hydroxyl  radicals 


and  other  reactive  oxygen  species  are  generated; 
these  in  turn  cleave  peptide  bonds  at  positions  in 
the  protein  chain  in  proximity  to  the  chelator. 
The  cleavage  sites  can  be  determined  by  peptide 
mapping  and  protein  sequencing.  The  reagent 
cleaves  native  proteins  at  a  number  of  solvent- 
accessible  sites  close  to  the  site  of  attachment. 
The  reagent  has  also  been  used  to  map  the  prox- 
imity of  several  residues  of  76  resolvase  to  its 
DNA-binding  sites. 

Analysis  of  Protein  Folding  Using  NMR 
Spectroscopy 

Protein  molecules  are  generally  thought  to 
adopt  a  final  tertiary  structure  where  all  back- 
bone and  side  chain  conformations  and  tertiary 
contacts  are  within  local  energy  minima.  Several 
well-refined  protein  molecules  display  excep- 
tions to  this  view,  where  significant  strain  is  in- 
duced at  a  particular  point  in  the  structure, 
usually  involving  a  residue  in  the  enzyme  ac- 
tive site.  Examples  include  c/s-peptide  bonds, 
eclipsed  side  chain  rotamers,  and  energetically 
unfavorable  van  der  Waals  contacts.  How  a  pro- 
tein imposes  the  stress  needed  to  favor  locally 
strained  conformations  remains  unclear. 

Staphylococcal  nuclease  provides  an  excellent 
system  to  examine  the  relationship  between 
stress  and  strain  in  a  globular  protein.  Residues 
115-118  adopt  a  type  Via  /3-turn,  containing  a 
c/5-peptide  bond,  forming  one  wall  of  the  nu- 
cleotide-binding  pocket  of  the  active  site.  Al- 
though the  conformation  of  the  Lysl  l6-Prol  17 
peptide  bond  of  this  jS-turn  is  predominantly  in 
the  c/5-configuration,  the  equilibrium  between 
cis-  and  ^ra«5-conformers  can  be  monitored  by 
NMR  spectroscopy.  Site-directed  mutants  within 
this  type  Via  ;8-turn  have  been  examined  by  NMR 
spectroscopy  and  x-ray  crystallography.  Limiting 
the  backbone  conformational  space  available  to 
the  residue  preceding  the  cw-proline  contributes 
to  the  stress  favoring  the  strained  c/5-peptide 
bond  conformation.  We  have  begun  a  collabora- 
tion with  Axel  Brunger  (HHMl,  Yale  University) 


139 


Biophysical  Genetics  of  Protein  Structure  and  Folding 


to  simulate  this  system  using  molecular  dynamics 
techniques. 

A  structural  analysis  of  early  protein-folding  in- 
termediates has  been  difficult  because  of  their 
transient  nature.  Amide  hydrogen  exchange  has 
been  used  by  others  to  examine  the  appearance  of 
hydrogen-bonded  protein-folding  intermediates 
for  a  number  of  proteins.  We  have  begun  to  use 
this  technique  to  examine  early  folding  interme- 
diates of  a  staphylococcal  nuclease  variant.  Par- 
tial protection  of  amides  occurs  early  in  the  fold- 
ing process,  despite  the  long  overall  folding  time 
for  nuclease. 

Genetic  Analysis  of  a  /S-Turn 

A  sharp  change  in  the  trajectory  of  a  polypep- 
tide chain  between  secondary  structure  elements 
in  a  globular  protein  has  been  defined  as  a  reverse 
turn  or  j8-turn.  These  structures  occur  in  a  num- 
ber of  defined  geometric  types  and  frequently 
contribute  side  chains  to  the  active  site  of  the 
enzymes,  such  as  staphylococcal  nuclease,  or  the 
combining  site  of  binding  proteins,  such  as  the 
immunoglobulins.  We  wish  to  determine  the  se- 
quence requirements  for  the  formation  of  differ- 
ent /3-turn  types  to  better  understand  the  detailed 
structure  of  globular  proteins  and  to  define  de- 
sign principles  for  protein  engineering. 

We  have  developed  a  genetic  approach  to  de- 
termine which  amino  acid  sequences  are  consis- 
tent with  a  particular  |S-turn  structure  in  staphylo- 
coccal nuclease.  Each  member  of  our  gene 
library  contains  a  unique  sequence  at  this  jS-turn. 


Only  a  small  fraction  of  the  sequences  examined 
are  consistent  with  an  enzymatically  active  and 
stable  protein  in  Escherichia  coli.  The  |8-turn 
under  consideration  is  well  removed  from  the  ac- 
tive site,  suggesting  that  the  modulation  in  the 
observed  enzyme  activity  is  due  to  changes  in  the 
stability  of  the  protein.  There  are  strong  biases  in 
the  amino  acids  occurring  at  each  position  in  the 
i8-turn.  Recently,  a  statistical  analysis  of  these  data 
has  led  to  a  predictive  model  for  this  /3-turn  type 
in  all  globular  proteins.  This  approach  may  be 
useful  in  defining  other  sequence-secondary 
structure  relationships.  Results  of  this  and  related 
experiments  should  provide  insight  into  the  rela- 
tionship between  amino  acid  sequence  and  struc- 
ture required  for  the  rational  design  and  engineer- 
ing of  protein  molecules. 

Structural  Studies  of  Trypanosome 
Calmodulin 

Calmodulin  serves  as  a  calcium-dependent  reg- 
ulatory subunit  of  a  variety  of  cytoskeletal  pro- 
teins and  cytoplasmic  enzymes,  including  a  num- 
ber of  protein  kinases.  The  novel  structure  of  rat 
calmodulin  was  determined  in  the  laboratory  of 
Charles  Bugg.  The  protein  is  composed  of  two 
largely  helical  domains,  each  with  two  calcium- 
binding  sites,  separated  by  an  extended  solvent- 
exposed  a-helix.  We  have  obtained  crystals  of 
calmodulin  from  Trypanosoma  brucei  rhode- 
siense,  in  collaboration  with  Curtis  Patton  (Yale 
University).  The  crystal  structure  in  progress 
should  serve  as  the  basis  for  rational  drug  design 
against  this  and  related  organisms. 


140 


Molecular  Basis  of  Genetic  Diseases 
and  Chromosome  Mapping 


Uta  Francke,  M.D. — Investigator 

Dr.  Francke  is  also  Professor  of  Genetics  and  Pediatrics  at  Stanford  University  School  of  Medicine.  She 
received  her  M.D.  degree  from  the  University  of  Munich,  Germany,  trained  in  pediatrics  at  Los  Angeles 
Children 's  Hospital,  and  carried  out  postdoctoral  research  and  clinical  training  in  medical  genetics 
at  the  University  of  California  (Los  Angeles  and  San  Diego).  Before  moving  to  Stanford,  Dr.  Francke 
was  Professor  of  Human  Genetics  and  Pediatrics  at  Yale  University  School  of  Medicine. 


THE  genetic  maps  of  humans  and  the  mouse 
are  undergoing  rapid  growth  and  develop- 
ment. Through  the  efforts  of  many  laboratories, 
including  ours,  several  hundred  homologous 
genes  have  been  mapped  in  both  species,  and 
over  60  chromosome  regions  have  been  delin- 
eated that  contain  conserved  groups  of  genes. 
Thus  it  has  become  possible,  after  mapping  a 
gene  in  one  species,  to  predict  the  location  of  its 
homologue  in  the  other.  Comparative  mapping 
information  is  used  to  evaluate  the  possibility  of  a 
mouse  mutation  being  a  true  model  of  a  human 
genetic  disorder. 

Our  laboratory  is  employing  in  situ  hybridiza- 
tion, with  multicolor  nonradioactive  detection  of 
chromosomal  signals,  as  well  as  somatic  cell  ge- 
netic approaches,  to  locate  cloned  genes  of 
known  function  on  human  and  mouse  chromo- 
somes. We  are  using  this  information  to  define 
candidate  genes  for  human  inherited  disorders  or 
for  phenotypic  mutations  in  mice  and  to  further 
delineate  regions  that  contain  homologous  genes 
in  both  species.  Our  goal  is  to  identify  genes  in- 
volved in  producing  phenotypic  abnormalities 
in  chromosomal  imbalance  syndromes  and  in  in- 
herited disorders,  to  understand  their  function, 
and  to  devise  precise  diagnostic  tests  and  rational 
treatment  strategies. 

Search  for  Genes  Involved  in  Diseases 

In  collaboration  with  the  laboratory  of  Eric 
Shooter,  Stanford  University,  we  have  mapped  a 
peripheral  myelin  protein  gene,  identified  by 
others  as  having  a  growth  arrest  function,  to 
mouse  chromosome  1 1  and  human  chromosome 
17.  It  thus  became  a  candidate  gene  for  involve- 
ment in  the  mouse  mutation  Trembler  and  the 
inherited  human  neuropathy  Charcot-Marie- 
Tooth  disease,  type  lA.  Point  mutations  in  this 
gene  were  indeed  demonstrated  in  Trembler 
mice,  and  the  work  on  the  human  disorder  is  in 
progress. 

In  collaboration  with  Stuart  Lefif  and  Tim  Don- 
Ion,  also  at  Stanford,  we  have  assigned  a  gene 
whose  product  is  involved  in  mRNA-processing 
(splicing)  events  to  the  region  of  human  chromo- 


some 15  that  is  commonly  deleted  in  patients 
with  the  Prader-Willi  syndrome.  In  this  deletion 
syndrome,  hypotonia,  hypogonadism,  mental  re- 
tardation, and  obesity  due  to  lack  of  appetite  con- 
trol are  associated  with  often  submicroscopic 
(micro-)  deletions  of  region  15qll.2-ql3.  Since 
no  gene  of  known  function  has  yet  been  mapped 
to  the  smallest  deletion  overlap  region,  our  as- 
signment of  a  gene  to  this  region  makes  it  a  candi- 
date for  contributing  to  the  microdeletion  pheno- 
type.  It  is  well  established  that  in  Prader-Willi 
syndrome  the  chromosome  with  the  deletion  is 
paternally  derived,  while  the  maternally  derived 
homologous  genes  do  not  appear  to  be  expressed 
(imprinting) .  If  we  can  show  that  our  candidate 
gene  is  also  imprinted  on  the  maternally  derived 
chromosome,  it  would  support  the  hypothesis 
that  this  gene  contributes  to  the  deletion 
phenotype. 

While  the  classical  forms  of  X-linked-recessive 
progressive  muscular  dystrophy  (Duchenne  and 
Becker  types)  are  due  to  deletions  or  mutations  in 
the  dystrophin  gene  on  Xp21,  there  is  a  distinct 
autosomal  recessive  form  of  muscular  dystrophy, 
affecting  both  sexes  and  clinically  resembling 
Duchenne  muscular  dystrophy,  for  which  the  de- 
fect is  unknown.  We  have  collected  and  studied 
several  families  with  more  than  one  affected  indi- 
vidual. The  dystrophin-like  gene  on  chromosome 
6  has  been  excluded  as  a  candidate,  and  linkage 
to  other  chromosomal  sites  is  being  tested.  We 
are  also  collaborating  with  Kevin  Campbell 
(HHMI,  University  of  Iowa,  Iowa  City),  whose 
laboratory  has  characterized  and  isolated  a  dys- 
trophin-associated  complex  of  glycoproteins  lo- 
cated at  the  sarcolemma  that  interacts  with  dys- 
trophin. We  have  begun  to  map  genes  for  these 
proteins  to  chromosomal  sites  as  a  prerequisite 
for  testing  them  as  possible  candidates  for  in- 
volvement in  autosomal  childhood-onset  progres- 
sive muscular  dystrophy. 

Search  for  Mutations  in  Candidate  Genes 

In  order  to  find  mutations  in  a  candidate  gene, 
we  are  employing  screening  methods  in  which 
the  gene  is  amplified  in  small  portions  of  DNA  of 


141 


Molecular  Basis  of  Genetic  Diseases  and  Chromosome  Mapping 


an  obligate  heterozygote.  The  amplified  DNA  du- 
plexes are  screened  for  the  presence  of  base  dif- 
ferences by  denaturing  gradient  gel  electrophore- 
sis (DGGE)  or  single-strand  conformational 
polymorphism  (SSCP)  studies.  Amplified  exons 
that  appear  to  have  base  differences,  identified  by 
altered  melting  behavior  or  conformational  prop- 
erties, are  sequenced  to  reveal  the  precise  base 
differences. 

In  a  rare  form  of  inherited  dv^^arfism,  the  Laron 
syndrome,  individuals  are  unresponsive  to 
growth  hormone,  lack  the  activity  of  a  specific 
growth  hormone-binding  protein  in  serum,  and 
have  low  levels  of  insulin-like  growth  factor  I. 
The  growth  hormone  receptor  is  a  likely  candi- 
date gene  for  mutations  leading  to  this  disorder. 
The  receptor  gene  consists  of  10  exons  that  are 
amplified  individually  from  flanking  primers.  In 
a  study  of  38  Laron  syndrome  individuals  from  a 
highly  inbred  population  in  the  mountains  of 
southern  Ecuador,  we  identified  a  base  substitu- 
tion in  the  extracellular  domain  of  the  receptor 
that  does  not  alter  the  encoded  amino  acid  but 
creates  a  new  donor  splice  site.  The  resulting  mu- 
tant mRNA  is  deleted  for  24  bases,  which  predicts 
a  protein  lacking  eight  amino  acids  in  a  highly 
conserved  region  of  the  molecule.  The  location 
of  the  deleted  eight  amino  acids  does  not  involve 
the  hormone-binding  or  receptor  dimerization 
sites.  The  most  likely  effect  of  this  mutation  is  a 
poorly  folded,  unstable  protein  that  might  de- 
grade rapidly.  Further  studies  are  under  way  to 
test  this  prediction. 

The  same  strategy  is  now  being  pursued  to  find 
mutations  in  the  growth  hormone  receptor  gene 
in  other  patients  with  Laron  syndrome  who  are 
from  different  ethnic  and  racial  populations.  The 
Ecuadorean  growth  hormone  receptor  mutation 
represents  the  first  instance  of  a  disease-causing 
base  substitution  that  acts  by  creating  a  new  do- 
nor splice  site  and  leads  to  its  exclusive  use  while 
the  original  site  is  unaltered. 

The  gonadotropin-releasing  hormone  (GnRH) 
is  produced  in  specialized  cells  in  the  hypothala- 
mus, travels  to  the  pituitary  gland,  and  causes  the 
release  of  the  gonadotropic  follicle-stimulating 
hormone  (FSH)  and  luteinizing  hormone  (LH). 
We  previously  mapped  the  gene  to  the  short  arm 
of  human  chromosome  8.  In  the  mouse,  partial 
deletion  of  this  gene  causes  a  recessive  pheno- 
type,  termed  hypogonadal,  leading  to  lack  of  sex- 
ual development.  In  human  families  with  in- 
herited incomplete  sexual  development  due  to 


low  levels  of  gonadotropin  hormones,  the  GnRH 
gene  is  a  candidate  for  involvement,  as  in  the 
mouse  model.  We  have  studied  a  family  with 
three  affected  siblings  that  show  a  sequence 
polymorphism  in  the  signal  peptide  of  the  GnRH 
gene,  but  have  found  that  this  polymorphism 
does  not  cosegregate  with  the  disease  phenotype, 
thus  excluding  a  GnRH  gene  defect  in  this  partic- 
ular family. 

The  Marfan  syndrome  is  an  autosomal  domi- 
nant condition  that  involves  abnormalities  in 
elastic  tissue  of  the  aorta  and  the  fibrils  that  sus- 
pend the  optic  lens  and  in  connective  tissue  of 
many  organs,  including  bones,  tendons,  and  lung. 
The  estimated  incidence  is  1  in  10,000,  and  clin- 
ical severity  is  highly  variable.  Dissection  and 
rupture  of  the  aorta  is  a  life-threatening  compli- 
cation. During  the  past  year,  linkage  studies  in 
other  laboratories  have  pinpointed  the  site  of  the 
Marfan  gene  on  human  chromosome  15,  and  co- 
incidentally  the  gene  encoding  fibrillin,  the  ma- 
jor microfibrillar  protein,  was  also  mapped  to  the 
same  location.  Subsequently,  a  mutation  in  the 
fibrillin  gene  was  documented  in  two  unrelated 
Marfan  patients. 

Stanford  University  is  a  center  for  Marfan  diag- 
nosis, treatment,  and  research.  Over  the  last  three 
years,  we  have  established  a  broad-based  research 
program  that  involves  a  detailed  clinical  and  ge- 
nealogical database  and  the  collection  of  blood 
and  tissue  samples  from  affected  persons  and  fam- 
ily members.  We  hypothesize  that  a  mutation  in 
the  fibrillin  gene  causes  the  Marfan  phenotype  by 
producing  an  abnormal  protein  that  when  in- 
corporated into  the  microfibrils  causes  their 
instability. 

We  are  now  screening  the  mRNA  produced  by 
skin  or  aortic  fibroblasts  from  patients  with  clas- 
sical Marfan  syndrome  and  related  connective  tis- 
sue disorders.  After  reverse  transcription  of  the 
10,000-bp  fibrillin  mRNA,  small  overlapping 
sections  are  amplified  and  screened  for  point  mu- 
tations by  DGGE  and  SSCP  studies.  Our  goal  is  not 
only  to  develop  a  molecular  diagnostic  test  for  a 
disorder  that  is  clinically  often  difficult  to  diag- 
nose, but  also  to  gain  insight  into  the  relation- 
ships between  particular  types  of  mutations  and 
the  phenotypes  they  produce. 

Since  there  are  fibrillin-related  genes  else- 
where in  the  genome  and  many  connective  tissue 
disorders  that  overlap  the  Marfan  phenotype  to 
some  degree,  this  will  be  a  large  fruitful  field  of 
investigation. 


142 


Molecular  Biology  of  Obesity  and  Diabetes 


Jeffrey  M.  Friedman,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Friedman  is  also  Associate  Professor  and  Head  of  Laboratory  at  the  Rockefeller  University.  He  received 
his  B.S.  and  M.D.  degrees  from  the  Renssalaer  Polytechnic  Institute-Albany  Medical  College.  After 
completing  a  residency  in  internal  medicine  at  Albany  Medical  College  and  a  gastroenterology  fellowship 
at  Cornell  University  Medical  College,  he  enrolled  in  the  graduate  program  at  Rockefeller,  where  he 
received  his  Ph.D.  degree  in  molecular  biology. 


EXTENSIVE  studies  of  humans  and  other  organ- 
isms have  suggested  that  body  weight,  body 
composition  (percent  body  fat) ,  and  food  intake 
are  under  strict  physiological  control.  The  "set 
point"  hypothesis  holds  that  both  the  intake  and 
expenditure  of  energy  are  physiologically  regu- 
lated in  the  individual  to  maintain  a  predeter- 
mined body  weight.  Implicit  in  this  hypothesis  is 
the  notion  that  signal  molecules  reflecting  the 
nutritional  state  are  synthesized  in  the  periphery 
and  sensed  by  brain  centers  whose  appropriate 
response  is  set  to  stabilize  body  weight. 

Studies  in  which  the  rodent  brain  has  been  se- 
lectively lesioned  suggest  that  this  feeding  con- 
trol center  resides,  at  least  in  part,  in  the  hypothal- 
amus. However,  the  site  of  synthesis  of  the 
molecules  that  signal  nutritional  state  is  un- 
known, though  fat  cells  or  cells  of  the  gastrointes- 
tinal tract  have  been  proposed.  Knowledge  of  the 
site  of  synthesis  and  the  molecular  nature  of  sig- 
nals aff'ecting  the  control  of  appetite  and  body 
composition  could  have  important  implications 
for  our  understanding  of  nutritional  disorders. 

To  learn  more  about  these  signaling  mecha- 
nisms, we  have  been  taking  a  variety  of  ap- 
proaches, both  genetic  and  molecular,  to  study 
the  role  of  specific  gene  products  in  the  control 
systems. 

Molecular  Basis  of  Obesity  in  oh/ ob 
and  db/db  Mice* 

If  one  wishes  to  understand  the  basis  for  differ- 
ences in  the  complicated  system  of  energy  ho- 
meostasis, there  are  a  number  of  experimental 
advantages  to  studying  mutant  mice,  including 
the  ability  to  control  for  environment  and  to  set 
up  genetic  crosses.  For  these  reasons,  we  have 
begun  a  study  of  mice  carrying  recessive  muta- 
tions that  result  in  profound  obesity.  At  least  four 
obesity-causing  mutations  are  available:  obese 
(ob),  diabetes  (db),fat  (fat),  and  tubby  (tub). 
In  each  case,  a  mouse  becomes  obese  because  of  a 
single-gene  defect.  We  have  focused  on  the  ob 
and  db  mutations  for  several  reasons.  The  mutant 
mice  become  very  obese,  often  three  times  nor- 
mal weight;  and  the  obese  phenotype  in  mutant 


animals,  as  in  humans,  appears  to  result  from 
both  increased  food  intake  and  diminished  en- 
ergy expenditure.  Furthermore,  Douglas  Cole- 
man at  the  Jackson  Laboratory  has  suggested  that 
ob  mice  lack  a  circulating  factor  that  suppresses 
appetite  and  that  db  mice,  which  are  unable  to 
respond  to  this  factor,  may  lack  its  receptor. 

Current  techniques  in  molecular  genetics, 
such  as  Southern  blots  and  chromosome  walking, 
make  it  possible  to  clone  genes  such  as  ob  and 
db  whose  function  is  known  on  the  basis  of  a  mu- 
tant phenotype  but  whose  gene  product  is  un- 
known. This  approach,  called  positional  cloning, 
utilizes  restriction  fragment  length  polymor- 
phisms (RFLPs) — genetic  markers  defined  by  spe- 
cific cloned  pieces  of  DNA.  These  markers  define 
genetic  differences  in  inbred  mouse  strains.  The 
first  step  in  attempts  to  clone  a  mutant  gene  uti- 
lizes genetic  crosses  between  normal  and  obese 
(or  diabetic)  mice.  Inheritance  of  the  obese  phe- 
notype is  compared  in  individual  animals  with 
that  of  individual  RFLPs,  which  if  inherited  along 
with  the  obese  phenotype,  are  said  to  be  linked 
genetically  to  the  obesity  gene. 

By  performing  this  analysis  on  several  thousand 
mice  with  several  dozen  different  RFLPs,  we  have 
been  able  to  identify  a  series  of  DNA  probes  that 
are  very  tightly  linked  to  the  ob  and  db  genes. 
RFLPs  linked  genetically  to  these  mutations  are  in 
physical  proximity  to  ob  and  db  and  can  be  used 
as  starting  points  to  characterize  the  adjacent 
DNA  and  clone  the  mutant  genes. 

In  the  case  of  ob  mice,  we  first  used  three  dif- 
ferent RFLPs  to  generate  a  detailed  genetic  map 
around  the  ob  locus.  The  genes  represented  were 
the  met  oncogene  carboxypeptidase  A,  which 
codes  for  a  pancreatic  enzyme;  the  trp  gene;  and 
the  cystic  fibrosis  gene.  Similarly,  the  mouse  db 
gene  has  been  mapped  relative  to  RFLPs  for  inter- 
feron-a  and  a  complement  gene.  We  have  also 
found  that  the  rat  obesity  mutation  fatty  (fa)  is 
also  flanked  by  the  genes  for  interferon-a  and 


*  Studies  aimed  at  cloning  the  mutant  obesity  genes  ob  and 
db  from  mice  have  been  supported  by  the  National  Institute 
of  Diabetes  and  Digestive  and  Kidney  Diseases. 


143 


Molecular  Biology  of  Obesity  and  Diabetes 


complement.  These  data  suggest  that  mutations 
in  the  same  gene  can  cause  obesity  in  two  differ- 
ent rodent  species  and  raise  the  possibility  that 
similar  mutations  can  cause  obesity  in  humans. 
We  are  endeavoring  to  test  this  possibility,  in  col- 
laboration with  Rudolph  Leibel,  by  using  the  in- 
terferon-a  and  complement  genes  to  characterize 
human  families  with  a  high  incidence  of  obesity. 

To  identify  other  RFLPs  that  are  more  tightly 
linked  than  the  probes  mentioned  above,  we  have 
used  the  technique  of  chromosomal  microdissec- 
tion, in  which  small  slices  of  individual  chromo- 
somes are  dissected  and  cloned.  Separate  libraries 
have  been  made  from  proximal  chromosome  6, 
where  ob  maps,  and  from  mid  chromosome  4, 
where  db  maps.  Two  probes  have  been  isolated 
from  the  chromosome  4  library  that  flank  db  and 
are  about  1  cM  apart.  Similarly,  two  probes  that 
flank  ob  and  are  about  0.2  cM  apart  were  isolated 
from  the  chromosome  6  library.  A  genetic  dis- 
tance of  0.2  cM  corresponds  to  about  400,000 
base  pairs  of  DNA. 

We  are  currently  attempting  to  clone  the  DNA 
near  these  probes  by  using  techniques  such  as 
pulsed-field  gel  electrophoresis  and  cloning  of 
large  fragments  in  yeast  artificial  chromosomes 
(YACs).  YACs  of  300,000  to  1  million  base  pairs 
have  already  been  isolated  for  each  of  the  probes 
that  flank  ob,  and  the  search  for  the  gene  in  these 
artificial  chromosomes  should  begin  shortly.  The 
cloning  of  these  genes  should  further  our  under- 
standing of  the  mechanisms  that  control  food  in- 
take and  body  weight. 

Polygenic  Inheritance  of  Type  II  Diabetes 

The  mice  carrying  the  ob  and  db  obesity  muta- 
tions develop  diabetes  of  adult  onset  that  is  quite 
similar  to  type  II  diabetes  in  humans.  However, 
differences  in  the  type  II  phenotype  are  seen  in 
mutant  mice,  depending  on  the  strain  carrying 
the  mutation.  C57BL/6J  ob/ob  mice  develop  a 
mild  insulin-resistant  diabetes  with  high  levels  of 
plasma  concentration  of  glucose  and  insulin.  In 
contrast,  DBA/2J  mice  develop  a  severe  diabetes 
characterized  by  very  high  plasma  levels  of  glu- 
cose and  relatively  low  plasma  insulin  concentra- 
tion. These  data  suggest  that  genetic  differences 
in  insulin  sensitivity  and  output  from  pancreatic 
/3-ceIls  can  influence  the  severity  of  the  diabetes 
in  genetically  obese  mice  of  different  inbred 
strains. 

Further  analyses  of  these  data  have  suggested 
that  the  genetic  differences  are  a  result  of  poly- 


genic inheritance — genetic  variation  in  several 
genes.  Advances  in  molecular  genetics  now  make 
it  possible  to  dissect  polygenic  traits  (such  as  dia- 
betes in  mice)  into  single  gene  components.  This 
requires  that  a  collection  of  genetic  markers, 
such  as  RFLPs,  be  available  for  genotyping  the 
diabetic  animals.  To  facilitate  the  analysis  of  poly- 
genic traits,  we  have  helped  Eric  Lander  and  Wil- 
liam Dietrich  (Massachusetts  Institute  of  Technol- 
ogy) generate  a  linkage  map  of  the  mouse 
genome,  using  a  new  type  of  genetic  marker 
known  as  the  SSR  (simple  sequence  repeat). 
These  markers  greatly  facilitate  genetic  mapping 
experiments.  We  are  now  analyzing  obese  prog- 
eny of  various  genetic  crosses  with  these  SSR 
markers  to  identify  novel  genes  that  predispose 
ob  animals  to  diabetes. 

Regulation,  Function,  and  Expression 
of  Cholecystokinin  in  Human  Tumors 

The  hormone  cholecystokinin  (CCK)  was  origi- 
nally found  in  the  small  intestine  by  virtue  of  its 
ability  to  stimulate  gallbladder  contraction  and 
pancreatic  secretion  in  response  to  feeding.  High 
levels  of  CCK  have  also  been  found  in  neurons  of 
the  mammalian  brain,  where  it  functions  as  a  neu- 
rotransmitter. The  first  demonstration  that  CCK 
could  affect  behavior  was  reported  by  Gerry  Smith 
and  Dick  Gibbs,  who  showed  that  peripherally 
administered  CCK  had  an  appetite-suppressing 
effect  on  rats.  It  has  also  been  demonstrated  that 
CCK  antagonists  increase  feeding  behavior  in  ro- 
dents. These  observations  suggest  that  the  regula- 
tion and  function  of  this  gene  is  important  in  the 
control  of  appetite. 

At  present,  we  are  using  a  variety  of  techniques 
to  explore  CCK's  function(s).  One  of  our  first 
objectives  was  to  determine  whether  any  human 
tumors  associated  with  weight  loss  overexpress 
this  peptide,  and  we  therefore  began  screening 
tumor  cell  lines  for  CCK  production.  Several  pe- 
diatric tumors  were  found  to  synthesize  the  appe- 
tite depressant,  including  peripheral  neuroepi- 
thelioma (a  rare  nerve  tumor  that  usually 
develops  in  the  chest  wall) ,  Ewing's  sarcoma  of 
bone,  and  rhabdomyosarcoma  (a  malignant  mus- 
cle tumor) .  These  data  suggest  that  measurement 
of  the  CCK  levels  in  the  blood  may  be  of  diagnos- 
tic and  prognostic  value  in  the  management  of 
these  tumors.  Studies  are  being  initiated  to  ascer- 
tain whether  overproduction  of  CCK  in  patients 
with  these  tumors  is  associated  with  excessive 
weight  loss. 


144 


Test-tube  filaments  made  from  genetically  engineered  human  epidermal  keratins.  On  the 
left  are  filaments  from  normal  keratins;  on  the  right,  filaments  from  keratins  with  a 
single  amino  acid  substitution  (arginine  to  cysteine).  This  point  mutation  was  discov- 
ered in  a  patient  with  epidermolysis  bullosa  simplex,  a  disease  involving  abnormalities  in 
the  keratin  filament  network. 

From  Coulombe,  P. A.,  Hutton,  M.E.,  Letai,  A.,  Hebert,  A.,  Paller,  A.S.,  and  Fuchs,  E. 
1991.  Cell  66:1301-131 1.  Copyright©  1991  by  Cell  Press. 


146 


Regulation  of  Keratin  Expression  During 
Differentiation  and  Development  in  Human  Skin 


Elaine  Fuchs,  Ph.D. — Investigator 

Dr.  Fuchs  is  also  Professor  in  the  Departments  of  Molecular  Genetics  and  Cell  Biology  and  of  Biochemistry 
and  Molecular  Biology  at  the  University  of  Chicago.  She  received  her  B.S.  degree  in  chemistry  from  the 
University  of  Illinois  and  her  Ph.D.  degree  in  biochemistry  from  Princeton  University,  where  she  studied 
with  Charles  Gilvarg.  Her  postdoctoral  research  was  done  with  Howard  Green  at  the  Massachusetts 
Institute  of  Technology.  Dr.  Fuchs  counts  among  her  honors  the  R.R.  Bensely  Award  from  the  American 
Association  of  Anatomists. 


THE  long-range  objective  of  our  research  is  to 
understand  the  biochemical  mechanisms  that 
operate  and  regulate  the  expression  of  human 
genes  during  development  and  differentiation  in 
skin.  Present  knowledge  of  the  biochemistry  of 
human  skin  and  its  diseases  is  limited.  Although 
dermatologists  have  always  directed  their  interest 
toward  human  skin  biology  and  skin  diseases,  the 
field  of  molecular  biology  has  only  recently  ap- 
proached a  level  of  understanding  that  permits 
the  complex  biochemistry  of  human  skin  to  be 
explored.  A  major  factor  facilitating  such  studies 
is  the  ability  to  grow  human  skin  cells  in  tissue 
culture,  including  epidermal  cells,  dermal  fibro- 
blasts, melanocytes,  and  dermal  papillae  cells. 
The  recent  development  of  technology  for  target- 
ing foreign  genes  to  transgenic  animals'  skin  has 
provided  a  valuable  in  vivo  model  system  for  the 
study  of  genetic  skin  diseases  and  skin  cancers. 

Much  of  our  research  on  human  skin  has  fo- 
cused on  the  epidermis,  which  comprises  about 
20  cell  layers  whose  outermost  is  the  skin  sur- 
face. Only  the  inner  or  basal  layer  of  the  epider- 
mis is  truly  living  and  undergoes  DNA  synthesis 
and  cell  division.  Under  an  influence  as  yet  un- 
identified, a  basal  cell  ceases  to  divide  and  makes 
a  commitment  to  differentiate  terminally.  As  the 
cell  moves  outward  to  the  skin  surface,  it  under- 
goes a  variety  of  morphological  and  biochemical 
changes.  The  most  pronounced  of  these  is  the  pro- 
duction of  a  dense  network  of  keratin  filaments, 
which  are  tough,  resilient  protein  fibers.  Many 
skin  diseases  of  the  epidermis,  including  psoria- 
sis and  basal  and  squamous  cell  carcinomas,  in- 
volve a  malfunctioning  of  the  differentiative  pro- 
cess that  is  frequently  associated  with  some 
abnormality  in  the  production  or  organization  of 
these  filaments.  Our  investigation  is  focused  on 
the  regulation  of  the  expression  of  keratin  pro- 
teins and  their  genes  in  human  epidermis  and  in 
epidermal  cells  differentiating  in  tissue  culture. 

The  keratins  are  a  group  of  1 0-20  related  pro- 
teins (40-70  kDa)  that  form  the  10-nm  keratin 
filaments  in  the  cytoplasm  of  epidermal  cells. 
Only  a  subset  (typically  2-6)  of  keratins  are  ever 
expressed  at  one  time.  As  a  normal  epidermal  cell 
differentiates,  it  changes  the  subset  of  keratins  it 


makes.  In  addition,  the  cell  increases  its  keratin 
synthesis,  leaving  the  fully  differentiated  epider- 
mal cell  with  85  percent  of  its  total  protein  as 
keratins.  In  diseases  of  the  skin  involving  epider- 
mal hyperproliferation,  including  psoriasis  and 
squamous  cell  carcinomas,  a  new  subset  of  kera- 
tins not  normally  made  in  the  epidermis  is  pro- 
duced, and  can  be  diagnostic. 

A  coordinated  genetic  and  biochemical  ap- 
proach is  necessary  to  determine  the  regulation 
of  the  multiple  keratins  and  to  decipher  their 
structural  and  functional  roles  in  the  differentiat- 
ing epidermal  cell.  A  number  of  years  ago,  we 
showed  that  expression  of  different  subsets  of 
epidermal  keratins  is  due  to  changes  in  the  syn- 
thesis of  different  mRNAs.  We  used  DNA  recombi- 
nant technology  to  show  that  these  mRNAs  are 
encoded  by  about  20  different  genes  of  two  dis- 
tinct types.  Type  I  encodes  small  keratins  (40-53 
kDa);  type  II,  larger  keratins  (53-67  kDa). 

Keratins  are  expressed  as  specific  pairs  of  type 
I  and  II  proteins.  The  basic  subunit  of  keratin  fila- 
ments is  a  heterodimer,  composed  of  the  two 
types.  Approximately  20,000  heterodimers  form 
a  single  10-nm  filament;  the  assembly  process  is 
energy  independent  and  does  not  appear  to  re- 
quire auxiliary  proteins  or  factors.  Using  DNA 
sequencing,  we  determined  the  amino  acid 
sequences  for  several  keratin  pairs.  The  cytoskele- 
tal  architecture  of  keratin  filaments  may  be  specif- 
ically tailored  to  suit  the  particular  structural 
needs  of  each  epidermal  cell  at  various  stages  of 
differentiation  and  development. 

To  determine  the  details  of  the  filament  assem- 
bly process  and  to  investigate  the  interactions  of 
keratin  filaments  with  other  proteins  and  organ- 
elles, we  used  deletion  and  site-directed  muta- 
genesis to  alter  the  coding  sequences  of  K5  and 
Kl4,  the  pair  expressed  in  the  living  cells  of  the 
epidermis.  We  generated  substantial  quantities  of 
keratins  for  filament  assembly  studies,  using  ge- 
netic engineering  to  overexpress  wild  type  and 
mutant  human  keratins  in  bacteria,  which  do  not 
have  keratin.  We  purified  these  keratins  and  iso- 
lated and  examined  the  consequences  of  muta- 
tions and  deletions  on  keratin  filament  assembly 
in  vitro. 


147 


Regulation  of  Keratin  Expression  During  Differentiation  and  Development 
in  Human  Skin 


In  the  past  few  years,  we  identified  those  se- 
quences involved  in  filament  elongation  and 
those  that  are  more  important  for  lateral  associa- 
tions. In  addition,  we  used  gene  transfection  of 
human  tissue  culture  cells  to  examine  the  dy- 
namics of  filament  assembly  in  vivo  and  to  deter- 
mine how  abnormal  expression  of  keratins  in  ma- 
lignant and  hyperproliferating  epidermal  cells 
might  change  their  biology  and  cytoskeletal  ar- 
chitecture. The  results  of  these  studies  have  be- 
gun to  yield  valuable  insights  into  the  complex 
assembly  process  of  keratin  filaments  and  the 
function  they  perform  in  providing  a  tough  skin 
surface. 

We  have  also  been  interested  in  determining 
whether  natural  mutations  in  human  epidermal 
keratin  genes  might  lead  to  genetic  skin  diseases. 
We  had  previously  shown  that  elevated  expres- 
sion of  epidermal  keratin  genes  is  a  relatively  late 
event  in  development  and  that  certain  keratin 
mutants  have  a  deleterious  effect  in  cultured 
cells,  disrupting  the  endogenous  keratin  filament 
network.  In  the  past  two  years,  we  have  made 
transgenic  mice  and  used  epidermal  keratin  pro- 
moters to  target  expression  of  some  of  these  domi- 
nant mutant  keratins  to  the  epidermis. 

Unexpectedly,  the  transgenic  mice  exhibit  a 
phenotype  resembling  that  of  humans  with  epi- 
dermolysis bullosa  simplex  (EBS) ,  a  class  of  blis- 
tering skin  diseases  that  are  dominant,  sometimes 
life-threatening,  and  of  previously  unknown  etiol- 
ogy. Mice  expressing  Kl4  mutants  that  severely 
disrupt  filament  assembly  exhibit  severe  blister- 
ing over  body  trunk  regions — lesions  resembling 
those  of  Dowling  Meara  EBS,  the  severest  form; 
while  mice  expressing  Kl4  mutants  that  mildly 
perturb  the  network  exhibit  blistering  only  over 
paws,  as  in  the  mildest  form  of  EBS,  or  Weber- 
Cockayne,  where  patients  show  blistering  pre- 
dominantly on  their  hands  and  feet.  Thus  we 
were  able  to  demonstrate  that  multiple  mutations 
in  a  single  gene,  namely  K14,  can  give  rise  to 
most  if  not  all  forms  of  EBS,  thus  strongly  suggest- 
ing that  these  diseases  may  be  genetically  linked. 
Further  studies  enabled  us  to  demonstrate  that 
skin  blistering  in  EBS  is  due  to  the  fragility  of 
basal  epidermal  cells  caused  by  an  abnormal  ker- 
atin filament  network,  thereby  compromising  the 
mechanical  strength  of  this  cell  layer. 

The  similarities  between  the  phenotypes  in 
mice  and  humans  prompted  us  to  focus  on  this 
disease.  In  the  past  year  we  characterized  the  Kl  4 
and  K5  mRNAs,  genes  and  proteins  from  two  pa- 


tients with  Dowling  Meara  EBS.  Both  patients 
have  point  mutations  in  an  amino  acid  of  the  Kl4 
protein  that  we  had  previously  shown  to  be  criti- 
cal for  filament  assembly.  When  we  engineered 
and  tested  the  two  mutations  in  our  wild-type 
K14  gene,  we  verified  that  these  defects  are  re- 
sponsible for  the  phenotype  of  the  EBS  patients.  It 
will  take  much  work  to  develop  better  diagnostic 
and  therapeutic  tools  for  EBS,  but  elucidating  the 
genetic  basis  is  an  important  first  step. 

While  EBS  is  a  disease  of  defects  in  the  coding 
portions  of  keratin  genes,  there  are  other  skin  dis- 
eases that  appear  to  arise  from  abnormalities  in 
the  control  of  epidermal  genes  or  genes  that  influ- 
ence growth  and/or  differentiation.  As  a  prerequi- 
site to  investigating  the  bases  for  these  types  of 
diseases,  we  are  1 )  analyzing  the  molecular  mech- 
anisms underlying  the  differential  expression  of 
epidermal  keratin  genes  and  2)  utilizing  our  Kl4 
promoter  to  make  transgenic  mice  that  overex- 
press  various  regulatory  factors — e.g.,  transform- 
ing growth  factor-a  (TGFa),  epidermal  growth 
factor  (EOF),  cytokines,  hormone  receptors,  and 
proto-oncogenes  in  the  epidermis. 

In  the  past  few  years,  we  have  identified  proxi- 
mal and  distal  domains  that  act  synergistically  to 
regulate  expression  of  the  human  Kl4  gene.  Our 
goal  is  to  identify  the  sequences  and  transcription 
factors  involved.  Analysis  of  the  factors  control- 
ling the  cell's  major  structural  genes  should  lead 
us  to  the  factors  determining  keratinocyte  fate. 
Unraveling  the  nature  of  promoter  and  enhancer 
sequences  involved  in  regulating  epidermal 
genes  will  be  important  not  only  for  understand- 
ing epidermal  development  but  also  for  targeting 
products  to  the  epidermis.  In  the  last  year,  we 
used  the  Kl4  promoter/enhancer  to  engineer 
transgenic  mice  that  overexpress  TGFa,  the  major 
autocrine  growth  factor  of  the  epidermis.  Our  re- 
sults have  shown  that  TGFa  plays  an  important 
role  in  controlling  the  thickness  of  the  epidermis 
and  that  certain  features  of  TGFa  overexpression 
are  similar  to  those  of  psoriasis,  a  hyperprolifera- 
tive  skin  disease. 

The  Kl4  promoter/enhancer,  so  effective  in 
transgenic  technology,  should  also  be  useful  for 
drug  therapy.  Because  epidermal  cells  can  be  re- 
moved from  a  patient,  cultured  in  vitro,  and 
grafted  back,  it  should  be  possible  to  introduce 
foreign  genes,  driven  by  the  Kl4  promoter/en- 
hancer, into  the  cultured  epidermal  cells  prior  to 
grafting.  Such  techniques  have  potentially  power- 
ful applications  for  future  medical  research. 


148 


The  Molecular  Basis  of  Viral  Replication 
and  Pathogenesis 


Donald  E.  Ganem,  M.D.  — Associate  Investigator 

Dr.  Ganem  is  also  Professor  of  Microbiology  and  Immunology  and  of  Medicine  at  the  University  of 
California,  San  Francisco.  He  received  an  A.B.  degree  in  biochemistry  from  Harvard  College  and  the  M.D. 
degree  from  Harvard  Medical  School.  Following  his  clinical  training  in  infectious  diseases,  he  did 
postdoctoral  research  training  in  the  laboratory  of  Harold  Varmus  at  UCSF. 


OUR  laboratory  studies  the  molecular  mecha- 
nisms by  which  pathogenic  human  viruses 
infect  the  host  and  cause  disease.  We  are  espe- 
cially interested  in  those  viruses  that  produce 
persistent  infections  and  engender  chronic  pa- 
thology. Several  fundamental  questions  underlie 
our  work:  How  are  persistent  infections  estab- 
lished and  maintained?  What  factors  regulate 
viral  replication  and  spread?  How  does  the  persis- 
tent presence  of  virus  evoke  disease? 

Most  of  our  work  centers  around  the  human 
hepatitis  B  virus  (HBV)  and  its  animal  homo- 
logues.  HBV  is  a  small  DNA  virus  that  replicates 
principally  in  liver  cells  (hepatocytes)  and  pro- 
duces acute  and  chronic  type  B  hepatitis.  Most 
initial  infections  are  transient:  following  a  rela- 
tively brief  period  of  liver  injury  (hepatitis),  the 
immune  system  eliminates  virus  from  the  liver 
and  invokes  lasting  immunity.  However,  5-10 
percent  of  infections  are  not  successfully  elimi- 
nated. In  these  cases,  viral  replication  persists  in 
the  liver  for  the  life  of  the  host,  evoking  various 
degrees  of  chronic  liver  injury  that  can  lead  to 
premature  death  from  liver  failure.  Most  strik- 
ingly, persistence  of  viral  replication  for  several 
decades  enormously  increases  the  risk  of  liver 
cancer. 

There  are  many  reasons  to  be  interested  in  this 
remarkable  infection.  First  is  its  great  public 
health  significance.  Worldwide,  there  are  over 
250  million  chronic  HBV  carriers.  In  large  re- 
gions of  Asia  and  Africa,  10-15  percent  of  all  hu- 
man inhabitants  are  persistently  infected  by  the 
virus.  Second,  the  virus  replication  cycle  is  dis- 
tinctly unusual:  replication  of  the  viral  DNA  ge- 
nome is  accomplished  via  reverse  transcription  of 
an  RNA  intermediate.  The  HBV  life  cycle  is  thus  a 
permuted  version  of  the  retroviral  life  cycle,  and 
a  fuller  understanding  of  its  details  allows  in- 
structive comparisons  to  be  made  with  cognate 
steps  in  retroviral  replication.  More  importantly, 
unraveling  the  mechanism  of  HBV  replication 
should  identify  new  potential  targets  for  antiviral 
therapy.  Finally,  the  nature  of  the  link  between 
chronic  HBV  replication  and  hepatocellular  carci- 
noma represents  one  of  the  great  unsolved  prob- 


lems in  human  cancer  biology,  and  a  deeper  un- 
derstanding of  HBV  persistence  is  expected  to 
provide  new  clues  to  its  solution. 

Our  studies  of  HBV  replication  have  examined 
many  different  steps  in  the  viral  life  cycle.  The 
initial  step  in  all  viral  infections  is  binding  of  the 
virus  to  the  cell  surface  receptor  and  its  entry  into 
the  cell.  Little  is  known  about  this  step  in  HBV 
infection,  but  we  have  begun  to  explore  it  in  a 
convenient  animal  model,  the  duck  hepatitis  B 
virus  (DHBV) .  By  using  recombinant  DHBV  sur- 
face proteins  to  look  for  cellular  proteins  that 
will  bind  them,  we  have  identified  a  single  host 
glycoprotein  that  will  interact  with  DHBV  parti- 
cles with  high  affinity  and  specificity.  Such  mole- 
cules are  good  candidates  for  the  receptor,  and 
we  are  actively  attempting  to  clone  the  gene  for 
this  host  protein  to  allow  its  more-detailed 
characterization. 

Next,  internalized  virus  particles  must  be  deliv- 
ered to  the  nucleus,  where  their  DNA  genome  is 
transcribed.  This  is  perhaps  the  most  poorly  un- 
derstood of  all  reactions  in  virology.  For  no  virus 
has  the  cellular  machinery  involved  in  this  essen- 
tial step  been  identified.  Recently,  we  discovered 
that  drugs  affecting  the  integrity  of  cellular  mi- 
crotubules block  this  step,  implying  that  these 
structures  are  involved  in  the  intracellular  trans- 
port of  subviral  particles.  This  raises  the  exciting 
possibility  that  such  particles  might  also  be  used 
to  identify  host  proteins  that  recognize  them  and 
perhaps  bind  not  only  virus  particles  but  normal 
cellular  components  (e.g.,  organelles)  to  cyto- 
skeletal  motors  for  transport  within  the  cell. 

Once  in  the  nucleus,  viral  genes  are  tran- 
scribed into  RNA  to  be  transferred  to  the  cyto- 
plasm for  translation.  Following  translation,  prog- 
eny particles  assemble  in  the  cytoplasm.  In  this 
remarkable  reaction,  viral  RNA  is  packaged  into 
subviral  particles  along  with  the  viral  reverse 
transcriptase.  Surprisingly,  this  RNA  packaging 
process  requires  the  participation  of  the  reverse 
transcriptase  itself.  To  understand  this  key  step 
better,  we  are  currently  attempting  to  reproduce 
this  RNA  recognition  in  vitro,  using  recombinant 
reverse  transcriptase  and  cloned  viral  RNA.  Sim- 


149 


The  Molecular  Basis  of  Viral  Replication  and  Pathogenesis 


ilar  in  vitro  reconstruction  reactions  are  being 
attempted  for  the  reverse  transcription  reaction 
itself.  This  enormously  complex  reaction  gener- 
ates the  duplex  DNA  genome  from  its  RNA  pre- 
cursor. By  fully  characterizing  the  molecules  that 
catalyze  the  reaction  and  the  intermediates 
through  which  it  proceeds,  we  hope  to  identify 
novel  targets  for  drugs  that  would  be  useful  in 
interrupting  virus  growth. 

The  development  of  liver  cancer  during  persis- 
tent infection  is  another  major  focus  of  interest. 
For  this  we  study  two  relatives  of  HBV:  the  wood- 
chuck  hepatitis  virus  (WHV),  which  is  potently 
oncogenic  in  its  normal  host,  and  the  ground 


squirrel  hepatitis  virus  (GSHV),  a  less-efficient 
carcinogenic  stimulus  in  woodchucks.  Our  stud- 
ies thus  far  suggest  fundamental  differences  in 
the  oncogenic  pathways  employed  by  these 
closely  related  viruses.  WHV-induced  tumors  fre- 
quently (30-45  percent)  contain  integrated 
WHV  genomes  adjacent  to  the  N-myc  oncogene, 
whose  expression  is  thereby  activated.  Such  in- 
sertional  activation  is  not  apparent  in  GSHV- 
induced  hepatomas.  We  are  currently  looking  for 
other  genes  that  are  regularly  disrupted  by  WHV 
integration,  with  the  view  that  such  loci  may  de- 
fine other  proteins  centrally  involved  in  the  con- 
trol of  normal  hepatocyte  growth. 


150 


Second  Messengers  and  Cell  Regulation 


David  L.  Carters,  Ph.D. — Investigator 

Dr.  Garters  is  also  Professor  of  Pharmacology  at  the  University  of  Texas  Southwestern  Medical  Center  at 
Dallas.  He  received  his  B.S.  degree  in  agriculture  and  his  Ph.D.  degree  in  biochemistry  at  the  University 
of  Wisconsin.  His  postdoctoral  research  was  done  at  Vanderbilt  University.  Before  assuming  his  present 
position,  Dr.  Garbers  was  Professor  of  Pharmacology  and  of  Molecular  Physiology  and  Biophysics 
at  Vanderbilt  University  School  of  Medicine. 


THE  focus  of  the  research  in  this  laboratory 
centers  on  the  mechanisms  by  which  ceils 
communicate  with  each  other — specifically  the 
mechanisms  by  which  sea  urchin  or  mammalian 
spermatozoa  detect  signals  from  the  egg.  These 
studies  have  turned  out  to  be  applicable  to  so- 
matic cells  and  have  led  to  the  identification  of  a 
new  cell  surface  receptor  family  in  humans  and 
other  mammals.  Members  of  this  family  serve  as 
receptors  for  molecules  that  regulate  blood  pres- 
sure, as  well  as  a  large  number  of  other  physiolog- 
ical processes. 

Around  1981  our  laboratory  reported  the  puri- 
fication of  a  peptide  that  can  stimulate  sperm  mo- 
tility. This  peptide  was  derived  from  media  in 
which  sea  urchin  eggs  had  been  allowed  to  stand. 
Subsequently  it  was  demonstrated  that  different 
species  of  sea  urchins  contain  different  peptides 
and  that  the  molecules  from  one  species  do  not 
necessarily  stimulate  the  sperm  cells  from  an- 
other. In  later  research  it  became  clear  that  sperm 
cells  detect  higher  concentrations  of  peptide  and 
swim  toward  them.  Since  the  highest  concentra- 
tions are  around  the  egg,  the  sperm  cell  swims 
directly  toward  the  egg  under  normal  conditions. 

This  laboratory  set  out  to  determine  how  the 
cell  detects  the  egg  peptide.  We  found  that  a  par- 
ticular protein  on  the  sperm  plasma  membrane 
specifically  bound  the  egg  peptide.  This  protein 
appeared  to  serve  as  the  detector  or  receptor  mol- 
ecule, but  how  it  signaled  to  spermatozoa  that  a 
specific  egg  peptide  had  been  bound  was  not 
clear. 

To  help  resolve  the  question,  we  purified  the 
receptor  protein  on  the  plasma  membrane.  It  was 
identified  as  the  enzyme  guanylyl  cyclase,  which 
catalyzes  the  formation  of  cyclic  GMP,  a  small 
molecule  that  causes  a  change  in  the  behavior  of 
many  different  cells.  It  seemed  possible  that  the 
receptor  is  situated  with  part  of  it  outside  the 
cell,  where  it  could  bind  the  egg  peptide,  and  the 
other  part  inside,  where  it  forms  cGMP.  The 
cGMP  thus  formed  would  then  serve  as  a  signal 
that  egg  peptide  is  being  detected,  lending 
greater  speed  and  direction  to  the  spermatozoon. 

To  provide  evidence  that  the  membrane  recep- 


tor protein  is  in  fact  guanylyl  cyclase,  we  isolated 
complementary  DNA  clones  for  this  enzyme. 
Such  clones  allow  one  not  only  to  predict  the 
primary  structure  of  a  protein,  the  receptor  in 
this  case,  but  to  direct  protein  synthesis  in  quan- 
tity. Unfortunately,  the  sea  urchin  sperm  receptor 
was  not  formed  in  the  proper  manner,  and  re- 
search continues  on  its  expression. 

Under  appropriate  conditions,  DNA  will  bind 
(hybridize)  to  closely  related  DNA.  Therefore  sea 
urchin  DNA  was  used  to  determine  whether 
mammals  contain  a  related  protein  that  might 
serve  as  a  receptor.  Clones  containing  comple- 
mentary DNA  were  isolated  from  rat  brain.  The 
DNA  sequence  revealed  that  a  rat  brain  guanylyl 
cyclase  is  a  component  of  the  plasma  membrane, 
with  approximately  one-half  of  the  protein  out- 
side and  one-half  inside  the  cell.  The  intracellu- 
lar region  of  the  sea  urchin  sperm  enzyme  is  very 
similar  to  the  part  of  the  rat  brain  enzyme  inside 
the  cell.  In  the  regions  outside  the  cell,  however, 
the  two  proteins  show  little  similarity.  This 
would  be  expected  if  guanylyl  cyclase  serves  as  a 
cell  surface  receptor  for  peptides  and  the  peptide 
of  mammals  is  different  from  that  of  the  sea  ur- 
chin. That  is,  the  detector  part  of  the  molecule 
would  need  to  change  to  recognize  a  different 
peptide,  but  intracellular  regions  of  the  receptor 
could  remain  unaltered. 

Subsequent  binding  studies  demonstrated  that 
the  cloned  rat  brain  guanylyl  cyclase  could  specif- 
ically bind  and  be  activated  by  certain  peptides 
synthesized  in  the  heart  and  brain.  These  atrial 
natriuretic  peptides  (ANPs)  regulate  blood  pres- 
sure, as  well  as  various  other  physiological 
events. 

Since  sea  urchin  sperm  cells  respond  to  pep- 
tides that  do  not  resemble  ANP,  other  animals 
may  be  expected  to  contain  yet  other  peptides 
that  interact  with  guanylyl  cyclase.  In  addition, 
multiple  membrane  forms  of  guanylyl  cyclase 
may  exist  within  mammals.  We  have  used  the 
DNA  that  encodes  the  ANP  receptor  to  determine 
whether  other  guanylyl  cyclase  receptors  exist. 
Another  receptor  with  properties  similar  to  the 
one  described  above  has  been  identified.  This 


151 


Second  Messengers  and  Cell  Regulation 


guanylyl  cyclase  receptor  is  similar  to  the  first 
ANP  receptor  within  intracellular  regions  but  is 
only  43  percent  identical  in  the  extracellular, 
ligand-binding  region.  It  now  appears  that  a  pep- 
tide other  than  ANP  regulates  this  receptor. 

We  subsequently  discovered  the  existence  of  a 
third  guanylyl  cyclase  receptor.  This  receptor 
binds  small  peptides  released  from  various  bacte- 
ria that  cause  acute  diarrhea.  This  form  of  diar- 
rhea (often  referred  to  as  traveler's  diarrhea)  is 
prevalent  in  infant  humans  and  young  domestic 
animals.  The  receptor  has  the  same  general  fea- 
tures as  the  two  receptors  described  above  but 
has  a  markedly  different  amino  acid  sequence 
within  the  toxin-binding  region.  Whether  a  regu- 
latory molecule  not  of  bacterial  origin  normally 
exists  that  binds  to  this  receptor  is  not  yet  known. 


During  the  past  year,  we  have  obtained  evi- 
dence to  suggest  that  at  least  two  other  guanylyl 
cyclase  receptors  exist.  The  hormones  that  nor- 
mally regulate  these  receptors  have  not  yet  been 
identified.  It  seems,  therefore,  that  different  cells 
contain  unique  guanylyl  cyclase  receptors, 
which  allow  them  to  respond  to  specific  hor- 
monal signals  with  an  increased  production  of 
the  messenger  molecule  cGMP.  The  cells  then 
change  their  behavior.  For  example,  one  receptor 
may  control  smooth  muscle  relaxation  and  be 
principally  involved  in  the  regulation  of  blood 
pressure,  another  may  be  involved  in  neural 
functions,  and  yet  another  may  regulate  epithe- 
lial cell  secretion.  These  results  also  suggest  that 
drugs  specific  to  different  forms  of  the  guanylyl 
cyclase  receptors  may  prove  clinically  relevant. 


152 


Molecular  Genetics  of  the  Major 
Histocompatibility  Complex 


Jan  Geliebter,  Ph.D. — Assistant  Investigator 

Dr.  Geliebter  is  also  Assistant  Professor  and  Head  of  the  Mammalian  Molecular  Genetics  Laboratory  at  the 
Rockefeller  University.  He  received  his  Ph.D.  degree  in  microbiology  and  immunology  from  the  State 
University  of  New  York,  Downstate  Medical  Center.  He  was  a  postdoctoral  fellow  and  research  associate 
in  the  laboratory  of  Stanley  Nathenson  at  the  Albert  Einstein  College  of  Medicine,  Bronx,  New  York. 


THE  immune  system  functions  to  rid  the  body 
of  foreign  objects  such  as  bacteria,  viruses, 
tumors,  and  transplants.  The  portion  of  such  mat- 
ter that  is  recognized  as  foreign  by  the  immune 
system  is  called  an  antigen.  Antigens  that  are 
found  on  cells  are  "presented"  to  the  immune 
system  by  cell  surface  molecules  called  histocom- 
patibility molecules  (also  called  HLA  molecules 
in  humans  and  H-2  molecules  in  the  mouse).  His- 
tocompatibility molecules  are  able  to  bind  anti- 
genic fragments  of,  for  example,  viruses,  and 
stimulate  white  blood  cells  (lymphocytes)  to 
attack  the  virus-infected  cell,  thereby  limiting 
the  spread  of  infection.  Without  these  antigen- 
presenting  molecules  the  host  would  be  unable 
to  mount  an  immune  response  against  pathogens 
and  would  not  survive. 

Different  H-2  molecules  can  bind  and  present 
different  types  of  antigens.  Because  inbred  mice 
have  about  three  different  types  of  H-2  molecules 
on  their  cells,  they  can  bind  and  present  a  large, 
but  limited,  number  of  antigens  to  the  immune 
system. 

To  ensure  the  survival  of  the  species,  it  is  bene- 
ficial that  many  varieties  of  H-2  molecules  be 
present  in  the  population.  In  this  way  there  will 
always  be  some  portion  of  the  population  that 
will  mount  an  immune  response  to  a  given  anti- 
gen. Indeed,  an  extraordinary  number  of  differ- 
ent histocompatibility  molecules  have  been 
found  in  almost  all  species  investigated.  In  hu- 
mans, the  large  variety  of  HLA  molecules  ensures 
our  survival  but  is  the  major  obstacle  confound- 
ing tissue  transplantation.  Our  interest  lies  in  the 
genetic  mechanism  that  generates  the  different 
histocompatibility  genes  in  mice  and  other 
species. 

The  H-2  genes  of  the  mouse  are  part  of  the 
larger  major  histocompatibility  complex  class  I 
multigene  family.  This  gene  family  also  contains 
genes  that  are  structurally  similar  to  H-2  genes 
and  have  unknown  functions.  The  genetic  mecha- 
nism that  generates  variety  in  H  2  genes  is  the 
microrecombination  process,  which  reassorts 
DNA  among  H-2  genes  and  other  related  class  I 
genes.  By  substituting  small  segments  of  class  I 


gene  sequences  into  //-2  genes,  the  microrecom- 
bination process  can  create  new  H-2  molecules 
that  have  different  antigen-presenting  capabili- 
ties, thereby  expanding  the  immune  responsive- 
ness of  the  population. 

Our  interest  is  to  understand  better  the  mecha- 
nism underlying  the  microrecombination  pro- 
cess. This  process  has  previously  been  studied  by 
identifying  microrecombinant  mice  that  differed 
from  their  otherwise  identical  siblings  by  altered 
H-2  genes.  Since  microrecombinant  H-2  mole- 
cules elicit  skin  graft  rejection,  these  studies 
were  accomplished  by  skin  graft  testing  thou- 
sands of  mice.  The  rejection  of  a  skin  graft  by  a 
sibling  mouse  signaled  an  alteration  in  H-2  mole- 
cules. These  labor-intensive  studies  found  that, 
on  the  average,  one  microrecombinant  mouse 
was  detected  for  every  5,000  skin  grafts 
performed. 

To  gain  further  insight  into  the  microrecom- 
bination process,  we  are  using  in  vitro-engi- 
neered  constructs  to  detect  microrecombinant 
H-2  genes.  We  have  constructed  a  fusion  gene  in 
which  /3-galactosidase  sequences  replace  two  cy- 
toplasmic exons  for  the  gene.  The  fusion  pro- 
tein can  be  detected  by  staining  for  |8-galactosi- 
dase  activity,  which  is  manifested  as  blue-colored 
cells.  We  have  also  site-directed  two  in-frame  ter- 
mination codons  in  the  gene  at  positions  that 
undergo  frequent  microrecombinations.  This 
prevents  the  expression  of  |S-galactosidase. 
/3-Galactosidase  expression  can  be  rescued  by  a 
microrecombination  with  a  linked  class  I  gene, 
Q4,  that  recombines  away  the  termination  co- 
dons. Thus  microrecombinations  can  be  scored  as 
blue  cells. 

This  microrecombination  construct,  once  in- 
troduced into  a  variety  of  cell  types,  will  help 
determine  the  microrecombination  frequencies 
of  different  cells.  Its  introduction  into  transgenic 
mice  will  help  determine  frequencies  in  germ 
cells.  Data  from  previous  studies  indicate  that  mi- 
crorecombinations occur  in  female  germ  cells. 
We  also  hope  to  determine  if  they  occur  in  sperm 
cells  as  well  and  at  what  frequency.  Some  strains 
of  mice  may  undergo  microrecombinations  more 


153 


Molecular  Genetics  of  the  Major  Histocompatibility  Complex 


frequently  than  others.  Placing  this  construct 
onto  different  genetic  backgrounds  may  help  to 
determine  microrecombination  frequencies  in 
different  mouse  strains  and  perhaps  identify  criti- 
cal parameters  in  the  process.  These  studies  will 
contribute  to  our  understanding  of  the  genetic 
processes  that  control  the  evolution  and  ulti- 
mately the  function  of  the  mammalian  immune 
system. 

Although  sequence  diversity  and  polymor- 
phism are  the  hallmark  of  genes,  region  genes  are 
characterized  by  sequence  conservation  among 
alleles  and  limited  polymorphism.  The  lack  of 
polymorphism  among  these  genes  has  been  sug- 
gested to  preclude  an  immunological  function 
for  the  gene  products.  We  have  identified  a  re- 
gion gene  w^hose  sequence  differs  greatly  be- 
tween alleles  of  the  C57BL/6  and  C3H  mice.  The 
sequence  differences  between  the  two  alleles  are 


manifested  in  both  scattered  and  clustered  nu- 
cleotide substitutions.  The  clustered  substitu- 
tions are  similar  to  those  observed  in  the  micro- 
recombinations  that  diversify  genes  and  may 
reflect  past  microrecombination  events  with  H-2 
and  other  Qa  region  genes.  These  data  may  pro- 
vide the  first  evidence  that  Qa  genes  can  be  recip- 
ients in  the  microrecombination  process. 

Polymerase  chain  reaction  (PGR)  analysis  has 
indicated  that  this  Qa  gene  is  transcribed  in  some 
strains  of  mice.  We  are  currently  engaged  in  an 
in-depth  analysis  of  the  transcription,  translation, 
and  cell  surface  expression  of  this  gene  and  its 
product  to  ascertain  its  function.  This  gene  is 
polymorphic  in  at  least  three  strains  of  mice,  and 
the  analysis  of  other  strains  is  under  way.  The  di- 
versity and  polymorphism  of  this  gene  suggest  an 
immunological  function  for  its  product,  perhaps 
the  first  Qa  gene  to  be  ascribed  such  a  function. 


154 


The  Decoding  Code  in  mRNA 


Raymond  F.  Gesteland,  Ph.D. — Investigator 

Dr.  Gesteland  is  also  Professor  of  Human  Genetics  at  the  University  of  Utah  School  of  Medicine  and 
Professor  of  Biology  and  Adjunct  Professor  of  Bioengineering  at  the  University  of  Utah.  He  received  his  B.S. 
degree  in  chemistry  and  his  M.S.  degree  in  biochemistry  from  the  University  of  Wisconsin.  He  earned  his 
Ph.D.  degree  in  biochemistry  from  Harvard  University,  where  he  studied  with  J.  D.  Watson.  Dr.  Gesteland 
was  an  NIH  postdoctoral  fellow  at  the  Institute  de  Biologie  Moleculaire,  Geneva,  Switzerland.  He  served 
as  Assistant  Director  and  Investigator  at  Cold  Spring  Harbor  Laboratory,  New  York,  before  assuming  his 
present  responsibilities. 


THE  genetic  code  precisely  specifies  the  iden- 
tification of  each  of  the  20  amino  acids  from 
the  set  of  6 1  triplets  in  mRNA,  with  three  triplets 
dedicated  to  termination.  As  a  ribosome  moves 
progressively  along  the  mRNA  deciphering  it,  the 
contiguous  triplets  contain  all  the  information 
necessary  to  specify  amino  acid  sequence.  How- 
ever, there  is  another  code,  also  encrypted  by  the 
linear  order  of  bases  in  mRNA,  that  carries  infor- 
mation about  the  mechanism  of  translation. 

This  code  specifies  how  the  common  genetic 
code  should  be  implemented  for  each  mRNA.  In 
some  cases  the  additional  information  may  be 
quite  simple,  consisting  of  instructions  that  tell 
the  ribosome  where  to  start  and  where  to  stop 
decoding.  In  other  cases  this  secondary  code  radi- 
cally changes  the  decoding  process  so  that  indi- 
vidual mRNAs  use  a  different  version  of  the  ge- 
netic code,  altered  either  in  the  meaning  of 
certain  codons  or  in  the  linear  readout  mecha- 
nism. This  set  of  instructions  could  be  called  the 
"decoding  code,"  in  that  it  operates  on  the  ge- 
netic code.  Different  decoding  codes  individual- 
ize mRNAs. 

A  decoding  code  operating  during  the  deci- 
phering of  a  specific  mRNA  often  results  in  a  vio- 
lation of  the  conventional  genetic  code.  This  can 
occur  with  a  set  frequency,  so  that  the  same 
mRNA  produces  two  different  protein  products; 
but  the  frequency  can  also  depend  on  metabolic 
state,  thus  providing  another  point  at  which  gene 
expression  is  controlled. 

For  example,  the  ribosomal  machinery  can  be 
instructed  to  read  a  nontriplet  number  of  bases  at 
one  site  so  that  an  alternate  reading  frame  will  be 
used  from  that  point  on.  Or  a  proportion  of  the 
ribosomes  can  be  instructed  to  jump  from  one 
site  to  another  on  the  same  mRNA  so  that  noncon- 
tiguous codons  will  be  read  out.  Or  the  meaning 
of  specific  codons  can  be  altered  so  that  a  ribo- 
some reads  a  stop  codon  as  an  amino  acid,  or  even 
as  a  new  amino  acid  that  is  not  a  member  of  the 
conventional  set  of  20.  Each  of  these  unusual 
events  is  specified  by  the  decoding  code 
information. 


The  diversity  of  schemes  for  encrypting  infor- 
mation for  the  decoding  code  in  individual 
mRNAs  is  just  beginning  to  be  appreciated.  In 
some  cases,  rather  simple  sequences  are  in- 
volved; in  others,  sequence  dictates  complex 
RNA  structures. 

The  signals  in  mRNA  that  carry  the  instructions 
for  programmed  ribosomal  frameshifting  usually 
include  a  shift  site  where  the  frame  changes  and  a 
stimulator  sequence  that  greatly  increases  the 
shift  site's  efficiency.  The  shift  site  consists  of 
4-7  nucleotides  that  allow  a  ribosomal-bound 
tRNA  decoding  in  the  first  frame  to  slip  forward 
or  backward  by  one  nucleotide  to  get  into  the 
new  frame.  The  stimulator  information  can  be 
upstream  of  the  shift  site,  as  in  the  case  of  the  RF2 
gene  in  Escherichia  coli,  where  5-6  nucleotides 
need  to  pair  directly  with  a  specific  sequence  in 
ribosomal  RNA  in  order  to  stimulate  the  shift.  Or 
it  can  be  downstream,  as  in  the  case  of  many  retro- 
viruses and  retroviral-like  genes  where  the  spe- 
cific interactions  are  less  clear  and  the  mRNA 
contains  secondary  and  tertiary  structures  that 
somehow  stimulate  frameshifting  at  the  upstream 
shift  site.  The  combination  of  shift  site  and  stimu- 
lator sequences  constitutes  the  decoding  code 
that  tells  how  the  standard  genetic  code  should 
be  corrupted  for  translation  of  a  specific  message. 

There  is  only  one  compelling  case  of  ribosomal 
jumping,  and  here  the  decoding  code  is  complex. 
The  mRNA  for  gene  60  of  bacteriophage  T4  has  a 
gap  of  50  nucleotides  that  is  very  efficiently  by- 
passed by  ribosomes.  The  mRNA  sequence  ele- 
ments involved  include  both  structural  informa- 
tion in  the  gap  and  upstream  information  in  the 
form  of  the  amino  acid  sequence  of  the  growing 
peptide  chain  that  must  interact  with  the  same 
part  of  the  complex. 

Other  versions  of  the  decoding  code  reprogram 
stop  codons  to  specify  amino  acids.  Some  retrovi- 
ruses (e.g. ,  murine  leukemia  virus,  MuLV)  make  a 
fusion  protein  from  two  in-frame  genes  separated 
by  a  UAG  stop  codon,  which  in  this  case  is  de- 
coded as  glutamine.  The  decoding  code  that  spe- 
cifies this  includes  a  downstream  element  whose 


155 


The  Decoding  Code  in  mRNA 


secondary  and  tertiary  structures  are  crucial.  In 
the  handful  of  known  cases  of  proteins  having  the 
unusual  amino  acid  selenocysteine,  a  UGA  stop 
codon  is  reprogrammed  to  be  decoded  as  this 
21st  amino  acid.  The  decoding  code  clearly  in- 
cludes structural  elements  that  are  downstream, 
in  one  case  150  nucleotides  away  in  the  non- 
translated  part  of  the  message. 

An  emerging  theme  of  the  decoding  code  is 
mRNA  secondary  and  tertiary  structural  elements. 
Stem-loop  structures  can  often  be  identified  by 
inspection  of  sequence,  and  evidence  for  their 
participation  can  be  obtained  by  making  disrup- 
tive and  restorative  mutations.  The  functional 
stems  can  be  short  in  some  contexts,  long  in 
others,  and  can  be  near  the  site  of  action  or  well 
downstream. 

In  an  increasing  number  of  cases,  the  crucial 
structure  is  more  complicated,  involving  a  pseu- 
doknot  where  a  sequence  downstream  of  a  stem- 
loop  folds  back  to  pair  with  the  loop  of  the 
stem,  resulting  in  two  stems  and  two  loops  that 
are  intertwined.  Physical-chemical  properties  of 
model  pseudoknots  show  that  the  two  stems  are 
coaxial,  with  considerable  stability  coming  from 
the  extended  base  stacking  that  is  achieved. 

To  test  the  biological  importance  of  each  of  the 
bases  of  these  complicated  structures  requires 


construction  of  a  very  large  number  of  mutants, 
and  analysis  of  the  resulting  structural  changes  is 
so  far  minimal.  In  some  cases  the  actual  sequence 
of  the  proposed  stems  is  unimportant  so  long  as 
base-pairing  is  maintained  and  the  sequence  of 
the  loops  is  not  crucial.  In  fact,  these  criteria  are 
used  to  define  the  importance  of  a  stem  or  pscu- 
doknot. In  at  least  one  case  (MuLV  stop  codon 
read-through) ,  a  few  bases  in  the  loop  region  of  a 
pseudoknot  are  crucially  important. 

Structures  in  ribosomal  RNA  are  beginning  to 
be  delineated  clearly,  largely  from  evolutionary 
comparisons  of  sequence.  We  are  still  quite  igno- 
rant, however,  about  the  complexity  of  structures 
in  mRNA.  At  least  for  the  elements  involved  in  the 
decoding  code,  there  is  a  biological  assay  for 
function. 

We  are  even  less  clear  about  how  these  struc- 
tures in  mRNA  alter  the  decoding  process.  The 
simplest  view — that  mRNA  structures  merely 
cause  a  ribosomal  pause  allowing  alternative  reac- 
tions— is  almost  certainly  naive.  But  whether  the 
structures  bind  protein  factors,  interact  with  ribo- 
somal proteins,  pair  with  ribosomal  RNA,  or  even 
interact  with  other  parts  of  mRNA  is  completely 
untested.  Investigation  of  these  possibilities  in 
the  context  of  the  decoding  code  will  be 
revealing. 


Ribosome  Interaction  Sequence 


GGA  UAG  ^  J-  GGA  UUA- 

■  -STOP  ■  \ 

Coding  Gap 


GLY  LEU 

u 


UCA  AAA  AAC  UUG 


Decoding  of  the  genetic  code.  Top:  Model  of  a 
ribosome  traversing  the  50-nucleotide  coding 
gap  in  mRNA  of  bacteriophage  T4 's  gene  60, 
illustrating  the  involvement  of  upstream  pep- 
tide sequences  and  sequences  in  the  gap. 

Bottom:  Model  of  a  ribosome  reading  a  stop 
codon  in  mRNA  of  a  murine  leukemia  virus, 
stimulated  by  a  downstream  pseudoknot 
structure. 

Research  of  Norma  Wills,  Robert  Weiss, 
Dianne  Dunn,  and  fohn  Atkins  in  the  labora- 
tory of  Raymond  Gesteland. 


UC  AAA  AAA  CUU  C 


156 


Molecular  Analysis  of  Proteins  Involved 
in  Human  Disease 


Mary-Jane  H.  Gething,  Ph.D. — Investigator 

Dr.  Gething  is  also  Professor  of  Biochemistry  at  the  University  of  Texas  Southwestern  Medical  Center  at 
Dallas.  She  received  her  bachelor  's  and  Ph.D.  degrees  from  the  University  of  Melbourne.  After  holding 
research  fellowships  in  Cambridge,  England,  with  Brian  Hartley  and  in  London  with  Michael  Waterfield, 
she  joined  the  scientific  staff  of  the  Imperial  Cancer  Research  Fund,  London.  After  that  she  was  a  senior 
staff  investigator  at  the  Cold  Spring  Harbor  Laboratory,  New  York,  before  going  to  the  Southwestern 
Medical  Center. 


OUR  studies  on  experimental  models  of  hu- 
man disease  involve  three  systems:  1)  hu- 
man tissue-type  plasminogen  activator  (t-PA),  a 
serine  protease  involved  in  fibrinolysis,  tissue  re- 
modeling, and  metastasis;  2)  the  hemagglutinin 
of  influenza  virus,  which  is  being  used  to  develop 
models  of  autoimmune  disease  in  transgenic 
mice;  and  3)  the  tumor-suppressor  protein  p53 
and  its  interaction  with  cytosolic  stress-70 
proteins. 

Role  of  t-PA  in  Thrombolysis  and  Metastasis 

Many  normal  and  abnormal  biological  pro- 
cesses that  require  extracellular  proteolysis,  in- 
cluding thrombolysis,  tissue  remodeling,  and 
metastasis,  are  mediated  by  plasminogen  activa- 
tors that  cleave  plasminogen  to  the  active  pro- 
tease plasmin.  One  such  activator,  t-PA,  is  the 
principal  thrombolytic  agent  in  the  circulation, 
and  its  elevated  expression  is  thought  to  be 
linked  to  increases  in  the  metastatic  potential  of 
some  types  of  tumor  cells,  including  malignant 
melanomas. 

The  t-PA  protein  is  composed  of  a  number  of 
independent  structural  domains.  The  finger  do- 
main and  an  epidermal  growth  factor  (EGF)-like 
domain  are  involved  in  the  initial,  high-affinity 
binding  of  t-PA  to  fibrin,  while  stimulation  of  t-PA 
activity  requires  secondary,  lower-affinity  inter- 
actions of  fibrin  with  either  of  two  kringle  do- 
mains of  the  molecule.  The  binding  of  t-PA  to 
specific  clearance  receptors  on  hepatic  cells  also 
involves  sequences  within  the  finger  and/or  EGF- 
like  domains.  Finally,  a  specific  inhibitor,  plas- 
minogen activator  inhibitor  !  (PAI-1),  interacts 
with  the  active  site  in  the  carboxyl-terminal  cata- 
lytic domain. 

Although  the  three-dimensional  structure  of 
t-PA  has  not  been  solved,  we  have  been  able  to 
model  all  the  domains  through  use  of  known 
structures  of  homologous  domains  in  other  pro- 
teins. Site-directed  mutants  using  these  proposed 
structures  have  provided  information  about  the 
role  of  individual  amino  acid  sequences  of  the 
protein,  and  variant  enzymes  have  been  gener- 
ated that  are  efficient,  fibrin-stimulated  plasmin- 


ogen activators  but  are  resistant  to  inhibition  by  a 
variety  of  serpins,  including  PAI- 1 ,  or  do  not  bind 
to  the  t-PA  receptor(s)  involved  in  clearance  of 
the  enzyme  in  the  liver.  Because  these  mutant 
enzymes  should  have  an  extended  effective  life  in 
the  circulation,  they  may  have  significant  poten- 
tial for  use  in  thrombolytic  therapy  of  patients 
with  myocardial  infarction. 

The  variant  enzymes  are  also  being  utilized  to 
test  the  role  of  t-PA  in  metastasis  of  malignant 
melanoma  cells.  Transgenic  mice  expressing  the 
T  antigen  oncogene  from  simian  virus  40  (SV40) 
under  the  control  of  the  mouse  tyrosinase  pro- 
moter develop  ocular  melanoma  with  high  fre- 
quency. These  tumors  are  transplantable  to  non- 
transgenic  animals  but  are  not  metastatic.  We  are 
currently  developing  additional  lines  of  trans- 
genic mice  that  express  wild-type  or  inhibitor- 
resistant  forms  of  t-PA  from  the  same  tyrosinase 
promoter.  Crossing  of  the  Ty-Tag  and  Ty-tPA 
transgenic  mice  will  reveal  whether  an  increased 
level  of  t-PA  production  in  melanoma  cells  would 
result  in  increased  metastatic  potential. 

Transgenic  Models  of  Immune  Tolerance 
and  Autoimmune  Disease 

We  are  using  transgenic  mice  to  study  the  de- 
velopment of  immunological  responses  to  the 
hemagglutinin  (HA)  of  influenza  virus.  RIPHA 
mice,  which  express  HA  from  the  rat  insulin  II 
promoter/enhancer  only  in  the  /3-cells  of  the  pan- 
creas, promise  to  provide  a  valuable  model  for 
the  study  of  immune  tolerance  and  autoimmune 
diabetes.  These  mice  display  no  physiological 
problems  until  4-5  months  of  age.  After  that,  in- 
creases in  the  blood  glucose  levels  begin  to  ap- 
pear, and  shortly  thereafter  these  mice  develop  a 
severe  hyperglycemia  that  is  responsive  to 
insulin. 

In  the  RIPHA-33  line,  both  male  and  female 
animals  can  develop  an  immune  response  to  HA 
and  other  antigens  of  pancreatic  |8-cells.  How- 
ever, male  mice  become  hyperglycemic  with 
much  higher  frequency  (45  percent  )  than  female 
mice  (  5  percent ) .  Recent  studies  have  shown  that 
the  amount  of  fat  in  the  diet  is  an  important  regu- 


157 


Molecular  Analysis  of  Proteins  Involved  in  Human  Disease 


lator  of  diabetes  in  RIPHA-33  mice  and  that  the 
sex  bias  may  reflect  a  decreased  ability  of  male 
mice  to  control  their  blood  glucose  levels. 
Current  studies  focus  on  a  comparison  of  the 
RIPHA  model  with  mice  of  the  non-obese  dia- 
betic (NOD)  line  and  on  the  role  played  by  cyto- 
kines, such  as  interleukin-1 ,  tumor  necrosis  fac- 
tor, interferon-7,  and  interleukin-6,  during  the 
progression  of  the  disease. 

Folding  and  Assembly 

of  the  Tumor-Suppressor  Protein  p53 

Until  recently  it  was  widely  assumed  that  the 
folding  and  oligomerization  of  newly  synthesized 
polypeptides  and  their  subsequent  molecular  re- 
arrangements are  spontaneous  processes  that  do 
not  require  the  intervention  of  other  cellular  pro- 
teins. However,  it  is  now  apparent  that  members 
of  the  stress-70  protein  family  are  "chaperones" 
intimately  involved  in  facilitating  protein  folding 
and  assembly  within  prokaryotic  and  eukaryotic 
cells. 

Our  previous  studies  of  the  interaction  of  BiP, 
the  endoplasmic  reticulum  stress-70  protein, 
with  newly  synthesized  membrane  and  secretory 


proteins  has  led  to  an  understanding  of  the  role  of 
stress-70  proteins  in  stabilizing  unfolded  or 
partly  folded  polypeptides  in  a  form  competent 
for  further  folding  and  oligomeric  assembly. 
Current  studies  focus  on  the  role  of  hsc70,  the 
cytosolic  stress-70  protein,  in  modulating  the 
structure  or  activity  of  mutant  forms  of  the 
tumor-suppressor  protein  p53. 

Other  workers  have  shown  that  p53  plays  a 
role  in  regulation  of  normal  cell  growth  and  that 
it  binds  to  a  number  of  viral-transforming  pro- 
teins, including  SV40  T  antigen.  Mutations  in  p53 
convert  the  protein  to  an  oncogenic  form  capable 
of  co-operating  with  another  oncogene,  activated 
ras,  to  transform  cells.  These  mutant  forms  of 
p53  appear  to  be  altered  conformationally,  as  in- 
dicated by  loss  of  T  antigen  recognition,  altered 
reactivity  with  monoclonal  antibodies,  and  co- 
translational  binding  to  hsc70.  We  are  analyzing 
the  interaction  between  p53  and  hsc70,  using  the 
fd  bacteriophage  expression  system.  By  screening 
p53-peptide-expressing  bacteriophage  for  bind- 
ing to  hsc70,  with  subsequent  enrichment  and 
amplification  in  bacteria  of  those  expressing 
hsc70-binding  epitopes,  the  region(s)  in  p53  rec- 
ognized by  hsc70  will  be  identified. 


158 


Signal  Transduction  Pathways 
in  B  Lymphocytes 


Sankar  Ghosh,  Ph.D. — Assistant  Investigator 

Dr.  Ghosh  is  also  Assistant  Professor  of  Immunobiology  and  of  Molecular  Biophysics  and  Biochemistry 
at  Yale  University  School  of  Medicine.  After  receiving  his  Ph.D.  degree  from  the  Albert  Einstein  College 
of  Medicine,  Bronx,  New  York,  he  conducted  postdoctoral  work  at  the  Whitehead  Institute 
in  Cambridge,  Massachusetts,  under  the  supervision  of  David  Baltimore. 


THE  focus  of  research  in  our  laboratory  is  to 
understand  the  signals  that  regulate  B  cell  de- 
velopment and  the  pathways  through  which  they 
are  transduced.  We  propose  to  address  these  ques- 
tions by  studying  the  regulation  of  developmen- 
tal-stage-specific expression  of  the  immunoglob- 
ulin K  light-chain  gene.  Primarily,  we  wish  to  find 
the  signal  that  initiates  k  gene  expression  during 
B  cell  development  and  determine  whether  ex- 
pression of  the  gene  is  necessary  for  rearrange- 
ment of  the  K  locus. 

The  expression  of  the  k  gene  in  mature  B  cells 
is  driven  by  an  enhancer  located  in  the  intron 
between  the  J/c  and  Ck  genes.  The  activity  of  this 
enhancer  depends  on  the  binding  of  a  transcrip- 
tion factor,  called  NF-/cB,  to  a  specific  site  in  the 
element.  In  keeping  with  its  role  as  a  regulator  for 
developmental-stage-specific  expression  of  the  k 
gene,  NF-/cB  can  only  be  detected  in  an  active 
form  in  the  nucleus  of  mature  B  cells.  However,  it 
is  present  in  the  cytoplasm  of  pre-B  cells  and 
other  cell  types  as  an  inactive  precursor,  bound 
to  an  inhibitory  protein  called  I/cB. 

NF-kB  activity  can  be  induced  by  treatment  of 
cells  with  agents  such  as  phorbol  esters  or  bacte- 
rial lipopolysaccharide.  These  agents  activate  sig- 
nal transduction  pathways  that  result  in  the  modi- 
fication of  I/cB  and  the  subsequent  dissociation  of 
the  NF-/cB:IkB  complex.  The  free  NF-zcB  then 
enters  the  nucleus  and  activates  gene  expression. 
Therefore  the  inducible  system  of  NF-/cB  behaves 
like  a  second  messenger,  transducing  cell  surface 
signals  to  the  nucleus. 

To  understand  the  change  in  IkB  that  results  in 
the  dissociation  of  the  NF-kB:IkB  complex,  we  es- 
tablished an  in  vitro  system  to  look  at  the  involve- 
ment of  protein  kinases  in  the  activation  process. 
The  results  strongly  suggested  phosphorylation 
of  I/cB  as  a  key  event  in  the  activation  of  NF-kB. 
However,  further  analysis  of  the  activation  pro- 
cess was  hampered  by  the  lack  of  reagents,  such 
as  antibodies,  or  clones  for  the  genes  encoding 
NF-kB.  Therefore  we  undertook  to  clone  the 
genes;  the  information  we  obtained  has  resulted 
in  significant  new  insights  into  the  function  of 
these  proteins. 


NF-kB  is  composed  of  two  subunits  of  50  kDa 
(p50)  and  65  kDa  (p65)  and  exists  as  a  hetero- 
dimer.  Cloning  the  cDNA  encoding  p50  revealed 
that  it  was  actually  synthesized  as  a  larger  protein 
of  105  kDa,  which  was  processed  to  the  mature 
50-kDa  size.  The  full-length  protein  did  not  bind 
to  DNA,  but  removal  of  the  carboxyl-terminal  re- 
gion revealed  the  DNA-binding  activity.  How- 
ever, the  most  interesting  feature  of  the  clone  was 
the  homology  for  over  300  amino  acids  in  the 
amino-terminal  half  (i.e.,  the  region  encoding 
p50)  to  the  oncogene  \-rel,  its  cellular  counter- 
part c-rel,  and  the  Drosophila  morphogen 
dorsal. 

Subsequent  cloning  of  the  p65  subunit  re- 
vealed a  similar  re/-homology  region,  which  con- 
tains both  DNA-binding  and  dimerization  do- 
mains. In  fact,  with  the  recent  cloning  of  other 
re/-homologous  proteins,  a  picture  has  begun  to 
emerge  in  which  the  different  members  associate 
with  one  another  to  form  transcription  factors 
that  can  activate  different  genes. 

The  homology  between  NF-kB  and  dorsal  also 
has  interesting  implications  for  understanding 
the  biology  of  both  proteins.  The  developmental 
morphogen  dorsal  is  responsible  for  the  estab- 
lishment of  the  dorsoventral  polarity  in  Drosoph- 
ila embryos.  The  basis  for  its  action  is  the  estab- 
lishment of  a  gradient  of  dorsal  protein  that  is 
progressively  localized  to  the  nucleus.  The  cyto- 
plasmic localization  of  dorsal  was  genetically  de- 
termined to  be  due  to  the  action  of  a  gene  called 
cactus,  which  behaves  like  IkB,  and  recent  clon- 
ing of  the  gene  has  shown  that  it  is  a  homologue 
of  mammalian  IkB. 

The  signal  for  the  nuclear  localization  of  dor- 
sal goes  through  a  pathway  involving  eight  up- 
stream maternal-effect  genes.  However,  for  those 
of  us  studying  NF-kB,  what  has  been  of  great  inter- 
est is  the  finding  that  one  of  these  upstream 
genes,  toll,  is  a  membrane  receptor  with  a  cyto- 
plasmic domain  that  is  homologous  to  one  of  the 
mammalian  interleukin-1  (IL-1)  receptors.  This 
is  significant  because,  among  the  different  inter- 
leukins,  only  IL-1  can  activate  NF-kB.  Therefore 
the  two  genes  that  lie  between  toll  and  dorsal 


159 


Signal  Transduction  Pathways  in  B  Lymphocytes 


(i.e.,  pelle  and  tube)  are  probably  components  of 
the  signal  transduction  pathway  that  resuh  in  the 
modification  of  cactus.  The  challenge  for  the  fu- 
ture is  to  find  the  analogues  of  pelle  and  tube  in 
mammalian  cells,  and  we  are  exploring  difl'erent 
approaches  in  our  attempt  to  elucidate  the  signal 
transduction  pathway  that  results  in  the  constitu- 
tive activation  of  NF-/cB  in  mature  B  cells. 

The  next  major  advance  was  the  cloning  of  I/cB, 
and  once  again  the  cloning  revealed  a  sequence 
homology  that  is  important  for  I/cB  function.  In 
this  case  the  sequence  elements  are  the  ankyrin 
repeats  that  are  found  in  a  diverse  group  of  pro- 
teins, including  the  pi 05  precursor  of  NF-/cB 
p50,  Drosophila  Notch,  yeast  cdclO,  and  the  hu- 
man erythrocyte  ankyrin  protein.  Because  all  of 
these  proteins  are  either  cytosolic  or  membrane 
associated,  the  initial  idea  was  that  ankyrin  se- 
quences mediate  cytosolic  retention  of  proteins 
by  binding  to  cytosolic  or  membrane  structures. 
However,  a  nuclear  transcription  factor  was  re- 
cently found  to  contain  these  sequences,  necessi- 
tating a  change  in  our  idea  of  the  role  of  ankyrin 
sequences.  A  more  comprehensive  model  envi- 
sions them  as  domains  responsible  for  protein- 
protein  interaction.  We  also  found  that  pp40,  a 
protein  associated  with  v-Rel  and  c-Rel  in 
chicken  lymphoid  cells,  is  a  homologue  of  mam- 
malian IkB.  This  observation,  along  with  the  find- 
ing that  cactus  is  also  homologous  to  I/cB,  sug- 
gests that  the  NF-KB/rel/dorsal  family  has 
evolved  over  millions  of  years  and  used  the  same 
general  principles  for  regulating  the  activity  of 
these  positive-acting  transcription  factors. 

One  of  the  most  interesting  questions  about 
NF-/(B  is  its  regulation  during  B  cell  development. 
NF-kB  is  an  inducible  cytosolic  protein  in  pre-B 
cells  but  changes  into  a  constitutively  active  nu- 


clear protein  in  mature  B  cells.  There  can  be  at 
least  two  simple  explanations  for  this:  either  a 
transcriptional  shutofl"  of  I/cB  synthesis  or  a  con- 
stitutive modification  of  I/cB  in  mature  B  cells. 
Preliminary  results  indicate  that  transcription  of 
the  major  form  of  I/cB  is  not  shut  off  in  mature  B 
cells,  but  the  UB  that  is  synthesized  is  somehow 
inactivated.  We  are  trying  to  determine  how  IkB 
activity  is  regulated  in  mature  B  cells.  We  also 
want  to  determine  the  signal  responsible  for  the 
developmental  transition  of  a  pre-B  cell  to  a  ma- 
ture B  cell. 

The  other  major  focus  of  our  research  is  to  un- 
derstand the  regulation  of  k  gene  expression  in 
plasma  cells.  Unlike  mature  B  cells,  plasma  cells 
express  large  amounts  of  the  immunoglobulin 
chains,  including  the  k  chains,  which  form  com- 
plete immunoglobulin  molecules  that  are  se- 
creted. The  high-level  expression  of  the  k  gene  is 
not  driven  by  the  intronic  enhancer;  instead,  an 
enhancer  located  9  kb  downstream  of  the  k  gene 
appears  to  be  the  dominant  element  in  plasma 
cells.  Therefore  k  expression  appears  to  be  con- 
trolled by  a  unique  dual  enhancer  system  during 
development:  the  intronic  enhancer  drives  low- 
level  expression  in  mature  B  cells,  while  the  3' 
enhancer  is  responsible  for  high-level  expression 
in  plasma  cells.  The  questions  we  wish  to  address 
in  this  system  are,  What  is  the  protein(s)  responsi- 
ble for  the  activity  of  the  3'  enhancer  in  plasma 
cells?  WTiat  is  the  signal  that  activates  the  3'  en- 
hancer? and  finally.  What  role  does  crosslinking 
of  the  surface  immunoglobulins  play  in  this  pro- 
cess? We  would  also  like  to  study  the  surface  im- 
munoglobulin complex  on  mature  B  cells  that  in- 
cludes proteins  such  as  mb-1  and  B-29  and 
elucidate  the  signal  transduction  pathway  that 
leads  to  the  activation  of  the  3'  enhancer. 


160 


Molecular  Genetics  of  Blood  Coagulation 


David  Ginsburg,  M.D. — Associate  Investigator 

Dr.  Ginsburg  is  also  Associate  Professor  in  the  Departments  of  Internal  Medicine  and  Human  Genetics 
at  the  University  of  Michigan  Medical  School.  He  received  his  B.A.  degree  in  molecular  biophysics 
and  biochemistry  from  Yale  University  and  his  M.D.  degree  from  Duke  University  School  of  Medicine. 
His  postdoctoral  research  training  was  done  in  the  laboratory  of  Stuart  Orkin  at  the  Children's 
Hospital,  Harvard  Medical  School.  While  in  Boston,  Dr.  Ginsburg  was  also  Instructor  in  Medicine 
at  Brigham  and  Women 's  Hospital,  Harvard  Medical  School. 


THE  major  research  activities  of  my  laboratory 
concern  two  important  blood  clotting  pro- 
teins, von  Willebrand  factor  and  plasminogen 
activator  inhibitor- 1,  and  their  associated  hu- 
man diseases.  In  addition,  we  are  applying  mo- 
lecular tools  to  the  study  of  bone  marrow 
transplantation. 

von  Willebrand  Factor 

One  major  function  of  von  Willebrand  factor 
(vWF) ,  which  is  an  important  part  of  the  body's 
blood  clotting  system,  is  to  serve  as  a  bridge  be- 
tween blood  platelets  and  the  wall  of  an  injured 
blood  vessel,  thereby  helping  to  control  bleed- 
ing. vWF  is  also  the  carrier  for  factor  VIII,  the 
missing  substance  in  patients  with  hemophilia. 
Abnormalities  in  vWF  result  in  von  Willebrand 
disease  (vWD),  the  most  common  inherited 
bleeding  disorder,  occurring  in  1-3  percent  of 
the  general  population.  Over  20  different  types 
of  vWD  have  been  described. 

Our  aim  is  to  understand  how  the  various  parts 
of  the  vWF  protein  work  in  the  body  and  how 
they  interact  with  other  factors  in  the  blood  clot- 
ting system.  Recently  we  have  made  considerable 
progress  in  identifying  the  defects  within  the 
vWF  gene  that  are  responsible  for  vWD.  In  addi- 
tion to  aiding  in  the  diagnosis  and  management  of 
vWD,  this  information  has  provided  important  in- 
sights into  the  function  of  vWF. 

Over  90  percent  of  patients  with  type  IIB  vWD 
have  one  of  four  specific  defects,  all  within  a 
small  region  of  the  vWF  gene  critical  for  its  inter- 
action with  blood  platelets.  By  introducing  one 
of  these  defects  into  the  DNA  of  tissue  culture 
cells,  we  have  shown  that  this  single  change  is 
responsible  for  the  type  IIB  variant.  In  similar 
studies  of  type  IIA  vWD,  we  have  identified  an- 
other set  of  defects  clustered  in  a  different  region 
of  the  vWF  gene.  We  have  introduced  these  de- 
fects into  cultured  cells  and  have  shown  that  type 
IIA  vWD  may  be  due  to  abnormalities  in  the  pro- 
cess whereby  vWF  is  manufactured  inside  the 
cell.  In  studies  of  a  patient  whose  vWF  is  unable 
to  bind  factor  VIII,  we  identified  a  single  change 
in  the  gene  that  has  helped  to  pinpoint  the  region 


of  vWF  responsible  for  factor  VIII  transport. 
We  have  also  identified  in  some  patients  with 
type  III  vWD  (the  most  severe  form)  a  defect  in 
the  ability  to  copy  the  vWF  gene  into  normal  mes- 
senger RNA. 

Despite  considerable  progress,  type  I  vWD,  the 
most  common  variant,  remains  a  mystery.  We 
have  recently  begun  a  new  project  to  study  the 
molecular  basis  for  a  disease  in  mice  that  closely 
resembles  type  I  vWD  of  humans.  Surprisingly,  it 
appears  that  the  defect  in  the  mouse  may  be  in  a 
gene  other  than  vWF.  If  we  are  successful  in  iden- 
tifying the  mutant  gene  in  the  mouse,  our  find- 
ings should  be  directly  transferable  to  the  study 
of  human  type  I  vWD.  Through  these  studies,  we 
hope  to  expand  our  understanding  of  vWF,  to  ad- 
vance our  ability  to  diagnose  and  classify  vWD, 
and  eventually  to  improve  the  medical  treatment 
for  this  common  human  disorder.  The  work  on 
von  Willebrand  factor  has  been  funded  in  part  by 
a  grant  from  the  National  Institutes  of  Health. 

Plasminogen  Activator  Inhibitor-1 

The  fibrinolytic  system  is  the  body's  mecha- 
nism for  breaking  down  blood  clots.  This  system 
must  be  precisely  balanced  with  the  clot-forming 
system,  since  an  imbalance  can  result  in  un- 
wanted blood  clotting  or  uncontrolled  bleeding. 
The  protein  that  turns  on  the  fibrinolytic  system 
is  plasminogen  activator.  Its  activity  is  controlled 
by  a  regulator  protein,  plasminogen  activator  in- 
hibitor! (PAI-1). 

Synthetic  plasminogen  activators,  such  as  re- 
combinant tissue-type  plasminogen  activator 
(t-PA)  and  urokinase,  are  now  used  in  patients  to 
dissolve  blood  clots,  particularly  in  the  early 
stages  of  a  heart  attack  when  a  major  blood  vessel 
to  the  heart  muscle  has  become  blocked.  There  is 
also  increasing  evidence  that  patients  with  abnor- 
mally high  blood  levels  of  PAI-1  (disrupting  the 
normal  clot-dissolving  activity  of  natural  plas- 
minogen activator)  are  at  particularly  high  risk 
for  heart  attacks  and  other  diseases  due  to  in- 
creased blood  clot  formation.  Thus  an  under- 
standing of  the  structure  and  function  of  PAI-1 
and  its  interaction  with  plasminogen  activators  is 


161 


Molecular  Genetics  of  Blood  Coagulation 


of  great  significance  in  the  study  of  the  fibrino- 
lytic system  and  its  role  in  a  number  of  important 
diseases. 

We  have  established  a  system  for  directing  bac- 
teria to  make  synthetic  PAI-1  in  the  test  tube.  This 
recombinant  PAI-1  has  all  of  the  important  prop- 
erties of  the  natural  protein.  We  have  made  over 
170  variants  of  PAI-1  in  which  a  small  part  of  the 
molecule  has  been  changed.  Studying  the  effects 
of  these  small  changes,  which  can  vary  consider- 
ably in  their  ability  to  regulate  the  different  types 
of  plasminogen  activators,  has  advanced  our  un- 
derstanding of  how  PAI-1  functions  and  how  it 
interacts  with  other  parts  of  the  blood  clotting 
system.  These  observations  might  eventually  aid 
in  the  design  of  new  drugs  for  the  treatment  of 
bleeding  and  clotting  diseases.  This  work  has 
been  funded  in  part  by  a  grant  from  the  National 
Institutes  of  Health. 

We  have  recently  identified  the  molecular  de- 
fect in  our  first  patient  with  complete  deficiency 
of  PAI-1 .  This  nine-year-old  girl  has  a  moderately 
severe  bleeding  disorder.  Studies  of  her  DNA  dem- 
onstrated complete  inactivation  of  the  gene,  ac- 
counting for  the  total  absence  of  PAI-1  in  her 
blood.  These  observations  have  provided  impor- 
tant insights  into  the  true  function  of  PAI- 1  and 
the  fibrinolytic  system  in  the  body.  Further  stud- 
ies will  provide  valuable  information  for  the  fu- 
ture treatment  of  this  and  other  patients  with 
PAI- 1  deficiency  and  for  studying  how  PAI- 1  actu- 


ally works  inside  the  body.  We  are  currently  at- 
tempting to  disrupt  the  PAI-1  gene  in  mice  to 
create  an  animal  model  for  this  human  disease. 

Bone  Marrow  Transplantation 

Bone  marrow  transplantation  is  being  used 
with  increasing  frequency  to  treat  a  variety  of  dis- 
eases, including  several  types  of  leukemia  and  a 
number  of  other  cancers.  In  this  procedure,  doses 
of  radiation  and  chemotherapy  are  given  that  are 
designed  to  destroy  the  patient's  diseased  bone 
marrow.  As  this  would  ordinarily  prove  fatal,  the 
patient  is  then  "rescued"  by  the  transplantation 
of  marrow  from  a  healthy  donor.  Our  laboratory 
has  had  a  long-standing  interest  in  bone  marrow 
transplantation  and  has  developed  techniques  to 
monitor  what  happens  to  the  blood — to  the  pa- 
tient's own  blood  cells  and  those  received  from 
the  donor — following  the  procedure. 

Currently  the  major  obstacle  to  the  more  wide- 
spread use  of  bone  marrow  transplantation  is  an 
often-fatal  complication  called  graft-versus-host 
disease  (GVHD).  This  is  caused  by  small  differ- 
ences between  the  genes  of  patient  and  donor 
that  cannot  yet  be  detected.  We  have  begun  a  new 
research  effort  to  identify  the  gene  or  genes  re- 
sponsible for  GVHD.  If  this  effort  is  successful, 
the  process  of  matching  patients  and  donors  will 
be  much  improved  and  bone  marrow  transplanta- 
tion may  become  considerably  safer  and  more 
widely  applied. 


162 


Uncovering  the  Molecular  Basis 
of  X-linked  Disorders 


Jane  M.  Gitschier,  Ph.D. — Assistant  Investigator 

Dr.  Gitschier  is  also  Associate  Professor  of  Medicine  ( Genetics)  at  the  University  of  California,  San 
Francisco.  She  received  a  B.S.  degree  in  engineering  science  from  Pennsylvania  State  University,  an  M.S. 
in  applied  physics  from  Harvard  University,  and  a  Ph.D.  in  biology  from  the  Massachusetts  Institute 
of  Technology.  She  did  postdoctoral  research  with  Richard  lawn  at  Genentech,  before  joining  the  faculty 
at  the  University  of  California. 


X-LINKED  disorders  result  from  mutations  in 
genes  on  the  X  cliromosome,  one  of  the  two 
sex  chromosomes  in  humans.  These  common  in- 
herited conditions  almost  exclusively  affect 
males  because  they  lack  the  protection  of  a  sec- 
ond, normal  X  chromosome.  Females,  though 
they  have  such  protection,  can  be  silent  carriers 
of  X-linked  diseases  and  genetically  transmit 
them  to  their  sons.  Consequently,  X-linked  dis- 
eases are  often  referred  to  as  "sex-linked." 

Our  laboratory  is  interested  in  uncovering  the 
molecular  biology  of  some  of  these  disorders.  We 
are  seeking  to  identify  both  the  genes  responsible 
for  several  diseases  of  unknown  biochemical 
basis  and  the  underlying  mutations. 

Much  of  our  effort  is  concentrated  on  a  particu- 
lar region  of  the  X  chromosome  called  Xq28,  the 
terminal  portion  of  the  chromosome's  long  arm. 
To  date,  24  inherited  disorders  have  been 
mapped  to  this  region,  which  consists  of  approxi- 
mately 9  million  base  pairs  of  DNA.  As  this  density 
of  disease  loci  is  unusually  high  compared  with 
that  found  in  other  regions  of  the  chromosome,  it 
suggests  that  Xq28  may  be  very  rich  in  genes. 

The  incidence  of  Xq28-linked  disorders  is  vari- 
able. Color  blindness  is  extremely  common,  af- 
fecting about  1  of  every  10  males.  However,  the 
majority  of  diseases  occur  infrequently,  making 
accurate  genetic  mapping  difficult.  These  rare 
diseases  are  clinically  diverse  and  of  unknown 
biochemical  basis.  They  include  Emery-Dreifuss 
muscular  dystrophy,  nephrogenic  diabetes  insi- 
pidus, adrenoleukodystrophy,  incontinentia  pig- 
menti,  and  dyskeratosis  congenita. 

We  are  attempting  to  isolate  genes  in  Xq28  and 
determine  whether  they  are  mutated  in  individ- 
uals affected  by  Xq28-linked  disorders.  To  date 
we  have  isolated  six  candidate  genes  and  have 
collected  5 1  patient  samples,  including  at  least 
one  example  of  most  of  the  rare  diseases.  In  some 
cases  the  sequence  of  the  cloned  gene  has  pro- 
vided a  clue  to  its  function  and  suggested  possi- 
ble involvement  in  a  particular  disease.  On  the 
other  hand,  one  of  the  isolated  genes  does  not 
appear  to  produce  a  protein.  Until  a  match  is 
made  between  candidate  gene  and  inherited  dis- 


ease, we  are  screening  all  patient  samples  for  mu- 
tations in  all  genes,  searching  for  gross  rearrange- 
ments in  genes  as  well  as  single-base  alterations. 

One  Xq28-linked  disorder  we  have  studied  in 
depth  is  classic  hemophilia,  or  hemophilia  A.  It 
results  from  mutations  in  the  gene  coding  for  a 
blood  coagulation  protein  called  factor  VIII.  The 
gene  was  identified  previously  because  part  of 
the  amino  acid  sequence  was  known.  Our  labora- 
tory is  interested  in  understanding  what  types  of 
mutations  lead  to  hemophilia  A  and  how  these  are 
generated. 

Hemophilia  is  well  suited  to  studies  on  muta- 
genesis because  it  is  clinically  heterogeneous, 
implicating  a  wide  variety  of  mutations  in  the  fac- 
tor VIII  gene.  Correlating  the  types  of  mutations 
with  particular  clinical  findings  may  be  very 
helpful  in  understanding  the  role  of  factor  VIII  in 
coagulation.  To  that  end  we  recently  completed  a 
study  of  mutations  that  lead  to  amino  acid 
changes  in  the  protein.  These  mutations,  which 
affect  a  single  base  pair  in  DNA,  were  revealed  by 
a  sensitive  technique  called  denaturing  gradient 
gel  electrophoresis.  By  comparing  the  amino  acid 
sequence  of  factor  VIII  with  sequences  from  evo- 
lutionarily  related  proteins,  we  were  able  to  infer 
that  some  mutations  are  likely  to  destroy  activity 
by  disrupting  the  protein's  structure,  while 
others  alter  amino  acids  that  play  a  role  unique  to 
factor  VIII  function. 

Hemophilia  can  occur  in  families  lacking  a  ge- 
netic history  of  hemophilia,  reflecting  newly  aris- 
ing mutations.  It  is  possible  to  uncover  the  origin 
of  factor  VIII  gene  mutations  in  these  cases.  In  the 
past,  mutations  were  assumed  to  occur  as  isolated 
events  in  either  eggs  or  sperm.  However,  we  have 
found  evidence  in  several  sporadic  cases  for  "mo- 
saicism"— i.e.,  a  mixture  of  cells  with  and  with- 
out mutation  within  the  individual.  These  results 
suggest  that  mutations  can  occur  during  embryo- 
logical  development.  In  addition,  these  findings 
demonstrate  that  the  unaffected  mother  of  a  child 
with  sporadic  disease  may  be  at  substantial  risk  of 
having  a  second  child  with  the  disease. 

Another  aspect  of  the  hemophilia-related  re- 
search has  been  a  continued  investigation  of  the 
factor  VIII  gene.  We  discovered  that  it  may  en- 


Uncovering  the  Molecular  Basis  of  X-linked  Disorders 


code  two  additional  proteins.  The  two  extra  mes- 
senger RNAs,  termed  factor  Vlll-associated  tran- 
scripts A  and  B,  emanate  from  an  intron,  one  of 
the  noncoding  regions  of  the  factor  VIII  gene. 
The  function  of  the  gene  is  unknown,  but  the 
RNAs  are  produced  at  high  levels  in  many  differ- 
ent cell  types,  suggesting  a  "housekeeping"  role. 
It  is  interesting  to  speculate  that  the  A  and  B  tran- 
seripts  influence  the  expression  of  factor  VIII  and 
that  mutations  in  them  may  in  turn  be  responsible 
for  some  instances  of  hemophilia. 

Finally,  we  are  attempting  to  create  a  hemophi- 


lic mouse.  Genes  on  the  X  chromosome  in  hu- 
mans are  located  on  the  X  in  other  mammals  as 
well,  and  hemophilia  has  been  recognized  in 
male  sheep,  dogs,  and  cats.  It  would  be  prefera- 
ble, however,  to  use  a  mouse  model  for  rapid 
testing  of  factor  VIII  products  and  for  future  gene 
therapy  experiments.  In  progress  are  experi- 
ments to  disrupt  the  normal  mouse  factor  VIII 
gene,  in  hopes  that  male  mice  bearing  the  disrup- 
tion will  have  hemophilia.  If  successful,  this  ap- 
proach will  be  extended  to  create  mouse  models 
for  other  Xq28-linked  disorders. 


164 


Membrane  Lipids  and  Cell  Regulation 


John  A.  Glomset,  M.D. — Investigator 

Dr.  Glomset  is  also  Professor  of  Medicine  and  Biochemistry  at  the  University  of  Washington  School  of 
Medicine.  He  received  his  M.D.  and  M.D. /Ph.D.  degrees  from  the  University  of  Uppsala,  Sweden.  He  then 
joined  the  Department  of  Medicine  at  the  University  of  Washington.  He  received  an  honorary  M.D.  degree 
from  the  University  of  Oslo  for  his  discovery  of  a  plasma  enzyme,  lecithin:cholesterol  acyltransferase 
(LCAT).  Dr.  Glomset  is  a  member  of  the  National  Academy  of  Sciences. 


ONE  of  the  major  research  areas  in  my  labora- 
tory concerns  the  phosphorus-containing 
lipids  (phospholipids)  of  animal  cell  mem- 
branes. In  particular,  we  have  been  focusing  at- 
tention on  the  biosynthesis  and  function  of  phos- 
pholipids that  contain  a  polyunsaturated  fatty 
acid,  arachidonic  acid.  This  fatty  acid  or  its  pre- 
cursor, linoleic  acid,  must  be  present  in  the  diet 
if  animals  are  to  reproduce,  develop,  grow,  and 
maintain  their  health.  The  basis  for  this  require- 
ment is  not  fully  understood,  but  most  of  the  ara- 
chidonic acid  that  becomes  available  to  animal 
cells  is  rapidly  incorporated  into  membrane  phos- 
pholipids. Furthermore,  in  response  to  various 
stimuli,  a  small  amount  of  the  arachidonic  acid  is 
released  from  the  phospholipids  and  converted 
into  products  that  can  trigger  many  different  cell 
functions. 

The  mechanisms  that  promote  the  incorpora- 
tion of  arachidonic  acid  into  cell  membrane 
phospholipids  remain  to  be  characterized,  but  it 
is  clear  that  the  metabolic  pathways  involved  are 
not  the  classical  ones  described  in  most  text- 
books. For  example,  phosphatidylinositol  (PI),  a 
phospholipid  that  plays  an  important  role  in  cell 
signaling,  typically  contains  high  amounts  of  ara- 
chidonic acid.  As  much  as  80  percent  of  the  PI 
present  in  animal  cell  membranes  consists  of  a 
molecular  species  that  contains  arachidonic  acid 
and  a  saturated  fatty  acid,  called  stearic  acid.  Ex- 
periments with  radioactive  precursors  have 
shown  that  cells  form  this  exceptional  species  of 
PI,  5n-l-stearoyl-2-arachidonoyl  (SA)  PI,  several 
hours  after  forming  Pis  that  contain  other  fatty 
acids. 

To  investigate  the  special  metabolic  pathway 
that  forms  SA  PI,  we  recently  examined  the  pos- 
sibility that  animal  cells  in  culture  might  be 
able  to  incorporate  a  radioactive  precursor,  sn-2- 
arachidonoyl  monoacylglycerol,  into  this  phos- 
pholipid. Our  experiments  showed  that  to  be  the 
case.  Furthermore,  follow-up  incubation  experi- 
ments with  cell  fractions  identified  two  new  en- 
zyme activities  that  seemed  to  be  involved.  A 
monoacylglycerol  kinase  activity  could  catalyze 
the  phosphorylation  of  sn-2-arachidonoyl  mono- 


acylglycerol to  form  the  phospholipid  sn-2- 
arachidonoyl  lysophosphatidic  acid,  and  a  stear- 
oyl  transacylase  could  convert  the  lysophospha- 
tidic acid  into  SA  phosphatidic  acid.  In  the 
presence  of  appropriate  cofactors,  other  enzymes 
could  convert  the  phosphatidic  acid  into  the 
corresponding  species  of  PI.  Thus  it  appeared 
that  we  had  discovered  a  new  pathway  of  PI 
biosynthesis. 

More  recent  experiments  have  suggested  that 
additional  steps  may  contribute  to  the  pathway. 
We  have  been  able  to  solubilize  the  stearoyl  trans- 
acylase from  membranes  and  examine  its  specific- 
ity. Surprisingly,  the  enzyme  can  use  SA  PI  as  a 
stearoyl  donor  in  the  transacylase  reaction.  When 
it  does,  a  major  product  of  the  reaction  is  sn-2- 
arachidonoyl  lysophosphatidylinositol.  If  the 
enzyme  forms  this  product  in  intact  cells,  the  ly- 
sophosphatidylinositol might  well  have  some  spe- 
cial function  or  metabolic  fate.  To  investigate  this 
possibility  we  are  currently  conducting  a  search 
for  enzymes  that  catalyze  the  conversion  of  lyso- 
phosphatidylinositol into  PI.  If  we  find  some,  we 
will  characterize  their  activities  in  order  to  de- 
fine the  pathway  more  completely.  Then  we  will 
attempt  to  localize  the  various  enzymes  within 
cells  in  order  to  explore  the  pathway's  intracellu- 
lar role. 

One  potential  role  of  pathways  that  form  ara- 
chidonic acid-containing  phospholipids  may 
relate  to  the  fine  structure  of  animal  cell  mem- 
branes. Thus  the  arachidonic  acid  in  phospho- 
lipids might  conceivably  affect  their  ability  to 
pack  tightly  with  one  another  in  membranes.  We 
began  to  investigate  this  possibility  some  time 
ago  in  parallel  with  our  studies  of  phospholipid 
biosynthesis.  We  used  a  computer-based  ap- 
proach to  examine  the  effects  of  arachidonic  acid 
on  the  conformation  and  packing  properties  of 
model  phospholipids  in  simulated  monolayers. 
The  results  of  this  molecular  modeling  approach 
have  suggested  that  arachidonic  acid-containing 
phospholipids  may  be  able  to  form  straight  con- 
formations and  pack  together  in  regular,  tight 
arrays. 

We  are  currently  attempting  to  test  this  possi- 
bility, in  collaboration  with  Howard  Brockman  of 


165 


Membrane  Lipids  and  Cell  Regulation 


the  Hormel  Institute.  If  we  find  that  semisyn- 
thetic arachidonic  acid-containing  phospho- 
lipids form  tightly  packed  arrays  in  experimen- 
tally produced  phospholipid  monolayers  and 
bilayers,  we  will  expand  our  experiments  to  in- 


clude animal  cell  membranes.  A  demonstration  of 
such  tightly  packed  arrays  in  cell  membranes 
would  raise  fundamental  questions  about  the  role 
of  phospholipid  domains  in  programming  mem- 
brane-associated events. 


Fruiting  bodies  of  the  slime  mold  Dictyostelium,  each  a  mass  of  spore  cells  supported  by  a  thin 
column  of  stalk  cells. 
Research  of  Richard  Gomer. 

166 


Determination  and  Maintenance  of  Cell  Type 


Richard  H.  Gomer,  Ph.D. — Assistant  Investigator 

Dr.  Gomer  is  also  Assistant  Professor  of  Biochemistry  and  Cell  Biology  at  Rice  University  and  Adjunct 
Assistant  Professor  of  Cell  Biology  at  Baylor  College  of  Medicine.  He  received  his  B.A.  degree  in  physics 
from  Pomona  College  and  his  Ph.D.  degree  in  biology  from  the  California  Institute  of  Technology.  He  was 
a  postdoctoral  fellow  in  the  laboratory  of  Richard  Firtel  at  the  University  of  California,  San  Diego. 


OUR  laboratory  is  interested  in  the  general 
problem  of  differentiation  and  morphogen- 
esis. We  are  trying  to  understand  at  a  molecular 
level  some  of  the  factors  that  determine  the  cell 
type  into  which  an  embryonic  cell  differentiates 
and  how  the  ratios  of  the  different  cell  types  are 
then  maintained  in  an  organism.  As  a  model  sys- 
tem, we  are  using  the  slime  mold  Dictyostelium 
discoideum. 

Dictyostelium  normally  exists  as  undifferen- 
tiated single  cells  called  amoebae  that  eat  bacte- 
ria in  soil  and  decaying  leaves  and  proliferate  by 
cell  division.  When  the  amoebae  eventually  over- 
grow their  food  supply  and  starve,  they  aggregate 
together  in  groups  of  about  100,000.  Roughly  80 
percent  of  these  cells  become  spores.  (A  spore  is 
a  cell  with  a  tough  outer  coat  that  forms  an 
"escape  capsule.")  The  remaining  20  percent  of 
the  cells  form  a  stalk  about  2  mm  high  that  holds 
the  spore  cells  off  the  ground.  A  spore,  dispersed 
by  the  wind,  will  crack  open  to  release  an 
amoeba,  which  may  luckily  find  itself  in  the 
midst  of  a  new  supply  of  bacteria.  The  advantage 
of  this  organism  is  its  simplicity:  cells  differen- 
tiate into  just  two  main  types. 

Determining  Cell  Fate 

In  the  presence  of  a  food  source,  Dictyoste- 
lium cells  grow  to  a  certain  size  and  then  separate 
their  chromosomes  to  opposite  sides  of  the  cell 
and  divide  in  two.  The  cycle  of  growth  and  divi- 
sion then  repeats.  In  a  field  of  cells,  there  will  be 
cells  at  all  different  phases  of  this  cycle. 

Dictyostelium  uses  a  simple  and  elegant  mech- 
anism based  on  this  cycle  to  determine  whether  a 
cell  will  become  stalk  or  spore.  When  the  cells 
starve,  those  cells  that  have  recently  separated 
their  chromosomes  and  divided  will  become 
stalk  cells;  the  remaining  cells  will  become 
spores.  As  long  as  the  cells  are  randomly  distrib- 
uted with  respect  to  the  phase  of  their  cell  cycles, 
there  will  always  be  the  proper  percentage  of 
cells  in  the  "stalk"  quadrant.  We  refer  to  this  as  a 
musical  chairs  mechanism,  since  the  decision  of 
any  given  cell  to  become  either  stalk  or  spore  is 


made  by  the  phase  of  the  cycle  that  the  cell  hap- 
pens to  be  in  at  the  time  of  the  differentiation 
signal  (starvation). 

Cell-type  choice  determination  mechanisms  of 
this  sort  may  operate  in  humans,  and  aberrations 
could  thus  lead  to  birth  defects.  We  are  develop- 
ing a  variety  of  techniques  that  use  DNA  inserted 
into  Dictyostelium  cells  to  identify  genes  in- 
volved in  cell-type  differentiation.  In  the  past 
year  we  have  engineered  DNA  constructs  to  mu- 
tate Dictyostelium  cells  three  ways:  by  having 
too  many  copies  of  a  sequence  of  DNA,  by  insen- 
ing  foreign  DNA  into  the  chromosomal  DNA,  and 
by  making  RNA  that  binds  to  and  renders  useless 
the  RNA  encoding  a  specific  protein.  In  prelimi- 
nary experiments  with  these  constructs,  we  have 
identified  mutants  that  may  have  abnormal  ratios 
of  the  two  cell  types.  In  addition,  to  examine  the 
extent  of  the  linkage  between  the  cell  cycle  and 
cell-type  choice,  we  have  treated  cells  with  drugs 
that  disrupt  the  cell  cycle.  These  experiments 
confirmed  that  the  cell-type  choice  mechanism  is 
linked  to  the  cell  cycle  and  does  not  use  a  sepa- 
rate timer. 

Sensing  Cell  Density 

During  Dictyostelium  development,  cells  turn 
on  the  stalk-  or  spore-specific  genes  only  when 
above  a  certain  cell  density.  Being  able  to  sense 
whether  other  cells  are  nearby  represents  a  para- 
digm for  possible  mechanisms  that  would  allow, 
for  instance,  liver  cells  or  others  to  sense  how 
much  of  the  body  is  composed  of  liver  cells.  At 
present,  little  is  known  about  the  molecular 
mechanisms  whereby  the  size  and  density  of  a 
tissue  are  sensed  by  individual  cells.  Such  mecha- 
nisms would  be  centrally  involved  in  the  regula- 
tion of  growth  during  development,  wound  heal- 
ing, and  regeneration.  In  addition,  an  important 
and  relevant  aspect  of  tumor  cells  is  that  they 
have  lost  their  ability  to  regulate  the  size  and/or 
density  of  the  tissue  and,  as  a  result,  proliferate. 
One  way  this  could  happen  would  be  if  the  tumor 
cells  could  no  longer  properly  sense  the  total 
mass  of  the  tissue. 


167 


Determination  and  Maintenance  of  Cell  Type 


In  experiments  funded  by  the  National  Insti- 
tutes of  Health,  we  have  found  that  Dictyoste- 
lium  cells  sense  whether  they  are  near  other 
starving  cells  by,  upon  starvation,  secreting  small 
quantities  of  a  protein  that  we  have  named  den- 
sity-sensing factor  (DSF).  Cells  are  sensitive  to 
DSF:  above  a  threshold  concentration  they  will 
express  cell-type-specific  genes.  We  have  done 
jheoretical  diffusion  calculations  and  have 
found,  in  agreement  with  our  observations,  that 
DSF  secreted  from  cells  that  are  quite  far  from 
other  DSF-secreting  cells  diffuses  away  so  quickly 
that  it  never  reaches  the  threshold  concentration. 
However,  at  a  sufficiently  high  density  of  starving 
cells,  the  DSF  concentration  will  be  above  the 
threshold  value.  We  have  purified  DSF  protein 
and  have  found  that  DSF  eventually  breaks  down 
to  small  protein  fragments  that  are  much  more 
effective  in  activating  differentiation.  This  might 
be  a  safety  mechanism  to  allow  cells  that  cannot 
find  other  starving  cells  to  stimulate  themselves 
and  perhaps  form  an  isolated  spore.  Interestingly, 


DSF  is  made  and  stored  in  growing  cells  and  is 
only  secreted  upon  starvation. 

We  have  cloned  and  sequenced  the  DNA  en- 
coding DSF.  This  has  been  used  in  turn  to  obtain 
the  sequence  of  amino  acids  for  the  DSF  protein. 
Computer  comparison  with  data  banks  of  other 
protein  sequences  indicates  that  DSF  is  not 
closely  related  to  any  known  protein.  We  have 
used  the  DSF  DNA  to  generate  Dictyostelium 
cells  that  do  not  make  DSF;  these  cells  do  not 
aggregate  unless  DSF  protein  is  added  to  their  me- 
dium. Dictyostelium  cells,  which  eat  bacteria 
that  are  almost  their  size,  starve  asynchronously. 
The  behavior  of  the  cells  lacking  DSF  suggests 
that  its  function  is  to  coordinate  development  by 
triggering  aggregation  when  most  of  the  cells  in  a 
given  area  have  starved,  as  signaled  by  DSF  secre- 
tion, and  are  ready  to  form  one  large  fruiting 
body.  Finally,  we  have  made  antibodies  that  will 
allow  us  to  examine  where  DSF  is  stored  in  Dic- 
tyostelium cells  and  to  search  for  similar  pro- 
teins in  other  organisms. 


These  two  photographs  show  the  expression  offasciclin  II  (brown  axon 
bundles)  in  the  developing  central  nervous  system  of  the  grasshopper 
(left)  and  T)Tosop\\i\2L  embryos  ( right).  Although  separated  by  well  over 
300  million  years,  these  two  insects  continue  to  express  the  protein  on 
a  specific  subset  of  longitudinal  axon  pathways.  The  brown  staining 
results  from  HRP  immunocytochemistry  using  anti-fasciclin  II  anti- 
bodies in  both  species. 

left  photograph  from  Allan  Harrelson  and  Corey  Goodman.  Right 
photograph  from  Gabriele  Grenningloh  and  Corey  Goodman. 


168 


Growth  Cone  Guidance  and  Neuronal 
Recognition  in  Drosophila 


Corey  S.  Goodman,  Ph.D. — Investigator 

Dr.  Goodman  is  also  Class  of  '33  Professor  of  Neurobiology  and  Genetics  in  the  Department  of  Molecular 
and  Cell  Biology  at  the  University  of  California,  Berkeley,  and  Adjunct  Professor  in  the  Department  of 
Physiology  at  the  University  of  California,  San  Francisco.  He  received  his  B.S.  degree  in  biology  from 
Stanford  University  and  his  Ph.D.  degree  in  developmental  neurobiology  from  the  University  of  California, 
Berkeley.  His  postdoctoral  work  in  developmental  neurobiology  was  done  at  the  University  of  California, 
San  Diego.  Prior  to  his  present  position,  Dr.  Goodman  was  a  faculty  member  at  Stanford  University.  His 
honors  include  the  Alan  T.  Waterman  Award  from  the  National  Science  Board. 


WE  are  interested  in  understanding  the  mo- 
lecular mechanisms  by  which  neuronal 
growth  cones  find  their  way  toward,  and  ulti- 
mately recognize,  their  correct  targets  during  de- 
velopment. Growth  cones  navigate  over  long  dis- 
tances and  often  through  a  series  of  complex 
choice  points,  appearing  to  follow  signals  on  the 
surfaces  of  cells  and  in  the  extracellular  environ- 
ment. Our  studies  are  aimed  at  uncovering  the 
molecules  and  mechanisms  that  impart  specific- 
ity to  the  developing  nervous  system  and  thus  al- 
low growth  cones  to  recognize  their  correct  path- 
ways and  targets.  To  address  these  issues,  we  use 
molecular  genetic  approaches  in  the  fruit  fly 
Drosophila. 


Guidance  of  Pioneers 

Within  and  just  outside  of  the  developing  cen- 
tral nervous  system  (CNS) ,  certain  glial  cells  and 
other  special  midline  cells  provide  instructive  in- 
formation for  the  differential  guidance  of  the  ini- 
tial, "pioneering"  growth  cones  as  they  choose 
which  cells  to  extend  toward  or  along.  For  exam- 
ple, a  specific  subset  of  cells  at  the  midline  ap- 
pears to  provide  an  attractive  cue  for  the  growth 
cones  that  extend  toward  the  midline  and  pio- 
neer the  commissural  axon  pathways  that  con- 
nect the  two  sides  of  the  developing  CNS.  Simi- 
larly, a  specific  pattern  of  longitudinal  glia 
appears  to  provide  an  important  substrate  for  the 
formation  of  the  longitudinal  axon  tracts  that 
connect  adjoining  segments  of  the  CNS.  We  have 
conducted  a  large-scale  screen  for  mutations  that 
perturb  the  guidance  of  pioneering  growth 
cones. 

Of  the  hundreds  of  new  mutations  identified  in 
this  screen,  mutations  in  three  genes  are  of  partic- 
ular current  interest.  Mutations  in  longitudinals 
lacking  (fold)  have  a  dramatic  phenotype.  al- 
though both  commissural  and  peripheral  path- 
ways are  normal,  as  are  most  other  aspects  of  em- 
bryogenesis,  the  CNS  of  these  mutants  lack  all 
longitudinal  axon  tracts.  In  lola  mutant  embryos, 
the  longitudinal  glia  are  born,  initially  migrate, 
and  divide  as  normal;  the  earliest  defect  is  seen 


about  the  time  that  the  first  growth  cones  contact 
these  glia  and  fail  to  extend  along  them. 

Mutations  in  the  second  gene,  commissure- 
less,  have  an  equally  dramatic  phenotype:  al- 
though all  other  axon  pathways  are  normal,  the 
CNS  of  these  mutants  lack  all  commissural  path- 
ways. They  also  have  normal  peripheral  nervous 
system  (PNS)  axon  pathways,  muscles,  sensory 
organs,  and  body  organization.  In  these  mutant 
embryos,  the  growth  cones  of  CNS  neurons  do  not 
extend  across  the  midline  and  commissures  never 
form,  although  other  aspects  of  embryonic  pat- 
tern formation  and  neuronal  development  appear 
normal.  The  commissureless  gene  product  is  a 
good  candidate  to  be  either  the  signal  or  the  re- 
ceptor for  the  guidance  of  growth  cones  toward 
the  midline. 

Mutations  in  a  third  gene,  roundabout  (robo) , 
lead  to  a  dramatic  misrouting  of  the  growth  cones 
that  normally  pioneer  the  MPl  pathway.  For  ex- 
ample, the  MPl  growth  cone  extends  across  the 
anterior  commissure  where  it  contacts  its  homo- 
logue  from  the  other  side,  rather  than  proceeding 
posteriorly.  In  contrast,  many  other  longitudinal 
pathways  develop  as  normal  in  robo  mutant 
embryos. 


Pathway  Recognition 

Once  the  initial  axon  pathways  are  established, 
the  predominant  guide  for  "follower"  growth 
cones  is  the  surface  of  the  earlier  axons  in  these 
pathways.  Growth  cones  are  able  to  distinguish 
one  particular  axon  bundle,  or  fascicle,  out  of  an 
array  of  many.  The  experimental  analysis  of  these 
phenomena  led  to  the  labeled  pathways  hypothe- 
sis, which  holds  that  axon  pathways  are  diff'eren- 
tially  labeled  by  recognition  molecules  that  en- 
able growth  cones  to  navigate  through  complex 
choice  points.  To  identify  such  recognition  mole- 
cules, we  used  an  immunological  approach  to 
identify  and  subsequently  clone  the  genes  encod- 
ing five  diff'erent  surface  glycoproteins:  fasciclin 
I,  fasciclin  II,  fasciclin  III,  fasciclin  IV,  and  neuro- 
glian  (a  sixth,  connectin,  has  recently  been 
cloned  using  a  different  method,  as  described  in 


169 


Growth  Cone  Guidance  and  Neuronal  Recognition  in  Drosophila 


the  following  section) .  Five  of  the  six  glycopro- 
teins are  dynamically  expressed  on  overlapping 
subsets  of  growth  cones,  axon  fascicles,  and  glia 
during  embryonic  development;  the  sixth,  neuro- 
glian,  is  more  broadly  expressed  on  the  surface  of 
most  axons  and  glia.  Fasciclin  II,  neuroglian,  and 
fasciclin  III  are  members  of  the  immunoglobulin 
superfamily;  connectin  is  a  member  of  the  leu- 
,cine-rich  repeat  family;  fasciclin  I  and  fasciclin 
rv  are  neither  related  to  each  other  nor  thus  far  to 
anything  else  in  the  data  bank.  Five  of  the  six 
proteins  (except  for  fasciclin  IV)  can  function  as 
homophilic  cell  adhesion  molecules. 

Genetic  analysis  has  shown  that  fasciclin  II  is 
indeed  a  neuronal  recognition  molecule  that 
plays  an  important  role  in  specific  growth  cone 
guidance.  The  fasciclin  II  protein  is  normally  ex- 
pressed on  a  subset  of  growth  cones  and  axons 
that  pioneer  and  selectively  fasciculate  in  the 
MPl  axon  fascicle;  later  it  is  expressed  on  several 
other  axon  pathways.  In  embryos  that  are  mutant 
for  the  fas  II  gene,  although  these  specific 
growth  cones  extend,  they  do  not  properly  recog- 
nize one  another  and  fail  to  fasciculate.  These 
studies  are  the  first  molecular  confirmation  of  the 
existence  of  functional  labels  on  specific  axon 
pathways  in  the  developing  nervous  system. 

A  major  current  goal  is  to  determine  the  molec- 
ular machinery  whereby  the  activation  of  fasci- 
clin II,  by  either  another  fasciclin  II  molecule  or 
a  different  ligand  on  the  surface  of  neighboring 
neurons,  instructs  a  fasciclin  Il-expressing 
growth  cone  to  extend  toward  and  along  these 
specific  cells.  We  have  established  a  highly  sensi- 
tive assay  that  utilizes  a  mutation  in  the  fas  II 
gene  that  reduces  the  amount  of  fasciclin  II  pro- 
tein to  the  minimal  amount  required  for  fas  II 
function.  Using  this  assay  we  are  looking  for  other 
genes  in  which  a  50  percent  reduction  in  the 
level  of  their  protein  product  results  in  a  failure 
of  fas  II  function.  In  this  way  we  are  in  the  pro- 
cess of  identifying  genes  that  appear  to  encode 
products  that  interact  with  the  fas  II  gene  and 
thus  function  in  the  events  of  neuronal 
recognition. 

Our  previous  studies  have  shown  that  the  ex- 
pression of  surface  recognition  molecules  is  dy- 
namic and  regional  on  the  surface  of  individual 
neurons;  i.e.,  parts  of  the  cell  are  differentially 
labeled  in  accordance  with  the  processes  around 
it  for  which  it  has  a  selective  affinity.  We  wish  to 
uncover  the  signals  that  instruct  a  neuron,  as  it 
navigates  from  one  pathway  to  another,  to  change 
the  expression  of  surface  recognition  molecules 
on  its  growth  cone.  For  example,  the  growth 


cones  of  many  commissural  interneurons  extend 
right  past  longitudinal  axon  pathways  on  their 
own  side;  once  they  cross  the  midline,  they  dis- 
play a  high  affinity  for  one  of  these  axon  pathways 
on  the  other  side  of  the  CNS.  This  change  in  the 
behavior  of  the  growth  cone  is  accompanied  by 
an  equally  dramatic  change  in  its  expression  of 
surface  recognition  molecules;  neurons  express 
different  molecules  on  the  surface  of  their  longi- 
tudinal and  commissural  processes.  We  are  inves- 
tigating whether  extension  across  the  midline  is 
required  for  growth  cones  to  make  the  appro- 
priate changes  in  surface  expression  and  subse- 
quent pathway  choices,  by  examining  the  behav- 
ior of  specific  growth  cones  in  commissureless 
mutant  embryos  in  which  guidance  toward  the 
midline  does  not  occur.  Thus  far  it  appears  that 
some  aspects  of  longitudinal  guidance  are  per- 
turbed in  these  mutant  embryos. 

Target  Recognition 

Having  navigated  along  a  series  of  pathways, 
growth  cones  are  ultimately  capable  of  recogniz- 
ing their  correct  target  cells.  In  the  Drosophila 
embryo,  the  specificity  of  neuronal  growth  cones 
for  target  cells  is  most  clearly  studied  in  the  abil- 
ity of  motoneuron  growth  cones  to  recognize  spe- 
cific muscle  fibers.  A  large-scale  molecular  ge- 
netic screen  has  led  to  the  identification  of 
several  genes  that  are  expressed  by  different  sub- 
sets of  undiff^erentiated  muscle  fibers  prior  to  in- 
nervation. A  new  gene,  connectin,  and  a  previ- 
ously identified  gene.  Toll,  encode  membrane 
proteins  that  are  members  of  the  leucine-rich  re- 
peat family  of  cell  adhesion  and  signaling  mole- 
cules. The  connectin  gene  encodes  a  protein  that 
is  expressed  on  the  surface  of  eight  (of  the  31) 
muscle  fibers  in  each  abdominal  segment  and  on 
the  grovvT:h  cones  and  peripheral  axons  of  the 
very  motoneurons  that  innervate  these  eight  mus- 
cle fibers.  The  growth  cones  of  this  subset  of  mo- 
toneurons, and  the  undifferentiated  muscle 
fibers  they  innervate,  express  the  connectin  pro- 
tein prior  to  contact  and  synapse  formation.  In 
vitro  studies  show  that  the  connectin  protein  is 
capable  of  functioning  as  a  homophilic  cell  adhe- 
sion molecule.  Thus  this  protein  is  a  good  candi- 
date to  be  a  bona  fide  target  recognition  molecule 
involved  in  the  formation  of  specific  synaptic 
connections.  A  genetic  analysis  of  connectin 
function  is  presently  in  progress.  In  addition,  a 
large-scale  genetic  screen  is  under  way  to  identify 
mutations  that  perturb  the  guidance  of  motoneu- 
ron growth  cones. 


170 


Mechanisms  of  Immunological  Self-Tolerance 
and  Autoimmunity 


Christopher  C.  Goodnow,  B.V.Sc,  Ph.D. — Assistant  Investigator 

Dr.  Goodnow  is  also  Assistant  Professor  of  Microbiology  and  Immunology  at  Stanford  University  School  of 
Medicine.  He  was  educated  in  the  United  States  and  Australia  and  received  B.S.  and  veterinary  degrees 
from  the  University  of  Sydney.  After  training  in  molecular  immunology  with  Mark  Davis  at  Stanford 
University,  he  returned  to  the  University  of  Sydney  to  complete  doctoral  and  postdoctoral  studies 
on  immunological  tolerance  in  the  laboratory  of  Antony  Basten. 


EACH  individual  B  and  T  lymphocyte  in  the 
immune  system  expresses  antigen  receptors 
of  one  type  on  its  surface,  which  confer  on  the 
cell  an  ability  to  recognize  one  of  the  millions  of 
different  antigens;  and  there  are  millions  of  dif- 
ferent lymphocytes  in  the  immune  system.  Given 
the  system's  annihilative  powers,  it  is  remarkable 
that  tissue  components  of  our  own  bodies  are 
spared  during  immunological  attacks  on  invading 
foreign  organisms.  Normally  the  immune  system 
can  recognize  one's  tissue  components  as  "self" 
and  tolerate  them.  Self-tolerance  is  lost,  however, 
in  a  variety  of  "autoimmune"  diseases,  such  as 
systemic  lupus  erythematosus,  type  I  diabetes 
mellitus,  and  rheumatoid  arthritis,  resulting  in 
inexorable  destruction  of  particular  organs  and 
tissues.  The  mechanisms  that  maintain  self- 
tolerance  in  healthy  individuals,  and  the  factors 
that  lead  to  its  breakdown  in  autoimmune  dis- 
ease, are  the  main  focus  of  our  laboratory. 

It  has  been  theorized  for  many  decades  that 
self-tolerance  might  somehow  result  from  the  si- 
lencing or  elimination  of  lymphocytes  bearing 
antigen  receptors  that  happen  to  recognize  self 
antigens.  To  determine  whether  this  idea  was 
correct,  however,  has  been  difficult,  since  it  is 
almost  impossible  to  track  the  life  of  any  one  cell 
among  millions. 

Advances  in  biotechnology,  in  particular  the 
advent  of  transgenic  mice,  have  opened  doors  to 
the  development  of  ways  to  follow  the  life  of  par- 
ticular immune  cells  in  vivo.  Transgenic  mice 
are  genetically  altered  at  the  outset  of  embryonic 
development  by  microinjecting  carefully  de- 
signed cassettes  (transgenes)  into  fertilized  oo- 
cytes. With  colleagues  in  the  laboratory  of  Antony 
Basten  at  the  University  of  Sydney,  we  produced 
transgenic  mice  in  which  most  of  the  B  lympho- 
cytes expressed  identical,  rather  than  widely  dif- 
fering, antigen  receptors. 

This  was  done  by  introducing  transgenes  that 
coded  for  a  single  antibody  molecule  (since  anti- 
body molecules  serve  as  antigen  receptors  on  B 
lymphocytes) .  The  particular  molecule  was  one 
that  recognized  and  bound  a  foreign  protein,  hen 
egg-white  lysozyme,  and  because  the  transgene 


was  expressed  in  essentially  all  the  B  lympho- 
cytes, they  all  now  recognized  lysozyme  in  an 
identical  fashion. 

The  extraordinary  abundance  of  lysozyme- 
binding  B  cells  in  the  transgenic  mice  has  made  it 
possible  to  track  the  development  and  fate  of 
these  cells  in  the  body.  To  determine  the  fate  of  B 
cells  that  might  happen  to  recognize  a  self  anti- 
gen rather  than  a  foreign  one,  we  prepared  addi- 
tional transgenic  mice  in  which  transgene  cas- 
settes led  to  the  synthesis  and  production  of 
lysozyme  by  the  mouse  itself.  When  the  two  types 
of  mice  are  mated,  a  fraction  of  their  offspring 
inherit  both  types  of  transgene  and  thus  contain 
large  numbers  of  lysozyme-binding  B  cells  that 
encounter  lysozyme  expressed  as  if  it  were  a  nor- 
mal self  constituent. 

The  self-reactive  B  cells  that  develop  in  these 
mice  undergo  one  of  three  distinct  fates.  First,  if 
lysozyme  is  present  at  too  low  a  concentration  or 
it  binds  to  the  B  cell's  receptors  with  too  low  an 
affinity,  no  signal  is  registered  by  the  cell.  As  a 
result,  the  B  cells  are  neither  activated  nor  ren- 
dered tolerant  but  remain  fully  capable  of  mount- 
ing an  antibody  response  if  they  receive  a 
stronger  stimulus  subsequently.  Second,  when 
higher  concentrations  of  soluble  lysozyme  are 
bound  with  sufficient  affinity,  the  B  cells  con- 
tinue to  follow  their  normal  maturation  program 
but  store  some  sort  of  negative  signal,  such  that 
they  are  unable  to  mount  an  antibody  response 
when  stimulated  to  do  so.  Third,  if  lysozyme  is 
encountered  in  a  form  where  it  is  displayed  on 
cell  surfaces,  and  thus  bound  by  the  B  cell  with 
extremely  high  avidity,  developing  B  cells  in  the 
bone  marrow  are  triggered  to  abort  their  matura- 
tion program  and  die. 

These  distinct  cellular  events  help  to  explain 
the  long-standing  observation  that  self-tolerance 
is  an  actively  acquired  but  incomplete  process 
and  suggest  a  number  of  possible  avenues  along 
which  autoimmunity  may  ultimately  develop. 
For  example,  our  laboratory  has  found  that  toler- 
ant B  cells  that  have  stored  a  negative  signal  can 
nevertheless  recover  the  ability  to  mount  an  effi- 
cient antibody  response.  Recovery  of  function 


171 


Mechanisms  of  Immunological  Self-Tolerance  and  Autoimmunity 


and  breakage  of  tolerance  depends,  however,  on 
a  balance  between  stimuli  that  promote  antibody 
formation  and  stimuli  that  reinforce  tolerance. 
One  line  of  research  in  our  laboratory  involves 
developing  transgenic  mouse  models  for  study- 
ing how  the  interplay  between  these  factors 
shapes  the  development  and  remission  of  autoim- 
mune disease  in  vivo.  This  research  is  funded  by 
the  National  Institutes  of  Health. 

Identifying  the  molecular  events  that  prevent 
tolerant  cells  from  mounting  an  antibody  re- 
sponse, or  lead  to  the  death  of  the  autoreactive 
cells,  will  be  crucial  for  fully  understanding  how 
self-tolerance  is  induced  and  maintained.  Our  ap- 
proach to  this  problem  is  severalfold.  First,  we 
are  continuing  to  delineate  the  precise  condi- 
tions under  which  tolerance  rather  than  activa- 


tion is  induced  in  B  cells  in  vivo.  Second,  we 
have  developed  tissue  culture  systems  that  repro- 
duce the  cellular  responses  observed  in  the 
whole  animal,  allowing  much  closer  scrutiny  of 
the  sequence  of  events  accompanying  tolerance 
induction.  In  autoreactive  B  cells  that  are  unable 
to  mount  an  antibody  response,  this  is  due  to  in- 
terference with  the  cell's  capacity  to  replicate 
and  to  differentiate  into  an  antibody-secreting 
cell.  For  B  cells  that  are  triggered  to  die,  the  pri- 
mary event  appears  to  be  an  arrest  of  cell  develop- 
ment at  an  immature,  short-lived  stage  of  develop- 
ment. Third,  we  are  using  a  combination  of 
biochemical,  molecular,  and  genetic  strategies  to 
search  for  the  key  differences  in  biochemical  sig- 
naling cascades  or  changes  in  gene  expression 
that  underlie  these  events. 


Self-reactive  B  lymphocytes  sorted  from  the  bone  marrow  and  visual- 
ized by  electron  microscopy.  The  antigen  receptors  on  these  cells  have 
already  been  triggered,  and  as  a  result  the  cells  are  destined  to  die 
within  a  few  days  in  vivo.  At  this  stage,  however,  the  process  is  revers- 
ible and  the  cells  show  none  of  the  ultrastructural  changes  that  precede 
programmed  cell  death. 

Research  of  Suzanne  Hartley  and  Christopher  Goodnow. 


172 


Developmental  Control  of  Gene  Expression 


Rudolf  Grosschedl,  Ph.D. — Assistant  Investigator 

Dr.  Grosschedl  is  also  Associate  Professor  of  Microbiology  and  Immunology  and  of  Biochemistry  and 
Biophysics  at  the  University  of  California,  San  Francisco.  He  completed  his  undergraduate  studies  on  the 
replication  of  lambdoid  bacteriophages  in  the  laboratory  of  Gerd  Hobom  in  Freiburg,  West  Germany.  His 
graduate  studies,  on  the  regulation  of  histone  gene  expression,  were  carried  out  in  the  laboratory  of  Max 
Birnstiel  in  Zurich,  Switzerland.  Dr.  Grosschedl  spent  his  postdoctoral  years  with  David  Baltimore  at  the 
Massachusetts  Institute  of  Technology  and  the  Whitehead  Institute. 


THE  process  of  terminal  differentiation  turns  a 
multipotential  cell  into  a  cell  that  carries  out 
a  particular  function  or  synthesizes  a  specific 
product.  The  lymphoid  B  cell  lineage  ultimately 
generates  a  cell  that  secretes  antibody.  During 
cell  differentiation,  genes  that  encode  the  anti- 
body or  associated  proteins  are  expressed  in  a  de- 
fined cell  type-specific  and  temporally  ordered 
pattern. 

Transcription  of  the  m  immunoglobulin  (Ig) 
gene  encoding  the  heavy  chain  of  the  antibody 
can  be  detected  in  virtually  all  lymphocytes.  By 
contrast,  the  k  immunoglobulin  light-chain  gene 
is  transcribed  only  in  late-stage  B  cells,  and  the 
mb  l  gene  encoding  an  antibody-associated  pro- 
tein is  expressed  only  in  early-stage  B  cells.  The 
goal  of  our  research  is  to  gain  some  insight  into 
the  molecular  mechanisms  that  mediate  the  de- 
velopmental control  of  lymphoid-specific  gene 
expression. 

Tissue-Specific  Regulation  of  Ig  Gene 
Expression  in  a  Transgenic  Model 

We  are  attempting  to  understand  how  multiple 
cis-acting  regulatory  information  of  a  gene  is  in- 
tegrated to  govern  the  correct  developmental 
pattern  of  expression.  Toward  this  goal,  we  trans- 
ferred rearranged  wild-type  and  mutated  Ig 
genes  into  the  mouse  germline  and  analyzed  ex- 
pression in  various  tissues  and  developmental 
stages.  Our  first  set  of  experiments  suggested  that 
lymphoid-specific  expression  is  dependent  upon 
multiple  tiers  of  regulation. 

We  found  that  the  intragenic  enhancer  directs 
high-level  expression  in  lymphoid  tissues  and  ba- 
sal expression  in  most  but  not  all  nonlymphoid 
tissues.  Basal  expression  appears  to  be  governed 
by  negative  regulation,  since  mutations  of  the 
juE5-  and  ME2-binding  sites  in  the  enhancer  in- 
creased At  gene  expression  in  many  nonlymphoid 
tissues  to  levels  similar  to  those  found  in  lym- 
phoid tissues.  However,  the  "off-state"  of  ex- 
pression observed  in  a  few  other  nonlymphoid 
tissues  was  not  affected  by  these  mutations,  sug- 
gesting that  another  tier  of  negative  regulation 


may  be  involved  in  further  decreasing  basal 
expression. 

An  additional  set  of  experiments  suggested  that 
the  Oct-binding  site  in  the  promoter  contributes 
to  the  positive  regulation  of  ^l  gene  expression 
during  B  cell  differentiation.  A  mutation  in  the 
Oct-binding  site  decreased  ti  gene  expression  in 
fetal  pre-B  cells  (which  represent  early  B  cells) 
by  a  factor  of  5,  but  decreased  ^L  gene  expression 
in  stimulated  splenocytes  (representing  late- 
stage  B  cells)  by  a  factor  of  100  to  200.  Together 
these  experiments  indicate  that  the  multiple 
modes  of  regulation  are  necessary  to  maximize 
the  difference  between  the  on-state  and  off-state 
of  gene  expression  in  vivo. 

In  a  second  set  of  experiments,  we  addressed 
the  regulation  of  the  accessibility  of  binding  sites 
for  nuclear  factors  in  native  chromatin.  With  the 
aim  of  uncoupling  changes  in  chromatin  struc- 
ture from  the  process  of  transcription,  we  linked 
DNA  fragments  comprising  the  m  enhancer  region 
to  the  promoter  for  bacteriophage  T7  RNA  poly- 
merase and  incorporated  the  gene  construct  into 
the  mouse  germline.  Subsequently,  we  examined 
the  accessibility  of  the  T7  promoter  in  isolated 
pre-B  cell  nuclei  by  adding  exogenous  T7  RNA 
polymerase  and  measuring  the  synthesis  of 
TT-specific  RNA. 

The  T7  promoter  was  accessible  in  7/7  lines 
when  linked  to  the  ti  enhancer.  By  contrast,  the 
T7  promoter  alone  was  only  weakly  accessible  in 
two  lines  and  not  accessible  in  six.  Because  the 
enhancer  fragment  used  in  this  experiment 
lacked  any  known  promoter  activity,  we  are 
currently  using  this  assay  to  examine  which  se- 
quences confer  accessibility  in  native  chromatin. 

Novel  Lymphoid-Specific  Regulators 
of  Gene  Expression 

To  identify  and  clone  novel  lineage-specific 
transcriptional  regulators,  we  adopted  two  ap- 
proaches. First,  we  included  in  our  analysis  of 
lymphoid-specific  gene  expression  the  mb-1 
promoter,  which  has  a  cell  type-specific  pattern 
of  activity  distinct  from  that  of  the  Ig  promoters. 
We  found  that  the  mb- 1  promoter  is  functional  in 


173 


Developmental  Control  of  Gene  Expression 


pre-B  and  surface  Ig-positive  B  cells  but  not  in 
later-stage  B  cells  or  T  cells.  In  our  study  of  the 
mb- 1  promoter,  we  identified  a  novel  nuclear 
factor,  termed  early  B  cell  factor  (EBF) . 

EBF  was  shown  to  bind  a  functionally  impor- 
tant sequence  in  the  distal  promoter  region.  The 
factor  was  found  to  be  present  specifically  in  pre- 
B  and  surface  Ig-positive  B  cells,  thus  paralleling 
the  pattern  of  activity  of  the  mb- 1  promoter.  Re- 
cently we  purified  EBF  to  homogeneity  and  ob- 
tained partial  amino  acid  sequences.  Based  on 
this  information,  we  isolated  cDNA  clones  that 
correspond  to  EBF.  Currently  we  are  characteriz- 
ing its  DNA-binding  and  regulatory  properties. 

The  second  approach  to  identify  lineage- 
specific  regulators  of  gene  expression  consisted 
in  differential  screening  of  a  pre-B  cell  (minus 
erythroid)  cDNA  library.  One  of  the  cDNA  clones 
was  found  to  encode  a  DNA-binding  protein  with 
homology  to  the  chromosomal  nonhistone  high- 
mobility  group  protein  HMG- 1  and  to  regulators 
of  cell  specialization.  This  pre-B  and  T  cell-spe- 
cific DNA-binding  protein,  termed  LEF-1  (lym- 
phoid enhancer-binding  factor  1),  was  found  to 
bind  a  functionally  important  site  in  the  T  cell 
receptor  (TCR)  a  gene  enhancer. 

LEF-1  was  shown  to  participate  in  the  regula- 
tion of  the  TCRa  enhancer.  First,  co-transfection 
of  cells  lacking  endogenous  LEF-1  with  TCRa  en- 
hancer-containing reporter  genes  together  with 
an  LEF-1  cDNA  expression  plasmid  increased  en- 
hancer function.  Second,  in  the  context  of  the 


intact  minimal  TCRa  enhancer,  the  LEF-1 -bind- 
ing site  is  required  for  full  enhancer  function. 

We  showed  that  the  HMG  domain  of  LEF- 1  en- 
compasses a  sequence-specific  DNA-binding  do- 
main that  interacts  with  the  minor  groove  of  DNA. 
Moreover,  we  defined  amino  acids  that  are  con- 
served among  various  members  of  the  family  of 
HMG-domain  proteins  as  residues  important  for 
DNA  binding. 

In  previous  experiments,  we  found  that  LEF-1 
by  itself  is  unable  to  augment  basal  promoter  ac- 
tivity. This  observation  raised  the  possibility  that 
LEF-1  functions  by  aiding  the  binding  and/or  ac- 
tion of  other  nuclear  factors.  Recently  we  demon- 
strated that  DNA  binding  by  the  HMG  domain  in- 
duces a  sharp  bend  in  the  DNA  helix.  Therefore, 
we  examined  whether  LEF- 1  -induced  DNA  bends 
can  facilitate  communication  between  proteins 
bound  at  distant  sites. 

We  replaced  one  binding  site  for  the  bacterial 
integration  host  factor  with  one  for  LEF- 1  in  the 
att?  locus  of  bacteriophage  X  and  found  that 
LEF- 1  is  capable  of  precisely  aligning  widely  sepa- 
rated integrase  (Int)  protein-binding  sites  and 
stimulating  Int-dependent  recombination.  These 
data  suggest  that  LEF-1  can  serve  as  an  "architec- 
tural" element  in  the  assembly  of  higher-order 
nucleoprotein  structures.  Currently  we  are  exam- 
ining the  role  of  DNA  bending  for  the  regulation 
of  TCRa  enhancer  function  and  are  attempting 
to  gain  insight  into  the  importance  of  LEF- 1  for 
lymphoid  differentiation  by  targeted  gene 
inactivation. 


174 


Polypeptide  Hormone  Gene  Regulation 


Joel  F.  Habener,  M.D. — Investigator 

Dr.  Habener  is  also  Professor  of  Medicine  at  Harvard  Medical  School  and  Associate  Physician  and  Chief 
of  the  Laboratory  of  Molecular  Endocrinology  in  the  Department  of  Medicine  at  Massachusetts  General 
Hospital,  Boston.  He  obtained  his  B.S.  degree  in  chemistry  at  the  University  of  Redlands  and  his  M.D. 
degree  at  the  University  of  California,  Los  Angeles.  After  medical  residency  training  at  the  Johns  Hopkins 
Hospital,  he  spent  two  years  in  research  at  the  National  Cancer  Institute.  Dr.  Habener  completed  his 
medical  training  in  endocrinology  and  metabolism  at  Massachusetts  General  Hospital. 


OUR  laboratory  seeks  an  understanding  of  the 
molecular  processes  involved  in  the  regula- 
tion of  gene  expression.  The  general  hypothesis 
being  tested  is  that  peptide  hormones  are  impor- 
tant regulatory  molecules  in  conveying  informa- 
tion among  cells  via  ligand-receptor  interactions 
and  corresponding  signal  transduction,  resulting 
in  the  expression  of  specific  genes.  These  pro- 
cesses are  important  in  determining  cellular  meta- 
bolic responses  such  as  secretory  activity,  cellu- 
lar differentiation,  and  growth. 

Peptides  in  Cellular  Metabolism 

Peptides  activate  metabolic  responses  in  cells 
by  way  of  interactions  with  specific  receptors  on 
distant  (endocrine),  adjacent  (paracrine),  or  the 
same  (autocrine)  cells.  These  ligand-receptor  in- 
teractions lead  to  the  activation  of  signal  trans- 
duction pathways  involving  postulated  cascades 
of  protein  phosphorylation  enzymatically  cata- 
lyzed by  protein  kinases,  eventuating  in  the  as- 
sembly of  active  transcriptional  complexes. 
Under  intensive  investigation  are  two  such  signal- 
ing pathways  mediated  by  cAMP-dependent  pro- 
tein kinase  A  and  by  phospholipid/diacylglyc- 
erol-stimulated  protein  kinase  C.  A  major  focus  of 
the  laboratory  is  to  understand  how  specific 
phosphorylated  DNA-binding  proteins  interact 
with  cognate  DNA  sequences  and  thereby  induce 
gene  expression. 

Genes  Encoding  Polypeptide  Hormones 

We  have  determined  the  structures,  organiza- 
tion, and  regulation  of  the  expression  of  genes 
encoding  several  of  the  polypeptide  hormones. 
Our  work  has  centered  on  the  genes  encoding  the 
glucagon  and  glucagon-related  peptides,  somato- 
statin, insulin,  angiotensinogen,  and  the  gonado- 
tropins. These  studies  have  progressed  through 
several  stages:  1)  cloning  of  the  genes  and  eluci- 
dation of  their  structures,  2)  determinations  of 
the  DNA  enhancer  and  suppressor  sequences  re- 
sponsible for  the  regulation  of  the  transcriptional 
expression  of  the  genes,  and  3)  isolation  and  char- 
acterization of  DNA-binding  proteins  involved  in 
the  regulation  of  expression.  These  studies  have 


led  to  the  identification  of  cell-specific  enhancer 
sequences  within  the  "promoter"  regions  of  the 
genes — sequences  that  determine  in  which  cel- 
lular phenorype  the  genes  are  expressed  and  how 
gene  transcription  responds  to  activator  sub- 
stances such  as  cAMP  and  phorbol  esters.  They 
have  also  led  to  the  identification  of  complex, 
cell-specific  post-translational  processing  of  pro- 
tein precursors  (prohormones)  encoding  the 
peptide  hormones. 

Recently  we  determined  that  specific  nuclear 
proteins  bind  to  these  important  DNA  enhancer 
sequences  and  that  the  binding  specificities,  as 
well  as  the  transactivation  activities,  of  these  pro- 
teins are  regulated  by  their  phosphorylation. 

Regulation  of  Glucagon  and  Somatostatin 
Gene  Expression 

Our  analyses  of  the  regulation  of  the  expres- 
sion of  the  glucagon  gene  in  pancreatic  islet  cell 
lines  reveal  that  islet  cell-specific  expression  re- 
sides in  at  least  two  enhancer-like  sequences  and 
that  A  cell  expression  in  the  islets  is  determined 
by  an  additional  enhancer/promoter  combina- 
tion. The  expression  of  the  somatostatin  gene 
is  restricted  to  the  D  cells  because  suppressor 
elements  prevent  expression  in  the  glucagon- 
producing  A  cells  and  insulin-producing  B  cells. 
Transcriptional  activation  of  the  glucagon  gene  is 
mediated  through  both  protein  kinase  C  and  pro- 
tein kinase  A  pathways,  whereas  activation  of  the 
somatostatin  gene  is  regulated  by  protein  kinase  A 
and  a  calcium-regulated  pathway. 

Cloning  and  Structure  of  a  cAMP-Dependent 
DNA-binding  Protein 

In  studies  of  the  somatostatin  and  gonadotro- 
pin genes,  we  have  determined  that  their  expres- 
sion is  stimulated  via  a  cAMP-dependent  signal 
transduction  pathway.  We  have  discovered  that 
DNA-binding  proteins  interact  with  specific,  short 
DNA  sequences  to  generate  cAMP-responsive 
complexes.  These  DNA-protein  complexes  that 
mediate  either  cAMP  or  phorbol  ester  control  of 
gene  transcription  share  certain  related  struc- 
tures. Cooperative  interactions  among  several 


175 


Polypeptide  Hormone  Gene  Regulation 


DNA-binding  proteins  and  with  adjacent  target 
DNA  sequences  appear  to  determine  cellular 
specificity  of  gene  expression  as  well  as  metaboli- 
cally  regulated  expression.  Recently  we  have 
cloned  several  members  of  a  family  of  cAMP- 
responsive  enhancer-binding  proteins  (CREBs) 
and  have  discovered  a  domain  on  one  of  the 
CREBs  that  is  phosphorylated  by  the  cAMP- 
dependent  protein  kinase  A,  as  well  as  by  addi- 
tional protein  kinases.  The  phosphorylation  of 
CREB  has  a  profound  effect  of  increasing  the  tran- 
sactivation  of  gene  transcription. 

Discovery  of  an  Insulinotropic  Peptide 

We  have  discovered  a  new  glucagon-like  pep- 
tide related  in  its  structure  to  pancreatic  gluca- 
gon and  co-encoded  with  glucagon  in  the  precur- 
sor protein  of  the  two  hormones  (proglucagon) . 
These  peptides  are  differentially  cleaved  from 
the  proglucagon,  the  initial  translation  product, 
in  the  pancreas  and  the  intestines.  One  of  the 
peptides,  glucagon-like  peptide  !  (7-37)  pro- 
duced in  the  intestine  and  released  in  response  to 
oral  nutrients  has  potent  insulinotropic  activi- 
ties. Concentrations  as  low  as  10""- 10"'^  M 
stimulate  insulin  secretion  in  the  perfused  rat 


pancreas  and  stimulate  both  cAMP  formation  and 
proinsulin  gene  transcription  and  mRNA  levels  in 
insulinoma  cell  lines.  We  have  determined  that 
the  glucagon-like  peptide  regulates  insulin  se- 
cretion in  humans  and  propose  that  it  may  be  in- 
volved in  the  pathogenesis  of  certain  types  of  dia- 
betes mellitus  due  to  a  faulty  regulation. 
Particular  emphasis  is  on  analyses  of  the  |8-cell 
receptor  for  the  glucagon-like  peptide  and  the 
mechanisms  operative  in  the  stimulation  of  insu- 
lin gene  transcription  in  response  to  the  actions 
of  the  peptide. 

Our  goals  are  1)  to  characterize  further  the 
genes  encoding  the  regulatory  (DNA-binding) 
proteins  that  interact  with  tissue-specific  en- 
hancers and  to  determine  how  cAMP-mediated 
expression  of  the  peptide  hormone-encoding 
genes  is  regulated  in  the  specific  cellular  pheno- 
types  and  2)  to  test  further  the  new  glucagon-like 
peptides  for  their  potential  regulatory  actions 
within  the  pancreatic  islets  and  the  intestinal 
tract  and  to  explore  the  possible  role  of  the  pep- 
tides in  diabetes  mellitus. 

Some  of  these  studies  were  also  supported  by 
funds  from  the  National  Institutes  of  Health. 


176 


Structural  Studies  of  Macromolecular  Assemblies 


Stephen  C.  Harrison,  Ph.D. — Investigator 

Dr.  Harrison  is  also  Professor  of  Biochemistry  and  Molecular  Biology  at  Harvard  University  and  Research 
Associate  in  Medicine  at  the  Children's  Hospital,  Boston.  He  received  his  A.B.  degree  in  chemistry 
and  physics  from  Harvard  College  and  his  Ph.D.  degree  in  biophysics  from  Harvard  University. 
Dr.  Harrison  is  a  member  of  the  National  Academy  of  Sciences. 


HOW  do  regulatory  proteins  activate  or  in- 
hibit transcription  of  particular  genes?  How 
do  viruses  leave  and  enter  cells?  How  do  recep- 
tors and  their  ligands  cycle  from  cell  surface  to 
ceil  interior  and  back?  These  questions  deal  with 
molecular  recognition  in  the  determination  of 
cell  organization.  They  represent  groups  of  proj- 
ects in  our  laboratory,  all  of  which  involve  eluci- 
dation of  detailed  atomic  structures  as  a  prerequi- 
site for  tackling  functional  problems. 


Transcriptional  Regulatory  Complexes 

A  common  characteristic  of  eukaryotic  tran- 
scriptional regulatory  elements  is  the  presence  of 
sites  in  multiple  copies  varying  slightly  in  se- 
quence, often  with  two  or  more  related  proteins 
that  can  bind  to  them.  The  best-understood  pro- 
karyotic  paradigm  is  in  the  immunity  region  of 
temperate  bacteriophages,  where  two  proteins, 
repressor  and  Cro,  bind  two  sets  of  three  sites, 
with  appropriately  graded  affinities.  We  have 
made  an  effort  to  understand  the  mechanism  of 
this  regulatory  switch,  by  determining  the  struc- 
tures of  a  series  of  specific  protein/DNA  com- 
plexes containing  the  Cro  protein  of  phage  434 
or  the  DNA-binding  domain  of  its  repressor.  We 
are  using  computational  approaches  to  link  ob- 
served structural  differences  among  these  various 
complexes  with  the  corresponding  free  energies 
of  binding. 

We  are  studying  structural  aspects  of  eukaryo- 
tic transcriptional  regulation,  initially  by  examin- 
ing the  DNA-binding  domains  of  regulatory  pro- 
teins in  complex  with  synthetic  binding  sites. 
GAL4,  the  prototype  of  a  class  of  such  proteins,  is 
a  regulator  of  galactose  metabolism  in  yeast.  It 
binds  as  a  dimer  to  the  upstream  activity  se- 
quences of  several  genes  involved  in  galactose 
and  melibiose  catabolism.  The  DNA-binding  re- 
gion is  at  the  amino  terminus  of  the  881 -residue 
polypeptide  chain.  We  have  determined  the 
structure  of  a  fragment  containing  residues  1-65, 
in  a  specific  complex  with  DNA.  A  small  domain 
containing  zinc  ions  (residues  8-40)  recognizes 


a  conserved  CCG  triplet  at  each  end  of  the  17- 
base  pair  binding  site,  through  direct,  major- 
groove  contacts.  A  short  a-helical  coiled-coil  ele- 
ment imposes  twofold  symmetry.  A  segment  of 
extended  polypeptide  chain,  linking  the  metal- 
binding  module  to  the  dimerization  element, 
specifies  the  length  of  the  site.  The  complex  has  a 
relatively  open  structure,  which  would  allow  an- 
other protein  to  bind  coordinately  with  GAL4 .  Co- 
ordinate binding  of  two  or  more  proteins  has 
been  shown  to  be  an  important  feature  of  many 
eukaryotic  control  elements. 

GCN4,  also  a  yeast  transcriptional  regulator, 
represents  yet  another  class  of  DNA-binding  pro- 
teins. It  contains  a  dimerization  element,  gener- 
ally called  a  leucine  zipper,  which  forms  an 
a-helical  coiled  coil  about  30  residues  in  length. 
This  segment  is  preceded  in  the  protein  sequence 
by  a  positively  charged  region,  which  has  little 
ordered  structure  in  the  free  protein  but  which 
also  acquires  a-helical  structure  when  it  binds 
to  DNA.  We  have  prepared  crystals  of  the  basic 
region/leucine  zipper  fragment  of  GCN4,  in 
complex  with  a  synthetic  binding  site  (having  a 
so-called  AP- 1  sequence) .  The  structure  determi- 
nation is  nearly  complete.  Preliminary  analysis 
shows  that  each  chain  is  a  continuous  a-helix. 
The  part  corresponding  to  the  basic  region  lies 
along  the  major  groove,  and  contacts  can  be  made 
by  suitable  side  chains  to  four  base  pairs  on  either 
side  of  a  central  GC. 

TflllA,  which  controls  5S  RNA  transcription  in 
Xenopus,  represents  the  so-called  zinc  finger 
class  of  regulatory  proteins.  The  finger  is  a  small, 
30-residue  domain,  stabilized  by  a  tightly  bound 
zinc  ion.  A  recombinant  fragment  comprising 
seven  of  the  nine  fingers  from  TflllA  binds  to  a 
30-base  pair  DNA  containing  an  appropriate  part 
of  the  total  binding  site,  and  we  have  crystallized 
this  complex.  The  fragment  also  binds  specifi- 
cally to  5S  RNA. 

Understanding  how  these  various  structures 
recognize  their  DNA  sites  is  only  a  beginning.  The 
specificities  of  interactions  between  other  do- 
mains of  these  proteins  and  additional  compo- 


177 


Structural  Studies  of  Macromolecular  Assemblies 


nents  of  a  transcriptional  initiation  complex  pres- 
ent even  more  challenging  puzzles  for  the  future. 

Viruses 

The  small,  double-stranded  DNA  viruses  SV40 
(simian  virus  40)  and  polyoma  have  given  us  a 
first  glimpse  of  virus  particles  that  package  a  mini- 
chromosome  in  one  cell  and  deliver  it  to  the  nu- 
cleus in  another.  The  shells  of  these  viruses  are 
composed  of  72  pentamers  of  the  major  struc- 
tural protein  VPl  and  30-60  copies  each  of  two 
internal  proteins,  VP2  and  VP3.  These  compo- 
nents package  the  viral  DNA.  The  VPl  polypep- 
tide chain  folds  in  such  a  w^ay  that  two  large 
j8-sheets  with  radially  directed  strands  form  a 
framework,  with  very  tight  interactions  between 
adjacent  subunits  in  a  pentamer.  The  carboxyl 
terminus  of  VPl  forms  an  extended  arm  that  in- 
teracts with  subunits  of  another  pentamer,  gener- 
ating three  kinds  of  interpentamer  contact  in  the 
virus  particle.  This  tying  together  of  standard 
building  blocks  allows  for  the  required  variabil- 
ity in  packing  geometry  without  sacrificing  speci- 
ficity. Flexibly  extended  arms,  which  form  or- 
dered structures  only  when  the  units  assemble 
into  a  particle,  appear  to  be  an  important  feature 
of  complex  assemblies. 

A  recently  determined  structure  for  the  murine 
polyoma  virus  shows  that  an  important  surface 
loop  is  larger  in  the  polyoma  subunit  than  in 
SV40.  Mutational  evidence  suggests  that  this  loop 
creates  a  shallow  pocket  for  binding  sialic  acid, 
required  for  cell  entry  by  polyoma  but  not  by 
SV40.  A  number  of  viruses  of  various  structural 
types  use  cell-surface  sialic  acid  for  attachment, 
and  it  is  of  broad  interest  to  understand  how  spe- 
cific viruses  accomplish  the  interaction.  Compar- 
ison of  SV40  and  polyoma  shows  that  this  func- 
tion can  readily  be  added  or  lost  by  small  changes 
at  the  surface  of  a  viral  coat  protein. 

We  have  recently  begun  to  study  the  double- 
stranded  RNA  viruses.  Crystals  of  rotavirus  single- 
shelled  particles  and  reovirus  cores  diflfract  to 
at  least  7-A  resolution.  These  particles  are  elab- 
orately organized  molecular  machines,  con- 


taining the  complete  transcription  and  RNA- 
modification  activities. 

Receptors 

The  receptor  for  human  immunodeficiency 
virus  (HIV)  is  the  lymphocyte  surface  antigen 
CD4.  Its  extracellular  portion  is  composed  of 
four  immunoglobulin-like  domains.  We  have  de- 
termined the  atomic  structure  of  a  two-domain, 
amino-terminal  fragment,  which  binds  HIV  as 
tightly  as  does  the  intact  receptor.  The  first  two 
domains  are  joined  by  a  continuous  (8-strand  con- 
nector, and  they  have  an  extensive  hydrophobic 
interface.  Thus  they  form  a  rigid,  rod-like  seg- 
ment. The  HIV-binding  site  appears  to  be  a  ridge 
along  one  edge  of  the  first  domain.  Binding  and 
mutational  studies,  carried  out  collaboratively, 
show  that  a  projecting  phenylalanyl  side  chain  is 
critical  for  the  interaction  with  HIV  gpl20  and 
that  various  positively  charged  residues  sur- 
rounding it  are  also  important.  The  same  region 
appears  to  be  involved  in  contacts  with  class  II 
MHC  (major  histocompatibility  complex)  mole- 
cules. We  have  also  crystallized  a  four-domain 
fragment  of  CD4,  corresponding  to  the  entire  ex- 
tracellular region,  and  the  structure  determina- 
tion is  in  progress. 

Many  important  receptors  are  taken  up  into 
the  cell  by  a  process  of  endocytosis  mediated 
by  clathrin-coated  vesicles.  The  transferrin  re- 
ceptor is  one  such  molecule.  It  undergoes  a  well- 
characterized  cycle  of  uptake  and  return  to  the 
cell  surface.  We  have  crystallized  the  extracellu- 
lar domain,  which  makes  up  about  three-fourths 
of  the  molecule.  This  domain  exhibits  reversible 
conformational  changes  at  low  pH  that  we  be- 
lieve to  be  significant  for  intracellular  sorting 
steps.  The  determination  of  the  structure  has  re- 
cently been  facilitated  by  the  observation  that 
much  better  diffraction  data  can  be  collected  by 
studying  crystals  at  liquid-nitrogen  temperatures. 
Such  freezing  techniques  are  now  being  broadly 
applied  in  our  laboratory,  enhancing  our  ability 
to  study  radiation-sensitive  crystals  of  very  com- 
plex structures. 


178 


Control  of  Gene  Expression  During  the  Cell  Cycle 
and  Development  of  the  Mammalian  Cerebellum 


Nathaniel  Heintz,  Ph.D. — Investigator 

Dr.  Heintz  is  also  Professor  at  the  Rockefeller  University.  He  received  his  Ph.D.  degree  at  the  State 
University  of  New  York  at  Albany,  where  he  studied  the  genetics  and  biochemistry  of  bacteriophage  SPOl 
gene  expression.  During  postdoctoral  studies  with  Robert  Roeder  at  Washington  University,  St.  Louis, 
he  initiated  his  work  on  histone  gene  expression  during  the  cell  cycle.  Continuation  of  these  studies 
and  examination  of  the  developing  mammalian  cerebellum  are  his  current  research  interests. 


OUR  studies  are  focused  on  the  identification 
of  molecular  mechanisms  controlling  gene 
expression  during  the  cell  cycle  and  in  the  devel- 
oping cerebellum.  The  elucidation  of  these  mech- 
anisms should  provide  fundamental  insights  into 
the  biological  transitions  that  underlie  the  con- 
trol of  cell  division  and  the  development  of  the 
mammalian  central  nervous  system. 

Control  of  Gene  Expression  During 
the  Cell  Cycle 

Transcription  of  histone  genes  during  the  S 
phase  of  the  eukaryotic  cell  cycle  is  achieved 
through  the  agency  of  subtype-specific  consensus 
elements  within  histone  gene  promoters  and 
their  cognate  distinct  transcription  factors.  Co- 
ordinate transcription  of  this  gene  family  is  ac- 
complished through  biochemically  distinct  tran- 
scription factors;  this  suggests  pleiotropic 
regulatory  mechanisms  that  control  the  activities 
of  these  factors  during  the  cell  cycle.  We  wish  to 
identify  these  mechanisms  at  the  molecular  level 
and  elucidate  their  importance  for  cell-cycle  pro- 
gression. Identification  of  such  mechanisms  may 
provide  highly  specific  targets  for  intervention  in 
cell  growth. 

Recently  we  have  examined  the  post-transla- 
tional  modifications  to  the  transcription  factor 
Octl,  which  participates  in  histone  H2b  expres- 
sion during  the  cell  cycle.  Both  monoclonal  and 
polyclonal  antisera  have  been  used  to  analyze 
changes  in  phosphorylation  of  Octl  as  cells  pro- 
ceed to  division.  We  have  discovered  that  multi- 
ple forms  of  Octl  exist  in  the  cell,  and  their  dis- 
tribution is  dramatically  regulated  during  the  cell 
cycle. 

Further  analysis  of  Octl  phosphorylation  has 
established  that  late  in  the  cell  cycle  this  tran- 
scription factor  appears  to  be  a  substrate  for  both 
CDC2  kinase  and  protein  kinase  A  (PKA).  Phos- 
phorylation of  Octl  by  PKA  occurs  within  its 
DNA-binding  domain  and  results  in  loss  of  DNA 
binding  in  vitro.  This  correlates  with  a  signifi- 
cant decrease  in  Octl  DNA  binding  during  mito- 
sis in  vivo.  These  results  indicate  that  modifica- 
tion of  Octl  during  the  cell  cycle  results  in 


modulation  of  its  function.  Inactivation  of  Octl 
by  phosphorylation  during  mitosis  also  provides 
a  possible  explanation  of  the  long-standing  ob- 
servation that  transcription  is  generally  sup- 
pressed during  this  time  in  the  cell  cycle.  Our 
present  efforts  are  to  extend  this  analysis  to  the 
histone  HI  transcription  factor  H1TF2,  to  deter- 
mine whether  its  control  during  the  cell  cycle 
may  occur  through  the  same  molecular  mecha- 
nisms. Demonstration  that  the  timing  and  nature 
of  the  post-translational  modifications  on  Octl 
and  H1TF2  are  similar  in  vivo  would  prove  the 
existence  of  the  proposed  pleiotropic  molecular 
mechanism  for  regulation  of  transcription  during 
the  S  phase. 

A  question  that  has  arisen  from  these  studies  is 
whether  S-phase-specific  transcription  and  DNA 
replication  are  mechanistically  coupled.  To  ad- 
dress this  issue  we  have  focused  on  two  specific 
questions:  Are  the  regulatory  proteins  for  S-phase 
histone  gene  transcription  directly  involved  in 
DNA  synthesis?  Might  proteins  that  regulate  DNA 
synthesis  at  specific  chromosomal  origins  of  repli- 
cation be  activated  by  the  same  mechanisms  that 
modulate  those  transcription  factors? 

In  collaboration  with  Nicholas  Heintz  (Univer- 
sity of  Vermont)  and  Lisa  Dailey  (Rockefeller  Uni- 
versity) we  have  identified  a  cellular  protein 
complex  with  several  properties  expected  of  rep- 
lication-initiation factors.  We  have  recently  pre- 
pared antibodies  and  obtained  primary  amino 
acid  sequences  from  one  of  these  proteins 
(RIP60)  and  are  using  these  tools  to  determine 
whether  this  factor  participates  in  cellular  DNA 
synthesis.  Analysis  of  this  factor  during  the  cell 
cycle  may  help  answer  the  two  questions  posed 
above. 

Development  of  the  Mammalian  Cerebellum 

The  mammalian  cerebellum  is  a  complex  and 
highly  stereotyped  structure  in  which  major  pat- 
tern formation  and  functional  organization  occur 
postnatally.  The  precise  description  of  the  cellu- 
lar events  occurring  during  cerebellar  develop- 
ment, and  the  existence  of  many  mutant  mouse 
strains  in  which  normal  development  of  the  cere- 


179 


Control  of  Gene  Expression  During  the  Cell  Cycle  and  Development 
of  the  Mammalian  Cerebellum 


bellum  is  perturbed,  recommend  it  as  an  amena- 
ble system  for  molecular  analysis  of  central  ner- 
vous system  development.  Our  initial  interests  in 
this  area  have  been  to  identify  genes  that  are  ei- 
ther essential  for  normal  development  of  the  cere- 
bellum or  that  serve  as  molecular  markers  for  spe- 
cific developmental  events  that  occur  during  its 
formation.  Our  ultimate  goal  is  to  utilize  these 
genes  to  identify  novel  proteins  that  are  crucial  to 
proper  development  of  the  cerebellum  and  to 
identify  molecular  mechanisms  that  participate 
in  specific  developmental  events  by  analysis  of 
the  pathways  that  result  in  their  correct  spatial 
and  temporal  expression. 

To  identify  genes  that  are  required  for  normal 
development  or  maintenance  of  cerebellar  struc- 
ture and  function,  we  have  initiated  efforts  to 
clone  the  genes  responsible  for  several  neurologi- 
cal mutants  of  mice.  Our  most  significant  pro- 
gress has  been  in  studies  concerning  the  Lurcher 
{Lc)  and  meander  tail  (mea)  loci.  Lc  is  a  semi- 
dominant  mutation  that  results  in  death  of  essen- 
tially all  cerebellar  Purkinje  cells,  beginning  at 
about  two  weeks  of  age.  Secondary  loss  of  cere- 
bellar granule  cells  and  olivary  neurons  is  also 
observed.  We  have  constructed  a  detailed  genetic 
map  surrounding  the  Lc  locus  on  chromosome  6 
and  have  identified  an  RLFP  (restriction  fragment 
length  polymorphism)  marker  approximately 
0.5  cM  from  the  gene.  Genomic  sequences  from 
this  closely  linked  marker  were  used  to  screen  a 
yeast  artificial  chromosome  (YAC)  library  from 
Shirley  Tilghman  (HHMI,  Princeton  University), 
resulting  in  isolation  of  a  280-kilobase  YAC  that 
maps  to  chromosome  6.  Using  sequences  isolated 
from  this  YAC  and  informative  recombinants  gen- 
erated during  genetic  mapping  of  the  Lc  locus, 
we  have  begun  a  chromosomal  walk  toward  the 
Lc  gene. 

The  gene  mea  is  a  recessive  mutation  resulting 
in  gross  perturbations  of  cerebellar  cytoarchitec- 
ture  that  are  confined  to  the  anterior  lobes  of  the 


cerebellum.  The  sharp  boundary  between  the 
normal  and  affected  area  of  the  mea/mea  cere- 
bellum is  reminiscent  of  the  discrete  boundaries 
evident  in  many  Drosophila  developmental  mu- 
tants, suggesting  that  the  mea  gene  may  influ- 
ence compartmental  cellular  organization  in 
mammalian  brain.  In  this  case  we  have  also  con- 
structed a  detailed  genetic  map  surrounding  the 
mea  gene  on  chromosome  4  and  have  begun  ef- 
forts to  identify  appropriate  genomic  sequences 
to  begin  isolation  of  YAC  clones  containing  the 
mea  locus.  The  identification  of  genes  responsi- 
ble for  these  and  other  mouse  neurological  muta- 
tions should  provide  fundamentally  important 
insights  into  cerebellar  structure  and  function. 

During  the  past  year  we  have  continued  to  pur- 
sue several  different  strategies  to  identify  cDNA 
clones  that  are  cell  specific  and  developmentally 
regulated  in  the  cerebellum.  Using  both  subtrac- 
tive  hybridization  and  differential  screening 
methods,  a  large  number  of  novel  cDNA  clones 
have  been  isolated  and  are  presently  being 
analyzed. 

One  particularly  successful  strategy  has  fo- 
cused on  the  use  of  antisera  to  purified  granule 
neuron  precursors  to  isolate  genes  that  define 
stages  in  granule  cell  differentiation  (in  collabo- 
ration with  Mary  Beth  Hatten,  Columbia  Univer- 
sity) .  A  large  number  of  novel  granule  cell  cDNAs 
have  been  isolated  that  define  discrete  stages  of 
granule  cell  neurogenesis.  One  biological  insight 
gained  in  these  studies  is  the  existence  of  a  tran- 
sient stage  in  development  of  granule  cell  neu- 
rons that  occurs  just  as  these  cells  complete  their 
migratory  journey  from  the  external  germinal 
layer  to  the  internal  granule  layer  of  the  develop- 
ing mouse  cerebellum.  Since  this  step  in  granule 
cell  differentiation  has  not  been  noted  before,  it 
will  be  interesting  to  determine  its  function,  its 
relevance  to  neurogenesis  in  other  areas  of  the 
central  nervous  system,  and  the  role  of  the  genes 
expressed  uniquely  at  this  stage  in  granule  cell 
development. 


180 


Structural  Biology  of  CD4  and  CDS  Involvement 
in  the  Cellular  Immune  Response 


Wayne  A.  Hendrickson,  Ph.D. — Investigator 

Dr.  Hendrickson  is  also  Professor  of  Biochemistry  and  Molecular  Biophysics  at  Columbia  University 
College  of  Physicians  and  Surgeons.  He  did  his  doctoral  studies  in  biophysics  at  the  Johns  Hopkins 
University  and  remained  for  a  year  of  postdoctoral  research  with  Warner  Love  before  going  to  the  Naval 
Research  Laboratory  for  continued  postdoctoral  study  with  Jerome  Karle.  He  stayed  on  at  NRL  until 
he  joined  the  faculty  of  Columbia  University. 


THYMUS-derived  lymphocytes,  or  T  cells,  dif- 
ferentiate upon  maturation  into  two  major 
cell  types.  These  are  principally  distinguished  by 
the  exclusive  occurrence  of  either  the  CD4  or 
CDS  glycoproteins  on  their  surfaces.  The  CD4- 
bearing  cells  are  known  as  helper  T  cells,  and  the 
CD8-bearing  cells  as  cytotoxic  or  killer  T  cells.  T 
cells  of  both  types  are  stimulated  into  action 
through  the  interplay  of  surface  receptor  mole- 
cules with  peptide  antigens,  which  must  be  pre- 
sented on  target  cell  surfaces  as  complexes  with 
molecules  of  the  major  histocompatibility  com- 
plex (MHC).  The  helper  T  cells  (CD4+  CDS") 
can  only  interact  with  class  II  MHC  molecules, 
which  occur  on  certain  immune  system  cells 
such  as  macrophages  and  B  cells,  whereas  the 
killer  T  cells  (CD4~  CDS+)  interact  with  the  class 
I  MHC  molecules,  found  on  all  cells. 

The  involvement  of  CD4  or  CDS  is  essential  for 
the  efficient  stimulation  of  the  respective  re- 
sponses. Stimulation  of  helper  T  cells  by  peptides 
from  an  invading  pathogen  leads  to  the  produc- 
tion of  cytokines,  which  in  turn  stimulate  the  pro- 
liferation of  selected  lymphocytes  for  antibody 
production  or  for  cellular  defense.  Subsequent 
stimulation  of  appropriate  killer  T  cells  by  pep- 
tide antigens  from  infected  cells  then  leads  to  the 
production  of  lytic  factors  that  can  eliminate  the 
infected  cell.  Thus  the  molecules  CD4  or  CDS 
can  properly  be  described  as  co-receptors  in  the 
cellular  immune  response. 

We  have  undertaken  a  series  of  structural  stud- 
ies on  CD4  and  CDS.  This  work  is  collaborative 
with  Richard  Axel  (HHMI,  Columbia  University) 
and  with  Ray  Sweet  and  his  co-workers  at  Smith- 
Kline  Beecham.  Results  from  these  studies,  when 
taken  in  conjunction  with  mutational  studies  by 
others,  are  beginning  to  provide  a  picture  of  the 
molecular  interactions  implied  by  the  specific 
stimulatory  processes  of  the  immune  response. 
Moreover,  the  structural  results  also  relate  to  the 
involvement  of  CD4  as  a  receptor  for  infection  by 
the  human  immunodeficiency  virus  (HIV). 

Crystals  of  Soluble  CD4 

Both  CD4  and  CDS  are  single-pass  transmem- 


brane proteins.  These  can  be  made  tractable  for 
crystallographic  study  by  the  expression  of  solu- 
ble extracellular  fragments  of  the  protein.  A  solu- 
ble human  CD4  fragment  containing  the  entire 
extracellular  portion  of  the  protein  was  ex- 
pressed in  mammalian  cells,  and  Peter  Kwong  has 
crystallized  this  protein  into  several  different 
crystal  lattices.  Unfortunately,  none  of  these  dif- 
fracts very  well.  From  a  characterization  of  these 
crystals,  it  is  possible,  nevertheless,  to  deduce 
some  interesting  features  of  the  CD4  molecule. 

It  appears  that  CD4  oligomerizes  at  the  high 
concentrations  needed  for  crystallization,  and  the 
lattice  dimensions  are  consistent  with  a  tetra- 
meric  model  of  1 25  A  in  length  along  a  diad  axis 
of  the  molecule.  This  propensity  to  associate  may 
be  relevant  to  signal  transduction  during  T  cell 
stimulation.  Thus  we  are  pursuing  a  crystallo- 
graphic analysis  of  one  of  these  crystals,  despite 
the  prospect  of  resolution  limited  to  approxi- 
mately 4  A. 

Structure  of  a  CD4  Fragment 

The  consistently  poor  diffraction  from  several 
polymorphs  of  a  chemically  homogeneous  pro- 
tein preparation  suggested  to  us  that  CD4  may  be 
somewhat  flexible.  This  possibility  would  be 
compatible  with  the  four-domain  structure  de- 
duced from  sequence  comparisons  and  intron 
positions,  and  limited  proteolysis  experiments 
produced  stable  fragments  corresponding  to  the 
first  two  domains  (D1D2)  and  the  second  two 
domains  (D3D4).  A  recombinant  construct  ex- 
pressing the  D 1 D2  domain  also  produced  a  stable 
molecule,  and  Seong-Eon  Ryu  obtained  diffrac- 
tion-quality crystals  of  this  fragment. 

The  resulting  structure  analysis  revealed  a  Dl 
domain  folded  as  in  the  variable  domains  of  im- 
munoglobulins and  a  D2  domain  in  a  variation  of 
the  immunoglobulin  constant-domain  topology. 
These  two  domains  are  intimately  connected, 
with  the  last  strand  of  Dl  running  directly  into 
the  first  strand  of  D2.  Despite  the  similarity  of  Dl 
to  immunoglobulin  variable  domains,  there  are 
appreciable  differences  in  the  loop  structures. 


181 


Structural  Biology  of  CD4  and  CDS  Involvement  in  the  Cellular 
Immune  Response 


One  of  these  is  the  major  determinant  for  HIV 
binding  to  CD4. 

Structure  of  a  CDS  Fragment 

Although  the  function  of  CDS  is  quite  analogous 
to  that  of  CD4,  the  architecture  of  the  two  mole- 
cules is  different.  CDS  is  a  disulfide-bridged  dimer, 
whereas  unactivated  CD4  is  monomeric,  and  each 
^CDS  chain  has  only  one  immunoglobulin-like  do- 
main rather  than  four.  This  single  domain  is  Hnked 
to  the  transmembrane  segment  by  a  50-residue 
stretch  that  is  highly  glycosylated. 

Dan  Leahy  has  expressed  a  soluble  fragment  of 
CDS  composed  of  the  immunoglobulin-like  do- 
main and  half  of  the  stalk  region.  He  has  recently 
solved  a  crystal  structure  of  this  molecule,  and 
this  shows  that  the  CDS  dimer  is  organized  very 
much  as  in  the  variable  domain  tips  of  antibodies. 
The  stalk  region  from  this  molecule  is  disordered 
in  these  crystals,  a  feature  compatible  with  a  role 
as  flexible  tether  to  the  membrane  surface. 

Interactions  with  MHC  Molecules 

Although  a  direct  physical  interaction  between 
isolated  CD4  or  CDS  molecules  and  their  respec- 
tive MHC  partners  has  not  been  demonstrated, 
these  interactions  are  expected  to  be  very  weak. 
Otherwise,  in  light  of  the  high  polyvalency  in 
cell-cell  interactions,  unwanted  adhesion  would 
be  expected.  Less-direct  evidence  for  these  inter- 
actions, however,  is  available  from  suitably  con- 
structed cell  biology  experiments. 

In  the  case  of  CD4,  Rafick  Sekaly  and  his  col- 


leagues at  the  Clinical  Research  Institute  of  Mon- 
treal have  used  point  mutations  on  human  CD4  to 
identify  residues  involved  in  the  interaction  with 
class  II  molecules.  These  mutated  residues  map 
to  exposed  residues  on  one  face  of  the  D1D2 
structural  model.  Further  analyses  are  still  in 
progress. 

In  the  case  of  the  CDS-class  I  interaction,  we 
have  been  able  to  model  a  plausible  mode  of  asso- 
ciation between  the  positively  charged  CDS  mol- 
ecule and  the  largely  negative  site  localized  to 
the  a3  domain  of  the  class  I  MHC  molecule.  This 
putative  interaction  is  also  consistent  with  results 
from  a  mutational  study  by  Paula  Kavathis  at  Yale 
University. 

CD4  and  AIDS 

The  hallmark  of  AIDS  (acquired  immune  defi- 
ciency syndrome)  is  the  immunodeficiency 
brought  on  by  elimination  of  CD4"^  T  cells.  We 
continue  in  our  efforts  to  understand  molecular 
details  of  the  interaction  between  CD4  and  HIV 
that  are  involved  in  viral  entry.  Seong-Eon  Ryu  is 
completing  his  refinement  of  the  D1D2  frag- 
ment, Reza  Beigi  is  analyzing  the  structures  of 
mutant  proteins  with  abnormal  HIV-binding 
properties,  and  Peter  Kwong  has  characterized  an 
antibody  fragment  that  permits  full  HIV  binding 
to  CD4  but  inhibits  infection.  This  antibody, 
which  binds  to  the  D3  domain,  indicates  an  in- 
volvement of  CD4  flexibility  in  HIV  entry.  Peter 
Kwong  is  also  attempting  to  crystallize  the  viral 
coat  protein  gpl20  and  various  complexes  with 
this  crucial  component  of  the  system. 


182 


Variegated  Position  Effects  in  Drosophila 


Steven  Henikoff,  Ph.D. — Investigator 

Dr.  Henikoff  is  also  a  member  of  the  Basic  Sciences  Division  of  the  Fred  Hutchinson  Cancer  Research 
Center,  Seattle.  He  received  a  B.S.  degree  in  chemistry  at  the  University  of  Chicago  and  a  Ph.D.  degree 
in  biochemistry  and  molecular  biology  at  Harvard  University,  working  in  the  laboratory  of  Matthew 
Meselson.  He  did  postdoctoral  work  with  Charles  Laird  at  the  University  of  Washington. 


EACH  individual  gene  occupies  a  fixed  posi- 
tion on  a  chromosome.  By  and  large,  moving 
a  gene  has  only  a  minor  effect  on  expression  of 
the  gene.  Thus  most  studies  of  gene  expression 
are  able  to  focus  on  the  gene  as  an  independent 
unit,  without  taking  into  account  larger  organiza- 
tional features.  However,  there  are  exceptional 
cases  in  which  the  relationship  between  a  gene 
and  its  environment  plays  a  role  in  expression  of 
the  gene. 

The  relationship  between  a  gene  and  its  chro- 
mosomal environment  is  especially  apparent  in 
examples  of  "position  effects"  associated  with 
chromosomal  rearrangements.  In  flies  a  well- 
known  class  of  position  effects  involves  inactiva- 
tion  of  genes  in  the  vicinity  of  rearrangement 
breakpoints.  Gene  inactivation  is  extremely  vari- 
able from  cell  to  cell,  such  that  the  affected  tissue 
shows  a  variegated  pattern  of  expression.  In  each 
case,  it  is  found  that  the  gene  has  been  juxtaposed 
to  heterochromatin,  the  deeply  staining  regions  of 
chromosomes  that  flank  the  centromere.  Although 
heterochromatin  contains  a  substantial  fraction  of 
DNA  in  all  higher  eukaryotes,  the  repetitive  se- 
quence structure  characteristic  of  heterochromatin 
and  the  near  absence  of  genes  have  hampered  at- 
tempts to  understand  its  role  in  the  genome.  Genes 
that  show  variegated  expression  when  placed  next 
to  heterochromatin  provide  a  reporter  function, 
allowing  us  to  investigate  these  poorly  understood 
regions  of  chromosomes. 

Variegated  position  effects  caused  by  juxtapo- 
sition to  heterochromatin  are  seen  for  a  large 
number  of  genes  in  Drosophila.  One  well-stud- 
ied example  is  the  brown  gene,  required  for  full 
pigmentation  of  the  eye.  Unlike  nearly  all  other 
genes,  however,  such  position  effects  on  the 
brown  gene  are  dominant  over  wild  type — that 
is,  placing  one  copy  of  brown  next  to  heterochro- 
matin can  lead  to  inactivation  of  the  other  copy. 

We  have  investigated  the  genetic  basis  for  this 
gene  inactivation  in  trans  and  have  found  that  a 
necessary  component  is  the  pairing  of  homo- 
logues  in  the  immediate  vicinity  of  the  brown 
gene.  These  findings  have  led  to  an  explanation 
for  "trans-inactivation,"  whereby  protein  compo- 


nents of  heterochromatin  make  direct  contact  with 
the  trans  copy  of  the  brown  gene  across  paired  ho- 
mologues.  In  support  of  this  hypothesis,  we  have 
been  able  to  reproduce  trans-inactivation  at  sites  of 
transposons  carrying  tht  brown  gene,  but  only  for 
paired  copies  of  the  gene.  In  addition,  we  have 
found  that  even  very  small  lesions  that  disrupt  pair- 
ing in  the  immediate  vicinity  of  the  gene  also  re- 
duce trans-inactivation. 

Mapping  of  the  sequence-specific  component 
necessary  for  trans-inactivation  to  occur  has  local- 
ized it  to  the  brown  gene  itself,  probably  to  the 
region  immediately  upstream.  This  supports  the 
notion  that  the  contact  between  homologues  is 
between  a  DNA-binding  protein  necessary  for 
normal  brown  gene  activity  and  a  protein  compo- 
nent of  heterochromatin. 

Our  current  efforts  are  aimed  at  identification 
of  the  protein  components  involved  in  trans- 
inactivation.  One  approach  is  to  focus  on  the  se- 
quences in  the  immediate  upstream  region  of  the 
brown  gene,  where  we  expect  that  the  sequence- 
specific  component  should  bind.  Another  is  to 
identify  genes  that  encode  proteins  involved  in 
the  process  by  screening  for  mutations  that  specif- 
ically reduce  the  degree  of  trans-inactivation.  We 
have  now  isolated  several  such  mutations  and  are 
in  the  process  of  precisely  mapping  them  to 
clone  the  corresponding  genes. 

A  new  research  direction  for  us  came  from  a 
serendipitous  finding  (during  a  screen  for  posi- 
tion-effect variegation  mutations)  of  an  unstable 
chromosome  that  causes  gene  markers  carried  on 
it  to  appear  variegated.  This  chromosome  derives 
from  a  fusion  between  a  chromosome  arm  carry- 
ing the  markers  and  a  centromere  from  another 
chromosome.  The  unstable  chromosome  is  inter- 
mediate in  size  among  wild-type  and  rearranged 
linear  Drosophila  chromosomes,  all  of  which  are 
quite  stable  in  somatic  cells.  The  instability  re- 
sults from  failure  of  the  two  products  of  replica- 
tion— called  sister  chromatids — to  come  apart 
reliably  at  mitosis,  leading  to  clones  and  single 
cells  that  have  either  gained  an  extra  copy  of  the 
chromosome  or  have  lost  it  entirely. 

The  appearance  of  the  unstable  chromosome 


183 


Variegated  Position  Effects  in  Drosophila 


during  mitosis  indicates  that  instability  is  asso- 
ciated with  premature  separation  of  sister  chro- 
matids; as  a  result,  the  sisters  might  sometimes 
independently  attach  to  the  spindle  apparatus 
that  pulls  them  apart.  Current  evidence  suggests 
that  the  defect  results  from  a  position  effect  on 
the  centromere,  because  genetic  suppressors  of 
position-effect  variegation  also  suppress  the  insta- 
bility phenotype.  To  understand  further  the  basis 
for  instability,  we  have  initiated  physical  map- 
ping studies  on  this  centromere.  Such  studies 
should  help  identify  sequences  necessary  for 
centromere  function  in  a  higher  eukaryote. 


Phenomena  that  depend  on  the  position  of  a 
sequence  in  the  chromosome  or  on  somatic  pair- 
ing of  homologues  are  easily  observed  in  Dro- 
sophila, where  powerful  tools  are  available  for 
genetic  dissection.  Related  phenomena  are 
known  to  occur  in  mammals,  such  as  X  chromo- 
some inactivation,  in  which  one  of  the  female's  X 
chromosomes  becomes  heterochromatic.  The 
many  similarities  between  chromosomes  in  or- 
ganisms as  diverse  as  flies  and  mammals  lead  to 
the  expectation  that  an  understanding  of  position 
effects  and  centromere  function  in  Drosophila 
will  have  general  implications. 


184 


Biological  Roles  and  Expression 
of  Complement  Receptors 


V.  Michael  Holers,  M.D. — Assistant  Investigator 

Dr.  Holers  is  also  Assistant  Professor  of  Medicine  and  Pathology  at  the  Washington  University  School 
of  Medicine  and  Assistant  Physician  at  Barnes  Hospital,  St.  Louis.  He  received  his  undergraduate  degree 
from  Purdue  University  and  his  M.D.  degree  from  Washington  University.  He  did  postdoctoral  research 
at  the  University  of  Colorado,  Denver,  and  then  at  Washington  University. 


THE  complement  system  was  initially  de- 
scribed as  an  activity  found  in  serum  that 
could  mediate  the  killing  of  foreign  infectious 
organisms  such  as  bacteria  or  viruses.  This  sys- 
tem, in  conjunction  with  specific  immunity  gen- 
erated by  vaccination,  was  found  to  be  critical  to 
preventing  and  fighting  infections.  Later  it  was 
realized  that  complement  also  facilitates  the  in- 
teraction of  antigen-antibody  complexes  with 
cells  of  the  immune  system,  which  greatly  en- 
hances the  specific  immune  response.  Therefore 
complement  not  only  helps  to  initiate  an  immune 
response  but  also  plays  an  important  role  in  the 
ability  to  clear  infections  and  foreign  antigens 
from  the  body. 

The  complement  system  consists  of  at  least  20 
serum  proteins  that  are  activated  in  a  cascade  fash- 
ion. As  part  of  the  activation  process,  protein 
fragments  are  released  that  attract  inflammatory 
cells.  In  addition,  antigen-antibody  complexes 
are  coated  with  specific  complement  fragments 
that  covalently  attach  to  this  target.  One  of  these 
fragments,  complement  component  C3,  is  able  to 
be  cleaved  proteolytically  after  attachment  to  a 
target.  This  cleavage  reaction  results  in  a  number 
of  different  conformations  of  C3,  which  allow  it 
to  interact  with  at  least  three  unique  cell  surface 
receptors.  After  binding  to  cells,  specific  trans- 
membrane signals  are  sent,  and  the  complement 
receptors  then  mediate  ingestion  and  processing 
or  killing  of  the  targets.  Also  as  part  of  this  pro- 
cess, C3  fragments  may  bind  to  self  tissues,  rather 
than  to  the  antibody-bound  target,  thereby  attack- 
ing at  inappropriate  sites.  Other  cell  membrane 
C3-binding  proteins  are  able  to  inactivate  this  C3 
and  prevent  inappropriate  damage  to  self  tissues. 

We  are  interested  in  the  interaction  of  C3  with 
its  specific  receptors  and  regulatory  proteins,  par- 
ticularly the  biological  aspects  of  complement 
receptor  2  (CR2).  In  addition,  we  are  studying 
mouse  homologues  of  CR2  and  other  proteins  of 
this  type.  Human  CR2  serves  as  the  receptor  for 
the  Epstein-Barr  virus  (EBV),  which  is  responsi- 
ble for  most  cases  of  infectious  mononucleosis 
and  is  causally  associated  with  a  number  of  hu- 
man tumors  of  B  lymphocytes  and  epithelial 


cells.  Patients  who  have  forms  of  congenital  or 
acquired  immunodeficiency  (such  as  AIDS  or 
after  organ  transplantation)  are  particularly  sus- 
ceptible to  tumors  associated  with  EBV. 

In  the  past  few  years  we  have  cloned  and  ana- 
lyzed the  structure  and  activities  of  human  CR2, 
its  mouse  homologues,  and  a  unique  mouse  pro- 
tein called  Crry/p65.  We  have  shown  that  ex- 
pression of  recombinant  forms  of  these  proteins 
in  other  cells  is  sufficient  to  mediate  the  binding 
of  ligands  or  to  control  inappropriate  comple- 
ment deposition.  By  using  other  recombinant 
techniques  and  creating  mutations  within  these 
proteins,  we  have  shown  that  specific  amino 
acids  in  small  domains  are  important  for  ligand 
interactions.  In  addition,  we  have  synthesized 
peptides  that  have  the  ability  to  block  binding  of 
some  ligands,  in  particular,  EBV  binding  to  CR2. 
These  studies  should  allow  us  to  devise  strategies 
to  alter  the  function  of  this  receptor  in  vivo.  For 
instance,  one  type  of  reagent  might  block  EBV 
binding  to  CR2  but  not  normal  binding  of  C3. 
This  could  be  useful  in  some  illnesses  associated 
with  EBV. 

We  have  analyzed  the  murine  homologues  of 
these  proteins  to  understand  further  the  biologi- 
cal role  of  human  CR2,  in  addition  to  other  com- 
plement receptors  and  regulatory  proteins.  Once 
the  activities  of  these  proteins  are  understood,  we 
should  be  able  to  utilize  murine  models  of  the 
normal  immune  response,  as  well  as  autoimmune 
diseases,  to  elucidate  the  in  vivo  roles  of  these 
proteins. 

Another  aspect  of  CR2  expression  is  also  under 
analysis.  Expression  of  CR2  varies  during  human 
B  lymphocyte  development:  it  is  expressed  only 
on  late  pre-B  cells  and  mature  B  cells  and  not  on 
very  early  pre-B  lymphocytes  or  on  late  immuno- 
globulin-secreting  cells.  The  molecular  mecha- 
nisms that  underlie  this  phenotype,  which  is  also 
found  among  other  B  cell-specific  markers,  are 
likely  fundamental  to  the  overall  processes  by 
which  B  cells  mature  and  are  activated.  We  are 
analyzing  these  mechanisms.  As  part  of  these  stud- 
ies we  have  defined  a  promoter  for  CR2  and  other 
sites  within  the  gene  that  are  likely  to  be  impor- 


185 


Biological  Roles  and  Expression  of  Complement  Receptors 


tant  in  gene  regulation.  We  have  determined  that 
the  receptor  levels  go  up  and  down  because  the 
gene  is  turned  on  and  off  during  development, 
we  have  identified  functionally  important  do- 
mains and  sites  of  protein  interaction  within  the 
CR2  promoter,  and  we  are  clarifying  the  role  of 


each  site  in  CR2  expression  and  B  cell  matura- 
tion. These  studies  will  increase  our  understand- 
ing of  specific  gene  expression  in  B  lymphocytes 
and  further  our  knowledge  of  how  to  alter  B  cell 
phenotypes  along  pathways  that  might  be  more 
beneficial  during  certain  disease  states. 


186 


Genetic  Control  of  Nematode  Development 


H.  Robert  Horvttz,  Ph.D. — Investigator 

Dr.  Horvitz  is  also  Professor  of  Biology  at  the  Massachusetts  Institute  of  Technology  and  Neurobiologist 
and  Geneticist  at  Massachusetts  General  Hospital,  Boston.  He  earned  his  undergraduate  degrees  in 
mathematics  and  in  economics  at  the  Massachusetts  Institute  of  Technology,  followed  by  the  M.A.  and 
Ph.D.  degrees  in  biology  from  Harvard  University.  His  postdoctoral  research  was  done  at  the  Medical 
Research  laboratory  of  Molecular  Biology,  Cambridge,  England.  Dr.  Horvitz  is  a  member  of  the  National 
Academy  of  Sciences. 


HOW  do  genes  control  animal  development? 
Taking  a  primarily  genetic  approach  to  an- 
swer this  question,  members  of  our  laboratory 
have  isolated  developmental  mutants  of  the 
roundworm  Caenorhabditis  elegans  and  have 
used  both  genetic  and  molecular  genetic  tech- 
niques to  characterize  these  mutants.  Because  the 
complete  cellular  anatomy  (including  the  com- 
plete wiring  diagram  of  the  nervous  system)  and 
the  complete  cell  lineage  of  C.  elegans  are 
known,  mutant  animals  can  be  studied  at  the 
level  of  single  cells  and  even  single  synapses. 
Genes  that  play  specific  roles  in  cell  lineage,  cell 
signaling,  cell  death,  and  cell  migration  have 
been  identified  and  analyzed. 

Cell  Lineage 

The  problem  of  cell  lineage — how  a  single  fer- 
tilized egg  cell  undergoes  a  complex  pattern  of 
cell  divisions  to  generate  a  multiplicity  of  dis- 
tinct cell  types — is  one  major  focus  of  the  re- 
search of  our  laboratory.  We  have  identified 
hundreds  of  genes  responsible  for  controlling 
aspects  of  the  C.  elegans  cell  lineage.  Many  of 
these  genes  function  in  generating  cell  diversity 
during  development.  For  example,  some  genes 
act  to  make  the  two  daughter  cells  generated  by  a 
single  cell  division  different  from  each  other,  and 
one  gene  acts  to  make  certain  daughter  cells  dif- 
ferent from  their  mothers.  The  action  of  some 
cell  lineage  genes  is  constrained  to  a  single  cell 
type,  tissue,  or  organ.  For  example,  one  gene  acts 
only  in  the  nervous  system,  and  another  acts  only 
in  the  hypodermis.  Other  genes  act  in  multiple 
tissues. 

We  have  analyzed  a  number  of  these  genes  at 
the  molecular  level.  These  studies  have  revealed 
that  many  genes  that  control  cell  lineage  in  C. 
elegans  are  strikingly  similar  to  genes  found  in 
other  organisms,  including  humans.  Thus  the 
analysis  of  developmental  control  genes  in  C.  ele- 
gans should  help  us  to  understand  aspects  of  the 
development  of  more-complex  organisms. 

Cell  Signaling 

Much  of  the  development  of  C.  elegans,  like 


that  of  other  organisms,  involves  intercellular 
communication.  We  have  studied  cell  interac- 
tions in  nematode  development  by  using  a  laser 
microbeam  to  kill  single  cells  in  living  animals:  if 
destruction  of  one  cell  alters  the  fate  of  a  second 
cell,  the  first  cell  must  normally  interact  with  the 
second.  We  have  analyzed  in  detail  the  cell  inter- 
actions involved  in  inducing  the  development  of 
the  vulva,  which  forms  the  external  genitalia, 
connects  the  uterus  with  the  outside  environ- 
ment, and  is  used  for  egg  laying  and  copulation. 
We  have  characterized  many  genes  that  function 
in  the  cell  interactions  of  vulval  development. 
One  gene  that  acts  as  a  switch  in  the  vulval  induc- 
tive signaling  pathway  is  a  member  of  the  ras 
gene  family.  Other  ras  genes  are  associated  with 
many  human  cancers;  the  same  mutations  that 
cause  extra  vulval  cell  divisions  in  C.  elegans  are 
oncogenic  in  mammals.  Another  gene  in  the  vul- 
val signaling  pathway  has  similarities  to  the  src 
gene,  which  is  also  associated  with  human 
cancers.  The  study  of  these  and  other  genes  that 
function  in  cell  signaling  in  C.  elegans  might 
provide  insights  relevant  to  cancerous  growth  in 
humans. 

Cell  Death 

Naturally  occurring  or  "programmed"  cell 
death  is  common  during  the  development  of  the 
nervous  system  of  many  animals,  including  C. 
elegans.  Why  organisms  generate  cells  only  to 
have  them  die  is  an  intriguing  question.  Further- 
more, the  mechanisms  responsible  for  cell  death 
might  be  of  medical  importance,  as  the  clinical 
features  of  many  human  disorders  (including 
trauma,  stroke,  and  a  variety  of  neurodegenera- 
tive diseases)  are  a  consequence  of  nerve  cell 
deaths. 

We  have  been  identifying  and  characterizing 
genes  that  function  in  programmed  cell  death  in 
C.  elegans.  Two  genes  cause  cells  to  die,  and 
seven  other  genes  are  involved  in  removing  the 
corpses  of  dead  cells.  The  two  genes  that  cause 
cells  to  die  must  be  expressed  by  the  dying  cells 
themselves,  indicating  that,  at  least  to  this  extent, 
programmed  cell  deaths  are  cell  suicides.  Al- 


187 


Genetic  Control  of  Nematode  Development 


though  many  cells  die  during  the  course  of 
C.  elegans  development,  most  cells  survive;  cell 
survival  requires  the  inactivation  of  the  cell  death 
process,  as  a  tenth  gene  functions  to  prevent  the 
action  of  the  nine  cell  death  genes  in  surviving 
cells.  In  addition,  this  regulatory  gene  is  itself 
controlled  in  a  cell-specific  fashion  by  other 
genes  that  determine  which  cells  are  to  live  and 
which  are  to  die.  Molecular  analyses  of  genes  in- 
volved in  programmed  cell  deaths  in  C.  elegans 
have  suggested  that  the  biochemical  processes  re- 
sponsible might  be  similar  to  those  suspected  to 
cause  nerve  cells  to  die  in  human  neurological 
disorders.  We  hope  that  knowledge  of  what 
makes  cells  die  and  of  what  can  block  the  cell 
death  process  in  C.  elegans  will  lead  to  methods 
that  will  prevent  the  cell  deaths  responsible  for 
human  disorders. 

Cell  Migration 

During  animal  development,  cells  are  often 
generated  far  from  their  final  positions  and  must 
migrate  considerable  distances  before  being  able 


to  function.  To  understand  what  causes  cells  both 
to  migrate  and  to  stop  migrating,  we  are  analyzing 
two  C.  elegans  cell  migrations.  The  first  involves 
a  pair  of  muscle  precursor  cells  that  are  born  in 
the  posterior  body  region  and  move  to  a  central 
position  along  the  animal's  length,  near  its  gonad. 
We  have  discovered  that  these  migrations  involve 
signaling  between  the  migrating  cells  and  go- 
nadal cells  located  at  the  termination  site  of  the 
migration.  We  are  characterizing  genes  that  func- 
tion in  this  signaling  process. 

The  second  migration  we  have  studied  involves 
a  pair  of  neuronal  cells  that  move  from  the  tail 
region  to  the  midbody  region  of  the  animal.  Thir- 
teen genes  have  been  identified  that  must  func- 
tion for  these  neuronal  migrations  to  occur  prop- 
erly. Some  of  these  genes  probably  act  in  the 
migration  process  per  se,  but  some  do  not.  The 
actions  of  some  of  these  latter  genes  allow  these 
neurons  to  acquire  their  identities;  if  these  genes 
do  not  function,  these  neurons  fail  to  express 
their  normal  characteristics,  including  their 
long-range  cell  migration. 


188 


Protein  Folding  in  Vivo 


Arthur  L.  Harwich,  M.D. — Associate  Investigator 

Dr.  Norwich  is  also  Associate  Professor  of  Human  Genetics  and  Pediatrics  at  Yale  University  School  of 
Medicine.  He  received  A.B.  and  M.D.  degrees  in  biomedical  sciences  from  Brown  University.  His  internship 
and  residency  training  in  pediatrics  were  done  at  Yale.  His  postdoctoral  research  training  was  at  the  Salk 
Institute  with  Walter  Eckhart  and  at  Yale  University  with  Leon  Rosenberg. 


UNTIL  recently  it  has  been  assumed  that 
newly  made  proteins  in  the  living  cell,  com- 
prising amino  acid  chains  with  characteristic  se- 
quences, are  able  to  fold  spontaneously  into  pre- 
cise three-dimensional  structures  that  exhibit 
biological  activity.  Such  folding  has  been  ob- 
served in  test-tube  experiments,  where  many 
proteins  unfolded  by  denaturing  agents  can  be 
diluted  from  these  agents  and  observed  to  refold 
into  their  biologically  active  forms.  Recent  stud- 
ies, however,  suggest  that  in  the  cell  the  process 
of  folding  is  assisted  by  other  specialized  pro- 
teins. We  originally  identified  such  a  protein 
while  studying  mitochondria,  the  intracellular 
organelles  that  carry  out  energy  metabolism. 

Most  of  the  proteins  of  mitochondria  are  first 
made  outside  the  organelles,  in  the  cytosol,  and 
then  imported  through  two  membranes  to  reach 
the  innermost  mitochondrial  "matrix"  compart- 
ment. To  traverse  the  membranes,  the  newly 
made  proteins  are  first  unfolded  on  the  cytosolic 
side.  After  import,  the  proteins  refold  on  the  in- 
side of  the  organelles  into  their  biologically  ac- 
tive conformations.  We  identified  a  mutant  cell 
in  which  mitochondrial  proteins  were  imported 
but  failed  to  fold  into  biologically  active  forms. 
The  mutation  was  found  to  affect  a  protein  that 
normally  resides  in  the  matrix  compartment, 
called  heat-shock  protein  60  (hsp60). 

This  protein  was  originally  identified  by  the 
observation  that  its  abundance  is  increased  about 
twofold  in  response  to  incubation  of  cells  at  high 
temperatures.  However,  it  is  an  abundant  protein 
even  before  heat  shock,  and  our  genetic  analysis 
demonstrated  that,  consistent  with  a  critical  base- 
line function,  hsp60  is  required  not  only  at  high 
temperatures  but  at  all  temperatures.  The  in- 
creased level  produced  in  response  to  heat  stress 
could  represent  an  effort  to  try  to  refold  effi- 
ciently mitochondrial  proteins  that  heat  has 
denatured. 

In  the  mitochondrial  matrix,  hsp60  is  found  in 
a  higher  order  structure,  a  complex.  Fourteen 
copies  of  the  protein  are  arranged  in  two  stacked 
rings,  a  "double  donut . ' '  Each  ring  contains  seven 
radially  arranged  copies  of  hsp60.  Our  studies 


have  demonstrated  that  unfolded  mitochondrial 
proteins  entering  the  matrix  space  become  asso- 
ciated with  the  surface  of  the  hsp60  complex. 
Then,  in  steps  requiring  both  energy  (ATP)  and  a 
second  protein,  the  polypeptides  are  folded  into 
their  active  forms  and  released  from  the 
complex. 

The  folding  pathway  must  be  dictated  by  the 
amino  acid  sequence  of  the  "substrate"  protein 
to  be  folded,  not  by  the  hsp60  complex,  because 
we  have  used  the  complex  to  fold  proteins  that 
normally  reside  outside  mitochondria.  It  seems, 
however,  that  hsp60  acts  by  speeding  up,  or  cata- 
lyzing, the  folding  of  proteins.  How  does  it  do 
this?  One  possibility  is  that  it  simply  prevents  do- 
mains of  proteins  from  wrongfully  interacting,  ei- 
ther with  each  other  or  with  nearby  proteins  in 
the  mitochondrial  matrix,  a  "chaperone"  func- 
tion. Another  possibility  is  that  the  complex  ac- 
tively promotes  the  progression  of  an  unfolded 
protein  through  a  series  of  folding  steps. 

Because  mitochondria  arose  from  bacteria  (one 
cell  ingested  another),  it  is  not  surprising  that  a 
structurally  related  component,  the  groEL  pro- 
tein, has  been  found  in  bacteria.  In  the  1970s  it 
was  observed  that  when  Escherichia  coli  cells 
partially  defective  in  this  protein  are  infected 
with  a  virus,  the  newly  made  viral  coat  proteins 
are  unable  to  assemble  to  make  new  virus 
particles. 

Additional  evidence  comes  from  our  reconsti- 
tution  experiments.  When  a  test  protein  unfolded 
in  denaturant  was  diluted  into  a  mixture  contain- 
ing purified  groEL  complex,  it  became  bound  to 
the  complex  and  was  thus  prevented  from  mis- 
folding  and  aggregating.  The  bound  protein  was 
found  as  a  folding  intermediate,  called  a  molten 
globule,  that  has  formed  its  local  structures  but 
lacks  the  three-dimensional  organization  of  the 
active  form.  When  both  a  cooperating  compo- 
nent, groES  (a  small  ring  structure  also  with  seven 
members),  and  ATP  were  added,  the  polypeptide 
was  observed  to  reorganize  its  structure  while  in 
association  with  the  complex  during  the  next 
minutes  and  to  be  released  in  its  active  form.  The 
cost  of  folding  a  single  polypeptide  was  approxi- 
mately 100  ATP  hydrolyzed,  amounting  to  about 


189 


Protein  Folding  in  Vivo 


one-tenth  of  the  amount  of  energy  needed  to  syn- 
thesize the  polypeptide. 

How  general  is  the  utilization  of  folding  ma- 
chinery outside  bacteria  and  mitochondria?  We 
thought  we  might  find  a  functionally  similar  com- 
ponent in  the  cytosol  of  higher  organisms  by  look- 
ing first  in  the  kingdom  of  archaebacteria.  These 
organisms  are  evolutionarily  distinct  from  bacte- 
ria and  have  recently  been  shown  to  contain  com- 
ponents closely  related  to  those  in  the  cytosol  of 
higher  organisms.  We  transferred  a  thermophilic 
archaebacterium  that  grows  normally  at  75  °C  to  a 
near-lethal  temperature  of  88°C  and  found  that  a 
single  major  heat-shock  protein  was  produced. 
This  protein ,  already  abundant  at  normal  tempera- 
ture, is  a  double-ring  complex  that  looks  much 
like  the  complexes  of  mitochondria  and  bacteria, 


except  that  each  ring  contains  nine  members. 
The  purified  ring  complex  binds  unfolded  pro- 
teins and  exhibits  ATPase  activity,  consistent  with 
the  idea  that  it  might  function  in  the  archaebac- 
teria similarly  to  the  hsp60  and  groEL  complexes. 
Analysis  of  the  protein's  amino  acid  sequence 
demonstrated  no  significant  relationship  to  ei- 
ther hsp60  or  groEL,  but  rather  a  relationship  to  a 
protein  called  TCPl ,  an  essential  cytosol  protein 
of  higher  organisms,  whose  function  has  been 
largely  unknown  but  which  has  been  implicated 
in  assembly  of  the  mitotic  spindle  apparatus.  Us- 
ing a  mutant  yeast  cell  defective  in  TCPl ,  we  are 
now  investigating  whether  the  protein  has  a  spe- 
cialized function  in  assembly  of  the  spindle  or  a 
more  general  function  as  a  "folding  machine"  for 
the  cytosol  of  higher  organisms. 


MgATP 

groEL 


Model  for  folding  of  polypeptides  by  groEL.  Polypeptide,  shown  in  yellow,  is  bound  by  groEL  and 
stabilized  in  "molten  globule"  conformation.  When  MgATP  and  groES  are  present,  polypeptide 
undergoes  conformational  changes  and  is  ultimately  released,  reaching  a  native,  active 
conformation. 

Derived  from  Martin,  f.,  Langer,  T.,  Boteva,  R.,  Scbramel,  A.,  Horwich,  A.L.,  and  Hartl,  F.-U. 
1991.  Nature  352:36-42. 


190 


Molecular  Mechanisms  in  the  Regulation 
of  Synaptic  Transmission 


Richard  L.  Huganir,  Ph.D. — Associate  Investigator 

Dr.  Huganir  is  also  Associate  Professor  of  Neuroscience  at  the  Johns  Hopkins  University  School  of 
Medicine.  He  completed  his  undergraduate  work  in  biochemistry  at  Vassar  College  and  received  his  Ph.D. 
degree  in  biochemistry  and  molecular  and  cell  biology  from  Cornell  University,  where  he  performed  his 
thesis  research  in  the  laboratory  of  Efraim  Packer.  After  completing  a  postdoctoral  fellowship  with  Paul 
Greengard  at  Yale  University  School  of  Medicine,  Dr.  Huganir  moved  to  the  Rockefeller  University. 


INFORMATION  processing  in  the  brain  de- 
pends on  the  transmission  of  signals  between 
neurons  at  specialized  areas  of  contact,  called 
synapses.  At  synapses,  ion  channel  proteins  in  the 
neuronal  cell  membrane  generate  an  electrical 
current,  which  triggers  the  release  of  chemical 
signals  from  the  first  neuron,  called  the  presynap- 
tic neuron.  These  chemical  signals,  or  neurotrans- 
mitters, bind  to  specific  receptor  proteins  in  the 
membrane  of  the  second  neuron,  called  the  post- 
synaptic neuron.  The  neurotransmitter  receptors 
then  generate  electrical  currents  in  the  postsyn- 
aptic neuron  and  thus  complete  the  process  of 
synaptic  transmission. 

The  efficiency  of  synaptic  transmission  at  any 
given  synapse  is  constantly  changing  in  response 
to  a  variety  of  factors;  this  synaptic  plasticity 
plays  a  major  role  in  the  function  of  the  nervous 
system.  Both  the  amount  of  neurotransmitter  re- 
leased from  the  presynaptic  neuron  in  response 
to  a  given  electrical  signal  and  the  sensitivity  of 
the  postsynaptic  receptor  system  for  a  given 
amount  of  neurotransmitter  can  be  modulated. 
The  molecular  mechanisms  that  underlie  the 
modulation  of  synaptic  transmission  have  only 
begun  to  be  defined.  Recent  studies  have  pro- 
vided evidence  that  protein  phosphorylation  is 
an  important  mechanism  in  the  regulation  of  syn- 
aptic transmission. 

Protein  phosphorylation  systems  consist  of 
three  primary  components,  a  protein  kinase,  a 
substrate  protein,  and  a  phosphoprotein  phos- 
phatase. Protein  kinases  are  enzymes  that  catalyze 
the  chemical  transfer  of  phosphate  molecules 
from  ATP  to  specific  substrate  proteins.  The  activ- 
ities of  many  protein  kinases  are  regulated  by  neu- 
rotransmitters and  hormones  through  the  actions 
of  substances  called  second  messengers,  such  as 
cAMP,  calcium,  and  diacylglycerol.  Substrate 
proteins  include  many  cellular  components, 
among  them  enzymes,  ion  channels,  and  neuro- 
transmitter receptors.  The  addition  of  the  nega- 
tively charged  phosphate  group  alters  the  struc- 
ture of  these  substrate  proteins,  thereby 
regulating  their  functional  properties.  Phospho- 
protein phosphatases  are  enzymes  that  reverse 


the  process  of  protein  phosphorylation,  remove 
the  phosphate  group  from  the  substrate  protein, 
and  return  it  to  its  basal  state. 

My  laboratory  is  concerned  with  the  structure 
and  function  of  neurotransmitter  receptors  and 
the  role  of  protein  phosphorylation  in  the  regula- 
tion of  the  properties  of  the  neurotransmitter  re- 
ceptors. We  have  used  as  a  model  system  the  best- 
characterized  neurotransmitter  receptor  and  ion 
channel  in  neurobiology  today,  the  nicotinic  ace- 
tylcholine receptor.  In  addition,  we  have  been 
studying  the  major  inhibitory  neurotransmitter 
receptors  in  the  brain,  the  GABA^  receptors,  and 
the  major  excitatory  neurotransmitter  receptors 
in  the  brain,  the  glutamate  receptors.  These  re- 
ceptors are  neurotransmitter-dependent  ion 
channels  that  generate  electrical  currents  in  the 
postsynaptic  membrane  of  the  synapse  in  re- 
sponse to  their  neurotransmitter. 

To  study  the  molecular  mechanisms  involved 
in  neurotransmitter  receptor  and  ion  channel 
function,  it  is  essential  to  identify  chemically  the 
specific  proteins  required  for  this  activity.  We 
began  by  defining  the  molecular  components  re- 
quired for  the  functioning  of  the  nicotinic  acetyl- 
choline receptor  ion  channel.  Using  membrane 
reconstitution  techniques,  we  solubilized  the  nic- 
otinic receptor  and  its  ion  channel  from  isolated 
postsynaptic  membranes,  purified  it,  and  recon- 
stituted it  into  phospholipid  vesicles.  These  stud- 
ies demonstrated  that  the  purified  receptor,  con- 
sisting of  four  types  of  protein  subunits  (a,  /?,  7, 
5) ,  contains  the  ion  channel  and  has  all  the  biolog- 
ical properties  of  the  nicotinic  receptor  in  the 
intact  cell. 

We  next  began  to  characterize  the  protein 
phosphorylation  of  these  structural  components. 
Postsynaptic  membranes  isolated  from  synapses 
highly  enriched  in  the  nicotinic  acetylcholine  re- 
ceptor contain  at  least  three  different  types  of 
protein  kinases  that  phosphorylate  the  nicotinic 
receptor  on  six  different  phosphorylation  sites: 
cAMP-dependent  protein  kinase  phosphorylates 
the  7-  and  6-subunits  of  the  receptor;  a  calcium- 
and  diacylglycerol-dependent  protein  kinase 
phosphorylates  the  5-subunit;  and  a  protein- 


191 


Molecular  Mechanisms  in  the  Regulation  of  Synaptic  Transmission 


tyrosine  kinase  phosphorylates  the  |8-,  7-,  and 
5-subunits.  These  postsynaptic  membranes  also 
contain  phosphoprotein  phosphatase  activity 
that  dephosphorylates  the  phosphorylated  nico- 
tinic acetylcholine  receptor. 

We  are  currently  using  protein  purification  and 
molecular  cloning  techniques  to  characterize  the 
protein-tyrosine  kinases  that  phosphorylate  the 
receptor  and  the  phosphotyrosine  protein  phos- 
phatases that  dephosphorylate  the  receptor.  We 
recently  identified  several  cDNA  clones  for  dif- 
ferent types  of  protein-tyrosine  kinases  that  are 
expressed  in  cells  enriched  in  the  nicotinic  re- 
ceptor and  are  attempting  to  determine  which  of 
these  protein-tyrosine  kinases  phosphorylate  the 
receptor.  In  addition,  we  recently  purified  the 
phosphotyrosine  protein  phosphatase  that  de- 
phosphorylates the  tyrosine-phosphorylated  ace- 
tylcholine receptor  and  are  using  molecular 
cloning  techniques  to  isolate  cDNA  clones  for 
this  phosphotyrosine  protein  phosphatase. 

What  are  the  functional  effects  of  phosphoryla- 
tion of  the  receptor  by  these  protein  kinases?  We 
have  examined  this  question  directly  by  studying 
the  properties  of  the  purified  and  reconstituted 
receptor  phosphorylated  to  different  degrees  by 
the  various  protein  kinases.  Phosphorylation  of 
the  receptor  on  the  7-  and  6-subunits  by  cAMP- 
dependent  protein  kinase  or  phosphorylation  of 
the  receptor  on  the  0-,  7-,  and  5-subunits  by  the 
protein-tyrosine  kinase  dramatically  increases  the 
rate  of  desensitization  of  the  receptor.  Desensiti- 
zation  is  the  process  by  which  the  receptor  is  re- 
versibly  inactivated  in  the  continued  presence  of 
the  neurotransmitter  acetylcholine.  These  stud- 
ies provide  direct  evidence  that  protein  phos- 
phorylation of  the  nicotinic  acetylcholine  recep- 
tor regulates  its  physiological  properties  and 
plays  a  role  in  modulating  its  sensitivity  to 
acetylcholine. 

To  analyze  the  effect  of  phosphorylation  on  the 
desensitization  of  the  receptor  in  more  detail,  we 
have  recently  used  site-specific  mutagenesis  tech- 
niques to  mutate  the  phosphorylation  sites  on  the 
receptor  subunits.  Mutant  receptor  subunits  lack- 
ing phosphorylation  sites  have  been  expressed  in 
Xenopus  oocytes,  and  the  regulation  of  desensiti- 
zation of  these  receptors  by  protein  phosphoryla- 
tion is  being  analyzed  and  compared  with  normal 
receptors. 

Using  muscle  cell  cultures  that  are  highly 


enriched  in  the  acetylcholine  receptor,  we  have 
investigated  the  regulation  of  the  phosphoryla- 
tion of  the  receptor  by  neurotransmitters,  hor- 
mones, and  neuropeptides.  Calcitonin  gene- 
related  peptide  (CGRP),  a  neuropeptide  that  is 
released  from  the  presynaptic  neuron  with  ace- 
tylcholine, increases  the  intracellular  levels  of 
cAMP  and  thereby  regulates  the  phosphorylation 
of  the  receptor  by  the  cAMP-dependent  protein 
kinase.  In  addition,  studies  in  our  laboratory  sug- 
gest that  acetylcholine  itself  regulates  intracellu- 
lar levels  of  calcium  and  thereby  regulates  the 
phosphorylation  of  its  own  receptor  by  the  cal- 
cium- and  diacylglycerol-dependent  protein  ki- 
nase. We  have  also  demonstrated  that  tyrosine 
phosphorylation  of  the  nicotinic  receptor  is  regu- 
lated by  the  neurons  that  synapse  on  muscle. 
More  recently,  in  collaboration  with  Bruce  Wal- 
lace (University  of  Colorado  Health  Sciences 
Center),  we  have  found  that  agrin,  an  extracellu- 
lar matrix  protein,  may  be  the  factor  from  neu- 
rons that  regulates  tyrosine  phosphorylation  of 
the  receptor.  Agrin  is  a  well-characterized  pro- 
tein that  is  secreted  from  neurons  and  induces 
receptor  clustering  under  the  nerve  during  syn- 
apse formation.  These  results  suggest  that  agrin- 
induced  tyrosine  phosphorylation  of  the  receptor 
may  be  involved  in  the  induction  of  clustering  of 
the  receptor  at  the  synapse. 

Our  recent  studies  on  GABA^  and  glutamate  re- 
ceptors have  supported  our  hypothesis  that  pro- 
tein phosphorylation  of  neurotransmitter  recep- 
tors plays  an  important  role  in  the  modulation  of 
their  function.  We  have  expressed  the  genes  for 
these  receptors  in  a  variety  of  cells  and  have  ana- 
lyzed the  effect  of  phosphorylation  on  their  func- 
tional properties.  Using  this  system,  we  have 
shown  that  phosphorylation  of  the  /3-subunit  by 
cAMP-dependent  protein  kinase  decreases  the  re- 
sponse of  the  GABA^  receptor  to  its  neurotransmit- 
ter and  alters  the  desensitization  of  the  receptor. 
In  addition,  recent  studies  have  demonstrated 
that  glutamate  receptors  are  phosphorylated  by  a 
protein-tyrosine  kinase.  The  functional  effect  of 
tyrosine  phosphorylation  of  the  glutamate  recep- 
tor is  currently  being  examined.  This  work,  com- 
bined with  our  studies  of  the  nicotinic  acetylcho- 
line receptor,  provides  strong  evidence  that 
protein  phosphorylation  of  neurotransmitter  re- 
ceptors is  a  primary  mechanism  in  the  regulation 
of  synaptic  transmission. 


192 


Molecular  Aspects  of  Signal  Transduction 
in  the  Visual  System 


James  B.  Hurley,  Ph.D. — Associate  Investigator 

Dr.  Hurley  is  also  Associate  Professor  of  Biochemistry  at  the  University  of  Washington  School  of  Medicine. 
He  received  his  undergraduate  degree  in  chemistry  from  the  State  University  of  New  York  College  of 
Environmental  Science  and  Forestry,  Syracuse,  and  his  Ph.D.  degree  in  physiology  and  biophysics  from 
the  University  of  Illinois,  Urbana,  where  he  worked  with  Thomas  Ebrey.  His  postdoctoral  research  included 
studies  with  Melvin  Simon  at  both  the  University  of  California,  San  Diego,  and  the  California  Institute  of 
Technology,  and  with  Lubert  Stryer  at  Stanford  University. 


OUR  laboratory  studies  molecular  mecha- 
nisms responsible  for  visual  transduction  in 
vertebrate  and  invertebrate  photoreceptors.  De- 
spite the  fact  that  these  two  types  of  cells  respond 
to  light  via  quite  diverse  mechanisms,  they  have 
many  general  features  in  common.  We  are  inves- 
tigating mechanisms  that  determine  such  photo- 
receptor characteristics  as  sensitivity,  rates  of  ac- 
tivation and  deactivation,  and  ability  to  adapt  to 
constant  light. 

Light  hyperpolarizes  vertebrate  photorecep- 
tors via  a  G  protein-mediated  cascade  that  culmi- 
nates in  cyclic  GMP  hydrolysis.  Depletion  of 
cGMP  reduces  the  activity  of  cGMP-gated  cation 
channels  in  the  photoreceptor  plasma  mem- 
brane. In  darkness  Ca^"^  enters  the  cell  through 
these  channels.  Light  blocks  this  entry,  and  the 
resulting  depletion  of  cytosolic  Ca^"^  promotes  re- 
covery by  stimulating  guanylate  cyclase  to  re- 
synthesize  cGMP. 

Invertebrate  photoreceptors  respond  to  light 
very  differently.  In  these  cells  light  activates 
phospholipase  C,  which  produces  inositol  tri- 
phosphate and  diacylglycerol  as  second  messen- 
gers. Few  biochemical  details  of  invertebrate 
phototransduction  are  well  understood. 

Vertebrate  Phototransduction 

Our  laboratory  recently  identified  a  novel 
Ca^"^-binding  protein  that  imparts  Ca^"^  sensitivity 
to  photoreceptor  guanylate  cyclase.  This  protein, 
named  recoverin,  promotes  recovery  by  stimulat- 
ing guanylate  cyclase  when  free  Ca^"^  concentra- 
tions fall  below  300  nM.  The  amino  acid  se- 
quence of  recoverin  reveals  three  Ca^"^-binding 
sites.  Ca^"^  influences  a  variety  of  physical  proper- 
ties of  recoverin,  including  fluorescence  and  mo- 
bility on  electrophoresis  gels.  We  cloned  re- 
coverin cDNA  and  expressed  recombinant 
recoverin  in  Escherichia  coli. 

The  effects  of  Ca^"^  on  recombinant  recoverin 
and  retinal  recoverin  are  quite  different.  To  ac- 
count for  these  differences,  we  compared  the 
masses  of  recombinant  and  retinal  recoverin  di- 
rectly by  ion-spray  mass  spectrometry.  To  our 
surprise,  the  modification  turned  out  to  be  a 


novel  type  of  heterogeneous  amino-terminal  acy- 
lation.  Each  recoverin  is  acylated  with  either  a 
CI4:0,  C14:1,  C14:2,  or  C12:0  fatty  acid 
residue. 

Following  stimulation  by  light,  transducin,  the 
photoreceptor  G  protein,  hydro lyzes  its  bound 
GTP  and  loses  its  ability  to  activate  phosphodies- 
terase. Photoreceptor  cells  recover  from  a  light 
flash  within  a  couple  of  seconds,  but  the  steady- 
state  hydrolysis  of  GTP  is  slower.  To  clarify  the 
role  that  GTP  hydrolysis  plays  in  recovery  from  a 
photoresponse,  we  produced  transgenic  mice 
that  express  a  mutant  transducin  that  hydrolyzes 
GTP  more  slowly  than  their  normal  counterparts. 
Preliminary  results  suggest  that  photoreceptors 
expressing  this  form  of  transducin  are  abnormally 
desensitized.  This  efi'ect  may  reflect  an  attempt 
by  the  cells  to  compensate  for  the  persistent 
phosphodiesterase  activity  of  the  mutant 
transducin. 


Drosophila  Vision 

Biochemical  and  physiological  evidence  sug- 
gests that  a  G  protein  mediates  invertebrate  pho- 
totransduction by  stimulating  phospholipase  C. 
We  characterized  several  Drosophila  G  proteins 
with  the  aim  of  understanding  their  role  in  inver- 
tebrate phototransduction.  In  addition  to  several 
G  protein  a-subunits,  two  G  protein  /3-subunits 
were  identified.  The  first  one  was  detected 
throughout  the  nervous  system  but  not  in  the 
eyes.  This  prompted  us  to  search  for  a  photore- 
ceptor G  protein  /3-subunit. 

Through  use  of  a  specific  type  of  cDNA  screen- 
ing method,  we  were  able  to  identify  a  novel  type 
of  G  protein  /S-subunit  that  is  expressed  specifi- 
cally in  the  Drosophila  compound  eye.  Recently, 
in  collaboration  with  Charles  Zuker  and  his  col- 
leagues (HHMI,  University  of  California,  San 
Diego) ,  two  mutant  Drosophila  strains  have  been 
identified  with  reduced  expression  of  this  eye- 
specific  |S-subunit.  Biochemical  analyses  of  eyes 
from  normal  Drosophila  and  from  these  mutants 
are  being  used  to  study  the  role  of  G  proteins  in 
phototransduction . 


193 


Molecular  Aspects  of  Signal  Transduction  in  the  Visual  System 


A  toxin  from  the  microorganism  Bordetella 
pertussis  specifically  inactivates  certain  G  pro- 
teins. To  study  the  physiological  importance  of  G 
proteins  in  Drosophila,  we  produced  transgenic 
flies  that  conditionally  express  endogenous  per- 
tussis toxin.  Pertussis  toxin  induction  in  adult 


Drosophila  alters  their  visual  response  and  eat- 
ing behavior.  We  are  investigating  the  molecular 
mechanisms  responsible  for  these  phenotypes. 

The  long-term  objective  addressed  in  these 
projects  is  to  identify  biochemical  mechanisms 
by  which  photoreceptors  respond  to  stimuli. 


194 


The  Molecular  Basis  of  Cell  Adhesion  in  Normal 
and  Pathological  Situations 


Richard  O.  Hynes,  Ph.D. — Investigator 

Dr.  Hynes  is  also  Professor  of  Biology  and  Director  of  the  Center  for  Cancer  Research  at  the  Massachusetts 
Institute  of  Technology.  He  received  his  undergraduate  degree  in  biochemistry  from  the  University  of 
Cambridge  and  his  Ph.D.  degree  in  biology  from  the  Massachusetts  Institute  of  Technology.  After  several 
years  of  postdoctoral  work  at  the  Imperial  Cancer  Research  Fund  laboratories  in  London,  where  he 
initiated  his  early  work  on  fibronectins,  he  returned  to  MIT  as  a  faculty  member.  Dr.  Hynes  has  been  the 
recipient  of  a  Guggenheim  Fellowship  and  is  a  Fellow  of  the  Royal  Society  of  London  and  the  American 
Association  for  the  Advancement  of  Science. 


MOST  cells  in  the  body  adhere  to  their  neigh- 
bors and  to  the  extracellular  matrix,  a  com- 
plex array  of  proteins  that  comprise  a  fibrillar 
meshwork  throughout  the  body.  Cell  adhesion 
plays  important  roles  in  the  normal  functions  of 
cells,  contributing  to  cellular  organization  and 
structure,  proliferation,  and  metabolism.  During 
embryological  development,  cell  adhesion  is  im- 
portant for  the  movements  of  cells  that  contrib- 
ute to  modeling  of  the  embryo.  In  the  adult,  ap- 
propriate cell  adhesion  is  necessary  for  numerous 
physiological  processes. 

For  example,  in  the  blood,  cells  known  as  plate- 
lets adhere  to  the  walls  of  blood  vessels  that  are 
damaged  and  help  to  prevent  bleeding.  This  ad- 
hesion process  is  essential  to  protect  against  hem- 
orrhage. On  the  other  hand,  it  is  equally  impor- 
tant that  platelets  should  not  adhere  at 
inappropriate  times.  If  they  do,  the  result  is 
thrombosis.  Thus  the  control  of  platelet  adhesion 
is  a  matter  of  life  and  death.  Other  blood  cells 
involved  in  defense  mechanisms  during  infection 
or  inflammation  need  to  adhere  to  the  walls  of 
blood  vessels  at  the  sites  of  infection  to  emigrate 
into  the  affected  tissues. 

Another  process  involving  cell  adhesion  and 
migration  is  wound  healing.  When  skin  is  dam- 
aged, the  skin  cells  migrate  in  over  the  wound  to 
cover  it.  The  processes  of  cell  migration  involved 
in  wound  healing  have  much  in  common  with 
those  occurring  during  development. 

A  final  example  is  that  of  cancer.  Tumor  cells 
exhibit  altered  adhesion  both  to  one  another  and 
to  their  surroundings.  This  altered  adhesion  is 
thought  to  be  involved  in  the  invasion  and  metas- 
tasis of  tumor  cells. 

These  examples  illustrate  the  importance  of 
appropriate  adhesion  of  cells  to  their  surround- 
ings. Our  laboratory  is  involved  in  molecular  anal- 
yses of  these  processes.  We  seek  to  understand 
the  proteins  involved  in  cell  adhesion  and  how 
they  control  adhesion  and  migration  of  cells  in 
both  normal  and  pathological  processes. 

Two  main  classes  of  proteins  interest  us.  The 
first  comprises  the  large  proteins  that  make  up 


the  extracellular  matrix.  These  proteins  cooper- 
ate to  build  a  fibrillar  meshwork  to  which  the 
cells  attach  and  on  and  through  which  they  mi- 
grate. We  have  investigated  several  of  these  pro- 
teins, which  we  refer  to  as  "nectins"  to  denote 
their  role  in  binding  to  cells.  Fibronectins,  a 
closely  related  group  of  proteins  all  encoded  by  a 
single  gene,  are  the  best  understood  of  these  nec- 
tins. The  different  forms  of  fibronectin  are  gener- 
ated by  alternative  RNA  splicing.  We  and  others 
have  analyzed  in  detail  the  functions  and  the 
structure  of  these  proteins.  This  work  is  leading 
to  a  deeper  understanding  of  their  roles  in  cell 
behavior.  For  example,  it  is  now  known  that  fibro- 
nectins have  several  sites  in  each  molecule  that 
bind  cells.  The  detailed  structure  of  these  bind- 
ing sites  is  being  elucidated.  One  intriguing  ob- 
servation is  that  fibronectins  share  with  many 
other  nectins  a  common  recognition  site  made  up 
of  only  three  amino  acids.  This  site  (designated 
RGD  in  the  single-letter  amino  acid  code)  is  rec- 
ognized by  receptor  molecules  on  cell  surfaces. 
This  interaction  can  be  blocked  by  antibodies  to 
the  nectins  or  to  the  receptors,  which  are  known 
as  integrins,  or  by  competitor  peptides  contain- 
ing the  RGD  sequence.  Such  blockades  interfere 
with  the  cell-adhesive  interactions  involved  in 
the  physiological  processes  discussed  above.  Re- 
cent work  has  identified  other  cell-binding  sites 
within  fibronectins,  which  are  recognized  by  dif- 
ferent receptors.  Cells  interact  with  these  mole- 
cules in  a  complex  fashion,  which  is  as  expected, 
given  the  participation  of  cell  adhesion  in  many 
diverse  cellular  functions. 

Our  second  major  focus  of  interest  is  the  family 
of  integrin  receptors.  These  comprise  a  family  of 
related  cell  surface  receptors,  each  composed  of 
two  subunits.  Each  integrin  receptor  has  a  partic- 
ular specificity  for  certain  nectins  and  mediates 
the  interactions  of  cells  with  the  extracellular 
matrix.  In  addition,  the  integrins  connect  to  the 
inside  of  the  cell,  where  they  mediate  interac- 
tions with  the  internal  structures  or  cytoskeleton 
of  the  cell  that  are  involved  in  the  shape,  organiza- 
tion, and  migration  of  cells.  This  integration  of 
the  organization  of  the  extracellular  matrix  with 


195 


The  Molecular  Basis  of  Cell  Adhesion  in  Normal 
and  Pathological  Situations 


the  cytoskeleton  inside  the  cells  is  one  of  the  ori- 
gins of  the  name  "integrins." 

Using  the  methods  of  cell  and  molecular  biol- 
ogy, we  are  studying  the  structure  and  function  of 
fibronectins  and  integrins,  their  interactions,  and 
their  roles  in  various  physiological  processes,  in- 
cluding development,  blood  clotting,  inflamma- 
jjon,  wound  healing,  and  cancer.  We  observe  reg- 
ulated expression  of  these  molecules  during 
these  processes,  and  it  is  clear  that  these  mol- 
ecules are  crucial  for  the  appropriate  behavior  of 
cells.  For  instance,  altered  expression  of  both  fi- 
bronectins and  integrins  in  tumor  cells  contrib- 
utes to  their  wayward  behavior,  and  expression  of 
these  proteins  is  altered  during  wound  healing. 
Using  recombinant  DNA  methods,  we  can  pro- 
duce specific  and  modified  forms  of  fibronectins 
and  integrins  and  thus  investigate  the  ways  in 
which  they  alfect  the  behavior  of  individual  cell 
types. 

We  have  recently  made  progress  in  analyses  of 
the  role  of  the  intracellular  portions  of  various 
integrin  receptors  in  interactions  with  the  cyto- 
skeleton. We  have  also  obtained  detailed  struc- 
tural information  about  the  cytoskeletal  protein 
talin,  which  is  a  primary  candidate  for  interac- 
tions with  integrins.  We  are  now  investigating 
possible  interactions  between  normal  and  mutant 
integrins  and  talin.  Further  progress  along  these 
lines  should  help  explain  the  effects  of  cell  adhe- 
sion on  cell  structure  and  behavior. 

Work  in  the  past  year  has  uncovered  evidence 
that  integrins  do  more  than  provide  a  physical 
connection  between  the  extracellular  matrix  and 
the  cytoskeleton.  Engagement  of  integrins  trig- 
gers tyrosine  phosphorylation  inside  the  cells, 
strongly  suggesting  signaling  via  integrins.  Inte- 
grin function  can  also  be  regulated  from  inside 
cells.  Cells  need  to  detach  as  well  as  attach.  How 
is  this  regulated?  We  find  that  cell  detachment 
can  apparently  be  triggered  by  phosphorylation 
of  the  cytoplasmic  domains  of  integrins.  Thus  our 
current  picture  of  integrins  is  that  they  can  medi- 
ate signaling  both  into  and  out  of  cells. 


To  extend  our  understanding  of  the  roles  of 
fibronectins  and  integrins  to  intact  organisms,  we 
use  genetic  analyses  in  two  animal  systems.  First, 
we  are  analyzing  the  role  of  integrins  during  the 
development  of  Drosophila  melanogaster,  a 
fruit  fly  that  is  suitable  for  genetic  analyses.  Flies 
with  mutations  in  genes  encoding  integrins  have 
defects  in  embryonic  development,  in  muscle 
function,  and  in  the  development  of  wings  and 
eyes.  Analyses  of  these  defects  provide  insights 
into  the  functions  of  these  proteins.  We  are  also 
identifying  new  integrin  species  in  Drosophila. 

In  the  second  genetic  project,  we  have  gener- 
ated strains  of  mice  that  are  mutant  for  fibronec- 
tins. When  both  normal  copies  of  the  fibronectin 
gene  are  ablated,  mouse  embryos  cannot  proceed 
normally  beyond  the  early  developmental  stage 
known  as  gastrulation.  We  have  also  made  more- 
subtle  mutations  in  the  fibronectin  gene  and  are 
currently  analyzing  their  effects.  These  mutant 
mice,  together  with  transgenic  mice  expressing 
different  forms  of  fibronectin,  should  allow  us  to 
dissect  the  functions  of  the  various  forms  of  fibro- 
nectin in  vivo.  Encouraged  by  the  progress  on 
fibronectin  molecular  genetics  in  mice,  we  have 
also  begun  to  generate  mutations  in  integrin  re- 
ceptor genes  and  in  other  cell  adhesion  mole- 
cules, particularly  selectins,  cell-cell  adhesion 
molecules  involved  in  the  early  steps  of  inflam- 
mation. As  our  analyses  proceed,  we  will  be  able 
to  investigate  the  effects  of  the  various  mutations 
on  hemostasis,  thrombosis,  wound  healing,  and 
tumor  development. 

These  studies  should  provide  a  deeper  under- 
standing of  the  molecular  basis  of  cell  adhesion 
and  its  involvement  in  physiological  and  patho- 
logical processes.  This  understanding,  in  turn, 
should  provide  opportunities  for  therapeutic 
treatments  of  diseases  such  as  thrombosis  and 
cancer. 

The  work  on  cultured  cells  and  some  of  the 
work  on  mice  are  supported  by  grants  from  the 
National  Institutes  of  Health. 


196 


Molecular  Genetics  of  Intracellular 
Microorganisms 


Ralph  R.  Isberg,  Ph.D. — Assistant  Investigator 

Dr.  Isberg  is  also  Assistant  Professor  of  Molecular  Biology  and  Microbiology  at  Tufts  University  School  of 
Medicine.  He  received  his  A.B.  degree  in  chemistry  from  Oberlin  College  and  his  Ph.D.  degree  in 
microbiology  and  molecular  genetics  from  Harvard  Medical  School.  He  conducted  postdoctoral  work  on 
bacterial  pathogenesis  in  the  laboratory  of  Stanley  Falkow  at  Stanford  University.  His  honors  include  a 
Searle  Scholars  Award  and  a  National  Science  Foundation  Presidential  Young  Investigator  Award. 


MANY  species  of  bacteria  are  capable  of  caus- 
ing diseases  by  colonizing  and  growing 
within  human  hosts,  using  tactics  that  avoid  nor- 
mal immune  responses.  As  part  of  a  general  strat- 
egy to  establish  an  infectious  niche,  a  variety  of 
microorganisms  cause  diseases  by  entering  and 
growing  inside  human  cells  soon  after  encounter. 
Bacteria  that  establish  infections  in  this  manner 
are  called  intracellular  microorganisms.  Among 
the  diseases  they  cause  are  tuberculosis  and  the 
most  common  types  of  sexually  transmitted  and 
food-borne  diseases  found  in  the  industrialized 
world.  Despite  the  prevalence  of  such  infections, 
there  was  little  information  until  recent  years  on 
the  factors  expressed  by  these  microorganisms 
that  allow  them  to  enter  host  cells  and  thrive. 

The  objectives  of  our  research  are  to  investi- 
gate two  important  aspects  of  the  life-style  of  in- 
tracellular microorganisms.  First,  we  would  like 
to  determine  at  the  molecular  level  how  these 
organisms  can  enter  human  cells  that  do  not  nor- 
mally internalize  bacteria.  Second,  we  want  to 
analyze  factors  they  encode  that  allow  them  to 
survive  and  grow  within  the  ordinarily  hostile  en- 
vironment of  human  cells.  Our  main  approach 
has  been  to  identify  bacterial  species  that  enter  or 
grow  particularly  well  within  host  cells  and  to 
develop  genetic  and  biochemical  techniques  for 
analyzing  their  strategies.  The  primary  rationale 
for  this  approach  is  that  it  provides  insights  into 
basic  processes  that  are  applicable  to  numerous 
intracellular  microorganisms. 

To  investigate  the  molecular  mechanism  of  bac- 
terial binding  and  entry  into  host  cells,  we  have 
been  analyzing  the  bacterium  Yersinia  pseudo- 
tuberculosis, an  organism  that  causes  an  intes- 
tinal disease  often  accompanied  by  infection  of 
multiple  organ  systems. 

To  investigate  intracellular  growth,  we  have 
been  analyzing  Legionella  pneumophila,  the 
causative  agent  of  Legionnaire's  disease  pneumo- 
nia. The  intracellular  growth  process  of  the  bacte- 
rium is  very  similar  to  that  of  a  wide  range  of 
intracellular  microorganisms,  and  development 
of  molecular  strategies  for  analyzing  it  has  been 
relatively  straightforward. 


Yersinia  pseudotuberculosis  Entry  Into 
Cultured  Human  Cells 

Y.  pseudotuberculosis  can  enter  host  cells  via 
three  different  paths.  For  each  path  the  microor- 
ganism apparently  encodes  a  unique  set  of  pro- 
tein factors  to  be  used  at  different  tissue  sites  dur- 
ing the  infection  process.  We  have  focused  on  the 
path  that  is  promoted  by  the  protein  invasin,  the 
product  of  the  bacterial  inv  gene.  Invasin  is  a 
108-kDa  protein  on  the  surface  of  the  bacterium 
that  allows  it  to  enter  human  cells  by  binding 
receptor  molecules  on  their  surface.  We  have 
shown  that  a  20-kDa  region  of  invasin  binds  the 
host  cells,  and  this  region  is  sufficient  to  promote 
uptake.  Evidence  indicates  that  after  the  binding 
occurs,  the  host  cells  do  most  of  the  work  in  in- 
ternalizing the  bacterium. 

Invasin  binds  at  least  four  different  receptors. 
Called  integrins,  these  had  been  previously  iden- 
tified by  investigators  interested  in  a  variety  of 
mammalian  cell  adhesion  processes.  The  particu- 
lar integrin  receptors  that  bind  invasin  can  adhere 
to  a  variety  of  mammalian  proteins,  such  as  fibro- 
nectin  and  molecules  that  allow  adhesion  of  im- 
mune response  cells  to  inflamed  tissues. 

Although  invasin  binds  these  well-character- 
ized receptors,  there  is  no  obvious  sequence  simi- 
larity between  invasin  and  other  proteins  that 
bind  integrins,  and  mutations  that  eliminate  the 
interaction  between  invasin  and  its  receptors 
identify  amino  acid  residues  not  previously 
shown  to  be  involved  in  integrin  binding. 

Integrins  are  clearly  not  the  only  host-encoded 
factors  necessary  for  internalization.  Analysis  of 
this  process  has  indicated  that  two  additional  fac- 
tors are  required.  Mutant  studies  of  one  of  these 
integrin  receptors  indicate  that  a  cell  structure 
called  a  clathrin  coat  directly  interacts  with  the 
integrin  receptor  during  the  internalization  of 
the  bacterium.  If  this  interaction  is  eliminated, 
the  bacterium  cannot  be  internalized.  A  second 
structure,  the  host  cell  cytoskeleton,  also  is  in- 
volved in  the  internalization,  but  we  believe  that 
this  structure  performs  an  indirect  role  and  does 
not  directly  bind  the  integrin  during  all  stages  of 
bacterial  uptake. 


197 


Molecular  Genetics  of  Intracellular  Microorganisms 


Our  investigation  of  invasin/integrin  has  led  to 
a  model  for  Yersinia  uptake  into  host  cells.  Bind- 
ing of  invasin  to  its  integrin  receptor  leads  to  rear- 
rangement of  the  cytoskeleton — rearrangement 
requisite  to  entry.  A  signal  must  be  sent  to  cause 
the  host  cell  to  internalize  the  microorganism, 
and  the  internalization  is  facilitated  by  the  ex- 
traordinary avidity  with  which  invasin  binds  its 
receptors.  Other  proteins  that  bind  the  identical 
integrins  cannot  produce  this  signal  so  effi- 
ciently, because  they  do  not  bind  the  receptors 
tightly. 

Thus  invasin  appears  to  promote  entry  of  the 
microorganism  because  it  binds  an  important  re- 
ceptor that  interacts  with  clathrin  coats  and  com- 
municates with  the  cell  cytoskeleton,  and  be- 
cause it  binds  so  tightly  to  this  receptor. 

Legionella  pneumophila  Growth 
in  Phagocytic  Cells 

Z.  pneumophila  causes  a  variety  of  diseases  in 
humans,  including  Legionnaire's  disease  pneumo- 
nia. The  bacterium  grows  in  lung  tissues  after  en- 
counter with  its  human  host.  Its  favorite  habitat  is 
within  alveolar  macrophages,  cells  that  normally 
function  to  kill  invading  microorganisms.  An  im- 
portant mechanism  for  macrophages  to  kill  or 
inhibit  the  growth  of  a  microorganism  is  to  inter- 
nalize it  and  sequester  it  in  a  compartment  called 
a  phagosome,  which  in  turn  fuses  with  a  lyso- 
somal compartment  filled  with  antibacterial  fac- 
tors. L.  pneumophila  is  able  to  grow  within  the 
phagosome,  convert  it  into  an  organelle  with  a 
unique  morphology,  and  prevent  the  introduc- 
tion of  the  antibacterial  lysosomal  components 
into  this  site. 

We  have  been  interested  in  determining  how  L. 
pneumophila  is  able  to  establish  and  grow  within 
this  protective  niche.  We  have  been  taking  two 
tactics  toward  analyzing  this  process.  Our  first 
approach  has  been  to  isolate  mutations  in  this  bac- 
terium that  prevent  it  from  growing  intracellu- 
larly.  Our  second  approach  has  been  to  identify 
factors  that  are  selectively  synthesized  by  the  bac- 
terium only  during  intracellular  growth. 

Using  the  first  approach,  three  easily  distin- 


guishable classes  of  mutants  have  been  isolated. 
The  first  class  causes  the  bacterium  to  be  internal- 
ized by  a  macrophage  via  a  novel  pathway,  and 
this  causes  an  extreme  defect  in  bacterial  growth. 
The  second  class,  and  most  easily  isolated,  con- 
sists of  mutants  that  are  no  longer  able  to  prevent 
the  lysosomal  contents  from  being  introduced 
into  the  phagosome.  The  third  class  appears  nor- 
mal for  uptake  as  well  as  for  shutting  out  the  lyso- 
somal components,  but  the  phagosome  contain- 
ing the  mutant  microorganism  no  longer  exhibits 
the  unique  morphology  usually  found  in  a  Le- 
gionella infection. 

These  classes  of  mutants  indicate  that  the  mi- 
croorganism performs  a  distinct  series  of  steps 
within  the  macrophage,  each  of  which  contrib- 
utes to  the  parasite's  efficient  growth.  To  investi- 
gate the  factors  missing  in  these  mutants  and  ana- 
lyze the  steps  in  growth  performed  by  this 
bacterium,  we  have  identified  a  small  region  of 
the  Legionella  chromosome  that  encodes  the  fac- 
tors missing  in  the  latter  two  mutant  classes.  We 
are  currently  analyzing  this  region  of  the  chromo- 
some intensively,  with  the  hope  of  purifying  the 
factors  encoded  by  this  region  in  order  to  de- 
scribe their  functions  in  molecular  detail. 

Our  second  approach  involves  using  a  novel 
scheme  to  identify  L.  pneumophila  genes  that  are 
regulated  in  a  fashion  such  that  they  are  turned 
off  when  the  bacterium  is  growing  outside  the 
host  cell  but  are  rapidly  turned  on  during  growth 
within  macrophages.  This  tactic  involves  cloning 
fragments  of  L.  pneumophila  chromosomal  DNA 
in  front  of  a  reporter  gene  and  introducing  these 
molecular  clones  into  the  bacterium.  All  molecu- 
lar clones  constructed  in  this  manner  are  killed 
unless  they  contain  genes  that  are  regulated  in  the 
desired  fashion.  Using  this  strategy,  we  have 
cloned  at  least  eight  genes  that  are  normally  only 
expressed  by  L.  pneumophila  when  the  microor- 
ganism is  within  a  host  cell. 

We  hope  that  these  two  strategies  will  allow 
identification  of  most  of  the  factors  encoded  by  L. 
pneumophila  that  mediate  the  striking  rearrange- 
ment of  host  cell  organelles  and  facilitate  groMT:h 
of  the  microorganism. 


198 


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i 


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Scanning  electron  micrograph  oflucerifase  reporter  mycobacteriophages  (LRMs )  adsorbed  to  the 
surface  o/Mycobacterium  bovis — BCG — cells.  This  is  a  model  for  the  use  of  LRMs  for  rapid  detec- 
tion o/ Mycobacterium  tuberculosis  cells  in  a  human  clinical  sample.  The  phage  particle,  once 
adsorbed,  injects  its  genome,  into  which  the  firefly  luciferase  gene  has  been  inserted.  Expression 
of  the  gene  causes  the  mycobacterial  cells  to  emit  light,  allowing  sensitive  and  rapid  detection  of 
M.  tuberculosis  cells  and  assessment  of  their  drug- susceptibility  patterns. 

Scanning  photograph  by  Rupa  Udani  and  Frank  Macalusa  in  the  laboratory  of  William  Jacobs. 


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200 


I 


Genetic  Approaches  to  the  Control 
of  Mycobacterial  Diseases 


William  R.  Jacobs,  Jr.,  Ph.D. — Assistant  Investigator 

Dr.  Jacobs  is  also  Associate  Professor  of  Microbiology  and  Immunology  and  of  Molecular  Genetics  at 
Albert  Einstein  College  of  Medicine.  He  received  a  B.A.  degree  in  mathematics  at  Edinboro  University  of 
Pennsylvania  and  a  Ph.D.  degree  in  molecular  cell  biology  from  the  University  of  Alabama  at 
Birmingham.  His  doctoral  work  on  Mycobacterium  leprae  was  performed  in  the  laboratory  of  Josephine 
Clark  Curtiss  and  Roy  Curtiss  III,  first  at  the  University  of  Alabama  and  then  at  Washington  University, 
St.  louis.  His  postdoctoral  studies  with  Barry  Bloom  focused  on  developing  systems  to  express  foreign 
antigens  in  the  tuberculosis  vaccine  strain  BCG  (bacille  Calmette-Guerin). 


TUBERCULOSIS,  caused  by  Mycobacterium 
tuberculosis,  continues  to  be  a  serious 
health  problem  throughout  the  world.  Even  in 
the  developed  countries,  this  plague  has  been 
escalating  in  recent  years.  The  World  Health  Orga- 
nization (WHO)  estimates  a  worldwide  inci- 
dence of  approximately  8  million  new  cases  and 
over  3  million  deaths  annually.  After  32  years  of 
steady  decline  in  the  United  States,  the  number  of 
new  cases  has  taken  a  surprising  and  alarming  up- 
ward turn  for  each  of  the  last  five  years.  In  1990 
there  was  a  34  percent  increase  in  New  York  City 
and  a  10  percent  increase  nationally.  More  re- 
cently a  strain  of  multidrug-resistant  M.  tubercu- 
losis has  resulted  in  numerous  deaths,  demon- 
strating the  dire  reality  that  tuberculosis  caused 
by  a  virtually  invincible  pathogen  is  now 
spreading. 

Other  mycobacteria  play  significant  roles  in 
world  health.  Mycobacterium  leprae  is  the  caus- 
ative agent  of  leprosy,  an  affliction  know  to  the 
ancients  that  affects  over  1 3  million  in  the  world 
today.  Another  agent  is  Mycobacterium  avium,  a 
major  opportunistic  pathogen  for  many  individ- 
uals with  AIDS. 

Our  ongoing  studies  are  aimed  at  developing 
novel  ways  of  treating  mycobacterial  disease,  par- 
ticularly tuberculosis,  using  molecular  genetic 
approaches.  In  addition,  we  have  taken  a  new 
look  at  the  vaccine  known  as  BCG  (bacille 
Calmette-Guerin),  which  has  been  used  widely 
since  1 924  to  prevent  tuberculosis.  We  are  genet- 
ically engineering  BCG  into  a  multivalent  vac- 
cine vector  that  can  elicit  a  protective  immune 
response  to  a  wide  variety  of  bacterial,  viral,  and 
parasitic  pathogens.  The  development  of  systems 
to  alter  mycobacteria  genetically  should  permit 
both  goals  to  be  achieved. 

Genetic  manipulation  of  these  microorganisms 
has  only  been  possible  in  the  last  few  years.  M. 
tuberculosis,  like  other  mycobacteria,  is  difficult 
to  analyze  genetically  for  a  variety  of  reasons. 
First,  the  tubercle  bacillus,  which  multiplies 
only  once  every  24  hours,  requires  3  weeks  to 
form  a  colony  from  a  single  cell.  In  contrast,  Esch 


erichia  coli  yields  visible  colonies  in  8  hours. 
The  leprosy  bacillus  has  yet  to  be  cultivated  in 
the  laboratory  and  can  only  be  grown  in  mouse 
footpads  or  the  nine-banded  armadillo.  Over  the 
last  five  years,  we  have  developed  a  series  of 
phage-  and  plasmid-based  vectors  that  have  en- 
abled the  efficient  introduction  of  recombinant 
DNA  into  mycobacteria.  Recombinant  DNA  tech- 
nologies have  opened  exciting  new  doors  to  basic 
knowledge  about  these  organisms  and  the  ways 
they  cause  infection.  In  addition,  these  technolo- 
gies offer  novel  reagents,  such  as  luciferase  re- 
porter phages,  and  recombinant  vaccines  that 
could  play  major  roles  in  combating  human 
disease. 

Epidemiological  Analysis 
of  Tuberculosis  Infections 

Use  of  restriction  fragment  length  polymor- 
phisms (RFLPs)  can  play  a  key  role  in  determin- 
ing the  mode  of  transmission  of  tuberculosis,  as 
individual  isolates  can  be  tracked  from  one  in- 
fected person  to  the  next.  The  recent  increases  in 
the  incidence  of  tuberculosis  in  the  United  States 
seem  to  be  largely  associated  with  the  AIDS  epi- 
demic. However,  it  was  unclear  whether  the  tu- 
berculosis seen  in  AIDS  patients  results  from 
reactivated  disease,  reflecting  pre -AIDS  expo- 
sure, or  from  a  first-time  infection.  In  collabora- 
tions with  researchers  in  San  Francisco,  including 
Gary  Schoolnik  (HHMI,  Stanford  University),  we 
have  undertaken  analyses  to  distinguish  these  two 
possibilities. 

Different  isolates  of  M.  tuberculosis  have  dif- 
ferent RFLP  patterns  when  probed  with  a  particu- 
lar DNA  element  found  in  M.  tuberculosis 
strains.  In  isolates  from  a  recent  tuberculosis  out- 
break among  AIDS  patients  in  a  group  home,  RFLP 
analysis  revealed  that  1 1  persons  had  all  been  in- 
fected with  the  same  strain  of  M.  tuberculosis. 
This  demonstrates  that  AIDS  patients  are  highly 
susceptible  to  M.  tuberculosis  infection  and  con- 
firms that  tuberculosis  is  highly  contagious. 
These  clear  results  should  be  translatable  into 
better  public  health  care  policies.  Similar  analy- 


201 


Genetic  Approaches  to  the  Control  of  Mycobacterial  Diseases 


ses  are  under  way  to  track  M.  tuberculosis  iso- 
lates that  are  multiply  drug  resistant. 

Luciferase  Reporter  Phages 

Accurate  diagnosis  of  M.  tuberculosis  infec- 
tion routinely  requires  4-6  weeks  to  allow  culti- 
vation of  the  tubercle  bacilli  and  time  to  perform 
tiie  appropriate  tests.  Assessment  of  the  drug- 
susceptibility  patterns  of  clinical  isolates  can  re- 
quire another  4-6  weeks  of  analysis.  In  light  of 
the  increasing  numbers  of  drug-resistant  speci- 
mens, we  set  out  to  devise  a  novel  diagnostic  test 
that  would  shorten  the  time  required  for  both 
procedures.  The  test  uses  a  mycobacteriophage,  a 
virus  that  infects  M.  tuberculosis,  into  which  we 
have  cloned  a  gene  that  encodes  a  reporter  en- 
zyme, the  luciferase  that  makes  fireflies  glow.  Lu- 
ciferase reporter  mycobacteriophage  (LRM)  par- 
ticles, when  mixed  with  bacterial  cells,  result  in 
the  production  of  light. 

The  LRM  test  is  extraordinarily  sensitive,  as 
photons  can  be  detected  at  extremely  low  levels. 
In  addition,  it  is  exquisitely  specific,  as  the  phage 
only  attaches  to  mycobacterial  cells.  Thus  the  pro- 
duction of  light  reveals  the  presence  of  M.  tuber- 
culosis in  the  sample.  We  have  undertaken  stud- 
ies in  clinical  application.  Since  the  luciferase 
reaction  requires  ATP  for  photon  production,  we 
also  hope  to  use  the  test  to  assess  the  metabolic 
activity  of  a  cell.  And  since  drug  treatment  of  my- 
cobacterial cells  abrogates  this  activity,  we  are 
also  exploring  the  use  of  the  test  to  distinguish 
drug-resistant  from  drug-sensitive  cells. 

Genetic  Analysis  of  Mycobacterial 
Virulence  Determinants 

We  would  like  to  know  why  pathogens  such  as 
M.  tuberculosis  or  M.  leprae  cause  severe  disease 
when  BCG  can  elicit  an  effective  immune  re- 
sponse against  these  organisms.  To  approach 
these  questions,  we  have  focused  on  developing 
systems  for  defining  and  characterizing  the  genes 
of  mycobacteria. 

The  first  approach  is  to  generate  specific  inser- 
tion mutations  within  the  virulent  M.  tuberculo- 
sis genome  and  to  screen  for  mutants  that  are  no 
longer  virulent  in  our  animal  models.  Toward 
this  goal,  we  are  doing  a  number  of  studies  de- 
signed to  generate  random  insertions  within  the 
genome  by  taking  advantage  of  natural  mycobac- 
terial transposons  or  recombination  systems. 
Complementary  to  this  mutant  isolation  is  the  de- 
velopment of  both  extrachromosomal  and  inte- 
grating vectors  that  permit  highly  efficient  in- 


troduction of  libraries  of  genes  from  virulent 
mycobacteria  into  avirulent  strains. 

The  combination  of  these  two  strategies  has  al- 
ready allowed  us  to  identify  genes  necessary  for 
the  biosynthesis  of  amino  acids,  purines,  and 
complex  polysaccharides  found  on  the  surface  of 
the  pathogenic  mycobacteria.  We  are  currently 
developing  a  variety  of  systems  to  screen  these 
libraries  for  genes  that  confer  virulence  charac- 
teristics to  the  avirulent  organisms.  By  identifying 
the  genes  and  their  products  responsible  for  spe- 
cific virulence  phenotypes,  we  hope  to  make  pos- 
sible the  design  of  strategies  to  prevent  or  control 
mycobacterial  disease.  In  addition,  the  geneti- 
cally engineered  avirulent  mutants  should  pro- 
vide novel  vaccine  candidates. 

Recombinant  BCG  Vaccines  and  Novel 
Vaccine  Strategies 

BCG  is  an  attenuated  mutant  of  bovine  tuber- 
culosis bacillus.  It  has  been  used  as  a  vaccine 
against  tuberculosis  in  humans  for  over  50  years. 
The  bacterium  possesses  several  unique  proper- 
ties that  make  it  ideal  for  use  as  a  live  vector  in 
generating  multivalent  vaccines.  BCG  is  a  safe 
vaccine,  having  been  used  in  2.5  billion  individ- 
uals with  a  mortality  rate  significantly  lower  than 
that  of  the  smallpox  vaccine.  It  is  the  only  live 
vaccine  other  than  oral  polio  that  WHO  recom- 
mends for  use  in  infants.  Also,  the  mycobacterial 
cell  wall  has  potent  adjuvant  properties  that  can 
engender  excellent  humoral  responses;  and  since 
BCG  is  stored  within  macrophages,  which  are  key 
antigen-presenting  cells,  it  follows  that  it  can  also 
elicit  cellular  immune  responses. 

In  the  last  few  years,  our  laboratory,  in  collabo- 
ration with  Barry  Bloom  (HHMI,  Albert  Einstein 
College  of  Medicine),  Medlmmune,  and  Graham 
Hatfull  at  the  University  of  Pittsburgh,  has  devel- 
oped a  series  of  expression  vectors  and  transfor- 
mation systems  whereby  foreign  genes  encoding 
antigens  from  virtually  any  pathogen  can  be 
cloned  and  expressed  in  BCG.  Mice  immunized 
with  these  recombinant  BCG  cells  have  been 
shown  to  elicit  both  humoral  and  cellular  im- 
mune responses  to  the  expressed  foreign  pro- 
teins. We  are  currently  cloning  and  expressing 
genes  from  pathogens  that  cause  leishmaniasis, 
schistosomiasis,  and  toxoplasmosis  in  BCG.  Im- 
munization of  mice  with  recombinant  BCG  will 
provide  useful  models  to  test  the  types  of  im- 
mune responses  that  can  be  elicited  with  these 
live  bacteria.  Ultimately  we  hope  to  engineer  a 
recombinant  BCG  that  could  protect  humans 
from  these  dread  diseases. 


202 


Mechanisms  of  Neurotransmitter  Storage 
and  Release 


Reinhard  John,  Ph.D. — Associate  Investigator 

Dr.  Jahn  is  also  Associate  Professor  of  Pharmacology  at  Yale  University  School  of  Medicine.  He  received  his 
Ph.D.  degree  at  the  University  of  Gottingen,  Germany.  His  postdoctoral  training  was  with  Hans  Dieter 
Soling  at  the  University  of  Gottingen  and  later  with  Paul  Greengard  at  Yale  University  and  the  Rockefeller 
University.  He  was  Assistant  Professor  at  the  Rockefeller  University  and  subsequently  headed  a  research 
group  at  the  Max  Planck  Institute  for  Psychiatry,  Martinsried. 


NERVE  cells,  or  neurons,  communicate  with 
each  other  and  with  other  cells  by  means  of 
small  molecules,  the  neurotransmitters.  This 
communication  occurs  at  specialized  contact 
zones,  the  synapses.  Upon  arrival  of  incoming  ac- 
tion potentials,  the  sender  cell  releases  its  neuro- 
transmitters, which  cross  the  synaptic  cleft.  The 
plasma  membrane  of  the  receiving  cell  has  spe- 
cific receptor  molecules  that,  in  turn,  translate 
the  signals  into  functional  changes. 

In  the  resting  state,  the  presynaptic  nerve  end- 
ings of  the  sender  cell  store  their  neurotransmit- 
ters in  small  membrane-enclosed  compartments, 
the  synaptic  vesicles.  When  an  action  potential 
arrives,  voltage-gated  calcium  channels  open  and 
calcium  ions  (Ca^"^)  enter  the  terminal  from  the 
extracellular  space.  Within  a  fraction  of  a  milli- 
second, synaptic  vesicles  fuse  with  the  plasma 
membrane,  releasing  their  contents.  The  vesicle 
membrane  protein  is  then  retrieved  by  endocyto- 
sis  for  use  in  regenerating  fusion-competent  syn- 
aptic vesicles. 

The  details  of  this  membrane  recycling  are  still 
largely  unclear,  and  the  enzymes  catalyzing  the 
major  steps  have  not  been  identified.  As  a  starting 
point  for  detailed  functional  analysis,  we  and 
others  have  characterized  the  major  membrane 
proteins  of  synaptic  vesicles.  This  was  facilitated 
by  the  fact  that  synaptic  vesicles  are  abundant  and 
can  easily  be  purified  in  large  amounts.  Due  to 
the  smallness  of  the  vesicles,  the  number  of  pro- 
tein species  per  individual  vesicle  is  inherently 
limited.  Therefore  it  should  be  possible  to  iden- 
tify most,  if  not  all,  of  the  major  protein  constitu- 
ents. To  date,  several  families  of  unique  vesicle 
proteins  have  been  characterized,  and  advanced 
tools  for  their  study  are  available.  We  are 
currently  involved  in  a  systematic  chemical  analy- 
sis of  the  vesicle  membrane  to  identify  the  re- 
maining proteins  of  this  organelle. 


Parallel  to  this  protein  analysis,  we  have  re- 
cently begun  to  establish  assay  systems  for  indi- 
vidual steps  of  the  vesicle  cycle.  One  of  the 
model  systems  accessible  for  biochemical  analy- 
sis is  the  isolated  nerve  terminal.  Upon  homogen- 
ization  of  nervous  tissue,  nerve  terminals,  though 
sheared  off  their  axons,  reseal  and  remain  func- 
tional for  several  hours  after  isolation.  Applica- 
tion of  depolarizing  stimuli  or  Ca^"^  ionophores 
causes  a  massive  exocytotic  transmitter  release 
and  a  parallel  increase  in  membrane  turnover. 

We  found  that  during  exo-endocytosis,  one  of 
the  vesicle  proteins,  the  GTP-binding  protein 
rab3A,  dissociates  from  the  vesicle  membrane 
and  reassociates  again  at  a  later  stage  of  the  mem- 
brane cycle.  This  dissociation-association  cycle  is 
probably  necessary  for  an  orderly  and  sequential 
processing  of  the  vesicle  membrane.  Thus  small 
GTP-binding  proteins  may  serve  in  the  nerve  ter- 
minal, as  in  other  organelles,  as  "status  indica- 
tors" for  the  recycling  membrane,  being  asso- 
ciated only  with  a  specific  step  in  the  life  cycle  of 
the  vesicle  membrane.  The  biochemical  events 
leading  to  GTPase  activation  and  membrane  disso- 
ciation and  reassociation  are  presently  under 
study.  In  addition,  the  individual  G  proteins 
should  allow  an  isolation  of  the  compartments 
representing  separate  steps  in  the  membrane 
cycle. 

Thus  we  have  isolated  clathrin-coated  vesicles 
from  nerve  terminals  by  conventional  procedures 
and  have  analyzed  their  membrane  and  coat  com- 
position. We  have  also  developed  immunoisola- 
tion  procedures  for  synaptic  vesicle-derived 
membrane  populations,  using  monoclonal  anti- 
bodies directed  against  individual  membrane 
components.  We  hope  these  studies  will  aid  in 
the  functional  definition  of  the  compartments  in- 
volved in  synaptic  vesicle  recycling  and  will  form 
the  basis  for  the  reconstitution  of  individual  steps 
of  the  membrane  cycle  in  cell-free  systems. 


203 


Molecular  Studies  of  Voltage-Sensitive 
Potassium  Channels 


Lily  Y.Jan,  Ph.D. — Investigator 

Dr.  Jan  is  also  Professor  of  Physiology  and  Biochemistry  at  the  University  of  California,  San  Francisco. 
During  her  graduate  study  at  the  California  Institute  of  Technology  with  Jean  Paul  Revel  and  Max 
Delbriick,  Dr.  Jan  localized  the  visual  pigment  rhodopsin  at  the  ultrastructural  level.  Because  she  studied 
high-energy  theoretical  physics  before  becoming  a  biology  student,  her  Ph.D.  degree  was  in  physics  and 
biophysics.  She  stayed  at  CalTech  to  do  postdoctoral  research  with  Seymour  Benzer  and  began  to 
collaborate  with  her  husband,  Yuh  Nungjan.  Their  first  collaboration  resulted  in  the  identification  of  the 
Shaker  locus  as  a  potential  structural  gene  for  a  potassium  channel.  Before  accepting  faculty  appointments 
at  UCSF,  the  Jans  worked  in  Stephen  Kuffler's  laboratory  at  Harvard  Medical  School. 


VOLTAGE-sensitive  potassium  channels  proba- 
bly constitute  the  most  diverse  and  wide- 
spread class  of  ion  channels.  More  than  30  differ- 
ent types  of  potassium  channels  have  been 
characterized.  They  dilFer  in  their  voltage  sensi- 
tivity, their  kinetic  properties,  and  their  sensitiv- 
ity to  second  messengers  within  the  cell.  Potas- 
sium channels  have  been  found  in  almost  every 
eukaryotic  cell  type  examined,  in  both  the  ani- 
mal and  the  plant  kingdoms.  They  are  important 
for  a  wide  range  of  physiological  functions,  in- 
cluding insulin  release  due  to  raised  glucose  lev- 
els, proliferation  of  lymphocytes  induced  by  mi- 
togens, and  the  movements  of  leaflets  in  plants  or 
the  opening  and  closing  of  leaf  stomatal  pores.  In 
the  mammalian  nervous  system,  potassium  chan- 
nels control  excitability  and  the  strength  of  sig- 
naling between  nerve  cells.  Indeed,  some  of  the 
potassium  channels  have  been  implicated  as 
playing  a  role  in  learning  and  memory. 

To  study  how  the  diversity  of  potassium  chan- 
nels arises  and  how  they  serve  the  wide  variety  of 
cellular  functions,  one  needs  to  study  these  chan- 
nels biochemically  as  well  as  biophysically.  How- 
ever, they  are  difficult  to  purify  because  they  are 
rather  heterogeneous  and  inaccessible.  For  this 
reason  we  have  taken  advantage  of  the  well- 
developed  genetic  technologies  applicable  to  the 
fruit  fly  Drosophila  melanogaster.  In  this  organ- 
ism, if  a  gene  (say,  coding  for  a  potassium  chan- 
nel) can  be  identified  by  the  abnormalities 
caused  by  its  mutations,  one  can  clone  it  for  mo- 
lecular studies  of  the  gene  product. 

More  than  a  decade  ago,  Yuh  Nungjan  (HHMI, 
University  of  California,  San  Francisco),  Mike 
Dennis,  and  I  found  that  mutations  at  the  Shaker 
locus  cause  prolonged  transmitter  release  from 
the  motor  nerve  terminal,  probably  because  of  a 
defect  in  potassium  channel  function.  The  locus 
was  subsequently  cloned  by  Diane  Papazian,  Tom 
Schwarz,  and  Bruce  Tempel  in  our  laboratory.  It 
codes  for  proteins  that  contain  multiple  stretches 
of  hydrophobic  amino  acids  that  can  potentially 
span  the  cell  membrane;  several  proteins  that 


diff^er  in  sequence  flanking  these  stretches  of  hy- 
drophobic amino  acids  are  generated  because  of 
alternative  splicing  of  the  primary  transcript. 
Leslie  Timpe  demonstrated  that  RNA  encoding 
for  these  Shaker  proteins  of  known  deduced  se- 
quence, when  injected  into  frog  oocytes,  causes 
the  functional  expression  of  potassium  channels 
of  different  kinetic  properties.  Since  the  different 
Shaker  proteins  are  present  in  different  regions 
of  the  fly  brain,  they  are  likely  to  give  rise  to  dif- 
ferent subtypes  of  A  channels. 

Starting  with  the  Shaker  gene  in  the  fruit  fly, 
Bruce  Tempel  isolated  a  gene  in  the  mouse  that 
codes  for  a  potassium  channel  in  the  mouse 
brain.  This  protein  is  65  percent  identical  in  its 
sequence  to  the  Drosophila  potassium  channel 
protein.  In  frog  oocytes,  the  gene  produces  potas- 
sium channels  that  do  not  inactivate  rapidly.  Anal- 
ysis of  the  distribution  of  amino  acid  residues  that 
appear  to  be  essential  and  have  been  totally  con- 
served over  600  million  years  has  provided  some 
clues  to  the  channel's  structure.  Now  over  1 5  dif- 
ferent mammalian  potassium  channel  genes  have 
been  characterized  by  a  number  of  laboratories. 
One  of  these  (the  rat  Shall  gene),  cloned  and 
characterized  by  Tim  Baldwin,  Meei-Ling  Tsaur, 
and  George  Lopez  in  our  laboratory,  produces  a 
rapidly  inactivating  potassium  channel  in  frog 
oocytes  and  is  expressed  in  the  heart  as  well  as 
the  brain.  The  high  degree  of  conservation  among 
these  mammalian  channels  and  the  fruit  fly  potas- 
sium channels  reiterates  the  point  that  any  experi- 
mental organism,  as  long  as  it  is  amenable  to  the 
specific  type  of  experimentation,  will  reveal  in- 
formation of  medical  interest. 

Having  cloned  some  of  the  potassium  channel 
genes,  we  can  now  ask  how  this  channel  works. 
How  does  it  detect  a  voltage  change  across  the 
cell  membrane  and,  responding,  open?  How  does 
it  "inactivate"  after  it  opens?  How  does  it  discrim- 
inate between  sodium  and  potassium  ions  and 
show  exquisite  selectivity?  To  probe  these  ques- 
tions, we  have  altered  specific  residues  of  the  po- 
tassium channel  to  see  how  the  various  functions 
are  affected.  Studies  in  our  laboratory  (Ehud 


205 


Molecular  Studies  of  Voltage-Sensitive  Potassium  Channels 


IsacofF,  George  Lopez,  Diane  Papazian,  and  Leslie 
Timpe;  this  work  is  supported  by  a  grant  from  the 
National  Institutes  of  Health)  and  others  have 
provided  evidence  for  the  involvement  of  spe- 
cific structural  elements  in  the  detection  of  volt- 
age changes  across  the  cell  membrane  and  in  the 
subsequent  conformational  changes  that  open 
the  channel  and  allow  the  cytoplasmic  mouth  of 
the  pore  to  interact  with  the  inactivation  gate, 
thereby  terminating  channel  opening. 

For  studies  of  the  biological  functions  of  potas- 
sium channels,  we  have  chosen  to  concentrate  on 
the  mammalian  heart  and  hippocampus.  A  variety 
of  cardiac  potassium  channels  have  been  charac- 
terized biophysically  and  are  important  in  con- 
trolling the  rhythmic  heartbeat.  Molecular  stud- 
ies of  these  channels  will  not  only  contribute  to 
our  understanding  of  channel  function  but  will 
also  be  relevant  clinically,  for  example,  in  the 
development  of  more-specific  drugs  for  cardiac 
arrhythmia.  The  hippocampus  is  a  region  of  the 
mammalian  brain  that  appears  to  play  an  impor- 
tant role  in  learning  and  memory.  It  has  also  been 
studied  extensively  in  experimental  paradigms 


that  induce  epileptic  activity.  By  cloning  and  ana- 
lyzing potassium  channel  genes  that  are  ex- 
pressed in  this  tissue,  we  hope  to  learn  about  the 
involvement  of  these  potassium  channels  in  the 
normal  function  and  pathology  of  the  nervous 
system. 

Using  specific  probes  for  individual  potassium 
channel  polypeptides  and  their  messenger  RNA, 
Morgan  Sheng  and  Meei-ling  Tsaur  have  found 
distinct  patterns  of  expression  in  the  mammalian 
brain.  In  addition  to  spatial  regulation  at  the  level 
of  brain  regions  as  well  as  specific  neuronal 
types,  dynamic  changes  in  potassium  channel 
gene  expression  have  also  been  observed  in 
the  adult  brain  following  pentylenetetrazole- 
induced  seizure.  (This  work  is  supported  by  a 
grant  from  the  National  Institute  of  Mental 
Health.)  The  observed  decrease  of  specific  potas- 
sium channel  transcripts  in  the  excitatory  neu- 
rons in  the  dentate  gyrus  of  the  hippocampus  is 
likely  to  lead  to  an  increase  of  excitability.  These 
observations  suggest  that  potassium  channel  gene 
regulation  may  contribute  to  long-term  neuronal 
plasticity. 


206 


Neural  Development  in  Drosophila 


Yuh  Nungjan,  Ph.D. — Investigator 

Dr.  Jan  is  also  Professor  of  Physiology  and  Biochemistry  at  the  University  of  California,  San  Francisco. 
Although  Dr.  Jan  went  to  the  California  Institute  of  Technology  to  study  theoretical  physics,  he  instead 
became  interested  in  biology  and  received  his  Ph.D.  degree  from  Caltech  in  biophysics  and  physics.  While 
there  he  studied  sensory  transduction  of  the  fungus  Phycomyces  with  Max  Delbriick.  Dr.  Jan  began  his 
study  of  the  nervous  system  during  postdoctoral  research  with  Seymour  Benzer  at  CalTech  and  continued 
this  line  of  research  with  Stephen  Kuffler  at  Harvard  Medical  School.  His  primary  interest  remains  the 
nervous  system. 


HOW  a  nervous  system  is  organized  during 
development  is  a  major  unresolved  problem 
in  biology.  For  several  years  our  laboratory  has 
been  interested  in  the  following  questions:  How 
do  neurons  arise  from  undifferentiated  ectoder- 
mal cells?  What  gives  the  neurons  their  individual 
identity  in  terms  of  shape  and  function? 

Our  long-term  goal  is  to  understand  these  pro- 
cesses at  the  molecular  level,  and  our  approach  is 
a  genetic  one.  We  first  isolate  mutations  that  af- 
fect neurogenesis,  neuronal  type,  or  axonal  path- 
way formation.  Identification  of  these  mutations 
can  lead  to  the  isolation  of  important  genes. 

During  the  last  few  years,  our  laboratory  has 
been  engaged  in  an  extensive  search  and  analysis 
of  mutants  affecting  neural  development  in  the 
fruit  fly  Drosophila.  To  identify  and  analyze  such 
mutants,  we  have  been  using  the  embryonic  sen- 
sory nervous  system,  which  has  been  character- 
ized in  considerable  detail  at  the  single-cell  level. 
Roughly  half  of  the  Drosophila  genome  has  been 
screened  for  mutations  that  alter  the  peripheral 
nervous  system  (PNS),  resulting  in  the  identifica- 
tion of  a  number  of  genes  that  specify  cell  fate  in 
the  embryo.  Analysis  of  those  genes  had  led  us  to 
propose  a  "progressive  determination  of  the 
PNS"  model. 

Early  during  embryogenesis,  cells  in  different 
locations  within  the  ectodermal  layer  acquire  un- 
equal developmental  potential  as  a  result  of  the 
actions  of  the  "prepattern  genes."  These  include 
genes  that  specify  dorsoventral  and  anteroposte- 
rior orientation  as  well  as  segmentation.  Cells 
from  some  of  the  domains  are  affected  by  the  ac- 
tion of  "proneural"  genes,  which  apparently  en- 
dow cells  with  the  competence  to  become  neuro- 
nal precursors.  Genes  of  the  achaete  scute 
complex  {AS-C)  and  daughterless  {da)  belong 
to  this  group.  Both  y45-C  and  da  encode  proteins 
with  the  helix-loop-helix  motif.  It  is  likely  that 
products  of  these  genes  may  form  homo-  or  heter- 
odimers  that  bind  to  DNA  and  regulate  the  tran- 
scription of  target  genes  in  order  to  initiate  neuro- 
nal precursor  development. 
As  a  neuronal  precursor  forms,  it  inhibits  neigh- 


boring cells  from  doing  so.  This  "lateral  inhibi- 
tion" involves  the  action  of  six  known  "neuro- 
genic" genes.  Removing  the  function  of  any  of 
the  six.  Notch,  Delta  (Dl),  the  Enhancer  of  split 
complex  [E(spl)-C\,  mastermind  (mam),  neu- 
ralized  (neu),  and  big  brain  {bib),  leads  to  hy- 
pertrophy of  both  the  central  nervous  system 
(CNS)  and  the  PNS,  presumably  as  a  result  of  los- 
ing lateral  inhibition. 

There  appear  to  be  at  least  two  independent 
cell-cell  interaction  pathways.  One  is  mediated 
by  the  gene  products  of  Notch  and  Dl,  both  cod- 
ing for  membrane  proteins  with  epidermal 
growth  factor  (EGF)-Iike  repeats.  E(spl)-C, 
mam,  and  perhaps  neu  are  involved  in  this  path- 
way. The  second  pathway  is  mediated  by  bib, 
which  encodes  a  membrane  protein  with  signifi- 
cant homology  to  the  bovine  major  intrinsic  pro- 
tein (MIP),  soybean  nodulin  26,  and  Escherichia 
coli  glycerol  facilitator,  which  allows  passive 
transport  of  small  molecules  such  as  glycine. 

The  commitment  of  neuronal  precursors  may 
involve  the  actions  of  a  group  of  "master  regula- 
tory" genes,  which  endow  the  precursor  with 
certain  unique  properties  of  the  nervous  system. 
The  identity  of  a  neuronal  precursor  is  further 
specified  by  "neuronal-type  selector"  genes.  For 
example,  the  cut  locus  is  required  for  external 
sensory  organs  to  acquire  their  correct  identity. 
In  the  absence  of  cut  function,  these  organs  are 
transformed  into  chordotonal  organs.  Normally 
cut  is  expressed  in  sensory  organ  precursors  but 
not  in  chordotonal  organ  precursors,  and  we 
think  that  the  cut  gene  determines  which  organ 
the  precursor  will  develop  into.  The  cm?  product 
contains  a  homeodomain  and  is  likely  to  act  as  a 
transcription  factor  regulating  the  expression  of 
downstream  differentiation  genes. 

Recently  we  began  to  work  on  the  problem  of 
axonal  pathfinding  and  target  recognition  in  Dro- 
sophila. Tracing  the  axonal  pathway  in  the  fly 
nervous  system  had  been  problematic  in  the  past. 
Because  of  the  small  size  of  the  neurons,  it  was 
difficult  to  use  traditional  methods  to  trace  path- 
ways, such  as  filling  neurons  with  fluorescent 
dyes  and  other  tracers.  However,  Ed  Giniger,  a 


207 


Neural  Development  in  Drosophila 


postdoctoral  fellow  in  our  laboratory,  found  a  so- 
lution to  this  difficulty. 

He  modified  the  enhancer  trap  method  we 
used  previously  by  replacing  the  /3-galactosidase 
(/3-gaI)  reporter  gene  with  a  kinesin-|8-gal  fusion 
gene.  The  idea  is  that  the  kinesin  part  of  the  fu- 
sion protein  will  drag  /3-gaI  down  the  axon  to  the 
terminal.  The  idea  works  very  nicely.  We  have 
-now  generated  over  a  thousand  enhancer  trap 
lines  with  kinesin-/«cZ  as  a  reporter  gene  and,  in 
more  than  a  hundred  of  those,  have  labeled  sub- 
sets of  CNS  and/or  PNS  pathways,  including  sen- 
sory and/or  motor  projections.  We  would  like  to 
choose  a  small  number  of  lines  that  reveal  simple 
axonal  pathways  (e.g.,  sensory  neuron  or  motor 
neuron  pathways)  as  a  basis  for  a  mutant  screen. 

Based  on  sequence  information  of  a  number  of 
genes  involved  in  neural  development,  it  seems 
that  the  majority  of  these  genes  contain  a  previ- 
ously identified  functional  motif — e.g.,  the  EGF 
repeat  found  in  Notch,  the  tyrosine  kinase  do- 
main in  sevenless,  the  homeodomain  in  cut,  and 
the  helix-loop-helix  motif  in  da  and  AS-C  In 
each  case  the  existence  of  such  a  motif  immedi- 
ately provides  strong  suggestions  for  the  likely 
mode  of  gene  action,  which  may  be  tested 
experimentally. 


The  existence  of  readily  identifiable  motifs  in 
the  majority  of  genes  involved  in  neural  develop- 
ment suggests  that  cells  have  a  limited  repertoire 
of  mechanisms  for  essential  regulatory  functions. 
These  include  various  signaling  systems,  such  as 
ligands,  receptors,  second  messenger  systems, 
and  regulators  of  gene  expression.  Perhaps  only  a 
modest  number  of  new  tools  had  to  be  invented 
for  the  formation  of  the  nervous  system.  Many  of 
the  cellular  mechanisms  used  in  neural  develop- 
ment may  already  have  been  available  before  the 
nervous  system  evolved.  Understanding  neural 
development  may  require  an  understanding  of 
the  usage  and  manipulations  of  these  basic  func- 
tional motifs. 

It  is  also  apparent  that  many  functional  motifs 
have  been  highly  conserved  during  evolution. 
Several  hundred  million  years  separate  the  in- 
sects and  vertebrates,  yet  the  aforementioned  mo- 
tifs (the  EGF  repeat,  tyrosine  kinase  domain,  ho- 
meodomain, helix-loop-helix)  are  common  to 
both  and  remarkably  enduring.  Thus  studying  de- 
velopment in  the  simpler  organisms  with  well- 
developed  genetics,  such  as  the  fruit  fly  and  the 
nematode,  may  provide  valuable  background  for 
the  investigation  of  corresponding  problems  in 
higher  forms. 


To  follow  the  development  of  a  nerve 
cell,  the  neurobiologist  will  typically 
inject  it  with  a  tracer  dye.  In  view  of  the 
minuteness  of  the  fruit fly  embryo,  how- 
ever, a  method  was  developed  to  cause 
the  fly  to  fill  its  own  neurons  with  a 
marker.  A  bacterial  enzyme  was  tar- 
geted to  nerve  processes  by  fusing  the 
gene  for  kinesin,  a  molecular  motor,  to 
the  gene  encoding  fi-galactosidase,  an 
enzyme  for  which  there  are  simple  col- 
orimetric  assays,  and  Drosophila  stocks  were  established  that  express  the  fusion  protein.  The 
example  shown  here  displays  the  pattern  seen  when  the  protein  is  made  in  all  the  neurons  of  the 
embryo.  The  central  ladder-like  structure  comprises  the  axons  of  the  ventral  nerve  cord,  and  the 
fine  lateral  extensions  are  the  peripheral  neurons  and  axons. 

Research  and  photograph  by  Ed  Giniger  in  the  laboratory  ofYuh  Nungjan. 


208 


Activation  ofCD4  T  Cells 


Charles  A.  Janeway,  Jr.,  M.D.  — Investigator 

Dr.  Janeway  is  also  Professor  of  Immunobiology  at  Yale  University  School  of  Medicine.  He  is  a  graduate  of 
Harvard  College  (B.A.,  chemistry)  and  of  Harvard  Medical  School.  He  had  research  training  during 
medical  school  at  the  National  Institute  for  Medical  Research  in  London,  England;  postdoctoral  training 
in  internal  medicine  at  the  Peter  Bent  Brigham  Hospital  in  Boston;  and  immunology  research  experience 
at  NIH  and  the  Biomedical  Center  in  Uppsala,  Sweden.  Dr.  Janeway  was  awarded  the  degree  of  Doctor 
Honoris  Causa  by  the  Copernicus  Medical  School  of  Jagellonian  University  in  Cracow,  Poland. 


THE  critical  event  in  most  immune  responses 
is  the  activation  of  specific  CD4  T  lympho- 
cytes. When  these  cells  are  lost,  as  happens  in 
AIDS,  the  immune  system  can  no  longer  defend 
the  host  against  infection.  Our  laboratory  is  study- 
ing the  mechanism  by  which  these  cells  become 
active.  This  is  a  complex  process  involving 
changes  in  both  the  cell  that  presents  the  activat- 
ing antigen  and  the  T  cell  itself. 

The  process  requires  two  events.  First,  the 
antigen-specific  receptor  on  the  T  cell  must  en- 
counter its  appropriate  ligand  on  the  surface  of 
an  antigen-presenting  cell.  This  results  in  spe- 
cific signaling  of  the  T  cell  that  will  cause  it  to 
become  immunologically  inert,  or  anergic.  Aner- 
gic T  cells  cannot  mount  responses.  In  order  for 
antigen  recognition  to  lead  to  an  eff'ective  re- 
sponse, the  CD4  T  cell  must  receive  a  second  sig- 
nal or  co-stimulator.  Studies  in  our  laboratory 
have  characterized  the  chemical  structure  of  sev- 
eral peptides  that  are  bound  by  class  II  molecules 
of  the  major  histocompatibility  complex  (MHC) 
and  thus  recognized  by  CD4  T  cells.  Second,  we 
have  identified  two  molecules  expressed  on  anti- 
gen-presenting cells  that  are  capable  of  co-stimu- 
lating the  activation  of  resting,  naive  CD4  T  cells. 
Finally,  we  have  shown  that  a  single  cell  must 
present  both  the  peptide-MHC  complex  and  the 
co-stimulator  in  order  for  normal  CD4  T  cells  to 
be  activated  eff'ectively. 

The  ligand  recognized  by  the  receptor  on  CD4 
T  cells  consists  of  a  peptide  fragment  of  a  foreign 
protein  antigen  bound  to  a  class  II  MHC-encoded 
molecule.  Our  laboratory  has  isolated  peptides 
from  such  molecules  and  characterized  their 
structure.  These  molecules  are  approximately  15 
amino  acids  in  length  and  have  specific  residues 
at  certain  positions  for  each  different  MHC  class 
II  molecule.  Although  the  cleavage  of  these  frag- 
ments from  the  native  proteins  has  not  been  char- 
acterized, the  results  of  our  analysis  suggest  that 
the  MHC  molecule  protects  the  peptide  fragment 
from  further  proteolytic  degradation. 

A  striking  result  in  these  studies  was  the  finding 
that  1 2  percent  of  normal  MHC  class  II  molecules 
on  antigen-presenting  cells  were  in  complex 


with  a  particular  self  peptide.  As  a  number  of  sim- 
ilarly dominant  peptides  were  observed,  it  seems 
possible  that  the  total  complexity  of  self  peptides 
to  which  the  immune  system  must  be  tolerant 
may  be  quite  limited.  This  is  important,  as  it  al- 
lows the  maximum  complexity  of  foreign  pep- 
tides to  be  recognized. 

Of  even  greater  interest  is  the  finding  that  this 
specific  complex  is  found  richly  represented  on 
cells  in  the  thymic  medulla,  including  bone 
marrow-derived  cells,  but  is  present  only  at  very 
low  levels  on  thymic  cortical  epithelium.  These 
two  tissues  are  known  to  carry  out  distinctive 
events  involving  self  MHC-peptide  complexes  in 
the  maturation  of  T  cells  within  the  thymus.  The 
finding  that  a  self  peptide  is  unevenly  distributed 
between  these  cell  types  suggests  that  T  cell  de- 
velopment occurs  under  different  selecting  envi- 
ronments for  different  processes.  The  antibody 
recognizing  this  processed  self  peptide  bound  to 
self  MHC  class  II  will  allow  our  laboratory  to 
probe  this  process  in  living  animals.  The  same 
technology  is  now  being  applied  to  the  analysis  of 
autoantigens  and  peptides  generated  by  intracel- 
lular infectious  agents  such  as  Salmonella 
typhimurium. 

In  addition  to  ligation  of  the  receptor,  the  same 
antigen-presenting  cell  must  also  express  co- 
stimulatory  molecules  on  its  surface  in  order  to 
activate  CD4  T  cells  optimally.  We  have  con- 
firmed the  importance  of  the  B7  molecule,  a 
known  co-stimulator  of  human  T  cells,  in  the  ac- 
tivation of  murine  CD4  T  cells  and  have  discov- 
ered that  the  heat-stable  antigen  can  also  serve  as 
a  co-stimulator  for  murine  T  cells.  Either  of  these 
proteins,  transfected  into  fibroblasts,  can  confer 
their  CD4  T  cell-activating  properties.  More- 
over, the  two  proteins  act  in  a  strongly  synergistic 
manner  on  normal  antigen-presenting  cells  to 
promote  the  proliferation  of  CD4  T  cells. 

Finally,  we  have  shown  that  B7  expression  is 
regulated  in  parallel  with  co-stimulatory  activity 
when  B  lymphocytes  respond  to  a  variety  of  mi- 
crobial products.  This  regulated  behavior  of  co- 
stimulators  allows  B  cells  to  present  antigen  to 
CD4  T  cells  for  tolerance  in  the  absence  of  micro- 


209 


Activation  ofCD4  T  Cells 


bial  infection  or  for  immunity  in  its  presence.  We 
documented  the  importance  of  this  by  showing 
that  tolerance  to  a  self  protein  could  be  broken  by 
inducing  the  expression  of  co-stimulator  mole- 
cules on  self  antigen-reactive  B  cells.  This  last 
system  may  help  to  explain  the  role  of  specific 
pathogens  in  the  initiation  of  autoimmunity. 
Parts  of  this  work  are  also  supported  by  grants 
from  the  National  Institutes  of  Health. 

The  T  cell  perceives  the  complex  of  foreign 
peptide  and  self  MHC  class  II  molecule  with  a 
receptor  resembling  the  antigen-binding  frag- 
ment of  an  immunoglobulin  molecule.  Although 
the  most  variable  portion  of  this  receptor  lies  in 
its  central  region,  generated  by  the  joining  of  sev- 
eral different  gene  segments,  some  variability 
is  also  expressed  around  the  periphery  of  the 
ligand-binding  site.  This  raises  the  question  of 
what  residues  on  the  peptide-MHC  complex  are 
contacted  by  these  peripheral  antigen-binding 
loops  on  the  T  cell  receptor. 

We  recently  showed  that  at  least  one  of  these 
loops  makes  contact  with  one  side  of  the  MHC 


molecule  binding  the  foreign  peptide.  Detailed 
mutagenesis  studies  of  both  the  T  cell  receptor 
and  the  MHC  protein  should  soon  reveal  the  de- 
finitive orientation  of  the  T  cell  receptor  to  its 
ligand. 

The  information  derived  from  our  studies  on  T 
cell  activation  is  being  applied  to  the  analysis 
of  a  model  autoimmune  disease,  the  insulin- 
dependent  diabetes  mellitus  that  occurs  sponta- 
neously in  non-obese  diabetic  mice.  We  have  iso- 
lated cloned  T  cells  capable  of  invading  the  islets 
of  irradiated  mice  and  destroying  (8-cells,  produc- 
ing diabetes.  In  addition,  we  have  identified 
other  cells  that  appear  to  protect  the  islet.  Analy- 
sis of  the  peptide-MHC  ligands  recognized  by 
these  various  cell  types,  and  of  the  events  in- 
volved in  their  activation,  should  permit  a  better 
understanding  of  diabetes  mellitus  and  lead  to  its 
specific  immunomodulation.  This  work  is  also 
supported  by  a  grant  from  the  National  Institutes 
of  Health.  Thus  the  basic  science  base  of  the  labo- 
ratory is  being  applied  to  disease  models  in  hopes 
of  improving  therapy  in  immunological  diseases. 


210 


Control  of  Cell  Pattern  in  the  Developing 
Nervous  System 


Thomas  M.  Jessell,  Ph.D. — Investigator 

Dr.  Jessell  is  also  Professor  of  Biochemistry  and  Molecular  Biophysics  at  Columbia  University  College  of 
Physicians  and  Surgeons  and  a  member  of  the  Center  for  Neurobiology  and  Behavior.  He  received  his 
Ph.D.  degree  in  neurobiology  from  Cambridge  University,  England,  and  was  elected  a  research  fellow  of 
Cambridge's  Trinity  College.  He  was  a  postdoctoral  fellow  in  Gerald  Fischbach's  laboratory  at  Harvard 
Medical  School.  Next  he  served  as  Assistant  Professor  of  Neurobiology  at  Harvard  Medical  School,  before 
moving  to  Columbia  University. 


OUR  research  is  aimed  at  understanding  the 
mechanisms  that  control  cell  patterning 
within  the  developing  vertebrate  nervous  system. 
The  major  focus  is  on  deciphering  how  discrete 
neural  cell  types  appear  at  defined  positions  in 
the  embryo.  In  addition,  we  are  examining  the 
roles  of  diffusible  and  cell  surface  molecules  in 
the  guidance  of  developing  axons  in  the  spinal 
cord.  Our  studies  over  the  past  several  years  have 
provided  evidence  that  the  floor  plate,  a  special- 
ized group  of  neuroepithelial  cells,  has  critical 
roles  in  both  the  control  of  cell  identity  and  in 
axon  guidance.  Within  the  past  year,  further  de- 
tails of  the  functions  and  molecular  properties  of 
the  floor  plate  have  become  apparent. 

Control  of  Motor  Neuron  DiflFerentiation 

Our  previous  studies  had  shown  that  signals 
originating  from  the  floor  plate  regulate  the  iden- 
tity of  specific  cell  types  within  the  neural  tube. 
In  order  to  examine  in  more  detail  the  actions  of 
the  floor  plate  on  neural  cell  patterning,  we  have 
begun  to  focus  on  one  well-characterized  neuron 
class,  the  spinal  motor  neuron.  There  is  extensive 
information  on  the  mechanisms  that  control  the 
pathfinding  of  motor  axons  and  the  formation  of 
synapses  at  the  neuromuscular  junction,  but  the 
events  that  control  the  generation  of  motor  neu- 
rons remain  largely  obscure. 

Insight  into  the  molecular  mechanisms  in- 
volved in  the  generation  of  motor  neurons  by 
floor  plate-derived  signals  requires  the  identifi- 
cation of  genes  that  are  expressed  at  the  initial 
stages  of  motor  neuron  differentiation.  In  collabo- 
ration with  Thomas  Edlund's  laboratory  in  Umea, 
Sweden,  we  have  found  that  embryonic  chick 
motor  neurons  express  a  homeobox  gene  called 
Islet- 1  (Isl-1).  This  is  a  member  of  the  subfamily 
of  homeobox  genes  that  contain  cysteine-rich  re- 
gions called  LIM  domains.  Other  members  of  the 
family  include  Lin- 11  and  Mec-3,  which  have 
been  shown  to  regulate  cell  fate  in  Caenorhab- 
ditis  elegans. 

Isl- 1  binds  to  enhancer  elements  in  the  rat  in- 
sulin gene  and  is  expressed  in  pancreatic  islet 
cells  and  in  a  subset  of  neurons,  including  motor 


neurons.  In  the  embryonic  chick  spinal  cord, 
Isl- 1  immunoreactivity  is  first  detected  in  the  nu- 
clei of  cells  in  the  ventral  region,  lateral  to  the 
floor  plate.  The  number  of  Isl- 1*  cells  in  the  ven- 
tral spinal  cord  increases  markedly  during  early 
spinal  cord  development.  We  established  that  the 
ventral  spinal  cord  cells  that  express  Isl- 1  are  mo- 
tor neurons  by  retrograde  injection  of  horserad- 
ish peroxidase  (HRP)  into  motor  axons  in  the 
ventral  root. 

Analysis  of  the  expression  of  motor  neuron 
markers  by  chick  spinal  cord  cells  in  vivo  has 
provided  evidence  that  the  differentiation  of  mo- 
tor neurons  is  dependent  on  inductive  signals 
from  the  floor  plate.  In  agreement  with  this,  the 
spinal  cord  of  embryos  that  had  received  floor 
plate  grafts  contained  additional  ectopic  Isl-1^ 
cells.  The  induced  Isl-1^  cells  also  expressed  the 
SCI  glycoprotein  and  sent  axons  out  of  the  spinal 
cord  consistent  with  their  identity  as  motor 
neurons. 

These  results  provide  evidence  that  signals 
from  the  floor  plate  can  induce  the  expression  of 
Isl-1  in  dorsal  neural  tube  cells.  Elimination  of 
the  notochord  and  floor  plate  before  neural  tube 
closure  results  in  the  development  of  a  spinal 
cord  devoid  of  ventral  neuronal  types,  including 
motor  neurons.  Isl- 1  expression  in  the  ventral  spi- 
nal cord  is  also  dependent  on  signals  from  the 
notochord  and  floor  plate.  These  results  support 
the  idea  that  elimination  of  the  notochord  and 
floor  plate  prevents  the  initial  steps  in  the  differ- 
entiation of  motor  neurons. 

Homeobox  genes  are  involved  in  many  aspects 
of  vertebrate  development.  The  expression  pat- 
tern of  Hox  genes  along  the  anteroposterior  axis 
of  the  neural  tube,  and  of  Pax  genes  along  the 
dorsoventral  axis,  together  with  the  phenotypes 
that  result  from  inactivation  of  some  of  these 
genes,  suggests  that  they  contribute  to  the  re- 
gional patterning  of  the  developing  nervous  sys- 
tem. In  contrast,  the  restricted  expression  of  Isl-1 
and  the  involvement  of  related  LIM-homeodo- 
main  proteins  in  the  determination  of  cell  fate  in 
C.  elegans  suggest  that  Isl- 1  may  be  involved  in 
specifying  the  fate  of  specific  neuronal  subtypes 
— in  particular,  motor  neurons. 


211 


Control  of  Cell  Pattern  in  the  Developing  Nervous  System 


Floor  Plate-Specific  Genes 
in  Axon  Guidance 

After  the  identity  of  motor  neurons  and  other 
spinal  cord  neurons  has  been  established,  the 
floor  plate  appears  to  provide  both  long-range 
and  local  guidance  cues  that  promote  the  growth 
of  axons  to  and  across  the  ventral  midline  of  the 
jspinal  cord.  First,  the  floor  plate  secretes  a  diffu- 
sible chemoattractant  that  can  orient  the  growth 
of  axons  of  commissural  neurons  in  vitro  and 
may  account  for  the  homing  of  these  axons  to  the 
floor  plate  in  vivo.  Second,  the  floor  plate  may 
contribute  to  the  change  in  trajectory  of  commis- 
sural axons  from  the  transverse  to  the  longitu- 
dinal plane  that  occurs  immediately  after  cross- 
ing the  ventral  midline.  In  support  of  this 
proposal,  genetic  mutations  in  mice  and  zebra 
fish  that  result  in  the  absence  of  the  floor  plate 
during  embryonic  development  lead  to  errors  in 
the  pathfinding  of  commissural  axons  at  the  mid- 
line of  the  spinal  cord.  Third,  the  floor  plate  may 
promote  the  fasciculation  of  commissural  axons 
that  occurs  after  they  cross  the  midline  of  the  spi- 
nal cord  by  regulating  the  expression  of  glyco- 
proteins of  the  immunoglobulin  superfamily. 
The  specialized  role  of  the  floor  plate  in  verte- 
brate neural  development  has  parallels  in  inver- 
tebrate organisms,  in  that  cells  at  the  midline  of 
the  embryonic  Drosophila  and  C.  elegans  central 
nervous  systems  have  been  implicated  in  neural 
patterning  and  axon  guidance. 

To  identify  molecules  that  may  mediate  the  di- 
verse functions  of  the  floor  plate  during  early 
neural  development,  we  have  used  subtractive  hy- 
bridization techniques  to  isolate  cDNA  clones  ex- 
pressed selectively  by  the  floor  plate.  One  of 
these  cDNA  clones  encodes  a  novel  secreted  pro- 
tein, F-spondin,  which  is  expressed  at  high  levels 
by  the  rat  floor  plate  during  embryonic  develop- 
ment. F-spondin  contains  domains  similar  to 
those  present  in  thrombospondin  and  other  pro- 
teins implicated  in  cell  adhesion  and  neurite  out- 
growth. In  vitro  assays  show  that  F-spondin 
promotes  neural  cell  adhesion  and  neurite 
outgrowth,  suggesting  that  its  secretion  by  the 
floor  plate  contributes  to  the  growth  and  guid- 
ance of  axons  in  the  developing  central  nervous 
system. 

The  F-spondin  protein  may  be  associated  with 
the  extracellular  matrix,  since  it  has  several  clus- 
ters of  basic  residues  that  function  as  glycosami- 


noglycan-binding  domains  in  other  secreted  pro- 
teins. The  restricted  distribution  of  F-spondin 
mRNA  in  the  embryonic  nervous  system  contrasts 
with  the  distribution  of  other  secreted  glycopro- 
teins that  promote  neural  cell  adhesion  and  neu- 
rite outgrov^h.  For  example,  the  expression  of 
F-spondin  mRNA  is  more  restricted  than  that  of 
thrombospondin  and  of  tenascin,  which  appear 
to  be  expressed  widely  in  the  embryonic  central 
nervous  system. 

The  prominent  expression  of  F-spondin  in  the 
floor  plate  suggests  that  the  protein  may  be  in- 
volved in  the  plate's  development  or  functions. 
Midline  neural  plate  cells  that  give  rise  to  the 
floor  plate  undergo  marked  changes  in  cell  shape 
during  the  closure  of  the  neural  tube.  Thus  one 
possible  function  of  F-spondin  could  be  to  medi- 
ate adhesive  plate  cell  interactions  that  maintain 
the  integrity  of  the  floor  plate  during  formation 
of  the  embryonic  spinal  cord. 

The  expression  of  F-spondin  mRNA  in  floor 
plate  cells  is  highest  at  the  time  of  the  plate's 
suggested  role  in  the  chemotropic  guidance  of 
commissural  axons.  However,  recombinant  F- 
spondin  does  not  mimic  the  ability  of  the  floor 
plate-derived  chemoattractant  to  promote  the 
outgrowth  of  commissural  axons  from  dorsal  spi- 
nal cord  explants.  This  suggests  that  F-spondin 
may  not  be  involved  in  the  long-range  guidance 
of  commissural  axons  to  the  floor  plate,  at  least 
through  chemotropism. 

F-spondin  is  more  likely  to  be  involved  in  the 
contact-dependent  guidance  of  commissural 
axons  once  they  reach  the  ventral  midline  of  the 
spinal  cord  under  the  influence  of  distinct  che- 
motropic guidance  cues.  The  growth  cones  of 
commissural  neurons  cross  the  midline  by  grow- 
ing between  the  basal  surface  of  floor  plate  cells 
and  the  underlying  basal  lamina.  Floor  plate- 
secreted  F-spondin  may  accumulate  at  high  levels 
in  association  with  the  basal  surface  of  floor  plate 
cells  or  with  the  underlying  basal  lamina,  thus 
generating  a  difference  in  adhesive  properties  of 
the  floor  plate  and  the  lateral  neuroepithelium. 
The  growth  cones  of  commissural  neurons  may 
adhere  preferentially  to  F-spondin,  prompting 
them  to  change  trajectory  at  the  boundary  of  the 
floor  plate  and  lateral  neuroepithelium.  It  is  also 
possible  that  F-spondin  has  a  more  active  signal- 
ing role  that  induces  changes  in  the  properties  of 
commissural  growth  cones,  permitting  them  to 
respond  to  other  midline  guidance  cues. 


212 


Energy-transducing  Membrane  Proteins 


H.  Ronald  Kaback,  M.D. — Investigator 

Dr.  Kaback  is  also  Professor  of  Physiology  and  of  Microbiology  and  Molecular  Genetics  in  the  Molecular 
Biology  Institute  of  the  University  of  California,  Los  Angeles.  He  received  his  M.D.  degree  from  the  Albert 
Einstein  College  of  Medicine,  interned  at  Bronx  Municipal  Hospital  Center,  and  did  postdoctoral  research 
in  physiology  at  Einstein.  Subsequently  he  conducted  research  in  membrane  biochemistry  at  the  National 
Heart  Institute  and  the  Roche  Institute  of  Molecular  Biology,  chairing  at  Roche  the  Department  of 
Biochemistry.  Dr.  Kaback  is  a  member  of  the  National  Academy  of  Sciences  and  the  American  Academy 
of  Arts  and  Sciences.  Among  his  honors  is  the  Distinguished  Alumnus  Award  from  the  Albert  Einstein 
College  of  Medicine. 


THE  molecular  mechanism  of  energy  trans- 
duction in  the  membranes  of  living  cells  is  an 
enigma.  Although  the  immediate  driving  force 
for  many  seemingly  unrelated  processes,  such  as 
active  transport,  oxidative  phosphorylation,  and 
bacterial  motility,  is  a  bulk-phase,  transmem- 
brane electrochemical  H"^  or  Na^  gradient,  the 
molecular  mechanism  (s)  by  which  free  energy 
stored  in  these  gradients  is  transduced  into  work 
or  into  other  forms  of  chemical  energy  (e.g., 
ATP)  remains  unknown.  In  order  to  gain  insight 
into  this  important  basic  problem,  studies  in  our 
laboratory  have  focused  on  an  enzyme,  the  lac- 
tose (lac)  permease  of  the  bacterium  Escherichia 
coli,  as  a  paradigm. 

The  ability  of  E.  coli  to  accumulate  the  sugar 
lactose  and  other  |8-galactosides  against  a  concen- 
tration gradient  is  dependent  upon  lac  permease, 
a  very  hydrophobic  plasma  membrane  protein 
that  catalyzes  the  coupled  translocation  of  a  sin- 
gle sugar  molecule  with  a  single  H"^  (i.e.,  symport 
or  co-transport) .  Under  physiological  conditions, 
lac  permease  utilizes  free  energy  derived  from 
downhill  translocation  of  to  drive  accumu- 
lated (S-galactosides  against  a  concentration  gra- 
dient or,  conversely,  uses  free  energy  released 
from  downhill  translocation  of  /J-galactosides  to 
drive  uphill  translocation  of  H^.  The  polarity  of 
the  reaction  reflects  the  direction  of  the  concen- 
tration gradient  of  the  substrate.  As  such,  lac  per- 
mease represents  a  huge  number  of  machines  that 
catalyze  similar  reactions  in  virtually  all  biologi- 
cal membranes,  from  archebacteria  to  the  mam- 
malian central  nervous  system. 

The  permease  is  encoded  by  the  lacY  gene, 
which  has  been  cloned  into  a  recombinant 
plasmid  and  sequenced.  By  combining  overex- 
pression  with  the  use  of  a  specific  photoaffinity- 
labeled  substrate  for  the  permease  and  recon- 
stitution  of  transport  activity  in  artificial 
phospholipid  vesicles  (i.e.,  proteoliposomes), 
the  permease  was  solubilized  from  the  mem- 
brane, purified  to  homogeneity,  and  shown  to  cat- 
alyze all  the  transport  reactions  typical  of  the  fi- 


galactoside  transport  system  in  vivo  with  similar 
turnover  numbers.  Therefore,  a  single  enzyme — 
the  product  of  lacY — is  solely  responsible  for  all 
of  the  translocation  phenomena  catalyzed  by  the 
iS-galactoside  transport  system.  In  addition,  evi- 
dence has  been  presented  that  the  permease  is 
functional  as  a  monomer. 

Based  on  circular  dichroic  measurements  indi- 
cating that  purified  permease  is  about  80  percent 
helical  and  on  hydropathy  analysis  of  the  de- 
duced amino  acid  sequence,  a  secondary  struc- 
ture was  proposed.  The  model  predicts  that  the 
protein  has  a  short  hydrophilic  amino  terminus, 
12  hydrophobic  domains  in  a-helical  conforma- 
tion that  traverse  the  membrane  in  zigzag  fashion 
connected  by  hydrophilic  loops,  and  a  17- 
residue  hydrophilic  carboxyl-terminal  tail.  Spec- 
troscopic, biochemical,  and  immunological  data 
are  consistent  with  the  general  features  of  the 
model  and  indicate  that  the  amino  and  carboxyl 
termini  are  on  the  cytoplasmic  surface  of  the 
membrane.  Studies  on  an  extensive  series  of  lac 
permease-alkaline  phosphatase  chimeras  have 
provided  strong  support  for  the  topological  pre- 
dictions of  the  12-helix  model. 

This  report  concentrates  on  current  experi- 
ments that  involve  the  use  of  site-directed  muta- 
genesis to  engineer  lac  permease  so  as  to  permit 
certain  biochemical  and  biophysical  approaches 
to  structure-function  relationships. 

Recent  studies  provide  definitive  support  for 
the  argument  that  cysteinyl  residues  do  not  play  a 
direct  role  in  the  lac  permease  mechanism.  Thus, 
when  site-directed  mutagenesis  is  used  to  replace 
each  of  the  eight  cysteinyl  residues  simulta- 
neously, the  "C-Iess"  permease  catalyzes  active 
lactose  transport  moderately  well  relative  to 
wild-type  permease  (about  35  percent  of  the 
maximum  velocity  and  55  percent  of  the  steady- 
state  level  of  accumulation).  Moreover,  active 
lactose  transport  in  right-side-out  vesicles  con- 
taining C-less  permease  is  not  inactivated  by 
the  alkylating  agent,  7V-ethylmaleimide,  in  dra- 
matic contrast  to  vesicles  containing  wild-type 
permease. 


213 


Energy-transducing  Membrane  Proteins 


Although  site-directed  mutagenesis  is  useful 
for  delineating  amino  acid  residues  that  are  im- 
portant for  lactose-H"^  symport  and/or  substrate 
binding  and  recognition,  it  has  become  apparent 
that  high-resolution  structure  is  required  to  be- 
gin to  determine  the  role  of  these  residues  in  the 
mechanism.  Moreover,  dynamic  information  at 
high  resolution  will  also  be  required  to  solve  the 
mechanism.  In  this  respect,  chemical  labeling 
and  spectroscopic  approaches  in  w^hich  reactive 
cysteinyl  residues  are  tagged  v^^ith  radioactive 
sulfhydryl  reagents,  electron  paramagnetic  la- 
bels, or  fluorescent  probes  are  potentially  power- 
ful means  for  examining  static  and  dynamic 
aspects  of  protein  structure-function  relation- 
ships at  high  resolution.  A  principle  difficulty 
with  the  general  approach,  however,  is  the  com- 
plexity resulting  from  the  presence  of  multiple 
cysteinyl  residues  in  most  proteins,  eight  in  the 
case  of  lac  permease.  Thus,  in  addition  to  the  im- 
portant conclusion  that  cysteinyl  residues  do  not 
play  an  important  role  in  the  mechanism  of  lac 
permease,  the  construction  of  a  functional  per- 
mease molecule  devoid  of  cysteinyl  residues  pro- 
vides the  basis  for  an  approach  to  the  analysis  of 


static  and  dynamic  aspects  of  permease  structure- 
function  relationships. 

By  using  the  lacY  gtne  encoding  C-less  per- 
mease, for  instance,  it  is  now  possible  to  design 
mutants  in  which  an  individual  amino  acid  resi- 
due in  a  putative  hydrophilic  or  hydrophobic 
domain  is  replaced  with  a  cysteinyl  residue. 
This  can  then  be  reacted  specifically  with  ei- 
ther permeant  or  impermeant  sulfhydryl  re- 
agents in  right-side-out  or  inside-out  membrane 
vesicles,  followed  by  solubilization  and  im- 
munoprecipitation.  In  addition,  single  Cys  mu- 
tants can  be  solubilized  and  purified,  tagged 
with  appropriate  electron  paramagnetic  or  fluo- 
rescent probes,  then  reconstituted  and  studied 
spectroscopically. 

Finally,  it  should  be  possible  to  study  proxim- 
ity relationships  between  transmembrane  do- 
mains by  placing  single  cysteinyl  residues  in  pairs 
of  helical  domains  predicted  to  lie  close  to  each 
other  within  the  membrane.  In  these  contexts,  it 
is  encouraging  that  more  than  200  single  Cys  re- 
placements have  been  constructed  in  the  C-less 
permease  and  that  the  great  majority  of  the  mu- 
tants exhibit  highly  significant  transport  activity. 

Lactose  transport  in  Escherichia  coli.  A: 
Uphill  lactose  (Lac )  transport  in  response  to 
generated  either  by  respiration  or  A  TP  hy- 
drolysis. B:  Uphill  transport  in  response 
to  an  inwardly  directed  lactose  gradient.  C: 
Uphill  transport  in  response  to  an  out- 
wardly directed  lactose  gradient. 

From  Kaback,  H.R.  1989.  Harvey  Lect 
83:77-105.  Copyright©  1989  Alan  R.  Liss, 
Inc.  Reprinted  by  permission  ofWiley-Liss,  a 
division  of  John  Wiley  and  Sons,  Inc. 


214 


Control  of  the  Immunoglobulin 
Heavy-Chain  Gene 


Thomas  R.  Kadesch,  Ph.D. — Associate  Investigator 

Dr.  Kadesch  is  also  Associate  Professor  of  Human  Genetics  at  the  University  of  Pennsylvania  School  of 
Medicine.  He  received  his  Ph.D.  degree  in  biochemistry  from  the  University  of  California,  Berkeley,  where 
he  studied  with  Michael  Chamberlin.  His  postdoctoral  research  was  done  with  Paul  Berg  at  the  Stanford 
University  School  of  Medicine. 


EXPRESSION  of  immunoglobulin  genes  is  lim- 
ited to  one  cell  type,  namely  B  lymphocytes. 
Only  B  cells  express  the  functions  required  for 
gene  rearrangement,  the  process  whereby  mature 
immunoglobulin  genes  are  formed  from  discrete 
gene  segments;  and  only  B  lymphocytes  possess 
the  components  necessary  for  immunoglobulin 
gene  transcription,  the  process  that  creates  an 
RNA  copy  of  the  rearranged  genes. 

Within  these  genes,  there  are  at  least  two  major 
transcriptional  regulatory  elements:  the  pro- 
moter, a  DNA  domain  located  close  to  the  site 
where  transcription  begins,  and  the  enhancer, 
another  DNA  domain  that  stimulates  initiation 
from  the  promoter.  The  activities  of  each  of  these 
elements  are  restricted  to  the  B  cells  and  are  con- 
trolled by  proteins  that  bind  them.  We  have  fo- 
cused our  studies  on  the  proteins  binding  the  im- 
munoglobulin heavy-chain  (IgH)  gene  enhancer, 
in  an  attempt  to  understand  how  they  elicit  the 
enhancer's  B  cell-specific  activity. 

Contributions  from  a  number  of  laboratories, 
including  our  own,  led  to  a  detailed  map  of  the 
IgH  enhancer.  The  map  indicates  the  protein- 
binding  locations  and  gives  some  information 
about  the  regulatory  mechanism.  The  enhancer, 
while  relatively  small  (200  base  pairs),  proves  to 
be  exceedingly  complex.  Interestingly,  many  of 
the  perhaps  nine  or  more  distinct  enhancer- 
binding  proteins  are  found  in  multiple  cell  types, 
even  those  in  which  the  enhancer  is  normally  in- 
active. To  explain  this,  it  has  been  argued  that 
some  of  the  proteins  may  serve  to  stimulate  the 
enhancer's  activity,  while  others  may  repress  it. 

During  the  past  few  years,  our  efforts  have  been 
directed  toward  the  isolation  of  cDNAs  that  en- 
code these  enhancer  binders.  Thus  far,  we  have 
identified  six.  We  are  presently  using  segments  of 
these  genes  (cDNAs)  to  manipulate  and  character- 
ize their  encoded  proteins  both  structurally  and 
functionally. 

We  have  shown  that  two  of  the  IgH  enhancer- 
binding  proteins,  E2-5  and  TFE3,  are  involved  in 
a  fascinating  transcriptional  regulatory  scheme. 
In  B  cells  the  situation  is  relatively  straightfor- 
ward, as  both  proteins  bind  the  enhancer  and  act 


in  concert  to  stimulate  its  activity.  In  non-B  cells 
the  situation  is  more  complicated.  In  vivo  exper- 
iments suggest  the  presence  of  an  additional  pro- 
tein, a  repressor,  that  binds  the  enhancer  and  pos- 
sibly precludes  E2-5  action. 

Binding  of  this  putative  repressor  has  two  ef- 
fects. First,  the  enhancer  is  less  active  as  a  result 
of  the  absence  of  bound  E2-5  protein.  Second,  the 
repressor  can  attenuate  at  a  distance  the  function 
of  TEE  3.  Hence  the  presence  of  the  repressor  in 
non-B  cells  results  in  the  shutdown  of  both 
E2-5-  and  TFE3-mediated  activation.  The  effects 
of  the  repressor  can  be  overcome  in  these  cells  by 
artificially  overproducing  the  E2-5  protein.  Pre- 
sumably such  overexpression  is  sufficient  to  dis- 
place the  bound  repressor. 

A  third  gene  segment  isolated  in  the  laboratory 
likely  encodes  the  repressor.  Although  we  have 
yet  to  prove  this  directly,  the  encoded  protein, 
denoted  Zeb,  is  structurally  distinct  from  the 
aforementioned  proteins  that  activate  transcrip- 
tion and,  moreover,  falls  into  the  family  of  so- 
called  zinc  finger  proteins,  many  of  which  have 
been  shown  to  repress  transcription  of  other 
genes.  The  DNA  sites  to  which  Zeb  binds  in  the 
test  tube  overlap,  but  are  distinct  from,  those  that 
the  activator  proteins  bind,  yet  are  identical  to 
those  that  confer  the  repressing  activity  within 
cells.  Ongoing  experiments  should  further  char- 
acterize Zeb's  mode  of  action  and  possible  roles 
in  other  gene  systems. 

E2-5  can  be  specifically  repressed  by  yet  an- 
other protein,  a  structural  relative  named  Id 
(identified  and  initially  characterized  by  Harold 
Weintraub  [HHMI,  Ered  Hutchinson  Cancer  Re- 
search Center]).  Unlike  Zeb,  Id  binds  directly  to 
E2-5  (and  related  proteins),  prevents  its  binding 
to  DNA,  and  thus  keeps  it  from  stimulating  tran- 
scription. Id  expression  was  observed  to  fall  off 
in  several  cell  lines  when  they  were  induced  to 
differentiate,  suggesting  that  it  may  serve  as  a  gen- 
eral antagonist  of  cellular  differentiation  by  in- 
hibiting DNA-binding  proteins  specifically  re- 
quired for  differentiation. 

The  importance  of  E2-5  for  IgH  enhancer  activ- 
ity led  to  our  asking  whether  Id  plays  a  role  dur- 


215 


Control  of  the  Immunoglobulin  Heavy-Chain  Gene 


ing  B  lymphocyte  maturation.  In  collaboration 
with  Stephen  Desiderio  (HHMI,  Johns  Hopkins 
University  School  of  Medicine),  we  analyzed  Id 
mRNA  expression  in  a  variety  of  cell  lines  repre- 
senting different  stages  of  B  lymphoid-cell  dif- 
ferentiation. Only  two  of  these  lines,  represent- 
ing the  earliest  stages  of  development,  were 
Jfound  to  express  Id.  We  confirmed  that  these 
cells  were  unable  to  support  the  activity  of  the 
IgH  enhancer,  whereas  later-stage  cells  could. 
Hence  Id  is  restricted  to  early  B  lymphoid-cell 
progenitors,  and  its  presence  correlates  inversely 
with  IgH  enhancer  activity.  Presumably  Id  serves 
to  keep  enhancer  activity  low  in  the  B  cell  pro- 
genitors. Further  differentiation  of  B  cells  would 
require  a  decrease  in  Id  expression  that,  in  turn, 
would  allow  the  IgH  enhancer  to  become  active. 


We  have  shown  that  Id  also  plays  an  important 
role  in  the  development  of  another  hematopoi- 
etic lineage,  the  myeloid  lineage.  In  collabora- 
tion with  Giovani  Rovera  (Wistar  Institute),  we 
demonstrated  that  Id  is  expressed  in  neutrophil 
precursors.  When  induced  to  differentiate  in  cul- 
ture, the  Id  levels  in  these  cells  were  observed  to 
decrease,  but  only  transiently.  Cells  bearing  an 
artificial,  constitutively  expressed  Id  gene  failed 
to  differentiate.  Hence  an  appropriate,  transi- 
ent shutoflf  of  Id  is  required  for  neutrophil 
development. 

An  important  corollary  to  these  results  is  that 
myeloid  cells  must  utilize  proteins  of  the  E2-5 
family  to  promote  their  differentiation.  We  are 
presently  attempting  to  identify  and  characterize 
these  proteins. 


216 


Genetic  Control  of  Hemoglobin  Synthesis 


Yuet  Wat  Kan,  M.D.,  D.Sc. — Investigator 

Dr.  Kan  is  also  Louis  K.  Diamond  Professor  of  Hematology  in  the  Departments  of  Laboratory  Medicine  and 
Medicine  at  the  University  of  California,  San  Francisco.  He  received  his  M.D.  and  D.Sc.  degrees  from  the 
University  of  Hong  Kong  Medical  School.  After  internship  and  residency  at  Queen  Mary  Hospital,  Hong 
Kong,  he  obtained  postdoctoral  training  in  hematology  at  Peter  Bent  Brigham  Hospital,  the  Massachusetts 
Institute  of  Technology,  Royal  Victoria  Hospital  at  McGill  University,  and  the  Children 's  Hospital,  Boston. 
Dr.  Kan  has  received  numerous  honors,  including  the  Gairdner  Foundation  International  Award  and  the 
Albert  Lasker  Clinical  Medical  Research  Award.  He  is  a  fellow  of  the  Royal  Society  (London )  and  a  member 
of  the  National  Academy  of  Sciences  and  of  the  Academia  Sinica  (Taiwan ). 


THE  focus  of  our  research  is  the  molecular 
basis  of  genetic  diseases  affecting  the  hemato- 
poietic or  blood-forming  cells.  The  two  diseases 
we  have  studied  in  depth  are  sickle  cell  anemia 
and  thalassemia.  Both  result  from  abnormal  glo- 
bin  production  and  constitute  important  health 
problems  in  the  Mediterranean  region,  Africa,  the 
Middle  East,  and  Asia.  In  the  United  States  these 
disorders  occur  frequently  among  people  of  Afri- 
can, Italian,  Greek,  and  Asian  descent.  We  have 
defined  the  mutations  that  give  rise  to  these  de- 
fects and  devised  DNA  analyses  for  their  detec- 
tion. In  addition,  we  are  studying  the  control  of 
globin  gene  expression  in  red  cell  precursors  and 
the  signals  that  switch  these  genes  from  fetal  to 
adult  globin  production. 

Previously  we  demonstrated  that  the  common 
genetic  defect  in  a-thalassemia  is  deletion  of  the 
a-globin  structural  gene.  We  also  defined  some  of 
the  molecular  lesions  in  jS-thalassemia.  These 
studies  led  to  our  ability  to  detect  thalassemia  by 
analysis  of  fetal  DNA. 

We  initiated  a  new  method  of  linkage  analysis 
using  restriction  endonucleases  to  detect  poly- 
morphism in  DNA  sequences  and  applied  it  to 
tracing  the  evolution  of  the  sickle  and  thalasse- 
mia mutations.  Restriction  enzyme  site  polymor- 
phism is  now  an  important  tool  for  detecting 
many  genetic  disorders  and  for  mapping  the  ge- 
netic loci  of  many  diseases. 

We  developed  a  method  for  prenatal  diagnosis 
of  sickle  cell  anemia  and  thalassemia.  Initially, 
fetal  samples  were  required.  With  the  advent  of 
recombinant  DNA  technology,  mutations  in  the 
human  genome  can  be  analyzed  directly  using 
DNA  obtained  by  amniocentesis  or  chorionic  vil- 
lus biopsy,  permitting  early  in  utero  diagnosis  of 
these  conditions. 

Prenatal  Diagnosis  of  Sickle  Cell  Anemia 
and  Thalassemia 

The  polymerase  chain  reaction  has  made  it  pos- 
sible to  diagnose  many  point  mutations  rap- 
idly. For  sickle  cell  anemia,  practical  nonradio- 


active tests  are  now  available.  In  the  case  of 
(S-thalassemia,  however,  the  need  to  diagnose 
multiple  mutations  makes  these  tests  somewhat 
tedious,  especially  in  the  developing  countries 
where  the  disease  is  common.  Hence  we  are  now 
devising  a  rapid  approach  using  the  reversed  dot 
blot  principle.  Oligonucleotides  corresponding 
to  the  mutations  common  in  a  given  area  are  im- 
mobilized on  a  filter;  the  test  DNA  is  amplified 
and  hybridized  to  the  oligonucleotides;  and  one 
of  several  nonradioactive  methods  is  then  used 
for  detection.  We  believe  this  procedure  will  fa- 
cilitate prenatal  diagnosis  of  i8-thalassemia  on  a 
broad  scale. 

Control  of  Globin  Gene  Expression 

We  are  studying  the  factors  that  control  the  tis- 
sue- and  development-specific  expression  of  the 
human  globin  genes.  Synthesis  of  the  globin 
chains  is  precisely  coordinated  during  develop- 
ment. The  embryonic  e-  and  f-globins,  which  are 
synthesized  in  the  early  embryo,  are  replaced  in 
the  fetus  by  the  a-  and  7-globin  chains.  Prior  to 
birth,  the  /3-globin  chain  is  known  to  become  pre- 
dominant over  the  7-globin  chain;  but  the  factors 
that  control  the  expression  of  the  /3-globin  gene 
in  the  bone  marrow  cells  and  coordinate  the  ex- 
pression of  the  globin  genes  during  development 
have  not  yet  been  elucidated.  We  are  now  study- 
ing the  DNA  sequences  and  protein  factors  that 
govern  the  expression  of  these  genes. 

Recently  DNA  elements  that  are  important  for 
the  control  of  the  tissue-specific  and  develop- 
ment-specific expression  of  the  globin  genes 
have  been  revealed.  This  sequence  is  known  as 
the  locus  control  region,  or  LCR.  In  the  jS-globin 
gene,  the  LCR  contains  four  hypersensitive  sites, 
which  contain  consensus  sequences  that  bind 
trans-acting  factors.  We  have  been  studying  the 
cis  sequences,  which  are  important  for  protein- 
DNA  interactions  in  two  ways. 

First,  we  have  determined  by  mutation  analysis 
of  these  sequences,  using  both  DNA-transfection 
and  transgenic  experiments,  that  important  core 


217 


Genetic  Control  of  Hemoglobin  Synthesis 


elements  called  NFE2-AP1 -binding  sites  are  es- 
sential for  these  enhancing  activities.  In  addition, 
using  in  vivo  footprint  experiments,  we  have  ex- 
amined the  consensus  sequences  that  are  impor- 
tant for  in  vivo  interaction.  We  have  studied  sev- 
eral cell  lines  that  express  different  amounts  of  6-, 
7-,  and  /3-globin  genes  and  have  found  that  the 
consensus  sequences  are  bound  differently  in 
these  cell  lines,  depending  on  the  type  of  fi- 
globin  gene  expressed.  We  are  now  characteriz- 
ing the  factors  responsible  for  this  binding  in 
order  to  understand  the  molecular  basis  of  hemo- 
globin switching. 

Red  Cell  Membrane  Disorders 

Many  hereditary  hemolytic  anemias  are  accom- 
panied by  spherocytosis  or  elliptocytosis.  The  de- 
fects in  these  disorders  lie  in  mutations  of  various 
cytoskeleton  proteins,  such  as  spectrin,  ankyrin, 
and  protein  4.1.  We  have  studied  families  with 


hereditary  elliptocytosis  due  to  protein  4.1  defi- 
ciency and  have  defined  three  different  defects 
due  to  gene  rearrangements. 

Protein  4. 1  is  found  in  all  cell  types  and  exists 
in  multiple  isoforms  generated  by  alternate  splic- 
ing in  at  least  five  different  regions  of  the  gene. 
Some  isoforms  are  more  abundant  in  nucleated 
cells,  others  in  mature  red  cells.  Although  pro- 
tein 4.1  maintains  the  integrity  of  the  cytoskele- 
ton in  the  red  cell,  its  function  in  nucleated  cells 
is  not  yet  known.  In  a  patient  who  has  no  protein 
4.1  in  the  red  cell,  severe  hemolytic  anemia  en- 
sues, while  other  cellular  functions  are  normal. 
Structural  analysis  showed  that  the  protein  4.1 
gene  is  rearranged  at  the  region  corresponding  to 
the  amino  terminal  of  the  erythroid  protein  4.1. 
Otherwise  it  continues  to  produce  functional 
protein  4.1  by  an  alternate  splicing  mechanism, 
which  skips  the  mutated  region.  This  example 
may  point  out  an  advantage  of  alternate  splicing. 


218 


Cell  Biological  Studies  of  Memory 


Eric  R.  Kandel,  M.D. — Senior  Investigator 

Dr.  Kandel  is  also  University  Professor  of  Physiology  and  Psychiatry  at  the  Center  for  Neurobiology  and 
Behavior  of  the  Columbia  University  College  of  Physicians  and  Surgeons.  He  was  born  in  Vienna,  Austria. 
He  graduated  from  Harvard  College,  having  majored  in  history  and  literature,  and  received  his  medical 
degree  from  the  New  York  University  School  of  Medicine.  He  took  postdoctoral  training  with  Wade 
Marshall  in  the  Laboratory  of  Neurophysiology  at  NIH  and  with  Ladislav  Tauc  at  the  Institut  Morey  in 
Paris.  Dr.  Kandel  was  the  founding  director  of  the  Center  for  Neurobiology  and  Behavior  at  Columbia. 
He  is  a  member  of  the  National  Academy  of  Sciences  and  counts  among  his  honors  the  Lasker  Award,  the 
Gairdner  Award,  and  the  National  Medal  of  Science. 


LEARNING  is  commonly  divided  into  two  ma- 
jor types:  declarative  and  reflexive.  Declara- 
tive learning  refers  to  the  acquisition  of  informa- 
tion about  people,  places,  or  things.  Reflexive 
learning  refers  to  the  acquisition  of  motor  skills 
and  strategies.  Our  laboratory  has  been  studying 
elementary  forms  of  reflexive  learning  in  the  gill- 
withdrawal  reflex  of  the  marine  snail  Aplysia.  In 
an  attempt  to  compare  these  mechanisms  with 
those  underlying  declarative  forms  of  learning, 
we  are  also  conducting  a  study  of  long-term  po- 
tentiation in  the  mammalian  hippocampus.  The 
present  discussion  is  limited  to  reflexive  studies 
in  Aplysia. 

We  have  shown  that  the  gill-withdrawal  reflex 
can  be  modified  by  nonassociative  and  associa- 
tive forms  of  reflexive  learning,  giving  rise  to 
both  short-  and  long-term  memory,  whose  dura- 
tion is  a  function  of  the  number  of  training  trials. 
We  have  recently  focused  on  sensitization,  a  non- 
associative  form  of  learning  in  response  to  a 
noxious  stimulus.  To  analyze  the  relationship  be- 
tween the  memory  for  short-  and  long-term  sensi- 
tization, we  have  studied  in  particular  detail  one 
component  of  the  neural  circuit  of  this  reflex:  the 
connections  between  the  siphon  sensory  neuron 
and  the  gill  motor  neurons.  These  connections 
can  be  studied  both  in  the  intact  animal  and  in 
dissociated  cell  culture. 

With  sensitization  the  connections  undergo  an 
increase  in  synaptic  effectiveness  (facilitation), 
whose  duration  is  a  function  of  the  number  of 
behavioral  reinforcing  stimuli  to  the  tail.  Simi- 
larly, in  culture,  the  duration  of  the  facilitation  is 
a  function  of  the  number  of  applications  of  seroto- 
nin (5-HT),  a  modulatory  transmitter  released  by 
tail  stimuli .  A  single  tail  stimulus  or  a  single  pulse 
of  5-HT  produces  short-term  facilitation  lasting 
minutes,  whereas  continuous  application  of  5-HT 
for  1 .5  h  or  four  or  five  pulses  over  a  1 .5-h  period 
elicit  long-term  facilitation  lasting  one  or  more 
days. 

In  both  the  behavioral  reflex  and  the  monosyn- 
aptic facilitation,  there  is  a  parallel  requirement 


for  protein  and  mRNA  synthesis  for  long-term  but 
not  for  short-term  effectiveness.  The  short-term 
process  reflects  enhanced  transmitter  release 
from  preexisting  synaptic  connections  due  to  co- 
valent  modification  of  preexisting  proteins.  The 
long-term  process  requires  new  protein  synthesis 
and  leads  to  the  grovvT:h  of  new  synaptic 
connections. 

Proteins  and  mRNAs  necessary  for  long-term 
memory  must  either  be  induced  during  the  brief 
(1 .5-h)  time  window  of  training  or  be  constituti- 
vely  expressed  and  transiently  accessible  to  cova- 
lent  modification  during  this  period.  The  experi- 
ments described  below  were  designed  to 
distinguish  between  these  two  possibilities. 

In  earlier  experiments,  Ari  Barzilai,  Tim  Ken- 
nedy, David  Sweatt,  and  I  found  that  repeated 
pulses  of  5-HT  induced  changes  in  the  synthesis 
of  specific  proteins  in  the  sensory  neurons.  Simi- 
lar changes  in  protein  synthesis  can  be  produced 
by  cAMP,  a  second  messenger  activated  in  the  sen- 
sory neurons  by  5-HT.  These  proteins  could  there- 
fore reflect  the  transcription  of  cAMP-inducible 
genes.  In  mammals,  genes  induced  by  cAMP  share 
a  control  element  called  the  cAMP  recognition 
element  (CRE),  which  binds  transcriptional  acti- 
vators called  the  CRE-binding  proteins  (CREBs) . 
Aplysia  neurons  contain  proteins  homologous  to 
mammalian  CREBs. 

These  findings  raise  three  questions  that  Bong- 
Kiun  Kaang,  Seth  Grant,  and  I  attempted  to  ad- 
dress: Can  the  facilitating  transmitter  5-HT  in- 
duce transcriptional  activation  of  reporter  genes 
in  the  sensory  neurons  that  are  driven  by  the  CRE? 
Does  this  transcriptional  activation  correlate 
with  the  graded  induction  of  long-term  facilita- 
tion? and  How  does  5-HT  activate  transcription 
by  CREB? 

Because  the  CRE  will  also  confer  cAMP  induc- 
ibility  when  placed  upstream  of  reporter  genes 
such  as  (8-galactosidase,  these  gene  constructs  can 
serve  as  an  assay  system  for  transcriptional  induc- 
tion by  cAMP.  Kaang,  Grant,  and  I  therefore  mi- 
croinjected  a  CKE-lacZ  reporter  plasmid  into 


219 


Cell  Biological  Studies  of  Memory 


Aplysia  sensory  neurons  and  measured  the  levels 
of  expression  driven  by  the  CRE  in  response  to 
stimulation  by  5-HT.  We  next  exposed  the  neu- 
rons to  five  repeated  (5-min)  pulses  of  5-HT,  the 
protocol  that  produces  long-term  synaptic  facili- 
tation, and  found  a  fourfold  induction  of  the  re- 
porter gene.  Similarly,  cAMP  induced  expression 
by  3-4-fold.  Thus  repeated  pulses  or  prolonged 
exposure  to  5-HT  stimulates  transcription,  and 
this  stimulation  can  be  simulated  by  elevating  lev- 
els of  cAMP.  The  increase  in  lacZ  expression  is 
mediated  specifically  through  the  CRE  sequence. 
A  plasmid  (ACRE-/«cZ)  from  which  the  CRE  ele- 
ment is  deleted  shows  no  expression. 

Does  the  stimulation  of  transcription  mediated 
by  5-HT  depend  on  the  binding  of  transcription 
factors  to  the  CRE  sequence?  To  address  this  ques- 
tion, we  co-injected  the  CRE  reporter  construct 
with  either  an  oligonucleotide  that  encodes  the 
wild-type  CRE  sequence  and  binds  CREB,  or  with 
a  mutant  oligonucleotide  that  does  not  bind 
CREB-like  factors.  The  wild-type  oligonucleo- 
tide, which  binds  to  and  titrates  out  CREB, 
blocked  the  5-HT-induced  expression  of  the  re- 
porter construct,  whereas  the  mutant  oligonucle- 
otide produced  no  inhibition. 

These  experiments  suggest  that  the  induction 
mediated  by  5-HT  requires  positively  acting 
CREB-like  cellular  factors  that  interact  with  the 
CRE,  and  provide  direct  evidence  that  5-HT  can 
stimulate  transcription  of  genes  containing  the 
CRE  sequences.  This  is  consistent  with  results 
that  Pramod  Dash,  Binyamin  Hochner,  and  I  ob- 
tained last  year  in  physiological  experiments,  in 
which  we  microinjected  the  CRE  into  the  nucleus 
of  sensory  neurons  and  selectively  blocked  the 
long-term  increase  in  synaptic  effectiveness  with- 
out affecting  short-term  facilitation.  Both  these 
sets  of  results  suggest  that  CREB-like  transcrip- 
tional activators  are  required  for  the  induction  of 
long-term  facilitation. 

Does  the  activation  of  transcription  by  5-HT 
have  a  clear  threshold  or  is  it  graded?  To  assess 
this  question,  Kaang,  Grant,  and  I  were  in- 
fluenced by  our  earlier  findings  discussed  above, 
that  a  single  (5-min)  pulse  of  5-HT  will  only 
elicit  short-term  facilitation.  To  generate  signifi- 
cant long-term  facilitation,  four  or  five  pulses  are 


required.  Using  the  same  pulse  protocol,  we 
found  that  a  single  pulse  of  5-HT  does  not,  in  fact, 
stimulate  expression  of  the  reporter  gene, 
whereas  four  to  five  pulses  of  5-HT/IBMX  pro- 
duced a  sevenfold  stimulation.  An  intermediate 
number  of  pulses  (two  or  three)  gave  an  interme- 
diate level  (twofold)  of  stimulation.  This  correla- 
tion, between  the  number  of  5-HT  trials  and  the 
level  of  transcriptional  induction  and  facilita- 
tion, suggests  that  the  graded  nature  of  long-term 
facilitation  may  reflect  the  graded  nature  of  tran- 
scriptional induction. 

Does  activation  by  5-HT  require  that  CREB  be 
phosphorylated?  If  so,  must  activation  of  CREB  in 
the  sensory  neuron  be  mediated  by  protein  kinase 
A  (PKA)?  To  address  these  questions,  Kaang, 
Grant,  and  I  next  microinjected  two  constructs 
into  the  sensory  neurons:  a  reporter  gene  as  well 
as  a  chimeric  transactivation  plasmid  that  ex- 
presses CREB.  Following  co-injection,  exposure 
to  5-HT  produced  a  10-fold  stimulation  of  tran- 
scription. By  contrast,  injection  of  either  trans- 
activation  or  reporter  plasmid  alone  showed 
no  expression.  This  transcriptional  stimulation 
depends  on  phosphorylation  of  the  PKA  consen- 
sus site  at  Ser'  in  CREB.  Microinjecting  a  transac- 
tivation plasmid  (pSAl  19)  containing  a  mutation 
that  converts  Ser"^  to  Ala"^  showed  no  stimula- 
tion with  5-HT. 

Thus  long-term  facilitation  induced  by  5-HT 
leads  to  activation  of  CRE-inducible  genes.  The 
induction  of  these  genes  by  the  cAMP  cascade  is 
graded  and  can  be  initiated  by  PKA  through  phos- 
phorylation of  CREB.  A  conventional  modulatory 
transmitter,  5-HT,  can  therefore  select  either  a 
cytoplasmic  or  a  genomic  program  of  cellular  ac- 
tion, depending  on  the  number  of  presentations 
of  5-HT.  By  being  able  to  activate  a  nuclear  signal 
(through  the  phosphorylation  of  CREBs  and  its 
action  on  the  CREs) ,  modulatory  transmitters  can 
activate  transcription  and  thereby  take  on  the 
properties  of  growth  factors.  In  the  case  of  sen- 
sory neurons,  transcriptional  activation  of  CREB- 
related  proteins  seemed  to  represent  one  compo- 
nent of  the  switch,  which  extends  the  short-term 
cytoplasmic  process  for  synaptic  facilitation  into 
the  genomic  changes  characteristic  of  the  long- 
term  facilitatory  process — one  that  includes  the 
growth  of  new  synaptic  connections. 


220 


The  T  Cell  Repertoire 


John  W.  Kappler,  Ph.D. — Investigator 

Dr.  Kappler  is  also  a  member  of  the  Division  of  Basic  Immunology  of  the  Department  of  Medicine  at  the 
National  Jewish  Center  for  Immunology  and  Respiratory  Medicine,  Denver,  and  Professor  of  Microbiology 
and  Immunology  and  of  Medicine  at  the  University  of  Colorado  Health  Sciences  Center,  Denver.  He  was 
educated  at  Lehigh  University  and  received  his  Ph.D.  degree  in  biochemistry  at  Brandeis  with  Gordon 
Sato.  He  did  postdoctoral  work  at  the  University  of  California,  San  Diego,  with  Richard  Dutton.  After 
holding  faculty  positions  at  the  University  of  Rochester,  he  moved  to  his  present  position  at  the  National 
Jewish  Center.  He  was  awarded  the  Wellcome  Foundation  Prize  by  the  Royal  Society  and  is  a  member  of 
the  National  Academy  of  Sciences. 


AS  protection  against  invasion  by  foreign  or- 
ganisms, higher  animals  have  evolved  a 
complex  collection  of  cells  and  chemicals 
broadly  termed  the  immune  system.  Components 
of  this  system  are  able  to  recognize  foreign  mate- 
rial in  the  body  and  give  rise  to  a  series  of  events 
that  cause  the  destruction  or  inactivation  of  the 
invader.  Cells  arising  in  the  thymus,  the  T  lym- 
phocytes, are  central  to  the  efficient  function  of 
this  system. 

T  cells  bear  receptors  on  their  surfaces  that  are 
able  to  interact  specifically  with  foreign  material. 
Such  interaction  stimulates  these  cells  to  pro- 
duce chemicals  that  allow  other  cells,  and  the  T 
cells  themselves,  to  respond  to  the  invader.  There 
are  two  kinds  of  T  cells,  bearing  or  76  recep- 
tors respectively.  The  a/?  receptors  are  made  up 
of  several  segments — Va,  Ja,  VjS,  D/3,  and  J(8 — 
each  of  which  can  differ  in  structure  from  one  T 
cell  to  another.  This  is  possible  because  the  DNA 
of  higher  mammals  contains  a  number  of  alter- 
nate genes  for  each  of  these  segments.  As  each  T 
cell  develops,  it  selects  a  different  combination 
of  these  genes,  and  therefore  eventually  ex- 
presses receptors  that  are  not  exactly  the  same 
in  structure  as  those  of  its  fellows.  It  is  these  varia- 
tions in  a/3  receptor  sequence  that  enable  one  T 
cell  to  recognize  influenza  virus,  for  example, 
and  another  poliovirus. 

In  order  for  T  cells  to  recognize  most  foreign 
materials,  they  must  bear  exactly  the  right  combi- 
nation of  variable  segments — Va,  Ja,  etc.  The 
proper  combinations  are  usually  rare,  so  when  an 
animal  is  confronted  with  an  invading  organism, 
the  cells  that  can  actually  recognize  the  invader 
are  few,  probably  about  one  in  100,000  or  one  in 
a  million  of  all  T  cells.  This  fact  does  not  hold 
true,  however,  for  special  types  of  foreign  mate- 
rial called  superantigens. 

Superantigens  bind  to  special  cell-surface  mol- 
ecules— class  II  proteins  of  the  major  histocom- 
patibility complex  (MHC).  They  then  interact 
with  the  VjS  portion  of  the  T  cell  receptor,  almost 
without  regard  to  the  composition  of  its  other 


variable  elements.  Since  there  are  only  about  75 
different  sequences  for  V(8  in  humans,  any  given 
superantigen  will,  theoretically,  react  with  at 
least  1  percent  of  all  T  cells.  In  the  mouse,  with 
fewer  different  V/3  sequences,  superantigens 
react  with  at  least  5  percent  of  T  cells.  In  fact,  a 
particular  superantigen  can  react  in  some  cases 
with  up  to  30  percent  of  all  T  cells  in  either  of 
these  species. 

The  fact  that  superantigens  can  react  with  so 
many  T  cells  causes  them  to  have  some  important 
pathogenic  properties.  For  example,  massive 
stimulation  of  T  cells  by  superantigens  causes 
toxic  shock  in  humans,  and  there  is  reason  to  be- 
lieve that  these  antigens  may  be  involved  in  cer- 
tain autoimmune  diseases. 

Our  laboratory  has  recently  been  studying  the 
interaction  among  superantigens,  the  V/3  portions 
of  T  cell  receptors,  and  the  class  11  MHC  proteins. 
A  staphylococcal  toxin,  SEB,  has  been  used  as  a 
model  for  these  experiments.  Amino  acids  have 
been  identified  in  SEB  that  control  the  binding  of 
this  protein  to  V/8  or  class  II. 

In  many  cases  proteins  that  must  bind  to  two 
different  target  molecules  express  their  binding 
sites  in  different  domains.  That  is,  the  different 
functions  of  the  protein  are  separated  spatially. 
Surprisingly,  this  does  not  seem  to  be  the  case  for 
SEB.  The  binding  sites  of  this  protein  for  V(8  and 
class  II  appear  to  be  interwoven,  as  though  they 
must  lie  in  close  proximity. 

The  structural  studies  on  SEB  have  led  to  the 
creation  of  a  collection  of  mutant  SEBs,  some  able 
to  bind  class  II  MHC  but  not  V/3,  some  with  a  more 
limited  range  of  V/3  specificities  than  SEB  itself, 
and  some  able  to  bind  neither  MHC  nor  the  T  cell 
receptor.  These  mutant  SEBs  are  now  being 
screened  as  vaccines.  Mice  preimmunized  with 
the  mutant  molecules  are  no  longer  sensitive  to 
the  toxic  effects  of  SEB  given  later. 

Our  laboratory  and  others  have  discovered  a 
second  class  of  superantigens,  encoded  by  retrovi- 
ruses that  cause  mammary  tumors  in  the  mouse. 
Although  the  genes  coding  for  these  superanti- 
gens have  been  known  for  some  time,  the  struc- 


221 


The  T  Cell  Repertoire 


ture  and  function  of  their  protein  products  have 
only  recently  been  determined.  They  turn  out  to 
be  unusual  proteins.  Although  they  are  bound  to 
the  surface  membranes  of  cells,  they  differ  from 
most  surface  proteins,  in  that  the  amino  acids  of 
their  carboxyl  terminal  rather  than  amino  termi- 
nal lie  outside  the  cell.  Only  a  small  amino- 
terminal  stretch  of  the  protein  lies  inside.  Experi- 
ments show  that  this  inside  portion  is  not 
essential  for  the  superantigenic  properties  of  the 
viral  proteins,  but  that  the  proteins  must  be  mem- 


brane bound  in  order  to  engage  T  cell  receptors. 
The  extreme  carboxyl-terminal  set  of  amino  acids 
are  among  those  that  bind  to  T  cell  receptor  V/Js. 

In  attempts  to  find  out  more  about  how  these 
viral  superantigens  operate,  monoclonal  antibod- 
ies have  been  raised  to  various  parts  of  the  pro- 
teins. The  antibodies  reveal  that  only  a  few  of 
these  proteins  are  expressed  on  the  cell 
surface — probably  about  1 ,000  per  cell.  Despite 
this  small  number,  the  viral  superantigens  are  ex- 
tremely effective  stimulators  of  T  cell  responses. 


222 


The  Genetic  Control  of  Morphogenesis 


Thomas  C.  Kaufman,  Ph.D. — Investigator 

Dr.  Kaufman  is  also  Professor  of  Genetics  in  the  Department  of  Biology  at  Indiana  University, 
Bloomington,  and  Adjunct  Professor  of  Medical  Genetics  in  the  Department  of  Medical  Genetics  at  Indiana 
University  Medical  Center.  He  received  his  M.A.  and  Ph.D.  degrees  from  the  University  of  Texas,  Austin, 
and  did  his  postdoctoral  research  at  the  University  of  British  Columbia  in  Vancouver. 


THE  long-term  goal  of  our  laboratory  is  to  con- 
tribute to  an  understanding  of  the  genetic 
basis  of  the  developmental  program  of  higher  or- 
ganisms. The  organism  chosen  for  our  studies  is 
the  fruit  fly  Drosophila  melanogaster,  and  our 
principal  focus  is  a  set  of  genes  called  homeotic, 
which  play  a  crucial  role  in  development. 

The  homeotic  genes  were  first  identified  by 
virtue  of  the  striking  phenotypes  observed  when 
flies  carried  mutations  at  these  loci.  Specifically 
these  homeotic  lesions  cause  one  portion  of  the 
animal  to  be  transformed  into  an  identity  nor- 
mally found  in  another  region.  Thus  mutations  at 
the  Antennapedia  locus  cause  a  transformation 
of  the  antennae  of  the  adult  fly  into  a  leg,  and 
lesions  in  the  proboscipedia  gene  result  in  the 
development  of  legs  in  place  of  the  adult  mouth- 
parts.  Both  genes  are  members  of  a  cluster  of  five 
homeotic  genes  called  the  Antennapedia  com- 
plex (ANT-C),  which  is  found  in  a  restricted 
domain  at  the  base  of  the  right  arm  of  chromo- 
some 3. 

The  aggregate  results  of  genetic,  developmen- 
tal, and  molecular  analyses  of  the  ANT-C  have  re- 
vealed that  the  role  of  the  resident  loci  is  best 
viewed  as  a  series  of  developmental  switches  for 
either/or  decisions  of  cellular  fate  in  the  embry- 
onic and  larval  stages  of  the  organism.  Further- 
more, DNA  sequence  analysis  of  the  homeotic 
loci  reveals  that  they  encode  proteins  containing 
a  motif,  dubbed  the  homeodomain,  that  endows 
the  proteins  with  DNA-binding  ability.  Indeed, 
the  homeotic  proteins  are  found  complexed  with 
the  nuclear  DNA  of  the  cells  in  which  they  are 
expressed.  Thus  it  appears  that  the  switch  activity 
of  the  loci  is  reflected  in  their  functioning  as  regu- 
lators of  specific  target  genes.  Not  entirely  clear 
at  this  point  is  how  each  homeotic  locus  is  re- 
stricted to  its  own  unique  pattern  of  expression 
and  which  sets  of  genes  are  the  targets  of  the 
switches. 

In  order  to  investigate  these  two  unknowns,  we 
have  concentrated  our  efforts  on  three  of  the  resi- 
dent members  of  the  ANT-C:  Sex  combs  reduced 
(5cr),  proboscipedia  (pb),  and  labial  (lab). 
Each  gene  was  chosen  for  the  unique  properties  it 


displayed  during  initial  characterization.  For  ex- 
ample, Scr  is  the  only  homeotic  gene  expressed 
at  the  juncture  between  the  head  and  trunk  of  the 
developing  animal,  and  there  were  indications 
that  the  regulatory  hierarchy  of  genes  expressed 
in  these  two  domains  is  different.  Additionally,  a 
genetic  analysis  revealed  pb  and  lab  to  be  small 
by  homeotic  standards.  This  meant  that  for  the 
two  genes,  a  complete  dissection  of  the  regula- 
tory elements  of  each  locus  was  feasible. 

The  Sex  combs  reduced  Gene 

Our  prior  genetic  and  molecular  analyses  of 
the  Scr  locus  had  shown  that  DNA  sequences  50 
kilobases  (kb)  distal  to  the  point  at  which  the 
RNA  product  is  initiated  were  necessary  for  nor- 
mal gene  expression.  Using  "enhancer  sniffers" — 
constructs  capable  of  detecting  DNA  fragments 
that  have  the  ability  to  regulate  gene  expression 
— we  surveyed  the  entire  Scr  locus  for  such  ele- 
ments. To  date  we  have  found  at  least  five  frag- 
ments that  specify  the  accumulation  of  gene 
product  in  the  posterior  head  and  anterior 
thorax.  These  are  scattered  over  a  30-kb  interval; 
consistent  with  our  earlier  genetic  results,  the  re- 
gions of  DNA  that  control  head  and  trunk  expres- 
sion appear  to  be  physically  separate. 

Genetic  analysis  has  also  shown  that  Scr  is  sub- 
ject to  the  regulatory  effects  of  "transvection." 
Normally  the  gene  is  only  expressed  in  the  first, 
or  most  anterior,  thoracic  segment;  however,  cer- 
tain mutants  in  the  gene  allow  its  product  to  ac- 
cumulate more  posteriorly,  in  the  second  and 
third  thoracic  segments.  We  have  shown  that  the 
normal  restriction  of  pattern  results  from  nega- 
tive regulation  and  requires  that  the  two  copies  of 
the  gene  in  the  cells  of  the  posterior  thorax  be 
paired  with  each  other.  If  this  pairing  is 
disrupted,  the  negative  effect  is  removed  and  ab- 
normal ectopic  expression  occurs. 

Using  sniffer  constructs  similar  to  those  above, 
we  have  identified  three  DNA  fragments  that  ap- 
pear to  be  associated  with  the  transvection  effects 
at  the  Scr  locus.  Two  of  these  three  elements  are 
located  approximately  40  and  10  kb  upstream  of 
the  gene,  while  the  third  is  located  within  an  in- 


223 


The  Genetic  Control  of  Morphogenesis 


tron  about  1 5  kb  downstream  of  the  transcription 
start  site.  These  analyses  of  Scr  are  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

The  location  of  these  three  elements  relative  to 
the  identified  enhancer  elements  is  intriguing. 
Two  flank  the  enhancers,  while  the  third  is  lo- 
cated in  the  midst  of  the  regulatory  elements.  Our 
current  hypothesis  is  that  the  transvection  ele- 
=^-ments  serve  to  define  a  chromatin  domain  con- 
trolling an  on/off  state  for  the  locus  and  that  the 
resident  enhancers  can  only  have  their  effects  in 
the  on  state.  We  are  currently  extending  our  anal- 
ysis to  define  more  precisely  the  boundaries  and 
sequences  associated  with  the  above-identified 
fragments.  We  also  hope  to  identify  further  the 
cellular  factors  that  act  through  these  elements. 

The  labial  Gene 

Using  enhancer  sniffers,  we  have  identified  a 
majority  of  the  sequences  needed  for  the  normal 
expression  of  the  lab  gene.  These  are  found  up- 
stream, either  within  3  .6  kb  of  the  transcription 
start  site  or  within  the  major  1 4-kb  intron.  Using  a 
minigene  construct  that  contains  all  of  the  up- 
stream sequences  but  lacks  the  intronic  ele- 
ments, we  have  succeeded  in  rescuing  the  lethal- 
ity and  morphological  anomalies  associated  with 
deletion  of  the  lab  gene  in  the  embryo.  Thus  it 
would  appear  that  the  regulatory  elements  found 
within  the  intron  are  not  necessary  to  lab's  nor- 
mal embryonic  functions. 

Although  the  lab  minigene  rescues  the  embry- 
onic defects  associated  with  /aft-deficient  geno- 
types, there  is  no  apparent  rescue  of  adults.  We 
have  shown  that  this  failure  does  not  result  from 
the  minigene's  inability  to  be  expressed  in  adult 
tissues,  but  rather  because  its  protein  product  is 
inappropriately  and  ectopically  accumulated. 
This  abnormal  expression  pattern  only  takes 
place  in  lab  mutant  animals  and  is  not  detected  in 
normal  minigene-bearing  hosts. 

We  have  found  that  the  normal  lab  gene  actu- 
ally encodes  two  polypeptides,  one  of  which  is 
six  amino  acids  longer  than  the  other.  The  mini- 
gene is  only  capable  of  directing  the  synthesis  of 
the  shorter  protein.  It  would  appear,  therefore, 
that  animals  capable  of  making  the  long  form 
show  correct  adult  expression,  while  short-form 
animals  cannot,  and  that  normal  adult  expression 
involves  autogenous  negative  regulation. 

Our  earlier  studies  revealed  that  lab  expres- 


sion in  the  embryo  also  involved  autogenous  regu- 
lation. In  that  case,  however,  the  effect  was  posi- 
tive. That  is,  lab  protein  served  to  keep  the  lab 
gene  turned  on,  not  to  turn  it  off.  Moreover,  the 
short-form  protein  was  capable  of  performing 
this  function. 

We  have  now  demonstrated  that  the  sequences 
at  the  lab  locus  necessary  for  the  positive  and 
negative  loops  are,  like  the  required  proteins, 
different.  The  positive  sequences  are  upstream  of 
the  transcription  initiation  site,  while  the  nega- 
tive targets  are  in  the  transcribed  portion  of  the 
gene  in  the  first  exon.  We  are  currently  investi- 
gating the  possibility  that  the  two  loops  are  af- 
fected by  the  direct  interaction  of  the  lab  protein 
isoforms  with  the  two  alternate  target  sites  in  the 
lab  gene  itself. 

The  proboscipedia  Gene 

As  in  the  case  of  lab,  we  have  used  a  minigene 
derived  from  the  pb  locus  and  have  obtained  full 
rescue  of  the  adult  homeotic  mutant  phenotype. 
Moreover,  fragments  extracted  from  one  of  the 
introns  and  placed  upstream  of  the  pb  promoter 
function  to  direct  a  normal  spatiotemporal  pat- 
tern of  expression.  Thus  sequences  both  up-  and 
downstream  of  the  transcription  start  site  are  re- 
quired for  normal  pb  expression. 

However,  tests  of  the  "enhancer"  sequences 
on  a  heterologous  promoter  element  in  the 
sniffer  constructs  demonstrate  no  activity.  On  the 
other  hand,  minigene  constructs  in  which  the  en- 
hancer elements  are  deleted  can  be  directed  by 
novel  "enhancer"  elements  derived  from  other 
loci.  Therefore  it  appears  that  there  are  specific 
promoter/enhancer  interactions  at  this  locus  and 
that  the  specificity  appears  to  lie  primarily  with 
the  "enhancer"  elements. 

We  have  also  found  a  second  regulatory  ele- 
ment at  the  pb  locus  upstream  of  the  transcrip- 
tion start  site.  However,  this  element,  unlike  the 
positive  enhancer  above,  appears  to  be  negative 
and  serves  to  prevent  ectopic  expression  of  the 
gene.  Moreover,  like  the  Scr  locus,  it  seems  to 
have  a  pairing-sensitive  component.  We  are  now 
beginning  a  dissection  of  this  system,  which 
offers  an  opportunity  to  make  direct  comparisons 
between  two  similar  regulatory  schemes  in  the 
ANT-C.  Our  work  on  the  proboscipedia  gene  is 
supported  by  a  grant  from  the  National  Institutes 
of  Health. 


224 


Protein  Folding  and  Macromolecular 
Recognition 


Peter  S.  Kim,  Ph.D. — Assistant  Investigator 

Dr.  Kim  is  also  Member  of  the  Whitehead  Institute  for  Biomedical  Research,  Associate  Professor  of  Biology 
at  the  Massachusetts  Institute  of  Technology,  and  Assistant  Molecular  Biologist  at  the  Massachusetts 
General  Hospital,  Boston.  His  undergraduate  degree  in  chemistry  was  obtained  at  Cornell  University, 
where  he  studied  with  George  Hess.  After  receiving  the  Ph.D.  degree  in  biochemistry  from  Stanford 
University,  where  he  studied  with  Robert  Baldwin,  Dr.  Kim  moved  to  the  Whitehead  Institute  for 
Biomedical  Research  as  a  Whitehead  Fellow. 


INFORMATION  transfer  in  biology  generally 
proceeds  from  DNA  to  RNA  (transcription)  and 
then  from  RNA  to  protein  (translation) .  The  lin- 
ear, unfolded  protein  chains  made  during  transla- 
tion must  fold  into  a  three-dimensional  shape  to 
be  functional.  Although  the  basic  mechanisms  of 
transcription  and  translation  are  understood,  at 
least  in  outline,  the  transfer  of  information  from 
one  to  three  dimensions — i.e.,  protein  folding — 
remains  a  major  unsolved  problem  in  molecular 
biology.  To  understand  protein  folding  is  a  prime 
objective  of  this  laboratory. 

A  second  effort  is  aimed  at  understanding  the 
principles  of  macromolecular  recognition:  spe- 
cific protein-protein  interactions  and  interac- 
tions between  protein  molecules  and  DNA.  These 
interactions  are  central  to  much  of  molecular 
physiology  and  developmental  biology.  We  have 
focused  on  a  structural  motif  called  the  leucine 
zipper,  which  occurs  in  several  different  DNA- 
binding  proteins,  including  the  products  of  some 
nuclear  oncogenes. 

A  third  and  new  effort  is  the  de  novo  design  of 
peptides  and  proteins. 

Protein  Folding 

Much  of  our  work  in  this  area  is  centered  on 
bovine  pancreatic  trypsin  inhibitor  (BPTI) ,  argu- 
ably the  protein  most  thoroughly  characterized 
in  biophysical  terms.  It  is  difficult  to  determine 
the  structures  of  protein-folding  intermediates, 
because  protein  folding  is  a  cooperative  process. 
Trapped  disulfide-bonded  intermediates,  such  as 
those  identified  in  the  early  folding  steps  of  BPTI, 
are  often  rather  insoluble;  this  hinders  detailed 
structural  characterization  by  nuclear  magnetic 
resonance  (NMR) .  We  have  developed  a  peptide 
model  approach  that  circumvents  the  problem  of 
cooperativity  and  improves  solubility,  so  that  the 
structures  contained  within  protein-folding  in- 
termediates can  be  characterized  in  detail. 

Peptide  models  that  simulate  two  crucial  early 
intermediates  in  the  folding  of  BPTI  have  been 
designed  and  synthesized  chemically.  By  using 
two-dimensional  NMR,  we  find  that  the  struc- 
tures contained  within  these  peptide  models  are 


remarkably  native- like,  corresponding  to  subdo- 
mains  of  BPTI.  These  results  suggest  that  a  large 
part  of  the  protein-folding  problem  can  be  re- 
duced to  identifying  and  understanding  subdo- 
mains  of  native  proteins. 

Earlier  work  by  others,  however,  concluded 
that  there  are  well-populated,  nonnative  states  in 
the  oxidative  folding  of  BPTI.  This  conclusion 
complicates  efforts  to  understand  protein  fold- 
ing. Recently  we  reexamined  the  spectrum  and 
population  of  intermediates  present  during  the 
folding  of  BPTI,  taking  advantage  of  improve- 
ments that  have  been  made  in  separation  technol- 
ogies in  the  years  since  the  original  BPTI-folding 
experiments.  In  contrast  to  earlier  studies,  we 
find  that  all  of  the  well-populated  intermediates 
in  the  folding  of  BPTI  contain  only  native  disul- 
fide bonds  and  that  the  essential  features  of  the 
BPTI-folding  reaction  are  determined  in  large 
part  by  native  structure.  These  results  emphasize 
the  importance  of  native  protein  structure  for  un- 
derstanding protein  folding. 

A  recombinant  model  for  an  early  predominant 
intermediate,  containing  a  single  disulfide  bond 
between  residues  5  and  55,  has  been  made  by 
replacing  the  cysteines  not  involved  in  the  disul- 
fide bond  with  alanine.  Remarkably,  this  model 
folds  essentially  into  the  same  conformation  as 
native  BPTI,  as  judged  by  two-dimensional  NMR, 
and  it  inhibits  trypsin.  These  findings  provide  an 
explanation  for  the  properties  of  this  interme- 
diate in  the  folding  of  BPTI  and  demonstrate  that 
the  native  fold  of  BPTI  can  be  obtained  without 
the  assistance  of  nonnative  disulfide  species.  The 
recombinant  model  also  provides  an  attractive 
model  system  for  studies  of  protein  folding.  This 
work  is  supported  by  a  grant  from  the  National 
Institutes  of  Health. 

Other  efforts  are  directed  at  evaluating  electro- 
static fields  at  the  ends  of  a-helices  and  develop- 
ing a  model  system  to  evaluate  the  /3-sheet  pro- 
pensities of  different  amino  acid  residues. 

Macromolecular  Recognition 

In  this  area,  we  have  focused  on  the  leucine 
zipper  class  of  DNA-binding  transcriptional  acti- 


225 


Protein  Folding  and  Macromolecular  Recognition 


vator  proteins,  originally  identified  by  Steven 
McKnight  and  his  co-workers  (HHMI,  Carnegie 
Institution) .  The  leucine  zipper  regions  of  these 
proteins  are  important  for  homodimer  or  specific 
heterodimer  formation. 

Our  approach  in  this  work  is  to  use  "protein 
dissection."  GCN4,  a  homodimeric  transcription 
factor,  serves  as  a  prototype  protein.  A  synthetic 
peptide  corresponding  to  the  33-residue  leucine 
zipper  region  folds  as  a  parallel  pair  of  helices. 
This  led  us  to  propose  that  leucine  zippers  are 
actually  short  coiled  coils.  X-ray  crystal lographic 
studies  of  this  peptide  (with  Tom  Alber's  group, 
University^  of  California,  Berkeley)  confirm  that 
the  leucine  zipper  of  GCN4  is  a  coiled  coil  and 
provide  the  first  high-resolution  structure  of  a 
two-stranded  parallel  coiled  coil.  The  effects  of 
amino  acid  replacements  on  the  stability,  struc- 
ture, and  dynamics  of  this  leucine  zipper  are  be- 
ing investigated. 

Proper  biological  function  requires  that  recog- 
nition between  many  different  macromolecules 
in  the  cell  occurs  with  exquisite  specificity.  We 
have  found  that  the  isolated  leucine  zipper  re- 
gions from  the  nuclear  oncogene  products  Fos 
and  Jun  are  sufficient  to  mediate  specific  hetero- 
dimer formation.  This  provides  a  very  simple 
model  system  for  studying  the  specificity  of 
protein-protein  interactions:  two  helices  that 


prefer  to  interact  with  each  other  rather  than  with 
themselves.  By  making  hybrid  leucine  zipper 
peptides,  we  found  that  eight  amino  acid  resi- 
dues from  each  of  the  leucine  zipper  sequences 
are  sufficient  to  mediate  specific  heterodimer 
formation.  The  predominant  mechanism  for  spec- 
ificity in  this  system  was  found  to  be  electrostatic 
in  nature. 

A  region  of  GCN4  rich  in  basic  amino  acid  resi- 
dues, immediately  adjacent  to  the  leucine  zipper, 
is  involved  in  DNA  recognition.  We  find  that  this 
basic  region  by  itself,  when  dimerized  via  a  flexi- 
ble disulfide  linker  in  place  of  the  leucine  zip- 
per, is  also  capable  of  sequence-specific  DNA 
binding.  In  addition  to  simplifying  structural 
analysis  of  this  new  DNA-binding  motif,  the  find- 
ing provides  a  new  strategy  for  the  design  of  DNA- 
binding  peptides.  This  work  was  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

Peptide  and  Protein  Design 

Knowledge  of  the  rules  involved  in  protein 
folding  and  macromolecular  recognition  can  be 
tested  by  trying  to  design  de  novo  amino  acid 
sequences  that  fold  into  specific  conformations 
and/or  that  interact  in  a  predetermined  manner 
with  other  molecules.  Toward  this  end,  we  have 
begun  to  analyze  the  structural  hierarchies  in  nat- 
ural proteins  and  to  design  simple  "building 
blocks"  of  peptide  structure. 


226 


RNA  Viral  Genetics 


KarlaA.  Kirkegaard,  Ph.D. — Assistant  Investigator 

Dr.  Kirkegaard  is  also  Assistant  Professor  of  Molecular,  Cellular,  and  Developmental  Biology  at  the 
University  of  Colorado  at  Boulder  and  Adjunct  Assistant  Professor  of  Cellular  and  Structural  Biology  at  the 
University  of  Colorado  Health  Sciences  Center,  Denver.  After  receiving  a  B.S.  degree  in  genetics  from  the 
University  of  California,  Berkeley,  she  developed  her  doctoral  thesis  in  the  Department  of  Biochemistry  and 
Molecular  Biology  at  Harvard  University  with  James  Wang.  Her  postdoctoral  work  in  virology  was  in 
association  with  David  Baltimore  at  the  Massachusetts  Institute  of  Technology  and  the  Whitehead  Institute. 


FOR  numerous  viruses  and  other  subcellular 
parasites,  RNA  rather  than  DNA  is  the  mole- 
cule used  for  storage  and  transmission  of  genetic 
information.  We  are  interested  in  the  conse- 
quences for  a  virus  of  having  an  RNA  genome  and 
in  any  mechanistic  similarities  or  differences  in 
genetic  processes  between  RNA  and  DNA  organ- 
isms. In  addition,  we  are  exploring  the  mecha- 
nisms of  RNA  packaging,  replication,  and  recom- 
bination in  the  genome  of  poliovirus  and  other 
RNA  viruses.  We  are  also  interested  in  the  interac- 
tions between  viruses  and  their  host  cells,  espe- 
cially in  the  realm  of  RNA-protein  biochemistry. 

Many  of  our  genetic  studies  utilize  poliovirus, 
a  small  icosahedral  virus  with  an  RNA  genome  of 
only  7,500  nucleotides.  We  have  shown,  for  ex- 
ample, that  RNA  recombination  occurs  among 
poliovirus  genomes  with  sufficient  frequency 
that  1  out  of  every  25  is  a  recombinant.  In  con- 
trast to  the  breaking  and  joining  of  molecules 
that  leads  to  DNA  recombination,  recombination 
of  RNA  occurs  during  its  synthesis.  Genetic  re- 
arrangement results  from  the  switching  of  paren- 
tal templates  by  the  viral  RNA  polymerase. 

Thale  Jarvis,  a  postdoctoral  fellow  in  our  labo- 
ratory, has  developed  a  sensitive  quantitative  as- 
say for  RNA  recombination  using  the  polymerase 
chain  reaction  (PGR).  Recombinants  can  be  de- 
tected at  a  frequency  as  low  as  1  in  1 0*^  parental 
genomes.  Using  this  assay,  we  have  been  able  to 
learn  a  great  deal  about  the  process  of  RNA  recom- 
bination in  poliovirus-infected  cells.  For  exam- 
ple, we  discovered  that  recombination  frequency 
increases  exponentially  throughout  the  course  of 
a  single  infectious  cycle.  It  was  surprising  that 
the  extensive  cytopathic  changes  during  the 
course  of  poliovirus  infection  do  not  inhibit  the 
access  of  the  parental  RNA  templates  to  each 
other.  This  work  was  also  supported  by  a  grant 
from  the  National  Institutes  of  Health. 

Taken  together  with  other  data  we  have  ob- 
tained, our  results  suggest  that  the  process  by 
which  recombinant  RNA  genomes  are  generated 
may  be  quite  simple,  and  possibly  universal 
among  RNA  viruses.  We  are  using  the  PGR  assay  to 


test  this  idea  by  looking  for  RNA  recombination 
among  other  RNA  viruses  that  are  less  amenable 
to  genetic  analysis  than  poliovirus.  We  hope  to 
gain  a  better  understanding  of  the  prevalence  and 
mechanism  of  genetic  recombination  among  RNA 
genomes,  a  process  that  is  certainly  responsible 
for  much  of  the  variability  and  rapid  evolution  of 
RNA  viruses. 

Using  x-ray  crystallography,  James  Hogle  at 
Scripps  Glinic  has  determined  the  three-dimen- 
sional structure  of  the  poliovirion.  However,  an 
appreciation  of  functional  interactions  between 
the  viral  RNA  and  the  virion  proteins  calls  for  the 
application  of  genetics  as  well  as  structural  bio- 
chemistry. We  do  not  know,  for  example,  exactly 
which  subviral  protein  particles  package  the  po- 
liovirus RNA  into  the  final  virion  structure,  nor 
do  we  know  the  structural  requirements  of  the 
participants  in  the  packaging  reactions.  Is  the 
viral  RNA  threaded  into  an  intact,  preformed  ico- 
sahedral capsid,  or  do  smaller  parts  of  the  capsid 
condense  around  the  viral  RNA  to  form  the  final 
icosahedral  structure? 

We  have  constructed  several  mutants  in  the  po- 
liovirus RNA  genome  and  have  characterized 
them  in  great  detail.  Two  of  these  mutants  have 
pointed  out  a  region  of  the  viral  capsid,  quite 
internal  to  the  virion,  that  is  involved  both  in  RNA 
packaging  and  RNA  uncoating.  We  are  examining 
the  RNA-binding  properties  of  subviral  particles 
from  both  mutant  and  wild-type  poliovirus- 
infected  cells  in  hopes  of  identifying  which  sub- 
viral particles  bind  to  RNA  during  intracellular 
assembly. 

To  investigate  the  RNA  uncoating  defects  dur- 
ing cell  entry  of  these  mutant  viruses,  we  have 
devised  an  assay  to  detect  interactions  between 
poliovirions  and  their  cellular  receptor  directly. 
The  laboratory  of  Vincent  Racaniello  (Golumbia 
University)  has  identified  the  poliovirus  receptor 
as  a  cellular  adhesion  protein  of  the  immunoglob- 
ulin superfamily.  Using  antibodies  prepared 
against  peptides  from  the  receptor's  amino  and 
carboxyl  termini,  we  can  analyze  proteolytic  di- 
gestion patterns  of  the  receptor  in  the  presence 


227 


RNA  Viral  Genetics 


and  absence  of  bound  wild-type  and  mutant  po- 
liovirions.  In  this  way  we  are  exploring  the  spe- 
cific protein-protein  contacts  made  betu^een  the 
viruses  and  the  receptor  during  binding  and  the 
subsequent  conformational  changes  during  entr}' 
of  the  virus  into  the  cell. 

We  have  extended  the  study  of  RNA  genetics  to 
she  interactions  between  yeast  cells  and  the  small 
double-stranded  RNA  genome  of  L-A,  a  cytoplas- 
mic virus-like  particle.  Yeast  cells,  unlike  the  pri- 
mate cells  in  which  poliovirus  and  other  RNA  vi- 
ruses of  medical  interest  are  propagated,  are 
amenable  to  elegant  genetic  analysis,  making  it 
possible  to  identif\'  quite  quickly  the  cellular 
molecules  that  are  involved  in  any  given  process. 
The  virus-like  particle  L-A  has  been  shown  by  the 
laboratory^  of  Reed  Wickner  (National  Institutes 
of  Health)  to  replicate  in  the  cytoplasm  of  yeast 
cells  in  large  numbers.  Genetic  analysis  of  L-A, 
however,  will  depend  on  our  ability  to  make  de- 
fined mutations  in  its  RNA  genome. 

In  the  past  year,  we  have  developed  the  techni- 
cal capabilities  to  pursue  this  goal.  First,  we  had 


to  develop  the  technology  to  introduce  RNA  di- 
rectly into  yeast  cells,  which  we  accomplished  by 
using  RNA  molecules  encoding  the  luciferase 
protein  of  fireflies.  Second,  we  needed  an  assay 
for  the  successful  introduction  of  the  L-A  genome 
into  yeast.  Since  the  particle  is  so  ubiquitous,  no 
phenotype  for  its  presence  or  absence  in  a  yeast 
cell  had  been  reported.  We  found  that,  under 
somewhat  unusual  laboratory  conditions,  certain 
yeast  strains  require  the  L-A  genome  for  growth! 
This  suggests  the  possibility  of  mutualistic  rela- 
tionships between  host  cells  and  virus-like  RNA 
genomes,  a  possibility  we  are  also  pursuing  in 
other  systems. 

Most  importantly  for  this  work,  we  now  have  a 
phenotype  for  the  presence  of  the  L-A  RNA  ge- 
nome in  yeast  cells.  We  have  recently  been  able 
to  initiate  L-A  "infections"  in  yeast  by  introduc- 
ing RNA  made  in  vitro.  We  are  now  in  a  position 
to  mutate  this  RNA  genome,  to  study  any  defect  in 
the  L-A  replication  cycle  caused  by  the  mutations, 
and  to  observe  the  effects  of  yeast  mutants  on  the 
replication  cycle  of  mutant  and  wild-type  RNA. 


228 


Adrenergic  Receptor  Structure  and  Function 


Brian  K.  Kobilka,  M.D. — Assistant  Investigator 

Dr.  Kobilka  is  also  Assistant  Professor  of  Medicine,  Cardiology,  and  Molecular  and  Cellular  Physiology  at 
the  Stanford  University  Medical  Center.  He  received  his  undergraduate  degree  in  biology  and  chemistry 
from  the  University  of  Minnesota,  Duluth,  and  his  M.D.  degree  from  Yale  University.  After  his  residency 
in  internal  medicine  at  Barnes  Hospital,  St.  Louis,  he  joined  the  laboratory  of  Robert  Lefkowitz  as  a 
research  fellow  in  cardiology  at  Duke  University.  Four  years  later  he  was  appointed  Assistant  Professor  in 
the  Department  of  Medicine  at  Duke  University,  and  the  following  year  he  assumed  his  present  position 
at  Stanford. 


THE  autonomic  nervous  system  serves  as  the 
master  control  center  for  the  cardiovascular 
system.  It  monitors  the  effectiveness  of  the  latter 
system  in  providing  nutrients  and  oxygen  to  the 
rest  of  the  body  and  appropriately  adjusts  the 
heart  rate,  blood  pressure,  and  blood  flow.  These 
adjustments  are  made  via  nerves  that  serve  the 
heart,  blood  vessels,  and  kidneys. 

Adrenergic  receptors  form  the  interface  be- 
tween these  nerves  (of  the  sympathetic  subsys- 
tem) and  the  organs  they  innervate.  Catechol- 
amines released  from  sympathetic  nerve 
terminals  bind  to  adrenergic  receptors  on  the  sur- 
face of  target  cells,  and  the  activated  receptors 
modify  the  function  of  these  cells. 

When  a  catecholamine  occupies  its  binding 
site,  the  receptor  activates  a  GTP-binding  protein 
(G  protein)  inside  the  cell.  The  activated  G  pro- 
tein may  then  modulate  the  activity  of  a  cellular 
en2yme  or  ion  channel.  The  genes  (or  corre- 
sponding cDNAs)  for  nine  types  of  adrenergic  re- 
ceptors have  been  cloned.  There  are  three  types 
of  ttj-adrenergic  receptors,  three  types  of 
adrenergic  receptors,  and  three  types  of  |8  recep- 
tors. All  of  these  receptors  are  structurally  simi- 
lar, having  seven  hydrophobic  domains  that  are 
thought  to  be  membrane  spanning.  These  fea- 
tures are  shared  by  other  receptors  that  activate  G 
proteins. 

a2-Adrenergic  Receptor  Subtype  Diversity 

The  role  played  by  the  aj-,  a2-,  and  /S-adrener- 
gic  receptors  in  the  function  of  the  sympathetic 
nervous  system  has  been  extensively  studied. 
Adrenergic  receptors  are  involved  in  blood  pres- 
sure control  and  in  directing  blood  flow  to  spe- 
cific tissues.  However,  the  physiological  role  of 
each  of  the  three  a2  receptor  types  is  not  known. 
This  is  in  part  due  to  the  lack  of  highly  selective 
drugs  that  can  activate  or  inhibit  each  type  of 
adrenergic  receptor.  We  are  attempting  to  iden- 
tify distinctive  functional  and  physiological  prop- 
erties for  each  of  the  different  a2  receptors.  These 
studies  may  provide  incentive  for  the  develop- 
ment of  more  highly  selective  a2'^drenergic  re- 


ceptor drugs  for  the  treatment  of  hypertension 
and  vascular  disease. 

Adrenergic  Receptor  Structure 

A  major  focus  in  my  laboratory  is  to  learn  more 
about  the  three-dimensional  structure  of  adrener- 
gic receptors  and  to  determine  how  they  transmit 
signals  across  the  cell  membrane's  lipid  bilayer. 
We  are  taking  several  approaches  to  study  the  re- 
ceptor structure.  Over  the  past  year  our  mutagen- 
esis studies  have  identified  a  specific  amino  acid 
in  the  /Jj-adrenergic  receptor  that  forms  part  of 
the  binding  site  for  a  large  class  of  /3  receptor- 
blocking  drugs.  When  this  amino  acid  is  placed  in 
an  a2  receptor,  it  confers  the  ability  to  bind  to 
these  (8  receptor  drugs.  We  have  also  used  muta- 
genesis studies  to  identify  intramolecular  interac- 
tions within  ^2-  ^rid  a2-adrenergic  receptors.  This 
information  is  useful  in  developing  models  for 
the  three-dimensional  structure  of  the  receptor. 

A  long-range  goal  is  to  study  the  three- 
dimensional  structure  of  the  /32-3drenergic  re- 
ceptor, using  techniques  that  provide  high- 
resolution  pictures  of  this  protein  or  detect 
changes  in  the  structure  of  the  receptor  during 
signal  transduction.  These  techniques  require 
large  quantities  of  pure,  functional  receptor  pro- 
tein. Using  recombinant  DNA  techniques,  we 
have  made  several  modifications  in  the  structure 
of  the  /32-adrenergic  receptor  that  lead  to  in- 
creased expression  in  cultured  cells.  Our  goal 
during  the  next  year  will  be  to  refine  the  purifica- 
tion procedure  to  optimize  the  yield  and  mini- 
mize the  cost  of  large-scale  production  of  recep- 
tor protein. 

Receptor  Biosynthesis 

The  primary  amino  acid  sequence  of  a  receptor 
contains  all  the  essential  information  needed  for 
the  receptor's  proper  folding,  post-translational 
processing,  and  cellular  targeting.  Understanding 
the  process  by  which  receptors  are  folded  and 
processed  should  provide  insights  into  receptor 


229 


Adrenergic  Receptor  Structure  and  Function 


structure  and  may  identify  factors  that  will  en- 
hance the  production  of  functional  protein. 

Using  a  cell-free  expression  system  developed 
in  my  laboratory,  we  have  characterized  the  in- 
sertion of  specific  membrane-spanning  domains 
into  the  lipid  bilayer.  The  proper  insertion  of  the 
first  membrane-spanning  domain  is  relatively  in- 
efficient. Using  recombinant  DNA  techniques, 
we  have  identified  a  structural  modification  that 
enhances  the  integration  of  this  domain  and  in- 
creases the  amount  of  functional  ^2  receptor  pro- 
tein expressed  in  cultured  cells. 

We  have  previously  shown  that  ^2  receptor  is 
nonfunctional  immediately  after  translation  and 
translocation  into  the  endoplasmic  reticulum. 
Our  studies  indicate  that  additional  cytosolic  and 
membrane  factors  are  required  to  process  the  re- 
ceptor to  a  functional  form.  We  are  attempting  to 
identify  structural  differences  between  a  func- 
tional receptor  and  newly  synthesized  receptors 
that  have  not  undergone  the  post-translational 
processing  necessary  to  produce  functional  pro- 


tein. We  hope  to  identify  the  requisite  cytosolic 
and  membrane  factors. 

Cellular  Biology  of  /82-Adrenergic  Receptors 

Following  prolonged  exposure  to  catechol- 
amines, the  ;82-adrenergic  receptor  becomes  de- 
sensitized and  is  less  efficient  in  activating  adeny- 
lyl  cyclase.  Several  mechanisms  contribute  to  the 
process  of  desensitization,  including  receptor 
phosphorylation  and  the  removal  of  receptors 
from  the  plasma  membrane.  Over  the  past  year 
we  have  used  immunocytochemical  techniques 
to  study  agonist-mediated  internalization  of  ^2- 
adrenergic  receptors.  These  studies  confirm  that 
these  receptors  are  rapidly  internalized  into  in- 
tracellular vesicles  called  early  endosomes.  We 
plan  to  continue  our  efforts  to  determine  the 
mechanism  by  which  this  occurs  and  to  identify 
the  cellular  proteins  that  mediate  this  process. 
Furthermore,  we  hope  to  gain  a  better  under- 
standing of  the  relative  importance  of  agonist- 
activated  internalization  in  the  physiology  of 
182-adrenergic  receptors. 


230 


Molecular  Genetics  of  Lymphocyte  Development 
and  Neoplasia 


Stanley  J.  Korsmeyer,  M.D. — Associate  Investigator 

Dr.  Korsmeyer  is  also  Professor  of  Medicine  and  Molecular  Microbiology  at  Washington  University  School 
of  Medicine,  St.  Louis.  He  received  his  B.S.  degree  in  biology  from  the  University  of  Illinois,  Urbana,  and 
his  M.D.  degree  from  the  University  of  Illinois,  Chicago.  He  did  his  internship  and  residency  in  internal 
medicine  at  the  University  of  California,  San  Francisco,  and  his  postdoctoral  research  with  Thomas 
Waldmann  and  Philip  Leder  at  NIH,  where  he  became  a  Senior  Investigator  at  the  National  Cancer 
Institute.  His  honors  include  membership  in  the  American  Society  for  Clinical  Investigation. 


GENES  that  encode  receptors  for  foreign  anti- 
gens have  provided  our  most  pivotal  insights 
into  early  lymphocyte  development  and  lym- 
phoid malignancies.  The  genes  for  immunoglobu- 
lin (Ig),  or  antibodies,  and  for  the  T  cell  receptor 
(TCR)  encode  the  antigen  receptors  for  B  cells 
and  T  cells,  respectively.  During  early  lympho- 
cyte development,  recombination  at  the  DNA 
level  assembles  these  genes  to  create  a  wide  re- 
pertoire of  receptor  specificities. 

Much  of  what  we  know  about  these  genes  has 
been  gleaned  from  studies  of  lymphoid  tumors. 
These  malignancies  are  clonal  expansions  of  a 
single  cell  and  provide  multiple  identical  copies 
of  these  genetic  events.  Provocatively,  the  char- 
acteristic interchromosomal  translocations  that 
typify  B  cell  malignancies  break  at  the  Ig  genes, 
while  those  of  T  cell  tumors  often  occur  at  the 
chromosomal  home  of  the  TCR  genes.  We  have 
exploited  this  geography  to  clone  the  DNA  at 
these  illegitimate  interchromosomal  junctures. 
This  serves  as  a  bridge  from  the  antigen  receptor 
loci  to  the  other  chromosomal  partner,  which  has 
often  introduced  a  new  cancer-promoting  gene. 

As  a  prototype,  we  have  cloned  the  juncture 
between  chromosomes  1 4  and  1 8  that  is  present 
in  the  most  frequent  form  of  human  lymphoma, 
follicular-type  B  cell  lymphoma.  This  transloca- 
tion occurs  early  in  the  development  of  a  B  cell 
and  introduces  a  newly  discovered  gene,  Bel- 2, 
into  the  Ig  locus.  A  hybrid  Bcl-2-lg  fusion  gene  is 
created,  resulting  in  the  overproduction  of  Bcl-2. 
Transgenic  mice  were  created  that  possess  a  copy 
of  the  abnormal  Bcl-2-lg  fusion  gene  in  their  ge- 
netic material.  They  progressed  from  an  indolent 
expansion  of  resting  B  cells  to  high-grade  life- 
threatening  lymphomas,  recapitulating  the  natu- 
ral course  of  the  human  disease  and  proving  that 


this  translocation  causes  malignancy.  The  Bcl-2 
protein  is  unique  among  proto-oncogenes  by  be- 
ing located  in  mitochondria.  Moreover,  it  has  a 
novel  function  in  that  it  blocks  the  programmed 
death  of  cells  independent  of  promoting  their 
growth.  When  deregulated,  Z?c/- 2  extends  the  sur- 
vival of  cells  normally  destined  to  die.  Bcl-2  con- 
stitutes the  first  member  of  a  new  oncogene  cate- 
gory, regulators  of  cell  death. 

In  a  parallel  set  of  experiments,  unanticipated 
rearrangements  into  the  6  TCR  locus  on  chromo- 
some 14  have  identified  the  interchromosomal 
translocation  sites  that  typify  early  T  cell  acute 
lymphoblastic  leukemias.  Two  of  these  new 
genes,  Ttg-1  from  chromosome  11  and  Hox-11 
from  chromosome  10,  are  not  normally  ex- 
pressed in  T  cells.  Instead  their  function  is  di- 
verted from  their  normal  sites  to  T  cells.  Trans- 
genic mice  reveal  that  redirecting  these 
regulatory  genes  into  T  cells  causes  malignancy. 

The  majority  of  chromosomal  defects,  how- 
ever, have  no  candidate  gene  at  either  side  of  the 
juncture.  The  responsible  gene  lies  a  consider- 
able distance  from  known  genes.  One  promising 
approach  utilizes  portions  of  chromosomes  from 
yeast  organisms  to  obtain  and  propagate  long 
stretches  of  human  DNA.  This  makes  it  possible  to 
clone  and  map  entire  segments  of  human  chro- 
mosomes, precisely  linking  known  genes,  and 
generating  probes  to  search  for  new  disease  loci. 
This  approach  has  pinpointed  the  chromoso- 
mal breakpoint  and  identified  a  candidate  onco- 
gene for  an  extremely  aggressive  leukemia  of 
childhood. 

This  group  of  studies  aims  to  improve  our  un- 
derstanding of  the  genetic  pathways  of  early  T 
and  B  cell  development,  as  well  as  the  aberran- 
cies  that  result  in  malignancy. 


231 


Bcl-2 
,^  Survival  Pathway 


r 


Apoptotic 
Death 


Centrocytes 


r      Light  Zone 

I  ().  Basal 
^J^^^^Light  Zone 


Bcl-2 


Dark^ 
Zonel 


B  Blast  <][ 


Plasma 
Cell 

Memory 


Centroblasts 


Distribution  of  the  Bcl-2  protein  that  blocks  cell  death  within  the  germinal  center  of  the  immune 
system.  Bcl-2  is  confined  to  survival  zones.  B  cells  in  the  follicular  mantle  are  longer  lived  and 
recirculate.  The  growing  centroblasts  in  the  dark  zone  and  the  dying  centrocytes  in  the  basal  light 
zone  lack  the  protein.  It  returns  in  the  apical  light  zone,  where  long-lived  memory  cells  emerge  to 
permit  recall  responses. 

Research  of  Stanley  Korsmeyer. 


232 


Molecular  Genetics  of  Neuromuscular  Disease 


Louis  M.  Kunkel,  Ph.D. — Investigator 

Dr.  Kunkel  is  also  Professor  of  Pediatrics  and  of  Genetics  at  Harvard  Medical  School.  He  received  his  B.A. 
degree  from  Gettysburg  College  and  his  Ph.D.  degree  in  biology  from  the  Johns  Hopkins  University.  He 
took  postdoctoral  training  with  Brian  McCarthy  at  the  University  of  California,  San  Francisco,  and  with 
Samuel  Latt  at  the  Children's  Hospital,  Boston.  He  held  appointments  at  Children's  Hospital/Harvard 
Medical  School  before  joining  HHMI.  His  honors  include  the  Gairdner  Award  and  election  to  the  National 
Academy  of  Sciences. 


WE  continue  to  study  the  underlying  cause 
of  Duchenne  and  Becker  muscular  dystro- 
phies. Our  identification  of  the  gene  led  rapidly 
to  our  description  of  the  encoded  protein  dystro- 
phin, which  was  found  to  be  an  unknown 
member  of  a  family  of  proteins  that  includes  the 
spectrins  and  a-actinins.  Located  at  the  inner  face 
of  the  plasma  membrane  of  myofibers,  dystro- 
phin is  thought  to  confer  strength  to  the  mem- 
brane during  muscle  contraction  and  relaxation. 
Absence  or  abnormality  of  dystrophin  at  this  loca- 
tion causes  the  myofiber  degeneration  of  Du- 
chenne and  Becker  dystrophy.  Our  work  has  led 
to  improved  diagnosis  of  the  diseases  and  to  test- 
able ideas  on  therapeutic  intervention. 

Over  the  past  year  the  laboratory  has  concen- 
trated on  identifying  new  members  of  the  dystro- 
phin family  of  proteins,  in  the  hope  that  these  can 
play  a  role  in  mitigating  disease  caused  by  dystro- 
phin deficiency.  These  dystrophin-related  pro- 
teins might  themselves  be  involved  in  other 
neuromuscular  diseases.  By  antigenic  cross- 
reactivity,  we  have  cloned  the  human  microtu- 
bule-associated  protein  IB  (MAP- IB)  and  have 
mapped  this  locus  in  close  proximity  to  muta- 
tions on  chromosome  5  that  cause  a  motor  neu- 
ron degenerative  disease,  spinal  muscular  atro- 
phy (SMA). 

We  have  sequenced  this  human  gene  and  have 
begun  its  direct  mutational  analysis  in  SMA  pa- 
tients. We  have  also  identified  at  the  locus  two 
separate  (CA)n  repeat  polymorphisms  that  have 
been  shown,  in  collaboration  with  Conrad 
Gilliam,  to  be  linked  tightly  to  SMA  mutations.  In 
this  genetic  analysis,  a  few  rare  recombinations 
have  been  detected  between  SMA  and  MAP- IB, 
implying  that  MAP- IB  may  be  very  close,  but  not 
involved  in  the  disease.  As  a  result  of  this  observa- 
tion, we  have  cloned  large  segments  of  human 
DNA  from  the  region  of  chromosome  5  as  yeast 
artificial  chromosomes  (YACs)  and  have  begun  a 
search  for  other  genes  near  MAP-  IB,  any  of  which 
might  be  the  SMA  gene. 

By  reduced  stringency  hybridization  with  dys- 
trophin cDNA  clones,  a  chromosome  6-encoded 
protein  was  identified  by  Kay  Davies  in  Oxford. 


This  dystrophin-related  protein  (DRP)  is  highly 
similar  to  dystrophin,  and  antibodies  against  it 
developed  in  our  laboratory  have  revealed  that 
the  two  proteins  are  almost  identical  in  size.  Im- 
munolocalization  of  DRP  has  shown  that  it  colo- 
calizes  with  dystrophin  in  a  developing  myofiber. 
In  a  mature  fiber,  however,  it  is  located  only  at 
the  neuromuscular  and  myotendinous  junctions 
of  muscle.  We  are  currently  attempting  to  clone 
the  entire  coding  sequence  of  DRP  as  cDNA.  We 
believe  that  the  sequence  of  this  protein  might 
reveal  why  it  has  such  a  specialized  location  in 
mature  muscle  and  what  role,  if  any,  it  might  play 
in  mitigating  some  of  the  effects  of  dystrophin 
absence  in  Duchenne  dystrophy.  We  are  also 
searching  for  other  neuromuscular  diseases  that 
might  be  caused  by  DRP  abnormalities. 

One  obvious  set  of  dystrophin  relatives  for  us 
to  attempt  to  characterize  were  the  a-actinins.  A 
smooth  muscle  form  had  been  cloned  and  local- 
ized to  chromosome  14,  but  there  was  at  least 
one  other  that  had  been  identified  from  chicken 
muscle.  Using  a  conserved  motif  of  the  known 
a-actinin  amino  acid  sequence,  we  designed  a  de- 
generate set  of  PGR  (polymerase  chain  reaction) 
primers  that  amplified  a  human  muscle  product 
with  the  sequence  of  an  a-actinin.  Using  this 
small  product  as  a  hybridization  probe,  we 
screened  a  muscle  cDNA  library  and  obtained  two 
classes  of  hybridizing  human  cDNA  clones.  Se- 
quence analysis  revealed  that  the  clones  con- 
tained two  different  a-actinins  that  were  unique 
for  humans.  They  are  both  muscle  specific  and 
are  encoded  from  chromosomes  1  and  1 1 ,  respec- 
tively. We  are  now  developing  antisera  specific 
for  each  of  the  muscle  a-actinin  gene  products 
and  attempting  to  identify  disease  phenotypes 
that  might  involve  a-actinin. 

Abnormalities  of  dystrophin  are  easily  detected 
at  the  protein  level,  and  nearly  70  percent  of  the 
mutations  that  cause  them  have  been  shown  to  be 
deletions  or  duplications  of  some  part  of  this  ex- 
tremely large  locus.  We  have  designed  primers 
from  dystrophin's  nucleotide  sequence  to  allow 
PGR  amplification  of  specific  regions  of  dystro- 
phin's transcript.  In  analyzing  these  PGR  prod- 


233 


Molecular  Genetics  of  Neuromuscular  Disease 


ucts  from  patients  who  do  not  have  a  detectable 
deletion,  we  have  found  at  least  one  mutation 
that  involves  splicing  of  dystrophin's  primary 
transcript. 

PGR  amplification  yielded  a  product  of  abnor- 
mal size  that  lacked  exon  5.  Sequence  analysis  of 
the  patient's  DNA  from  the  region  of  this  exon 
revealed  an  identical  sequence  to  that  of  normals. 
We  are  currently  cloning  other  surrounding  DNA 
to  identify  the  exact  point  where  a  mutation 
might  effect  the  splicing  of  exon  5.  We  believe 
that  mutations  in  splicing  signals  may  lead  to  a 
better  understanding  of  how  this  huge  locus  is 
processed  into  a  mature  mRNA  transcript. 

During  our  screening  of  different  dystrophin 
antibodies  for  finding  cross-reactive  proteins,  our 
carboxyl  domain-directed  antibodies  detected  a 
small  protein  (approximately  70  kDa).  At  the 
same  time,  David  Yafife's  group  in  Israel  reported 
observing  a  short  dystrophin  transcript  in  non- 
muscle  tissues.  Our  laboratory  cloned  this  tran- 
script from  our  human  brain  cDNA  library,  and 
sequencing  of  the  cDNA  clones  revealed  a  diver- 
gence from  expected  dystrophin  sequence  at  ap- 
proximately exon  62.  We  are  currently  preparing 
antibodies  against  novel  peptide  sequences  to  de- 
termine if  the  shorter  protein  detected  in  brain 


tissue  with  antidystrophin  antibodies  is  indeed  a 
product  of  this  novel  transcript. 

In  addition  to  the  published  shorter  transcript, 
we  have  also  detected  a  slightly  larger  protein 
(approximately  90  kDa)  in  sciatic  nerve.  cDNA 
clones  have  been  prepared  and  shown  to  diverge 
from  expected  dystrophin  sequence  at  exon  54. 
Similar  studies  are  under  way  for  this  novel  tran- 
script to  determine  the  role  these  smaller  pro- 
teins might  play  in  the  phenotype  of  Duchenne 
dystrophy. 

Our  aim  for  the  future  year  is  to  build  on  work 
already  in  progress.  We  will  continue  to  identify 
dystrophin-related  proteins  and  attempt  to  deter- 
mine their  normal  function  in  muscle.  We  will 
determine  what  role,  if  any,  they  play  in  other 
neuromuscular  diseases  and  whether  they  miti- 
gate the  effects  of  abnormal  dystrophin.  We  will 
continue  our  analysis  of  dystrophin-alternative 
transcripts  and  what  role  they  might  play  in  nor- 
mal development  and  the  phenotype  of  Du- 
chenne dystrophy.  As  more  human  disease  genes 
are  mapped  to  specific  human  chromosomes,  we 
believe  that  our  candidate  gene  approach,  which 
builds  upon  knowledge  of  abnormal  dystrophin, 
should  help  in  the  rapid  characterization  of  these 
diseases. 


234 


Structural  Studies  on  DNA-Replication 
Enzymes,  src- related  Oncogene  Products, 
and  Oxidoreductases 


John  Kuriyan,  Ph.D. — Assistant  Investigator 

Dr.  Kuriyan  is  also  Associate  Professor  of  Molecular  Biophysics  at  the  Rockefeller  University.  He  received  a 
B.S.  degree  in  chemistry  from  Juniata  College,  Pennsylvania,  and  a  Ph.D.  degree  from  the  Massachusetts 
Institute  of  Technology,  where  he  worked  with  Gregory  Petsko  and  Martin  Karplus  on  the  dynamics  of 
proteins.  He  continued  with  Martin  Karplus  at  Harvard  University  for  a  year  and  then  moved  to  the 
Rockefeller  University  as  a  University  Fellow.  He  is  also  a  Pew  Scholar  in  the  Biomedical  Sciences. 


OUR  interests  are  in  understanding  the  struc- 
tural determinants  of  protein  function.  To 
this  end  we  carry  out  x-ray  diffraction  experi- 
ments and  computer  simulations  aimed  at  charac- 
terizing the  three-dimensional  structures  of  pro- 
teins. We  apply  the  knowledge  gained  from  these 
studies  toward  the  design  of  mutations  and  inhibi- 
tors to  modify  protein  activity,  and  seek,  from  the 
particular  cases  in  hand,  to  extrapolate  to  fami- 
lies of  related  proteins.  Our  current  efforts  are 
focused  on  three  main  areas:  DNA  replication, 
5rc-related  oncogene  products  (tyrosine  ki- 
nases), and  redox  enzymes  and  oxidative  stress. 

Structure  of  the  /S-Subunit 
of  DNA  Polymerase  III 

DNA  polymerases  are  enzymes  that  duplicate 
the  information  content  of  DNA  by  catalyzing  the 
template-directed  polymerization  of  nucleic 
acids.  Polymerases  that  are  involved  in  chromo- 
somal replication,  such  as  DNA  polymerase  III 
(PolIII)  of  Escherichia  colt,  are  distinguished  by 
their  high  processivity — i.e.,  their  rapid  replica- 
tion (1 ,000  bases/second)  of  very  long  stretches 
of  DNA  without  dissociation.  Processivity  is  con- 
ferred upon  the  enzyme  by  one  of  its  subunits,  /3 
(processivity  factor),  which  acts  to  clamp  the 
polymerase  onto  DNA.  The  /3-subunit  binds  very 
strongly — indeed,  cannot  be  easily  separated 
from  circular  DNA.  It  has  been  shown,  however, 
to  slide  freely  along  duplex  DNA,  consistent  with 
its  role  as  a  clamp  that  tethers  the  polymerase 
core  to  the  template  and  advances  with  the  poly- 
merase during  replication. 

We  have  crystallized  the  |S-subunit  and  deter- 
mined its  structure  by  x-ray  diffraction  to  2.5-A 
resolution  (Xiang- Peng  Kong,  Rockefeller  Univer- 
sity; Michael  O'Donnell  [HHMI]  and  Rene  Onrust, 
Cornell  University  Medical  College) .  Two  mole- 
cules of  the  /3-subunit  are  tightly  associated  to 
form  a  donut-shaped  structure  that  forms  a  closed 
ring  around  duplex  DNA.  An  unexpected  feature 
of  the  structure  is  that  it  is  highly  symmetric. 
Each  monomer  consists  of  three  domains  of  iden- 
tical chain  topology.  Each  domain  is  roughly 
twofold  symmetric  in  its  architecture,  with  an 


outer  layer  of  two  (8-sheets  providing  a  scaffold 
that  supports  two  a-helices.  Replication  of  this 
motif  around  a  circle  results  in  a  rigid  molecule 
with  1 2  a-helices  lining  the  inner  surface  of  the 
ring  and  with  six  jS-sheets  forming  the  outer 
surface. 

The  high  symmetry  of  the  structure  is  well 
suited  to  interact  with  the  cylindrically  symmet- 
ric DNA  duplex,  and  the  hole  in  the  middle  of  the 
ring  is  large  enough  to  accommodate  either  A  or  B 
forms  of  DNA  with  no  steric  repulsion.  Although 
the  overall  charge  on  the  protein  is  negative,  a 
number  of  basic  side  chains  on  the  inner  surface 
generate  a  positive  electrostatic  field  localized  in 
the  hole  of  the  donut,  precisely  where  the  nega- 
tively charged  phosphate  backbone  of  DNA  is  ex- 
pected to  be.  We  are  carrying  out  molecular  dy- 
namics simulations  of  a  duplex-DNA  and 
(8-subunit  complex,  which  is  expected  to  provide 
information  not  readily  accessible  by  x-ray  crys- 
tallography. Other  extensions  of  this  project  in- 
clude crystallization  attempts  on  the  eukaryotic 
processivity  factor  PCNA  (proliferating  cell  nu- 
clear antigen) ,  other  subunits  of  PolIII,  and  other 
proteins  acting  at  the  replication  fork. 

Oncogene  Products  Related  to  src 

Tyrosine  kinases  such  as  the  product  of  the  src 
gene  play  a  central  role  in  signal  transduction 
pathways  in  the  cell,  and  abnormalities  in  their 
function  can  result  in  dramatic  changes  in  cellu- 
lar differentiation  and  life  cycles.  Our  approach 
is  to  work  on  these  proteins  in  isolation,  and  we 
are  collaborating  with  Hidesaburo  Hanafusa 
(Rockefeller  University)  and  Marilyn  Resh 
(Sloan-Kettering  Institute)  to  crystallize  various 
functional  domains  of  these  oncogene  products 
and  their  cellular  equivalents.  Initial  success  has 
been  obtained  for  the  src  SH2  domain,  which  is 
responsible  for  binding  to  proteins  containing 
phosphorylated  tyrosine  residues.  Starting  from 
src-containing  plasmids  (provided  by  Resh), 
Dorothea  Kominos  and  Gabriel  Waksman  in  our 
laboratory  have  cloned,  overexpressed,  purified, 
and  crystallized  SH2.  The  crystals  diffract  to  2.5-A 
resolution,  and  the  x-ray  structure  determination 


235 


Structural  Studies  on  DNA-Replication  Enzymes,  src- related  Oncogene 
Products,  and  Oxidoreductases 


of  a  complex  of  SH2  with  a  phosphotyrosyl  pep- 
tide has  been  completed  at  1.5 -A  resolution. 

Redox  Proteins  and  the  Transcriptional 
Response  to  Oxidative  Stress* 

We  have  recently  solved  and  refined  the  three- 
dimensional  structures  of  two  related  redox  en- 
zymes, thioredoxin  reductase  and  trypanothione 
-reductase.  Crystallographic  investigations  of 
enzyme-substrate  complexes  are  now  in  progress 
(G.  Waksman).  Work  has  also  begun  on  a  new 
member  of  the  disulfide  reductase  family.  Pro- 
teins related  to  thioredoxin  have  been  implicated 
in  the  process  by  which  disulfide-containing  pro- 
teins fold  up  rapidly  without  scrambling  their 
disulfide  pairings. 

James  Bardwell,  Karen  McGovern,  and  Jon 
Beckwith  (Harvard  Medical  School)  have  re- 
cently identified  an  E.  colt  protein  that  is  re- 
quired for  the  correct  folding  of  disulfide- 
containing  proteins  in  vivo.  This  21-kDa  protein, 
the  product  of  the  dsbA  gene,  has  no  detectable 
sequence  similarity  to  any  known  protein  except 
for  a  short  stretch  of  amino  acids  with  similarity 


to  the  active  site  of  thioredoxin  (including  the 
redox-active  disulfide  bond).  The  dsbA  protein 
has  been  purified  in  our  laboratory,  from  cells 
provided  by  Bardwell  and  Beckwith,  and  single 
crystals  that  diffract  to  2-A  resolution  have 
been  obtained.  The  determination  of  the  three- 
dimensional  structure  is  under  way,  and  we  are 
also  pursuing  the  construction  of  site-specific 
mutants  at  the  active  site  (Jennifer  Martin) . 

We  are  interested  in  determining  the  mecha- 
nism of  the  bacterial  oxidative  stress  sensor 
OxyR.  The  OxyR  protein  responds  to  oxidizing 
agents  by  elevating  the  expression  of  such  redox 
enzymes  as  catalase,  superoxide  dismutase,  and 
glutathione  reductase.  The  intact  OxyR  molecule 
proved  to  be  unstable  in  the  absence  of  DNA.  In 
collaboration  with  Gisela  Storz  (National  Insti- 
tutes of  Health),  Scott  Robertson  in  our  labora- 
tory has  cloned,  overexpressed,  and  purified  the 
regulatory  domain  alone,  and  its  crystallization  is 
being  pursued. 


'  This  work  is  being  supported  by  a  grant  from  the  National 
Institutes  of  Health. 


Representations  of  the  structure  of 
fi-subunit — the  sliding  clamp — of 
DNA  polymerase  III  of  Escherichia 
coli,  determined  by  x-ray  crystallog- 
raphy. The  atomic  structure  is 
shown  in  yellow  and  its  electrostatic 
potentials  in  red  ( negative )  and 
blue  (positive).  The  top  two  images 
are  different  views  of  the  dimer,  and 
the  bottom  is  a  single  monomer.  The 
electrostatic  potential  generated  by 
the  protein  helps  stabilize  the  dimer 
and  its  interactions  with  the  DNA 
molecule  ( not  shown ),  which 
threads  through  the  clamp  formed 
by  the  protein. 

From  Kong,  X.-P.,  Onrust,  R., 
O'Donnell,  M.,  and  Kuriyan,  f.  1992. 
Cell  69:425-437.  Copyright©  1992 
by  Cell  Press. 


236 


Molecular  Analysis  of  Down  Syndrome 


David  M.  Kumit,  M.D.,  Ph.D. — Investigator 

Dr.  Kumit  is  also  Professor  of  Pediatrics  and  Human  Genetics  at  the  University  of  Michigan  Medical 
School.  He  received  his  M.D.  and  Ph.D.  degrees  in  cell  biology  from  Albert  Einstein  College  of  Medicine. 
He  did  his  internship  and  residency  in  pediatrics  at  the  University  of  Pittsburgh  and  his  fellowship  in 
medical  genetics  at  the  University  of  Washington.  After  seven  years  as  a  Harvard  Medical  School  faculty 
member  at  Children's  Hospital,  he  joined  the  University  of  Michigan  Medical  Center. 


WE  are  primarily  interested  in  the  etiology 
and  pathogenesis  of  Down  syndrome. 
Why  does  the  risk  of  having  an  offspring  with 
Down  syndrome  increase  dramatically  as  women 
age?  There  are  two  competing  theories.  The 
"older  egg"  model  states  that  the  older  a  woman 
becomes,  the  more  abnormal  (aneusomic)  eggs 
she  produces.  The  "relaxed  selection"  model 
states  that  as  women  age,  they  lose  the  ability  to 
abort  abnormal  conceptuses.  To  determine  the 
correct  hypothesis,  or  whether  a  combination  of 
the  two  applies,  it  is  necessary  to  study  a  large 
number  of  Down  syndrome  cases. 

Given  our  important  (and  unexpected)  finding 
that  94  percent  of  nondisjunction  errors  are  ma- 
ternal in  origin,  resolution  of  the  above  contro- 
versy requires  an  ability  to  distinguish  the  stage 
of  maternal  cell  division  (meiosis  I  or  II)  in 
which  the  error  occurs.  To  accomplish  this,  we 
will  analyze  four  distinct  probes  (H6-5-6,  1 26-4- 
1,  D21S120,  and  GT14)  that  detect  polymorphic 
oligo  d(A,C)  sequences  in  the  pericentromeric 
region  of  chromosome  2 1 .  Conditions  and  sizes 
have  been  achieved  that  allow  each  of  these 
probes  to  be  multiplexed  in  a  single  lane  without 
co-interference.  Using  four  different  fluorescent 
markers,  it  is  feasible  to  analyze  electrophoreti- 
cally  in  a  single  gel  lane  a  molecular  weight  stan- 
dard and  a  family  consisting  of  father,  mother, 
and  their  child  with  Down  syndrome.  Pouring  the 
gels  is  the  labor-intensive  procedure  in  this  pro- 
tocol, and  the  ability  to  multiplex  three  family 
members  and  four  polymorphisms  in  a  single 
lane  will  yield  a  12-fold  savings  in  gel-pouring 
effort.  This  savings  is  made  worthwhile  by 
the  fact  that  we  must  study  at  least  500  fami- 
lies to  comprehend  the  biological  basis  for  non- 
disjunction. This  work  was  also  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

To  study  pathogenesis,  we  first  isolated  genes 
encoded  by  chromosome  21,  using  a  novel  re- 
combination-based methodology.  Genomic  frag- 
ments isolated  from  yeast  artificial  chromosomes 


(YACs)  that  map  to  chromosome  21  are  cloned 
into  a  plasmid  vector  with  the  genetic  marker 
sup¥.  RNA  isolated  from  a  variety  of  20-week  fetal 
human  tissues  was  used  to  construct  bacterio- 
phage X  cDNA  libraries,  which  are  then  infected 
into  cells  harboring  a  supY  plasmid  carrying  a 
nonrepeated  genomic  fragment  on  chromosome 
21 .  (Using  tissues  recently  obtained  from  earlier 
abortus  specimens,  we  will  now  be  able  to  make 
complex  cDNA  libraries  from  small  amounts  of 
these  tissues  by  polymerase  chain  reaction  tech- 
niques.) If  any  member  of  the  genie  cDNA  library 
in  X  shares  homology  with  the  genomic  DNA  frag- 
ment in  the  plasmid,  then  recombination  me- 
diated by  that  homology  will  ensue. 

Following  recombination  between  the  bacte- 
riophage and  the  plasmid,  selection  for  bacterio- 
phages carrying  a  given  plasmid  with  sup¥  will 
result  in  selection  for  bacteriophages  carrying  a 
cDNA  that  is  homologous  to  a  genomic  DNA 
cloned  in  the  plasmid.  In  other  words  the  system 
is  designed  to  select  for  genomic  sequences  that 
are  transcribed.  The  system  is  designed  to  stand 
alone  or  to  interdigitate  with  the  genomic  initia- 
tive as  it  proceeds.  In  the  latter  case,  as  sequenc- 
ing detects  open  reading  frames  that  represent 
candidates  for  transcription,  the  recombination- 
based  assay  is  designed  to  delineate  analytically 
the  tissue  and  timing  of  transcription  and  to  re- 
sult preparatively  in  isolation  of  the  transcribed 
sequence. 

The  bulk  of  cDNA  (gene)  libraries  are  contami- 
nated with  small  amounts  of  ubiquitous  DNA  se- 
quences from  plasmid  pBR322.  To  circumvent 
this  problem,  we  have  cloned  the  sup¥  gene  into 
an  R6K-derived  plasmid  that  lacks  homology 
with  pBR322  and  have  made  other  improvements 
that  should  increase  the  applicability  of  this  pro- 
cedure. We  should  now  be  able  to  screen  a  wide 
variety  of  extant  cDNA  libraries  for  transcription 
by  chromosome-specific  elements. 

This  systematology  applied  in  model  experi- 
ments has  demonstrated  that  we  can  perform 
both  selection  and  counterselection  appropri- 


237 


Molecular  Analysis  of  Down  Syndrome 


ately.  We  are  currently  analyzing  transcribed  se- 
quences on  distal  chromosome  21q  (where  the 
Down  syndrome  phenotype  maps)  and  have  iso- 
lated multiple  candidates.  The  technique  can 
also  be  applied  to  determine  the  timing  and  tis- 


sues of  transcription  of  likely-to-be-transcribed 
sequences,  as  determined  by  other  methodolo- 
gies. We  will  collaborate  with  researchers  per- 
forming these  other  techniques  to  make  such 
determinations. 


238 


Replication  and  Pathogenesis  of  RNA  Viruses 


Michael  M.-C.  Lai,  M.D.,  Ph.D. — Investigator 

Dr.  Lai  is  also  Professor  of  Microbiology  and  Neurology  at  the  University  of  Southern  California  School  of 
Medicine,  Los  Angeles.  He  obtained  his  M.D.  degree  from  National  Taiwan  University.  He  studied 
retroviruses  with  Peter  Duesberg  at  the  University  of  California,  Berkeley,  where  he  obtained  his  Ph.D. 
degree  in  molecular  biology  and  continued  for  postdoctoral  work. 


MEDICAL  history  is  marked  by  extraordinary 
successes  against  viral  infections,  but  it  is 
also  punctuated  by  the  continual  emergence  of 
new  viruses.  Since  viruses,  in  general,  contain 
very  limited  genetic  information,  they  must  rely 
on  host  cells  for  their  own  growth.  How  they 
cause  diseases  and  how  they  continue  to  flourish 
in  nature  are  not  only  interesting  subjects  in 
themselves  but  off^er  a  lesson  in  the  everyday 
workings  of  normal  cells.  Our  laboratory  is  inter- 
ested in  RNA  viruses,  replicating  entities  in 
which  RNA,  in  contrast  to  DNA,  is  the  genetic 
material.  We  are  exploring  how  these  viruses  rep- 
licate and  cause  diseases. 

One  of  those  we  are  studying  is  the  corona- 
virus,  named  for  its  similar  appearance  to  the  cor- 
ona of  the  sun.  The  virus  causes  the  common  cold 
in  humans  and  a  variety  of  gastrointestinal  and 
respiratory  problems  in  animals.  It  also  causes 
symptoms  very  similar  to  those  of  multiple  sclero- 
sis, thus  providing  a  model  system  for  studying 
this  disease.  The  virus  has  an  RNA  genome  of 
31,000  nucleotides,  which  is  the  longest  known 
stable  RNA.  We  are  interested  in  learning  how 
this  unusually  large  RNA  expresses  its  genes  and 
maintains  its  genetic  stability,  despite  an  over- 
whelmingly high  frequency  of  error  in  RNA  syn- 
thesis. We  have  recently  determined  the  com- 
plete sequence  of  the  genome,  giving  us  a 
glimpse  of  how  the  viral  genes  express 
themselves. 

The  virus  utilizes  a  novel  RNA  synthesis  mecha- 
nism, a  discontinuous  process  that  fuses  a  leader 
RNA  to  a  gene  located  some  distance  from  it.  This 
unusual  mechanism  allows  the  leader  RNA  to 
control  the  expression  of  viral  genes.  We  have 
recently  found  that  the  synthesis  of  one  of  the 
viral  surface  proteins  indeed  can  be  altered  by 
minor  changes  in  the  leader  RNA  sequences,  re- 
sulting in  the  variation  of  the  viral  pathogenicity. 
Furthermore,  this  variation  can  be  observed  dur- 
ing natural  viral  evolution.  These  observations 
suggest  that  the  enzyme  catalyzing  coronaviral 
RNA  synthesis  is  unusual,  which  is,  indeed,  sug- 
gested from  the  sequence  of  the  gene  encoding 
the  enzyme.  Our  laboratory  is  investigating  this 


novel  RNA  synthesis  mechanism.  A  research  grant 
from  the  National  Institutes  of  Health  provides 
support  for  this  part  of  the  research  program. 

Another  unusual  characteristic  of  coronavirus 
RNA  has  been  revealed  in  our  findings:  it  can  un- 
dergo genetic  exchange  (RNA-RNA  recombina- 
tion) at  an  extraordinarily  high  rate.  RNA-RNA  re- 
combination was  previously  thought  to  be  a  rare 
event  in  nature.  We  demonstrated,  however,  that 
it  occurs  readily  between  coronaviruses.  This  re- 
combination can  take  place  almost  anywhere  in 
the  RNA  genome,  both  in  tissue  culture  cells  and 
during  animal  infections.  We  recently  succeeded 
in  establishing  that  with  the  coronavirus,  recom- 
bination can  occur  not  only  between  two  replicat- 
ing viral  RNAs  but  between  viral  RNA  and  a  piece 
of  an  RNA  fragment  existing  inside  the  cell,  thus 
providing  a  model  system  for  RNA  recombination 
between  viral  and  cellular  RNAs.  This  type  of  re- 
combination is  one  of  the  mechanisms  by  which 
some  animal  viruses  become  pathogenic. 

We  have  demonstrated  that  a  recombinant 
virus  could  become  a  predominant  viral  popula- 
tion under  certain  conditions,  replacing  the  pa- 
rental virus  by  a  simple  natural  selection  process. 
Thus  recombination  represents  a  powerful  evolu- 
tionary tool  for  RNA  viruses.  From  the  standpoint 
of  viral  biology,  RNA  recombination  may  be  the 
genetic  mechanism  by  which  coronaviruses  weed 
out  defective  RNA  sequences  generated  by  errors 
in  RNA  synthesis.  Coronavirus  is  thus  able  to 
maintain  an  RNA  genome  larger  than  was  thought 
theoretically  possible.  RNA  recombination  has 
now  been  demonstrated  in  many  different  vi- 
ruses, suggesting  its  important  role  in  virus  evo- 
lution in  nature. 

This  genetic  phenomenon  also  has  an  impor- 
tant implication  in  vaccine  development  for  dis- 
eases such  as  AIDS  (acquired  immune  deficiency 
syndrome) ,  since  genetic  exchanges  between  vi- 
ruses may  lead  to  genetic  instability  of  live,  atten- 
uated virus  vaccines.  We  are  continuing  to  study 
the  RNA  recombination  mechanism  and  attempt- 
ing to  use  it  as  a  genetic  tool  in  determining  how 
viruses  cause  diseases. 

Another  virus  we  are  studying  in  our  laboratory 


239 


Replication  and  Pathogenesis  of  RNA  Viruses 


is  the  hepatitis  delta  virus  (HDV) ,  a  human  hepati- 
tis virus  commonly  associated  with  a  severe  form 
of  the  disease.  HDV,  by  itself,  does  not  infect  hu- 
mans, because  it  is  defective  and  requires  another 
viral  agent,  hepatitis  B  virus  (HBV),  to  supply  an 
essential  envelope  protein  to  infect  liver  cells. 
HDV  has  been  shown  to  cause  epidemics  of  fulmi- 
nant hepatitis  in  many  parts  of  the  world.  In  the 
-United  States,  it  is  prevalent  among  intravenous 
drug  abusers. 

The  virus  contains  a  circular,  single-stranded 
RNA  genome  of  only  1,700  nucleotides.  It  is  the 
only  animal  virus  with  a  circular  RNA.  This  ge- 
nome structure  is  reminiscent  of  a  group  of  plant 
pathogens,  viroids  or  virusoids,  that  cause  a  vari- 
ety of  plant  diseases.  Indeed,  the  similarity  be- 
tween HDV  RNA  and  plant  viroid  RNAs  goes 
beyond  their  circular  RNA  structure.  There  are 
several  structural  and  biochemical  features  that 
suggest  a  close  evolutionary  relationship  be- 
tween HDV  and  plant  viroid  RNAs.  Both  RNA 
groups  contain  a  "ribozyme"  activity,  in  which 
the  RNA  serves  as  an  enzyme  that  cleaves  and  li- 
gates  the  RNA  itself.  Thus  HDV  RNA  stands  at  a 
peculiar  place  in  the  evolutionary  ladder:  it  may 
have  been  derived  from  a  plant  pathogen  by  re- 
combination with  a  gene  that  gave  it  the  ability  to 
infect  human  cells  and  cause  diseases.  Our  labora- 
tory is  studying  the  properties  of  this  ribozyme 
activity.  We  have  determined  its  structural  and 
sequence  requirements,  which  are  distinct  from 
those  of  other  known  ribozymes.  Furthermore, 
we  have  begun  to  examine  whether  ribozyme  ac- 
tivity is  the  same  in  infected  cells  as  in  the  test 
tube.  The  data  obtained  thus  far  suggest  the  inter- 
esting possibility  that  some  cellular  factors  could 


participate  in  the  ribozyme  activity.  We  are  pur- 
suing these  factors. 

One  important  difference  between  HDV  RNA 
and  plant  viroid  RNAs  is  the  ability  of  the  former 
to  synthesize  a  protein,  hepatitis  delta  antigen 
(HDAg),  the  HDV  signature  protein.  HDAg  is  re- 
quired for  RNA  synthesis.  We  have  been  studying 
this  protein's  properties  and  functions  and  have 
shown  that  HDAg  is  a  phosphoprotein  that  resides 
in  the  nuclei  of  the  cells.  It  can  interact  with  itself 
to  form  a  protein  complex,  and  it  can  also  inter- 
act with  HDV  RNA  in  a  specific  way.  We  have 
shown  that  HDAg  utilizes  a  set  of  novel  sequence 
motifs  to  allow  itself  to  bind  to  RNA. 

What  is  the  role  of  this  protein,  HDAg,  in  HDV 
RNA  synthesis? — an  unusual  synthesis  because 
HDV  RNA  is  so  small  (1,700  nucleotides)  that  it 
lacks  the  capacity  to  provide  its  own  synthesizing 
enzymes.  Therefore,  HDV  most  likely  borrows 
cellular  enzymes  to  do  the  job,  which  is  unchar- 
acteristic of  RNA  viruses.  Most  RNA  viruses  have 
to  make  their  own  enzymes,  since  normal  cells  do 
not  appear  to  have  this  type.  We  are  studying  how 
HDV  modifies  the  cellular  enzymes  to  perform 
this  rather  atypical  RNA  synthesis  and  how  HDV 
RNA  synthesis  initiates  this.  It  appears  that  HDAg 
participates  in  these  processes. 

HDV  thus  provides  a  perspective  on  viral  strate- 
gies from  the  small  end  of  the  RNA  spectrum.  Our 
laboratory  is  studying  one  of  the  largest  RNA  vi- 
ruses (coronavirus)  and  the  smallest  (HDV), 
which  utilize  different  principles  for  viral  repli- 
cation. Our  studies  not  only  offer  insights  into 
how  these  viruses  cause  diseases  but  also  into 
fundamental  mechanisms  of  RNA  synthesis  and 
RNA  evolution. 


240 


Molecular  Biology  of  Human  Papillomaviruses 


LaimonisA.  Laimins,  Ph.D. — Assistant  Investigator 

Dr.  Laimins  is  also  Associate  Professor  of  Molecular  Genetics  and  Cell  Biology  at  the  University  of  Chicago. 
He  received  his  Ph.D.  degree  in  biophysics  and  theoretical  biology  from  the  University  of  Chicago.  His 
postdoctoral  research  was  done  with  George  Khoury  at  the  National  Cancer  Institute. 


MY  laboratory  is  studying  the  molecular  biol- 
ogy of  human  papillomaviruses  types  16 
and  18.  HPV- 1 6  and  - 1 8  are  the  etiological  agents 
of  the  many  malignancies  of  the  urogenital  re- 
gion, and  in  particular  those  of  the  cervix.  More 
than  70  different  types  of  HPV  are  known  to  in- 
fect cutaneous  and  mucosal  epithelia  in  various 
body  locations.  Some  types  induce  warts  on  the 
hands  or  soles  of  the  feet,  but  are  never  found  in 
genital  lesions.  Similarly,  the  viruses  that  induce 
warts  in  the  genital  region  are  never  found  on  the 
hands  or  feet. 

The  majority  of  HPVs  cause  benign  warts,  and 
about  one-third  of  the  types  are  specific  for  geni- 
tal epithelium.  A  subset  of  these  viruses  (HPV- 16, 
-18,  -31,  and  -51)  infect  the  cervix  and  are 
strongly  associated  with  the  development  of 
cervical  cancer.  Although  the  number  of  HPV- 
positive  individuals  has  increased  10-fold  in  the 
last  10  years,  effective  monitoring  procedures 
such  as  the  PAP  smear  have  limited  the  increase  in 
cervical  cancers  in  the  United  States  and  Europe; 

Up  to  20  percent  of  the  population  in  these 
countries  is  infected  by  some  type  of  genital  HPV, 
particularly  the  non-oncogenic  types  6  and  1 1 . 
However,  many  cases  of  infection  by  HPV- 1 6  and 
-18  without  visible  lesions  have  been  reported.  It 
therefore  appears  that  although  infection  by 
HPVs  is  necessary  for  the  development  of  cervical 
cancer,  infection  alone  is  not  sufficient  to  induce 
malignancy.  In  low-grade  lesions,  or  condylomas, 
viral  DNA  is  present  in  the  cell  as  episomes,  and 
progeny  viruses  are  produced.  In  contrast,  in  cer- 
vical cancers  the  viral  genome  is  found  integrated 
into  the  host  chromosome,  and  no  viral  particles 
are  synthesized.  One  model  for  progression  from 
a  low-grade  lesion  to  a  cancer  suggests  that  inte- 
gration is  important  in  the  development  of  the 
cancer  and  that  it  may  act  by  the  removal  of  a 
dominant  inhibitor  of  transformation.  Viral  inte- 
gration into  the  chromosome  may  thus  be  the  first 
step  of  a  multistep  carcinogenic  process.  My  labo- 
ratory is  currently  studying  the  molecular  mecha- 
nisms by  which  HPVs  contribute  to  this  process. 

A  major  impediment  to  the  study  of  HPVs  has 
been  an  inability  to  propagate  these  viruses  in 
culture.  Papillomaviruses  are  unusual  in  that 


their  life  cycle  is  tightly  coupled  to  the  differen- 
tiation program  of  epithelial  cells.  Although  most 
viruses  infect  one  cell  type  and  undergo  a  produc- 
tive infection  in  the  same  cell,  HPVs  infect  basal 
epithelial  cells,  establish  their  genomes  as  epi- 
somes, and  only  replicate  upon  cellular  differen- 
tiation, with  amplification  of  viral  DNA  and  ex- 
pression of  late  genes.  Inability  to  duplicate  this 
differentiation  process  in  vitro  long  prevented 
the  successful  propagation  of  virus.  We  have  re- 
cently duplicated  several  features  of  these  pro- 
ductive infections  of  HPV  in  culture,  and  current 
studies  are  directed  at  identifying  how  these  dif- 
ferentiation-specific properties  are  controlled. 
This  work  is  supported  by  a  grant  from  the  Ameri- 
can Cancer  Society. 

My  laboratory  has  found  that  HPV  can  immorta- 
lize epithelial  cells  derived  from  either  human 
foreskin  or  cervix  in  tissue  culture.  Normally  pri- 
mary cells  have  only  a  limited  life  span  in  vitro, 
but  the  presence  of  the  HPV  E7  protein  is  suffi- 
cient to  allow  for  unlimited  grov^h  in  culture. 

The  loss  of  differentiation  is  usually  a  charac- 
teristic of  many  epithelial  cancers,  including 
those  of  the  cervix.  We  have  been  studying  how 
HPV  viral  proteins  alter  epithelial  cell  differen- 
tiation. In  low-grade  lesions  in  which  infectious 
viruses  are  produced,  these  proteins  only  slightly 
alter  epithelial  differentiation.  In  cervical  can- 
cers, on  the  other  hand,  infected  cells  have  lost 
all  ability  to  differentiate. 

We  have  also  used  a  system  that  accurately  mim- 
ics the  differentiation  properties  of  epithelial 
cells  in  vitro,  to  show  that  HPV  sequences  alter 
the  ability  of  epithelial  cells  to  differentiate.  The 
morphological  changes  that  are  seen  in  this  tissue 
culture  system  are  very  similar  to  those  seen  in 
low-grade  cervical  neoplasias.  In  tissue  culture, 
our  HPV  cell  lines  quickly  lose  the  ability  to  dif- 
ferentiate and  develop  the  appearance  of  high- 
grade  neoplasias  or  cancers.  Two  HPV  genes,  E6 
and  E7,  seem  to  be  required  for  this  transforma- 
tion process.  We  believe  this  loss  of  differentia- 
tion may  be  an  in  vitro  model  for  the  develop- 
ment of  cervical  cancer.  In  the  future,  we  hope  to 
utilize  this  model  to  identify  other  important  fac- 
tors involved  in  controlling  the  development  of 


241 


Molectilar  Biology  of  Human  Papillomaviruses 


malignancy.  These  observations  also  strongly 
support  the  etiological  role  of  HPV  in  the  devel- 
opment of  cervical  cancer.  This  work  was  sup- 
ported in  part  by  a  grant  from  the  National  Insti- 
tutes of  Health. 

My  laboratory  has  been  able  to  identify  factors 
involved  in  the  cell  type-specific  regulation  of 
viral  gene  expression  and  replication.  We  have 
-identified  viral  enhancer  sequences  that  are  re- 
sponsible for  regulating  viral  expression  in  ma- 
turing keratinocytes.  The  function  of  one  of  these 
sequences  is  dependent  on  the  action  of  a  cellu- 
lar protein  that  is  only  found  in  epithelial  cells. 
We  have  designated  this  protein  keratinocyte- 
stimulating  factor,  KRF- 1 ,  and  are  currently  clon- 
ing the  gene  that  encodes  it. 


Eventually  we  will  examine  how  the  action  of 
cellular  transcription  factors  is  altered  by  the 
presence  of  viral  transforming  genes.  We  have 
shown  that  the  products  of  both  E6  and  E7  can 
indirectly  regulate  the  transcription  of  a  variety 
of  genes.  In  addition,  we  believe  that  a  major  de- 
terminant of  the  tissue  spectrum  of  papillomavi- 
ruses is  specified  through  the  regulation  of  tran- 
scription. Understanding  the  mechanisms  of 
tissue-specific  expression  will  facilitate  our  anal- 
ysis of  why  different  HPV  types  only  induce  le- 
sions in  particular  kinds  of  epithelial  cells.  Our 
studies  on  the  biology  of  HPV  should  provide 
information  on  mechanisms  of  transformation 
and  tumor  progression,  as  well  as  tissue-  and 
difi'erentiation-specific  expression. 


242 


Genetic  Studies  in  Cardiovascular  Disease 


Jean-Marc  Lalouel,  M.D.,  D.Sc. — Investigator 

Dr.  Lalouel  is  also  Professor  of  Human  Genetics  at  the  University  of  Utah  School  of  Medicine.  He  obtained 
a  medical  doctorate,  a  master's  degree  in  microbiology  and  genetics,  and  a  doctorate  of  sciences  in 
genetics  at  the  University  of  Paris,  France.  He  furthered  his  training  as  a  postdoctoral  fellow  and  a  research 
associate  with  Newton  Morton  at  the  University  of  Hawaii  and  was  Professor  of  Human  Biology  at  the 
University  of  Paris  before  joining  the  faculty  of  the  University  of  Utah. 


COMMON  cardiovascular  disorders  such  as 
coronary  artery  disease  and  essential  hyper- 
tension exhibit  a  familial  tendency.  Such  broad 
clinical  classes  represent  complex  etiological  en- 
tities, where  many  genes  and  environmental  de- 
terminants are  likely  to  be  involved.  Multiplicity 
and  heterogeneity  in  causation  stretch  the  ability 
of  genetic  methods  to  the  limit  of  their  investiga- 
tive power.  The  consideration  of  biochemical  pa- 
rameters can  reduce  this  degree  of  complexity. 
Before  the  global  picture  can  be  comprehended, 
however,  details  of  the  landscape  will  need  to  be 
scrutinized  first.  This  perspective  is  highlighted 
in  two  examples  from  our  laboratory.  Work  on 
lipoprotein  lipase  was  led  by  Mitsuru  Emi  and 
Akira  Hata,  while  work  on  hypertension  was  led 
by  Richard  Lifton. 

Abnormal  lipoprotein  concentrations  in 
plasma  are  commonly  observed  in  the  relatives  of 
patients  with  early  coronary  disease,  yielding 
various  patterns  of  hypercholesterolemia  and/or 
hypertriglyceridemia  within  families.  Such  com- 
plex phenotypes  are  thought  to  result  either  from 
the  variable  expression  of  a  single-gene  defect  or 
from  the  independent  contribution  of  two  or 
more  genes.  The  contribution  of  such  genes  to 
the  clinical  expression  of  hyperlipidemia  is  fur- 
ther blurred  by  the  fact  that  hormonal  influences, 
diet,  and  habitus  exert  major  influences  on  the 
regulation  of  lipid  metabolism.  A  severe  form  of 
familial  hypercholesterolemia,  which  accounts 
for  about  5  percent  of  myocardial  infarction,  has 
been  linked  to  molecular  defects  of  a  lipoprotein 
cellular  receptor.  More  than  90  percent  of  di- 
etary fat  is  hydrolyzed  by  lipoprotein  lipase  (LPL) 
in  an  initial  step  controlling  its  delivery  to  periph- 
eral tissues.  The  enzyme,  secreted  by  adipocytes 
and  muscle  cells,  acts  at  a  distance  from  its  site  of 
synthesis,  becoming  anchored  to  the  luminal  sur- 
face of  capillaries  by  an  ionic  interaction  with 
heparan  sulfate.  In  the  presence  of  a  specific  co- 
factor,  apolipoprotein  C-II,  the  enzyme  hydro- 
lyzes  triglycerides  of  intestinal  or  hepatic  origin 
by  binding  to  the  surface  of  circulating  lipopro- 
teins, thereby  releasing  fatty  acids  for  uptake  in 
the  tissues  where  they  can  be  used  as  fuel  or  rees- 


terified  for  storage.  Defective  functional  enzyme 
is  the  diagnostic  feature  of  a  rare  recessive  chylo- 
micronemia  syndrome,  familial  LPL  deficiency. 

This  condition  is  characterized  by  massive  chy- 
lomicronemia  in  the  fasting  state,  episodes  of  ab- 
dominal pain,  life-threatening  acute  pancreatitis, 
and  eruptive  xanthomas.  By  investigating  the  rela- 
tives of  a  homozygous  subject,  we  showed  pre- 
viously that  heterozygotes  for  such  molecular 
defects  tend  to  express  a  common  form  of  hyper- 
triglyceridemia. However,  when  we  screened 
unrelated  subjects  for  molecular  defects  of  the 
LPL  gene,  we  found  that  mutations  of  this  gene 
probably  account  for  3-5  percent  of  such  hyper- 
lipidemias.  This  situation,  similar  to  receptor  de- 
fects in  familial  hypercholesterolemia,  again 
stress  that  most  forms  of  hyperlipidemia  are  of 
heterogeneous  origin. 

We  and  others  have  now  identified  a  host  of 
mutations  in  the  LPL  gene  of  subjects  with  LPL 
deficiency,  including  a  large  number  of  simple 
atnino  acid  substitutions.  Such  mutations  may 
provide  natural,  specific  probes  of  functional  do- 
mains of  the  enzyme.  When  they  are  superim- 
posed to  the  known  three-dimensional  structure 
of  the  homologous  enzyme,  pancreatic  lipase,  it 
becomes  clear  that  they  are  spread  over  the  pro- 
tein's first  folding  domain,  which  includes  the 
triad  of  residues  directly  involved  in  catalysis 
(see  figure) . 

We  reproduced  four  such  mutations  in  vitro, 
expressed  them  in  cultured  cells,  and  analyzed 
the  corresponding  mutant  proteins.  A  common 
feature  of  these  mutations  is  that  they  aff^ected  the 
assembly  and  stability  of  the  two  identical  sub- 
units  characterizing  the  active  enzyme.  Hence 
the  majority  of  these  mutations  lead  to  loss  of 
catalytic  activity  through  rather  nonspecific 
mechanisms. 

To  probe  other  domains  of  the  enzyme,  it  be- 
came clear  that  we  could  not  rely  solely  on  natu- 
rally occurring  mutations.  LPL  binds  to  heparan 
sulfate,  a  member  of  a  family  of  ubiquitous  and 
abundant  complex  polysaccharides.  Interactions 
of  this  nature  play  a  role  in  many  biological  pro- 
cesses, such  as  lipolysis,  hemostasis,  cell  adhe- 
sion, cell  proliferation,  or  angiogenesis.  The  pro- 


243 


Genetic  Studies  in  Cardiovascular  Disease 


tein  determinants  involved  in  these  interactions 
are  poorly  characterized,  but  the  high  density  of 
negative  charges  on  heparan  sulfate  indicates  that 
the  protein  domains  should  include  positively 
charged  residues. 

After  generating  many  artificial  mutations  at 
such  positions,  we  have  produced  the  corre- 
sponding proteins  by  expression  in  cultured  cells 
and  analyzed  their  affinity  for  heparin.  We  have 
identified  six  basic  amino  acids  that  mediate  the 
high  affinity  of  the  active,  dimeric  enzyme  with 
heparin.  They  form  a  domain  of  unique  structure, 
which  we  are  attempting  to  characterize  further. 

Essential  hypertension  presents  a  yet  greater 
challenge,  for  little  biochemical  insight  is 
usually  available.  Glucocorticoid-remediable  al- 
dosteronism (GRA)  is  a  rare  autosomal  dominant 
disorder  marked  by  severe  hypertension  and 
hyperaldosteronism  with  high  levels  of  abnor- 
mal adrenal  steroids.  All  these  manifestations 
can  be  corrected  by  the  administration  of 
glucocorticoids. 

Aldosterone  is  a  steroid  involved  in  the  regula- 
tion of  the  balance  of  sodium  and  potassium  ions 
and  produced  in  the  zona  glomerulosa  of  the  adre- 
nal gland  under  the  primary  control  of  the  renin- 


angiotensin  system.  By  contrast,  glucocorticoids 
exert  their  effects  on  carbohydrate  metabolism, 
are  produced  by  the  zona  fasciculata  of  the  adre- 
nals, and  are  regulated  by  the  adenohypophyseal 
hormone  adrenocorticotropin  (ACTH).  Their  syn- 
thetic pathways  share  several  enzymes,  including 
lljS-hydroxylase.  Aldosterone  synthesis,  how- 
ever, requires  a  unique  enzymatic  step  catalyzed 
by  aldosterone  synthase,  which  is  normally  ex- 
pressed only  in  the  zona  glomerulosa. 

In  a  subject  with  GRA,  we  found  that  unequal 
crossing  over  between  1 1  (8-hydroxylase  and  aldo- 
sterone synthase,  in  close  proximity  on  chromo- 
some 8,  had  created  a  new  chimeric  gene  com- 
prising regulatory  sequences  of  1  lj8-hydroxylase 
and  sequences  responsible  for  the  catalytic  speci- 
ficity of  aldosterone  synthase.  This  observation 
explains  the  ectopic  production  of  aldosterone  in 
the  adrenal  tissue  responsible  for  synthesis  of 
glucocorticoids,  and  the  corresponding  hor- 
monal control  observed  in  GRA.  Although  this 
finding  provides  a  clear  interpretation  of  the 
complex  physiology  of  a  rare  form  of  hyperten- 
sion, its  significance  for  our  understanding  of  the 
more  common  forms  of  essential  hypertension 
remains  to  be  determined. 

Mutations  of  human  lipoprotein  lipase  that 
impair  the  enzyme's  ability  to  regulate  lipid 
metabolism  are  shown  superimposed  upon  a 
schematic  rendering  of  the  backbone  of  a  ho- 
mologous enzyme,  human  pancreatic  li- 
pase. The  three  circled  residues  define  the 
triad  directly  involved  in  catalysis. 

From  Lalouel,  f.-M.,  Wilson,  D.E.,  and  Iver- 
ius,  P.-H.  1992.  Curr  Opin  Upidol  3:86-95. 


244 


Structure  and  Replication  of  Influenza  Virus 
and  Paramyxoviruses 


Robert  A.  Lamb,  Ph.D.,  Sc.D. — Investigator 

Dr.  Lamb  is  also  John  Evans  Professor  of  Molecular  and  Cellular  Biology  and  Professor  of  Microbiology- 
Immunology  at  Northwestern  University.  He  received  his  undergraduate  degree  reading  biochemistry  at 
the  University  of  Birmingham,  England,  and  his  Ph.D.  and  Sc.D.  degrees  from  the  University  of  Cambridge. 
He  came  to  the  United  States  to  do  postdoctoral  work  with  Purnell  Choppin  at  the  Rockefeller  University, 
where  he  later  became  a  faculty  member.  Ten  years  ago  he  joined  the  faculty  of  Northwestern  University. 


ANIMAL  viruses  provide  a  unique  tool  with 
which  to  study  the  complex  biochemical 
processes  involved  in  the  biosynthesis  and  main- 
tenance of  eukaryotic  cells.  Our  laboratory  is  in- 
vestigating the  molecular  structure  and  the  mech- 
anism of  replication  of  two  enveloped  RNA 
viruses,  influenza  virus  and  the  paramyxovi- 
rus SV5. 

Influenza  virus  causes  important  diseases  in 
humans  and  animals.  It  has  tremendous  socioeco- 
nomic consequences,  for  influenza  continues  to 
occur  in  regular  epidemics  and  occasional  pan- 
demics and  is  a  leading  cause  of  morbidity  and 
mortality.  Paramyxoviruses  cause  many  biologi- 
cally and  economically  important  diseases  of  hu- 
mans and  lower  animals.  Besides  SV5,  these  vi- 
ruses include  parainfluenza  virus  types  1-5, 
mumps  virus,  measles  virus,  canine  distemper 
virus,  Newcastle  disease  virus  of  chickens,  and 
rinderpest  of  cattle. 

We  have  been  elucidating  the  wide  range  of 
mechanisms  used  by  these  RNA  viruses  to  maxi- 
mize the  amount  of  encoded  protein  in  their 
compact  genomes.  We  have  identified  overlap- 
ping reading  frames,  splicing  of  mRNAs,  the  use 
of  bicistronic  mRNAs,  transcriptional  stuttering 
to  add  nontemplated  nucleotides  to  an  RNA  tran- 
script (and  hence  yield  a  separate  mRNA) ,  and  a 
coupled  stop-start  translation  of  tandem  cistrons. 

Influenza  virus  and  SV5  were  selected  for  study 
not  only  because  of  their  importance  as  the  caus- 
ative agents  of  major  diseases  but  also  because 
they  provide  excellent  models  for  examining  a 
variety  of  properties  of  integral  membrane  pro- 
teins. Since  these  proteins  are  the  major  antigenic 
determinants  of  the  viruses,  knowledge  about 
their  structure  should  enhance  our  understand- 
ing of  how  they  act  as  immunological  targets, 
thus  aiding  in  developing  new  vaccines.  In  addi- 
tion, some  of  the  biochemical  activities  of  the 
influenza  virus  are  specialized  to  the  virus,  mak- 
ing them  attractive  as  points  of  intervention  in 
the  virus  life  cycle  to  which  rationally  designed 
therapeutic  agents  can  be  developed.  We  are  ana- 
lyzing biochemical  properties  of  the  viral 
glycoproteins. 


We  are  also  investigating  the  mechanism  by 
which  integral  membrane  proteins  are  trans- 
ported to  the  cell  surface  in  the  exocytotic  path- 
ways and  are  internalized  from  the  surface  by  the 
endocytotic  pathways.  We  are  studying  the  seven 
integral  membrane  proteins  encoded  by  influ- 
enza virus  and  SV5 — three  of  which  were  discov- 
ered in  our  laboratory — because  they  provide  a 
diverse  group  of  membrane  proteins  that  span  the 
cell  membrane  once. 

Virus  Cation  Channels 

Influenza  virus  protein  M2  is  a  small  (97-resi- 
due)  type-Ill  integral  membrane  protein  that 
forms  a  disulfide-linked  tetramer.  The  sensitivity 
of  influenza  virus  to  the  drug  amantadine  hydro- 
chloride, the  coupling  of  antiviral  action  to  the 
M2  transmembrane  domain,  and  the  premature 
acid-induced  conformational  change  in  the  viral 
hemagglutinin  in  the  presence  of  the  drug  sug- 
gest that  M2  is  an  ion  channel,  that  it  is  essential 
for  virus  uncoating  in  secondary  endosomes,  and 
that  it  can  alter  the  intracellular  pH  of  the  trans 
Golgi  network.  In  collaboration  with  Lawrence 
Pinto,  Northwestern  University,  we  have  tested 
the  M2  protein  for  ion  channel  activity  by  inject- 
ing M2  mRNA  into  Xenopus  oocytes  and  measur- 
ing surface  currents  with  a  two-electrode  patch- 
clamp  apparatus.  We  have  shown  that  expression 
of  the  protein  is  associated  with  an  ion  channel 
activity  selective  for  monovalent  ions. 

Amantadine  hydrochloride  significantly  atten- 
uated the  inward  current  induced  by  hyperpolar- 
ization  of  oocyte  membranes,  and  mutations  in 
the  M2  membrane-spanning  domain  that  confer 
viral  resistance  to  amantadine  produced  currents 
that  were  resistant  to  the  drug.  Thus  we  have  pro- 
vided direct  data  on  the  antiviral  drug's  mecha- 
nism of  action. 

The  M2  protein  does  not  have  the  molecular 
structure  of  most  ion  channels  cloned  to  date.  We 
had  to  perform  a  large  number  of  experiments  to 
eliminate  the  possibility  that  the  M2  protein  was 
not  a  regulator  that  activates  a  normally  silent 
channel  endogenous  to  oocytes.  Our  analysis  of 
distinguishing  characteristics  of  the  currents  pro- 


245 


Structure  and  Replication  of  Influenza  Virus  and  Paramyxoviruses 


duced  by  various  M2  proteins  with  changes  in  the 
transmembrane  domain  suggests  that  the  domain 
forms  the  channel  pore,  and  that  the  M2  protein  is 
therefore  a  channel  per  se. 

Our  experiments  have  also  indicated  that  the 
M2  protein  channel  activity  is  activated  by  lov^ 
pH,  suggesting  that  the  channel  is  only  switched 
on  in  endosomes  and  the  trans  Golgi  network — 
-intracellular  compartments  with  lowered  pH. 
We  are  currently  beginning  a  detailed  structure- 
function  analysis  of  this  channel  to  characterize 
further  the  residues  involved  in  drug  sensitivity 
and  ion  specificity. 

We  have  also  shown  that  the  influenza  B  virus 
NB  glycoprotein,  which  has  a  similar  overall 
structure  but  no  obvious  amino  acid  homology  to 
the  influenza  A  virus  M2  protein,  has  ion  channel 
activity  when  expressed  in  oocytes.  However,  the 
conductance  of  the  NB  ion  channel  activity  is  spe- 
cific for  chloride  ions  and  is  regulated  by  calcium 
ion  concentration. 

The  proposed  pivotal  role  of  ion  channels  in 
the  replicative  cycle  of  the  influenza  viruses  sug- 
gests that  the  proteins  present  an  important  target 
for  a  point  of  intervention  by  drugs  (in  addition 
to  amantadine)  in  the  prophylaxis  and  therapy  of 
virus  infections.  We  are  currently  testing  whether 
other  viral  proteins  with  similar  structures  to  M2 
and  NB  have  ion  channel  activities — e.g.,  the 
paramyxovirus  SH  protein,  the  vpu  integral  mem- 
brane protein  of  the  human  immunodeficiency 
virus  I,  and  the  C3  protein  of  the  coronavirus 
avian  infectious  bronchitis. 


Intracellular  Transport  of  Glycoproteins 

To  elucidate  the  rules  that  govern  protein  ori- 
entation in  the  lipid  bilayer,  we  are  examining 
how  polypeptides  are  initially  inserted  into  the 
endoplasmic  reticulum  (ER)  and  are  determining 
the  signals  necessary  for  the  protein-bilayer  inter- 
action. One  of  the  major  factors  is  the  presence  of 
positively  charged  residues  flanking  the  hydro- 
phobic membrane-spanning  domain  to  retain  a 


region  of  the  protein  in  the  cytoplasm.  We  have 
also  been  focusing  on  the  factors  and  signals 
needed  to  fold  the  primary  polypeptide  chain 
once  it  has  been  translocated  across  the  mem- 
brane of  the  ER. 

The  cellular  glucose-regulated  protein  GRP78- 
BiP  is  a  member  of  the  HSP70  stress  family  of 
gene  products  and  is  a  resident  component  of  the 
ER,  where  it  is  thought  to  play  a  role  in  the  fold- 
ing and  oligomerization  of  secretory  and  mem- 
brane-bound proteins.  GRP78-BiP  also  binds  to 
malfolded  proteins  and  this  may  be  one  mecha- 
nism for  preventing  their  intracellular  transport. 
During  folding,  the  SV5  hemagglutinin-neuramin- 
idase (HN)  glycoprotein  specifically  and  tran- 
siently associates  with  GRP78-BiP.  This  complex 
formation  can  only  be  detected  prior  to  oligo- 
merization of  the  immature  HN  molecules  to 
form  the  native  tetramer,  suggesting  that  GRP78- 
BiP  acts  as  a  chaperone  to  promote  correct  fold- 
ing of  the  molecule. 

Internalization  and  Degradation 
of  Glycoproteins 

The  SV5  HN  glycoprotein  is  extensively  inter- 
nalized from  the  virus-infected  cell  surface  and 
degraded  in  lysosomes.  We  are  making  an  exten- 
sive study  of  the  mechanism  of  HN  internaliza- 
tion. This  is  of  considerable  interest  because  HN 
lacks  an  aromatic  amino  acid  in  its  cytoplasmic 
tail  that  has  been  found  necessary  for  the  internal- 
ization of  several  well-characterized  receptor 
molecules  internalized  by  the  clathrin-coated  ves- 
icle pathway.  Examination  of  chimeric  mole- 
cules constructed  between  HN  and  another  type- 
II  integral  membrane  protein  that  is  not 
internalized,  influenza  virus  neuraminidase,  sug- 
gests that  the  HN  transmembrane  domain  signals 
internalization  from  the  cell  surface  and  specifies 
targeting  to  lysosomes. 

Some  aspects  of  research  in  our  laboratory  on 
influenza  virus  and  paramyxoviruses  are  sup- 
ported by  grants  from  the  National  Institutes  of 
Health. 


246 


Cancer  and  Genetic  Modification 


Philip  Leder,  M.D. — Senior  Investigator 

Dr.  Leder  is  also  John  Emory  Andrus  Professor  in  the  Department  of  Genetics  at  Harvard  Medical  School. 
He  received  his  M.D.  degree  from  Harvard  Medical  School.  He  has  also  received  three  honorary  D.Sc. 
degrees.  Dr.  Leder  held  several  positions  at  NIH  before  returning  to  Harvard.  His  many  honors  include  the 
Albert  Lasker  Medical  Research  Award,  the  National  Medal  of  Science,  and  the  Heinekin  Prize  awarded  by 
the  Royal  Netherlands  Academy  of  Arts  and  Sciences.  He  is  a  member  of  the  National  Academy  of  Sciences. 


THE  growth  of  cells  in  an  organism  is  far  too 
delicate  a  process  to  be  left  to  chance. 
Rather,  as  with  all  biologic  processes,  it  is  subject 
to  a  very  stringent  set  of  rules  that  are  pro- 
grammed into  the  makeup  of  the  organism.  The 
basis  for  the  control  of  growth  is  genetic.  The 
genes  set  the  parameters  that  allow,  say,  the  liver 
to  take  the  shape  it  does  or  the  kidney  to  assume 
its  particular  size  and  function.  Genes  establish 
the  rules  whereby  an  organ  grows  in  an  orderly 
fashion  and  reaches  a  prescribed  and  limited  size. 
Thus  growth  can  proceed  so  far  but  no  farther, 
attaining  a  programmed  equilibrium  compatible 
with  life. 

Cancer  as  a  Disease  of  Genes 

Cancer  is  a  profound  disorder  of  cell  growth. 
The  delicate  balance  established  by  a  genetically 
encoded  program  is  overturned.  Instead  of  reach- 
ing an  equilibrium,  the  cancer  cell  no  longer  re- 
sponds to  signals  that  would  limit  its  ability  to 
divide.  It  is  out  of  control,  and  its  unlimited 
growth  has  profoundly  dangerous  consequences 
for  the  organism. 

Over  the  past  decade  or  so,  it  has  become  in- 
creasingly clear  that  many  cancers  can  be  ac- 
counted for,  at  least  in  part,  by  damage  occurring 
to  genes  that  encode  the  rules  for  control  of  cell 
growth.  Genetic  damage,  or  mutation,  can  inacti- 
vate a  gene  or  cause  it  to  function  at  the  wrong 
time  or  in  the  wrong  place  or,  indeed,  even  cause 
it  to  make  the  wrong  product.  The  set  of  genes 
whose  mutation  can  give  rise  to  cancer  is  often 
just  those  that  normally  regulate  cell  growth.  Ge- 
neticists refer  to  the  damaged  genes  that  contrib- 
ute to  the  development  of  malignancy  as  onco- 
genes (from  the  Greek  ovkoc,  or  tumor). 

Transgenic  Mice  and  the  Genetic  Basis 
of  Cancer 

For  some  time  my  colleagues  and  I  have  been 
interested  in  genes  that  control  cell  growth.  Our 
work  has  been  considerably  advanced  by  the  tech- 
nique of  introducing  active  oncogenes  into  the 
hereditary  makeup  of  special  strains  of  laboratory 
mice.  Called  "transgenic,"  such  mice  carry  on- 


cogenes created  in  the  laboratory,  pass  on  these 
cancer-causing  genes  to  offspring,  and  therewith 
transmit  a  strong  tendency  to  develop  cancer. 
Thus,  in  many  ways,  transgenic  mice  become  use- 
ful models  of  human  malignancy. 

For  example,  we  have  designed  specific  mice 
that  develop  cancer  of  the  breast  and  others  that 
develop  cancer  of  the  blood  cells — specific  leu- 
kemias  and  lymphomas.  Some  of  these  mice  even 
develop  benign  prostatic  hypertrophy,  a  condi- 
tion that  aff^ects  up  to  85  percent  of  men  by  the 
eighth  decade  of  life.  These  experiments  have 
taught  us  that  certain  cancers  can  be  caused  by 
specific  oncogenes  and  that  many,  but  not  neces- 
sarily all,  cancers  are  the  result  of  a  collaboration 
between  two  or  more  oncogenes.  This  suggests 
that  cancer  is  often  a  "multihit"  process,  one  that 
requires  several  activating  events. 

A  Binary  System  for  Activating 
and  Silencing  Transgenes 

During  the  past  year  we  have  extended  the 
power  of  transgenic  technology  by  creating  a  sys- 
tem that  gives  us  much  better  control  over  the 
transgene  we  have  introduced.  For  example,  we 
often  introduce  genes  that  dramatically  increase 
the  incidence  of  certain  cancers  in  our  mice. 
Cancers  obviously  influence  the  ability  of  our  an- 
imals to  pass  their  genes  on  to  succeeding  genera- 
tions, as  such  genes  often  preclude  survival.  To 
overcome  this  problem  and  to  assure  that  no 
more  cancer-prone  mice  are  produced  than  we 
need  for  our  experiments,  we  have  designed  a 
binary  system  in  which  "target"  genes  can  be 
held  in  an  inactive  state  in  one  line  of  mice  and 
become  active  only  when  the  mice  are  bred  to  a 
second  line  that  carries  an  "activator"  gene.  The 
system  is  suggestive  of  epoxy  cement  that  is  held 
in  two  tubes,  the  contents  of  which  must  be 
mixed  to  become  functional. 

The  binary  system  has  the  further  advantage 
that  it  can  be  "multiplexed,"  or  used  in  a  variety 
of  combinations,  such  that  a  target  can  be  one  of 
many  different  transgenes  that  is  in  turn  com- 
bined with  one  or  more  activator  genes.  For  exam- 
ple, the  activators  could  specify  expression  of  a 


247 


Cancer  and  Genetic  Modification 


target  in  many  different  organs  at  different  times. 
A  further  interesting  aspect  of  this  system  is  that 
the  unity  of  biology  permits  elements  of  the  sys- 
tem that  have  been  derived  from  the  regulatory 
machinery  of  a  simple  unicellular  organism, 
yeast,  to  be  used  quite  readily  in  mammals. 

Host  Defenses  Against  Cancer 

While  transgenic  mice  are  very  useful  in  analyz- 
ing the  action  of  oncogenes,  they  are  also  useful 
in  exploring  the  host  defense  mechanism  mobi- 
lized to  prevent  the  development  and  spread  of 
cancer.  The  immune  system  is  one  of  the  organ- 
ism's chief  instruments  against  the  spread  of  in- 
fectious disease  and  for  the  rejection  of  foreign 
tissues.  For  instance,  the  body's  immune  system 
must  be  neutralized  to  accomplish  effective  heart 
or  kidney  transplants.  The  role  that  the  immune 
system  plays  in  tumor  rejection  is  poorly  under- 
stood, but  important  discoveries  in  immunology 
indicate  that  the  immune  response  is  regulated 
by  an  array  of  hormone-like  agents  called  lym- 
phokines.  These  are  released  by  cells  of  the  im- 
mune system  to  influence  the  growth  and  devel- 
opment of  other  cells. 

We  are  particularly  interested  in  how  lympho- 
kines  influence  the  host's  response  to  cancer.  In 
the  course  of  this  work,  we  have  focused  on  the 
action  of  two  particularly  interesting  lympho- 
kines,  IL-4  and  IL-7.  (These  biologic  response- 
modifying  agents  are  frequently  called  IL,  de- 
rived from  "interleukin,"  an  agent  that  mediates 
signals  between  white  blood  cells,  or  leuko- 
cytes.) Several  cell-signaling  functions  have  been 
recognized  in  IL-4.  Although  its  precise  role  in 
the  body  has  not  been  proved,  IL-4  is  suspected  of 
playing  a  role  in  modulating  the  immune  re- 
sponse. IL-7  is  thought  to  be  important  for  the 
orderly  development  of  antibody-producing 
cells,  the  so-called  B  cells. 

We  have  been  able  to  show  that  both  IL-4  and 
IL-7  are  potent  antitumor  agents,  acting  to  induce 
host  defense  mechanisms.  Further  studies  of  the 


action  of  IL-4  have  allowed  us  to  identify  two  par- 
ticular cell  types  that  may  mediate  this  antitumor 
effect.  One  of  these  is  the  eosinophil,  a  cell  in- 
volved in  many  allergic  responses.  Another  is  the 
macrophage,  a  scavenger  cell  concerned  with 
cell-killing  functions.  Our  most  recent  work  has 
focused  on  identifying  the  active  regions  of  the 
IL-4  molecule,  with  a  view  to  distinguishing 
those  structures  required  for  antitumor  activity 
from  those  that  give  rise  to  unwanted  side  effects. 

New  Directions:  Distinct  Genetic 
Contributions  from  Mothers  and  Fathers 

Mendel's  vision  of  genetics  held  that  a  particu- 
lar gene  carried  by  an  organism  behaves  in  ex- 
actly the  same  way  whether  it  is  inherited  from 
the  mother  or  the  father.  For  the  most  part,  this  is 
true.  Nevertheless,  in  mammals,  the  highest  or- 
ganisms, it  has  proved  impossible  to  induce  de- 
velopment artificially  from  the  egg  alone  (a  pro- 
cess known  as  parthenogenesis) ,  although  this  is 
possible  in  many  lower  forms.  Some  experiments 
using  transgenic  mice  have  helped  us  to  under- 
stand this  phenomenon  and  why  it  is  that,  at  least 
for  mammals,  mother  and  father  (that  is,  both  egg 
and  sperm)  are  necessary  for  development  of  the 
offspring. 

It  turns  out  that  a  small  number  of  genes  are 
expressed  differently  if  they  are  inherited  from 
one  or  the  other  parent.  In  a  particular  example 
that  we  created,  a  transgene  is  only  expressed  if  it 
is  inherited  from  the  father.  The  very  same  gene 
inherited  from  the  mother  is  silent.  We  have  now 
correlated  this  so-called  "parentally  imprinted" 
expression  with  a  chemical  modification  of  the 
gene  whereby  it  is  heavily  altered  (but  not  ex- 
pressed) if  inherited  from  the  mother  or  not  al- 
tered (but  expressed)  if  inherited  from  the  fa- 
ther. During  the  past  year  we  have  worked  out  the 
rules  that  govern  the  modification  of  this  gene 
during  embryogenesis.  Our  most  recent  work  is 
directed  toward  identifying  the  encoded  signal 
that  evokes  this  parental  effect. 


248 


From  Molecular  Biology  to  Therapy 
of  Human  Disease 


Fred  D.  Ledley,  M.D. — Assistant  Investigator 

Dr.  Ledley  is  also  Associate  Professor  of  Cell  Biology  and  Pediatrics  at  Baylor  College  of  Medicine.  He 
received  his  B.S.  degree  in  physical  sciences  from  the  University  of  Maryland,  College  Park,  and  his  M.D. 
degree  from  Georgetown  University.  He  trained  in  pediatrics  and  medical  genetics  at  the  Children 's 
Hospital,  Boston,  and  Harvard  Medical  School.  His  postdoctoral  research  was  conducted  with  David 
Baltimore  at  the  Massachusetts  Institute  of  Technology  and  with  Savio  Woo  at  Baylor  College  of  Medicine. 


SINCE  its  inception  this  laboratory  has  focused 
on  genetic  deficiency  of  the  enzyme  methyl- 
malonyl  CoA  mutase  (MCM)  as  a  model  for  molec- 
ular genetic  investigations  of  human  disease.  We 
have  cloned  and  sequenced  human  and  murine 
cDNAs  for  MCM,  have  mapped  and  cloned  the 
genomic  locus  in  mice  and  humans,  and  have  de- 
scribed a  series  of  mutations  causing  interesting 
and  informative  phenotypes  of  this  disorder. 

While  continuing  these  studies,  we  have  begun 
to  address  the  technologies  that  will  be  essential 
in  moving  toward  somatic  gene  therapy  for  MCM 
deficiency.  We  have  focused  our  efforts  on  un- 
derstanding the  consequences  of  metabolic  engi- 
neering of  MCM  activity  in  human  cells,  on  devel- 
oping methods  for  efficient  manipulation  and 
transduction  of  human  hepatocytes,  and  on  as- 
sessing methods  for  hepatocellular  transplanta- 
tion in  large  animals  and  humans. 

Retroviral-mediated  Correction 
of  MCM  in  Vitro 

We  constructed  a  high-titer,  amphotropic  re- 
troviral vector  containing  the  full-length  normal 
human  MCM  gene.  MCM-deficient  fibroblasts 
were  transduced  with  this  vector,  and  the  effi- 
ciency of  transduction  was  assessed  by  semiquan- 
titative identification  of  the  recombinant  pro- 
virus.  The  restoration  of  MCM  activity  was 
measured  by  the  ability  of  cells  to  metabolize 
['^C]-propionate.  The  metabolic  capacity  of  fibro- 
blasts was  restored  to  normal  levels  by  transduc- 
tion, even  though  only  1 0-30  percent  of  the  cells 
were  effectively  transduced.  No  further  increase 
in  metabolic  capacity  was  evident  when  MCM 
apoenzyme  activity  was  increased  above  normal 
levels  by  varying  transduction  or  transfection 
conditions.  Transduction  of  normal  fibroblasts  or 
hepatoma  cells  increased  MCM  apoenzyme  activ- 
ity, but  not  the  capacity  for  propionate  metabo- 
lism, suggesting  that  other  steps  on  these  path- 
ways are  normally  limiting. 

Metabolic  cooperation  between  cells  was 
shown  to  increase  the  flux  of  propionate  through 
subpopulations  of  metabolically  competent 
cells.  The  capacity  for  propionate  metabolism  of 


hepatic  cells  was  also  found  to  be  more  than  10- 
fold  greater  than  the  maximal  capacity  of  fibro- 
blasts, suggesting  that  the  phenotypic  impact  of 
metabolic  engineering  in  hepatic  cells  would  be 
greater  than  in  other  potential  targets  for  gene 
therapy. 

Transduction  of  Primary  Human 
Hepatocytes 

One  strategy  for  targeting  gene  therapy  to  the 
liver  involves  harvesting  and  culturing  hepato- 
cytes from  patients,  transducing  these  cells  ex 
vivo  with  retroviral  vectors,  and  returning  them 
to  patients  by  hepatocellular  transplantation.  We 
demonstrated  the  feasibility  of  hepatocellular 
harvest  from  human  liver  segments  preserved  in 
UW  (University  of  Wisconsin)  solution,  demon- 
strated proliferation  of  hepatocytes  and  preserva- 
tion of  differentiated  hepatocellular  functions  in 
cells  cultured  in  hormonally  defined  media,  and 
established  methods  for  transducing  human  cells 
with  recombinant  retroviral  vectors. 

The  efficiency  of  transduction  of  human  cells 
with  amphotropic  vectors  (1-10  percent)  was 
significantly  less  than  that  observed  in  various  an- 
imal models.  Vectors  containing  MCM  were 
shown  to  constitute  expression  of  recombinant 
mRNA  from  the  5'  long  terminal  repeat  (5'LTR) 
promoter.  Higher-efficiency  transduction  was 
obtained  using  retroviral  vectors  with  xenotropic 
or  gibbon  ape  enf  determinants.  Ongoing  studies 
are  aimed  at  optimizing  conditions  for  hepato- 
cyte  cultivation  and  transduction. 

Hepatocellular  Transplantation 

One  of  the  major  factors  limiting  the  applica- 
tion of  ex  vivo  strategies  for  hepatic  gene  therapy 
is  that  hepatocellular  transplantation  has  never 
been  attempted  in  clinical  practice.  We  have  as- 
sessed the  feasibility  in  large  animal  models,  us- 
ing a  novel  method  for  tracking  transplanted 
cells.  The  cells  are  stained  with  a  fluorescent  dye, 
Dil.  This  dye  is  not  metabolized  or  exchanged 
between  cells,  and  engraftment  can  be  identified 
in  recipient  tissues  by  fluorescent  microscopy  or 
flow  cytometry. 


249 


From  Molecular  Biology  to  Therapy  of  Human  Disease 


Studies  in  mice  and  rats  confirm  previous  re- 
ports that  the  efficiency  of  engraftment  is  rela- 
tively poor  (less  than  1  percent) .  We  have  stud- 
ied autologous  hepatocellular  transplantation  in 
baboons  as  a  model  for  human  anatomy  and  surgi- 
cal methods.  Transplantation  resulted  in  consti- 
tution of  approximately  5  percent  of  the  host 
liver  from  the  hepatocellular  graft  without 
,  complications. 

We  have  explored  the  possibility  of  hepatocel- 
lular transplantation  and  gene  therapy  in  utero. 
Heterologous  hepatocellular  transplantation  was 
performed  in  fetal  lambs  at  80-85  percent  gesta- 
tion by  infusion  of  cells  into  the  umbilical  vein. 
Flow  cytometry  of  hepatocytes  recovered  from 
transplanted  animals  demonstrated  that  1.5-4.5 
percent  of  hepatocytes  originated  from  the  graft. 

Transient  Gene  Therapy  by  in  Vivo 
Gene  Delivery 

Other  laboratories  have  reported  that  DNA 
coupled  to  asialoglycoproteins  can  be  targeted  to 
the  liver  in  vivo,  but  that  expression  of  these 
genes  is  transient  (days) .  We  are  interested  in  the 
possibility  that  transient  expression  might  be  the 
most  efficacious  approach  to  treating  MCM  defi- 


ciency, which  is  characterized  by  periods  of  rela- 
tive stability  punctuated  by  life-threatening  epi- 
sodes of  acidosis.  We  have  demonstrated  in  vivo 
delivery  of  MCM  to  the  livers  of  experimental 
mice  using  asialoorosomucoid-polylysine-DNA 
conjugates  and  are  currently  studying  the  conse- 
quences of  this  gene  delivery.  In  particular,  we 
are  concerned  with  demonstrating  that  DNA  is 
completely  eliminated  after  the  period  of  tran- 
sient expression,  since  damage  to  chromoso- 
mal DNA  from  inadvertent  integrations  is  one 
of  the  major  potential  risks.  Vectors  will  be  con- 
structed with  suicide  sequences  to  eliminate  in- 
tegrated DNA. 

Future  Directions 

Successful  gene  therapy  requires  attention  not 
only  to  methods  for  gene  delivery  and  gene  ex- 
pression, but  consideration  of  the  metabolic,  cel- 
lular, clinical,  and  social  consequences  of  ge- 
netic manipulation.  We  are  attempting  to 
establish  a  broad  base  of  expertise  and  experi- 
ence, using  MCM  deficiency  as  a  model.  This 
should  enable  rational  development  of  clinical 
trials  involving  somatic  gene  therapy  in  the 
future. 


250 


Molecular  Biology  of  Hormone  and  Drug  Receptors 
in  Health  and  Disease 


Robert  J.  Lefkowitz,  M.D. — Investigator 

Dr.  Lefltowitz  is  also  James  B.  Duke  Professor  of  Medicine  and  of  Biochemistry  at  the  Duke  University 
Medical  Center.  He  received  his  B.A.  ( chemistry)  and  M.D.  degrees  from  Columbia  University  and  clinical 
and  research  training  at  Columbia- Presbyterian  Medical  Center  in  New  York,  NIH,  and  Massachusetts 
General  Hospital.  As  a  molecular  pharmacologist  he  has  focused  on  the  molecular  structure  and 
regulatory  mechanisms  controlling  the  function  of  the  adrenergic  receptors  that  mediate  the  actions  of 
catecholamines.  Dr.  Lefkowitz  has  received  numerous  awards,  including  the  Gairdner  Foundation 
International  Award.  He  is  a  member  of  the  National  Academy  of  Sciences. 


OUR  research  program  is  concerned  with  the 
molecular  properties  and  regulatory  mecha- 
nisms that  control  the  function  of  plasma  mem- 
brane receptors  for  hormones  and  drugs  under 
normal  and  pathological  circumstances.  Recep- 
tors are  the  cellular  macromolecules  with  which 
biologically  active  substances  (e.g.,  hormones, 
drugs,  neurotransmitters,  growth  factors,  viruses, 
lipoproteins)  initially  interact.  Such  receptors 
perform  two  essential  functions:  1)  They  receive 
or  bind  these  biologically  active  substances  at  the 
surface  of  the  cell.  2)  They  transmit  the  sub- 
stance's message  into  the  cell,  thus  influencing 
its  metabolic  activity  and  function. 

We  have  utilized  the  receptors  for  epinephrine 
(adrenaline)  and  related  compounds,  which  are 
generally  termed  adrenergic  receptors,  as  models 
for  the  study  of  receptors.  Such  receptors  are 
found  throughout  the  brain,  heart,  smooth  mus- 
cle cells,  and  most  other  cells  of  the  human  organ- 
ism. There  are  at  least  nine  distinct  subtypes  of 
adrenergic  receptors  (aj,  a-^,  182,  etc.).  These 
receptors  interact  not  only  with  endogenous  epi- 
nephrine and  norepinephrine  but  with  a  variety 
of  clinically  important  drugs. 

We  have  studied  these  particular  receptors  for 
several  reasons:  they  are  more  widely  distributed 
than  perhaps  any  other  type  of  receptor,  they  are 
exemplary  of  each  of  the  major  biochemical 
pathways  by  which  receptors  are  known  to  signal 
to  the  interior  of  the  cell,  and  they  are  clinically 
and  therapeutically  significant.  These  receptors 
mediate  physiological  responses  as  diverse  as 
changes  in  blood  pressure,  changes  in  heart  rate 
and  contractility,  and  alterations  in  the  metabo- 
lism of  glucose.  Drugs  that  interact  with  these 
receptors  are  among  the  most  effective  agents 
used  to  treat  various  forms  of  heart  disease,  hy- 
pertension, asthma,  shock,  and  depression.  Re- 
search on  these  exemplary  receptors  has  impor- 
tant implications  for  understanding  hormone  and 
drug  receptor  interactions  generally  and  the 
mechanisms  by  which  they  are  regulated. 

Our  research  is  focused  on  several  intimately 
related  goals.  First,  we  wish  to  understand,  in  de- 


tailed molecular  terms,  the  biochemical  nature 
of  the  receptors.  This  is  being  accomplished  by 
the  application  of  recombinant  DNA  or  gene- 
cloning  techniques.  These  permit  isolation  of  the 
genes  for  the  receptors,  which  in  turn  permits 
their  complete  primary  amino  acid  sequences  to 
be  deduced. 

We  have  isolated  the  genes  for  all  of  the  known 
adrenergic  receptors,  as  well  as  a  number  of 
closely  related  receptors,  and  determined  their 
complete  sequences.  Remarkably,  the  structures 
of  these  receptors  are  similar  to  each  other  and  to 
that  of  the  visual  light  receptor  rhodopsin.  This 
insight  is  helping  to  clarify  the  general  mecha- 
nisms by  which  signals  as  divergent  as  a  photon  of 
light  and  a  drug  molecule  like  epinephrine  elicit 
their  characteristic  biochemical  and  physiologi- 
cal responses.  By  varying  the  structure  of  the  gene 
and  hence  the  receptor  protein  that  it  produces, 
we  can  define  which  structural  features  of  the 
receptor  molecule  determine  its  characteristic 
functions. 

An  unexpected  result  of  the  cloning  of  the 
known  types  of  adrenergic  receptor  genes  was 
the  discovery  of  the  genes  for  several  novel  sub- 
types of  adrenergic  receptors  not  previously 
known  to  exist.  Knowledge  of  these  new  recep- 
tors opens  the  way  for  the  development  of  new 
drugs  with  greater  selectivity  and  fewer  side  ef- 
fects. Such  drugs  might  conceivably  have  appli- 
cations in  the  treatment  of  such  disparate  diseases 
as  hypertension,  asthma,  diabetes,  and  prostat- 
ism, or  even  as  novel  anesthetic  agents. 

A  second  current  research  goal  is  to  elucidate 
the  patterns  of  receptor  regulation.  One  of  the 
most  important  insights  to  come  from  our  studies 
of  receptors  is  that  their  properties  are  not  fixed. 
Rather,  the  properties  of  the  receptors  are  in- 
fluenced by  the  hormones  and  drugs  with  which 
they  interact,  as  well  as  by  a  variety  of  disease 
states. 

There  are  important  clinical  implications  of 
the  ever-changing  nature  of  the  receptors.  For  ex- 
ample, this  provides  a  basis  for  beginning  to  un- 
derstand the  phenomenon  of  drug  tolerance  or 
desensitization,  the  diminishing  eff^ect  of  drugs 


251 


Molecular  Biology  of  Hormone  and  Drug  Receptors  in  Health  and  Disease 


over  time.  This  phenomenon  markedly  compro- 
mises the  therapeutic  efficacy  of  epinephrine  and 
many  other  drugs.  When  drugs  like  epinephrine 
combine  with  their  receptors,  they  not  only  stim- 
ulate them  but  also  produce  changes  that  impair 
their  function,  thus  leading  to  desensitization. 
These  changes  involve  an  actual  loss  of  receptors 
from  the  cell  surface  (they  move  inside  the  cell 
where  they  cannot  function)  and  a  chemical 
change  of  those  receptors  remaining  at  the  cell 
surface  so  that  they  function  less  effectively.  With 
fewer  functioning  receptors  present  at  their  sur- 
face, cells  are  less  able  to  respond  to  drugs  or 
hormones. 

Our  recent  research  is  increasing  our  under- 
standing, in  molecular  terms,  of  how  the  recep- 
tors become  functionally  desensitized.  We  have 
discovered  two  new  proteins  that  function  to  de- 
sensitize the  receptors.  The  first  is  an  enzyme,  the 
jS-adrenergic  receptor  kinase  (/3ARK) ,  which  mod- 
ifies the  structure  of  the  receptors  by  introducing 


a  phosphate  group  when  the  receptors  are  stimu- 
lated. The  second  is  the  protein  /3-arrestin,  which 
binds  to  the  phosphorylated  receptors  and  pre- 
vents them  from  acting.  Both  proteins  are  widely 
distributed  at  synapses  throughout  the  central 
nervous  system,  suggesting  their  actions  are  not 
limited  to  the  j8-adrenergic  receptors. 

The  implications  of  such  fundamental  research 
on  receptors  for  clinical  medicine  are  profound. 
Elucidation  of  the  detailed  structure  of  the  recep- 
tors will  allow  the  precise  design  of  drugs  that  are 
more  potent  and  specific.  Unraveling  of  the  mo- 
lecular basis  of  desensitization  will  allow  the  de- 
velopment of  strategies  for  interdicting  the  basic 
reactions  that  lead  to  loss  of  hormone  and  drug 
effect.  An  example  is  the  design  of  specific  en- 
zyme inhibitors  for  ^ARK  that  could  block  the 
reactions  leading  to  desensitization.  Successful 
conclusion  of  such  research  may  lead  to  methods 
for  greatly  prolonging  and  augmenting  the  thera- 
peutic actions  of  diverse  types  of  drugs. 


252 


Axis  Formation  and  Germline  Determination 
in  Drosophila 


Ruth  Lehntann,  Ph.D. — Assistant  Investigator 

Dr.  Lehmann  is  also  Associate  Member  of  the  Whitehead  Institute  of  Biomedical  Research,  Associate 
Professor  of  Biology  at  the  Massachusetts  Institute  of  Technology,  and  Assistant  Molecular  Biologist  at 
Massachusetts  General  Hospital,  Boston.  She  received  her  M.  Sc.  degree  from  the  University  of  Freiburg, 
where  she  worked  with  Jose  Campos-Ortega  on  early  neurogenesis  in  Drosophila.  She  received  her  Ph.D. 
degree  from  the  University  of  Tubingen,  where  she  worked  with  Christiane  Ntisslein-Volbard  at  the  Max 
Planck  Institute  for  Developmental  Biology  on  the  genetics  of  pattern  formation  in  the  Drosophila  embryo. 
After  postdoctoral  training  in  Tiibtngen  and  at  the  MRC  in  Cambridge  with  Michael  Wilcox,  she  joined 


the  Whitehead  Institute. 


HOW  does  a  developing  embryo  know  where 
to  form  a  head  and  where  to  put  a  tail?  In 
Drosophila,  basic  information  about  the  "coordi- 
nates" of  the  embryo  is  supplied  to  the  egg  cell 
by  the  mother.  Mutations  in  maternal  genes  have 
led  to  the  identification  of  a  small  number  of 
genes  that  are  required  for  the  establishment  of 
anterior-posterior  (head-to-tail)  polarity.  In  these 
mutants  the  lack  of  a  particular  gene  product  in 
the  mother  is  lethal  to  its  progeny. 

Three  signals  are  required  for  the  establish- 
ment of  pattern  along  the  anterior-posterior  axis. 
The  anterior  signal  controls  the  development  of 
head  and  thorax,  the  posterior  signal  determines 
the  abdominal  region,  and  the  terminal  signal  is 
required  at  both  ends  of  the  embryo.  The  anterior 
and  posterior  signals  are  localized  to  the  respec- 
tive poles  (see  figure) . 

We  have  concentrated  on  the  dissection  of  the 
pathway  leading  to  normal  development  of  the 
posterior  region.  Nine  maternal  genes,  called  the 
posterior  group,  set  the  basic  posterior  pattern. 
These  genes  share  the  abdominal  phenotype:  ho- 
mozygous mutant  females  produce  offspring  that 
lack  abdominal  segmentation.  One  gene,  nanos, 
encodes  the  localized  signal  required  for  the  de- 
velopment of  the  abdomen.  Another  gene,  pumi- 
lio,  regulates  the  activity  of  NANOS  protein, 
while  the  remaining  seven  genes — oskar,  vasa, 
tudor,  valois,  staufen,  cappuccino,  and  spire — 
are  required  for  the  localization  of  nanos  RNA  to 
the  posterior  pole. 

These  seven  genes  are  also  required  for  germ 
cell  formation.  Embryos  from  mutant  females 
lack  the  specialized  posterior  pole  plasm  that 
normally  contains  the  polar  granules.  These  em- 
bryos therefore  fail  to  produce  pole  cells,  the 
Drosophila  germ  cell  precursors.  Our  molecular 
analysis  of  the  posterior  group  is  aimed  at  under- 
standing how  nanos  and  other  RNA  species  be- 
come localized  to  the  posterior  pole  and  how  the 
pole  plasm,  composed  of  RNA  and  protein,  is 
assembled. 


Synthesis  and  Function 
of  the  Abdominal  Signal 

Through  genetic  experiments  as  well  as  cyto- 
plasmic transfers  between  wild-type  and  mutant 
embryos,  we  concluded  that  the  specialized  cyto- 
plasm at  the  posterior  pole  is  the  source  of  an 
abdominal  signal,  and  that  embryos  derived  from 
females  with  mutant  posterior  group  genes  lack 
this  signal  in  the  abdominal  region.  The  signal  is 
encoded  by  nanos.  We  have  isolated  and  cloned 
this  gene  and  have  shown  that  nanos  mRNA  is 
transcribed  during  oogenesis  and  becomes  local- 
ized to  the  posterior  pole  plasm  of  the  mature 
oocyte.  The  role  of  nanos  as  a  signal  for  posterior 
pattern  formation  is  demonstrated  by  the  finding 
that  injection  of  synthetic  nanos  mRNA  into  the 
anterior  pole  of  early  embryos  leads  to  suppres- 
sion of  head  formation  and  induction  of  a  second 
abdomen  in  mirror  image  to  the  normal  "poste- 
rior" abdomen. 

Seven  of  the  posterior  group  genes  affect  the 
localization  of  nanos  mRNA  to  the  posterior 
pole.  Since  these  seven  mutants  also  lack  pole 
plasm,  we  can  conclude  that  their  abdominal  seg- 
mentation defect  is  a  consequence  of  a  lack  of 
localized  nanos  mRNA.  We  have  identified  se- 
quences within  the  nanos  mRNA  required  for  its 
localization.  In  future  experiments  we  will  deter- 
mine which  of  the  posterior  group  genes  are  di- 
rectly involved  in  tethering  nanos  mRNA  to  the 
posterior  pole  plasm. 

Regulation  of  Posterior  Activity 

Several  lines  of  evidence  indicate  that  the  pu- 
milio  gene  is  required  to  regulate  the  activity  of 
NANOS  protein.  After  fertilization  of  the  egg, 
NANOS  is  translated  from  the  localized  mRNA  and 
emanates  anteriorly  toward  the  abdominal  region 
to  form  a  gradient  of  protein  concentration.  Cyto- 
plasmic transplantation  experiments  and  analysis 
of  the  distribution  of  nanos  RNA  and  NANOS  pro- 
tein in  pumilio  mutant  embryos  show  that  pumi- 


253 


Axis  Formation  and  Germline  Determination  in  Drosophila 


Ho  does  not  interfere  with  nanos  RNA  localiza- 
tion or  NANOS  protein  distribution,  synthesis, 
and  stability.  We  conclude  that  pumilio  is  re- 
quired for  optimal  NANOS  activity  in  regions  of 
the  embryo  where  NANOS  protein  concentrations 
are  low. 

We  have  cloned  the  pumilio  gene  and  have 
shown  that  pumilio  mRNA  is  localized  to  the  pos- 
terior pole  in  early  embryos.  This  observation  is 
consistent  with  a  possible  interaction  between 
the  pumilio  and  nanos  gene  products.  Localiza- 
tion of  pumilio  mRNA,  like  that  of  nanos  mRNA, 
is  disrupted  in  mutants  that  lack  the  specialized 
posterior  pole  plasm  (see  below) .  It  is  thus  possi- 
ble that  the  same  mechanism  of  RNA  localization 
acts  on  both  mRNAs. 

Assembly  of  the  Pole  Plasm 

The  remaining  seven  genes  in  the  posterior 
group  are  associated  with  pole  plasm  defects.  We 
are  studying  these  genes  from  two  perspectives. 
One  is  their  role  in  pattern  formation  and  body 
segmentation  (see  above) ;  the  other,  their  role  in 
pole  plasm  assembly  and  germ  cell  formation 
(see  below) . 

During  the  first  hour  of  embryogenesis,  the 
pole  plasm  appears  as  a  distinct  clear  zone  at  the 
posterior  end  of  the  newly  fertilized  egg.  Closer 
examination  reveals  that  this  zone  contains  spe- 
cialized cytoplasm  packed  with  mitochondria 
and  numerous  donut-shaped  organelles,  called 
polar  granules,  that  do  not  occur  anywhere  else 
in  the  embryo.  The  zone  of  specialized  cytoplasm 
coincides  with  the  site  of  germ  cell  formation, 
and  polar  granules  may  therefore  contain  factors 
that  control  germ  cell  fate.  Polar  granules  were 
first  recognized  in  Drosophila  embryos  30  years 
ago,  but  until  recently  little  was  known  about 
their  biochemical  structure  or  function.  Now  the 
link  between  defects  in  abdomen  formation  and 


the  absence  of  germ  cells  has  provided  a  new  ave- 
nue for  research. 

We  have  begun  extensive  studies  of  the  pole 
plasm  in  mutant  and  normal  embryos,  using 
probes  designed  to  recognize  mRNA  and  proteins 
derived  from  the  posterior  group  genes.  We  sus- 
pect that  oskar,  one  of  the  genes  required  for 
abdominal  and  germline  development  in  the  Dro- 
sophila embryo,  is  an  essential  component  of  the 
pole  plasm.  We  have  isolated  this  gene.  In  situ 
hybridization  to  whole-mount  embryos  reveals 
that  oskar  mRNA  distribution  is  restricted  to  the 
posterior  pole  of  early  embryos.  Genetic  studies 
suggest  that  pole  plasm  formation  occurs  in  a  se- 
ries of  steps,  with  each  step  dependent  on  the 
previous  one,  and  that  oskar  is  required  early  in 
the  assembly  pathway.  Future  experiments  will 
address  questions  about  the  role  of  oskar  mRNA 
and  protein  for  pole  plasm  formation  and  for  lo- 
calization of  the  abdominal  signal. 

We  are  optimistic  that  new  information  about 
the  assembly  and  composition  of  the  pole  plasm 
and  polar  granules  will  lead  to  better  understand- 
ing of  their  functions  in  the  early  embryo.  Their 
primary  role  may  be  to  protect  or  sequester  infor- 
mation required  by  the  future  germ  cells.  In  addi- 
tion, however,  they  could  provide  a  convenient 
anchor  for  substances  like  the  nanos  mRNA  that 
must  be  retained  in  the  posterior  region.  Struc- 
tures similar  to  the  polar  granules  in  Drosophila 
have  been  observed  in  association  with  germ 
plasm  in  many  different  invertebrate  and  verte- 
brate animals.  Thus  the  Drosophila  model  could 
reveal  basic  concepts  underlying  the  establish- 
ment of  germline  tissues  in  all  species. 

The  National  Institutes  of  Health  provided  fi- 
nancial support  for  two  projects  that  are  summa- 
rized here.  These  projects  focus  on  the  molecular 
and  functional  characterization  of  the  nanos  and 
oskar  genes. 

Localization  of  positional  signals  in  the  Dro- 
sophila embryo.  In  situ  hybridization  experi- 
ment, using  biotinylated  cDNA  probes,  local- 
izes RNA  of  the  anterior  signal  bicoid  and  of  the 
posterior  signal  nanos  respectively  to  the  ante- 
rior and  posterior  poles  of  an  early  ( stage  2 ) 
embryo.  Hybridization  to  nanos  at  the  posterior 
also  demarcates  the  position  of  the  germ  plasm. 

Research  by  Laura  Dickinson.  Photograph  by 
Ruth  Lehmann. 


254 


Regulation  of  Gene  Expression  During  Cellular 
Differentiation  and  Activation 


Jeffrey  M.  Leiden,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Leiden  is  also  Associate  Professor  of  Internal  Medicine  at  the  University  of  Michigan  Medical  School.  He 
received  his  M.D.  and  Ph.D.  degrees  from  the  University  of  Chicago.  His  residency  training  was  at  Brigham 
and  Women's  Hospital,  Boston,  and  his  postdoctoral  fellowship  was  in  the  laboratory  of  Jack  Strominger 
at  Harvard  University. 


THE  processes  of  cellular  differentiation  and 
activation  are  accompanied  by  complex  and 
precisely  orchestrated  changes  in  gene  expres- 
sion. Abnormalities  in  the  patterns  of  expression 
of  these  specific  genes  may  be  involved  in  the 
etiology  of  a  number  of  pathologic  states,  includ- 
ing autoimmune  disease  and  malignancy.  My  labo- 
ratory is  studying  gene  regulation  during  T  lym- 
phocyte and  muscle  cell  differentiation  in  order 
to  increase  understanding  of  the  molecular  mech- 
anisms that  regulate  gene  expression  during  both 
normal  and  pathologic  development. 

Regulation  of  the  T  Cell  Receptor  Gene 
During  T  Cell  Development 

Human  T  lymphocytes  recognize  foreign  anti- 
gens, such  as  virus- infected  cells  and  tumor  cells, 
via  a  specific  cell-surface  molecule,  the  T  cell 
receptor  (TCR) .  T  cells  can  be  divided  into  two 
subsets  based  on  their  expression  of  two  distinct 
types  of  antigen  receptor.  The  majority  of  T  cells 
that  circulate  in  peripheral  blood,  including  all 
helper  and  cytotoxic  T  cells,  express  the  a//3 
heterodimer  receptor,  while  a  small  but  distinct 
T  cell  subset  of  unknown  function  expresses  the 
7/6  receptor.  The  a//3  and  7/6  T  cells  appear  to 
develop  as  separate  lineages  during  thymic 
ontogeny. 

During  the  last  several  years,  my  laboratory  has 
been  interested  in  identifying  and  characterizing 
the  molecular  mechanisms  that  regulate  the  ex- 
pression of  these  different  TCR  genes  during  T 
cell  development.  In  an  initial  set  of  studies,  we 
identified  the  transcriptional  enhancer  elements 
that  control  the  expression  of  the  TCR  a  and  /5 
genes.  These  two  enhancers  were  shown  to  be 
required  for  the  expression  of  the  a  and  genes 
and  to  function  equally  well  in  both  mouse  and 
human  cells.  The  identification  and  localization 
of  the  human  TCR  enhancers  led  us  to  propose 
that  certain  T  cell  tumors  that  had  previously 
been  shown  to  contain  chromosomal  trans- 
locations into  the  human  TCR  a  and  loci 
might  be  caused  by  the  apposition  of  the 
TCR  gene  enhancers  with  translocated  human 
proto-oncogenes. 


Our  more  recent  studies  have  focused  on  pre- 
cisely identifying  and  characterizing  the  en- 
hancer DNA  sequences  and  the  nuclear  proteins 
they  bind  that  are  responsible  for  controlling 
TCR  a  and  /3  gene  expression.  These  experiments 
have  demonstrated  that  both  enhancers  contain 
4-5  different  binding  sites  for  nuclear  proteins. 
At  least  two  of  these  sites  in  each  enhancer  are 
absolutely  required  for  enhancer  activity.  Several 
of  the  nuclear  protein-binding  sites  in  each  en- 
hancer correspond  to  previously  defined  en- 
hancer motifs,  while  others  represent  novel  se- 
quence elements.  At  least  one  site  in  each 
enhancer  was  shown  to  bind  T  cell-specific  nu- 
clear proteins. 

Both  enhancers  were  shown  to  contain  a  bind- 
ing site  that  was  identical  to  the  previously  de- 
scribed cAMP  response  element  (CRE).  To  learn 
more  about  the  function  of  this  element  in  regu- 
lating T  cell  gene  expression,  we  have  cloned 
two  novel  CRE-binding  proteins,  CREB-2  and 
CREB-3,  that  specifically  bind  to  the  TCR  a  CRE 
and  to  CREs  from  several  other  eukaryotic  pro- 
moters. Both  of  these  new  CREB  proteins  contain 
similar  basic  DNA-binding  domains  and  a  leucine 
zipper  region  that  allows  them  to  form  dimeric 
complexes  both  with  themselves  and,  poten- 
tially, with  other  members  of  the  CREB  protein 
family. 

During  the  past  year,  we  have  shown  that  ets-\, 
a.  previously  described  human  proto-oncogene,  is 
actually  a  DNA-binding  protein  that  specifically 
recognizes  one  of  the  nuclear  protein-binding 
sites  in  the  TCR  a  enhancer.  These  studies  have 
helped  to  define  the  Ets  proteins  as  transcrip- 
tional regulators  that  may  play  an  important  role 
in  controlling  T  cell  gene  expression.  More  re- 
cently, we  have  cloned  several  new  members  of 
the  Ets  family  of  transcription  factors.  One  of 
these,  which  we  call  Elf- 1 ,  regulates  a  set  of  genes 
during  the  process  of  T  cell  activation.  In  addi- 
tion, it  appears  to  play  an  important  role  in  regu- 
lating the  expression  of  HIV-2,  one  of  the  AIDS 
viruses,  in  T  cells.  Finally,  in  collaboration 
with  Stuart  Orkin  (HHMI,  Children's  Hospital, 
Harvard  Medical  School),  we  have  identified  a 


255 


Regulation  of  Gene  Expression  During  Cellular  Differentiation  and  Activation 


novel  T  cell-specific  transcription  factor  called 
hGATA-3  that  binds  to  a  third  nuclear  protein- 
binding  site  within  the  TCR  a  enhancer.  Ongoing 
studies  are  designed  to  determine  the  role  of  each 
of  these  transcription  factors  in  T  cell  tumors,  as 
well  as  in  normal  T  cell  development  and 
activation. 

Genetically  Engineered  Myoblasts 

as  a  Recombinant  Protein  Delivery  System 

A  variety  of  acquired  and  inherited  diseases  are 
currently  treated  by  repeated  intravenous  or  sub- 
cutaneous infusions  of  recombinant  or  purified 
proteins.  In  addition  to  hemophilia  A,  which  is 
treated  with  intravenous  infusions  of  human  fac- 
tor VIII,  these  include  diabetes  mellitus,  treated 
with  subcutaneous  or  intravenous  injections  of 
insulin,  and  pituitary  dwarfism,  treated  with  sub- 
cutaneous injections  of  growth  hormone.  The  de- 
velopment of  cellular  transplantation  systems 
that  could  stably  produce  and  deliver  such  recom- 
binant proteins  into  the  systemic  circulation 
would  represent  an  important  advance  in  our  abil- 
ity to  treat  such  diseases. 

The  ideal  recombinant  protein  delivery  system 
would  utilize  a  cell  that  could  be  easily  isolated 
from  the  recipient,  grown  and  transduced  with 
recombinant  genes  in  vitro,  and  conveniently 
reimplanted  into  the  host  organism.  Such  a  cell 
should  produce  large  amounts  of  secreted  recom- 
binant protein,  and  following  secretion,  this  pro- 
tein should  gain  access  to  the  circulation.  Finally, 
these  implanted,  genetically  engineered  cells 
should  survive  for  long  periods  and  continue  to 
secrete  the  transduced  protein  product  without 
themselves  interfering  with  the  function  of  the 
tissue  into  which  they  were  implanted.  Several 
different  cellular  systems  have  been  used  to  pro- 
duce recombinant  proteins  in  vivo.  These  in- 
clude keratinocytes,  skin  fibroblasts,  hepato- 
cytes,  lymphocytes,  and  bone  marrow.  Although 
some  of  these  systems  have  yielded  detectable  lev- 
els of  circulating  proteins  briefly,  stable  physio- 
logical levels  of  circulating  proteins  have  proved 
difficult  to  produce  in  normal  animals. 

Genetically  engineered  myoblasts  represent  a 


potentially  useful  system  for  the  in  vivo  delivery 
of  recombinant  proteins  into  the  circulation. 
Myoblasts  can  be  readily  isolated  from  a  muscle 
biopsy  and  expanded  in  vitro  to  very  large  cell 
numbers.  Cultured  myoblasts  can  be  transfected 
in  vitro  and  will  synthesize  large  amounts  of  re- 
combinant proteins.  Most  importantly,  previous 
studies  have  demonstrated  that  cultured  myo- 
blasts can  be  injected  intramuscularly  and  will 
survive  and  fuse  into  adjacent  normal  muscle 
fibers  at  the  site  of  injection.  Finally,  skeletal 
muscle  is  a  highly  vascular  tissue.  Thus  proteins 
secreted  from  myoblasts  should  readily  enter  the 
circulation. 

We  have  recently  explored  the  feasibility  of  us- 
ing genetically  engineered  myoblasts  as  a  recom- 
binant protein  delivery  system.  To  this  end, 
stable  transfectants  of  the  murine  C2C12  myo- 
blast cell  line  were  produced  that  synthesize  and 
secrete  high  levels  of  human  growth  hormone 
(hGH)  in  vitro.  These  stably  transfected  myo- 
blasts were  injected  intramuscularly  into  normal 
syngeneic  C3H  mice,  and  serum  and  muscle  lev- 
els of  hGH  were  measured  5  days  to  3  weeks  after 
injection. 

Mice  injected  with  the  growth  hormone- 
transfected  myoblasts  produced  significant  and 
physiological  levels  of  hGH  both  locally  in  mus- 
cle and  in  serum,  as  compared  with  control  mice 
injected  with  nontransfected  myoblasts.  Human 
growth  hormone  levels  in  both  muscle  and  serum 
were  stable  for  at  least  three  months  following 
injection  and  exceeded  those  measured  in 
serum  from  normal  human  volunteers.  Histologi- 
cal examination  of  muscles  injected  with  /3- 
galactosidase-expressing  C2C12  myoblasts  dem- 
onstrated that  many  of  the  injected  cells  had 
fused  to  form  multinucleated  myotubes.  Thus 
these  studies  demonstrated  that  genetically  engi- 
neered myoblasts  represent  a  novel  and  powerful 
system  for  the  stable  delivery  of  recombinant 
proteins  into  the  circulation. 

Dr.  Leiden  is  now  Professor  of  Medicine  and 
Pathology  at  the  University  of  Chicago. 


256 


Chemical  Communication 


Michael  R.  Lemer,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Lemer  is  also  Associate  Professor  in  the  Departments  of  Internal  Medicine  and  of  Pharmacology  and 
the  Child  Study  Center  at  Yale  University  School  of  Medicine.  He  obtained  his  B.A.  degree  in  chemistry 
from  the  University  of  Pennsylvania  and  his  M.D.  and  Ph.D.  degrees  from  Yale.  His  doctoral  research,  with 
Joan  Steitz,  was  on  small  nuclear  ribonucleoproteins  (snRNPs).  He  interned  in  internal  medicine  at 
Barnes  Hospital,  St.  Louis,  and  did  postdoctoral  research  in  neurobiology  at  Washington  University  with 
Gerald  Fischbach  before  returning  to  Yale.  His  honors  include  the  George  Herbert  Hitchings  Award  for 
innovative  methods  in  drug  design. 


INTRASPECIES  communication  via  specific 
chemical  messengers  is  widely  employed 
throughout  the  animal  kingdom.  Among  the  com- 
mon uses  of  chemical  communication  are  mark- 
ing of  territory,  signaling  danger,  and  indicating 
sources  of  food.  A  particularly  striking  example  is 
the  use  of  sex  pheromones.  Here,  animals  release 
a  defined  blend  of  related  molecules  that  trigger 
distinct  mating  behaviors  in  members  of  the  op- 
posite sex.  For  detection  to  occur,  three  criteria 
must  be  satisfied.  Molecules  of  the  pheromone 
must  reach  olfactory  receptors,  must  interact 
with  them,  and  must  be  inactivated  so  that  subse- 
quent molecules  can  be  detected. 

Past  research  has  focused  on  the  problems  of 
chemical  transport  to  olfactory  receptors  and  in- 
activation  of  these  molecules.  Moths — particu- 
larly Manduca  sexta  and  Antheraea  polyphe- 
mus — have  provided  excellent  models.  Many  of 
the  olfactory  receptor  cells  of  the  male  M.  sexta 
and  most  of  those  from  the  male  A.  polyphemus 
are  specialized  for  detecting  sex  pheromone.  For 
both  animals,  the  pheromone-binding  proteins, 
which  solubilize  molecules  of  the  pheromone 
blend  and  carry  them  to  receptors,  have  been 
characterized.  In  addition,  a  family  of  general 
odorant-binding  proteins,  which  are  related  to 
the  ones  employed  by  the  moths  to  carry  phero- 
mone, has  been  discovered  and  characterized. 
Likewise,  the  enzymes  that  rapidly  and  specifi- 
cally inactivate  pheromone  molecules  and  are  ap- 
parently requisite  to  the  sensory  apparatus  em- 
ployed by  males  to  locate  females,  have  been 
investigated  at  the  biochemical  level.  For  M. 
sexta,  in  which  both  major  components  of  the 
pheromone  blend  are  aldehydes,  a  single  en- 
zyme, an  aldehyde  oxidase,  suffices,  while  A. 
polyphemus  employs  both  an  aldehyde  oxidase 
and  an  esterase  because  its  pheromone  compo- 
nents include  both  an  aldehyde  and  an  ester.  Our 
research  has  now  turned  to  developing  methods 
for  investigating  olfactory  receptors  themselves. 

Over  the  past  few  years  many  laboratories  have 
conducted  biochemical,  electrophysiological, 
and  molecular  cloning  experiments  concerned 


with  the  nature  of  signal  transduction  by  olfac- 
tory receptors.  Depending  on  the  animal  whose 
sense  of  smell  is  being  investigated,  the  results 
indicate  that  either  cAMP,  IP3/DAG  (IP3,  inositol 
1,4,5-trisphosphate;  DAG,  diacylglycerol) ,  or 
both  second  messenger  systems  are  involved.  It 
now  appears  that  the  study  of  olfaction,  and 
hence  a  major  aspect  of  chemical  communication 
between  animals,  is  part  of  the  general  problem 
of  how  G  protein-coupled  receptors  work. 

At  the  present  time,  several  methods  are  em- 
ployed to  study  how  receptors  that  regulate  intra- 
cellular concentrations  of  cAMP  or  IP3/DAG 
work,  such  as  adenylate  cyclase  assays,  radioim- 
munoassays, measurements  of  IP3  or  DAG,  moni- 
toring the  flow  of  ions  through  channels  in  frog 
oocyte  membranes,  or  looking  at  changes  in  in- 
tracellular calcium  concentrations  via  appro- 
priate fluorescent  indicators.  To  study  G  pro- 
tein-coupled receptors,  and  ones  relevant  to 
olfaction  in  particular,  we  are  developing  a  new 
method  for  monitoring  receptor  stimulation  that 
can  track  changes  in  intracellular  concentrations 
of  cAMP  or  IP3/DAG  in  over  10,000  individual 
cells  simultaneously. 

To  follow  changes  in  cAMP  or  IP3/DAG  levels 
in  many  individual  cells  at  the  same  time,  we 
have  turned  to  that  ability  of  some  animals  to 
change  colors  rapidly.  In  nature,  color  changes 
are  used  for  such  purposes  as  camouflage  and  the 
communication  of  states  of  emotion.  Among  ver- 
tebrates, quick  color  alterations  are  brought 
about  by  the  controlled  movement  of  pigment 
granules  within  chromatophores.  When  pigment 
granules  in  melanophores  (a  particular  type  of 
chromatophore)  are  aggregated,  the  animal  ap- 
pears light,  and  when  pigment  is  dispersed,  the 
animal  appears  dark.  It  turns  out  that  the  pigment 
translocation  apparatus  is  controlled  via  second 
messenger  systems  that  are  themselves  regulated 
by  G  proteins.  As  a  result,  the  state  of  pigment 
disposition  within  melanophores  reflects  the 
state  of  activity  of  G  protein-coupled  receptors. 

Recently  the  laboratory  has  successfully  har- 
nessed frog  melanophores  as  the  center  of  an 


257 


Chemical  Communication 


assay  for  monitoring  the  activity  of  G  protein- 
coupled  receptors  that  act  to  raise  or  lower  intra- 
cellular cAMP  or  that  raise  intracellular  IP3/DAG. 
The  ability  of  exogenous  receptors  to  couple  to 


and  control  the  pigment  translocation  appa- 
ratus within  melanophores  should  lead  to  im- 
proved methods  for  studying  G  protein-coupled 
receptors. 


258 


Structural  Determinants  of  Human  a-Glohin 
Gene  Expression 


Stephen  A.  Liebhaber,  M.D. — Investigator 

Dr.  Liebhaber  is  also  Professor  of  Genetics  and  Medicine  (Hematology)  at  the  University  of  Pennsylvania 
School  of  Medicine.  He  received  his  B.A.  degree  in  chemistry  from  Brandeis  University  and  his  M.D.  degree 
from  Yale  University.  He  took  clinical  training  in  internal  medicine  and  hematology  at  Case  Western 
Reserve,  the  University  of  Colorado,  Washington  University,  and  the  University  of  California,  San 
Francisco.  As  a  postdoctoral  fellow  with  David  Schlessinger  at  Washington  University,  Dr.  Liebhaber 
examined  ribosomal  RNA  processing,  and  with  Yuet  Wai  Kan  at  the  University  of  California,  San 
Francisco,  he  studied  human  globin  gene  expression  and  genetic  defects  in  a-thalassemia.  Before  moving 
to  Philadelphia,  he  was  a  faculty  member  of  the  Department  of  Medicine  at  UCSF. 


OUR  laboratory  has  largely  concentrated  on 
studying  the  expression  of  the  human  glo- 
bin genes.  These  genes  encode  hemoglobin,  the 
major  red  cell  protein  responsible  for  transport  of 
oxygen  from  the  lungs  to  peripheral  tissues. 
Since  the  hemoglobin  molecule,  a2i^2>  is  com- 
posed of  an  equal  number  of  a-  and  ;8-globin 
chains,  normal  synthesis  demands  balanced  ex- 
pression of  both  sets  of  genes,  which  are  located 
on  different  chromosomes.  Defects  in  either  set 
result  in  an  imbalance  of  expression  and  conse- 
quent hereditary  anemia:  a-  or  (S-thalassemia. 
Thalassemias  result  from  more  than  150  different 
mutations  in  the  globin  genes,  affecting  the 
health  of  millions  worldwide. 

Certain  characteristics  of  globin  gene  expres- 
sion make  it  particularly  interesting  for  study. 
The  extremely  high  level  of  globin  mRNA  in  the 
differentiating  red  cell  (over  95  percent  of  total 
cellular  mRNA)  has  no  equal  in  any  other  cell 
type.  This  abundance  reflects  both  high  levels  of 
globin  gene  transcription  and  an  unusual  stability 
of  the  mature  globin  mRNA. 

An  additional  interesting  aspect  of  globin  ex- 
pression is  that  the  genes  in  the  a-  and  |S-globin 
gene  clusters  follow  an  orderly  sequence  of  ex- 
pression during  embryologic  development.  This 
results  in  a  well-defined  switch  from  embryonic 
to  adult  globin  gene  expression  during  develop- 
ment of  the  fetus.  The  switching  results  in  the 
synthesis  of  successive  hemoglobins  with  oxygen 
affinities  that  match  changes  in  the  uterine  envi- 
ronment. The  active  transcription,  the  clearly  de- 
fined pattern  of  developmental  switching,  and 
the  unusual  mRNA  stability  are  areas  of  special 
focus  in  our  laboratory. 

The  loss  of  a-globin  expression  observed  in 
a-thalassemia  usually  results  from  deletion  or  ab- 
normal structure  of  one  or  more  of  the  a-globin 
genes.  We  have  recently  studied  three  unrelated 
patients  from  Germany,  Portugal,  and  Italy  who 
have  an  unusual  form  of  a-thalassemia.  In  each 
case  the  loss  of  a-globin  synthesis  reflects  loss  of 
expression  of  a  structurally  normal  a-globin  clus- 


ter. In  other  words,  one  of  the  a-globin  gene  clus- 
ters in  each  of  these  patients  is  not  functioning, 
even  though  the  genes  in  these  clusters  are  struc- 
turally normal  and  synthesize  normal  levels  of 
a-globin  when  isolated  and  expressed  in  tissue 
culture  cells.  Although  initially  puzzling,  the 
mechanism  of  this  remarkable  expression  defect 
was  eventually  defined  by  extensive  DNA  map- 
ping. In  each  of  the  three  independent  cases  we 
demonstrated  a  large  deletion  5'  to  the  inacti- 
vated cluster  itself.  In  one  case  this  deletion  be- 
gan as  much  as  50,000  bases  5'  to  the  silenced 
a-globin  genes.  By  comparing  the  maps  of  each  of 
these  deletions,  a  region  of  common  overlap  was 
noted.  These  studies  define  a  new  category  of 
a-thalassemia  and  demonstrate  a  critical  determi- 
nant of  a-globin  gene  expression  located  entirely 
external  to  the  a-globin  gene  cluster.  A  similar  set 
of  transcriptional  signals  has  been  localized  adja- 
cent to  the  j8-globin  gene  cluster.  One  can  there- 
fore speculate  that  such  signals  serve  coordi- 
nately  to  activate  and  balance  the  expression  of 
the  a-  and  ;8-globin  clusters  in  the  red  cell. 

The  human  a-globin  gene  cluster  contains  a 
globin  gene  expressed  specifically  in  the  embryo 
and  two  a-globin  genes,  al  and  a2,  expressed  in 
the  fetus  and  the  adult.  The  switch  from  embry- 
onic f-globin  to  adult  a-globin  expression  occurs 
at  the  end  of  embryonic  development  (7-8 
weeks  of  gestation) .  This  critical  developmental 
switch,  which  occurs  widely  in  mammals,  pres- 
ents a  well-defined  model  system  for  studying  de- 
velopmental control  of  gene  expression. 

To  establish  a  system  in  which  to  study  switch- 
ing within  the  human  a-globin  gene  cluster,  we 
have  injected  the  human  embryonic  and  adult 
a-globin  genes  into  fertilized  mouse  eggs  to  gen- 
erate transgenic  mice.  The  red  cells  of  these 
transgenic  mice  appropriately  express  the  human 
transgenes  during  development.  In  the  embry- 
onic period,  there  is  a  parallel  expression  of  the 
human  and  mouse  f-globin  genes  and,  by  day  1 2 
of  development,  parallel  expression  of  the 
a-globin  genes.  These  data  suggest  that  1 )  the  hu- 


259 


Structural  Determinants  of  Human  a-Globin  Gene  Expression 


man  transgenes  contain  the  necessary  informa- 
tion for  appropriate  developmental  control  and 
2)  the  factors  responsible  for  developmental 
switching  in  mouse  red  cells  have  been  suffi- 
ciently conserved  during  evolution  to  substitute 
in  the  control  over  the  human  transgenes.  By  gen- 
erating transgenic  mice  that  carry  only  the  human 
a-globin  gene  in  the  absence  of  the  f-globin  gene, 
we  have  further  demonstrated  that  the  appro- 
priate expression  of  this  gene  is  not  dependent  on 
competition  between  the  two  developmentally 
specific  genes.  By  introducing  into  the  mouse  ge- 
nome more  limited  gene  fragments,  as  well  as 
genes  with  specific  alterations,  and  by  studying 
their  pattern  of  developmental  expression  in  the 
transgenic  model  system,  we  should  be  able  to 
define  the  signal  (s)  critical  to  globin  gene 
switching. 

Selective  stabilization  of  globin  mRNA  is  essen- 
tial to  its  accumulation  in  terminally  differentiat- 
ing red  cells  to  95  percent  of  total  cellular  mRNA. 
We  are  attempting  to  define  the  structural  basis 
for  the  stability  of  a-globin  mRNA  through  inves- 
tigation of  an  a-thalassemia  mutation  in  which 
this  stability  is  lost:  a  Constant  Spring  (aCS) .  This 
mutation,  the  most  common  cause  of  nondele- 
tional  a-thalassemia  in  Southeast  Asia,  is  a  CAA  for 
UAA  substitution  at  the  normal  termination  codon 
of  the  a2-globin  gene.  As  a  result  of  this  single- 
base  substitution,  the  ribosome  translates  into  the 


normally  untranslated  3'  region  and  destabilizes 
the  mRNA. 

To  study  the  basis  for  this  destabilization  in  de- 
tail, we  have  established  an  experimental  system 
that  reproduces  the  selective  instability  of  aCS 
mRNA  in  tissue  culture  cells.  Remarkably,  we 
find  that  the  instability  of  the  aCS  mRNA  relative 
to  normal  a-globin  mRNA  observed  in  the  patient 
is  recapitulated  faithfully  when  the  a-  and  aCS- 
globin  genes  are  expressed  in  an  erythroid  tissue 
culture  cell  line.  In  contrast,  we  find  that  the 
mRNAs  from  these  two  genes  are  expressed  at 
equivalent  levels  when  expressed  in  nonery- 
throid  cell  lines.  These  data  suggest  that  the  sta- 
bility of  globin  mRNA  may  depend  on  interaction 
with  one  or  more  erythroid-specific  factors. 

By  specifically  altering  the  structure  of  the  a- 
globin  genes  prior  to  expression,  we  have  demon- 
strated that  the  region  critical  to  the  stability  of 
the  a-globin  mRNA  is  located  in  a  segment  of  the 
3'-nontranslated  region  just  past  the  translation 
termination  codon.  This  determinant  can  be  de- 
stabilized by  the  translating  ribosome  if  it  is  al- 
lowed to  enter  this  region,  as  is  the  case  in  the 
aCS  mutation,  or  by  site-specific  alterations  in 
the  primary  sequence  of  this  segment.  These  data 
suggest  that  it  should  now  be  possible  to  map  the 
limits  of  this  determinant  and  subsequently  iden- 
tify critical  cellular  factors  mediating  this 
response. 


260 


The  Heat-Shock  Response 


Susan  L.  Lindquist,  Ph.D. — Investigator 

Dr.  Lindquist  is  also  Professor  in  the  Department  of  Molecular  Genetics  and  Cell  Biology  and  in  the 
Committees  on  Developmental  Biology  and  Genetics  at  the  University  of  Chicago.  She  received  her  B.S. 
degree  in  microbiology  from  the  University  of  Illinois,  where  she  worked  with  John  Drake  on 
bacteriophage  T4.  Her  graduate  research  was  done  with  Matthew  Meselson  at  Harvard  University,  where 
she  began  her  studies  on  the  heat-shock  response.  She  continued  this  work  during  her  postdoctoral 
research  with  Hewson  Swift  at  the  University  of  Chicago. 


THE  causes  of  heat-induced  lethality  and  the 
mechanisms  that  cells  employ  to  protect 
themselves  from  heat  damage  are  poorly  under- 
stood. Over  the  past  decade,  a  great  deal  of  re- 
search has  focused  on  a  small  group  of  mole- 
cules, the  heat-shock  proteins  (HSPs).  These  are 
induced  in  response  to  temperature  elevation 
and  a  wide  variety  of  other  stresses.  No  known 
genetic  induction  is  more  highly  conserved  in 
evolution,  which  underscores  its  fundamental 
importance  in  biology.  Archaeobacteria,  eubac- 
teria,  plants,  and  animals  all  produce  similar  pro- 
teins. Several  of  these  proteins  show  very  high 
levels  of  conservation,  commonly  with  40-50 
percent  amino  acid  identity  between  HSPs  of  hu- 
man and  bacterial  cells. 

The  induction  of  HSPs  1)  allows  cells  to  grow 
at  the  upper  end  of  their  normal  growth  range,  2) 
potentiates  survival  during  long  exposures  to  tem- 
peratures just  beyond  the  normal  growth  range, 
and  3)  protects  cells  from  lethality  at  tempera- 
ture extremes.  Interestingly,  different  proteins 
are  required  for  each  of  these  functions.  HSPs 
also  protect  cells  from  heavy  metal  ions,  ethanol, 
and  many  other  sources  of  stress.  The  importance 
of  different  proteins  in  protecting  against  differ- 
ent forms  of  stress  also  varies. 

The  stress  inductions  of  HSPs  are  of  interest  to 
human  biology  and  medicine  for  several  reasons. 
First,  studies  of  cultured  cells  in  vitro  and  of  tu- 
mors in  vivo  demonstrate  that  many  cancer  cells 
are  more  readily  killed  by  heat  than  are  untrans- 
formed  cells.  For  this  reason,  hyperthermia,  in 
conjunction  with  radiation  and  chemotherapy,  is 
emerging  as  an  important  new  tool  in  cancer  ther- 
apy. Second,  high  temperatures  are  associated 
with  a  number  of  developmental  anomalies  in  a 
wide  variety  of  plants  and  animals,  including 
spina  bifida  in  humans.  In  those  organisms  that 
have  been  subjected  to  experimental  manipula- 
tion, mild  preheat  treatments,  which  induce  the 
HSPs,  provide  protection.  Third,  the  induction  of 
HSPs  is  associated  with  a  variety  of  human  patho- 
logical states,  including  strokes,  heart  attacks, 
and  kidney  disease.  Interest  in  the  proteins  in- 
cludes both  their  putative  protective  functions  in 


affected  tissues  and  the  possibility  of  quantifying 
them  as  disease  markers.  Fourth,  the  proteins  in- 
teract with  and  potentiate  the  function  of  many 
other  vital  proteins  in  the  cell. 

The  heat-shock  response  also  provides  a  superb 
model  system  in  which  to  study  the  cellular 
mechanisms  involved  in  regulating  protein  syn- 
thesis. Because  HSPs  are  required  for  survival,  a 
number  of  regulatory  mechanisms  are  employed 
to  ensure  that  the  proteins  will  be  produced  as 
rapidly  as  possible  after  exposure  to  stress.  Thus 
studies  of  the  response  have  provided  funda- 
mental insights  on  the  nature  of  nuclear  and  cy- 
toplasmic regulation  in  both  eukaryotes  and 
prokaryotes. 

The  recent  discovery  that  the  HSPs  themselves, 
or  close  relatives  produced  at  normal  tempera- 
tures, play  vital  roles  during  normal  growth  and 
development  has  opened  up  a  whole  new  field  of 
investigation.  The  specific  molecular  functions 
of  the  HSPs  are  only  beginning  to  be  elucidated, 
but  they  play  a  role  in  a  remarkable  number  of 
basic  cellular  processes,  including  secretion,  sig- 
nal transduction,  and  ribosome  assembly.  Deter- 
mining the  roles  that  HSPs  play  in  these  processes 
will  provide  fundamental  insights  in  cell  biology. 

We  are  investigating  the  regulation  and  func- 
tion of  these  proteins.  Our  research  focuses  on 
the  yeast  Saccharomyces  cerevisiae  and  the  fruit 
fly  Drosophila  melanogaster,  because  tech- 
niques of  genetic  manipulation  and  molecular 
analysis  are  so  advanced  in  these  organisms.  For 
the  past  few  years  our  investigations  of  the  regula- 
tion of  the  response  have  concentrated  on  post- 
transcriptional  mechanisms  that  are  employed  to 
maximize  the  synthesis  of  the  HSPs  during  heat 
shock  or  to  shut  off  synthesis  after  heat  shock. 

Tom  McGarry  and  Bob  Petersen  found  that 
heat-shock  mRNAs  in  Drosophila  cells  are  prefer- 
entially translated  during  heat  shock  by  virtue  of 
sequences  in  their  5'-untranslated  leaders  and  are 
preferentially  repressed  during  recovery  through 
sequences  in  their  3'-untranslated  tails.  The  latter 
sequences  are  shared  by  certain  normal  cellular 
messages,  which  have  the  common  property  of 
being  rapidly  degraded  at  normal  temperatures. 


261 


The  Heat-Shock  Response 


Heat-shock  regulation  takes  advantage  of  this 
common  pathway  to  control  HSP  expression.  The 
mechanism  is  inactivated  during  heat  shock  and 
restored  during  recovery. 

Joseph  Yost  demonstrated  that  heat  shock 
blocks  the  processing  of  mRNA  precursors, 
which  explains  why  heat-shock  genes  generally 
do  not  have  intervening  sequences.  (If  they  had, 
the  block  in  splicing  would  prevent  expression.) 
Sudden  high-temperature  heat  shocks  also  inhibit 
transcription  termination  (discovered  by  Robert 
Dellavalle).  By  some  mechanism  we  do  not  yet 
understand,  heat-shock  genes  are  more  likely  to 
be  terminated  correctly  than  normal  cellular 
genes. 

Although  our  studies  of  HSP  function  are  in 
many  ways  independent  of  our  studies  on  regula- 
tion, in  one  important  respect  they  overlap.  HSPs, 
and  hsp70  in  particular,  play  an  important  role  in 
restoring  normal  gene  expression  patterns  after 
heat  shock.  They  are  required  at  the  level  of 
translation,  RNA  turnover,  RNA  processing,  and 
transcription. 

To  investigate  the  function  of  the  HSPs,  we  cre- 
ated a  series  of  mutations  in  the  genes  of  both 
yeast  and  Drosophila.  Kathy  Borkovich  found 
that  hsp82  is  essential  for  growth  at  all  tempera- 
tures in  yeast  cells,  but  is  required  at  higher  con- 
centrations for  growth  at  high  temperatures.  Thus 
induction  is  required  for  cells  to  grow  at  the  up- 
per end  of  their  normal  temperature  range.  We 
believe  the  protein  is  needed  to  regulate  the  activ- 
ity of  a  wide  variety  of  other  cellular  proteins  and 
that  it  is  needed  at  higher  concentrations  at  high 
temperatures  in  order  to  drive  the  equilibrium  of 
these  interactions  toward  complex  formation. 

In  collaboration  with  Keith  Yamamoto's  labora- 
tory, Bushra  Khursheed  and  Marc  Fortin  demon- 
strated that  hsp82  interacts  with  the  steroid  hor- 
mone family  of  receptors  and  helps  these 
proteins  fold  into  an  active  conformation.  Most 
recently,  Yang  Xu  found  that  hsp82  is  also  re- 
quired for  the  maturation  of  oncogenic  proteins 
in  the  src  family. 


Yolanda  Sanchez  created  mutations  in  the 
HSP  104  gene  of  yeast.  The  mutations  have  no  ef- 
fect on  growth  at  high  or  low  temperatures.  How- 
ever, the  cells  are  unable  to  acquire  tolerance  to 
extreme  temperatures  when  given  a  mild  preheat 
treatment.  Thus  this  mutation  confirms  the  long- 
standing assumption  that  HSPs  play  a  vital  role  in 
establishing  thermotolerance.  Moreover,  it  pro- 
vides protection  from  many  other  forms  of  stress, 
such  as  exposure  to  ethanol  and  sodium  arsen- 
ite.  Dawn  Parsell  found  that  hspl04  is  highly 
conserved  in  mammals  and  in  prokaryotic  cells 
and  contains  two  essential  nucleotide-binding 
domains. 

In  Drosophila  our  mutational  analysis  has  con- 
centrated on  hsp70.  Janice  Rossi  found  that  vary- 
ing the  level  of  hsp70  expression  in  Drosophila 
cells  varies  the  rate  at  which  the  cells  recover 
from  heat  shock.  Jonathan  Solomon  found  that 
expressing  hsp70  from  independently  regulated 
promoters,  in  the  absence  of  heat  shock,  helps 
cells  to  survive  extreme  temperatures  but  in- 
hibits their  growth.  Thus  hsp70  helps  to  protect 
cells  from  the  ravages  of  extreme  temperatures 
but  is  actually  disadvantageous  at  normal 
temperatures. 

To  study  the  role  of  hsp70  in  whole  flies,  Kent 
Golic  developed  a  new  system  for  manipulating 
the  Drosophila  genome.  He  created  flies  that  ex- 
press the  FLP  recombinase  gene  of  yeast  under 
the  control  of  heat-shock  regulatory  sequences. 
When  flies  that  also  carry  a  recombinase  target 
sequence  are  given  a  very  mild  heat  shock,  the 
recombinase  is  induced  and  catalyzes  rearrange- 
ments of  the  target  sequence. 

Michael  Welte  and  Joan  Tetrault  have  em- 
ployed this  site-specific  recombination  system  to 
study  the  role  of  hsp70  in  induced  thermotoler- 
ance. Strains  that  carry  several  extra  copies  of  the 
hsp70  gene  were  constructed.  Embryos  from 
these  lines  survive  heat  treatments  much  better 
than  wild-type  embryos.  This  suggests  that  it  will 
be  possible  to  alter  the  stress  tolerance  of  even 
developmentally  complex  species. 


262 


T  Cell  Surface  Glycoproteins  in  Development 
and  Viral  Infections 


Dan  R.  Littman,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Littman  is  also  Associate  Professor  of  Microbiology  and  Immunology  and  of  Biochemistry  and 
Biophysics  at  the  University  of  California,  San  Francisco.  Following  undergraduate  studies  on  the 
structure  of  microtubules  in  Marc  Kirschner's  laboratory  at  Princeton  University,  he  completed  his 
M.D./Ph.D.  program  at  Washington  University  in  St.  Louis,  working  with  Benjamin  Schwartz  and  Susan 
Cullen  on  the  function  of  histocompatibility  molecules  in  antigen  presentation.  His  postdoctoral  research 
was  done  in  Richard  Axel's  laboratory  at  Columbia  University,  where  he  isolated  the  genes  for  CD4 
and  CDS. 


THE  shaping  of  a  mature  repertoire  of  T  lym- 
phocytes capable  of  responding  to  patho- 
genic microorganisms  involves  a  complex  pro- 
cess of  differentiation  within  the  thymus.  In  this 
process,  self-reactive  cells  are  eliminated  and 
cells  that  can  react  to  foreign  antigen  complexed 
to  molecules  of  the  major  histocompatibility 
complex  (MHC)  are  selected  to  survive  and  mi- 
grate to  peripheral  lymphoid  organs. 

The  signals  involved  in  the  different  phases  of 
thymic  selection  require  the  interaction  of  sev- 
eral thymocyte  surface  proteins  with  components 
of  the  thymic  microenvironment.  These  cell  sur- 
face molecules  include  the  clonally  restricted  T 
cell  receptors  and  the  CD4  and  CDS  glycopro- 
teins. Both  CD  A  and  CDS  are  expressed  on  imma- 
ture thymocytes,  but  the  gene  for  one  or  the  other 
is  shut  off  upon  maturation.  Cells  that  have  re- 
ceptors for  self-MHC  class  I  molecules  continue 
to  express  CDS,  but  shut  off  CD4;  cells  v^^ith  re- 
ceptors for  MHC  class  II  molecules  express  CD4 
and  shut  off  CDS.  Our  laboratory  is  studying  the 
mechanism  of  regulation  of  the  CD4  and  CDS 
genes  as  well  as  the  molecular  basis  of  the  cell- 
cell  interaction  resulting  in  the  selection  of  cells 
that  have  appropriate  specificity. 

The  CD4  and  CDS  molecules  interact  directly 
with  MHC  class  II  and  class  I  molecules,  respec- 
tively. Effective  T  cell  activation  requires  co- 
recognition  of  MHC  by  both  the  T  cell  receptor 
and  either  CD4  or  CDS.  To  test  whether  corecog- 
nition  is  also  required  in  transmembrane  signal- 
ing during  T  cell  development,  we  have  analyzed 
transgenic  mice  that  express  a  mutant  form  of 
MHC  class  I  incapable  of  binding  to  CDS  but  in- 
teractive with  T  cell  receptors.  These  mice  were 
shown  to  be  defective  in  developing  a  mature  T 
cell  repertoire  specific  for  the  mutant  MHC  class 
I  molecule. 

This  study  indicates  that  CDS:MHC  binding  is 
required  for  both  intrathymic  deletion  of  self- 
reactive  T  cells  and  positive  selection  of  useful  T 
cells.  Moreover,  since  endogenous  MHC  class  I 
molecules  were  competent  to  bind  CDS  but  un- 
able to  rescue  the  defect,  coordinate  recognition 


of  antigen/MHC  by  a  complex  of  the  T  cell  recep- 
tor and  CDS  is  required  for  both  positive  and  neg- 
ative selection. 

To  study  the  roles  of  CD4  and  CDS  in  greater 
detail,  we  are  preparing  animals  defective  in  the 
expression  of  these  molecules.  We  have  utilized 
gene-targeting  technology  to  mutate  the  CD4 
gene  in  embryonal  stem  cells.  These  have  been 
injected  into  mouse  blastocysts,  resulting  in  the 
birth  of  chimeric  animals.  The  mutant  CD4  gene 
has  been  propagated  in  the  mouse  germline,  and 
we  are  currently  studying  the  immune  system  of 
mice  lacking  CD4  expression.  These  mice  lack 
helper  T  cells  and  have  essentially  no  antibody 
response  to  T-dependent  antigens  and  no  T  cell 
response  to  antigen  presented  by  MHC  class  II 
molecules. 

Transgenic  mice  expressing  mutant  forms  of 
CD4,  predicted  to  be  defective  in  signal- 
transducing  functions  (as  a  result  of  defective  in- 
teraction with  cytoplasmic  tyrosine  kinases  or 
with  the  T  cell  receptor  complex),  have  been 
prepared  and  are  being  used  to  analyze  the  role  of 
CD4  signaling  during  development  in  the  ab- 
sence of  endogenous  CD4  expression.  We  have 
also  prepared  transgenic  mice  that  express  the 
human  CD4  gene  and  will  examine  the  ability  of 
the  human  molecule  to  function  during  T  cell 
development  in  mice  that  lack  murine  CD4.  Such 
animals  may  be  especially  useful  for  studies  of 
pathogenesis  and  therapy  in  autoimmune  dis- 
eases and  AIDS. 

The  CD4  and  CDS  glycoproteins  have  been 
shown  to  be  involved  in  the  activation  of  periph- 
eral T  lymphocytes.  For  example;,  artificial  cross- 
linking  of  CD4  or  CDS  to  the  T  cell  receptor  com- 
plex results  in  T  cell  activation.  Moreover,  T  cells 
that  have  lost  expression  of  either  CD4  or  CDS 
but  continue  to  express  the  T  cell  receptor  are 
usually  no  longer  responsive  to  antigen.  We  are 
performing  a  variety  of  structure/function  stud- 
ies to  determine  the  mechanism  through  which 
CD4  and  CDS  facilitate  signal  transduction  in  T 
cells. 

It  is  known  that  CD4  and  CDS  are  associated 


263 


T  Cell  Surface  Glycoproteins  in  Development  and  Viral  Infections 


with  a  cytoplasmic  tyrosine  kinase,  p56'''*,  a 
member  of  the  src  kinase  family.  The  sites  of 
interaction  between  these  molecules  map  to 
cysteine-containing  regions  in  the  cytoplasmic 
domains  of  CD4  and  CDS  and  in  the  unique 
amino-terminal  domain  of  p56'''*.  We  have  dem- 
onstrated that  only  CD4  molecules  that  can  asso- 
ciate with  the  kinase  are  functional  in  antigen- 
specific  T  cell  hybridomas,  which  normally 
produce  interleukin-2  upon  stimulation  with  the 
appropriate  antigen. 

Since  interaction  of  CD4  with  the  Ick  kinase  is 
essential  for  T  cell  activation,  we  are  now  begin- 
ning to  characterize  the  proteins  that  serve  as 
substrates  for  tyrosine  phosphorylation.  One  of 
the  substrates  appears  to  be  a  tyrosine  kinase  as- 
sociated with  the  T  cell  receptor  complex.  Cross- 
linking  of  p56'''*  to  the  receptor-associated  kinase 
results  in  tyrosine  phosphorylation  and  activation 
of  phospholipase-C7 1 ,  an  enzyme  that  is  also  as- 
sociated with  the  T  cell  receptor  complex.  Cleav- 
age of  phosphoinositol-containing  phospholipid 
by  this  activated  enzyme  generates  second  mes- 
sengers that  activate  the  transcriptional  machin- 
ery in  the  T  cell. 

The  laboratory  is  also  studying  the  mechanism 
involved  in  the  developmental  switch  from 
double-positive  (CD4"^CD8'^)  to  single-positive 
thymocytes.  The  switch  may  be  due  to  differen- 
tial signaling  through  CD4  or  CDS  (hence  due  to 
instruction)  or  to  random  (stochastic)  shutoff  of 
either  CD4  or  CDS,  followed  by  selection  of  cells 
that  have  the  appropriate  T  cell  receptor  and  co- 
receptor.  Several  experiments  are  being  per- 
formed to  discriminate  between  these  two  basic 
mechanisms  of  differentiation. 

An  understanding  of  the  mechanism  involved 
should  also  shed  light  on  the  transcriptional  regu- 
lation that  thymocytes  employ,  first  to  turn  on 
both  the  CD4  and  CDS  genes  and  subsequently  to 
shut  off  only  one  of  the  genes.  We  have  character- 
ized a  sequence  upstream  from  the  CD4  pro- 
moter with  T  cell-specific  transcriptional  en- 
hancing activity.  The  ability  of  this  and  other 
sequences  to  provide  subset-specific  regulatory 
signals  in  thymocyte  differentiation  is  being  stud- 
ied in  transgenic  mice. 


The  CD4  glycoprotein  is  doubly  important  be- 
cause it  is  the  receptor  for  the  human  immunode- 
ficiency virus  (HIV) .  We  have  found  that  several 
cell  types  that  express  CD4  can  bind  virus  but 
cannot  be  infected,  indicating  that  host  factors 
other  than  CD4  are  involved  in  viral  entry.  Resis- 
tance of  these  cells  to  infection  is  due  to  the  in- 
ability of  the  viral  envelope  to  fuse  to  the  cellular 
plasma  membrane.  It  is  therefore  likely  that 
plasma  membrane  molecules  other  than  CD4  are 
required  for  fusion  of  virus  to  target  cells.  We  are 
using  genetic  and  biochemical  approaches  to 
identify  such  molecules. 

Identification  of  additional  molecules  in- 
volved in  HIV  entry  may  permit  design  of  novel 
agents  to  interfere  with  the  spread  of  HIV.  In  ad- 
dition, we  hope  that  this  information  will  facili- 
tate the  design  of  a  mouse  model  system  for  HIV 
disease.  The  currently  available  mice  that  express 
the  human  CD4  transgene  are  resistant  to  infec- 
tion, but  expression  of  additional  genes  involved 
in  HIV  entry  may  permit  infection  of  these  ani- 
mals with  HIV. 

Using  a  genetic  system  for  studying  HIV  entry, 
we  have  shown  that  the  envelope  glycoprotein  of 
HIV  can  be  replaced  by  that  of  another  patho- 
genic human  retrovirus,  human  T  cell  leukemia 
virus  (HTLV),  forming  HIV(HTLV)  pseudotypes. 
HTLV  causes  T  cell  leukemias  and  lymphomas 
and  myelopathies.  Individuals  infected  with  both 
HIV  and  HTLV  have  more  rapid  progression  of 
HIV  disease  than  those  infected  with  HIV  alone. 
Since  both  viruses  infect  T  lymphocytes,  it  is 
likely  that  mixed  viral  particles  can  form  in  vivo. 
We  have  demonstrated  that  mixed  particles 
(pseudotypes)  that  form  in  vitro  have  an  ex- 
panded host  range — i.e.,  HIV  particles,  endowed 
with  the  HTLV-I  envelope  glycoprotein,  can 
readily  infect  CD4-deficient  cells.  Such  mixed 
particles  may  have  an  important  role  in  HIV 
pathogenesis,  particularly  in  infection  of  cells 
that  do  not  have  HIV  receptors,  such  as  cells  of 
the  central  nervous  system.  In  addition  to  investi- 
gating HIV  infection,  we  are  using  the  hybrid  par- 
ticles to  study  the  yet  uncharacterized  HTLV 
receptor. 


264 


The  Biology  of  T  Lymphocyte  Development 


Dennis  Y.-D.  Loh,  M.D. — Associate  Investigator 

Dr.  Loh  is  also  Professor  of  Medicine,  Genetics,  and  Molecular  Microbiology  at  Washington  University 
School  of  Medicine  and  Chief  of  the  Division  of  Allergy  and  Immunology  and  Associate  Physician  at 
Barnes  Hospital,  St.  Louis.  He  received  his  undergraduate  degree  in  biology  and  chemical  engineering  from 
the  California  Institute  of  Technology  and  his  medical  degree  from  Harvard  Medical  School.  After  finishing 
his  clinical  residency  in  internal  medicine  at  the  Peter  Bent  Brigham  Hospital,  Boston,  he  studied  as  a 
postdoctoral  fellow  with  David  Baltimore  at  the  Massachusetts  Institute  of  Technology. 


THE  immune  system  is  involved  intimately  in 
our  body's  defense  against  invading  microor- 
ganisms and  tumors.  In  addition,  it  plays  a  central 
role  in  organ  graft  rejection  and  autoimmune  dis- 
eases such  as  systemic  lupus  erythematosus, 
rheumatoid  arthritis,  and  diabetes.  Its  critical 
role  in  maintaining  health  is  best  manifested  in 
the  acquired  immune  deficiency  syndrome 
(AIDS) ,  in  which  destruction  of  a  specific  portion 
of  the  immune  system  results  in  a  potentially  fatal 
disease. 

Our  investigation  is  focused  on  the  molecular 
mechanisms  that  allow  normal  and  abnormal  de- 
velopment of  the  antigen-specific  T  cells.  T  cells 
are  those  lymphocytes  (a  type  of  white  cell)  that 
depend  on  the  presence  of  the  thymus  gland  for 
maturation.  An  antigen  may  be  viewed  as  any 
marker  that  these  cells  recognize.  T  cells  are 
thought  to  play  a  central  role  in  the  regulation  of 
the  immune  response.  T  cells  recognize  antigens 
by  means  of  a  cell  surface  structure  called  the  T 
cell  receptor  (TCR) .  The  genes  that  are  responsi- 
ble for  the  expression  of  the  TCR  undergo  DNA 
gene  rearrangement  and  gene  activation  specifi- 
cally in  the  thymus  during  the  individual's  early 
development.  Once  the  TCR  is  expressed  as  pro- 
tein, it  is  the  interaction  of  the  TCR  with  its  anti- 
gen that  triggers  the  activation  of  T  cells,  result- 
ing in  an  immune  response.  The  ultimate  result  of 
such  a  response  may  be  either  defense  against 
invading  organisms  or  tissue  destruction,  as 
seen  in  transplantation  rejection  and  autoim- 
mune phenomena. 

My  laboratory  initially  concentrated  on  identi- 
fying the  genetic  elements  that  encode  the  TCR 
genes.  We  then  shifted  our  efforts  to  study  the 
function  of  T  cells  in  the  intact  animal,  especially 
during  its  development.  Two  important  ques- 
tions were  addressed.  1)  Why  are  we  tolerant  of 
our  own  tissues  and  organs?  2)  Why  are  trans- 
planted organs  rejected  readily  (unless  they  are 
carefully  cross-matched)?  We  have  used  both  re- 
combinant DNA  technology  and  our  ability  to 
create  transgenic  mice  (mice  with  cloned  genes 
incorporated  in  their  own  chromosomes)  to 
study  these  questions.  Two  kinds  of  transgenic 


mice  have  been  created.  One  kind  bears  trans- 
genic TCR  genes;  the  other  has  transgenic  major 
histocompatibility  complex  (MHC)  genes  (a 
marker  that  distinguishes  us  individually  during 
transplantation) .  By  introducing  these  genes  back 
into  the  mouse  itself  and  into  the  mouse  germ- 
line,  we  can  determine  how  normal  T  cells  de- 
velop by  studying  how  the  TCR  and  MHC  interact 
during  development. 

This  strategy  has  been  very  successful.  By  creat- 
ing mice  of  appropriate  genetic  background,  we 
discovered  that  T  cells  that  are  self-recognizing 
and  hence  self-reactive  are  deleted  in  the  thymus 
during  development.  This  implies  that  part  of 
self-tolerance  is  accomplished  by  physical  elimi- 
nation of  self-reactive  T  cells.  Using  mice  con- 
taining transgenic  MHC,  we  showed  a  second 
mechanism  of  self-tolerance  that  does  not  involve 
physical  deletion.  In  this  case,  self-reactive  cells 
are  not  physically  eliminated  but  are  functionally 
paralyzed.  These  studies  allow  us  to  lay  the 
foundation  to  study  how  T  cells  acquire  self- 
tolerance.  Since  distinguishing  what  is  self  and 
nonself  is  a  central  problem  in  immunology,  we 
hope  that  these  studies  will  lead  to  a  better  un- 
derstanding of  transplantation  rejection  and  au- 
toimmune phenomena. 

The  detailed  study  of  T  cell  development  also 
allows  us  to  investigate  the  cellular  and  molecu- 
lar requirements  of  normal  cellular  developmen- 
tal processes.  For  example,  we  still  do  not  under- 
stand how  certain  T  cells  are  eliminated  while 
others  are  selected  to  survive.  To  elucidate  the 
exact  molecular  mechanisms  that  underlie  this 
highly  regulated  process,  we  have  most  recently 
focused  on  the  structural  and  signaling  basis  that 
controls  differential  cell  fate  of  the  developing 
thymocytes.  To  do  this  we  have  combined  the 
two  powerful  technologies  of  transgenic  mice 
and  gene  knock-out  mice.  In  the  latter  mice,  se- 
lected genes  can  be  targeted  to  be  destroyed  in 
the  germline  so  that  mice  bearing  selected  muta- 
tions can  be  created.  Once  these  mice  have  been 
prepared,  mutant  molecules  can  be  introduced  to 
replace  the  "knocked-out"  genes.  Current  efforts 
are  concentrated  on  signaling  molecules  such  as 
CD4  and  CDS  that  are  important  in  T  cell  devel- 


265 


The  Biology  of  T  Lymphocyte  Development 


opment.  By  such  an  approach,  we  hope  to  under-  mechanisms  by  which  T  cells  undergo  the  pro- 
stand  the  signaling  requirements  that  determine  grammed  cell  death  that  leads  eventually  to  self- 
cell  fate  during  development  as  well  as  the  exact  tolerance. 


266 


Molecular  Genetics  of  Mammalian 
Glycosyltransferases 


John  B.  Lowe,  M.D. — Assistant  Investigator 

Dr.  Lowe  is  also  Associate  Professor  of  Pathology  at  the  University  of  Michigan  Medical  School.  He  received 
his  bachelor's  degree  in  mathematics  from  the  University  of  Wyoming  and  his  M.D.  degree  from  the 
University  of  Utah  College  of  Medicine,  Salt  Lake  City.  He  was  trained  in  clinical  pathology  and  molecular 
genetics  at  Washington  University  School  of  Medicine,  St.  Louis.  He  was  later  Assistant  Professor  in  the 
Departments  of  Pathology  and  Medicine  at  Washington  University  and  also  served  as  Assistant  Medical 
Director  of  the  Barnes  Hospital  blood  bank,  St.  Louis,  before  moving  to  Michigan. 


THE  primary  long-range  goal  of  our  research  is 
to  understand  the  functions  of  oligosaccha- 
rides that  are  found  on  the  surface  of  mammalian 
cells  and  to  explain  how  the  cells  regulate  their 
expression.  Oligosaccharide  molecules  consist 
of  many  different  single-sugar  structures  linked 
together  in  complex  linear  and  branching  arrays. 
Quantitative  and  structural  changes  in  such  mole- 
cules have  been  shown  to  correlate  with  morpho- 
logic changes  that  occur  during  the  embryonic 
development  of  animals  and  in  association  with 
neoplastic  transformation.  These  and  other  ob- 
servations suggest  that  cell  surface  oligosaccha- 
rides may  function  as  information  bearers  in  me- 
diating interactions  between  cells  during  the 
developmental  process. 

Mammalian  cells,  in  constructing  these  mole- 
cules, use  special  proteins  called  glycosyltrans- 
ferase  enzymes.  With  few  exceptions,  a  unique 
glycosyltransferase  is  responsible  for  the  synthe- 
sis of  each  linkage  between  the  sugar  molecules 
in  an  oligosaccharide.  The  enormous  number  of 
different  oligosaccharides  dictates  that  many  dif- 
ferent glycosyltransferases  will  enter  the  con- 
struction of  the  complex  cell  surface  carbohy- 
drates on  any  particular  cell  or  tissue. 

In  many  instances,  changes  in  cell  surface  car- 
bohydrate structure  observed  during  differentia- 
tion or  in  association  with  malignant  transforma- 
tion have  been  shown  to  correlate  with  changes 
in  the  glycosyltransferase  repertoire.  The  mecha- 
nisms by  which  cells  coordinate  and  regulate  the 
expression  of  these  enzymes,  and  thus  the  ex- 
pression of  oligosaccharide  structures  at  the  cell 
surface,  are  largely  unknown.  During  the  past  few 
years,  the  main  focus  of  our  work  has  been  in 
establishing  systems  that  will  allow  molecular 
analysis  of  the  mammalian  genes  responsible  for 
glycosyltransferase  synthesis. 

The  human  ABO,  H,  and  Lewis  blood  group  an- 
tigens are  actually  cell  surface  oligosaccharides. 
The  determinant  genes  encode  particular  glyco- 
syltransferases that  are  able  to  construct  the 
"blood  group"  molecules.  These  glycosyltrans- 
ferases provide  convenient  genetic  and  biochemi- 
cal models  for  studying  how  the  processes  regu- 


late cell  surface  oligosaccharide  expression.  The 
blood  group  antigens  are  not  restricted  in  their 
expression  to  blood  cells.  They  are  found  on  a 
number  of  other  tissues  in  the  body,  suggesting 
that  tissue-specific  mechanisms  regulate  their  ex- 
pression. Moreover,  their  expression  changes 
during  human  embryonic  development  and  is  of- 
ten altered  in  malignancy. 

Our  initial  efforts  focused  on  developing  sys- 
tems to  isolate  glycosyltransferase  genes  without 
the  benefit  of  purified  enzyme  protein.  Using 
gene  transfer  approaches,  we  have  been  able  to 
isolate  several  of  these  genes.  They  include  hu- 
man genes  encoding  the  H  blood  group  a(l  ,2)fu- 
cosyltransferase  and  the  Lewis  blood  group 
q;(1,3/1,4)  fucosyltransferase . 

The  cloned  gene  segments  in  each  case  repre- 
sent tools  for  investigating  the  genetics  of  these 
enzymes  and  for  studying  the  function  and  regula- 
tion of  their  corresponding  cell  surface  oligosac- 
charides. For  example,  we  have  recently  used  the 
H  blood  group  gene  to  investigate  the  molecular 
basis  for  the  Bombay  blood  group  phenotype.  In- 
dividuals with  this  blood  group  are  extraordi- 
narily rare  and  are  cross-match  incompatible  with 
virtually  all  other  humans,  excepting  other  Bom- 
bay individuals.  This  incompatibility  is  due  to  the 
fact  that  these  persons  apparently  lack  a  func- 
tional H  blood  group  locus.  As  a  consequence, 
they  are  unable  to  construct  A,  B,  or  H  blood 
group  determinants,  and  thus  maintain  high  titers 
of  antibodies  directed  against  the  ABH  blood 
group  structures  found  on  red  cells  from  virtually 
all  other  humans. 

The  molecular  basis  for  the  defect  in  Bombay 
individuals  had  not  been  defined.  By  analyzing 
the  structure  of  the  H  gene  in  Bombay  pedigrees, 
we  identified  point  mutations  in  both  alleles  of 
the  gene  in  affected  individuals.  We  subse- 
quently demonstrated  that  these  mutations  inac- 
tivate the  enzyme  encoded  by  the  gene  and  are 
thus  responsible  for  the  Bombay  phenotype.  We 
have  also  analyzed  this  gene  in  para-Bombay  indi- 
viduals, whose  red  cells  are  deficient  in  ABH 
structures  but  whose  secretory  tissues  express  es- 
sentially normal  levels  of  these  molecules  (under 


267 


Molecular  Genetics  of  Mammalian  Glycosyltransferases 


the  control  of  the  Secretor  blood  group  locus). 
We  also  found  inactivating  point  mutations  in  the 
H  gene  in  these  persons,  thus  providing  strong 
evidence  that  the  human  Secretor  blood  group 
locus  corresponds  to  a  distinct  a(l  ,2)fucosyl- 
transferase  gene. 

We  have  also  used  glycosyltransferase  gene 
segments  to  identify  specific  cell  surface  oligosac- 
charide molecules  that  play  pivotal  functional 
roles  in  the  inflammatory  process.  One  of  the  pri- 
mary events  in  inflammatory  conditions  involves 
a  process  whereby  circulating  white  cells  leave 
the  interior  of  blood  vessels  and  become  local- 
ized in  inflammatory  foci  outside  the  vascular 
system.  This  process  begins  when  the  endothelial 
cells  lining  the  blood  vessels  become  "activated" 
by  substances  that  accompany  an  incipient  in- 
flammatory condition.  Circulating  white  cells 
known  as  neutrophils  adhere  tightly  to  activated 
endothelium,  insinuate  themselves  into  the  endo- 
thelial cell  pavement  lining  the  blood  vessel,  and 
ultimately  come  to  occupy  areas  outside  the  vas- 
cular tree. 

Neutrophil  adhesion  to  activated  endothelium 
is  mediated  in  part  by  a  protein  known  as  E- 
selectin,  or  endothelial  leukocyte  adhesion  mole- 
cule 1  (ELAM- 1 ) ,  which  is  found  on  the  surface  of 
activated  endothelium.  Structural  features  exhib- 
ited by  this  protein  suggested  that  it  might  inter- 
act with  an  oligosaccharide  molecule  specific  to 
the  surface  of  neutrophils.  By  transfecting  diff'er- 
ent  glycosyltransferase  gene  segments  into  mam- 
malian host  cells,  we  were  able  to  recapitulate 
the  biosynthesis  of  several  distinct  sets  of  cell  sur- 
face oligosaccharide  molecules  and  to  demon- 
strate that  one  set  allowed  transfected  cells  to  ad- 
here to  E-selectin.  We  further  demonstrated  that 
the  oligosaccharide  molecules  were  one  or 
more  members  of  a  family  of  oligosaccharides 
containing  sialic  acid  and  fucose  and  were  rep- 
resented by  a  molecule  known  as  the  sialyl  Lewis 
X  determinant.  More  recently,  we  have  shown 
that  E-selectin  maintains  a  relatively  high  specific- 
ity for  oligosaccharide  ligands  with  a(l,3)- 
linked  fucose  residues  in  terminal  positions,  ver- 
sus subterminal  locations.  We  have  also  shown 


that  terminal  «(!  ,3)fucosylation  may  be  cata- 
lyzed by  some,  but  not  all,  human  a(l,3)- 
fucosyltransferases . 

Aberrant  or  overexuberant  recruitment  of  neu- 
trophils to  sites  of  inflammation  can  contribute, 
after  tissue  hypoxia  and  in  other  pathological  cir- 
cumstances, to  undesirable  tissue  damage  in  au- 
toimmune disease.  Initial  events  in  this  process 
require  adhesive  interactions  between  E-selectin 
and  its  oligosaccharide  counter-receptors  on  the 
surfaces  of  neutrophils.  It  thus  seemed  possible 
that  purified  carbohydrate  counter-receptors 
might  function  as  anti-inflammatory  molecules 
by  preventing  pathologic  neutrophil  recruit- 
ment. We  have  tested  this  notion  in  collaboration 
with  Peter  Ward  here  at  the  University  of  Michi- 
gan. We  have  prepared  therapeutic  quantities 
of  sialyl  Lewis  X-containing  oligosaccharides, 
using  recombinant  «(!  ,3)fucosyltransferases. 
When  tested  in  an  animal  model  of  E-selectin- 
dependent  lung  inflammation,  these  carbohy- 
drates exhibit  potent  anti-inflammatory  activity. 
This  work  suggests  that  a  new  class  of  antiin- 
flammatory pharmaceuticals  may  be  developed 
from  such  molecules  or  from  their  chemical 
analogues. 

These  studies  have  been  aided  by  our  recent 
identification  of  several  novel  human  a(l,3)fu- 
cosyltransferase  genes.  We  have  shown  that  these 
encode  enzymes  with  shared  and  unique  primary 
sequence  domains,  as  well  as  distinctive  and  use- 
ful catalytic  properties.  For  example,  one  of 
these  works  extremely  well  for  in  vitro  synthesis 
of  the  sialyl  Lewis  X  tetrasaccharide,  and  others 
can  efficiently  construct  analogues  of  this  mole- 
cule. Work  is  in  progress  to  identify  sequence 
domains  within  the  enzymes  that  dictate  their 
distinctive  substrate  specificities. 

Circumstantial  evidence  gathered  by  other  in- 
vestigators suggests  that  oligosaccharides  are  im- 
portantly involved  in  cell  adhesion  during  mam- 
malian embryogenesis.  We  are  now  directing  our 
efi'orts  toward  exploring  this  hypothesis  and 
characterizing  the  genes  that  determine  these  in- 
teractions, through  genetic  manipulation  of  the 
murine  genome. 


268 


Mechanisms  of  Embryonic  Induction 
in  Vertebrates 


Richard  L.  Matts,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Maas  is  also  Assistant  Professor  of  Medicine  at  Harvard  Medical  School  and  Associate  Physician  at 
Brigham  and  Women's  Hospital,  Boston.  He  received  his  A.B.  degree  in  chemistry  from  Dartmouth  College 
and  an  M.D.  -Ph.D.  degree  from  Vanderbilt  University  School  of  Medicine.  Following  his  thesis  work  with 
John  Oates,  he  trained  as  a  medical  house  officer  at  Brigham  and  Women's  Hospital  and  completed  a 
postdoctoral  fellowship  in  Philip  Leder's  laboratory  in  the  Department  of  Genetics  at  Harvard  Medical  School. 


THE  goal  of  our  research  is  to  understand  the 
role  that  the  homeobox  genes  play  in  control- 
ling vertebrate  organogenesis.  These  genes  are 
defined  by  their  expression  of  a  60-amino  acid, 
helix-turn-helix,  DNA-binding  domain.  Highly 
conserved  in  evolution,  they  are  present  in  spe- 
cies as  divergent  as  Drosophila,  yeast,  and  hu- 
mans. Mutations  in  homeobox  genes  of  fruit  fly 
and  mouse  result  in  specific  developmental  de- 
fects. Our  work  thus  far  has  focused  on  the  char- 
acterization of  two  such  genes  that  appear  to  play 
important  roles  in  the  formation  of  the  mamma- 
lian kidney  and  eye,  respectively.  A  long-term 
goal  is  to  understand  the  target  genes  that  these 
homeobox  genes  interact  with,  using  a  combina- 
tion of  biochemical,  embryologic,  and  genetic 
techniques. 

Murine  Homeobox  Genes  Expressed  in 
Mouse  Embryonic  Kidney 

To  determine  which  homeobox  genes  are  ex- 
pressed in  the  developing  mouse  kidney,  we  un- 
dertook a  polymerase  chain  reaction  (PGR) 
screen  of  reverse-transcribed,  microdissected 
kidney  RNA  from  mouse  embryos  at  day  1 5 .  This 
experiment  yielded  27  different  homeobox- 
containing  genes,  some  11  of  which  correspond 
to  new  genes.  Among  these  novel  sequences,  we 
identified  several  with  85-98  percent  sequence 
similarity  to  known  murine  Hox  genes  at  the  nu- 
cleotide level.  In  addition,  we  identified  two 
other  genes,  closely  related  to  each  other,  that 
appear  to  be  new  members  of  the  Hox-1  and 
Hox- 3  clusters  in  the  mouse.  Mapping  experi- 
ments indicate  that  the  Hox-1  member  is 
Hox- 1.8. 

The  structures  of  several  cDNA  clones  of  Hox- 
1.8  are  being  determined.  Thus  far,  five  different 
alternatively  processed  forms  have  been  identi- 
fied. Surprisingly,  all  these  forms  share  a  com- 
mon feature:  due  to  the  presence  of  upstream  ter- 
mination codons,  none  would  actually  encode  a 
translatable  homeodomain.  We  suspect  that  a 
homeodomain-encoding  form  exists,  because  the 
homeodomain  is  preserved  intact  at  the  sequence 
level.  Current  efforts  are  aimed  at  securing  the  5' 


end  of  the  Hox- 1.8  gene  in  order  to  determine 
whether  splice  forms  exist  that  would  encode  a 
functional  homeodomain. 

A  current  working  hypothesis  of  our  laboratory 
is  that  many  murine  Hox  genes  may,  as  a  general 
rule,  encode  both  homeobox-containing  and 
homeobox-less  forms.  Such  forms  may  interact  in 
heterodimeric  combinations  with  one  another,  or 
with  other  Hox  genes,  to  alfect  the  capacity  of  the 
homeobox-containing  form  to  bind  to  DNA. 

The  expression  of  the  //ox- 7. S  transcripts  has 
been  analyzed  as  a  function  of  mouse  embryogen- 
esis.  As  determined  by  Northern  blot  analysis,  sig- 
nificant expression  appears  at  day  1 0  of  embryo- 
genesis,  peaks  at  day  13,  and  subsides  by  day  15. 
Expression  in  adults  is  confined  to  the  kidney  and 
to  skeletal  muscle.  Interestingly,  the  adult  kidney 
appears  to  express  a  smaller  transcript  form  of 
approximately  1.5  kb,  in  addition  to  the  larger 
class  of  2.8-3.9  kb  observed  in  both  embryos  and 
kidney.  This  size  range  corresponds  to  extensive 
alternate  processing,  as  noted  above. 

We  have  further  analyzed  the  expression  of 
Hox- 1 .8  duTing  embryogenesis  by  in  situ  hybrid- 
ization. Regionally  restricted  expression  is  ob- 
served in  the  submucosa  of  the  foregut  and  mid- 
gut and  also  in  somites.  Of  particular  interest  to 
the  potential  role  of  this  gene  in  nephrogenesis  is 
its  expression  in  the  condensing  collecting  duct 
system,  in  the  region  that  comprises  the  develop- 
ing calyces.  Three-dimensional  reconstruction  of 
the  Hox- 1.8  expression  pattern  shows  that  ex- 
pression is  localized  to  this  part  of  the  developing 
kidney  at  embryonic  day  1 3  • 

Identification  of  a  Pax  Gene  Involved 
in  Formation  of  the  Vertebrate  Eye 

The  formation  of  the  vertebrate  eye  has  long 
served  as  an  attractive  model  system  for  studying 
basic  features  of  embryonic  induction.  The  eye 
forms  as  a  consequence  of  outgrowth  of  the  dien- 
cephalon  and  a  subsequent  interaction  of  this 
neuroectoderm-derived  structure  with  the  sur- 
face ectoderm,  resulting  in  an  invagination  of  the 
latter  to  form  the  lens  vesicle.  Additional  neuroec- 
toderm and  mesodermal  ingrowth  anterior  to  the 


269 


Mechanisms  of  Embryonic  Induction  in  Vertebrates 


lens  results  in  formation  of  the  anterior  structures 
of  the  eye,  specifically  the  iris,  cornea,  and  ciliary 
body.  An  additional  interaction,  between  the  de- 
veloping lens  and  retina,  is  suggested  by  experi- 
ments involving  transgenic  mice:  ablation  of  the 
developing  lens  results  in  an  abnormal  prolifera- 
tion of  retina,  perhaps  suggesting  the  presence  of 
an  inhibitory  factor  from  lens. 

In  both  mouse  and  human,  there  are  naturally 
occurring  mutations  that  affect  eye  development. 
In  the  mouse,  a  mutation  on  chromosome  2, 
called  Small  eye  {Sey),  results  in  deficient  eye 
formation.  The  portion  of  mouse  chromosome  2 
to  which  Sey  maps  is  homologous  to  a  portion  of 
human  chromosome  11,  llpl3.  Interestingly,  a 
semidominant  human  disorder  called  aniridia, 
which  also  affects  basic  eye  development,  is  lo- 
cated in  this  region  of  1 1 .  The  observation  that  a 
mouse  paired  box  and  homeobox  gene.  Pax- 6, 
maps  to  the  Sey  locus  prompted  us  to  examine 
the  role  of  this  gene  and  its  human  counterpart  in 
basic  ocular  development.  Recently  it  has  been 
shown  that  point  mutations  in  the  mouse  Pax- 6 
gene  are  in  fact  responsible  for  the  Sey  mutation. 


We  have  cloned  the  human  PAX6 gene,  which 
maps  to  within  200  kb  or  less  of  the  human  ani- 
ridia locus,  as  defined  by  various  translocation 
breakpoints.  This  gene  is  more  than  95  percent 
conserved  over  422  amino  acids  with  a  related 
gene  cloned  from  zebra  fish,  which  is  expressed 
in  both  the  diencephalon  and  lens  vesicle  during 
ocular  development.  Thus  both  the  chromosomal 
location  and  the  expression  pattern  of  this  gene 
are  consistent  with  its  involvement  in  aniridia. 

We  have  determined  the  genomic  structure  of 
the  PAX6  gene,  which  consists  of  at  least  13 
exons  spread  over  some  30  kb  of  genomic  DNA, 
and  have  sequenced  the  intron-exon  boundaries. 
This  has  permitted  the  design  of  PGR  primers  to 
each  exon,  which  are  conveniently  sized  for  anal- 
ysis by  PGR.  Thus  far  we  have  identified  a  non- 
sense mutation  located  in  helix  2  of  the  homeo- 
box of  the  human  PAX6  gene  in  one  patient  and, 
preliminarily,  a  small  insertion  in  the  PAX6  gene 
of  another  patient.  Although  analysis  of  more  mu- 
tations will  be  required  to  establish  that  muta- 
tions in  PAX6  indeed  account  for  aniridia,  these 
findings  argue  persuasively  that  they  do. 


270 


Cell  Cycle  Control 


James  L.  Mailer,  Ph.D. — Investigator 

Dr.  Mailer  is  also  Professor  of  Pharmacology  at  the  University  of  Colorado  School  of  Medicine.  He  received 
his  B.S.  degree  in  biochemistry  from  Cornell  University  and  his  Ph.D.  degree  in  molecular  biology  from 
the  University  of  California,  Berkeley,  where  he  worked  with  John  Gerhart.  He  then  carried  out 
postdoctoral  studies  with  Edwin  Krebs  at  both  the  University  of  California,  Davis,  and  the  University 
of  Washington  before  moving  to  Colorado. 


TWO  events  mark  the  reproductive  life  of  a 
cell:  replication  of  the  DNA,  and  its  distribu- 
tion to  daughter  cells  at  mitosis.  Because  of  the 
central  importance  of  cell  reproduction  to  or- 
dered cell  growth  and  to  birth  of  the  next  genera- 
tion, cells  have  evolved  rigorous  controls  to  en- 
sure that  both  events  are  carried  out  with  high 
fidelity  and  at  the  appropriate  time.  My  labora- 
tory is  interested  in  understanding  the  nature  and 
regulation  of  these  controls  with  respect  to  how  a 
cell  commits  itself  to  replicate  its  DNA  and  how  it 
knows  when  to  divide. 

The  cell  cycle  has  four  main  phases:  Gi,  S  (syn- 
thesis), G2,  and  mitosis.  The  decision  to  synthe- 
size DNA  (to  enter  the  S  phase)  is  made  in  Gj ,  and 
the  decision  to  begin  cell  division  (to  enter  the  M 
phase  of  mitosis)  is  made  in  G2.  There  is  abun- 
dant evidence  that  these  decisions  are  made  at 
checkpoints,  or  restriction  points,  in  the  cycle. 
The  nature  of  these  Gj  and  G2  decision-making 
periods  in  the  cell  cycle  underlies  fundamental 
processes  operative  in  early  embryonic  develop- 
ment and  in  malignant  cells. 

G2  ^  M  Regulation 

Our  laboratory  developed  a  G2-phase  extract 
from  frog  eggs  in  which  synthetic  nuclei  entered 
mitosis  at  the  addition  of  mitotic  signals.  We  then 
purified  the  mitosis-signaling  enzyme  (called 
maturation-promoting  factor,  or  MPF)  and  found 
that  it  was  composed  of  a  protein  kinase  com- 
plexed  to  a  G2  cyclin.  Kinases  have  the  ability  to 
attach  a  phosphate  group  to  many  different  cellu- 
lar proteins,  modifying  their  function  and  caus- 
ing profound  changes  in  cellular  biochemistry. 
The  protein  kinase  was  identified  as  a  vertebrate 
homologue  of  the  cdc2  gene,  which  had  been 
genetically  implicated  in  the  control  of  mitosis 
by  the  study  of  certain  mutants  in  yeast. 

G2  cyclins  are  proteins  that  accumulate  during 
interphase,  reach  high  levels  in  late  G2,  and  are 
then  degraded  near  the  metaphase  anaphase 
transition  in  mitosis.  This  degradation  is  required 
in  order  for  cells  to  complete  mitosis  successfully 
and  enter  Gj .  In  most  cells  there  are  two  classes  of 
G2  cyclins,  termed  A  and  B  cyclins,  that  differ  in 


sequence  similarity  and  have  different  kinetics  of 
accumulation  and  degradation.  Both  bind  cdc2 
kinase,  but  A- type  complexes  are  activated  much 
earlier  in  the  cell  cycle  than  B-type  complexes, 
and  only  B-rype  cyclins  are  found  in  puri- 
fied MPF. 

To  investigate  the  role  of  cyclin  A,  we  utilized 
extracts  from  metaphase-arrested  eggs  that  are 
able  to  exit  mitosis  in  vitro,  to  undergo  DNA  syn- 
thesis, and  then  to  reenter  mitosis.  These  extracts 
retain  the  characteristic  dependence  of  mitosis 
upon  completion  of  DNA  synthesis — that  is,  will 
not  enter  mitosis  if  DNA  synthesis  has  not  been 
completed,  which  can  result  from  an  excess  of 
DNA  in  the  system  or  the  presence  of  aphidicolin, 
an  inhibitor  of  DNA  polymerase.  By  using  anti- 
sense  oligodeoxynucleotides  to  ablate  cyclin  A 
mRNA  from  the  system,  we  were  able  to  show  that 
activation  of  MPF  (cyclin  B/cdc2)  occurred  even 
when  DNA  synthesis  had  not  been  completed. 
Readdition  of  recombinant  cyclin  A  protein  to  the 
antisense-ablated  extracts  restored  the  depen- 
dence of  mitosis  on  DNA  synthesis  by  causing  a 
lengthening  of  S  phase  until  DNA  synthesis  was 
complete. 

This  provides  evidence  that  one  of  the  func- 
tions of  cyclin  A  is  in  the  crucial  checkpoint  that 
prevents  the  activation  of  cyclin  B/cdc2  (MPF) 
until  DNA  synthesis  has  been  completed.  It  also 
explains  why  cyclin  A/cdc2  complexes  are  acti- 
vated earlier  in  the  cell  cycle  than  cyclin  B/cdc2 
complexes  and  why  cyclin  A  is  degraded  before 
cyclin  B.  The  latter  phenomenon,  long  known,  is 
clearly  appropriate,  since  cyclin  A  exerts  an  inhib- 
itory function  on  cyclin  B.  A  question  that  now 
merits  attention  concerns  what  substrates  exist 
for  phosphorylation  by  cyclin  A/cdc2  that  are  in- 
volved in  this  feedback  control  mechanism. 

We  are  interested  in  the  mechanism  of  activa- 
tion of  MPF  in  oocytes  during  the  cell  cycles  of 
meiosis  I  and  II.  In  these  cycles  the  synthesis  of 
proteins  other  than  cyclin  are  required  for  MPF 
activation.  One  protein  required  for  meiosis  I  and 
II  is  the  product  of  the  mos  proto-oncogene. 
Proto-oncogenes  are  the  normal  cellular  counter- 
part of  mutated  oncogenes  found  in  cancer  cells, 


271 


Cell  Cycle  Control 


suggesting  that  oncogenes  act  by  perturbing  nor- 
mal cellular  pathways.  In  general,  very  little  is 
known  about  how  proto-oncogenes  work,  but  the 
specific  involvement  of  mos  in  cell  cycle  control 
is  the  clearest  example  of  a  specific  function  for 
any  proto-oncogene  in  a  defined  cellular  process. 
The  mos  gene  encodes  a  serine/threonine  pro- 
tein kinase,  indicating  the  existence  of  a  substrate 
for  phosphorylation  by  mos  that  can  lead  to  acti- 
vation of  MPF  as  well  as  stabilization  of  cyclin  in 
the  metaphase  arrest  of  meiosis  II.  The  transition 
between  meiosis  I  and  II  is  also  perhaps  the  only 
well-documented  case  in  which  cdc2  kinase  ac- 
tivity declines  without  accompanying  cyclin  B 
degradation. 

This  year  Linda  Roy  observed  that  introduction 
of  protein  synthesis  inhibitors  into  the  system  at 
the  transition  between  meiosis  I  and  II  leads  to 
the  immediate  destruction  of  cyclin  B,  which 
would  otherwise  be  stable,  thereby  establishing 
that  the  stability  of  cyclins  between  meiosis  I  and 
II  requires  continuous  synthesis  of  protein.  Ini- 
tially we  expected  that  the  protein  synthesis  re- 
quired for  cyclin  stability  would  be  exemplified 
by  the  synthesis  of  the  mos  proto-oncogene  ki- 
nase itself,  since  that  kinase  is  known  to  comprise 
one  of  the  major  newly  synthesized  proteins  dur- 
ing maturation.  However,  Dr.  Roy  found  that,  in 
fact,  the  mos  kinase  remains  fully  active  even 
when  B-type  cyclins  are  degraded.  This  implies 
that  there  is  another  protein  whose  synthesis  is 
required  for  cyclin  stability  besides  the  mos 
proto-oncogene . 

Dr.  Roy  also  did  the  converse  experiment  by 
microinjecting  antisense  oligodeoxynucleotides 
against  the  mos  kinase  mRNA  into  cells  just  prior 
to  entry  into  meiosis  I.  By  this  procedure  she  was 
able  to  ablate  greatly  the  level  of  mos  proto- 
oncogene  expression.  However,  under  these  con- 
ditions, cyclin  B  does  not  undergo  degradation 
between  meiosis  I  and  II,  thereby  establishing 
that  mos  is  not  necessary  for  the  stability  of  cyclin 
B  between  meiosis  I  and  II.  These  results  suggest 
that  other  components  besides  the  mos  kinase  are 
required  for  the  unusual  cyclin  stability  between 
meiosis  I  and  II,  and  clearly  the  identification  of 
these  other  components  merits  attention. 

Gj  -*■  S  Regulation 

The  cell  cycle  restriction  point  in  Gj  governing 
the  Gi  ^  S  transition  that  involves  cdc2-like  ki- 
nases has  been  termed  START  in  yeast  and  the  R 
point  in  mammalian  cells.  In  both  budding  yeast 


and  fission  yeast,  it  is  quite  clear  that  the  genuine 
cdc2  gene  product  mediates  both  the  Gj  and  G2 
control  points.  Recently  it  has  become  evident 
that  the  regulation  of  the  Gj  ^  S  transition  in 
vertebrate  cells  is  considerably  more  compli- 
cated than  in  yeast.  One  aspect  of  this  complexity 
involves  the  presence  of  another  cdc2-like  gene 
in  the  Gj  phase  that  may  mediate  events  at  the 
Gj  -*  S  transition.  This  gene  was  originally  discov- 
ered in  Xenopus  and  given  the  name  Egl  but  has 
since  been  renamed  cyclin-dependent  kinase  2, 
or  cdk2.  The  idea  that  cdk2  might  be  a  form  of 
cdc2  specialized  for  Gj  control  has  come  from 
the  finding  by  others  that  cdk2  itself  will  not 
complement  mutations  in  the  Saccharomyces  cere- 
visiae  cdc2  cognate  gene  CDC28  that  affect  the 
G2  ^  M  restriction  point,  but  will  substitute  at  least 
partially  for  the  Gj  function  of  when  coex- 

pressed  with  a  Gj  cyclin  from  human  cells. 

This  year  we  have  made  a  major  effort  to  study 
the  biochemistry  and  regulation  of  cdk2  in  the 
Xenopus  embryonic  cell  system.  Toward  this  end 
we  have  developed  an  antibody  against  the  pro- 
tein and  used  it  to  show  that  the  protein  kinase 
activity  of  cdk2  oscillates  in  the  cell  cycle  with  a 
periodicity  similar  to  cdc2.  In  the  Xenopus  em- 
bryonic cell  cycle,  cdk2  is  not  associated  with 
either  cyclin  A  or  cyclin  B,  but  instead  with  two 
proteins  of  36  and  48  kDa.  This  suggested  the 
possibility  that  cdk2  would  be  regulated  by  phos- 
phorylation, perhaps  in  ways  similar  to  the 
known  regulation  by  phosphorylation  of  cdc2  ki- 
nase itself. 

Recently  we  showed  that  in  fact  cdk2  is  a  phos- 
phoprotein,  that  it  is  phosphorylated  on  tyrosine 
and  serine  residues,  and  that  phosphorylation  of 
these  residues  changes  during  the  cell  cycle.  In 
particular,  the  presence  of  phosphotyrosine  cor- 
relates with  less-active  forms  of  the  protein,  but 
the  major  site  of  phosphorylation  was  determined 
by  tryptic  phosphopeptide  mapping  to  be  in  a 
phosphopeptide  distinct  from  the  one  containing 
tyrosine  15,  so  characteristic  of  cdc2  kinase.  De- 
spite this  diff^erence  in  the  sites  of  phosphoryla- 
tion, however,  it  would  appear  that  the  same  en- 
zyme responsible  for  the  dephosphorylation  of 
cdc2  is  also  active  on  cdk2. 

By  continuing  to  study  both  cdc2  kinase  in  the 
G2  ^  M  transition  and  cdk2  kinase  in  the  Gj  ^  S 
transition,  it  should  be  possible  to  achieve  a  com- 
prehensive understanding  of  cell  cycle  control  at 
the  two  main  restriction  points  present  in  eukar- 
yotic  cells. 


272 


The  Role  of  T  Cells  in  Health  and  Sickness 


Philippa  Marrack,  Ph.D. — Investigator 

Dr.  Marrack  is  also  a  member  of  the  Division  of  Basic  Immunology  of  the  Department  of  Medicine  at  the 
National  Jewish  Center  for  Immunology  and  Respiratory  Medicine,  Denver,  and  Professor  of  Biochemistry, 
Biophysics  and  Genetics,  of  Microbiology  and  Immunology,  and  of  Medicine  at  the  University  of  Colorado 
Health  Sciences  Center,  Denver.  She  took  her  Ph.D.  in  biological  sciences  at  the  MRC  Laboratory  for 
Molecular  Biology  in  Cambridge,  England,  and  then  did  postdoctoral  work  with  Richard  Dutton  at  the 
University  of  California,  San  Diego.  From  there  she  moved  to  the  University  of  Rochester,  and,  after  seven 
years,  to  her  present  position.  Dr.  Marrack  is  a  member  of  the  National  Academy  of  Sciences  and  was 
recently  awarded  the  Christopher  Columbus  Discovery  Award  for  Biomedical  Science. 


T cells  are  essential  to  the  ability  of  higher  ver- 
tebrates to  resist  disease.  These  cells  are  not 
only  able  to  destroy  invading  organisms  by  killing 
cells  in  which  such  organisms  live,  but  they  also 
produce  hormone-like  substances  that  contribute 
to  protection  against  invasion,  by  stimulating  pro- 
duction of  protective  antibodies,  for  example. 

T  cells  bear  receptors  for  antigen  on  their  sur- 
face. There  are  about  20,000  receptors  on  each 
cell.  These  are  composed  of  tw^o  polypeptide 
chains,  ol  and  (8,  each  made  up  of  a  number  of 
variable  elements:  Va,  Ja,  V/3,  D/?,  andJjS.  In  each 
T  cell  the  receptors  are  composed  of  different 
combinations  of  these  elements,  so  that  each  T 
cell  has  receptors  with  a  somewhat  different 
structure. 

As  far  as  we  know,  receptors  are  assembled  ran- 
domly from  the  available  Vas,  Jas,  and  so  on, 
while  T  cells  are  developing.  Consequently  there 
is  a  distinct  possibility  that  some  T  cells  will  bear 
receptors  able  to  interact  with  self  antigens,  i.e., 
components  of  the  individual  containing  the  T 
cells.  These  cells  are  a  potential  threat,  since  they 
could  attack  and  destroy  their  own  host.  Usually, 
however,  they  themselves  are  destroyed  or  inacti- 
vated before  they  become  mature  enough  to 
cause  damage. 

There  is  reason  to  believe  that  some  potentially 
self-reactive  T  cells  avoid  the  processes  of  toler- 
ance and  are  allowed  to  mature.  Even  though  they 
could  attack  tissues  of  their  host,  they  seem  not 
to,  perhaps  because  the  self  antigens  with  which 
they  could  interact  are  sequestered  in  a  tissue 
that  they  cannot  reach.  Occasionally,  however, 
these  self-reactive  cells  are  activated  by  en- 
counter with  an  environmental  antigen.  After 
stimulation  the  cells  become  more  motile  and  ac- 
tive and  are  then  able  to  interact  with  and  destroy 
the  tissues  of  their  host,  causing  a  so-called  au- 
toimmune disease. 

A  number  of  human  diseases — juvenile  dia- 
betes, lupus  erythematosus,  and  multiple  sclero- 
sis among  others — may  be  due  to  T  cell  malfunc- 
tion similar  to  that  described  above.  Our  own 


work,  in  collaboration  with  Brian  Kotzin  (Depart- 
ment of  Pediatrics,  National  Jewish  Center),  has 
concentrated  on  rheumatoid  arthritis.  About  2 
million  people  in  the  United  States  suffer  from 
this  disease,  which  seems  to  be  due  to  an  immune 
attack  on  material  in  joints.  We  have  shown  that 
many  of  those  afflicted  have  unexpectedly  low 
levels  of  T  cells  bearing  V|8 1 4  in  their  blood,  and 
some  of  the  missing  cells  are  present  in  the  fluid 
bathing  the  rheumatic  joints. 

We  have  suggested  that  rheumatoid  arthritis  in- 
volves chronic  invasion  of  the  host  by  a  foreign 
antigen  able  to  interact  with  T  cells  bearing 
V/314.  This  antigen  would  first  stimulate  target  T 
cells,  and  then  cause  their  disappearance,  proba- 
bly by  the  processes  of  tolerance  described 
above.  Perhaps  the  subset  of  V(8 14 -bearing  cells 
that  can  recognize  self  antigens  is  waylaid  and 
rescued  from  death  by  sequestration  in  the  joints. 
Here  they  cause  inflammation  and  the  symptoms 
of  arthritis. 

At  the  moment  all  these  are  simply  ideas  upon 
which  to  base  future  experiments  on  the  cause, 
treatment,  and  prevention  of  rheumatoid  arthri- 
tis. Meanwhile,  we  have  begun  a  search  for  the 
foreign  antigen  that  may  interact  with  V|8l4- 
bearing  T  cells  and  thus  start  the  disease.  There  is 
every  indication  that  this  is  a  superantigen,  a  spe- 
cial kind  of  antigen  that  interacts  with  T  cells, 
primarily  via  the  VjS  portion  of  their  receptors.  It 
is  also  likely,  as  mentioned  above,  that  the  anti- 
gen is  produced  by  a  chronic  infectious  agent, 
such  as  a  chronic  virus. 

Until  recently  the  only  infectious  agents 
known  to  produce  superantigens  were  myco- 
plasma and  bacteria,  such  as  staphylococci  and 
streptococci.  These  agents  do  not  usually  infect 
their  hosts  in  a  chronic  fashion.  Early  in  1991, 
however,  we  and  several  other  groups  showed 
that  certain  viruses,  a  collection  of  retroviruses 
present  in  mice,  can  also  express  superantigens. 
Encouraged  by  this  result,  we  decided  to  screen 
human  viruses  for  such  expression.  Our  prelimi- 
nary results  suggest  that  Epstein-Barr  virus,  the 


273 


The  Role  of  T  Cells  in  Health  and  Sickness 


cause  of  a  common  chronic  infection  and  of  in-  that  Epstein-Barr  virus  is  the  causative  agent  of 
fectious  mononucleosis  in  humans,  may  indeed  rheumatoid  arthritis,  it  is  possible  that  other 
encode  a  superantigen.  While  we  do  not  think    members  of  the  Herpesviridae  family  may  be. 


274 


Cell  Regulation  by  Transforming 
Growth  Factors 


Joan  Massague,  Ph.D. — Investigator 

Dr.  Massague  is  also  a  member  of  the  Cell  Biology  and  Genetics  Program  at  Memorial  Sloan- Kettering 
Cancer  Center  and  Professor  of  Cell  Biology  at  Cornell  University  Graduate  School  of  Medical  Sciences, 
New  York.  He  received  his  Ph.D.  degree  in  biochemistry  from  the  University  of  Barcelona,  Spain,  and  was 
a  postdoctoral  fellow  with  Michael  Czech  at  Brown  University.  He  was  Assistant  and  Associate  Professor 
of  Biochemistry  at  the  University  of  Massachusetts  Medical  School  before  assuming  his  present  position. 


THE  proliferation  of  cells  is  controlled  by  a 
balance  of  positive  and  negative  signals.  The 
machinery  that  conveys  growth  inhibitory  signals 
is  similar  in  design  to  that  which  signals  cell 
growth.  Both  involve  1)  factors  that  circulate  be- 
tween cells  and  2)  membrane  receptors  that  are 
coupled  to  signal  transduction  circuitry  inside 
the  cell.  The  signals  carried  by  growth-promoting 
factors  have  been  extensively  studied  for  the  past 
two  decades.  The  growth  inhibitors,  however, 
have  come  to  the  attention  of  biologists  only  re- 
cently. Yet  they  include  some  of  the  most  wide- 
spread and  versatile  regulators  of  cell  growth  and 
phenotype.  Some  of  them  are  implicated  in  pro- 
cesses of  development,  tissue  repair,  and  recy- 
cling, and  their  study  may  show  us  ways  to  con- 
strain the  unrestricted  growth  of  cancer  cells. 

Multifunctional  Growth  Inhibitors 
and  Their  Receptors 

Ranking  high  in  the  list  of  growth  inhibitors, 
and  in  our  research  interests,  is  the  polypeptide 
TGF-(S  (transforming  growth  factor-/?) .  In  reality, 
TGF-(S  represents  a  large  family  of  growth  and  dif- 
ferentiation factors  that  also  includes  the  acti- 
vins,  the  bone  morphogenetic  proteins,  the 
Miillerian  inhibiting  substance,  and  others.  The 
evolutionary  conservation  of  these  factors  is  un- 
usually strict,  and  they  are  broadly  multifunc- 
tional. For  example,  TGF-)8  can  inhibit  cell  prolif- 
eration, regulate  cell  differentiation,  affect  how 
cells  organize  tissue  structures,  and  perform 
various  other  functions  in  cells  from  virtually 
every  lineage.  Likewise,  other  members  of  the 
family,  such  as  activin  and  decapentaplegic,  are 
involved  in  body  axis  formation  during  embryo 
development. 

Over  the  past  year  our  research  program  has 
centered  on  identifying  and  isolating  genes  that 
encode  receptors  for  TGF-(8  and  related  factors. 
We  had  previously  determined  that  TGF-|8  binds 
to  various  types  of  receptor  proteins  on  the  cell 
membrane,  and  we  have  recently  cloned  genes 
for  two  classes  of  such  receptors.  One  class  com- 
prises receptor  membrane  proteins  that  can  trig- 


ger a  biochemical  reaction  inside  the  cell  upon 
binding  a  factor  on  the  outside.  The  receptors  in 
this  class  probably  mediate  most  cellular  re- 
sponses to  these  factors.  Like  the  factors  they 
bind,  these  receptors  exist  in  many  variants,  each 
probably  representing  a  discrete  adaptation  to 
achieve  optimal  control  of  cell  functions. 

One  member  of  this  receptor  class  binds  the 
TGF-/3-related  factor  activin.  The  biochemical  re- 
action carried  out  by  this  receptor  is  the  transfer 
of  phosphate  from  ATP  to  serine  and  threonine 
groups  present  in  certain  intracellular  proteins. 
Phosphorylation  of  serines  and  threonines  repre- 
sents a  significant  departure  from  previously 
known  receptor  signals.  In  contrast  to  the  recep- 
tors for  activin  and  TGF-|8,  those  for  growth- 
promoting  factors  typically  trigger  phosphoryla- 
tion of  tyrosine  groups  in  proteins.  Thus  the  tyro- 
sine phosphorylation  signals  of  mitogens  are 
challenged  by  the  serine/threonine  phosphoryla- 
tion signals  of  antimitogens.  These  findings  offer 
new  insight  into  the  counteracting  signals  that 
preserve  the  normal  balance  of  cell  growth.  This 
work  has  been  supported  by  a  grant  from  the  Na- 
tional Cancer  Institute. 

Receptor  Accessory  Molecules 

The  other  TGF-/?  receptor  class  recently  cloned 
by  our  group  is  interesting  for  other  reasons.  This 
protein,  called  betaglycan,  is  thought  to  act  as  a 
helper  of  the  signaling  receptors.  Rather  than  me- 
diate cell  responses  directly,  betaglycan  seems  to 
regulate  the  access  of  cells  to  TGF-|8  by  either 
helping  present  this  factor  to  the  signaling  recep- 
tors or  storing  it  for  later  use  by  the  cell. 

The  structure  of  betaglycan  is  unusual  for  a 
growth  factor-binding  protein.  It  consists  of  a 
core  protein  that  carries  a  large  mass  of  negatively 
charged  carbohydrate.  TGF-/3  binds  the  core  pro- 
tein, whereas  the  carbohydrate  can  bind  the  so- 
called  heparin-binding  growth  factors.  Work  is 
under  way  to  map  the  portion  of  this  molecule 
that  binds  TGF-jS  and  to  test  its  ability  as  a  modula- 
tor of  TGF-|8  activity. 

With  the  cloning  of  these  genes,  it  is  now  possi- 


275 


Cell  Regulation  by  Transforming  Growth  Factors 


ble  to  explore  their  properties  in  detail  and  to 
identify  key  pieces  of  the  machinery  that  transfers 
growth  inhibitory  signals  from  the  membrane  to 
the  nucleus.  Furthermore,  it  should  be  possible 
to  determine  to  what  extent  the  genetic  loss  of 
TGF-jS  receptors  might  cause  loss  of  constraint  in 
cell  proliferation  and  thus  incite  tumor  cell 
outgrowth. 

Cell-Cell  Stimulation  by  Membrane-bound 
Growth  Factors 

Paracrine  growth  factors  and  polypeptide  hor- 
mones are  generally  synthesized  as  larger  soluble 
precursors  that  are  later  fragmented  to  yield  the 
bioactive  forms.  In  a  recently  found  variation  of 
this  theme,  factors  such  as  TGF-a  (no  structural 
relationship  to  TGF-jS)  are  generated  from  mem- 
brane-anchored proteins  rather  than  from  soluble 
precursors.  The  TGF-a  precursor  can  accumulate 
on  the  cell  membrane  and  bind  to  receptors  lo- 
cated on  the  surface  of  adjacent  cells.  This  inter- 
action can  sustain  cell-cell  adhesion  and  stimu- 
late DNA  replication  by  cell-cell  contact. 


In  work  supported  by  a  grant  from  the  National 
Cancer  Institute,  we  have  shown  that  generation 
of  TGF-a  by  cleavage  of  its  precursor  occurs  at 
the  cell  surface  by  a  highly  regulated  enzymatic 
system.  This  system  is  strongly  activated  by 
tumor-promoting  phorbol  esters  and  growth  fac- 
tors via  mechanisms  involving  protein  kinase  C 
and  calcium  influx  into  the  cell.  Thus  the  precur- 
sor cleavage  process  functions  as  a  regulated 
switch  between  two  active  forms  of  the  grouT:h 
factor,  one  membrane-bound  and  the  other  diffus- 
ible. The  membrane-bound  forms  could  be  im- 
portant in  tissue  development  processes  whose 
guidance  depends  on  discrete  cell-cell  interac- 
tions incompatible  with  the  diffusible  nature  of 
soluble  factors.  The  regulated  nature  of  the  pro- 
TGF-a  cleavage  process  renders  it  susceptible  to 
exogenous  control  with  pharmacologic  agents. 
Furthermore,  it  provides  a  way  to  identify  the 
pieces  of  the  general  machinery  controlling  the 
release  of  this  and  other  membrane  proteins  that 
mediate  cell-to-cell  interaction. 


In  this  molecular  model,  a  benzene  molecule 
(in  yellow)  is  shown  buried  within  the  core  of 
T4  lysozyme  in  a  cavity  created  by  replacing 
leucine  99  with  alanine.  The  inner  dotted  form 
represents  the  van  der  Waals  surface  of  the  ben- 
zene, and  the  outer  envelope,  the  van  der  Waals 
surface  of  the  cavity. 

Research  and  photograph  by  A.  Elisabeth 
Eriksson  and  Xue-jun  Zhang  in  the  laboratory 
of  Brian  Matthews. 


276 


Structural  Basis  of  Interactions  Within 
and  Between  Macromolecules 


f 


Brian  W.  Matthews,  Ph.D.,  D.Sc. — Investigator 

Dr.  Matthews  is  also  Professor  of  Physics  and  Director  of  the  Institute  of  Molecular  Biology  at  the 
University  of  Oregon  and  Adjunct  Professor  of  Biochemistry  at  the  Oregon  Health  Sciences  University, 
Portland.  He  received  his  undergraduate  and  graduate  training  at  the  University  of  Adelaide,  Australia. 
He  did  postdoctoral  research  at  the  MRC  Laboratory  of  Molecular  Biology,  Cambridge,  England  (with 
David  Blow)  and  at  the  National  Institutes  of  Health  ( with  David  Davies ).  Dr.  Matthews  is  a  member 
of  the  National  Academy  of  Sciences. 


OUR  laboratory  uses  x-ray  crystallography,  in 
concert  with  other  techniques,  to  try  to  ad- 
dress some  of  the  fundamental  problems  in  biol- 
ogy: How  do  proteins  spontaneously  fold  into 
their  biologically  active  three-dimensional  con- 
figurations? What  determines  the  stability  of 
these  folded  proteins,  and  can  stability  be  im- 
proved? How  do  proteins  interact  with  each 
other?  How  do  they  interact  with  DNA?  How  do 
enzymes  act  as  catalysts? 

The  Protein-folding  Problem 

An  area  of  long-standing  interest  is  the  so-called 
protein-folding  problem.  How  does  a  newly  syn- 
thesized, extended  peptide  chain  "know"  how 
to  fold  spontaneously  into  its  active  three- 
dimensional  shape? 

Although  it  has  long  been  recognized  that  the 
amino  acid  sequence  of  a  protein  determines  its 
three-dimensional  structure,  recent  work  from 
several  laboratories  has  made  it  clear  that  certain 
amino  acids  are  more  important  than  others  in  the 
folding  process.  At  some  positions,  typically  the 
solvent-exposed  mobile  sites  in  the  folded  pro- 
tein, amino  acids  can  be  interchanged  almost  at 
random  with  little  apparent  effect  on  folding  or 
stability.  On  the  other  hand,  interchange  of 
amino  acids  in  buried  or  rigid  parts  of  a  folded 
protein  can  destabilize  it,  suggesting  that  the 
amino  acids  at  these  positions  are  important  in 
determining  the  folded  conformation. 

One  of  the  encouraging  developments  has 
been  the  relative  freedom  with  which  amino  acid 
replacements  can  be  introduced  in  a  protein  of 
interest.  To  try  to  simplify  the  complexity  of  the 
protein-folding  problem,  we  are  attempting  to 
replace  some  of  the  "nonessential"  amino  acids 
in  phage  T4  lysozyme  with  alanine.  Such  a 
"polyalanine  protein"  would,  in  principle,  trun- 
cate all  nonessential  side  chains  and  allow  one  to 
focus  on  those  parts  of  the  amino  acid  sequence 
that  are  critical  for  the  folding  process. 

In  experiments  to  date,  a  series  of  alanines  has 
been  introduced  within  two  different  of-helices  of 
T4  lysozyme.  The  somewhat  surprising  result  is 
that  alanines  are  not  only  tolerated  at  most  posi- 


tions in  the  a-helix;  they  can  sometimes  increase 
the  protein's  stability.  In  an  extreme  case  it  has 
been  found  that  1 0  alanines  can  be  introduced  in 
sequence,  yet  the  protein  still  folds  normally  and 
has  full  activity.  This  illustrates  that  the  informa- 
tion in  the  amino  acid  sequence  of  a  protein  is 
highly  redundant. 

Understanding  the  Interactions  That 
Stabilize  Protein  Structures 

It  is  generally  agreed  that  the  major  factor  in 
stabilizing  the  folded  structures  of  globular  pro- 
teins is  the  hydrophobic  effect.  Until  recently  it 
has  also  been  generally  agreed  that  the  strength  of 
the  hydrophobic  effect — i.e.,  the  energy  of  stabi- 
lization provided  by  the  transfer  of  hydrocarbon 
surfaces  from  solvent  to  the  interior  of  a  protein 
— is  about  25-30  cal  mol~'  for  each  square  ang- 
strom of  surface  area  buried  within  the  protein. 
However,  some  recent  studies  using  site-directed 
mutagenesis  and  protein  denaturation  have  sug- 
gested that  the  strength  of  the  hydrophobic  effect 
might  be  much  higher. 

A  principal  difficulty  in  addressing  this  prob- 
lem has  been  the  lack  of  relevant  structural  data. 
How  does  a  protein  structure  respond  when  a 
bulky  hydrophobic  residue  such  as  leucine  is  re- 
placed by  a  smaller  residue  such  as  alanine?  Does 
the  protein  structure  remain  essentially  un- 
changed or  is  there  structural  rearrangement  to 
avoid  the  creation  of  a  cavity?  If  cavities  are  cre- 
ated, do  they  contain  solvent? 

To  address  these  questions,  six  "cavity-creat- 
ing" mutants  in  which  a  large  hydrophobic 
amino  acid  was  replaced  by  a  smaller  one  were 
constructed  within  the  hydrophobic  core  of 
phage  T4  lysozyme.  All  variants  were  crystallized 
and  the  structures  determined  at  high  resolution. 

The  structural  consequences  of  the  mutations 
differ  from  site  to  site.  In  some  cases  the  protein 
structure  hardly  changes  at  all.  In  other  cases, 
however,  both  side-chain  and  backbone  shifts  up 
to  0.8-1.0  A  were  observed.  In  every  case  re- 
moval of  the  wild-type  side  chain  allowed  some 
of  the  surrounding  atoms  to  move  toward  the  va- 
cated space,  but  a  cavity  always  remained. 


277 


Structural  Basis  of  Interactions  Within  and  Between  Macromolecules 


This  suggests  a  way  to  reconcile  the  different 
values  for  the  apparent  strength  of  the  hydropho- 
bic effect.  One  can  imagine  two  extreme  situa- 
tions. In  one  case  a  leucine  alanine  replace- 
ment is  constructed,  and  the  protein  structure 
remains  completely  unchanged.  In  this  situation 
the  size  of  the  created  cavity  is  large,  and  the  mu- 
tant protein  is  maximally  destabilized.  In  the 
other  extreme,  the  protein  structure  relaxes  in 
response  to  the  leucine  -•-  alanine  substitution, 
fills  the  space  occupied  by  the  leucine  side 
chain,  and  so  avoids  the  formation  of  any  cavity 
whatsoever.  In  this  case  the  decrease  in  energy  of 
the  mutant  protein  relative  to  wild  type  drops  to  a 
constant  energy  term  that  is  characteristic  for  a 
leucine      alanine  replacement. 

Ligand  Binding  Within  Cavities 

We  have  shown  by  crystallographic  and  thermo- 
dynamic analysis  that  the  cavity  created  by  the 
replacement  leucine  99  alanine  in  T4  lyso- 
zyme  is  large  enough  to  bind  benzene  and  that 
ligand  binding  increases  the  melting  temperature 
of  the  protein  by  5.7°C.  This  shows  that  cavities 
can  be  engineered  within  proteins  and  suggests 
that  such  cavities  might  be  tailored  to  bind  spe- 
cific ligands.  The  binding  of  benzene  at  an  inter- 
nal site  7  A  from  the  molecular  surface  also  illus- 
trates the  dynamic  nature  of  proteins,  even  in 
crystals. 

Receptor-Ligand  Interaction 

To  develop  an  understanding  of  the  mode  of 
action  of  growth  factors  and  their  interactions 
with  their  receptors,  we  have  crystallized  and  de- 
termined the  high-resolution  structure  of  human 
fibroblast  growth  factor.  The  structure  is  very  sim- 
ilar to  that  of  interleukin-I/3.  Clearly,  many 
growth  factors  have  similar  overall  structures. 


but  the  exact  relationship  of  these  factors  in  the 
vicinity  of  their  receptor-binding  regions  remains 
to  be  clarified.  Structural  studies  of  human  nerve 
growth  factor  are  in  progress. 

Protein-DNA  Interaction 

We  have  been  interested  for  some  time  in  the 
interaction  between  proteins  and  nucleic  acids. 
In  1981  we  determined  the  structure  of  the  Cro 
repressor  protein  of  X  bacteriophage  (bacteria- 
infecting  virus),  one  of  the  prototypical  exam- 
ples of  a  DNA-interacting  protein.  The  structure 
of  Cro,  as  determined  crystallographically,  sug- 
gested that  a  characteristic  part  of  the  protein, 
now  known  as  the  helix-tum-helix  motif,  is  espe- 
cially important  in  DNA  binding.  The  helix-turn- 
helix  unit  can  be  considered  as  a  "reading  head" 
that  fits  into  the  grooves  of  the  DNA  and  matches 
the  DNA  structure  at  the  specific  recognition  site. 
The  helix-turn-helix  motif  is  now  known  to  occur 
in  a  large  number  of  DNA-binding  proteins,  and 
its  functional  role  has  been  confirmed  by  struc- 
tures of  several  DNA-protein  complexes. 

We  have  subsequently  determined  the  crystal 
structure  of  Cro  protein  in  complex  with  a  tight- 
binding  1 7-base  pair  DNA  operator.  In  general 
terms  the  structure  of  the  complex  supports  the 
model  for  Cro-DNA  interaction  that  was  proposed 
on  the  basis  of  the  uncomplexed  protein,  al- 
though the  Cro  dimer  undergoes  a  substantial 
conformational  change  relative  to  the  uncom- 
plexed crystal  structure. 

Recently  we  have  determined  the  structure  of 
the  biotin  repressor  from  Escherichia  coli.  This  is 
a  more  complicated  protein  that  requires  the 
presence  of  an  effector  molecule  to  bind  DNA.  It 
also  acts  as  an  enzyme. 

Studies  of  protein  stability  and  protein-DNA  in- 
teraction were  supported  in  part  by  grants  from 
the  National  Institutes  of  Health. 


278 


What  Viruses  Are  Telling  Us  About  Gene  Regulation 
in  Mammalian  Cells 


Steven  Lanier  McKnight,  Ph.D. — Investigator 

Dr.  McKnight  is  also  a  staff  member  in  the  Department  of  Embryology  at  the  Carnegie  Institution  of 
Washington,  Baltimore,  and  Adjunct  Professor  in  the  Departments  of  Biology  and  of  Molecular  Biology 
and  Genetics  at  the  Johns  Hopkins  University  School  of  Medicine.  He  earned  his  Ph.D.  degree  in  biology 
from  the  University  of  Virginia  and,  except  for  four  years  with  the  Fred  Hutchinson  Cancer  Research 
Center  in  Seattle,  has  been  with  the  Carnegie  Institution  ever  since.  Dr.  McKnight  was  recently  elected  to 
the  National  Academy  of  Sciences. 


VIRUSES  that  attack  mammalian  cells  rely  on 
preexisting  enzymes,  factors,  and  cellular 
functions  to  negotiate  their  infectious  cycle.  Mo- 
lecular studies  of  virus  infection  have  thereby 
provided  key  insights  into  normal  cellular  pro- 
cesses. For  example,  studies  of  the  processing 
and  transport  of  membrane  glycoproteins  that 
form  the  exterior  coats  of  influenza  virus  and  ve- 
sicular stomatitis  virus  have  helped  to  explain 
\\oy<i  proteins  are  selectively  transported  to  the 
appropriate  cellular  compartment. 

Studies  of  viruses  have  also  illuminated  com- 
plex phenomena  regarding  selective  gene  ex- 
pression. RNA  splicing  was  first  discovered  in 
studies  of  human  cells  infected  by  adenovirus. 
Likevi'ise,  the  capacity  of  DNA  segments  known  as 
enhancers  to  regulate  gene  expression  from  re- 
mote locations  was  discovered  in  studies  of  sim- 
ian virus  40. 

Work  from  our  laboratory  has  focused  on  the 
mechanisms  of  herpesvirus  gene  regulation.  The 
herpesvirus  chromosome  contains  roughly  50- 
100  genes  that  are  expressed  in  a  tightly  con- 
trolled temporal  cascade.  Early  during  the  in- 
fectious cycle,  five  immediate-early  (IE)  genes 
are  activated.  The  IE  genes  encode  protein  prod- 
ucts, termed  transcription  factors,  that  act  to  regu- 
late subsequent  viral  gene  expression.  Several 
hours  later  about  25  delayed-early  (DE)  genes  are 
activated.  Transcription  of  DE  genes  is  strictly  de- 
pendent on  the  prior  production  of  IE  proteins. 
DE  genes  encode  proteins  required  for  replica- 
tion of  viral  DNA.  Following  viral  DNA  replica- 
tion, about  25  late  (L)  genes  are  activated.  Her- 
pesvirus L  genes  encode  structural  proteins  that 
form  the  intact  virus  particle,  including  a  com- 
plex set  of  membrane  glycoproteins  and  struc- 
tural proteins  of  which  the  viral  capsid  is 
composed. 

Interestingly,  one  of  the  L  gene  products  en- 
capsidated  in  the  mature  virus  is  a  potent  and 
specific  transcription  factor  dedicated  to  the  acti- 
vation of  IE  genes  in  the  subsequent  infectious 
cycle.  Roughly  1,000  molecules  of  this  protein, 
termed  viral  protein  16  (VP  16),  are  packaged 
into  the  mature  virus  particle.  Upon  infection  of 


an  otherwise  healthy  cell,  VP  16  is  released  from 
the  infecting  virus  and  comes  to  be  associated 
with  enhancer  elements  located  upstream  from 
each  IE  gene.  Thus  emplaced,  VP  16  acts  as  a  po- 
tent "trigger"  for  the  rapid  and  prolific  expres- 
sion of  IE  genes. 

Early  studies  in  our  laboratory  were  focused  on 
the  herpesvirus  DE  gene  encoding  thymidine  ki- 
nase (TK) .  In  order  to  probe  the  mechanisms  of 
activation  by  IE  proteins,  the  TK  gene  was  system- 
atically mutated  with  the  aim  of  defining  specific 
regulatory  switch  points  within  or  around  the 
gene.  A  region  of  35  base  pairs  encompassing  the 
site  of  transcription  initiation  was  identified  as 
being  responsive  to  activation  by  the  IE  transcrip- 
tion factors.  Surprisingly,  however,  such  studies 
identified  three  additional  regulatory  DNA  se- 
quences located  upstream  of  the  TK  gene.  These 
supplementary  regulatory  elements  were  identi- 
fied as  binding  sites  for  host  cell  proteins,  includ- 
ing the  Spl  (selectivity  protein  1)  transcription 
factor  discovered  by  Robert  Tjian  (HHMI,  Univer- 
sity of  California,  Berkeley)  and  his  colleagues. 

The  involvement  of  host  cell  transcription  fac- 
tors in  viral  gene  expression  has  since  been  ob- 
served in  numerous  cases.  An  exciting  recent  dis- 
covery along  such  lines  has  come  from  Joseph 
Nevins  (HHMI,  Duke  University  Medical  Center). 
Dr.  Nevins  and  his  colleagues  have  identified  a 
human  transcription  factor,  termed  E2F  (early  re- 
gion 2  factor) ,  that  plays  a  pivotal  role  in  the  tran- 
scriptional induction  of  certain  adenovirus 
genes.  Remarkably,  the  E2F  factor  has  been 
shown  to  form  a  specific  complex  with  the  prod- 
uct of  a  recessive  oncogene  encoded  by  the  reti- 
noblastoma locus.  Such  observations  are  begin- 
ning to  provide  mechanistic  insight  into  the  role 
of  the  retinoblastoma  protein  in  growth  control 
and  cancer. 

More  recent  studies  in  our  laboratory  have  fo- 
cused on  the  activation  of  herpesvirus  IE  genes 
during  the  early  stage  of  infection  of  cultured 
mammalian  cells.  As  mentioned  previously,  IE 
gene  activation  is  stimulated  by  VP  16,  a  virally 
encoded  L  protein  that  is  encapsidated  in  mature 
virus  particles.  Like  the  soldiers  sequestered  in 


279 


What  Viruses  Are  Telling  Us  About  Gene  Regulation  in  Mammalian  Cells 


the  proverbial  Trojan  horse,  VPl6  molecules  are 
deposited  in  the  newly  infected  cell  as  "ready- 
made"  transcription  factors.  In  an  elegant  series 
of  experiments,  Bernard  Roizman  and  his  col- 
leagues at  the  University  of  Chicago  showed  that 
the  activating  specificity  of  VP  16  for  herpesvirus 
IE  genes  is  dictated  by  regulatory  DNA  sequences 
located  upstream  of  each  IE  gene.  Such  IE  "en- 
hancers," when  linked  onto  another  gene  other- 
wise unresponsive  to  VP  16,  conferred  a  direct 
and  specific  response. 

On  its  own,  VP  1 6  is  incapable  of  direct  associa- 
tion with  the  IE  enhancers.  In  order  to  develop  an 
understanding  of  the  molecular  mechanisms  con- 
trolling transcriptional  activation  by  VP  16,  ef- 
forts have  been  undertaken  to  identify  and  purify 
cellular  proteins  that  bind  to  IE  enhancers.  At 
least  four  different  host  cell  proteins  are  required 
for  maximal  activation  of  IE  gene  expression  by 
VPl  6.  The  most  recent  work  in  our  laboratory  has 
entailed  the  identification  and  purification  of  a 
DNA-binding  complex  that  associates  avidly  and 
specifically  with  a  critical  switch  point  within  IE 
enhancers.  This  particular  switch  point,  termed 
in  the  jargon  of  the  transcription  field  a  "cis- 
regulatory  element,"  is  largely  composed  of  gua- 
nine (G)  and  adenine  (A)  residues.  The  cellular 
DNA-binding  protein  that  interacts  specifically 
with  the  GA-rich  cis-regulatory  element  has  been 
termed  GA-binding  protein  (GABP) . 

Kelly  LaMarco,  a  postdoctoral  fellow  in  our  lab- 
oratory, purified  GABP  and  found  it  to  be  com- 
posed of  two  polypeptide  subunits.  After  deriv- 
ing the  partial  amino  acid  sequence  from 
proteolyzed  fragments  of  each  subunit.  Dr.  La- 
Marco,  along  with  postdoctoral  associates  Cath- 
erine Thompson  and  Tom  Brown,  succeeded  in 
cloning  the  genes  encoding  each  GAPB  subunit. 
The  amino  acid  sequence  of  one  subunit,  termed 
GABPa,  exhibits  significant  similarity  to  the  prod- 


uct of  the  ETS  proto-oncogene.  Indeed,  the  ETS- 
related  region  of  GABPa  represents  the  part  of  the 
protein  that  mediates  direct  contact  with  DNA. 

This  same  region  of  the  protein  is  also  neces- 
sary for  protein-protein  contact  with  the  other 
subunit  of  the  complex,  termed  GABP/?.  Fortu- 
nately, the  sequence  of  GABP^  also  exhibits  a  re- 
gion of  amino  acid  sequence  similarity  with  previ- 
ously studied  proteins.  GABP/5  contains  four 
imperfect  repeats,  33  amino  acids  in  length,  that 
are  related  to  similarly  sized  repeats  present  in 
the  products  of  a  number  of  interesting  proteins. 

One  such  relative  is  the  product  of  the  Notch 
gene  of  fruit  flies  studied  by  Spyridon  Artavanis- 
Tsakonas  (HHMI,  Yale  University).  The  Notch 
gene  product  is  a  membrane  protein  that  plays  an 
important  role  in  cell-cell  communication  dur- 
ing fruit  fly  development.  Another  protein  that 
contains  the  33  amino  acid  repeats  is  ankyrin,  a 
cytoskeletal  protein  in  red  blood  cells  discovered 
by  G.  Vann  Bennett  (HHMI,  Duke  University  Med- 
ical Center).  Drs.  Thompson  and  Brown  found 
that  the  33  amino  acid  repeats  of  GABP/?  consti- 
tute the  part  of  the  protein  required  for  direct 
interaction  with  its  matching  subunit  (GABPa), 
thus  providing  the  first  conclusive  evidence  for 
the  mechanist  role  of  this  protein  structural 
motif. 

Many  questions  regarding  the  properties  and 
function  of  this  protein  complex  remain  unre- 
solved. For  example,  how  does  binding  of  the 
complex  facilitate  activation  of  herpesvirus  IE 
genes?  What  role  does  GABP  play  in  the  control  of 
cellular  gene  expression?  Might  GABP  in  some 
way  influence  the  decision  of  herpesvirus  to  exe- 
cute its  lytic  cycle  (as  it  does  in  epithelial  cells) 
or  the  latent  state  it  enters  when  the  virus  infects 
neuronal  cells?  Given  a  critical  set  of  molecular 
reagents,  including  recombinant  DNA  clones  and 
specific  antibodies,  it  should  now  be  possible  to 
address  these  questions  directly. 


280 


Fundamental  Mechanisms  of  Ion  Channel  Proteins 


Christopher  Miller,  Ph.D. — Investigator 

Dr.  Miller  is  also  Professor  of  Biochemistry  at  Brandeis  University  and  Adjunct  Professor  of  Molecular 
Biology  at  Massachusetts  General  Hospital,  Boston.  He  received  his  B.A.  degree  in  physics  from 
Swarthmore  College  and  his  Ph.D.  degree  in  molecular  biology  from  the  University  of  Pennsylvania.  He 
carried  out  postdoctoral  work  in  membrane  biochemistry  with  Efraim  Racker  at  Cornell  University  for 
two  years  and  then  joined  the  Graduate  Department  of  Biochemistry  at  Brandeis. 


ION  channels  are  the  most  fundamental  ele- 
ments of  molecular  hardware  in  the  nervous 
system.  They  are  the  membrane-spanning  pro- 
teins that  directly  mediate  the  transmembrane 
ionic  fluxes  by  which  electrical  signals  are  gener- 
ated, propagated,  and  integrated  in  neurons, 
muscle,  and  other  electrically  active  cells.  By 
forming  aqueous  pores  through  the  heart  of  the 
channel  protein  (and  hence  through  the  mem- 
brane that  the  protein  spans),  channels  act  as 
"leakage"  pathways  for  ions  down  their  preestab- 
lished  thermodynamic  gradients.  These  proteins 
make  intelligent  leaks.  Channels  can  discrimi- 
nate fiercely  among  the  different  species  of  inor- 
ganic ions  present  in  the  aqueous  solutions  bath- 
ing the  cell  membrane.  They  can  also  rapidly 
open  and  close  their  conduction  pores  in  re- 
sponse to  external  signals,  such  as  binding  of  neu- 
rotransmitters or  changing  of  the  transmembrane 
electric  field. 

Work  here  is  directed  toward  questions  of  basic 
molecular  mechanisms  of  ion  channel  operation 
and  of  the  underlying  protein  structures.  Since  no 
high-resolution  structures  have  been  obtained 
(or  are  soon  likely  to  be)  for  this  class  of  proteins, 
it  is  necessary  to  draw  structural  inferences  from 
close  examination  of  ion  channel  function.  This 
can  be  done  because  ion  channels,  unique  among 
proteins,  can  be  studied  quantitatively  at  the 
single-molecule  level.  In  this  laboratory,  heavy 
use  is  made  of  the  technique  of  "single-channel 
reconstitution,"  in  which  individual  ion  channel 
molecules  are  inserted  into  an  artificial  mem- 
brane under  simple,  chemically  controllable 
conditions. 

This  approach  has  allowed  us  to  develop  crude 
physical  pictures  of  several  ion  channels  in 
which  crucial  dimensions  have  been  deduced: 
the  conduction  pore's  width  and  length,  the  dis- 
tance of  its  entryway  from  the  lipid  bilayer  sur- 
face, and  the  number  of  ions  inside  the  channel 
during  the  conduction  process.  We  are  currently 
complementing  these  purely  functional  and 
mechanistic  studies  with  recently  developed 
methods  of  membrane  protein  biochemistry  and 
manipulation  of  ion  channels  at  the  genetic  level. 


Use  of  Peptide  Neurotoxins  as  Probes 
of      Channel  Structure 

Charybdotoxin  (CTX)  is  a  scorpion  venom- 
derived  peptide  that  blocks  a  small  family  of 
K^-specific  channels.  Having  shown  that  it  acts  by 
physically  plugging  the  channel's  externally  fac- 
ing mouth,  we  are  now  utilizing  CTX  as  a  probe 
of  this  important  region.  We  are  using  two  differ- 
ent channels  for  these  efforts:  the  high- 
conductance  Ca^^-activated  channel  from  skel- 
etal muscle  reconstituted  into  planar  lipid 
bilayers,  and  the  Shaker  channel  expressed  in 
Xenopus  oocytes.  Each  of  these  channels  has  a 
particular  advantage,  the  former  lending  itself  to 
close  mechanistic  study  and  the  latter  to  molecu- 
lar genetic  manipulation. 

Using  a  synthetic  gene  for  CTX  expressed  in 
Escherichia  colt,  in  combination  with  the  known 
structure  of  the  peptide,  we  have  mapped  the  in- 
teraction surface  of  the  CTX,  using  both  types  of 
channels  as  CTX  receptors.  As  a  result,  we 
know  that  the  peptide  makes  intimate  contact 
with  the  channel  on  a  well-defined  surface  built 
from  7-8  residues.  One  of  these  residues  inter- 
acts electrostatically  with  a  ion  residing  in  the 
channel's  conduction  pore. 

In  parallel  with  mapping  the  toxin,  we  have 
used  site-directed  mutagenesis  with  the  Shaker 
channel  to  identify  channel  residues  making 
up  the  CTX  receptor  located  in  the  channel's  ex- 
ternal mouth.  With  this  information  in  hand,  we 
are  currently  trying  to  identify  channel-toxin  in- 
teraction partners  by  constructing  complemen- 
tary mutants  of  peptide  and  receptor.  This  will 
allow  us  to  deduce  physical  distances  between 
residues  lining  the  mouth  of  the  K"^  channel. 

Purification  and  Reconstitution 
of  CI"  Channels 

The  electric  ray  Torpedo  californica  carries  in 
its  electric  organ  a  CP-specific  channel  with  an 
unusual  structural  characteristic.  The  channel  is 
built  as  a  dimeric,  or  "double-barreled"  com- 
plex, with  two  identical  CI"  diffusion  pathways 
in  a  single  molecular  unit.  We  have  developed  a 
functional  assay  for  this  channel  protein  in  a  solu- 


281 


Fundamental  Mechanisms  of  Ion  Channel  Proteins 


bilized  state  and  are  performing  conventional 
membrane  purification  studies.  The  channel  is 
expressed  in  its  natural  tissue  at  high  density,  so 
milligram  quantities  should  be  easily  obtained. 
The  isolation  of  this  channel  will  enable  us  to 
study  the  molecular  family  of  voltage-dependent 
anion  channels  at  the  protein-biochemical  level. 

-Structure-Function  Relations  in  a  Minimal 
K+  Channel 

We  are  beginning  a  structure-function  analysis 
on  a  K^-specific  channel,  first  cloned  from  a  kid- 
ney cDNA  library,  that  shows  a  remarkable  molec- 
ular property:  a  very  small  polypeptide  size  of 
only  130  amino  acids,  some  1 0-fold  smaller  than, 
for  example,  the  voltage-dependent  Na"^  channel. 
We  have  constructed  and  expressed  a  fully  syn- 


thetic gene  for  this  channel,  using  the  degeneracy 
of  the  genetic  code  to  build  a  large  number  of 
unique  restriction  sites  throughout  the  coding  se- 
quence. Thus  prepared  to  perform  routine  cas- 
sette mutagenesis,  we  are  initiating  a  search  for 
functional  domains  of  the  channel  and  develop- 
ing direct  tests  to  determine  whether  this  gene 
does  in  fact  code  for  an  ion  channel  at  all,  a  basic 
question  that  has  yet  to  be  answered  rigorously. 

We  have  found  point  mutations  in  the  single 
hydrophobic  domain  that  specifically  alter  ion  se- 
lectivity among  close  analogues  as  well  as  the 
affinities  of  several  channel  blockers.  The  results 
not  only  demonstrate  that  this  cDNA  does  code 
for  a  structural  gene  for  a  channel  but  also 
provide  initial  intimations  about  the  way  the  Re- 
conducting pore  is  formed. 


282 


Neural  Foundations  of  Vision 


J.  Anthony  Movshon,  Ph.D. — Investigator 

Dr.  Movshon  is  also  Professor  of  Neural  Science  and  Psychology  at  New  York  University  and  Adjunct 
Professor  of  Physiology  and  Biophysics  at  New  York  University  Medical  Center.  He  received  his  B.A.  degree 
and  his  Ph.D.  degree  in  experimental  psychology  from  Cambridge  University,  where  he  worked  with  Colin 
Blakemore.  After  joining  the  faculty  of  NYU,  Dr.  Movshon  has  remained  there  except  for  a  sabbatical  year 
at  Oxford  University.  He  was  founding  director  of  the  NYU  Center  for  Neural  Science.  Among  his  honors 
are  the  Young  Investigator  Award  from  the  Society  for  Neuroscience  and  the  Rank  Prize  in  Optoelectronics. 


OUR  research  concerns  the  function  and  de- 
velopment of  the  visual  system,  especially 
the  visual  areas  of  the  primate  cerebral  cortex. 
Our  main  experimental  tools  are  electrophysio- 
logical recording  and  quantitative  analysis  of  the 
visually  evoked  activity  of  single  neurons.  We 
also  draw  importantly  from  related  work  in  visual 
psychophysics,  computational  modeling,  and 
complementary  neuroanatomy. 

Presently  we  are  involved  in  two  broad  groups 
of  studies.  The  first  concerns  the  functional  prop- 
erties of  single  neurons  in  the  extrastriate  visual 
areas  of  the  macaque  monkey's  cerebral  cortex, 
with  special  emphasis  on  the  processing  of  infor- 
mation about  visual  motion,  space,  form,  and 
color.  The  second  group  of  studies  concerns  the 
development  of  cortical  visual  function  in  mon- 
keys and  the  way  that  development  is  affected  by 
abnormal  early  visual  experience. 

An  important  organizing  theme  derives  from 
the  discovery  of  two  functional  streams  in  the 
monkey's  geniculo-cortical  visual  pathway.  One 
stream,  the  P  system,  originates  in  the  dense  and 
numerous  P|8  ganglion  cells  of  the  retina,  contin- 
ues through  the  parvocellular  layers  of  the  lateral 
geniculate  nucleus  (LGN),  and  extends  to  layer 
4Ci8  of  the  striate  (or  primary  visual)  cortex,  VI . 
A  second  stream,  the  M  system,  originates  in  the 
large,  fast-conducting  but  relatively  sparse  Pa  ret- 
inal ganglion  cells,  continues  through  the  mag- 
nocellular  layers  of  the  LGN,  and  enters  the 
striate  cortex  through  layer  4Ca. 

Signals  from  the  P  system  are  passed  preferen- 
tially into  a  set  of  cortical  areas  that  seem  to  be  of 
special  importance  for  the  processing  of  form  and 
color,  especially  visual  areas  2  and  4,  and  into  the 
inferior  temporal  cortex.  Signals  from  the  M  sys- 
tem pass  rather  selectively  into  another  set  of  cor- 
tical areas  that  seem  to  be  essential  for  the  analy- 
sis of  visual  motion  and  visual  space,  especially 
the  middle  temporal  area  (MT  or  V5),  and  into 
the  posterior  parietal  cortex.  Our  working  hy- 
pothesis is  that  these  streams  subserve  different, 
albeit  overlapping  visual  functions,  and  also  that 
different  forms  of  developmental  visual  disorder 
may  reflect  abnormalities  primarily  affecting  one 
stream  or  the  other. 


To  study  the  functions  of  cortical  visual  areas, 
we  analyze  the  responses  evoked  in  single  neu- 
rons by  visual  stimuli  carefully  selected  to  permit 
formal  characterization  of  underlying  neuronal 
mechanisms.  The  class  of  properties  in  which  we 
are  generally  interested  concerns  the  selectivity 
with  which  neurons  respond  to  variations  along 
one  or  another  visual  dimension.  We  also  try  to 
examine  the  neuroanatomical  distribution  and 
functional  properties  of  neurons  providing  affer- 
ent signals  to  a  particular  area,  so  that  we  can 
attempt  to  understand  the  computational  trans- 
formations of  the  visual  signal  executed  by  the 
circuits  in  each  area. 

An  important  concern  is  to  establish  the  partic- 
ular dimensions  of  the  visual  stimulus  for  which 
neurons  in  that  area  show  an  invariant  selectivity 
— that  is,  for  which  their  selectivity  is  unaffected 
by  parametric  variation  in  other,  unrelated  di- 
mensions. For  example,  neurons  in  VI  have  in- 
variant selectivity  for  the  spatial,  temporal,  and 
chromatic  structure  of  visual  stimuli.  Neurons  in 
MT  transform  afferent  spatiotemporal  signals  into 
invariant  representations  of  an  object's  speed  and 
direction.  Neurons  in  V4,  on  the  other  hand,  may 
transform  simple  afferent  chromatic  signals  into 
invariant  representations  of  the  object's  surface 
properties. 

A  critical  issue  in  cortical  sensory  physiology  is 
to  relate  perceptual  experience  and  judgment  to 
the  activity  of  neurons  and  neuron  assemblies.  In 
collaboration  with  William  Newsome  at  Stanford 
University,  we  have  used  statistical  methods 
based  on  the  theory  of  signal  detection  to  com- 
pare the  performance  of  single  neurons  with  psy- 
chophysical measures  of  performance  obtained 
concurrently  from  an  awake,  behaving  monkey. 
The  goal  is  to  deduce  the  associations  between 
the  computation  of  perceptual  features  and  the 
activity  of  particular  groups  of  neurons.  The  re- 
sults suggest  that  small  groups  of  neurons  in  area 
MT  may  carry  the  signals  upon  which  behaving 
monkeys  make  judgments  of  the  motion  content 
of  visual  targets.  This  approach  allows  us  to  form 
a  common  language  in  which  to  consider  psycho- 
physical, computational,  and  neurobiological 


283 


Neural  Foundations  of  Vision 


analyses  of  visual  cortical  function.  We  are  also 
applying  methods  derived  from  statistical  deci- 
sion theory  to  the  question  of  the  absolute  effi- 
ciency of  visual  representations  in  the  brain. 

In  addition  to  its  purely  visual  functions,  the  M 
stream  provides  signals  that  drive  eye  movements 
of  pursuit,  the  slow,  smooth  eye  movements  with 
which  primates  stabilize  on  the  retina  the  image 
of  a  moving  visual  target.  In  collaboration  with 
Stephen  Lisberger  (University  of  California,  San 
Francisco),  we  have  studied  several  aspects  of  the 
relationship  between  visual  and  visuomotor  pro- 
cesses. In  a  series  of  neurophysiological  studies, 
we  have  explored  the  responses  of  MT  neurons  to 
the  dynamic  motion  profiles  used  to  characterize 
pursuit  and  have  documented  the  suitability  of 
the  motion-related  signals  in  these  neurons  for 
the  task  of  initiating  pursuit.  In  psychophysical 
work,  we  have  begun  to  explore  the  kinds  of  vi- 
sual signals  that  pass  into  the  oculomotor  system 
by  examining  the  relationship  between  the  de- 
tectability  of  particular  visual  patterns  and  the 
pursuit  eye  movements  they  elicit. 

Our  overall  ambition  for  these  studies  is  to 
"turn  the  sensory-motor  corner"  and  relate  the 
particulars  of  visual  processing  to  the  higher 
mechanisms  that  produce  voluntary  motor  com- 
mands. To  this  end,  we  are  developing  computa- 
tional models  designed  to  explain  the  signal 


transformations  that  take  place  at  a  series  of  stages 
between  the  initial  registration  of  the  visual 
image  and  the  formulation  of  the  final  oculomo- 
tor command. 

To  analyze  development,  in  a  project  sup- 
ported by  the  National  Eye  Institute,  we  study  the 
vision  of  monkeys  reared  either  with  an  artificial 
strabismus  (deviation  of  one  eye)  or  anisometro- 
pia (difference  in  the  refractive  state  of  the  two 
eyes) .  Both  of  these  manipulations  lead  to  condi- 
tions resembling  human  amblyopia,  a  common 
visual  deficit  of  central  nervous  system  origin.  In 
behavioral  experiments,  we  learn  how  experi- 
mental amblyopia  affects  perceptually  defined 
mechanisms  that  support  visual  sensitivity  to 
form,  contrast,  and  position.  Neurophysiological 
studies  in  the  same  animals  then  reveal  alter- 
ations in  cortical  neuron  properties  that  seem  to 
be  related  to  the  psychophysical ly  measured  vi- 
sual deficits. 

Using  this  strategy,  we  seek  to  uncover  the  rela- 
tionship between  the  neural  changes  that  under- 
lie amblyopia  and  the  perceptual  consequences 
of  the  disorder.  We  are  currently  pursuing  the 
idea  that  the  relatively  mild  type  of  amblyopia 
typically  produced  by  anisometropia  (having  un- 
symmetric  parts)  involves  a  deficit  in  the  P  sys- 
tem, while  the  more  complex  syndrome  that  of- 
ten follows  strabismus  also  involves  important 
deficits  in  the  M  system. 


284 


Human  Retroviral  Gene  Expression 
and  Cellular  Transcription 


Gary  J.  Nabel,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Nabel  is  also  Associate  Professor  of  Internal  Medicine  and  Biological  Chemistry  at  the  University  of 
Michigan  Medical  School.  He  received  his  bachelor  's  degree  from  Harvard  College,  his  M.D.  degree  from 
Harvard  Medical  School,  and  his  Ph.D.  degree  in  cell  and  developmental  biology  from  Harvard  University. 
He  was  a  research  fellow  at  the  Whitehead  Institute,  Massachusetts  Institute  of  Technology,  in  the 
laboratory  of  David  Baltimore,  before  moving  to  the  University  of  Michigan. 


T lymphocytes  protect  the  body  from  invasion 
by  foreign  organisms  but  can  also  serve  as  tar- 
gets of  infection  by  viruses.  One  such  agent  is  the 
human  immunodeficiency  virus  (HIV),  which 
causes  the  acquired  immune  deficiency  syn- 
drome (AIDS).  Under  normal  circumstances,  T 
cells  become  activated  in  response  to  infection 
and  begin  to  synthesize  a  set  of  proteins  that  acti- 
vate the  immunologic  defense  system.  In  T  cells 
that  contain  the  AIDS  virus,  cellular  activation 
provides  a  signal  to  HIV  to  stimulate  viral  replica- 
tion. Our  laboratory  has  characterized  regulatory 
proteins  that  stimulate  T  cell  and  retroviral  gene 
expression.  These  cells  provide  a  model  to  study 
coordinate  gene  expression  during  development 
and  following  viral  infection. 

We  have  identified  proteins  that,  in  binding  to 
control  regions,  regulate  the  expression  of  HIV 
and  other  immune  system  proteins.  At  the  same 
time,  we  have  begun  to  use  our  knowledge  of 
cellular  and  viral  transcription  to  deliver  recom- 
binant genes  in  vivo.  This  system  has  allowed  us 
to  learn  more  about  the  biology  of  these  genes, 
particularly  in  endothelial  and  vascular  smooth 
muscle  cells  of  the  vessel  wall,  and  has  provided 
new  opportunities  for  studies  on  gene  transfer. 

Regulation  of  HIV  Gene  Expression 
in  T  Cells  and  Monocytes 

The  expression  of  HIV  can  be  activated  in  T 
cells  treated  with  phorbol  esters  or  other  immune 
system  activators.  We  have  previously  shown  that 
stimulation  of  these  cells  increases  the  binding 
activity  of  a  protein  that  binds  to  a  DNA  control 
region,  called  NF-kB  (nuclear  factor  that  recog- 
nizes a  sequence  in  the  k  immunoglobulin  light 
chain  of  B  cells) .  NF-kB  is  responsible  for  stimula- 
tion of  HIV  transcription  in  activated  T  cells.  The 
DNA  sequence  recognized  by  this  transcription 
factor  is  twice  repeated  in  the  HIV  control  region, 
and  mutation  of  these  sites  abolishes  inducibility 
of  HIV.  NF-kB  acts  in  synergy  with  HIV  products, 
such  as  the  tat-\  gene,  to  enhance  HIV  gene  ex- 
pression in  an  infected  cell. 

The  transcription  factor  NF-kB  is  a  protein  com- 
plex composed  of  a  DNA-binding  subunit  and  an 


associated  transactivation  protein  (of  relative  mo- 
lecular masses  50  and  65  kDa,  respectively). 
Both  subunits  have  similarity  with  the  rel  onco- 
gene and  the  Drosophila  maternal-effect  gene 
dorsal.  The  5  0-kDa  DNA-binding  subunit  was  pre- 
viously thought  to  be  a  unique  protein,  derived 
from  the  105-kDa  gene  product  (pi 05).  We  have 
recently  reported  the  isolation  of  a  cDNA  that  en- 
codes an  alternative  DNA-binding  subunit  of  NF- 
kB.  It  is  more  similar  to  pi 05  NF-/cB  than  other 
family  members  and  defines  a  new  subset  of  rel- 
related  genes.  It  is  synthesized  as  a  protein  of 
about  100  kDa  (pi 00)  that  is  expressed  in  differ- 
ent cell  types,  contains  cell  cycle  motifs,  and  like 
pi 05,  must  be  processed  to  generate  a  50-kDa 
form. 

A  49-kDa  product  (p49)  can  be  generated  inde- 
pendently from  an  alternatively  spliced  tran- 
script. It  has  specific  /cB  DNA-binding  activity  and 
can  form  heterodimers  with  other  rel  proteins.  In 
contrast  to  the  50-kDa  protein  derived  from 
pl05,  p49  acts  in  synergy  with  p65  to  stimulate 
the  HIV  enhancer  in  transiently  transfected  Jurkat 
cells.  Thus  p49/plOO  NF-kB  could  be  important 
in  the  regulation  of  HIV  and  other  KB-containing 
genes.  The  above  studies  are  supported  in  part  by 
grants  from  the  National  Institutes  of  Health. 

In  addition  to  HIV  type  1  (HIV-1),  a  second 
related  virus,  HIV  type  2  (HIV-2),  can  induce 
AIDS.  HIV-2,  a  distinct  retrovirus,  shares  nucleic 
acid  and  protein  similarity  with  HIV-1.  First  de- 
scribed in  West  Africa,  HIV-2  has  begun  to  appear 
throughout  the  world.  Although  HIV-1  and  -2 
both  cause  AIDS,  the  length  of  the  asymptomatic 
period  following  infection  differs  for  the  two 
viruses. 

Because  increased  viral  replication  is  asso- 
ciated with  progression  of  HIV-related  disease, 
the  rate  of  disease  progression  may  be  influenced 
by  host  cell  regulatory  proteins  that  activate  virus 
replication.  Such  proteins  could  be  regulated  by 
distinct  cofactors  that  selectively  stimulate  cellu- 
lar activation  pathways.  These  T  cell  activation 
pathways  regulate  specific  transcription  factors 
that  may  contribute  to  the  regulation  of  the  latent 
phase  of  HIV  infection. 


285 


Human  Retroviral  Gene  Expression  and  Cellular  Transcription 


We  recently  defined  the  transcriptional  regula- 
tion and  induction  of  these  retroviruses  and 
found  that  their  regulation  differs.  A  distinct  T 
cell  activation  pathway — triggering  of  the  CD3 
component  of  the  T  cell  antigen  receptor  com- 
plex— stimulates  HIV- 2  gene  expression  but 
does  not  affect  HIV- 1 .  The  response  to  T  cell  re- 
-<:eptor  stimulation  in  HIV- 2  is  mediated  by  an  up- 
stream regulatory  element,  CD3R,  which  a  se- 
quence-specific DNA-binding  protein  of  the  ets 
family  recognizes. 

In  addition,  at  least  three  other  cis-acting  regu- 
latory sequences  contribute  to  HIV- 2  gene  ex- 
pression, including  /cB,  another  ets  binding  site, 
and  an  associated  element.  Jurkat  T  leukemia  cell 
lines  containing  HIV-2  provirus  also  show  in- 
creased viral  replication  following  stimulation  of 
the  T  cell  receptor  complex,  in  contrast  to  HIV-1 . 
These  findings  suggest  that  HIV-2  and  HIV-1 
differ  in  their  transcriptional  regulation  and  in- 
duction. These  studies  also  raise  the  possibility 
that  different  cofactors  contribute  to  the  activa- 
tion of  AIDS  associated  with  HIV-1  and  HIV-2. 

Expression  of  Cellular  and  Retroviral 
Vector  Genes  in  Vivo 

Despite  recent  advances  in  the  understanding 
of  eukaryotic  gene  regulation,  a  major  obstacle  to 
the  therapeutic  management  of  human  disease 
remains  the  site-specific  expression  of  genes  in 
vivo.  Using  our  knowledge  of  retroviral  gene  ex- 


pression, we  have  developed  systems  that  utilize 
viral  vectors  to  express  biologically  active  pro- 
teins in  cells  and  tissues  in  vivo.  We  have  devised 
methods  that  allow  a  recombinant  gene  to  be  ex- 
pressed efficiently  at  a  specific  site  in  vivo  by 
direct  introduction  of  genetic  material  at  the  time 
of  catheterization.  A  recombinant  jS-galactosidase 
gene  was  expressed  in  a  specific  arterial  segment 
in  vivo  by  direct  infection  with  a  retroviral  vector 
or  by  DNA  transfection  using  liposomes.  Several 
cell  types  in  the  vessel  wall  have  now  been  trans- 
duced with  recombinant  genes,  including  endo- 
thelial and  vascular  smooth  muscle  cells.  Site- 
specific  gene  expression  can  therefore  be 
achieved  by  direct  gene  transfer  in  vivo  and 
could  be  applied  to  the  treatment  of  such  human 
diseases  as  atherosclerosis,  cancer,  or  AIDS. 
These  studies  have  been  supported  by  grants  from 
the  National  Institutes  of  Health  and  the  Ameri- 
can Heart  Association. 

Biologically  active  proteins  are  now  being  in- 
troduced into  cells,  including  disparate  histocom- 
patibility antigens,  growth  factors,  growth  inhibi- 
tors, or  immune  system  proteins.  The  goal  of  this 
research  is  not  only  to  understand  basic  mecha- 
nisms of  gene  regulation,  transcriptional  activa- 
tion, and  viral  gene  expression,  but  also  to  define 
the  biological  significance  of  factors  that  regulate 
gene  expression  in  complex  organisms  and  to  de- 
velop novel  molecular  interventions  for  human 
disease. 


286 


Molecular  Analysis  of  Mtiscle  Development 
and  Function 


Bernardo  Nadal-Ginard,  M.D.,  Ph.D. — Investigator 

Dr.  Nadal-Ginard  is  also  the  Alexander  S.  Nadas  Professor  of  Pediatrics  and  Professor  of  Cellular  and 
Molecular  Physiology  at  Harvard  Medical  School  and  Cardiologist- in- Chief  at  the  Children's  Hospital, 
Boston.  He  received  his  M.D.  degree  from  the  University  of  Barcelona,  Spain,  and  his  Ph.D.  degree  in 
biology  from  Yale  University.  After  training  in  internal  medicine  and  cardiology,  he  was  a  student  and 
postdoctoral  fellow  with  Clement  Markert  at  Yale.  He  was  a  professor  of  cell  biology  at  Albert  Einstein 
College  of  Medicine  before  assuming  his  present  position. 


OUR  laboratory  continues  to  be  interested  in 
elucidating  the  molecular  mechanisms  that 
regulate  the  production  and  function  of  the  con- 
tractile system  in  muscle  cells.  This  apparatus 
converts  the  chemical  energy  generated  by  food- 
stuffs and  stored  in  the  form  of  ATP,  and  is  thus 
the  molecular  motor  for  locomotion  and  for  the 
heartbeat.  One  of  its  fundamental  roles  is  in 
maintaining  cell  shape.  There  are  variations  of 
the  contractile  system  in  every  cell  of  the 
organism. 

The  functional  unit  of  the  contractile  system  is 
the  sarcomere,  which  is  composed  of  a  precisely 
arranged  set  of  proteins.  These  are  produced  by  a 
small  family  of  genes,  each  making  a  different 
isoform.  In  addition,  a  single  gene  can  in  many 
cases  produce  several  kinds  of  proteins  by  a  pro- 
cess called  alternative  splicing.  Combinations  of 
the  different  isoforms  arising  from  these  two 
mechanisms  can  lead  to  millions  of  different 
types  of  sarcomeres.  Thus  significant  functional 
differences  among  sarcomeres  are  produced  by 
changing  their  components,  either  through  gene 
switching  at  the  transcriptional  level  or  alterna- 
tive splicing  from  the  same  gene.  In  addition,  the 
performance  of  a  given  sarcomere  can  be  affected 
by  changing  the  availability  of  ions  in  the  muscle 
cells.  These  three  aspects  of  muscle  cell  biology 
continue  to  be  the  focus  of  our  research. 

Transcriptional  Regulation  of  Contractile 
Protein  Genes 

Which  sarcomeres  assemble  in  a  cell  depends 
on  which  member  of  the  multigene  family  of 
contractile  protein  genes  is  expressed  at  that  par- 
ticular time.  To  analyze  the  mechanisms  involved 
in  switching  from  one  gene  to  another  in  the  same 
gene  family,  we  have  concentrated  on  those  cod- 
ing for  the  myosin  heavy  chain  (MHC) .  This  is  the 
most  important  protein  of  the  sarcomere,  since  it 
contains  the  enzymatic  activity  responsible  for 
generating  force.  We  are  currently  exploring  two 
main  questions  in  the  regulation  of  the  MHC 
genes:  What  determines  which  one  is  expressed 
in  a  given  cell  type  at  a  particular  developmental 
stage?  What  determines  the  level  of  expression? 


To  date,  the  best-characterized  muscle-specific 
regulatory  factors  are  the  myogenic  basic  helix- 
loop-helix  (bHLH)  proteins  of  the  MyoD  family. 
Muscle-specific  induction  by  these  proteins  de- 
pends on  specific  DNA  binding  to  a  particular  se- 
quence, called  an  E  box,  present  in  many  muscle 
enhancers  and  promoters.  The  MyoDs  interact 
with  the  DNA  in  conjunction  with  other  HLH  pro- 
teins that  are  distributed  more  widely  and  are 
present  in  limiting  amounts. 

During  the  past  year  we  have  cloned  and  char- 
acterized two  new  proteins  that  function  as  he- 
terodimeric  partners  of  MyoD.  These  are  alterna- 
tively spliced  products  of  gene  £2.2  and  are 
particularly  abundant  in  skeletal  muscle,  heart, 
and  brain.  Several  lines  of  evidence  indicate  that 
in  vivo  these  proteins  are  the  partners  of  MyoD  in 
muscle  and  that  they  also  play  an  important  role 
in  other  tissues.  The  fact  that  these  proteins  inter- 
act with  the  product  of  the  retinoblastoma  gene 
suggests  that  they  are  involved  in  the  regulation 
of  cell  proliferation. 

One  of  the  paradoxes  of  the  MyoD  paradigm  for 
muscle-specific  gene  regulation  is  that  not  all 
muscle  genes  contain  E  boxes,  although  they  are 
uniformly  required  for  efficient  muscle-specific 
expression.  In  addition,  many  of  the  genes  in- 
duced by  MyoD  in  skeletal  muscle  are  also  ex- 
pressed in  cardiac  and,  in  some  cases,  smooth 
muscle,  where  myogenic  bHLH  proteins  have  not 
been  found.  It  was  expected,  therefore,  that  other 
transcription  factors,  which  in  skeletal  muscle 
might  be  regulated  directly  or  indirectly  by  the 
MyoD  family,  mediate  activation  and  high-level 
expression  of  these  genes.  A  candidate  for  such  a 
factor  is  the  myocyte-specific  enhancer-binding 
factor  2  (MEF2),  which  binds  to  a  specific  DNA 
sequence  known  to  be  important  for  high-level 
expression  in  skeletal  and  cardiac  muscle. 

During  the  past  year  we  have  cloned  and  char- 
acterized a  family  of  MEF2  factors.  In  humans 
these  factors  are  encoded  by  a  family  of  at  least 
four  genes,  which  through  alternative  splicing 
generate  a  larger  number  of  proteins.  All  these 
proteins  share  domains  that  are  strikingly  similar 
to  the  DNA-binding  and  dimerization  domains  of 


287 


Molecular  Analysis  of  Muscle  Development  and  Function 


2l  recently  identified  MADS  gene  family,  which 
includes  yeast  transcription  factors,  plant  homeo- 
tic  genes  involved  in  floM^er  morphogenesis,  and 
the  serum  response  factor. 

MEF2  mRNAs  accumulate  preferentially  in  skel- 
etal muscle,  heart,  and  brain,  but  post-transcrip- 
tional  mechanisms  must  also  contribute  to  the 
,  MEF2  tissue  specificity.  Remarkably,  cardiac  and 
smooth  as  well  as  skeletal  muscle  contain  saturat- 
ing levels  of  MEF2  trans-activating  factors  that  are 
absent  in  nonmuscle  cells.  We  have  shown  that 
MEF2  is  induced  in  skeletal  muscle  cells  by  the 
MyoD  gene  family,  but  that  this  factor,  by  itself,  is 
insufficient  to  produce  the  whole  muscle  pheno- 
type.  Therefore,  although  MEF2  is  induced  in 
skeletal  muscle  by  bHLH  proteins,  other  lineage- 
determining  pathways  must  lead  to  MEF2  expres- 
sion in  nonskeletal  muscle  tissues. 

In  this  context,  the  finding  that  both  muscle 
and  brain  have  high-level  expression  of  the  same 
isoforms  strongly  suggests  that  MEF2  also  has  an 
important  role  in  neuronal  gene  regulation. 
Whether  bHLH  or  other  factors  induce  MEF2 
genes  in  cardiac  and  smooth  muscle,  as  well  as 
nerve,  the  regulatory  sequences  of  these  genes 
will  serve  as  powerful  tools  for  the  identification 
of  the  lineage-determining  pathways  in  these  cell 
types. 

Regulation  of  Protein  Diversity 
by  Alternative  Splicing 

One  of  the  fundamental  aspects  of  pre-mRNA 
splicing  that  is  particularly  important  for  the  gen- 
eration of  different  proteins  from  a  single  gene  is 
the  mechanism  by  which  splice  sites  are  identi- 
fied. We  have  previously  suggested  a  scanning 
model  for  the  location  of  the  3'  splice  site  of 
mammalian  introns.  We  proposed  that  this  site  is 
located  by  a  scanning  mechanism  searching  for 
the  first  AG  downstream  of  the  branch  point/ 
polypyrimidine  tract.  During  the  past  year  we 
have  further  confirmed  and  extended  this  model. 

Recent  experiments  show  that  scanning  for  the 
3'  splice  site  starts  at  the  branch  point,  not  the 
pyrimidine  tract,  and  proceeds  until  an  AG  is  rec- 
ognized. Failure  to  recognize  the  most  proximal 


AG  can  arise  from  extreme  proximity  to  the 
branch  point,  or  the  AG  can  be  sequestered 
within  a  hairpin  loop.  Once  the  AG  has  been  en- 
countered, scanning  stops,  but  the  spliceosome 
can  still  see  a  stretch  of  about  1 2-35  nucleotides 
downstream.  The  most  competitive  AG  within 
this  scanning  window  is  then  selected  as  the  3' 
splice  site.  The  strongest  determinant  of  compe- 
titiveness remains  the  proximity  to  the  branch 
point.  Thus,  in  many  respects,  the  scanning 
model  for  the  3'  splice  site  closely  resembles  the 
model  for  translation  initiation,  both  in  its  sim- 
plest formulation  and  in  the  predictable  excep- 
tions to  the  general  rule. 

To  analyze  the  mechanisms  involved  in  alterna- 
tive splicing,  we  have  continued  to  focus  on  the 
a-tropomyosin  (TM)  gene.  This  gene  generates  a 
minimum  of  1 0  different  isoforms  that  are  tissue 
specific  and  developmentally  regulated.  We  have 
concentrated  on  the  mutually  exclusive  exons  2 
and  3  to  elucidate  the  elements  involved  in  this 
type  of  regulation. 

In  the  past  we  identified  the  mechanisms  in- 
volved in  the  production  of  the  default  pattern, 
which  results  in  the  exclusion  of  exon  2  and  in- 
clusion of  exon  3  in  all  cell  types  but  smooth 
muscle  cells.  During  the  past  year,  we  have  con- 
centrated on  the  production  of  the  regulated  pat- 
tern in  smooth  muscle  cells  that  results  in  the 
inclusion  of  exon  2  and  exclusion  of  exon  3-  This 
pattern  is  the  result  of  negative  regulation  by  inhi- 
bition of  the  default  pattern  and  is  attributable  to 
two  well-defined  sequences  located  5'  and  3' 
from  exon  3-  These  sequences  are  the  binding 
site  for  protein  factors  present  in  smooth  muscle 
but  not  in  HeLa  cells.  Experiments  are  in  progress 
to  clone  this  factor. 

The  search  for  splicing  factors  that  interact 
with  the  polypyrimidine  tract  has  resulted  in  the 
cloning  and  characterization  of  a  novel  essential 
splicing  factor.  This  is  a  protein  of  about  1 00  kDa 
that  is  closely  associated  with  the  polypyrimidine 
tract-binding  protein  previously  described  by 
our  laboratory  and  others.  Experiments  are  in  pro- 
gress to  determine  the  particular  stage  of  spliceo- 
some assembly  in  which  this  factor  is  involved. 


288 


The  Genomic  Response  to  Growth  Factors 


Daniel  Nathans,  M.D. — Senior  Investigator 

Dr.  Nathans  is  also  University  Professor  of  Molecular  Biology  and  Genetics  at  the  Johns  Hopkins  University 
School  of  Medicine.  He  received  his  B.S.  degree  in  chemistry  from  the  University  of  Delaware  and  his  M.D. 
degree  from  Washington  University.  His  postdoctoral  research  was  done  at  the  National  Cancer  Institute 
and  the  Rockefeller  University.  Dr.  Nathans  is  a  member  of  the  National  Academy  of  Sciences  and  serves 
on  the  President's  Council  of  Advisers  on  Science  and  Technology.  He  received  the  Nobel  Prize  in  medicine 
or  physiology  in  1978 for  the  application  of  restriction  enzymes  to  problems  in  molecular  genetics. 


THE  proliferation  of  mammalian  cells  is  regu- 
lated by  extracellular  proteins  called  growth 
factors.  When  a  growth  factor  interacts  with  its 
specific  cell  surface  receptor,  a  cascade  of  bio- 
chemical reactions  leads  to  the  sequential  activa- 
tion of  specific  genes.  Research  in  my  laboratory 
concerns  the  analysis  of  this  induced  genetic  pro- 
gram in  a  mouse  fibroblast  cell  line. 

Among  the  genes  expressed  in  the  first  wave  of 
gene  activation  induced  by  growth  factors  are 
many  that  encode  transcriptional  regulatory  pro- 
teins. We  have  previously  described  several  of 
these  "immediate-early"  transcription  factors, 
including  members  of  the  Jun  and  Fos  families, 
zinc  finger  proteins,  and  a  helix-loop-helix  pro- 
tein. During  the  past  year  we  have  continued  our 
characterization  of  these  proteins  and  their 
genes. 

Several  of  the  immediate-early  transcription 
factor  genes  are  activated  by  platelet-derived 
growth  factor  (PDGF)  and  other  ligands  through 
one  or  more  serum  response  elements  (SREs)  up- 
stream of  each  gene.  In  the  case  of  jun-B,  the 
upstream  sequence  near  the  start  of  the  gene  has 
no  functional  SRE,  nor  other  signals  essential  for 
activation  of  the  gene.  By  analyzing  a  series  of 
mutants,  a  graduate  student  in  the  laboratory, 
Evelio  Perez-Albuerne,  has  found  that  jun-B  has 
regulatory  elements  downstream  of  the  gene,  in- 
cluding a  functional  SRE  and  a  cyclic  AMP  re- 
sponse element  that  mediates  the  activation  of 
jun-B  by  agents  that  elevate  intracellular  cAMP. 
Thus  the  mechanism  of  activation  of  jun-B  by 
serum  or  PDGF  appears  to  be  similar  to  that  previ- 
ously found  for  a  number  of  other  immediate- 
early  genes,  except  that  the  response  elements 
are  farther  away  from  the  start  of  transcription 
and  downstream  of  the  gene. 


The  Jun  and  Fos  family  of  proteins  are  DNA- 
binding  transcription  factors  that  form  dimers 
through  interacting  domains  called  leucine  zip- 
pers. Pierre  Chevray,  another  graduate  student, 
has  initiated  a  search  for  other  proteins  that  inter- 
act with  Jun  and  Fos  and  regulate  their  activities. 
For  this  purpose  he  used  a  previously  described 
yeast  genetic  system  that  allows  one  to  detect 
protein-protein  interactions  and  to  clone  the 
gene  for  an  interacting  protein.  By  this  means  he 
has  identified  several  proteins  that  interact  with 
the  Jun  segment  that  forms  a  leucine  zipper. 
Among  these  proteins  are  another  leucine  zipper 
transcription  factor,  two  previously  unidentified 
proteins,  and  the  cytoskeletal  proteins  a-  and 
/3-tropomyosin,  which  are  known  to  form  leucine 
zippers.  We  are  now  exploring  the  physiological 
significance  of  these  interactions. 

After  the  appearance  of  immediate-early  tran- 
scription factors  in  growth  factor-treated  cells, 
another  set  of  genes  comes  into  play.  Activation 
of  these  "delayed-early"  genes  is  thought  to  be 
mediated  by  immediate-early  transcription  fac- 
tors. Associates  Anthony  Lanahan  and  John 
Williams  have  cloned  and  characterized  a  num- 
ber of  cDNAs  corresponding  to  delayed-early 
genes.  Some  of  the  genes  are  induced  by  mitogens 
in  nonfibroblastic  cell  lines  also.  Among  the  pro- 
teins they  encode  are  a  chromosomal  protein,  a 
transmembrane  channel  protein,  an  enzyme  in- 
volved in  adenine  nucleotide  biosynthesis,  a  pro- 
tein related  to  a  known  cytokine,  and  several  pre- 
viously unidentified  proteins.  We  are  further 
characterizing  some  of  these.  In  addition,  we  are 
studying  the  role  of  immediate-early  transcrip- 
tion factors  in  activating  their  genes. 

Research  on  the  Jun  proteins  is  supported  by  a 
grant  from  the  National  Institutes  of  Health. 


289 


I 


The  commonest  rhodopsin  mutation  among  patients  with  autosomal  dominant  retinitis  pigmen-  | 

tosa  has  a  substitution  of  the  amino  acid  histidine  for  proline  23-  Antibody-stained  human  opsin  | 

(the  apoprotein  of  rhodopsin),  expressed  by  transient  transfection  in  tissue  culture  cells,  is  i 

shown  here.  A:  Wild-type  opsin  accumulates  in  the  plasma  membrane.  B:  The  mutant  opsin  is  \ 

predominantly  in  the  endoplasmic  reticulum.  | 

From  Sung,  C.  -H.,  Schneider,  B.G.,  Agarwal,  N.,  Papermaster,  D.S.,  and  Nathans,  f.  1991.  Proc  I 

Natl  Acad  Sci  USA  88:8840-8844.  j 

i 


I 
i 

I 
I 


I 

I 

I 
I 


290 


Molecular  Biology  of  Visual  Pigments 


Jeremy  Nathans,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Nathans  is  also  Associate  Professor  of  Molecular  Biology  and  Genetics  and  of  Neuroscience  at  the  Johns 
Hopkins  University  School  of  Medicine.  His  undergraduate  work  was  in  biology  and  chemistry  at  the 
Massachusetts  Institute  of  Technology.  He  received  a  Ph.D.  degree  in  biochemistry  and  later  his  M.D. 
degree  at  Stanford  University.  Before  joining  the  staff  at  Johns  Hopkins,  Dr.  Nathans  spent  a  year  as  a 
postdoctoral  fellow  at  Genentech. 


VISUAL  pigments  are  the  light-absorbing  pro- 
teins that  initiate  phototransduction.  Each 
consists  of  a  chromophore,  1  l  -cis  retinal,  joined 
to  an  integral  membrane  protein,  opsin.  The  vi- 
sual pigments  constitute  one  branch  of  a  large 
family  of  cell  surface  receptors  that  transduce  ex- 
ternal stimuli  by  activating  G  proteins.  In  the  vi- 
sual system,  the  activated  G  protein  stimulates  a 
cGMP  phosphodiesterase,  and  the  resulting  tran- 
sient decline  in  cGMP  closes  plasma  membrane 
cation  channels. 

Photon  absorption  by  1  \  -cis  retinal  causes  it  to 
isomerize  from  W-cis  to  all-fraws.  The  attached 
protein  then  undergoes  a  series  of  conforma- 
tional changes,  leading  ultimately  to  a  form  that 
interacts  with  the  G  protein.  The  changes  under- 
lying visual  pigment  activation  are  likely  to  re- 
semble those  that  accompany  hormone-receptor 
binding  among  the  other  members  of  this  recep- 
tor family. 

Our  laboratory  is  interested  in  three  general 
areas  related  to  the  visual  pigments:  their  struc- 
ture and  function,  the  control  of  their  expression, 
and  their  variation  within  the  human  population. 

Structure/Function  Studies 

Several  years  ago  we  developed  a  system  for 
producing  large  quantities  of  bovine  rhodopsin 
by  expression  of  cloned  cDNA  in  tissue  culture 
cells.  As  described  below,  we  are  using  this  sys- 
tem in  conjunction  with  site-directed  mutagene- 
sis to  define  those  residues  involved  in  protein 
conformational  changes,  in  protein-chromophore 
interactions,  and  in  protein  stability. 

Because  the  retinal  chromophore  is  sensitive  to 
changes  in  its  immediate  environment,  the 
various  conformations  of  light-activated  rhodop- 
sin each  possess  distinctive  absorption  spectra. 
By  following  the  changes  in  spectral  absorbance 
following  photoactivation,  one  can  determine 
the  quantity  and  rates  of  formation  and  decay  of 
each  conformational  intermediate.  Charles  Weitz, 
a  postdoctoral  fellow,  has  used  this  assay  to  map 
those  amino  acids  that  play  an  important  role  in 
rhodopsin's  transition  to  the  active  conforma- 


tion, i.e.,  the  conformation  that  interacts  with  the 
G  protein. 

In  one  set  of  experiments,  we  sought  to  exam- 
ine the  mechanism  responsible  for  the  pH  depen- 
dence of  this  transition.  Thirty  years  ago  George 
Wald  and  his  colleagues  observed  that  low  pH 
favors  this  transition  and  that  the  pH  dependence 
was  consistent  with  a  mechanism  in  which  pro- 
tonation  of  one  or  more  histidines  was  tightly 
coupled  to  the  transition.  We  therefore  mutated 
one-by-one  each  of  the  six  histidine  residues  to 
phenylalanine  and  monitored  the  ability  of  the 
mutant  rhodopsin  to  form  the  active  conforma- 
tion. All  of  the  mutant  proteins  could  bind  to  11- 
cis  retinal  and  form  a  light-sensitive  pigment,  but 
mutants  in  which  histidine^''  was  replaced 
either  by  phenylalanine  or  cysteine  were  unable 
to  assume  the  active  conformation. 

The  simplest  interpretation  of  this  experiment 
is  that  the  histidine'^"  is  the  site  at  which  proton- 
ation  drives  rhodopsin  into  its  active  conforma- 
tion. Dr.  Weitz  has  pursued  this  observation  by 
identifying  mutants  at  other  sites  that  have  the 
converse  property:  they  form  the  active  confor- 
mation more  efficiently  than  the  normal  protein. 
This  second  type  of  mutant  therefore  identifies 
amino  acids  that  normally  act  to  keep  the  protein 
in  the  inactive  conformation.  As  most  amino  acids 
have  little  or  no  effect  on  this  conformational 
transition,  it  should  be  possible  to  identify  the 
handful  of  critical  residues  that  control  it. 

In  the  human  retina,  rhodopsin  mediates  vision 
in  dim  light,  whereas  a  related  set  of  visual  pig- 
ments, the  cone  pigments,  mediate  bright  light 
vision  as  well  as  color  vision.  The  spectral  proper- 
ties of  the  cone  pigments  have  been  of  great  inter- 
est to  physiologists  and  psychologists  and  have 
been  the  object  of  investigation  for  over  a  cen- 
tury. Unfortunately,  the  instability  and  scarcity  of 
these  proteins  make  them  difficult  to  study.  Sev- 
eral years  ago  we  isolated  the  genes  that  encode 
the  human  cone  pigments.  Using  these  reagents, 
Shannath  Merbs,  a  graduate  student,  has  recently 
succeeded  in  producing  large  quantities  of  the 
pigments  and  determining  their  precise  absorp- 
tion spectra. 


291 


Molecular  Biology  of  Visual  Pigments 


Interestingly,  the  human  gene  pool  contains 
two  versions  of  the  red  pigment.  One  carries  an 
alanine  at  position  180  and  absorbs  maximally  at 
552  nm,  whereas  the  second  version  carries  a  ser- 
ine at  position  180  and  absorbs  maximally  at  557 
nm.  This  spectral  difference  explains  a  number  of 
long-standing  observations  regarding  differences 
in  color  vision  among  otherwise  color-normal  in- 
dividuals. We  are  now  using  this  system  to  study 
hybrid  pigments  encoded  by  hybrid  genes  that 
were  generated  by  recombination  between  the 
red  and  green  pigment  genes.  Hybrid  genes  of 
this  type  are  carried  by  7  percent  of  human  X 
chromosomes  and  account  for  most  of  the  com- 
mon forms  of  red-green  color  blindness. 

Control  of  Visual  Pigment  Gene  Expression 
We  have  been  interested  for  some  time  in  the 
general  question  of  how  different  cells  in  the  ret- 
ina assume  their  correct  identities.  As  an  initial 
approach,  we  have  examined  the  control  of  vi- 
sual pigment  gene  expression.  Each  photorecep- 
tor cell  appears  to  produce  only  a  single  type  of 
visual  pigment:  rhodopsin  in  the  rods,  and  the 
red,  green,  and  blue  pigments  in  their  respective 
cone  types.  As  a  working  model,  we  assume  that 
this  specificity  in  protein  production  reflects  a 
corresponding  specificity  at  the  level  of  gene 
transcription. 

One  region  of  DNA  that  is  important  for  activa- 
tion of  the  red  and  green  pigment  genes  has  re- 
cently been  identified.  Over  the  past  several 
years,  we  have  studied  families  with  a  rare  X- 
linked  disorder  called  blue  cone  monochromacy, 
in  which  both  red  and  green  cone  sensitivities  are 
absent.  In  many  families  a  DNA  deletion  is  ob- 
served in  which  sequences  are  removed  adjacent 
to  the  cluster  of  red  and  green  pigment  genes  on 
the  X  chromosome.  This  set  of  deletions  defines  a 
region  of  0.6  kilobases  that  appears  to  be  re- 
quired for  normal  red  and  green  visual  pigment 
gene  function,  even  though  the  start  sites  of  tran- 
scription of  these  genes  are,  respectively,  3  kilo- 
bases  and  42  kilobases  away. 

Yanshu  Wang,  a  graduate  student,  has  recently 
shown  that  a  segment  of  human  DNA  encompass- 
ing this  essential  region  and  including  the  red 


pigment  gene  promoter  directs  expression  of  a 
linked  reporter  gene  to  cone  cells  in  the  mouse 
retina.  An  otherwise  identical  construct  from 
which  the  0.6-kilobase  segment  has  been  re- 
moved is  inactive.  We  have  observed  that  within 
this  essential  segment,  there  is  a  smaller  region 
that  has  a  high  degree  of  DNA  sequence  homol- 
ogy across  species.  Most  likely,  this  region  repre- 
sents a  binding  site  for  one  or  more  transcription 
factors  present  in  the  red  and  green  cones. 

Retinitis  Pigmentosa 

During  the  past  several  years,  we  have  begun  to 
work  on  a  group  of  inherited  retinal  diseases 
called  retinitis  pigmentosa  (RP).  The  hallmarks 
of  RP  are  night  blindness  and  a  slow  progressive 
loss  of  peripheral  vision,  leading  in  most  cases  to 
complete  blindness  by  the  fifth  or  sixth  decades 
of  life.  RP  affects  one  person  in  4,000  in  all  popu- 
lations examined.  Last  year  Ching-Hwa  Sung,  a 
postdoctoral  fellow,  reported  finding  rhodopsin 
mutations  in  one-quarter  of  patients  with  autoso- 
mal dominant  RP,  an  inheritance  pattern  that  is 
found  in  approximately  one-fifth  of  the  patient 
population.  In  a  group  of  l6l  unrelated  patients, 
13  different  mutations  were  identified,  and  in  all 
cases  the  mutations  co-inherited  with  the  disease 
in  affected  families. 

Dr.  Sung  has  produced  each  of  the  mutant  op- 
sins, as  well  as  normal  human  opsin,  in  tissue  cul- 
ture cells  and  has  analyzed  their  biochemical 
properties.  The  mutant  proteins  fall  into  two 
classes.  Members  of  one  class  resemble  wild-type 
opsin  in  yield,  ability  to  bind  1  l-cis  retinal,  and 
efficient  transport  to  the  cell  surface.  By  contrast, 
members  of  the  second  class  are  produced  in  low 
yield,  bind  1 1  -cis  retinal  variably  or  not  at  all,  and 
are  transported  inefficiently  to  the  cell  surface. 
These  characteristics  suggest  that  the  second 
class  of  mutant  proteins  are  either  incorrectly 
folded  or  unstable.  It  seems  reasonable  to  sup- 
pose that  production  of  large  quantities  of  an  un- 
stable opsin  may  be  deleterious  to  the  photore- 
ceptor. The  biochemical  defect  in  the  first  class 
of  mutant  proteins  is  not  apparent  from  the  ex- 
periments performed  thus  far  and  is  currently 
under  investigation. 


292 


Gene  Regulation  in  Animal  Cells 


Joseph  R.  Nevins,  Ph.D. — Investigator 

Dr.  Nevins  is  also  Professor  of  Genetics  and  of  Microbiology  and  Immunology  at  Duke  University  Medical 
Center.  He  received  his  Ph.D.  degree  in  virology  at  Duke  University,  where  he  studied  with  Wolfgang  Joklik. 
His  postdoctoral  studies  as  a  Jane  Cofftn  Childs  fellow  focused  on  the  mechanisms  of  mRNA  biogenesis 
and  were  conducted  with  James  Darnell  at  the  Rockefeller  University,  where  he  later  became  a  faculty 
member. 


REGULATION  of  gene  expression  is  central  in 
the  determination  of  cellular  phenotype  and 
in  the  complex  transformations  that  take  place 
during  such  events  as  oncogenesis.  The  goal  of 
our  laboratory  is  to  elucidate  the  molecular  mech- 
anisms of  gene  control  pathways. 

Adenovirus  Transcriptional  Regulation — 
A  Role  in  Oncogenesis 

Complex  cellular  events  are  often  best  studied 
through  the  use  of  simple  model  systems.  The 
control  of  transcription  (transfer  of  DNA-coded 
information  to  RNA  to  guide  protein  synthesis) 
mediated  by  viral  regulatory  proteins  is  instruc- 
tive as  regards  the  v^^orkings  and  control  of  tran- 
scription factor  activity  in  eukaryotic  cells. 

Work  in  our  laboratory  has  focused  on  the  elu- 
cidation of  transcription  control  by  viral  pro- 
teins, using  in  vitro  assays.  We  have  found  that  a 
cellular  transcription  factor  termed  E2F  is  regu- 
lated by  other  cellular  proteins  and  that  these  in- 
teractions prevent  E2F  from  being  co-opted  by 
the  viral  genome.  The  adenovirus  regulatory  pro- 
tein El  A,  however,  can  dissociate  these  com- 
plexes, releasing  E2F,  which  can  then  be  utilized 
by  another  viral  product,  the  E4  protein.  This  re- 
directs the  cellular  transcription  factor  for  viral 
purposes. 

These  findings  are  significant  for  several  rea- 
sons. First,  they  provide  insight  into  the  evolu- 
tion of  viral  regulatory  events  that  manipulate  the 
host  cell  for  the  virus's  benefit.  In  particular,  they 
highlight  the  interplay  between  two  viral  pro- 
teins (El A  and  E4)  that  together  redirect  the  E2F 
factor  to  a  viral-specific  role. 

Second,  this  activity  of  ElA  correlates  with  the 
oncogenic  activity  of  the  protein.  The  sequences 
in  ElA  that  are  responsible  for  this  activity  are 
shared  with  other  viral  oncogene  products,  such 
as  the  SV40  T  antigen  and  the  human  papilloma- 
virus E7  protein.  Each  of  these  viral  proteins  has 
been  shown  to  activate  transcription,  dependent 
on  E2F. 

Third,  our  recent  experiments  have  shown  that 
the  viral  T  antigen  and  E7  protein  can  also  dissoci- 
ate complexes  containing  the  E2F  transcription 
factor.  In  short,  this  latest  discovery  has  defined  a 


common  biochemical  activity  of  these  viral  regu- 
latory proteins  that  is  likely  part  of  their  onco- 
genic activity. 

The  identification  of  the  cellular  proteins  that 
are  complexed  to  the  E2F  factor  has  provided  im- 
portant insight  into  the  role  of  ElA  as  an  onco- 
gene. We  have  found  that  there  are  multiple  such 
E2F  complexes  and  that  their  formation  is  regu- 
lated during  the  cell  cycle.  One  complex  con- 
tains the  cell  cycle-regulated  protein  cyclin  A, 
and  another  the  retinoblastoma  gene  product. 

The  product  of  the  retinoblastoma  susceptibil- 
ity gene  {RBI)  is  a  1 10-kDa  nuclear  phosphopro- 
tein  that  is  expressed  at  equivalent  levels  in  all 
cell  types  examined  except  certain  tumor  cells  in 
which  the  RBI  gene  has  been  inactivated  by  mu- 
tation or  deletion.  Given  that  it  is  the  loss  of  RBI 
function  that  is  correlated  with  the  development 
of  certain  human  tumors,  it  is  widely  believed 
that  the  Rb  protein  functions  to  limit  or  constrain 
cell  proliferation. 

It  now  appears  that  the  ability  of  E 1  A,  as  well  as 
T  antigen  and  E7,  to  dissociate  the  E2F-Rb  com- 
plex, releasing  free  E2F,  is  at  least  one  of  the 
events  of  Rb  inactivation.  The  E2F-Rb  complex, 
like  the  E2F-cycIin  complex,  is  also  regulated  by 
cell  proliferation.  Moreover,  the  E2F-Rb  complex 
is  absent  in  cells  that  express  a  nonfunctional 
form  of  the  Rb  protein. 

This  latter  result  strongly  argues  that  E2F  is  a 
functional  target  for  Rb  action.  Thus  the  conse- 
quence of  either  ElA  action,  the  deletion  or  mu- 
tation of  Rb,  or  the  phosphorylation  of  Rb  is  the 
loss  of  the  E2F-Rb  interaction  and  the  generation 
of  free  E2F  molecules.  In  short,  "inactivation"  of 
Rb  can  be  viewed  as  the  loss  of  the  E2F-Rb  inter- 
action. Moreover,  it  would  appear  that  the  conse- 
quence of  disruption  of  either  the  E2F-cyclin  A 
complex  or  the  E2F-Rb  complex  is  the  release  of 
transcriptionally  active  E2F  and  that  this  event 
may  be  important  for  stimulating  the  transcrip- 
tion of  genes  whose  products  are  critical  for  en- 
hancing cell  proliferation. 

Molecular  Mechanisms  for  Polyadenylation 

The  generation  of  the  3'  terminus  of  the  mature 
mRNA,  commonly  termed  poly(A)  site  formation. 


293 


Gene  Regulation  in  Animal  Cells 


is  a  critical  event  in  the  biogenesis  of  most  mRNA 
molecules.  Moreover,  since  many  transcription 
units  encode  multiple  mRNAs  that  utilize  alterna- 
tive poly(A)  sites,  there  is  a  potential  regulatory 
role  for  polyadenylation.  Indeed,  analysis  of  the 
formation  of  the  immunoglobulin  heavy-chain 
transcripts  during  B  cell  differentiation  suggests 
-that  alternative  poly(A)  site  choice  is  a  contribut- 
ing factor  in  the  switch  from  production  of  the  Ig 
mRNA  that  encodes  the  membrane-bound  form  of 
the  protein  to  the  mRNA  encoding  the  secreted 
form  of  the  protein.  Thus  an  understanding  of  the 
mechanism  of  poly(A)  site  utilization,  including 
the  sequences  directing  the  processing  event  and 
the  factors  involved  in  processing,  is  an  impor- 
tant focus  of  our  work. 

We  have  recently  used  a  biochemical  approach 
to  investigate  the  molecular  mechanism  of  poly- 
adenylation. Factors  have  been  purified  from 
HeLa  cell  nuclear  extracts  that  can  reconstitute 
accurate  and  sequence-specific  poly(A)  site  pro- 
cessing in  vitro.  Two  of  these  factors  interact  spe- 
cifically with  the  pre-mRNA,  and  an  analysis  of 


this  interaction  suggests  a  pathway  of  assembly  of 
a  functional  poly  (A)  site-processing  complex. 

The  interaction  of  one  factor  with  the  con- 
served AAUAAA  element  is  specific,  but  the  RNA- 
protein  complex  that  forms  is  very  unstable  and 
rapidly  dissociates.  The  interaction  of  a  second 
factor  with  the  RNA  requires  a  distinct  sequence 
element  but  also  requires  the  prior  interaction  of 
the  first  factor  with  the  AAUAAA  element.  Most 
importantly,  the  resulting  ternary  complex  is 
stable  and  does  not  readily  dissociate. 

These  observations  suggest  a  pathway  of  assem- 
bly of  poly(A)  site  factors  that  involves  an  initial 
recognition  of  the  AAUAAA  element  followed  by  a 
commitment  step  in  which  the  second  factor  sta- 
bilizes the  overall  complex.  Since  the  relative  sta- 
bility of  this  complex  reflects  the  relative  effi- 
ciency of  processing,  it  appears  that  these 
interactions  may  be  important  regulatory  targets 
for  the  control  of  polyadenylation. 

This  work  on  the  mechanisms  of  polyadenyla- 
tion is  supported  by  a  grant  from  the  National 
Institutes  of  Health. 


294 


Molecular  Genetics  ofX-linked  Disease 


Robert  L.  Nussbaum,  M.D. — Associate  Investigator 

Dr.  Nussbaum  is  also  Associate  Professor  of  Human  Genetics,  Pediatrics,  and  Medicine  at  the  University  of 
Pennsylvania  School  of  Medicine  and  Consultant  in  Clinical  Genetics  at  the  Children's  Hospital  of 
Philadelphia.  He  received  his  undergraduate  training  in  applied  mathematics  at  Harvard  College  and  his 
M.D.  degree  at  Harvard  Medical  School  in  the  Harvard-MIT  Joint  Program  in  Health  Sciences  and 
Technology.  After  his  residency  in  internal  medicine  at  Barnes  Hospital,  Washington  University  School  of 
Medicine,  he  moved  to  Baylor  College  of  Medicine,  first  for  a  genetics  fellowship  with  Thomas  Caskey  and 
Arthur  Beaudet  and  later  as  a  faculty  member.  He  then  moved  to  the  University  of  Pennsylvania,  where 
he  developed  his  research  program  in  molecular  genetics  and  its  application  to  the  diagnosis  and 
elucidation  of  human  genetic  disease. 


THE  research  in  my  laboratory  is  directed  to- 
ward elucidating  the  molecular  bases  for  a 
number  of  human  genetic  diseases.  Each  disease 
under  investigation  is  known  to  be  caused  by  a 
gene  on  the  X  chromosome,  but  the  molecular 
mechanism,  the  gene  involved,  and  the  nature  of 
the  underlying  mutations  have  been  hitherto  un- 
known. Recombinant  DNA  techniques  have  been 
employed  to  isolate  the  responsible  genes,  with 
the  aim  of  furthering  our  understanding  of  the 
normal  processes  that  when  disrupted  result  in 
each  of  these  diseases. 

Choroideremia 

Choroideremia  is  a  rare  X-linked  disease  of  the 
retina  that  produces  blindness  in  affected  males. 
The  gene  responsible  and  the  mechanism  of  reti- 
nal damage  have  until  recently  been  unknown. 
Our  laboratory  is  using  information  about  where 
the  choroideremia  gene  is  to  identify  it  and  ex- 
plain why  mutations  in  this  gene  cause  the 
disease. 

We  have  isolated  a  gene  from  the  region  around 
a  chromosomal  translocation  in  a  female  patient 
with  choroideremia.  Her  disease  resulted  from 
disruption  of  the  choroideremia  gene  by  the  chro- 
mosomal break  in  the  X  chromosome  in  this  re- 
gion. A  transcribed  gene  that  is  disrupted  by  this 
chromosomal  translocation  has  been  identified 
and  found  to  be  very  similar,  although  not  identi- 
cal, to  one  isolated  in  the  laboratory  of  Frans 
Cremers  by  his  study  of  males  with  choroi- 
deremia and  submicroscopic  deletions.  The  ex- 
pression of  this  gene,  at  the  level  of  mRNA, 
is  abnormal  in  75  percent  of  patients  with 
choroideremia. 

The  predicted  protein  sequence  of  the  gene 
identified  in  both  laboratories  has  a  subtle  similar- 
ity with  a  protein  isolated  from  platelets  that  may 
be  involved  in  regulation  of  the  G  proteins,  a  very 
large  and  heterogeneous  group  involved  in  carry- 
ing certain  intracellular  signals.  The  cellular 


mechanism  by  which  vision  occurs,  known  as  vi- 
sual transduction,  is  the  entire  pathway  by  which 
light,  the  initial  signal,  is  translated  first  into  bio- 
chemical reactions  and  then  nervous  impulses  in 
the  retina.  A  growing  body  of  evidence  suggests 
that  abnormalities  in  the  proteins  that  carry  out 
visual  transduction  contribute  to  a  variety  of  de- 
generative retinal  diseases  that  lead  to  blindness. 

The  plan  is  to  investigate  the  exact  role  of  the 
choroideremia  gene  product  in  visual  transduc- 
tion, in  an  attempt  to  understand  how  abnormali- 
ties in  that  gene  lead  to  retinal  degeneration.  This 
work  is  supported  by  a  grant  from  the  National 
Institutes  of  Health. 

Lowe's  Syndrome 

Lowe's  syndrome  is  an  uncommon  X-linked 
disease  that  causes  mental  retardation,  cataracts, 
and  kidney  dysfunction.  The  cause  is  unknown. 
As  with  choroideremia,  our  strategy  is  to  identify 
the  Lowe's  syndrome  gene  through  information 
about  its  location. 

We  have  isolated  two  cDNA  sequences  from  the 
region  around  a  translocation  breakpoint  in  a  fe- 
male with  Lowe's  syndrome.  The  transcript  de- 
tected by  one  of  these  cDNAs  is  disrupted  by  the 
translocation  and  is  absent  or  abnormal  in  nine 
unrelated  males  with  the  disease.  This  gene  en- 
codes a  protein  involved  in  the  metabolism  of 
inositol  phosphates,  a  very  heterogeneous  but 
important  class  of  compounds  with  a  complex 
biochemistry.  These  structural  components  of 
normal  cell  membranes  also  play  an  important 
role  in  anchoring  proteins  to  cell  membranes  and 
in  intracellular  signaling  pathways. 

Our  goal  is  to  understand  the  biochemical  pro- 
cesses that,  when  defective,  lead  to  brain,  lens, 
and  kidney  dysfunction  and  damage.  Insights  into 
normal  lens  formation  and  normal  brain  and  kid- 
ney function  could  result,  and  methods  for  im- 
proved diagnosis  and  therapy  for  the  disease  may 
be  found.  This  work  is  supported  by  a  grant  from 
the  National  Institutes  of  Health. 


295 


Expression  pattern  of  theWnt-5  gene  in  the  developing  mouse  brain.  In 
situ  hybridization  at  10  days  of  gestation  shows '^m-i  expression  in 
one  of  the  compartments  of  the  diencephalon,  D2,  and  in  the  develop- 
ing midbrain-hindbrain  junction  that  give  rise  respectively  to  the  dor- 
sal thalamus  and  the  cerebellum. 

Research  and  photograph  by  Patricia  Salinas  in  the  laboratory  of 
Roel  Nusse. 


Function  of  Oncogenes  in  Early  Emhryogenesis 


Roel  Nusse,  Ph.D. — Associate  Investigator 

Dr.  Nusse  is  also  Associate  Professor  of  Developmental  Biology  at  the  Stanford  University  School  of 
Medicine.  He  obtained  his  Ph.D.  degree  from  the  University  of  Amsterdam  and  was  a  postdoctoral  fellow 
with  Harold  Varmus  at  the  University  of  California,  San  Francisco,  before  returning  to  Amsterdam,  where 
he  became  head  of  the  Department  of  Molecular  Biology  at  the  Netherlands  Cancer  Institute.  Several  years 
ago  he  moved  to  the  Beckrnan  Center  of  Stanford  University  and  the  Howard  Hughes  Medical  Institute. 
Dr.  Nusse  is  a  member  of  the  European  Molecular  Biology  Organization  (EMBO ). 


IT  is  now  well  established  that  genes  whose  al- 
tered expression  can  lead  to  cancer  (generally 
called  oncogenes  or  proto-oncogenes)  are  indis- 
pensable regulators  of  normal  cell  proliferations. 
Many  oncogenes  participate  in  the  cells'  deci- 
sions whether  to  divide  or  remain  quiescent. 
Apart  from  the  regulation  of  cell  proliferation, 
growth  in  a  normal  organism  obviously  needs 
control  at  another  level:  during  the  formation  of 
patterns  in  which  cells  become  properly 
arranged.  Over  the  past  few  years,  it  has  become 
increasingly  clear  that  oncogenes  are  among  the 
key  regulators  of  this  aspect  of  growth  control 
also. 

The  evidence  for  such  controlling  functions 
has  primarily  come  from  the  genetic  dissection  of 
morphogenesis  in  organisms  such  as  Drosophila 
and  Caenorhabditis  elegans.  Many  of  the  devel- 
opmental genes  isolated  from  those  animals  are 
highly  homologous  to  mammalian  oncogenes,  in 
particular  those  that  encode  signaling  molecules 
involved  in  cell-to-cell  communications  during 
embryogenesis.  Now,  with  the  increasing  possi- 
bilities to  identify  developmental  genes  in  the 
mouse  as  well,  additional  examples  have 
emerged  for  a  link  between  cancerous  growth 
and  the  control  of  normal  development. 

The  Wnt/wingless  gene  family  is  a  paradigm 
for  the  connections  between  tumorigenesis  and 
embryogenesis.  The  prototypic  member  of  this 
group,  Wnt-l,  is  an  oncogene  frequently  acti- 
vated in  mouse  mammary  cancer.  The  Wnt-l 
gene  is  normally  not  expressed  in  the  mammary 
gland  or  in  most  other  adult  tissues.  In  tumors, 
however,  its  transcription  is  induced  by  nearby 
insertion  of  proviral  DNA  of  a  retrovirus,  the 
mouse  mammary  tumor  virus.  Proof  that  Wnt-l  is 
an  oncogene  came  from  transfection  experiments 
and  from  the  finding  that  introduction  of  Wnt-l 
as  a  transgene  into  the  germline  of  mice  can  lead 
to  tumor  induction.  Wnt-l  is  part  of  a  family  of 
genes  that  in  the  mouse  consists  of  at  least  10 
members.  All  of  these  genes  encode  secreted  pro- 
teins rich  in  cysteine  residues. 

Over  the  past  few  years,  evidence  that  the  Wnt 
genes  are  prime  determinants  for  early  develop- 


ment has  accumulated  from  many  different 
corners.  For  example,  most  of  these  Wnt  genes 
have  a  very  restricted  pattern  of  expression  dur- 
ing early  developmental  stages,  in  organisms 
ranging  from  mice  to  fruit  flies.  More  telling  are 
the  findings  that  Wnt  gene  mutations  prevent 
normal  development  of  the  mouse  brain  and  nor- 
mal segmentation  of  Drosophila  embryos.  More- 
over, it  has  been  shown  that  ectopic  expression 
of  Wnt  genes  induces  axis  duplication  in  frog 
embryos. 

The  work  in  our  group  is  focused  on  the  role  of 
several  members  of  the  Wnt  gene  family  in  the 
development  of  the  mouse,  along  with  investiga- 
tions of  Wnt  genes  in  the  fruit  fly.  In  the  mouse 
we  perform  detailed  in  situ  RNA  hybridization 
analysis  of  the  expression  of  Wnt-5  and  a  highly 
related  gene,  Wnt-5A.  Both  genes  are  expressed 
in  the  developing  neural  tube. 

In  particular,  the  anterior  boundary  of  expres- 
sion of  Wnt-5  and  Wnt-5A  is  interesting.  The 
genes  are  expressed  in  the  diencephalon  and  in 
the  cerebral  hemispheres,  suggesting  that  they 
play  important  roles  in  establishing  these  com- 
partments in  the  developing  brain.  For  example, 
Wnt-5  is  expressed  in  the  D2  neuromere  of  the 
developing  diencephalon  during  day  9  and  1 2  of 
embryogenesis.  The  expression  precedes  the  sub- 
division of  the  diencephalon  into  the  ventral  and 
dorsal  thalamus  (see  figure),  suggesting  a  func- 
tional role  for  Wnt-5  in  this  process.  Another  re- 
markably restricted  expression  pattern  of  Wnt-5 
is  seen  in  the  cerebellum,  where  the  gene  is  ex- 
clusively expressed  in  the  Purkinje  cells. 

Understanding  the  mechanism  of  action  of  the 
Wnt-l  gene  family  during  embryogenesis  has 
been  complicated  by  difficulties  in  characteriz- 
ing the  Wnt  proteins.  We  wish,  for  example,  to 
identify  the  receptors  for  these  secreted  mole- 
cules, but  as  no  biologically  active  Wnt  gene 
product  is  available,  the  nature  of  the  receptors 
remains  elusive.  In  parallel  to  these  biochemical 
experiments,  we  study  the  interactions  between 
Wnt  and  other  genes  by  taking  advantage  of  the 
extensive  genetic  analysis  of  Drosophila  embryo- 
genesis. Some  years  ago  we  made  the  surprising 
finding  that  the  Drosophila  Wnt-l  homologue  is 


297 


Function  of  Oncogenes  in  Early  Embryogenesis 


identical  to  the  segment  polarity  gene  wingless. 
This  observation  has  allowed  us  to  study  the  in- 
teractions of  Wnt-\/ wingless  with  other  segmen- 
tation genes. 

The  basic  body  plan  of  the  fruit  fly  is  estab- 
lished by  several  classes  of  genes  that  progres- 
sively divide  the  embryo  into  smaller  compart- 
^-ments:  the  gap  genes,  the  pair-rule  genes,  and  the 
segment  polarity  genes.  Whereas  the  gap  and 
pair-rule  genes  encode  nuclear  proteins  and  are 
active  before  the  Drosophila  embryo  becomes 
cellularized,  the  segment  polarity  genes  are  the 
first  that  control  cell-cell  interactions.  The  wing- 
less gene  is  a  good  example,  encoding  a  secreted 
factor,  but  other  segment  polarity  genes  are 
thought  to  interact  with  wingless.  To  study  the 
properties  of  the  wingless  protein,  we  have  made 
antibodies  that  recognize  it  in  whole-mount  em- 
bryos and  in  individual  cells.  The  protein  is  seen 
on  the  surface  of  cells  and  in  intracellular  vesicu- 
lar structures.  We  have  characterized  the  distri- 
bution of  the  wingless  protein  in  embryos  that 
are  mutant  for  wingless  itself  and  have  obtained 
evidence  that  lack  of  secretion  of  the  protein  is 
the  primary  defect  in  these  mutants. 


In  an  additional  approach,  we  have  overex- 
pressed  the  wingless  gene  in  embryos,  from  a 
heat-shock  promoter.  The  phenotype  of  the  heat- 
shock  wingless  embryos  is  the  opposite  of  the 
phenotype  of  embryos  lacking  wingless.  Surpris- 
ingly, heat-shock  wingless  embryos  are  very  simi- 
lar to  embryos  that  are  mutant  for  two  other 
segment  polarity  genes,  called  naked  and  zeste- 
white-3,  suggesting  common  biochemical  path- 
ways of  wingless  and  those  genes. 

We  have  also  found  that  wingless  in  Drosoph- 
ila is  part  of  a  gene  family,  with  at  least  two  addi- 
tional members.  These  genes,  called  DWnt-2  and 
T>Wnt-5  for  the  time  being,  are  also  expressed 
during  early  embryogenesis,  in  characteristic  pat- 
terns that  differ  from  wingless.  Mutants  at  these 
genes  have  not  yet  been  obtained,  but  the  un- 
usual character  of  the  DWnt-5  protein,  which  is 
three  times  as  long  as  other  Wnt  proteins,  may 
give  us  new  clues  as  to  the  biochemical  mecha- 
nism of  action  of  the  genes. 

In  the  future  we  hope  to  extrapolate  our  in- 
creasing understanding  of  the  function  of  the 
Wnt  genes  in  embryogenesis  to  the  mechanism  of 
action  of  the  genes  in  cancer. 


298 


Molecular  Mechanisms  That  Regulate 
B  Cell  Development 


Michel  C.  Nussenzweig,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Nussenzweig  is  also  Assistant  Professor  and  Head  of  Laboratory  at  the  Rockefeller  University.  He 
received  his  undergraduate  and  M.D.  degrees  from  New  York  University  and  his  Ph.D.  degree  from 
Rockefeller.  He  completed  his  residency  and  clinical  fellowship  at  Massachusetts  General  Hospital 
and  conducted  postdoctoral  research  at  Harvard  Medical  School  with  Philip  Leder. 


WE  are  interested  in  understanding  the  mo- 
lecular mechanisms  that  regulate  B  lym- 
phocyte activation  and  differentiation.  Our  ap- 
proach has  been  to  focus  on  one  important 
transition  in  B  lymphocytes,  allelic  exclusion. 

The  immune  system  is  responsible  for  protect- 
ing vertebrates  from  both  invasion  by  infectious 
organisms  and  deregulated  growth  of  endoge- 
nous malignancies.  To  accomplish  this  task,  the 
immune  system  must  be  able  to  distinguish  self 
from  non-self.  Evolution  has  solved  this  problem 
in  higher  vertebrates  by  providing  a  network  of 
cell  types  and  humoral  agents.  It  is  the  lympho- 
cytes— T  cells  and  B  cells — that  direct  the  speci- 
ficity of  immune  responses. 

Although  the  mechanism  of  antigenic  recogni- 
tion differs  for  these  two  cell  types,  the  genera- 
tion of  receptor  diversity  is  achieved  in  a  similar 
fashion.  In  both  cases  the  business  end  of  the  re- 
ceptor is  created  in  individual  somatic  cells  by  a 
series  of  genetic  recombinations  at  a  minimum  of 
two  loci.  For  example,  immunoglobulin  mole- 
cules that  serve  as  the  B  cell  receptor  are  com- 
posed of  two  sets  of  rearranging  genes  that  en- 
code the  heavy-  and  light-chain  immunoglobulin 
proteins.  Furthermore,  the  same  genes  that  en- 
code the  B  lymphocyte  receptor  also  direct  the 
production  of  secreted  antibodies  that  are  an 
important  component  of  humoral  immune  re- 
sponses. Thus  the  regulation  of  T  cell  and  B  cell 
receptor  rearrangements  is  a  central  feature  of 
the  generation  of  immune  responses. 

The  joining  events  that  bring  together  the  im- 
munoglobulin segments  occur  in  an  ordered  and 
regulated  fashion.  In  B  lymphocytes,  rearrange- 
ments begin  at  the  heavy-chain  locus  with  the  re- 
combination of  D  and  J  segments.  This  is  followed 
by  joining  of  DJ  with  one  of  100-1 ,000  variable- 
region  segments.  After  a  functional  immunoglob- 
ulin heavy-chain  transcription  unit  is  created,  the 
light-chain  genes  undergo  a  similar  set  of 
rearrangements . 

One  poorly  understood  aspect  of  these  events 
is  the  ability  of  lymphocytes  to  limit  themselves 
to  the  production  of  a  single  receptor.  Since  pro- 
ductive rearrangements  could  occur  in  two 


heavy-chain  and  four  light-chain  alleles,  a  single 
B  lymphocyte  could  potentially  make  several 
types  of  receptors,  including  hybrid  molecules. 
The  mechanism  that  ensures  that  only  one  recep- 
tor is  produced  is  referred  to  as  allelic  exclusion. 
It  is  an  important  safeguard  for  the  immune  sys- 
tem, since  production  of  multiple  receptors  by  a 
lymphocyte  would  dilute  the  specificity  of  any 
given  immune  response. 

Much  of  the  early  work  in  the  area  of  allelic 
exclusion  was  based  on  examining  the  status  of 
immunoglobulin  genes  in  transformed  B  cells. 
The  transformed  cells  are  frozen  in  one  stage  of 
lymphocyte  development  and  for  this  reason 
offer  only  a  static  picture  of  important  regulatory 
events.  Unfortunately  there  is  no  in  vitro  system 
that  faithfully  reproduces  regulated  immuno- 
globulin gene  rearrangements.  To  study  how  im- 
munoglobulin genes  can  regulate  allelic  exclu- 
sion, we  turned  to  transgenic  mice. 

Our  approach  has  been  to  introduce  into  the 
germline  of  mice  human  immunoglobulin  genes 
that  have  been  modified  to  direct  the  synthesis  of 
either  membrane-associated  or  secreted  immuno- 
globulin heavy  chains.  We  found  that  the  expres- 
sion of  human  membrane-bound  immunoglobu- 
lin M  (IgM)  results  in  the  exclusion  of  most 
endogenous  mouse  immunoglobulins.  The  se- 
creted version  of  the  same  transgene  has  little 
effect. 

This  initial  observation  raises  two  important 
questions.  First,  how  does  membrane  immuno- 
globulin signal?  Second,  how  is  exclusion  regu- 
lated at  the  genetic  level?  During  the  past  year  we 
have  made  significant  progress  toward  resolving 
both  questions.  To  understand  signaling  by  the 
membrane-associated  immunoglobulin  mole- 
cule, we  have  developed  a  system  for  complete 
functional  reconstitution  of  the  immunoglobulin 
receptor  from  cloned  components  in  heterolo- 
gous cells.  Transport  of  IgM  to  the  surface  of  T 
cells  requires  coexpression  of  the  immunoglobu- 
lin heavy  and  light  chains  with  B29,  an  immuno- 
globulin-associated  polypeptide.  In  addition,  the 
transfected  receptor  is  fully  active  in  the  pres- 
ence of  B29.  MBI ,  a  second  IgM-associated  poly- 


299 


Molecular  Mechanisms  That  Regulate  B  Cell  Development 


peptide,  is  not  required  for  transport  or  signal 
transduction.  Progress  in  understanding  the  func- 
tional role  of  the  immunoglobulin  receptor  sub- 
units  in  signaling  has  been  hindered  by  the  multi- 
subunit  nature  of  the  receptor.  In  addition, 
transfected  immunoglobulin  expressed  on  the 
surface  of  fibroblasts  does  not  appear  to  be  func- 
tional for  signal  transduction,  even  in  the  pres- 
ence of  B29  and  MBl.  Our  ability  to  produce  a 
functional  receptor  by  transfection  in  T  cells  es- 
tablishes that  there  is  enough  structural  similarity 
between  the  T  and  B  cell  signal  transduction 
pathways  to  allow  recognition  of  the  IgM-B29 
complex  in  T  cells.  This  observation  should  sim- 
plify the  structural  and  functional  analysis  of 
the  IgM  antigen  receptor  and  ultimately  allow 
us  to  understand  how  membrane  immunoglobu- 
lin signals  for  antigen  recognition  and  allelic 
exclusion. 

In  addition  to  our  ability  to  study  the  structural 
requirements  for  immunoglobulin  function,  our 
experiments  also  have  potential  clinical  implica- 
tions. One  major  difference  between  the  T  cell 
receptor  and  immunoglobulin  antigen  receptors 
is  the  nature  of  the  antigen  recognized.  Immuno- 
globulins recognize  antigens  directly;  the  major 
histocompatibility  complex  (MHC)  restricts  rec- 


ognition of  antigen  by  the  T  cell  receptor.  The 
requirement  for  MHC  recognition  severely  limits 
the  targets  recognized  by  T  cells  and  restricts 
transfer  of  cellular  immunity.  Our  reconstitution 
experiments  offer  one  potential  solution  to  this 
difficult  clinical  problem — the  production  of  T 
cells  that  utilize  immunoglobulins  as  antigen  re- 
ceptors. In  T  cells  that  recognize  antigen  with 
immunoglobulin  receptors  there  is  no  MHC  re- 
striction, and  thus  this  barrier  to  transfer  of  cellu- 
lar immunity  is  potentially  abrogated. 

Our  second  goal  has  been  to  understand  allelic 
exclusion  at  the  genetic  level.  We  have  docu- 
mented that  chromosomal  position  plays  an  im- 
portant role  in  the  regulation  of  gene  rearrange- 
ments in  the  immunoglobulin  locus.  In  addition 
we  have  started  to  investigate  the  regulatory 
function  of  the  recombinase  genes  RAG-1  and 
RAG-2  in  allelic  exclusion.  Deregulating  the  ex- 
pression of  the  RAG  genes  in  the  lymphocytes  of 
transgenic  mice  has  profound  effects  on  lympho- 
cyte differentiation  and  function.  Animals  that 
carry  /MG- 1  and  /2/4G'-2transgenes  develop  hepa- 
tosplenomegaly  and  a  profound  lymphoprolif- 
erative  disorder  that  is  rapidly  lethal.  We  are 
pursuing  a  molecular  understanding  of  the  patho- 
genesis of  this  disorder. 


300 


Mechanism  of  DNA  Replication 


Michael  E.  O'Donnell,  Ph.D. — Assistant  Investigator 

Dr.  O'Donnell  is  also  Associate  Professor  of  Microbiology  at  Cornell  University  Medical  College,  New  York 
City.  He  received  his  Ph.D.  degree  from  the  University  of  Michigan,  Ann  Arbor,  where  his  research  on 
electron  transfer  in  the  flavoprotein  thioredoxin  reductase  was  conducted  with  Charles  H.  Williams,  Jr. 
He  performed  postdoctoral  work  on  Escherichia  coli  replication  with  Arthur  Kornberg  and  then  on  herpes 
simplex  virus  replication  with  Bob  Lehman,  both  in  the  Biochemistry  Department  at  Stanford  University. 


MY  laboratory  is  studying  the  duplication  of 
genetic  information.  By  understanding  the 
fundamental  mechanisms  of  cell  growth,  or  the 
replication  of  DNA,  we  may  obtain  insights  into 
the  development  of  abnormal  cells,  including  tu- 
mor cells. 

The  genetic  material  of  our  cells,  the  chromo- 
somes, is  a  library  with  all  the  information 
needed  for  the  multitude  of  duties  required  to 
maintain  the  cell's  life.  Included  in  these  duties 
is  the  buildup  of  complete,  new  cellular  machin- 
ery for  the  synthesis  of  another  cell  (reproduc- 
tion). The  chromosome  library  is  made  of  two 
long  interwound  helical  fibers  of  DNA  (deoxyri- 
bonucleic acid  polymers).  Before  a  cell  can  di- 
vide to  form  two  new  cells,  it  must  duplicate  the 
genetic  library  so  that  each  cell  has  a  complete 
copy  of  instructions  on  how  to  live. 

The  process  of  duplicating  DNA  is  intricate, 
and  the  cell  has  evolved  a  precision  machine  to 
carry  out  this  important  task.  Its  several  protein 
parts  are  like  gears  of  a  machine,  which  coordi- 
nate their  actions  to  unzip  and  unwind  the  dou- 
ble-helical strands  of  DNA.  The  machinery  then 
uses  the  separated  single  strands  as  templates  to 
synthesize  two  double-helical  daughter  chromo- 
somes. Subsequently  these  will  segregate  in  two 
newly  formed  cells. 

Our  goal  is  to  understand,  at  a  molecular  level, 
the  workings  of  proteins  in  the  mechanics  of  DNA 
duplication.  The  system  we  are  studying  is  the 
bacterium  Escherichia  coli,  a  relatively  simple 
organism.  The  protein  machine  that  duplicates 
the  E.  coli  chromosome  is  called  DNA  polymer- 
ase III.  The  DNA  polymerase  III  of  E.  co/i  has  nine 
accessory  proteins  plus  the  polymerase.  Like  the 
E.  CO/?  polymerase  III,  the  DNA  polymerases  that 
replicate  the  chromosomes  of  higher  organisms 
such  as  yeast,  Drosophila,  and  humans  are  also 
composed  of  a  DNA  polymerase  protein  and  sev- 
eral other  "accessory"  proteins. 

The  function  of  the  DNA  polymerase  protein 
(the  a-subunit)  is  to  synthesize  the  DNA.  One  of 
the  accessory  proteins,  the  e-subunit,  is  an  exonu- 
clease  that  "proofreads"  the  product  of  the  poly- 
merase protein.  Very  little  is  known  about  the 


functions  of  the  other  eight  accessory  proteins. 
However,  since  the  several  accessory  proteins  to 
the  DNA  polymerase  are  conserved  in  evolution 
from  bacteria  to  humans,  it  seems  reasonable  to 
expect  their  individual  functions  to  serve  very 
important  roles  in  the  process  of  chromosome 
duplication.  Analysis  of  the  E.  coli  DNA  polymer- 
ase III  system  will  likely  extend  and  generalize 
the  understanding  of  the  replication  process  in 
all  organisms. 

We  have  recently  developed  methods  to  obtain 
pure  preparations  of  each  protein,  or  subunit,  of 
the  E.  coli  DNA  polymerase  III,  and  from  these 
the  whole  complex  can  be  reassembled.  We  have 
studied  the  individual  subunits  for  biochemical 
activities  and  for  their  physical  interactions.  Two 
subunits,  7  and  b,  bind  to  each  other  to  form  a 
complex  that,  upon  binding  to  primed  DNA,  hy- 
drolyzes  ATP.  In  the  presence  of  the  /3-subunit, 
the  75  heterodimer  couples  the  hydrolysis  of  ATP 
to  clamp  a  dimer  of  the  |8-subunit  onto  primed 
DNA.  One  molecule  of  the  76  heterodimer  can 
clamp  many  (8-dimers  onto  primed  DNA.  Our  bio- 
chemical studies  indicated  that  the  |8-dimer  is 
clamped  to  DNA  by  encircling  it  like  a  doughnut. 
The  x-ray  structure  of  this  /3-clamp  has  recently 
been  solved  in  a  collaboration  with  John  Kuriyan 
(HHMI,  Rockefeller  University).  It  appears  as  a 
thin  disk  with  a  hole  through  the  middle  to  ac- 
commodate the  DNA.  These  studies  on  the  /?- 
clamp  and  the  76  heterodimer  were  funded  by 
the  National  Institutes  of  Health. 

The  |8-clamp  on  DNA  binds  the  polymerase  sub- 
unit,  tethering  it  to  the  DNA  template.  Whereas 
the  polymerase  alone  is  slow  (20  nucleotides/ 
second),  it  is  greatly  accelerated  upon  binding 
the  jS-clamp  (700  nucleotides/second)  and  repli- 
cates an  entire  8-kb  single-strand  circular  DNA 
without  coming  off  (processive).  This  fits  nicely 
with  the  fact  that  the  E.  coli  cell  duplicates  its  4 
million-base  chromosome  within  30  minutes. 

Another  function  of  the  7-,  6-,  and  j8-subunits  is 
to  rapidly  deliver  the  polymerase  subunit  from  a 
completely  replicated  DNA  molecule  to  a  new 
primed  DNA  template.  This  rapid  delivery  of  poly- 
merase is  important  because  one  strand  of  the 


301 


Mechanism  of  DNA  Replication 


DNA  duplex  (lagging  strand),  as  a  result  of  the 
geometry  of  the  DNA  helix,  must  be  replicated  in 
fragments.  Synthesis  of  these  fragments  requires 
that  the  polymerase  subunit  be  used  over  and 
over  every  1-2  seconds  by  repeatedly  delivering 
it  from  a  finished  fragment  to  a  new  one.  Studies 
on  the  mechanism  of  rapid  polymerase  delivery 
-j;o  new  primed  templates  were  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

Two  T-subunits  bind  tightly  to  each  other  (di- 
meric),  and  each  binds  a  polymerase  molecule. 
Hence  the  r-subunit  dimer  serves  as  a  scaffold  to 
form  a  twin  polymerase.  Since  the  chromosome 
has  two  strands  of  DNA,  both  of  which  must  be 
replicated,  the  twin  polymerase  likely  serves  the 
function  of  coordinately  replicating  both  DNA 
strands  at  the  same  time.  These  studies  on  the 
dimeric  polymerase  were  also  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

These  studies  on  four  (7,  5,  /?,  t)  of  the  eight 
accessory  proteins  of  E.  coli  DNA  polymerase  III 
have  been  greatly  aided  by  having  the  genes  (the 
informational  area  in  the  DNA)  for  three  of  them 
(7,  fi,  r).  This  has  provided  large  quantities  of 


proteins  for  studies  via  molecular  cloning  and 
overproduction  techniques.  We  have  recently  dis- 
covered the  genes  for  each  of  the  remaining  five 
proteins  (5,  5',  x,  0)  of  DNA  polymerase  III  and 
have  used  these  genes  to  produce  and  purify  large 
quantities  of  these  proteins.  Identification  of  the 
genes  encoding  6',  x,  4^,  and  6  was  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

Toward  a  goal  of  understanding  the  overall 
structure  of  the  holoenzyme,  we  are  using  the 
pure  subunits  to  define  the  various  subunit  con- 
tacts within  the  holoenzyme  and  to  assemble  the 
entire  replicating  machine  from  its  separate 
parts.  Our  plans  to  identify  the  individual  func- 
tion of  each  subunit  include  biochemical  analysis 
and  also  use  of  the  newly  discovered  genes  for  the 
holoenzyme  subunits  to  construct  genetic  mu- 
tants of  E.  coli  for  further  clues  to  the  function  of 
each  subunit.  In  addition  to  the  polymerase  holo- 
enzyme, the  helicase  and  priming  proteins  are 
also  central  to  the  process  of  chromosome  repli- 
cation, and  we  have  initiated  studies  to  examine 
how  these  proteins  coordinate  their  actions  with 
the  holoenzyme. 

Model  of  the  13-clamp  of  the  en- 
zyme DNA  polymerase  III.  The 
function  of  the  clamp  is  to 
tether  the  enzyme  to  the  DNA, 
greatly  accelerating  DNA  syn- 
thesis. The  two  parts  of  the  ^- 
subunit  dimer,  represented  in 
red  and  yellow,  encircle  the 
DNA  ( in  green  and  gray ), 
which  has  been  modeled  in  the 
central  cavity. 

From  Kong,  X.-P.,  Onrust,  R., 
O'Donnell,  M.,  and  Kuriyan,  J. 
1992.  Cell  69:425-437.  Copy- 
right ©  1992  by  Cell  Press. 


302 


Large-Scale  Analysis  of  Yeast  and  Human  DNA 


Maynard  V.  Olson,  Ph.D. — Investigator 

Dr.  Olson  is  also  Professor  of  Genetics  at  the  Washington  University  School  of  Medicine.  He  was  trained  as 
a  chemist,  receiving  his  B.S.  degree  from  the  California  Institute  of  Technology  and  his  Ph.D.  degree  from 
Stanford  University.  After  five  years  on  the  faculty  at  Dartmouth  College,  he  moved  to  the  University  of 
Washington  and  changed  fields  from  chemistry  to  genetics.  He  has  served  on  the  National  Research 
Council  Committee  on  the  Mapping  and  Sequencing  of  the  Human  Genome  and  presently  serves  on  the 
NIH  Program  Advisory  Committee  of  the  National  Center  for  Human  Genome  Research. 


MOST  human  cells  contain  6  billion  base 
pairs  of  DNA.  Embedded  therein  are  an  un- 
known number  of  genes,  perhaps  100,000,  that 
direct  the  biochemical  events  in  the  cells.  At  pres- 
ent, well  over  95  percent  of  this  DNA  is 
unexplored. 

Geneticists  have  developed  powerful  methods 
with  which  to  study  DNA  in  small  packets.  Gene- 
splicing  techniques,  DNA  sequencing,  and  meth- 
ods of  reintroducing  altered  DNA  molecules  into 
cells  allow  the  detailed  structural  and  functional 
analysis  of  DNA  molecules  containing  up  to  tens 
of  thousands  of  base  pairs.  Our  laboratory  seeks 
to  extend  these  approaches  to  encompass  mole- 
cules ranging  up  to  millions  of  base  pairs  in  size. 

In  the  short  run,  this  research  should  allow  the 
analysis  of  larger  functional  units  of  DNA — for 
example,  large  human  genes,  clusters  of  coregu- 
lated  genes,  and  such  structures  as  centromeres 
and  telomeres,  which  govern  the  behavior  of  hu- 
man chromosomes  during  the  cell  division  cycle. 
In  the  long  run  these  methods  should  allow  the 
systematic  analysis  of  the  whole  human  genome 
— the  entire  complement  of  DNA  sequences — 
thereby  creating  tools,  such  as  detailed  maps,  that 
would  be  of  permanent  value  in  biology. 

Our  approach  has  been  to  build  on  progress  in 
the  genetic  analysis  of  microorganisms,  particu- 
larly yeast.  The  common  laboratory  yeast  Sac- 
charomyces  cerevisiae,  familiar  from  its  use  in 
baking  and  wine  making,  is  an  ideal  model  for 
studies  of  human  cells,  because  its  genetic  organi- 
zation and  biochemical  pathways  are  similar  to 
those  in  higher  organisms.  It  is  a  powerful  tool  as 
well,  because  human  DNA  can  be  altered  by 
gene-splicing  techniques  into  a  form  that  is  stably 
propagated  in  yeast. 

We  are  just  completing  a  long-term  project  to 
map  the  1 4  million  base  pairs  of  DNA  present  on 
the  16  natural  yeast  chromosomes.  The  yeast 
chromosomes  have  been  mapped  at  a  resolution 
(i.e.,  the  average  spacing  between  landmarks)  of 
3,000  base  pairs,  and  nearly  200  genes  have  been 
localized  on  the  map.  New  genes  can  now  be 
placed  on  this  map  in  only  a  few  hours.  This  pro- 
cess, which  replaces  mapping  techniques  that  re- 


quired weeks  of  effort,  is  now  in  use  in  more  than 
100  laboratories. 

Despite  this  success,  it  has  long  been  apparent 
that  no  straightforward  extension  of  these  meth- 
ods would  be  successful  on  the  greatly  expanded 
scale  of  the  human  genome.  For  this  reason,  we 
developed  a  method  to  import  manageable  seg- 
ments of  the  human  chromosomes  into  yeast, 
where  they  can  be  propagated  as  yeast  artificial 
chromosome  (YAC)  clones.  One  advantage  of 
YACs  over  previous  cloning  systems  is  that  there 
is  no  absolute  upper  size  limit  to  the  clones.  We 
can  now  prepare  large  collections  of  YAC  clones, 
each  containing  a  different  segment  of  human 
DNA,  averaging  hundreds  of  thousands  of  base 
pairs  in  size,  a  10-fold  improvement  over  the  ca- 
pacities of  previous  systems.  Another  advantage  is 
that  the  methods  of  packaging,  maintaining,  and 
replicating  DNA  are  more  similar  in  yeast  than  in 
bacteria  to  the  analogous  processes  in  human 
cells.  Consequently,  a  higher  fraction  of  the  hu- 
man genome  can  be  successfully  propagated  in 
yeast  than  in  the  more-conventional  bacterial 
hosts. 

Methods  of  recovering  any  desired  segment  of 
human  DNA  as  a  YAC  have  become  standard  dur- 
ing the  past  three  years.  YAC  clones  played  a  cen- 
tral role  in  two  recent  major  successes  in  human 
genetics:  discovery  of  the  molecular  basis  of  the 
fragile  X  syndrome,  carried  out  in  part  in  the  labo- 
ratories of  Thomas  Caskey  (HHMl,  Baylor  College 
of  Medicine)  and  Stephen  Warren  (HHMI,  Emory 
University) ;  and  the  discovery  of  the  gene  that  is 
mutated  in  familial  adenomatous  polyposis, 
carried  out  in  part  in  the  laboratory  of  Raymond 
White  (HHMI,  University  of  Utah).  The  fragile  X 
syndrome  is  a  common  heritable  cause  of  mental 
retardation;  familial  adenomatous  polyposis  is  a 
genetic  predisposition  to  colon  cancer. 

Now  that  YAC  cloning  is  a  proven  method  for 
recovering  large  blocks  of  human  DNA,  our  atten- 
tion has  turned  to  the  analysis  of  YACs.  A  need  for 
efficiency  arises  from  the  sheer  scale  of  human 
chromosomes:  approximately  10,000  YACs 
would  be  required  to  recover  the  DNA  present  in 
all  the  human  chromosomes.  To  meet  this  chal- 
lenge, it  will  be  necessary  to  develop  a  new  area 


503 


Large-Scale  Analysis  of  Yeast  and  Human  DNA 


of  analytical  biochemistry.  Although  the  ultimate 
goal  is  fully  automated  methodology,  the  princi- 
ples that  underlie  DNA  analysis  are  too  poorly 
understood  to  support  immediate  instrumenta- 
tion development.  One  key  will  be  to  obtain  a 
better  grasp  of  the  information  flow  that  accom- 
panies the  mapping  and  sequencing  of  DNA.  In 
collaboration  with  Will  Gillett  (Washington  Uni- 
versity), we  have  analyzed  the  computational 
problems  posed  by  DNA-mapping  methods  in 
which  large  blocks  of  DNA  are  broken  down  into 
smaller  segments  by  subcloning.  Powerful,  flexi- 
ble software  devoted  to  this  problem  is  under  de- 
velopment. Parallel  efforts  to  achieve  a  better  un- 
derstanding of  the  experimental  steps  are  also 
under  way. 


It  is  difficult  to  overestimate  the  extent  to 
which  the  analysis  of  DNA — cloning,  mapping, 
and  sequencing — has  come  to  underlie  progress 
in  biology  and  biomedical  research.  Biological 
research  has  been  driven  by  advances  in  method- 
ology, and  the  systematic  analysis  of  DNA  is  one 
of  the  field's  critical  technologies.  However,  in 
proportion  to  the  challenges  posed  by  the  genetic 
complexity  and  diversity  of  organisms,  the  devel- 
opment of  this  technology  is  still  in  its  infancy. 


Dr.  Olson  is  now  Professor  of  Molecular  Bio- 
technology at  the  University  of  Washington, 
Seattle. 


304 


Molecular  Genetic  Studies  of  Hematopoietic  Cells 


Stuart  H.  Orkin,  M.D. — Investigator 

Dr.  Orkin  is  also  Leland  Pikes  Professor  of  Pediatric  Medicine  at  Harvard  Medical  School.  He  received  his 
B.S.  degree  in  biology  from  the  Massachusetts  Institute  of  Technology  and  his  M.D.  degree  from  Harvard 
Medical  School.  His  postdoctoral  research  was  in  the  Laboratory  of  Molecular  Genetics  at  NIH  under  the 
supervision  of  Philip  Leder.  Upon  returning  to  Harvard,  Dr.  Orkin  received  specialty  training  in  pediatric 
hematology  at  the  Children 's  Hospital,  where  he  later  joined  the  faculty.  His  many  honors  include  the 
Clinical  Investigator  Award  from  the  American  Federation  for  Clinical  Research  and  the  Dameshek  prize 
of  the  American  Society  of  Hematology.  He  is  a  member  of  the  National  Academy  of  Sciences. 


ALL  mature  blood  cells  are  derived  from  plur- 
ipotem  hematopoietic  stem  cells,  which 
constitute  a  rare  population  in  the  bone  marrow. 
The  decision  of  stem  cells  to  differentiate  leads  to 
the  production  of  a  heterogeneous  array  of  cells 
with  varying  developmental  potentials  and  with 
commitment  to  expression  of  lineage-specific 
protein  products.  A  major  goal  of  this  laboratory 
is  an  improved  understanding  of  hematopoietic 
cell  development  and  the  expression  and  func- 
tion of  specific  genes  that  relate  to  the  normal 
biology  of  hematopoietic  cells. 

Efforts  are  directed  to  analyses  of  both  red  and 
white  blood  cells.  These  cell  types  are  important 
in  severe,  clinically  significant  genetic  disorders 
in  which  the  capacity  to  produce  specific  pro- 
teins is  impaired  by  mutation.  In  these  studies  we 
seek  to  describe  the  molecular  basis  of  inherited 
disorders,  understand  the  normal  regulation  of 
the  affected  genes,  and  utilize  the  findings  from 
this  basic  work  to  formulate  novel  treatments 
based  on  molecular  biologic  considerations. 

One  of  the  major,  classical  disorders  of  red 
cells  is  ;8-thalassemia  (also  known  as  Cooley's  ane- 
mia) ,  in  which  the  synthesis  of  hemoglobin  is  de- 
fective. Through  molecular  cloning  and  gene  ex- 
pression, the  molecular  basis  of  the  disease  was 
determined  in  this  laboratory  several  years  ago. 
Now  the  unsolved  problems  are  related  to  how 
globin  genes  are  normally  regulated  in  develop- 
ing erythroid  precursor  cells.  Specifically,  how 
are  the  globin  genes  activated  only  in  red  cells? 
How  are  different  globin  genes  regulated  in  devel- 
opment? How  do  erythroid  precursor  cells  arise 
during  development  from  progenitor  cells  that 
have  the  potential  to  yield  either  red  or  white 
cells? 

To  approach  these  general  problems,  we  have 
concentrated  on  identifying  and  characterizing 
unique  DNA-binding  proteins  that  appear  to  be 
major  transcriptional  regulators  in  erythroid 
cells.  A  prominent,  apparently  erythroid-specific 
DNA-binding  protein  was  first  discovered  that  rec- 
ognizes a  small  DNA  motif  (GATA)  found  in 
the  promoters  or  enhancers  of  all  erythroid- 


expressed  genes.  Through  molecular  cloning, 
mammalian,  avian,  and  amphibian  homologues 
were  characterized. 

The  protein  is  modular,  consisting  of  a  novel 
two-finger  structure  required  for  DNA-binding 
and  other  domains  that  serve  as  potent  positive 
activators  of  gene  transcription.  Expression  of  the 
protein  in  two  other  hematopoietic  cell  types, 
megakaryocytes  and  mast  cells,  suggests  that  it  is 
first  expressed  in  a  multipotential  progenitor  cell 
and  may  regulate  genes  in  those  cell  types  as 
well.  Recent  data  have  supported  these  conclu- 
sions. Attention  has  been  directed  to  how  this 
transcription  factor  is  itself  regulated  in  hemato- 
poietic cells.  An  improved  understanding  may 
provide  important  insights  into  the  initial  events 
involved  in  erythroid  decision-making  and 
maturation. 

Studies  of  the  gene  revealed  an  element  in  the 
promoter  region  that  serves  as  a  site  for  positive 
autoregulation.  In  this  manner,  expression  of  the 
factor  tends  to  maintain  its  own  expression  and 
stabilize  the  differentiated  state.  Furthermore, 
the  promoter  for  the  receptor  for  the  erythroid- 
specific  growth  factor,  erythropoietin,  is  under 
control  by  this  transcription  factor.  By  such  cir- 
cuitry, expression  of  the  factor  tends  to  guarantee 
subsequent  erythroid  development  and  viability. 

Site-specific  gene  disruption  in  mouse  embryo- 
derived  stem  cells  and  generation  of  chimeric  an- 
imals has  also  revealed  that  the  protein  is  essen- 
tial for  normal  erythroid  development  and  that 
related  proteins  that  bind  the  GATA  motif  cannot 
compensate  for  its  absence.  Using  in  vitro  differ- 
entiation of  embryo-derived  stem  cells  into  hema- 
topoietic cell  types,  we  have  developed  an  exper- 
imental system  that  permits  assessment  of  the 
role  of  GATA-transcription  factor  in  erythroid  de- 
velopment and  systematic  testing  of  various 
aspects  of  the  function  and/or  regulation  of  the 
protein. 

In  separate  but  conceptually  related  studies,  a 
gene  that  encodes  an  essential  component  of  the 
white  blood  cell  (phagocytic)  system  responsi- 
ble for  killing  ingested  microorganisms  is  being 
examined  in  an  effort  to  understand  how  this 


505 


Molecular  Genetic  Studies  of  Hematopoietic  Cells 


clinically  important  host  defense  system  is  regu- 
lated and,  more  generally,  how  cell-specific  gene 
expression  is  achieved  in  this  lineage,  also  de- 
scendent  from  the  pluripotent  stem  cell.  The 
gene  under  study  encodes  a  subunit  of  a  unique 
cytochrome  that  is  defective  in  an  X-linked  con- 
dition, chronic  granulomatous  disease.  By  posi- 
tional cloning,  we  previously  isolated  the  gene, 
determined  its  structure,  and  demonstrated  the 
presence  of  the  protein  product  in  the  cy- 
tochrome complex  of  phagocytic  cells. 

In  addition,  since  interferon-7  stimulates 
phagocytic  cell  function  generally  as  well  as  ex- 
pression of  the  cytochrome,  it  was  also  possible 
to  show  that  this  lymphokine  is  clinically  effec- 
tive in  chronic  granulomatous  disease.  Studies 
have  identified  several  point  mutations  in  the  cy- 
tochrome that  interfere  with  protein  function  in 
vivo  and  mutations  in  an  associated  cytochrome 
subunit  encoded  by  an  autosome  in  rarer  cases  of 
chronic  granulomatous  disease. 

More  current  efforts  are  directed  toward  defin- 
ing the  elements  of  the  gene  responsible  for  ap- 
proaching regulation  in  phagocytic  cells. 
Through  the  use  of  transgenic  mice,  we  identi- 
fied a  DNA  fragment  sufficient  for  targeting  re- 
porters and  oncogenes  to  a  subset  of  phagocytes 
in  vivo.  Coupled  to  an  oncogene,  this  fragment 


leads  to  the  consistent  development  of  an  in- 
herited malignancy  of  phagocytes  in  mice. 

In  the  promoter  we  identified  a  negative  regula- 
tory site  that  binds  a  protein  whose  concentration 
falls  dramatically  during  phagocyte  differentia- 
tion. This  factor  corresponds  to  a  putative  repres- 
sor termed  CCAAT  displacement  protein  (CDP) . 
Through  molecular  cloning,  we  have  character- 
ized human  CDP  and  shown  that  it  is  highly  re- 
lated to  a  novel  Drosophila  homeobox  protein,  a 
product  of  the  cut  gene,  which  controls  cell  fate 
decisions  in  several  tissues  during  fly  develop- 
ment. Although  this  repressor  does  not  explain 
the  restriction  of  cytochrome  expression  to 
phagocytic  cells,  its  loss  combined  with  the  pres- 
ence of  unknown  positive,  white  cell-specific 
regulators  very  likely  accounts  for  the  temporal 
pattern  of  cytochrome  expression  during  cellular 
maturation. 

More  generally,  the  remarkable  similarity  of 
human  CDP  and  the  fly  cut  protein  suggests  func- 
tional correlates  and  predicts  that  CDP  will  partic- 
ipate in  cell  fate  decisions  during  mammalian 
embryogenesis.  Current  efforts  are  directed  to 
analysis  of  the  role  and  function  of  CDP  in  mam- 
malian cells  and  to  identification  of  the  critical 
cis-  and  trans-components  responsible  for  white 
cell-specific  gene  expression. 


306 


Albinism  and  Tyrosinase 


Paul  A.  Overbeek,  Ph.D. — Assistant  Investigator 

Dr.  Overbeek  is  also  Associate  Professor  of  Cell  Biology  at  Baylor  College  of  Medicine  and  has  adjunct 
appointments  in  the  Institute  for  Molecular  Genetics,  the  Division  of  Neuroscience,  and  the  Department 
of  Ophthalmology  at  Baylor  College  of  Medicine.  He  received  his  B.A.  degree  in  chemistry  from  Kalamazoo 
College,  his  Ph.D.  degree  in  cellular  and  molecular  biology  from  the  University  of  Michigan,  and  an  M.B.A. 
degree  from  the  University  of  Chicago.  His  postdoctoral  research  was  done  in  the  laboratory  of  Heiner 
Westphal  at  NIH. 


THE  genetic  disorder  of  albinism,  or  loss  of 
pigmentation,  has  been  identified  in  many 
species.  Albinism  typically  is  caused  by  the  loss 
of  production  of  the  black  pigment  termed  mela- 
nin. Specialized  cells  known  as  melanocytes  are 
responsible  for  melanin  synthesis  in  the  skin, 
hair,  and  iris.  In  addition  to  melanocytes,  there 
are  cells  at  the  posterior  of  the  retina,  referred  to 
as  retinal  pigment  epithelial  cells,  that  normally 
synthesize  melanin.  When  these  two  types  of 
cells  lose  their  melanin-synthesizing  ability,  al- 
binism results.  Since  melanin  helps  protect  the 
skin  from  ultraviolet  radiation  and  the  visual  sys- 
tem from  bright  light,  albinism  is  often  associated 
with  secondary  health  problems,  including  in- 
creased risk  of  skin  cancer  and  visual  system 
deterioration. 

Melanin  is  derived  from  the  amino  acid  tyro- 
sine. An  enzyme  known  as  tyrosinase  converts  ty- 
rosine to  dopaquinone,  which  is  then  polymer- 
ized to  produce  melanin.  Studies  of  albino 
individuals,  including  humans  and  laboratory 
mice,  have  revealed  that  albinism  in  often  asso- 
ciated with  a  decrease  or  loss  of  tyrosinase  enzy- 
matic activity.  This  observation  has  led  to  the  hy- 
pothesis that  albinism  may  be  caused  by  a 
mutation  in  the  gene  that  encodes  tyrosinase.  A 
mutation  could  result  in  synthesis  of  an  abnormal 
tyrosinase  protein  that  no  longer  has  enzymatic 
activity.  We  have  recently  undertaken  a  series  of 
experiments  to  test  this  hypothesis  in  albino  labo- 
ratory mice. 

One  important  prediction  of  this  hypothesis  is 
that  the  nucleic  acid  sequence  of  the  tyrosinase 
gene  in  pigmented  mice  will  be  different  from 
the  sequence  in  albino  mice.  A  procedure  known 
as  the  polymerase  chain  reaction  was  used  to  am- 
plify specific  regions  of  the  tyrosinase  gene  from 
more  than  20  different  pigmented  and  albino 
mouse  strains.  The  nucleic  acid  sequences  were 
then  determined,  and  a  computer  program  was 
used  to  compare  them.  Interestingly,  every  pig- 
mented strain  was  found  to  encode  a  cysteine  at 
amino  acid  103  of  the  tyrosinase  gene,  while  the 
albino  mice  all  had  a  single  base  pair  change  that 
would  cause  amino  acid  103  to  become  a  serine. 
These  results  suggested  that  conversion  of  cys- 


teine to  serine  at  amino  acid  103  was  sufficient  to 
inactivate  the  tyrosinase  enzyme  and  produce 
albinism. 

In  order  to  confirm  this  prediction,  we  used 
recombinant  DNA  techniques  to  construct  two 
miniature  versions  of  the  tyrosinase  gene:  one 
with  a  cysteine,  the  other  with  a  serine  at  amino 
acid  103.  The  two  constructs  were  tested  in  an 
albino  strain  of  mice.  Embryos  of  the  albino  strain 
were  harvested  at  the  one-cell  stage  and  injected 
under  the  microscope  with  purified  DNA.  After 
injection  the  embryos  were  reimplanted  into  the 
reproductive  tracts  of  pregnant  females  and  al- 
lowed to  develop  to  birth. 

The  newborn  mice  were  then  screened  to  iden- 
tify those  mice  (termed  transgenic)  in  which  the 
injected  DNA  had  become  stably  incorporated 
into  the  genome.  Mice  that  had  incorporated  the 
cysteine  103  version  of  tyrosinase  became  pig- 
mented, while  all  of  the  mice  that  had  integrated 
the  serine  103  version  of  tyrosinase  were  still  al- 
bino. These  experiments  have  confirmed  that  al- 
binism can  be  caused  by  a  mutation  in  the  tyro- 
sinase gene  and  that  a  single  base  pair 
substitution  is  sufficient  to  inactivate  the  gene. 

These  studies  of  albinism  have  provided  an  im- 
portant new  strategy  for  the  identification  of 
transgenic  mice.  Prior  to  the  design  of  the  minia- 
ture tyrosinase  gene,  transgenic  mice  were  iden- 
tified by  techniques  that  required  genomic  DNA 
isolation.  The  miniature  tyrosinase  gene  allows 
transgenic  mice  to  be  recognized  by  simple  vi- 
sual inspection  for  pigmentation. 

To  test  the  general  usefulness  of  this  system, 
experiments  were  done  in  which  two  different 
recombinant  DNAs  were  co-injected  into  em- 
bryos. One  was  the  miniature  tyrosinase  gene;  the 
other  was  designed  to  generate  a  mouse  model 
for  predisposition  to  kidney  cancer.  The  newborn 
mice  were  first  checked  for  pigmentation,  then 
genomic  DNA  was  isolated  and  tested  for  integra- 
tion of  the  co-injected  DNA.  Altogether  five  pig- 
mented mice  were  obtained,  and  all  five  had  inte- 
grated both  DNAs.  Breeding  studies  showed  that 
the  two  different  DNAs  were  always  transmitted 
together. 

When  the  kidneys  were  checked  for  cancer  in- 


307 


Albinism  and  Tyrosinase 


duction,  only  the  pigmented  mice  were  found  to 
have  kidney  lesions.  Neoplastic  changes  were  lim- 
ited to  the  kidney  and  did  not  occur  in  the  skin. 
These  co-injection  experiments  demonstrate  that 
two  unrelated  genes  can  become  located  in  close 
proximity  to  each  other  in  the  genome  and  still 
function  in  an  independent  fashion.  Moreover, 
this  strategy  simplifies  long-term  maintenance 
-of  experimental  transgenic  mice,  since  the  de- 
sired mice  can  be  readily  identified  by  their 
pigmentation. 

An  additional  question  was  whether  the  synthe- 
sis of  tyrosinase  in  some  cell  type  other  than  mela- 
nocytes or  retinal  pigment  epithelial  cells  would 
lead  to  melanin  synthesis  and  pigment  formation. 
To  answer  this  question,  regulatory  sequences 
that  were  known  to  be  active  specifically  in  lens 
cells  were  linked  to  the  tyrosinase  coding  se- 
quences using  recombinant  DNA  techniques,  and 
transgenic  mice  were  generated  in  an  albino 
strain  of  mice.  The  transgenic  mice  all  devel- 
oped black  eyes.  Synthesis  of  tyrosinase  in  the 
lens  led  to  pigment  production  in  the  lens,  con- 
firming that  the  reason  lens  cells  do  not  nor- 
mally make  melanin  is  because  they  do  not 
normally  make  the  enzyme  tyrosinase.  Previous 
studies  had  suggested  that  pigment  synthesis  in 


inappropriate  cell  types  might  be  harmful  for 
those  cells.  In  the  transgenic  mice,  the  pig- 
mented lens  cells  show  histological  evidence  of 
cellular  injury  and  inhibition  of  normal  growth. 

When  transgenic  DNA  integrates  into  the  ge- 
nome, it  can  insert  into  the  middle  of  an  endoge- 
nous gene,  causing  insertional  inactivation  of  the 
gene.  Since  our  transgenic  tyrosinase  mice  were 
easy  to  identify  and  breed,  80  families  were 
tested  for  the  presence  of  insertional  mutations. 
Eight  families  were  found  that  have  defects,  rang- 
ing from  embryonic  lethality  to  male  sterility  to 
anemia  and  premature  kidney  failure. 

One  of  the  mutations  is  particularly  fascinating 
because  the  homozygous  transgenic  mice  all  have' 
an  inversion  of  the  left-to-right  organization  of 
their  internal  organs.  This  condition  in  humans  is 
known  as  situs  inversus.  Since  the  factors  that 
control  polarity  in  the  developing  mammalian 
embryo  have  not  yet  been  identified,  we  have  be- 
gun experiments  that  will  make  use  of  the  trans- 
genic insert  to  try  to  isolate  the  regulatory  factor 
for  situs  inversus.  The  identification  and  charac- 
terization of  such  a  factor  could  greatly  enhance 
our  understanding  of  embryonic  development 
and  guide  future  efforts  to  help  prevent  analo- 
gous birth  defects  in  humans. 


308 


Structural  Studies  of  DNA-binding  Proteins 


Carl  O.  Pabo,  Ph.D. — Investigator 

Dr.  Pabo  is  also  Professor  of  Biophysics  and  Structural  Biology  at  the  Massachusetts  Institute  of 
Technology.  He  received  his  undergraduate  degree  from  Yale  University,  where  he  majored  in  molecular 
biophysics  and  biochemistry.  He  did  his  graduate  work  in  Mark  Ptashne's  laboratory  at  Harvard 
University,  where  he  continued  his  research  as  a  Jane  Coffin  Childs  fellow  in  the  laboratories  of  Stephen 
Harrison  and  Don  Wiley.  Prior  to  accepting  a  position  at  MIT,  Dr.  Pabo  was  Professor  of  Molecular 
Biology  and  Genetics  and  of  Biophysics  at  the  Johns  Hopkins  University  School  of  Medicine. 


WE  are  interested  in  understanding  how  pro- 
teins recognize  specific  sites  on  double- 
stranded  DNA  and  how  the  bound  proteins  regu- 
late gene  expression.  We  would  like  to  know 
what  structural  motifs  are  used  by  DNA-binding 
proteins,  what  side  chains  make  sequence-spe- 
cific contacts,  and  whether  there  are  any  recur- 
ring patterns  or  rules  for  recognition  of  sites  on 
double-stranded  DNA.  Much  of  our  current  re- 
search has  focused  on  characterizing  the  major 
structural  motifs  found  in  DNA-binding  proteins. 
We  hope  to  use  this  information  to  design  novel 
DNA-binding  proteins  for  research,  diagnosis, 
and  therapy. 

Prokaryotic  repressors  provide  useful  model 
systems  for  the  study  of  protein-DNA  interactions, 
and  we  are  continuing  to  study  several  bacterial 
repressors.  The  repressor  from  the  bacteriophage 
X  uses  a  helix-turn-helix  motif  and  an  extended 
amino-terminal  arm  to  contact  sites  in  the  major 
groove.  The  «rc  repressor  from  5a/mo«e//a  bacte- 
riophage P22  uses  a  |8-sheet  for  site-specific  rec- 
ognition. The  major  developments  in  our  labora- 
tory during  the  past  two  years,  however,  have 
involved  studies  of  two  of  the  key  motifs — the 
homeodomain  and  the  zinc  finger — that  are  used 
by  eukaryotic  regulatory  proteins. 

Crystal  Structures  of  Homeodomain-DNA 
Complexes 

The  homeodomain  is  a  conserved  structural 
motif  found  in  many  eukaryotic  proteins  that  reg- 
ulate development  and  cell  fate.  To  understand 
how  this  motif  recognizes  DNA  and  how  this  is 
related  to  the  helix-turn-helix  motif  seen  in  pro- 
karyotic repressors,  we  have  determined  the 
crystal  structures  of  two  homeodomain-DNA 
complexes. 

We  began  by  studying  the  homeodomain  from 
the  engrailed  protein,  which  plays  a  key  role  in 
Drosophila  development.  (This  project  is  a  col- 
laboration with  Thomas  Kornberg  at  the  Univer- 
sity of  California,  San  Francisco.)  We  were  able  to 
grow  good  cocrystals  of  the  homeodomain-DNA 
complex,  and  Chuck  Kissinger  solved  the  struc- 
ture of  this  complex.  The  homeodomain  makes 


contacts  in  both  the  major  and  minor  grooves. 
The  helix-turn-helix  unit  makes  critical  contacts 
in  the  major  groove,  but  the  orientation  of  this 
helix-turn-helix  unit  with  respect  to  the  DNA  is 
different  than  the  arrangements  observed  with 
the  prokaryotic  repressors.  Residues  near  the 
amino-terminal  end  of  the  homeodomain  form  an 
extended  "arm"  that  fits  into  the  minor  groove 
and  makes  additional  site-specific  contacts. 

We  also  have  been  studying  a  complex  contain- 
ing the  homeodomain  from  the  a2  protein, 
which  helps  to  regulate  mating  type  in  yeast. 
(This  project  is  a  collaboration  with  Alexander 
Johnson  at  the  University  of  California,  San  Fran- 
cisco.) Cynthia  Wolberger  recently  solved  this 
structure,  and  comparison  with  engrailed  re- 
vealed that  1 )  the  structures  of  these  two  homeo- 
domains  are  very  similar  (despite  a  3 -residue  in- 
sertion in  a2  and  despite  significant  amino  acid 
sequence  differences)  and  2)  the  orientation  of 
the  helix-turn-helix  unit  with  respect  to  the  DNA 
also  is  conserved.  This  conserved  docking  ar- 
rangement is  maintained  by  side  chains  that  are 
identical  in  a2  and  engrailed.  Because  these  resi- 
dues tend  to  be  conserved  among  all  homeodo- 
mains,  these  structures  may  provide  a  general 
model  for  homeodomain-DNA  interactions. 

Our  studies  of  the  homeodomain  are  supported 
by  a  grant  from  the  National  Institutes  of  Health. 

Crystal  Structures  of  Zinc  Finger-DNA 
Complexes 

The  zinc  finger  domain,  which  contains  about 
30  amino  acids,  is  another  key  DNA-binding  motif 
that  is  found  in  a  large  family  of  eukaryotic  regula- 
tory proteins.  Studies  from  other  groups  have 
shown  that  each  finger  contains  an  antiparallel 
jS-sheet  and  an  a-helix,  but  little  has  been  known 
about  how  these  fingers  recognize  DNA.  Nikola 
Pavletich  recently  solved  the  structure  of  a  com- 
plex containing  three  zinc  fingers  from  a  murine 
transcription  factor.  Starting  with  cDNA  for  the 
zif 268 gene  (provided  by  Daniel  Nathans,  HHMI, 
Johns  Hopkins  University),  he  cloned  and  ex- 
pressed a  three-finger  peptide  and  crystallized 
the  peptide-DNA  complex.  The  zinc  fingers  rec- 


309 


Structural  Studies  of  DNA- binding  Proteins 


ognize  B-DNA  and  fit  into  the  major  groove.  Each 
finger  makes  its  primary  contacts  with  a  3 -base 
pair  "subsite,"  and  side  chains  near  the  amino- 
terminal  end  of  the  a-helix  make  the  critical  con- 
tacts with  the  bases. 

Since  the  zinc  fingers  are  used  in  a  modular 
fashion,  they  may  be  the  ideal  motif  to  use  as  we 
try  designing  novel  DNA-binding  proteins.  How- 
ever, we  need  to  determine  the  structure  of  addi- 
tional zinc  finger-DNA  complexes  so  that  we  can 
see  how  this  motif  is  used  in  other  proteins.  (Do 
all  zinc  fingers  dock  against  the  DNA  in  the  same 
way?)  Nikola  Pavletich  has  cloned  the  zinc  finger 
regions  from  a  number  of  other  proteins  and  has 
recently  solved  the  structure  of  a  complex  that 
contains  the  five  zinc  fingers  from  GLI,  a  protein 
that  is  amplified  in  a  subset  of  human  tumors. 
Finishing  this  structure  and  comparing  it  with  the 
zif  complex  should  provide  a  firmer  basis  for  the 
design  projects. 

Structural  Studies  of  Other  Protein-DNA 
Complexes 

It  is  important  to  obtain  structural  information 
about  the  other  major  DNA-binding  motifs  that 
occur  in  eukaryotic  regulatory  proteins.  We  are 
focusing  on  the  helix-loop-helix  proteins  and  the 
POU  domain,  which  have  very  important  roles  in 
development.  We  also  are  working  with  the 
TFIID  protein  (because  of  its  central  role  in  tran- 
scription) and  with  the  p53  protein,  which  is  the 
most  common  site  of  mutations  in  human  tumors. 


Because  structural  analysis  often  is  limited  by 
the  ability  to  obtain  suitable  crystals,  we  are  try- 
ing to  improve  methods  for  the  cocrystallization 
of  protein-DNA  complexes.  Our  initial  approach 
involved  systematic  changes  in  the  length  of  the 
DNA  site  and  required  that  the  entire  site  be 
resynthesized  for  each  experiment.  We  now  have 
encouraging  preliminary  results  with  a  linker  co- 
crystallization  scheme  that  combines  the  protein, 
the  binding  site,  and  a  library  of  DNA  linkers  that 
can  be  used  with  any  complex.  This  strategy  may 
allow  a  dramatic  increase  in  the  number  of  co- 
crystallization  conditions  that  can  be  tested. 

Design  of  Novel  DNA-binding  Proteins 

We  are  attempting  to  use  the  zinc  finger  struc- 
tures as  a  basis  for  designing  novel  DNA-binding 
proteins.  Two  major  approaches  are  being  tested: 

1 )  genetic  strategies  for  selecting  zinc  finger  pro- 
teins that  recognize  desired  target  sequences  and 

2)  strategies  for  computer-aided  protein  design. 
These  programs,  which  can  systematically  con- 
sider a  large  number  of  sequences  and  conforma- 
tions, are  being  used  in  our  attempts  to  design 
zinc  finger  proteins  that  will  recognize  novel 
binding  sites.  This  combination  of  structural  anal- 
ysis, computer-aided  protein  design,  and  genetic 
selection  should  provide  a  better  understanding 
of  protein-DNA  recognition  and  allow  rapid  de- 
sign of  zinc  finger  proteins  that  recognize  novel 
target  sites. 


310 


Image  of  an  x-ray  crystal  structure  showing  how  regulatory  proteins  of  a  key  class  known  as  zinc 
finger  bind  to  DNA.  The  protein  contains  three  "fingers"  ( shown  in  orange,  yellow,  and  purple) 
that  bind  to  the  double-helical  DNA  (blue).  Each  finger  has  an  a-helix  that  fits  into  the  major 
groove  of  the  DNA  and  makes  critical  contacts  that  help  the  protein  recognize  its  proper  bind- 
ing site. 

From  Pavletich,  N.P.,  and  Pabo,  CO.  1991.  Science  252:809-817.  Copyright©  1991  by  the 
AAAS. 


311 


Hypothesis:  XX  Males  and  XY  Females  Result 
from  X-Y  Interchange  at  Paternal  Meiosis 

'  ^  TDF 


X 


1/ 


I  I 

g +TDF  -TDF 


I 


XX  male  XY  female 


The  X-Y  interchange  hypothesis:  XX  males  and  XY  females  receive  reciprocal 
products  of  similar  aberrant  XY  exchanges  occurring  in  the  fathers  during 
the  production  of  sperm  ( meiosis).  TDF  is  testis-determining  factor,  the  sex- 
determining  gene  normally  located  on  the  Y  chromosome. 
Research  of  David  Page. 


312 


The  X  and  Y  Chromosomes  in  Mammalian 
Development 


David  C.  Page,  M.D. — Assistant  Investigator 

Dr.  Page  is  also  Associate  Member  of  the  Whitehead  Institute  for  Biomedical  Research,  Associate  Professor 
of  Biology  at  the  Massachusetts  Institute  of  Technology,  and  Assistant  Professor  at  the  Harvard 
University-MIT  Division  of  Health  Sciences  and  Technology.  He  received  his  undergraduate  degree  in 
chemistry  from  Sivarthmore  College  and  a  medical  degree  from  Harvard  Medical  School  and  the 
Harvard-MIT  Health  Sciences  and  Technology  Program.  After  training  with  Raymond  White,  at  the 
University  of  Massachusetts,  and  David  Botstein,  at  MIT,  Dr.  Page  became  one  of  the  first  Fellows 
of  the  Whitehead  Institute.  He  subsequently  joined  the  faculties  of  Whitehead  and  MIT. 


TO  a  large  degree,  human  individuals  differ  in 
physical  characteristics  because  of  the  im- 
pact of  genetic  variation  on  the  course  of  embry- 
onic development.  The  human  genome  is  orga- 
nized into  23  pairs  of  chromosomes,  each 
believed  to  carry,  on  average,  about  5,000  genes. 
When  considering  genetic  differences  among  in- 
dividuals, it  is  important  to  distinguish  between 
variation  in  a  single  gene,  called  Mendelian,  and 
massive  multigene  variation,  as  found  in  chromo- 
somal disorders. 

As  discussed  elsewhere  in  this  volume,  single- 
gene  defects  are  responsible  for  certain  condi- 
tions such  as  color  blindness,  cystic  fibrosis,  and 
muscular  dystrophy.  Other  conditions,  such  as 
Down  syndrome,  appear  to  be  the  result  of 
"wholesale"  abnormalities  affecting  an  entire 
chromosome.  As  reductionists  and  molecular  bi- 
ologists, we  proceed  on  the  assumption  that  the 
developmental  consequences  of  chromosomal  ab- 
normalities will  ultimately  be  understood  in 
terms  of  individual  genes  and  their  particular 
functions. 

My  colleagues  and  I  are  seeking  to  understand 
how  massive  variability  in  one  chromosome  pair 
— the  sex  chromosomes — dramatically  affects 
the  course  of  development.  Embryos  normally  in- 
herit one  sex  chromosome  from  each  parent.  The 
mother  contributes  an  X  chromosome,  and  the 
father  contributes  either  an  X  or  a  Y.  Thus  normal 
embryos  have  one  of  two  sex  chromosome  consti- 
tutions, XX  or  XY. 

Sex  Determination 

In  both  humans  and  mice  the  presence  or  ab- 
sence of  the  Y  chromosome  determines  whether 
an  embryo  develops  as  a  male  or  as  a  female.  XX 
embryos  become  females,  XY  embryos  males.  For 
years  scientists  wondered  whether  the  Y  chromo- 
some carried  few  or  many  sex-determining  genes 
and  how  those  were  distributed  along  the 
chromosome. 

We  found  that  the  entire  sex-determining  func- 
tion can  be  traced  to  one  tiny  portion  of  the  hu- 
man Y  chromosome.  This  sex-determining  region 


was  identified  by  studying  DNA  from  "XX  males" 
and  "XY  females."  XX  males  have  small  testes 
and  are  sterile.  XY  females  are  also  sterile  and  do 
not  develop  secondary  sexual  characteristics.  We 
found  that  almost  all  XX  males  had  inherited  a 
small  bit  of  the  Y  chromosome  attached  to  one  of 
their  X  chromosomes.  Conversely,  some  XY  fe- 
males lacked  the  same  segment  of  the  Y  that  was 
present  in  XX  males.  On  the  basis  of  the  chromo- 
somal deletions  found  in  such  patients,  we  con- 
structed a  map  of  the  Y  chromosome.  It  was  then 
we  came  to  recognize  that  the  presence  or  ab- 
sence of  one  small  region,  about  0.4  percent  of 
the  chromosome,  correlated  well  with  gender. 

Detailed  analysis  of  XX  males  suggests  that  one 
or  more  genes  within  this  relatively  small  seg- 
ment of  the  Y  chromosome  determine  the  out- 
come of  sexual  development.  Laboratories 
around  the  world  have  scoured  this  relatively 
small  segment  of  the  Y  chromosome  searching  for 
such  sex-determining  genes.  We  are  now  con- 
ducting a  variety  of  experiments  to  characterize 
the  functions  of  two  genes  in  the  region:  ZFY, 
which  we  identified  in  1987,  and  SRY,  a.  gene 
described  by  British  scientists  in  1990. 

Both  ZFY  and  SRY  appear  to  encode  DNA- 
binding  proteins  that  are  likely  to  regulate  the 
transcriptional  activity  of  particular  but  un- 
known target  genes.  Experiments  clearly  demon- 
strate that  SRY  is  a  sex-determining  gene.  Much 
less  clear  is  the  role,  if  any,  that  ZFY  plays  in  the 
process.  We  hope  to  understand  better  the  func- 
tion of  the  ZFF gene  by  simultaneously  analyzing 
a  closely  related  gene,  ZFX,  that  we  identified  on 
the  X  chromosome. 

In  some  human  XY  females,  we  have  identified 
mutations  near  or  within  the  SRY  gene.  By  con- 
trast, other  XY  females,  some  human  and  some 
mouse,  appear  to  have  intact  Y  chromosomes  but 
may  have  mutations  elsewhere,  perhaps  in  auto- 
somal genes  that  play  important  roles  in  sex  deter- 
mination. Identification  of  such  autosomal  sex- 
determining  genes  is  a  future  goal.  Our  studies  of 
sex  determination  arc  supported  by  a  grant  from 
the  National  Institutes  of  Health. 


313 


The  X  and  Y  Chromosomes  in  Mammalian  Development 


Turner  Syndrome 

As  mentioned  earlier,  embryos  normally  have 
two  sex  chromosomes.  However,  about  1-2  per- 
cent of  all  human  embryos  have  only  one.  The 
vast  majority  of  such  XO  embryos  are  lost  to  spon- 
taneous miscarriage,  but  a  few  survive.  The  sur- 
viving XO  embryos  develop  as  females  with  a  par- 
ticular set  of  physical  features  known  as  Turner 
syndrome,  which  includes  short  stature,  webbing 
of  the  neck,  puffiness  of  the  hands  and  feet,  and 
failure  of  secondary  sexual  development.  It  had 
been  postulated  that  Turner  syndrome  might  be 
the  result  of  having  a  single  copy  of  one  or  more 
genes  common  to  the  X  and  Y  chromosomes. 
Nothing  was  known  as  to  the  number  or  nature  of 
these  hypothetical  Turner  genes. 

We  began  to  focus  our  attention  on  this  dis- 
order when  it  was  noticed  that  certain  XY  females 
exhibit  the  same  anatomic  abnormalities  as  XO 
females.  A  pivotal  finding  was  that  all  such  XY 
Turner  females  lacked  a  portion  of  the  Y  chromo- 
some. We  postulated  that  the  Y  chromosomal  de- 
letions in  these  individuals  might  encompass  not 
only  a  sex-determining  gene  or  genes  but  also  a 
nearby  Turner  gene  or  genes. 

Pursuing  this  hunch,  we  discovered  two  candi- 
date Turner  genes,  one  on  the  Y  chromosome  and 
one  on  the  X  chromosome.  These  genes,  named 
RPS4Y2in<S.  RPS4X,  appear  to  encode  slightly  dif- 
ferent forms  of  a  protein  constituent  of  the  ribo- 
some,  a  structure  required  for  protein  synthesis 
and  vital  to  all  cells.  In  embryos  lacking  a  second 
RPS4  gene  (i.e.,  having  a  single  RPS4),  the  rate  at 
which  ribosomes  are  constructed  maybe  slowed, 
in  turn  reducing  the  embryo's  capacity  to  synthe- 
size other  proteins.  We  are  currently  testing  the 
highly  speculative  hypothesis  that  such  a  reduc- 
tion in  protein  synthetic  capacity  is  the  cause 
of  some  of  the  physical  features  of  Turner 
syndrome. 


An  interesting  analogy  can  be  found  in  the  fruit 
fly  Drosophila  melanogaster.  There,  deficien- 
cies in  ribosomal  protein  genes  are  associated 
with  a  particular  "syndrome"  called  the  "Mi- 
nute" (pronounced  mi-NUTE)  phenotype,  which 
includes  reduced  body  size,  diminished  viability 
and  fertility,  and  specific  anatomic  abnormali- 
ties. The  Drosophila  Minutes  may  serve  as  a  use- 
ful model  system  in  which  to  study  effects  of 
RPS4  gene  dosage.  We  are  also  exploring  poten- 
tial mouse  models  of  Turner  syndrome. 

The  very  existence  of  related  but  nonidentical 
ribosomal  protein  genes  on  the  X  and  Y  chromo- 
somes raises  the  possibility  that  the  ribosomes  of 
human  males  may  differ  slightly  from  those  of 
females.  Experiments  still  in  their  early  stages 
suggest  that  this  is  the  case.  Thus  the  differences 
between  the  sexes  may  extend  all  the  way  down 
to  the  most  fundamental  and  vital  of  intracellular 
machines! 

The  Human  Y  Chromosome 

As  mentioned  above,  we  constructed  a  map  of 
the  human  Y  chromosome  by  characterizing  natu- 
rally occurring  deletions,  such  as  those  found  in 
XY  females  and  XX  males.  We  are  continuing  to 
refine  this  map,  which  is  useful  not  only  in  study- 
ing sex  determination  and  Turner  syndrome,  but 
also  in  examining  the  role  of  Y  chromosomal 
genes  in  other  processes,  including  the  develop- 
ment of  certain  cancers  and  the  making  of  sperm. 
We  recently  set  out  to  "clone"  the  human  Y 
chromosome  as  a  series  of  overlapping  segments, 
each  segment  constituting  about  1  percent  of  the 
chromosome.  Such  an  ordered  array  of  cloned 
segments  should  facilitate  the  process  of  locat- 
ing and  characterizing  all  genes  on  the  Y  chromo- 
some. Our  efforts  to  map  and  clone  the  Y  chro- 
mosome are  supported  by  a  grant  from  the  Na- 
tional Institutes  of  Health. 


314 


Mammalian  Development  and  Disease 


Richard  D.  Palmiter,  Ph.D. — Investigator 

Dr.  Palmiter  is  also  Professor  of  Biochemistry  at  the  University  of  Washington.  He  received  his  Ph.D.  degree 
from  Stanford  University  and  did  postdoctoral  work  at  Stanford,  Searle  Research  Laboratories  in  England, 
and  Harvard  University.  Prior  to  his  current  work  with  transgenic  animals.  Dr.  Palmiter  studied  the 
mechanism  of  steroid  hormone  action  in  the  chick  oviduct  and  the  regulation  of  metallothionein  gene 
expression  in  mice.  He  is  a  member  of  the  National  Academy  of  Sciences  and  the  American  Academy 
of  Arts  and  Sciences. 


ABOUT  1 0  years  ago  we  began  a  fruitful  col- 
laboration with  Ralph  Brinster's  laboratory 
at  the  University  of  Pennsylvania.  Together  we 
helped  develop  methods  for  introducing  func- 
tional genes  into  all  cells  of  the  mouse.  The  genes 
under  study  are  manipulated  and  grown  in  bacte- 
rial plasmids,  using  standard  recombinant  DNA 
techniques.  Then  the  regions  of  interest  are  ex- 
cised from  the  plasmid,  and  a  few  hundred  copies 
are  injected  into  the  pronucleus  of  a  fertilized 
mouse  egg  (or  that  of  any  other  mammal) . 

Remarkably,  the  DNA  integrates  about  30  per- 
cent of  the  time  into  one  of  the  chromosomes 
prior  to  replication,  and  the  genes  are  inherited 
by  all  daughter  cells,  as  any  other  gene  would  be. 
Furthermore,  many  of  the  genes  are  functional, 
imparting  new  genetic  characteristics  to  the  ani- 
mal. Mice  and  other  animals  carrying  foreign  DNA 
are  referred  to  as  transgenic.  Because  the  new 
genes  are  also  in  the  germ  cells,  they  are  usually 
transmitted  to  subsequent  generations. 

One  of  our  goals  has  been  to  discover  what 
parts  of  a  gene  determine  when,  where,  and  how 
efficiently  it  will  be  utilized.  We  often  start  by 
testing  a  large  piece  of  DNA  that  includes  the 
gene  of  interest.  In  transgenic  animals,  the  gene 
will  usually  be  expressed  in  the  appropriate  time 
and  place,  even  though  it  has  integrated  at  an  ab- 
normal chromosomal  location  and  may  be  de- 
rived from  a  different  mammalian  species.  Then 
we  delete  various  regions  of  the  genes,  and  with 
each  variant  we  make  transgenic  mice  to  deter- 
mine what  regions  are  essential  for  appropriate 
expression. 

For  example,  we  have  delineated  a  small  re- 
gion (125  base  pairs)  of  the  rat  elastase  I  gene 
that  is  essential  for  the  expression  of  the  gene  in 
acinar  cells  of  the  pancreas.  Furthermore,  this  se- 
quence (often  called  an  enhancer)  can  be  used  to 
direct  the  expression  of  another  gene  (e.g.,  the 
growth  hormone  gene)  to  the  acinar  cells,  and 
the  sequence  will  function  when  positioned  al- 
most anywhere  in  the  vicinity  of  the  growth  hor- 
mone gene.  In  similar  experiments  we  have  been 
identifying  sequences  responsible  for  directing 
appropriate  expression  of  globin  genes  in  red 


blood  cells,  albumin  in  hepatocytes,  and  prota- 
mine I  in  male  germ  cells. 

Because  regulatory  elements  from  one  gene 
can  often  be  used  to  control  another;  the  expres- 
sion of  many  interesting  genes  can  be  directed  to 
a  specific  cell  type  and  the  consequences  on  the 
development  and  function  of  those  cells  can  be 
assessed.  For  example,  using  the  elastase  en- 
hancer element,  we  have  been  able  to  make 
strains  of  mice  that  reproducibly  develop  pancre- 
atic cancer  as  a  consequence  of  expressing  the 
transforming  gene  from  simian  virus  40,  the 
mouse  myc  gene,  or  the  human  Yi-ras  oncogene. 
Similarly,  we  have  developed  models  of  liver 
cancer  by  directing  the  expression  of  these  genes 
to  hepatocytes  with  the  albumin  enhancer. 

Significantly,  each  of  these  genes  results  in  a 
characteristic  morphological  transformation  of 
the  organ,  which  probably  reflects  the  particular 
cellular  events  that  these  genes  mediate.  By 
means  of  simple  genetic  crosses,  mice  carrying 
any  pair  of  these  transforming  genes  can  be  cre- 
ated. They  develop  tumors  that  appear  more  rap- 
idly and  grow  more  aggressively  than  those  in 
mice  carrying  a  single  gene,  suggesting  that  these 
genes  act  cooperatively. 

Some  genes  that  are  not  generally  considered 
oncogenes  may  also  predispose  cells  to  malignant 
transformation  and  cancerous  growth.  In  one  ex- 
ample, we  expressed  the  surface  antigens  of  hepa- 
titis B  virus  (HBV)  in  the  liver,  using  the  albumin 
enhancer.  In  transgenic  mice,  expression  of  this 
gene  resulted  in  the  synthesis  of  the  viral  surface 
antigen  and  envelope  protein,  which  aggregated 
within  the  secretory  apparatus  of  the  liver  cells, 
causing  cellular  injury  and  death.  This  is  accom- 
panied by  liver  cell  regeneration.  However, 
when  the  mice  were  more  than  a  year  old,  they 
developed  liver  cancer.  Because  HBV  infects  mil- 
lions of  people  worldwide,  and  the  incidence  of 
liver  cancer  among  them  is  very  high,  this  result 
may  indicate  that  chronic  expression  of  HBV  sur- 
face antigens  may  be  a  contributing  factor.  Simi- 
larly, expression  of  plasminogen  activator  in  the 
liver  using  the  albumin  enhancer  results  in  defec- 
tive blood  clotting  and  liver  injury.  In  this  case, 


315 


Mammalian  Development  and  Disease 


liver  cells  that  have  deleted  the  transgene  repopu- 
late  the  liver,  and  when  the  mice  are  a  few 
months  old  they  seem  normal.  However,  they 
also  succumb  to  liver  cancer  when  they  are  one  to 
two  years  old.  In  both  cases  we  postulate  that 
liver  regeneration  in  the  toxic  environment  of 
liver  injury  results  in  genetic  damage  that  predis- 
poses the  liver  cells  to  malignant  transformation. 

It  is  also  possible  to  develop  transgenic  mice 
that  mimic  some  human  genetic  diseases.  For  ex- 
ample, we  made  a  model  of  human  sickle  cell 
disease.  By  introducing  into  mice  both  human  a- 
and  /8-globin  genes  under  control  of  the  locus 
control  region  (a  newly  discovered  genetic  ele- 
ment essential  for  high-level  expression  of  globin 
genes) ,  we  have  made  mice  that  produce  as  much 
human  hemoglobin  as  mouse  hemoglobin.  When 
the  mutant  /3-globin  gene  from  people  with  sickle 
disease  is  substituted  for  the  normal  gene  in  these 
experiments,  the  red  blood  cells  of  the  mice 
sickle  under  appropriate  conditions.  These  mice 
may  be  a  valuable  resource  for  testing  experimen- 
tal therapies. 

A  long-range  goal  is  to  use  transgenic  mice  to 
study  aspects  of  neural  development.  We  have 
started  by  cloning  the  genes  involved  in  the  syn- 
thesis of  the  catecholamine  neurotransmitters: 
dopamine,  norepinephrine,  and  epinephrine. 
The  control  elements  from  these  genes  are  being 


tested  in  conjunction  with  genes  whose  products 
can  be  easily  visualized,  to  assess  when  and 
where  they  are  expressed  during  development. 
For  example,  we  have  shown  that  the  regulatory 
elements  from  the  gene  responsible  for  mak- 
ing norepinephrine  direct  the  expression  of  ^- 
galactosidase  to  certain  neurons  of  the  central 
nervous  system,  the  peripheral  nervous  system, 
the  enteric  nervous  system,  and  the  adrenal  me- 
dulla (see  figure).  Because  this  marker  gene  is 
expressed  very  early  during  neural  development, 
it  allows  us  to  visualize  the  cells  while  they  are 
still  migrating  to  their  final  destinations  and  be- 
fore they  acquire  properties  of  mature  neurons. 

By  mating  these  mice  to  mice  carrying  a  genetic 
defect  that  affects  the  innervation  of  the  bowel 
and  results  in  a  condition  similar  to  Hirsch- 
sprung's disease  in  humans,  we  have  shown  that 
neuronal  precursors  fail  to  migrate  into  the  distal 
portion  of  the  gut.  The  lack  of  innervation  of  the 
colon  results  in  chronic  impaction  of  fecal  mate- 
rial and  ultimately  death. 

We  are  also  using  the  control  elements  from 
these  genes  to  direct  the  expression  of  other 
genes  (e.g.,  encoding  neurotransmitters,  growth 
factors,  hormones,  proteases,  or  oncogenes)  to 
these  neurons  with  the  aim  of  affecting  the  deci- 
sions they  make  during  the  process  of  forming 
functional  connections  with  target  cells. 


Blue  staining  reveals  the  location  of  cells  that  ex- 
press a  ^  galactosidase  transgene  in  neural  precur- 
sors in  a  10. 5- day  mouse  embryo. 

Research  and  photograph  by  Raj  Kapur  in  the  lab- 
oratory of  Richard  Palmiter. 


316 


Regulation  of  Gene  Expression 
in  Steroid  Hormone  Biosynthesis 


Keith  L.  Parker,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Parker  is  also  Associate  Professor  of  Medicine  and  Biochemistry  at  Duke  University  Medical  Center. 
After  attending  Williams  College,  he  earned  his  M.D.  and  Ph.D.  degrees  in  genetics  at  Washington 
University,  studying  with  Donald  Shreffler.  He  served  as  intern  and  resident  in  internal  medicine  at 
Parkland  Memorial  Hospital,  Dallas.  He  then  moved  to  the  Department  of  Genetics  at  Harvard  Medical 
School,  where  he  was  a  postdoctoral  fellow  with  Jonathan  Seidman.  Dr.  Parker  's  next  move  was  to  the 
faculty  of  Duke  University  Medical  Center. 


THE  adrenal  gland  plays  an  essential  role  in  the 
body's  ability  to  respond  to  stress.  Two  dif- 
ferent parts  of  the  adrenal  gland,  an  outer  cortex 
and  an  inner  medulla,  produce  distinct  compo- 
nents of  this  response.  The  medulla  produces  epi- 
nephrine and  norepinephrine,  which  are  re- 
leased very  rapidly,  preparing  the  organism  for 
immediate  physical  activity.  In  contrast,  the  cor- 
tex produces  steroid  hormones,  which  are  re- 
leased more  slowly  and  exert  prolonged  effects. 

These  adrenal  steroids  constitute  two  major 
classes:  glucocorticoids,  which  are  made  in  the 
inner  zone  of  the  cortex  and  control  carbohydrate 
metabolism,  and  mineralocorticoids,  which  are 
made  in  the  outer  zone  and  regulate  salt  and 
water  balance.  Both  classes  of  steroid  hormones 
are  formed  from  cholesterol  by  the  sequential  ac- 
tion of  a  related  group  of  steroidogenic  enzymes. 
One  of  these,  the  cholesterol  side-chain  cleavage 
enzyme,  is  expressed  in  all  steroidogenic  tissues. 
A  second,  2 1 -hydroxylase,  is  expressed  through- 
out the  adrenal  cortex.  Finally,  there  are  distinct 
forms  of  1 1/3-hydroxylase:  one  form  produces 
mineralocorticoids  in  the  outer  zone,  and  the 
other  produces  glucocorticoids  in  the  inner 
zone.  The  physiological  regulators  of  these  two 
classes  of  adrenal  steroids  differ  markedly,  de- 
spite the  shared  role  of  the  same  enzymes  in  their 
biosynthesis. 

We  are  interested  in  defining  the  events  that 
control  the  expression  of  the  adrenal  steroido- 
genic enzymes.  These  studies  have  addressed  two 
major  questions.  First,  what  mechanisms  direct 
the  expression  of  these  related  genes  within  adre- 
nocortical cells?  Second,  what  determines  the 
functional  differentiation  of  the  adrenal  cortex 
into  mineralocorticoid-  and  glucocorticoid- 
producing  zones? 

Our  studies  of  gene  regulation  have  focused  on 
the  5'-flanking  regions  of  these  genes.  This  part, 
the  promoter  region,  contains  most  sequences 
important  in  transcriptional  regulation  of  other 
genes.  These  promoter  analyses  identified  a  pro- 
tein, steroidogenic  factor  1,  that  plays  a  major 
role  in  regulating  the  expression  of  all  three  ste- 
roidogenic enzymes.  We  only  found  this  protein 


in  cells  that  made  steroid  hormones,  suggesting 
that  it  contributes  to  the  cell-selective  expression 
of  these  genes.  The  global  role  of  this  protein  in 
the  expression  of  three  distinct  genes  suggests 
that  it  coordinates  the  adrenocortical  expression 
of  a  network  of  enzymes. 

We  next  used  cow  adrenal  glands,  providing 
much  greater  amounts  of  protein,  and  purified 
the  bovine  form  of  steroidogenic  factor  1.  The 
presently  available  amounts  of  purified  protein 
should  be  sufficient  to  determine  its  amino  acid 
sequence  and  to  raise  specific  antibodies.  The 
combination  of  specific  antibodies  and  amino 
acid  sequence  data  should  allow  us  to  clone  the 
gene  encoding  this  key  regulatory  protein.  By 
comparing  the  primary  structures  of  steroido- 
genic regulatory  protein  and  the  previously  de- 
scribed transcriptional  regulatory  proteins  (such 
as  the  steroid  hormone  receptor  proteins),  we 
may  gain  new  insights  into  the  mechanisms  that 
regulate  the  adrenal  steroidogenic  enzymes.  The 
specific  probes  and  antibodies  to  steroidogenic 
factor  1  will  further  permit  us  to  study  the  mecha- 
nisms that  regulate  its  expression.  These  studies 
will  provide  new  insights  into  the  basis  for  tissue- 
specific  differences  in  the  production  of  steroid 
hormones  and  may  identify  important  avenues  for 
therapeutic  intervention  in  clinical  settings  of 
abnormal  adrenal  steroidogenesis. 

In  a  related  effort,  we  are  trying  to  define  the 
potential  role  of  the  steroidogenic  enzymes  in  hy- 
pertension. Extremely  prevalent,  hypertension 
affects  approximately  20  percent  of  the  adult 
population  and  is  a  major  risk  factor  for  heart  at- 
tacks and  strokes,  the  leading  causes  of  death  in 
developed  nations.  Although  the  underlying  de- 
fect is  unknown  in  most  cases,  family  studies  indi- 
cate a  significant  genetic  component.  Certain  an- 
imal models  of  hypertension  have  directly 
implicated  disordered  adrenal  steroidogenesis  as 
an  important  contributor  to  hypertension.  We  are 
therefore  investigating  in  more  detail  the  role  of 
adrenal  steroids  in  hypertension. 

Initial  studies  implicated  a  single  1 1(8- 
hydroxylase  protein  in  the  biosynthesis  of  both 
mineralocorticoids  and  glucocorticoids,  but  we 


317 


Regulation  of  Gene  Expression  in  Steroid  Hormone  Biosynthesis 


subsequently  defined  two  mouse  1 1  jS-hydroxy- 
lase  genes  located  on  chromosome  1 5 .  Although 
the  overall  structures  of  the  two  genes  are  quite 
similar,  selected  regions  diverge  significantly, 
suggesting  that  the  proteins  encoded  by  these 
two  genes  might  have  different  activities.  More- 
over, the  5'-flanking  regions  of  the  two  genes  di- 
verge significantly,  suggesting  that  their  regula- 
tion may  differ. 

To  assess  the  enzymatic  activities  of  the  pro- 
teins produced  by  these  two  genes,  we  analyzed 
their  activities  following  gene  transfer  into  COS- 
7  cells,  which  normally  do  not  make  any  steroid- 
metabolizing  enzymes.  One  of  the  two  proteins, 
designated  1  l/S-hydroxylase,  was  able  to  produce 
glucocorticoids  but  could  not  make  mineralocor- 
ticoids.  In  contrast,  the  other  protein,  designated 
aldosterone  synthase,  performed  all  of  the  reac- 
tions needed  to  make  aldosterone.  Thus  there  are 
significant  differences  in  the  enzymatic  activities 
of  the  two  mouse  1 1  |S-hydroxylase  homologues. 

To  see  where  these  two  proteins  are  made  in 
the  adrenal  cortex,  we  used  the  technique  of  in 
situ  hybridization  to  examine  sites  of  expression 
in  the  sections  of  the  mouse  adrenal  gland.  The 
aldosterone  synthase  protein  is  only  present  in 
the  outer  zone  of  the  cortex,  where  mineralo- 
corticoids  are  made.  In  contrast,  the  11(8- 
hydroxylase  protein  is  only  present  in  the  sites  of 
glucocorticoid  production.  These  results  docu- 
ment an  exquisite  coupling  between  enzymatic 


activities  and  sites  of  expression.  The  ability  to 
maintain  this  segregation  of  the  two  proteins  is 
undoubtedly  an  important  part  of  the  adrenal 
gland's  ability  to  regulate  separately  the  produc- 
tion of  mineralocorticoids  and  glucocorticoids. 

Based  on  these  results,  we  plan  to  use  these 
promoter  regions  in  transgenic  mice  to  target 
gene  expression  to  specific  cortical  zones.  Ini- 
tially we  are  using  the  two  promoters  to  direct  the 
expression  of  renin,  a  gene  previously  shown  to 
cause  genetic  hypertension  when  expressed  in 
multiple  tissues  of  transgenic  animals.  If  success- 
ful, these  experiments  will  validate  the  zone-spe- 
cific expression  of  the  two  promoter  regions  and 
will  establish  that  adrenal  expression  of  renin  is 
relevant  to  this  hypertensive  state. 

We  will  also  express  the  aldosterone  synthase 
protein  in  the  inner,  glucocorticoid-producing 
zone.  To  this  end,  we  have  prepared  a  hybrid 
gene  with  the  inner-zone-specific  promoter 
driving  expression  of  the  aldosterone  synthase 
gene.  Based  on  the  gene  transfer  experiments  de- 
scribed above,  we  anticipate  that  this  hybrid  gene 
will  synthesize  large  amounts  of  mineralocorti- 
coids, thereby  creating  a  genetic  form  of  hyper- 
tension. Moreover,  treatment  with  glucocorti- 
coids should  alleviate  the  hypertension  by 
suppressing  the  expression  of  the  hybrid  gene  in 
the  inner  zone.  Recent  studies  strongly  suggest 
that  just  such  a  mechanism  is  responsible  for  a 
subset  of  human  patients  with  a  glucocorticoid- 
remediable  hypertension. 


318 


Molecular  Neuroimmunology 


Donald  G.  Payan,  M.D. — Assistant  Investigator 

Dr.  Payan  is  also  Associate  Professor  of  Medicine  and  of  Microbiology  and  Immunology  at  the  University 
of  California,  San  Francisco.  He  received  his  B.S.  degree  in  physics  and  mathematics  from  Stanford 
University.  He  went  on  to  do  graduate  work  in  physics  at  the  Massachusetts  Institute  of  Technology  and 
then  returned  to  Stanford  Medical  School,  where  he  received  his  M.D.  degree.  His  medical  residency  at 
Massachusetts  General  Hospital,  Boston,  was  followed  by  fellowships  in  infectious  diseases  at  MGH  and 
in  allergy-immunology  at  Brigham  and  Women's  Hospital. 


MY  laboratory  continues  to  study  the  interac- 
tions between  the  nervous  and  immune  sys- 
tems at  the  molecular  level.  Ongoing  work  is  fo- 
cusing on  the  signal  transduction  pathways  that 
are  activated  in  cells  into  which  we  have  trans- 
fected  a  number  of  neuropeptide  receptors,  in 
particular  the  tachykinin  receptor  for  substance 
P.  An  additional  effort  is  directed  at  understand- 
ing the  biochemical  properties  of  agrin,  the  nico- 
tinic acetylcholine  receptor-clustering  mole- 
cule, which  was  cloned  and  sequenced  during 
sabbatical  studies  with  Richard  Scheller  (HHMI, 
Stanford  University) . 

The  two  main  projects  currently  under  way  are 
the  analysis  of  the  signal  transduction  capabilities 
of  the  substance  P  receptor  (SPR)  and  accessory 
molecules  that  mediate  that  signaling,  and  an 
analysis  of  agrin's  protease  inhibitor  domains  and 
their  potential  function  in  modulating  neuronal 
plasticity  in  the  developing  brain.  With  Julie 
Sudduth-Klinger,  Christine  Christian,  and  Mark 
Gilbert,  we  have  transfected  SPR  cDNA  into  a 
number  of  different  immune  cells,  in  particular 
Jurkat  and  Reh,  in  order  to  study  the  effects  of  this 
receptor's  expression  on  the  cells'  immunologic 
properties.  We  can  get  approximately  100,000 
functional  receptors  per  cell  and  can  demon- 
strate a  very  brisk  mobilization  of  intracellular 
calcium  and  inositol  phosphate  (IP3)  metabo- 
lites following  stimulation  with  substance  P. 

Of  great  interest  are  many  differences  we  have 
observed  in  comparing  the  Jurkat  SPR  with  the 
Jurkat  muscarinic  receptor.  Activation  of  the  mus- 
carinic receptor  results  in  desensitization  of  sub- 
sequent T  cell  receptor  activation  with  an  anti-T 
cell  receptor  antibody;  conversely,  activation  of 
the  T  cell  receptor  does  not  result  in  desensitiza- 
tion of  the  transfected  muscarinic  receptor.  In 
contrast,  we  find  that  activation  of  Jurkat  SPRs  by 
substance  P  does  not  desensitize  subsequent  T 
cell  activation,  but  activation  of  the  T  cell  recep- 
tor does  result  in  desensitization  of  SPR.  We  are 
currently  investigating  whether  some  heterolo- 
gous desensitization  mechanism  is  taking  place 
and  whether  a  particular  kinase  is  involved. 
In  collaboration  with  Phyllis  Gardner  at  Stan- 


ford University,  we  have  also  been  able  to  demon- 
strate, using  patch-clamp  techniques,  that  activa- 
tion of  the  SPR  in  these  transfected  lymphocytes 
results  in  the  opening  of  a  chloride  channel.  In 
addition,  these  changes  can  be  nullified  by  in- 
jecting the  cells  with  a  calcium/calmodulin  ki- 
nase inhibitor  peptide. 

Our  early  observations  that  stimulation  of  these 
cell  lines  by  substance  P  resulted  in  both  IP3  and 
cyclic  AMP  activation  have  now  been  extended. 
Looking  at  the  cells'  respective  nuclear  regula- 
tory elements,  we  have  demonstrated  that  sub- 
stance P  stimulation  results  in  increased  expres- 
sion of  the  AP-1-  and  the  cAMP-responsive 
elements  in  these  cells.  Furthermore,  using  West- 
ern and  Northern  blotting  techniques,  we  have 
also  been  able  to  demonstrate  that  the  proto- 
oncogenes /os  and^wn  are  also  up-regulated  fol- 
lowing substance  P  stimulation.  Consequently, 
signal  transduction  analysis  suggests  that  a  dual 
activation  signal  pathway  may  be  activated  when 
the  SPR  is  expressed  in  lymphoid  cells. 

We  have  studied  the  functional  consequences 
of  stimulating  these  cells  with  the  peptide  sub- 
stance P  by  examining  the  expression  of  a  num- 
ber of  cell  surface  antigens  and  their  modulation 
on  Jurkat  SPRs.  We  find  that  in  Jurkat  SPR-positive 
cells,  substance  P  stimulation  results  in  a  down- 
regulation  of  the  LFA- 1  (integrin)  surface  antigen 
and  an  up-regulation  of  the  CD2  surface  antigen. 
In  addition,  when  combined  with  the  mitogen 
phytohemagglutinin,  substance  P  stimulation  re- 
sults in  an  increased  expression  of  the  interleu- 
kin-2  receptor.  We  continue  to  examine  these  re- 
sults in  order  to  delineate  further  how  tachykinin 
peptides  may  modulate  immune  responses. 

Joseph  Fisher  and  Sandra  Biroc  have  now  begun 
an  extensive  in  situ  hybridization  study  of  the 
expression  of  agrin  and  alternate  agrin  transcripts 
in  the  developing  rat.  Preliminary  results  suggest 
that  agrin  is  extensively  expressed  in  unique  sites 
within  the  developing  brain.  Moreover,  Dr. 
Fisher  has  now  expressed  full-length  recombi- 
nant agrin  in  a  number  of  cell  types  and  is  examin- 
ing the  molecule's  biochemical  properties.  In 
particular,  the  amino  terminus  of  the  molecule 


319 


Molecular  Neuroimmunology 


has  some  unique  sequences  characterized  by  pro- 
tease inhibitor  domains.  He  has  begun  a  study  of 
the  molecule's  protease  inhibitor  activity  to  see  if 
it  plays  a  role  during  the  remodeling  process  in 
neuronal  development.  We  expect  the  studies  re- 


garding receptor  signal  transduction  mechanisms 
and  early  developmental  remodeling  processes  in 
the  nervous  system  to  help  us  further  understand 
how  the  immune  and  nervous  systems  develop 
and  interact. 


Cell  surface  receptors  on  lymphocytes  some- 
times share  intracellular  signaling  mecha- 
nisms. In  this  case,  the  receptor  for  a  T  cell 
growth  factor  (interleukin-2  or  IL-2)  and  the 
protein  molecule  CD4,  which  assists  these  cells 
in  the  recognition  of  foreign  antigens,  both  in- 
teract with  a  lymphocyte- specific  protein- 
tyrosine  kinase,  p56^'^^ — a  potent  signaling 
molecule  that  induces  proliferation.  A  highly 
acidic  region  of  the  IL-2  receptor's  fS-chain  me- 
diates this  interaction,  while  a  very  different 
domain  in  the  CD4  protein,  containing  two 
critical  cysteine  residues  (c ),  performs  a  simi- 
lar function  for  this  receptor. 

Diagram  from  Roger  Perlmutter,  based  on 
work  in  his  laboratory. 


IaCIDIC 
:-  -  =7/--  '  proline-rich 


\\   tv  Signal 
V:~y  Transduction 


320 


I 


Molecular  Basis  of  Lymphocyte  Signaling 


Roger  M.  Perlmutter,  M.D.,  Ph.D. — Investigator 

Dr.  Perlmutter  is  also  Professor  of  Immunology,  Medicine,  and  Biochemistry  at  the  University  of 
Washington  School  of  Medicine.  He  received  his  B.A.  degree  from  Reed  College  and  his  M.D.  and  Ph.D. 
degrees  from  Washington  University,  St.  louis,  where  he  studied  with  Joseph  Davie.  After  clinical  training 
in  internal  medicine  at  Massachusetts  General  Hospital,  Boston,  and  the  University  of  California,  San 
Francisco,  he  became  Senior  Research  Fellow  and  later  Instructor  in  Biology  at  the  California  Institute 
of  Technology,  where  he  worked  with  lee  Hood. 


IMMUNE  recognition  of  potentially  injurious 
foreign  macromolecules  requires  the  elabora- 
tion of  an  enormous  repertoire  of  clonally  re- 
stricted receptors  (antigen  receptors)  on  the  sur- 
faces of  lymphoid  cells.  These  receptors  are 
sufficiently  heterogeneous  to  permit  recognition 
of  virtually  the  entire  universe  of  infectious  or- 
ganisms. Interaction  of  these  receptors  with  cog- 
nate antigen  provokes  a  stereotyped  response 
leading  to  cell  proliferation  and  the  production 
of  soluble  mediators  of  inflammation.  During  the 
past  several  years,  analysis  of  the  mechanisms  re- 
sponsible for  antigen  receptor  diversification  has 
stimulated  interest  in  a  related  question:  How  is 
the  signal  from  a  lymphocyte  antigen  receptor 
transmitted  to  the  cell  interior?  Our  laboratory 
has  adopted  a  molecular  genetic  approach  to  the 
dissection  of  signaling  pathways  in  immune  cells. 

Initially  we  identified  a  lymphocyte-specific 
enzyme,  similar  in  structure  to  proteins  known  to 
transmit  growth-promoting  signals  in  nonlym- 
phoid  cells,  that  modifies  the  behavior  of  target 
proteins  by  catalyzing  the  addition  of  phosphate 
groups  onto  certain  tyrosine  amino  acids  in  sub- 
strate proteins.  The  gene  that  encodes  this  lym- 
phocyte-specific kinase  was  identified  by  virtue 
of  its  overexpression  in  a  murine  lymphoid  malig- 
nancy. Moreover,  we  were  able  to  demonstrate 
that  a  single  point  mutation  in  this  Ick  gene  en- 
abled it  to  confer  malignant  properties  on  cells 
maintained  in  culture.  Thus  the  Ick  gene  encodes 
a  protein  that  is  capable  of  altering  the  growth 
properties  of  at  least  some  cell  types.  Since  Ick  is 
normally  expressed  only  in  lymphocytes,  there  is 
reason  to  believe  that  its  product  assists  in  regu- 
lating lymphocyte  proliferation. 

Biochemical  studies  support  this  view.  In  par- 
ticular, we  and  others  have  recently  demon- 
strated that  the  Ick-cncoded  kinase  is  physically 
associated  with  proteins  that  form  part  of  the  an- 


tigen receptor  on  T  lymphocytes.  Our  studies 
also  enabled  us  to  identify  two  additional  pro- 
tein-tyrosine  kinases  that  are  specifically  ex- 
pressed in  immune  cells.  In  each  case  there  is 
reason  to  believe  that  the  kinase  is  physically 
coupled  to  a  cell  surface  receptor  involved  in  im- 
mune recognition. 

To  investigate  the  functional  importance  of 
these  protein-tyrosine  kinase  signaling  elements, 
we  have  developed  methods  for  manipulating  the 
expression  of  each  gene  in  its  appropriate  cellu- 
lar context.  Using  this  approach,  we  have  begun 
to  dissect  the  hierarchy  of  signal  transduction 
events  precipitated  by  normal  immune  recogni- 
tion. For  example,  we  found  that  overexpression 
of  an  activated  Ick  gene  leads  to  extraordinarily 
rapid  development  of  thymic  tumors  in  mice. 
Hence  altered  expression  of  the  Ick  gene  can  di- 
rectly affect  lymphocyte  proliferation.  In  a  re- 
lated series  of  experiments  we  learned  that  inhibi- 
tion of  the  function  of  the  Ick  gene  completely 
disrupts  normal  mechanisms  that  permit  develop- 
ment of  T  lymphocytes.  In  fact  the  level  of  Ick- 
encoded  protein  must  be  maintained  within  a 
very  narrow  range.  Even  very  modest  (twofold) 
changes  in  the  abundance  of  this  kinase  are  in- 
compatible with  normal  T  lymphocyte  develop- 
ment. These  observations  probably  reflect  the 
fact  that  the  /cfe-encoded  kinase  participates  in  a 
large  number  of  receptor  signaling  pathways, 
including  some  pathways  that  are  activated 
by  lymphocyte-specific  growth  factors  such  as 
interleukin-2. 

Disturbances  in  lymphocyte  signaling  almost 
certainly  contribute  to  the  pathogenesis  of  lym- 
phoproliferative  and  immunodeficiency  diseases 
in  humans.  A  detailed  understanding  of  immune 
cell  signaling  mechanisms  should  permit  the  de- 
sign of  more-effective  therapeutic  strategies  for 
the  treatment  of  immune  system  dysfunction. 


321 


wildtype 


"'-iVilv. 


corkscrew 


Darkfield  preparation  of  a  wild-type  and  a  corkscrew  embryo.  The  twist  in 
the  mutant  embryo  is  due  to  a  perturbed  establishment  of  cell  fates  at  the 
termini  of  the  blastoderm. 

From  Perkins,  L.A.,  Larsen,  I.,  and  Perrimon,  N.  1992.  Cell  70:225- 
236.  Copyright  ©  1992  by  Cell  Press. 


Genetic  Dissection  of  a  Signal  Transduction 
Pathway  in  Drosophila  melanogaster 


Norbert  Perrimon,  Ph.D. — Assistant  Investigator 

Dr.  Perrimon  is  also  Assistant  Professor  of  Genetics  at  Harvard  Medical  School.  Of  French  nationality,  he 
tvas  educated  at  the  University  of  Paris  VI,  where  he  majored  in  biochemistry.  His  thesis,  with  Madeleine 
Gans  as  advisor,  was  on  Drosophila  genetics.  He  moved  to  Case  Western  Reserve  University  as  a 
postdoctoral  research  fellow  with  Anthony  Mahowald.  He  became  a  Lucille  P.  Markey  Scholar  in 
Biomedical  Sciences  while  in  Cleveland.  He  then  assumed  his  present  position  at  Harvard  Medical  School. 


INTERCELLULAR  communication  is  a  major 
player  in  the  establishment  of  developmental 
patterns.  For  example,  in  the  early  Drosophila 
embryo,  determination  of  cell  fates  at  the  termini 
requires  the  normal  activities  of  genes  from  two 
different  cell  types,  the  maternal  follicle  cells 
and  the  oocyte.  The  current  model  is  that  the 
transmembrane  tyrosine  kinase  receptor  encoded 
by  the  gene  torso  is  locally  activated  at  the  egg 
termini  by  cues  emanating  from  the  follicle  cells. 
This  localized  activation  of  torso  is  believed  to 
trigger  a  phosphorylation  cascade  in  the  egg 
which  ultimately  controls  the  expression  of  the 
transcription  factors  tailless  and  huckebein. 
Knocking  out  this  signaling  pathway  has  detri- 
mental effects  on  embryonic  development.  Since 
cell  fates  at  both  termini  are  perturbed,  the  re- 
sulting embryos  lack  most  head  and  all  tail 
structures. 

Our  laboratory  has  focused  on  identifying  the 
genetic  components  involved  in  transduction  of 
the  signal  from  torso,  the  membrane-bound  tyro- 
sine kinase,  to  the  nucleus.  Thus  far  we  have 
characterized  two  genes,  1(1  )pole  hole  and 
l(  1  )corkscrew,  that  are  involved  in  this  process. 
Genetic  epistasis  experiments  have  demonstrated 
that  both  these  genes  act  downstream  of  the  torso 
protein  activity.  Furthermore,  l( l)corkscrew 
acts  by  up-regulating  the  activity  of  1(1  )pole 
hole. 

We  previously  showed  that  the  l(  1  Jpole  hole 
gene  product  is  the  homologue  of  the  mamma- 
lian Raf- 1  proto-oncogene  and  encodes  a  serine/ 
threonine  kinase.  Recently  we  discovered  that 
l(  1 ) corkscrew  encodes  a  protein-tyrosine  phos- 
phatase similar  to  the  mammalian  PTPIC  protein. 
Identification  of  the  l(  1  )pole  hole  and  1(1  Jcork- 
screw  gene  products  has  strengthened  the 
current  model  that  torso  signaling  involves  a 


phosphorylation  cascade,  since  both  genes  en- 
code proteins  that  have  the  ability  to  affect 
the  level  of  phosphorylation  of  intracellular 
components. 

To  identify  additional  molecules  involved  in 
torso  signaling,  we  have  taken  a  genetic  ap- 
proach. Screens  for  second-site  suppressors  and 
enhancers  of  l(  1  Jpole  hole  and  l(  1  Jcorkscrew 
mutations  have  successfully  identified  a  number 
of  loci  involved  in  the  torso  signaling  pathway. 
Future  work  will  involve  a  detailed  characteriza- 
tion of  these  suppressors. 

In  addition  to  the  genetic  approach  described 
above,  we  are  utilizing  a  biochemical  screen  to 
identify  and  characterize  other  components  of 
this  signal  transduction  pathway.  Involved  is  the 
cloning  of  genes  encoding  proteins  that  bind  di- 
rectly to  activated  receptor  tyrosine  kinase.  In 
this  way  we  are  isolating  proteins  that  respond 
directly  to  the  ^or50-encoded  receptor. 

Establishment  of  cell  fate  at  the  termini  of  the 
embryo  provides  a  unique  genetic  system  to  dis- 
sect the  cascade  by  which  activation  of  a  receptor 
tyrosine  kinase  controls  the  expression  of  tran- 
scription factors.  A  combination  of  classical  ge- 
netics and  molecular  and  biochemical  tech- 
niques will  allow  characterization  of  the 
components  involved  in  the  various  steps  of  re- 
ceptor tyrosine  kinase  signaling. 

In  addition,  the  homology  between  torso  and 
the  mammalian  gene  for  platelet-derived  growth 
factor  (PDGF),  between  l(  1  Jpole  hole  and  the 
mammalian  Raf  l  proto-oncogene,  and  between 
l(  1  Jcorkscrew  and  the  human  PTPIC  enzyme 
suggests  that  biochemically  this  signal  transduc- 
tion pathway  may  have  been  conserved  in  evolu- 
tion between  organisms  as  diverse  as  Drosophila 
and  humans.  Thus  characterization  of  this  path- 
way in  Drosophila  may  help  elucidate  the  func- 
tions of  the  homologous  mammalian  proteins. 


525 


Gene  Regulation  and  Immunodeficiency 


B.  Matija  Peterlin,  M.D. — Associate  Investigator 

Dr.  Peterlin  is  also  Associate  Professor  of  Medicine  and  of  Microbiology  and  Immunology  at  the  University 
of  California,  San  Francisco.  He  obtained  his  undergraduate  degree  in  chemistry  and  physics  at  Duke 
University  and  his  M.D.  degree  from  Harvard  Medical  School.  His  postdoctoral  work  was  performed  with 
facob  Maizel  and  Philip  leder  at  NIH  and  with  Hugh  McDevitt  at  Stanford  University.  As  a  rheumatology 
fellow  at  Stanford,  he  chanced  upon  a  family  with  the  bare  lymphocyte  syndrome,  which  stimulated  his 
interest  in  this  genetic  disorder.  He  is  a  member  of  the  American  Society  for  Clinical  Investigation. 


SOME  years  ago  we  described  a  variant  of  the 
genetic  disorder  called  the  bare  lymphocyte 
syndrome  (BLS),  in  which  the  patient's  lympho- 
cytes fail  to  express  either  class  I,  class  II,  or  both 
major  histocompatibility  determinants  on  their 
cell  surfaces.  These  transplantation  antigens  are 
essential  for  the  development  of  the  immune  sys- 
tem, for  tumor  surveillance,  for  eradication  of 
viral  infections,  and  for  normal  immune  re- 
sponses. Thus  it  is  not  surprising  that  BLS  patients 
are  severely  immunocompromised,  fail  to  make 
antibodies,  or  have  autoimmune  diseases.  In  ad- 
dition, this  autosomal  recessive  syndrome  is  one 
of  only  two  known  inherited  deficiencies  of  a  reg- 
ulatory gene  in  humans. 

By  fusing  defective  cells  from  different  patients 
and  those  obtained  by  mutagenesis  in  tissue  cul- 
ture, four  genetic  complementation  groups  of 
BLS  were  found.  The  isolation  of  their  defective 
genes  should  make  possible  prenatal  diagnoses 
through  use  of  specific  genetic  probes  and  possi- 
bly lead  to  the  cure  of  BLS  by  the  targeting  of 
normal  genes  into  the  bone  marrow  of  alfected 
patients. 

To  study  the  defective  gene  in  BLS,  we  first  ex- 
amined regions  that  regulate  B  cell-specific  and 
interferon-7  (IFN-7) -inducible  expression  of 
class  II  genes.  Next,  we  looked  at  proteins  that 
bind  to  these  DNA  sequences  and  compared  class 
Il-specific  factors  in  various  cell  types.  Distinct 
patterns  of  DNA-binding  proteins  were  found  in  B 
cells,  IFN-7-inducible  cells,  and  T  cells. 

We  cloned  several  cDNAs  that  code  for  proteins 
that  bind  to  B  cell-specific  and  IFN-7-inducible 
sequences  in  class  II  promoters.  One  cDNA  codes 
for  Jun,  which  forms  active  Jun/Fos  heterodimers 
in  cells  that  do  not  express  class  II  determinants. 
Of  the  remaining  two  cDNAs,  one  codes  for  a  B 
cell-specific  helix-loop-helix  protein  and  the 
other  for  an  ETS-like  protein.  We  are  currently 
studying  their  genetic  organization,  expression, 
structure,  and  function.  By  expressing  one  of 
these  full-length  cDNAs  in  human  cells,  we  hope 
to  rescue  class  II  gene  expression  in  one  type  of 
BLS.  In  parallel  with  direct  biochemical  studies, 
we  are  also  using  genetic  approaches  to  rescue 
regulatory  defects  in  BLS. 


In  setting  up  these  genetic  approaches,  we  first 
tested  a  well-known  viral  trans-regulatory  system 
— namely  trans-activation  of  the  human  immuno- 
deficiency virus  (HFV)  by  the  virally  encoded  Tat 
protein.  The  precise  mechanism  of  Tat  action  had 
not  been  defined.  We  discovered  that  Tat  acts 
slightly  downstream  from  the  promoter  to  modify 
HIV  transcription  so  that  efficient  copying  of  the 
viral  genome  ensues.  Factors  assembled  near  the 
site  of  initiation  of  HIV  transcription  tether  the 
transcription  complex  to  the  promoter.  The  addi- 
tion of  Tat,  which  binds  to  an  RNA  stem-loop  in 
the  process  of  nascent  transcription,  releases  this 
transcription  complex.  Efficient  elongation  of 
transcription  and  clearance  of  the  promoter  fol- 
low. New  transcription  complexes  can  then  as- 
semble, interact  with  Tat,  and  move  quickly 
through  the  viral  genome.  Interactions  between 
Tat,  the  RNA  stem-loop,  and  cellular  proteins 
have  been  defined.  For  example,  using  a  heterolo- 
gous RNA-tethering  mechanism  (that  of  the  coat 
protein  of  bacteriophage  Rl  7  that  binds  to  its  op- 
erator), we  mapped  activation  and  RNA-binding 
domains  of  Tat.  By  studying  rodent  cells  and  so- 
matic cell  hybrids  between  rodent  and  human 
cells,  we  defined  a  cellular  RNA-binding  com- 
plex that  facilitates  interactions  between  Tat  and 
TAR  and  is  encoded  on  human  chromosome  12. 
We  hope  that  interfering  with  trans-activation  by 
Tat  will  lead  to  new  therapies  for  AIDS  (acquired 
immune  deficiency  syndrome)  and  AIDS-related 
disorders. 

Since  upstream  promoter  sequences  are  also 
essential  for  HIV  replication,  we  clarified  inter- 
actions between  host  cell  factors  and  viral  se- 
quences (long  terminal  repeat,  LTR).  Increased 
rates  of  initiation  of  HIV  transcription  were  ob- 
served in  activated  T  cells  and  macrophages. 
These  result  from  actions  of  nuclear  proteins  that 
are  also  required  for  T  cell  and  macrophage  ef- 
fector functions  and  for  T  cell  proliferation. 

Differences  between  LTRs  of  HIV  types  1  and  2 
were  observed  that  might  explain  the  longer  la- 
tency and  attenuated  clinical  course  of  HIV-2  in- 
fection. Furthermore,  effects  of  trans-activators 
encoded  by  several  DNA  viruses  on  HIV  transcrip- 


325 


Gene  Regulation  and  Immunodeficiency 


tion  were  examined.  Since  effects  of  these  ago- 
nists and  Tat  were  synergistic,  infection  by  DNA 
viruses  might  be  an  important  cofactor  in  progres- 
sion from  latent  disease  to  clinical  AIDS. 

Thus  the  quest  to  rescue  the  expression  of  class 


II  major  histocompatibility  genes  in  a  rare  human 
congenital  disease  has  led  to  genetic  approaches 
to  the  study  of  transcription  by  RNA  polymerase 
II  and  to  the  elucidation  of  a  potent  new  tran- 
scriptional regulatory  mechanism. 


326 


Mechanism  of  Action  of  Polypeptide 
Growth  Factors 


Linda  J.  Pike,  Ph.D. — Associate  Investigator 

Dr.  Pike  is  also  Associate  Professor  in  the  Department  of  Biochemistry  and  Molecular  Biophysics  at 
Washington  University  School  of  Medicine,  St.  Louis.  She  received  her  B.S.  degree  in  chemistry  from  the 
University  of  Delaware  and  her  Ph.D.  degree  in  biochemistry  from  Duke  University,  where  she  studied  with 
Robert  Lefkowitz.  Her  postdoctoral  training  was  done  in  the  laboratory  of  Edwin  Krebs  at  the  University 
of  Washington,  Seattle. 


A number  of  low-molecular-weight  polypep- 
tides have  been  shown  to  regulate  cell 
growth.  These  growth  factors  bind  to  specific  re- 
ceptors on  the  surface  of  cells.  Through  a  com- 
plex series  of  reactions,  the  binding  of  the  growth 
factor  to  its  receptor  stimulates  the  cell  to  grow 
and  divide.  Until  recently,  little  was  known  of  the 
mechanism  by  which  growth  factors  induce  cell 
division.  It  is  now  recognized,  however,  that  the 
receptors  not  only  bind  the  appropriate  growth 
factor  but  also  have  an  enzymatic  activity. 

The  receptors  catalyze  the  transfer  of  a  phos- 
phate group  from  adenosine  triphosphate  to  tyro- 
sine residues  in  selected  protein  substrates.  The 
enzyme  possessing  this  activity  is  called  a  tyro- 
sine protein  kinase.  Typically  the  phosphoryla- 
tion of  a  protein  by  a  kinase  leads  to  changes  in 
the  activity  of  the  protein.  Although  much  is 
known  about  the  growth  factor  receptor  kinases, 
the  substrates  for  these  enzymes  have  not  been 
identified. 

My  laboratory  is  involved  in  studies  of  the 
mechanism  by  which  the  binding  of  epidermal 
growth  factor  (EGF)  to  the  outside  of  the  cell 
elicits  a  biological  response  inside.  This  is  re- 
ferred to  as  signal  transduction.  Because  they  are 
extremely  responsive  to  EGF,  the  A431  line  of 
human  epidermal  carcinoma  cells  is  used  as  the 
model  system  in  most  of  our  studies. 

Phosphatidylinositol  Metabolism 

One  of  the  earliest  responses  of  A431  cells  to 
EGF  is  an  increase  in  the  metabolism  of  a  particu- 
lar phospholipid,  phosphatidylinositol.  This  im- 
portant lipid  serves  as  a  precursor  for  the  genera- 
tion of  two  intracellular  compounds  that  activate 
various  enzymes  and  thereby  mediate  the  effects 
of  EGF  within  the  cell.  One  of  the  enzymes  in- 
volved in  phosphatidylinositol  metabolism  is 
phosphatidylinositol  kinase.  Since  the  activity  of 
this  enzyme  is  stimulated  by  EGF,  it  represents  a 
potential  substrate  for  phosphorylation  by  the 
EGF  receptor  tyrosine  protein  kinase. 

We  have  purified  the  phosphatidylinositol  ki- 
nase from  both  A431  cells  and  human  placenta. 
This  55-kDa  enzyme  is  active  as  a  monomer — 


that  is,  as  a  single  polypeptide  chain.  It  phos- 
phorylates  phosphatidylinositol  on  the  4  position 
of  the  inositol  ring  and  hence  is  distinct  from  an- 
other phosphatidylinositol  kinase  that  phosphor- 
ylates  the  ring  on  the  3  position.  Information  re- 
garding the  sequence  of  the  amino  acids  that 
make  up  the  phosphatidylinositol  4-kinase  (PI 
4-kinase)  was  obtained.  Molecular  biology  tech- 
niques were  used  to  isolate  and  characterize  the 
cDNA  encoding  the  PI  4-kinase.  From  this  cDNA 
the  amino  acid  sequence  of  the  entire  PI  4-kinase 
was  deduced  and  was  found  to  be  unique.  The 
sequence  showed  limited  similarity  to  other  pro- 
teins that  bind  inositol  phosphate  or  carry  out  the 
phosphorylation  of  other  sugars,  suggesting  that 
the  PI  4-kinase  may  be  derived  from  enzymes  that 
metabolize  sugars.  Analysis  of  the  types  of  mRNAs 
present  in  a  variety  of  tissues  that  encode  the  PI 
4-kinase  indicated  that  at  least  two  types  of  mRNA 
are  present  and  that  the  amount  of  message  is  reg- 
ulated by  physiological  stimuli.  Efforts  are  now 
directed  toward  producing  a  cell  line  in  which 
this  PI  4-kinase  cDNA  is  overexpressed,  leading 
to  elevated  levels  of  PI  4-kinase  activity  within 
the  cell.  The  effects  of  this  overexpression  on 
the  growth  properties  of  the  cells  will  be  investi- 
gated. This  work  is  supported  by  a  grant  from  the 
National  Institutes  of  Health. 

Another  enzyme  involved  in  phosphatidylino- 
sitol metabolism  is  a  phosphatidylinositol  mono- 
phosphate phosphatase.  This  enzyme  catalyzes 
the  reverse  of  the  reaction  catalyzed  by  the  PI 
4-kinase — that  is,  it  removes  the  phosphate  from 
the  4  position  of  the  inositol  ring  of  phosphatidyl- 
inositol  monophosphate.  Although  this  enzyme 
has  not  been  studied  previously,  its  position  in 
the  metabolic  pathway  of  phosphatidylinositol 
suggests  that  it  may  be  important  in  the  overall 
regulation  of  the  pathway.  We  have  characterized 
it  with  respect  to  its  kinetic  properties,  substrate 
specificity,  and  response  to  various  inhibitors. 
We  have  purified  the  enzyme  to  a  high  degree  and 
have  shown  that  it  is  a  95-kDa  glycoprotein.  Un- 
like other  enzymes  in  this  pathway,  it  appears  to 
have  an  extracellular  domain.  The  possibility  that 
its  activity  is  regulated  through  the  binding  of  an 
extracellular  mediator  is  being  investigated. 


327 


Mechanism  of  Action  of  Polypeptide  Growth  Factors 


It  has  recently  been  postulated  that  the  inositol 
phospholipids  may  function  in  the  regulation  of 
cell  shape  via  interactions  with  proteins  that  con- 
trol the  polymerization  of  actin.  Using  EGF  and 
A43 1  cells,  we  have  shown  that  there  is  no  corre- 
lation between  EGF-induced  changes  in  cell 
shape  and  changes  in  the  levels  of  the  classical 
phosphoinositides,  phosphatidylinositol  4-phos- 
phate  and  phosphatidylinositol  4,5-bisphos- 
phate.  Further  experiments  have  also  failed  to 
document  the  involvement  of  novel  inositol  phos- 
pholipids, phosphorylated  on  the  3  position  of 
the  inositol  ring,  in  the  regulation  of  cell  shape 
by  EGF.  These  data  suggest  that  EGF  may  control 
the  actin  cytoskeleton  simply  by  a  mechanism 
that  involves  phosphorylation  of  cytoskeletal 
proteins  rather  than  indirectly  by  causing  alter- 
ations in  inositol  phospholipid  levels. 

Desensitization  of  the  EGF  Receptor 

When  A43 1  cells  are  treated  with  large  doses  of 
EGF,  washed,  and  subsequently  rechallenged 
with  EGF,  they  fail  to  respond  to  the  grovvT:h  fac- 
tor. This  phenomenon  is  known  as  desensitiza- 
tion. Our  studies  have  shown  that  when  the  EGF 
receptor  becomes  desensitized,  it  is  no  longer  in- 
ternalized into  the  cells,  and  EGF  no  longer  stimu- 
lates phosphatidylinositol  metabolism.  This  EGF- 


induced  desensitization  is  specific  for  the  EGF 
receptor,  because  the  responsiveness  of  other  re- 
ceptors is  not  decreased  after  EGF  treatment. 

The  EGF  receptor  itself  is  a  monomeric  pro- 
tein, a  single  chain.  Upon  binding  of  EGF  to  its 
receptor,  two  of  the  receptor  monomers  come 
together  to  form  an  EGF  receptor  dimer.  This 
dimer  is  the  form  that  is  active  in  signal  transduc- 
tion. We  have  shown  that  desensitized  EGF  re- 
ceptors do  not  transduce  a  signal  because  they 
cannot  undergo  this  EGF-induced  dimerization. 

Our  data  suggest  that  the  desensitization  of  the 
EGF  receptor  results  from  its  phosphorylation  by 
a  protein  kinase.  We  have  identified  a  protein  ki- 
nase in  A43 1  cell  cytosol  that  is  activated  by  EGF 
and  appears  to  be  involved  in  receptor  desensiti- 
zation. The  kinase  catalyzes  the  phosphorylation 
of  the  EGF  receptor  in  vitro.  Consistent  with 
what  has  been  observed  in  whole  cells,  phos- 
phorylation of  EGF  receptor  monomers  by  this 
kinase  leads  in  vitro  to  an  inhibition  of  the  ability 
of  the  phosphorylated  monomers  to  dimerize. 

The  kinase  phosphorylates  the  EGF  receptor  on 
a  serine  residue  in  the  second  half  of  the  receptor 
molecule.  Using  techniques  of  molecular  biol- 
ogy, we  have  altered  this  site  and  are  in  the  pro- 
cess of  characterizing  the  properties  of  cells  ex- 
pressing this  mutated  form  of  the  EGF  receptor. 


328 


Protein  Structures,  Molecular  Recognitions, 
and  Functions 


Florante  A.  Quiocho,  Ph.D. — Investigator 

Dr.  Quiocho  is  also  Professor  of  Biochemistry  and  Structural  Biology  and  of  Molecular  Physiology  and 
Biophysics  at  Baylor  College  of  Medicine.  He  obtained  his  Ph.D.  degree  in  biochemistry  at  Yale  University 
and  then  did  postdoctoral  research  in  chemistry  at  Harvard  University.  He  was  a  member  of  the  Rice 
University  faculty  before  joining  the  Baylor  faculty.  Dr.  Quiocho  has  been  a  visiting  research  scientist  at 
Oxford  University,  a  research  fellow  of  the  European  Molecular  Biology  Organization  (EMBO),  and  a 
Guggenheim  fellow. 


LIGAND  specificity  and  the  activity  of  pro- 
teins are  derived  from  their  precise  three- 
dimensional  structures.  Using  mainly  x-ray 
crystallographic  techniques,  our  laboratory  is  en- 
gaged in  atomic-level  elucidation  of  the  struc- 
tures and  functions  of  several  proteins  (including 
enzymes)  involved  in  biologically  important  pro- 
cesses. To  complement  our  work,  w^e  also  employ 
biochemical  and  recombinant  DNA  techniques. 

Adenosine  Deaminase 

Adenosine  deaminase  (ADA)  is  one  of  the  major 
enzymes  in  purine  metabolism,  catalyzing  the  ir- 
reversible hydrolysis  of  adenosine  or  deoxyaden- 
osine  to  the  respective  inosine  product  and  am- 
monia. The  enzyme  is  found  in  nearly  all 
mammalian  cells  and  plays  a  central  role  in  main- 
taining competency  of  the  immune  system, 
among  several  other  functions.  Lack  or  deficiency 
of  ADA  is  associated  with  severe  combined  immu- 
nodeficiency disease  (SCID),  a  genetically  in- 
herited disorder  usually  fatal  within  two  years  of 
birth  if  left  untreated. 

Last  year  we  determined  the  crystal  structure  of 
ADA  with  bound  6-i?-hydroxyl-l,6-dihydropu- 
rine  ribonucleoside  (HDPR) ,  a  nearly  ideal  tran- 
sition-state analogue  inhibitor.  We  have  since 
elucidated  the  structures  of  complexes  of  the  en- 
zyme with  the  following  ligands:  1 -deazaadeno- 
sine,  a  substrate  analogue;  2'-i?-deoxycoformycin, 
a  potent  transition-state  analogue  and  a  chemo- 
therapeutic  agent  for  the  treatment  of  hairy  cell 
leukemia;  and  inosine,  the  product  of  the  deami- 
nation  of  adenosine.  All  these  structures  have 
provided  us  with  a  molecular  anatomy  of  the 
various  steps  associated  with  ADA's  catalytic 
activity. 

All  of  the  four  crystal  structures  of  the  deami- 
nase indicated  above  have  been  determined  and 
refined  at  pH  4.2,  where  the  enzyme  is  only  20 
percent  active.  We  have  also  carried  out  the  re- 
finement of  the  structure  of  ADA  complexed  with 
HDPR  at  pH  6,  where  the  enzyme  is  fully  active. 
The  structures  at  either  pH  are  essentially  the 
same. 


Antibody- Antigen  Interactions 

Because  monoclonal  antibodies  against  extra- 
cellular polysaccharide  antigens  exhibit  very 
stringent  specificity,  they  have  been  used  in 
blood-grouping  and  in  differentiating  bacterial 
serogroups  and  serotypes.  We  previously  ob- 
tained crystals  of  the  Fab  fragment  of  the  antibody 
raised  against  the  surface  polysaccharide  O-anti- 
gen  of  Shigella  flexneri.  In  the  past  year  we  de- 
termined and  refined  the  structures  of  the  Fab 
and  its  complexes  with  a  trisaccharide,  a-Rha(l- 
3)Q!-Rha(l-3);8-GlcNAc,  and  a  pentasaccharide, 
a-Rha(l-2)a-Rha(l-3)a-Rha(l-3)i3-GlcNAc-(l- 
2)a;-Rha.  Both  oligosaccharides  contain  determi- 
nants of  the  O-antigen  serotype  of  the  bacteria. 

This  structural  work  is  in  line  with  our  interest 
in  protein-carbohydrate  interactions.  Moreover  it 
is  relevant  to  clinical  problems,  as  oligosaccha- 
ride epitopes  of  bacterial  and  tumor  cell  surfaces 
are  considered  to  be  disease  markers  and  targets 
for  therapeutic  antibodies. 

Aldose  Reductase 

Aldose  reductase  catalyzes  the  NADPH-depen- 
dent  reduction  of  a  wide  variety  of  carbonyl- 
containing  compounds  to  their  corresponding  al- 
cohols, with  a  broad  range  of  catalytic  efficien- 
cies. Steroids  are  the  best  substrates  and  sugars 
the  least  favorable.  Although  the  enzyme  is  found 
in  a  variety  of  cells,  its  physiological  function  has 
not  been  firmly  established.  A  role  in  reducing 
the  hyperglycemia  of  diabetes  mellitus  has  been 
linked  to  diabetic  complications  aff"ecting  the 
lens,  retina,  peripheral  nerves,  and  kidney.  Drugs 
designed  to  control  these  complications  have  not 
been  clinically  successful  to  date  because  of  lack 
of  specificity  or  inefficacy. 

In  collaboration  with  Kurt  Bohren  and  Kenneth 
Gabbay  of  the  Baylor  College  of  Medicine,  we 
have  obtained  excellent  diffracting  crystals  of  re- 
combinant aldose  reductase  (from  human  pla- 
centa) with  bound  NAD  PH.  We  have  determined 
and  refined  the  enzyme's  three-dimensional, 
1.65-A  resolution  structure.  The  enzyme  has  a 
parallel  jS/a-barrel  motif,  with  eight  central  /?- 
strands  connected  by  eight  peripheral  a-helices. 


329 


Protein  Structures,  Molecular  Recognitions,  and  Functions 


This  establishes  a  new  motif  for  NADP/NAD-bind- 
ing  oxidoreductases  and  the  first  structure  of  the 
superfamily  of  aldo-keto  reductases. 

The  substrate-binding  site  is  located  in  an  ex- 
tremely hydrophobic  elliptical  pocket  at  the  car- 
boxyl-terminal  end  of  the  (8-barrel.  The  nicotin- 
amide group  of  the  NADPH  is  at  the  bottom  of  the 
,deep  pocket.  Although  the  hydrophobic  nature  of 
the  active  site  greatly  favors  aromatic  (e.g.,  ste- 
roids) and  apolar  substrates,  it  is  not  wcW  suited 
for  binding  of  highly  polar  monosaccharides, 
which  are  believed  to  figure  in  the  pathogenesis 
of  diabetic  complications.  The  determination  of 
the  structure  of  aldose  reductase  paves  the  way 
for  rational  design  of  specific  inhibitors  that 
might  provide  molecular  understanding  of  the 
catalytic  mechanism,  as  well  as  possible  thera- 
peutic agents  for  the  prevention  of  diabetic 
complications. 

Periplasmic  Receptors  for  Active  Transport 
and  Chemotaxis 

The  family  of  binding  proteins  that  serve  as  ini- 
tial periplasmic  receptors  for  bacterial  active 
transport  and  chemotaxis  continues  to  be  a  gold 
mine  for  detailed  study  of  protein  structure  and 
molecular  recognition  of  a  variety  of  ligands.  We 


continue  to  push  the  structure  refinements  of  the 
seven  different  periplasmic  receptors  to  much 
higher  resolutions — the  sulfate-binding  protein 
(at  1 .7-A  resolution),  phosphate-binding  protein 
(1.17  A),  L-arabinose-binding  protein  (1.7  A), 
D-galactose/D-glucose-binding  protein  (1.5  A), 
maltodextrin-binding  protein  (1.7  A),  leucine/ 
isoleucine/valine-binding  protein  (1.7  A),  and 
leucine-specific-binding  protein  (1.7  A). 

Electrostatic  Interactions  in  Molecular 
Recognition  of  Ligands 

Electrostatic  interactions  are  among  the  key 
factors  determining  the  structure  and  function  of 
proteins.  The  refined  structure  of  the  liganded 
form  of  sulfate-binding  protein  shows  that  the 
bound  sulfate  dianion  is  completely  buried  and 
bound  by  hydrogen  bonds  and  van  der  Waals  con- 
tacts. The  bound  sulfate  is  adjacent  to  the  amino 
termini  of  three  helices  and  is  coupled  via  a  pep- 
tide unit  to  a  positively  charged  His  residue.  Nev- 
ertheless, using  site-directed  mutagenesis  and 
theoretical  analysis,  we  have  shown  that  helix 
macrodipoles  and  the  His  residue  contribute  al- 
most nothing  to  ligand  fixation  and  charge  com- 
pensation. It  is  the  collection  of  local  dipoles  im- 
mediately surrounding  the  sulfate  that  is 
responsible  for  charge  compensation. 


330 


Molecular  Approaches  to  Olfaction 


Randall  R.  Reed,  Ph.D. — Associate  Investigator 

Dr.  Reed  is  also  Associate  Professor  in  the  Departments  of  Molecular  Biology  and  Genetics  and  of 
Neuroscience  at  the  Johns  Hopkins  University  School  of  Medicine.  He  received  his  bachelor's  degree  in 
biophysics  from  Johns  Hopkins  and  his  Ph.D.  degree  from  Yale  University.  His  postdoctoral  research  was 
done  with  Philip  Leder  at  Harvard  Medical  School. 


OLFACTION  is  among  the  oldest  of  the  sen- 
sory systems.  All  multicellular  and  many  un- 
icellular organisms  have  evolved  sensitive  che- 
mosensory  systems  able  to  detect  and  identify 
natural  chemical  substances.  The  olfactory  sys- 
tem of  vertebrates  and  analogous  systems  for  the 
other  senses — vision,  hearing,  taste,  and  touch 
— allow  the  conversion  of  external  stimuli  into 
nerve  impulses.  In  mammals,  the  olfactory  sys- 
tem is  exquisitely  sensitive,  capable  of  detecting 
some  odorants  present  at  a  concentration  of  only 
a  few  parts  per  trillion. 

The  ability  of  the  olfactory  system  to  discrimi- 
nate thousands  of  different  odorants  suggests  a 
complex  coding  mechanism.  However,  the  bio- 
logical basis  for  this  coding  remains  a  mystery. 
Unlike  the  visual  and  auditory  systems,  which 
need  only  encode  information  on  frequency  (or 
wavelength)  and  intensity,  the  olfactory  sense  re- 
quires multidimensional  information.  These  con- 
siderations suggest  a  complex  signal  transductory 
process. 

The  signal  transductory  pathway  for  olfaction 
can  be  divided  temporally  and  spatially  into  sev- 
eral distinct  steps.  The  first  consists  of  the  solubi- 
lization and  concentration  of  airborne  odorants. 
Considerable  experimental  data  suggest  that  com- 
ponents of  the  mucus  are  able  to  concentrate 
odorants  several  thousandfold.  We  previously 
identified  cDNA  clones  encoding  proteins  that 
are  present  at  high  concentration  in  the  mucus 
and  appear  to  bind  odorants.  These  proteins, 
from  rat  and  frog,  are  members  of  the  retinol- 
binding  protein  family,  many  of  which  have  been 
shown  to  solubilize  hydrophobic  ligands  in 
serum.  They  are  likely  to  play  a  similar  role  in  the 
olfactory  system. 

The  recognition  of  the  chemical  structure  of  an 
odorant  and  transduction  of  that  information 
across  the  plasma  membrane  is  a  poorly  under- 
stood process.  Some  investigators  hypothesize 
that  odorants  interact  directly  with  the  lipid 
membrane,  but  it  is  difficult  to  see  how  the  abil- 
ity to  discriminate  stereoisomeric  compounds 
could  be  accommodated  by  such  a  system.  Sev- 
eral years  ago  we  began  to  test  an  alternative  hy- 


pothesis: that  binding  of  odorants  to  specific 
membrane-associated  proteins  leads  to  intracel- 
lular changes  in  the  primary  sensory  neuron. 

The  detection  of  odorant-stimulated  activation 
of  second  messengers  in  olfactory  neurons  sug- 
gested an  analogy  to  sensory  systems.  In  visual 
transduction,  which  is  the  best  characterized  of 
these  systems,  sensitivity  is  achieved  through  a 
second  messenger  cascade  consisting  of  the 
membrane-bound  receptor  rhodopsin,  a  rod 
photoreceptor-specific  GTP-binding  protein, 
transducin,  and  a  cyclic  GMP  phosphodiesterase. 
Light-stimulated  decrease  in  the  concentration  of 
intracellular  second  messenger  leads  to  modula- 
tion of  the  cellular  membrane.  Activation  of 
adenylyl  cyclase  in  preparations  of  rat  olfactory 
cilia  depends  on  the  presence  of  guanine  nucleo- 
tides. The  observation  of  odorant-stimulated 
GTP-dependent  adenylyl  cyclase  activity  argues 
strongly  for  a  similar  GTP-binding  protein- 
coupled  signal  transductory  pathway  in  olfaction. 

We  have  identified  a  GTP-binding  protein  as 
well  as  an  adenylyl  cyclase  expressed  exclusively 
in  olfactory  sensory  neurons.  Moreover,  these 
components  are  localized  to  the  olfactory  cilia, 
where  the  initial  events  in  olfactory  signal  trans- 
duction are  thought  to  occur.  The  Go,f  protein, 
which  is  highly  homologous  to  a  GTP-binding 
protein  that  stimulates  adenylyl  cyclase  in  other 
systems,  is  expected  to  interact  directly  with  the 
olfactory  receptors.  Additionally,  we  have  demon- 
strated that  Golf  can  couple  receptor  activation  to 
increases  in  intracellular  cAMP  in  cell  lines  defi- 
cient for  the  stimulatory  G  protein,  G^. 

Identification  of  genes  encoding  olfactory  re- 
ceptors may  reveal  how  these  structures  are  able 
to  detect  thousands  of  different  odors.  Recently, 
Linda  Buck  and  Richard  Axel  (HHMI,  Columbia 
University  College  of  Physicians  and  Surgeons) 
have  described  a  gene  family  that  encodes  pro- 
teins expressed  in  olfactory  tissue.  We  have  iden- 
tified additional  members  of  this  large  family, 
utilizing  the  polymerase  chain  reaction  tech- 
nique. These  experiments  reveal  that  expression 
of  the  mRNA  that  encodes  these  putative  olfac- 
tory receptors  is  confined  to  the  sensory  neurons 
of  the  olfactory  epithelium. 


531 


Molecular  Approaches  to  Olfaction 


We  are  now  engaged  in  characterizing  these  re- 
ceptors biochemically  and  in  screening  for  the 
specific  ligands  that  activate  them.  The  subcellu- 
lar localization  of  the  receptor  proteins  is  being 
examined  by  means  of  antibodies  directed  against 
conserved  regions.  These  molecular  tools  should 
allow  us  to  address  some  important  questions. 
T)oes  each  of  the  several  million  olfactory  sensory 
cells  express  a  single  receptor  protein  species? 
How  are  the  genes  that  encode  these  receptors 
organized,  and  how  is  their  expression  regulated? 

The  final  step  in  the  transduction  of  odorant 
stimuli  is  the  generation  of  the  intracellular  sig- 
nal and  the  firing  of  an  action  potential.  Special- 
ized forms  of  second  messenger-generating  en- 
zymes and  novel  ion  channels  play  important 
roles  in  this  process.  We  have  identified  cDNA 
clones  encoding  three  distinct  forms  of  adenylyl 
cyclase  and  are  investigating  the  regulation  of 
this  important  enzyme  in  olfactory  tissue.  Olfac- 
tory neuronal  adenylyl  cyclase  type  III  has  bio- 
chemical properties  that  would  be  advantageous 
for  an  enzyme  involved  in  sensory  transduction. 
Electrophysiologic  experiments  have  identified 
cyclic  nucleotide-gated  ion  channels  in  olfac- 
tory neurons,  and  we  have  recently  isolated  and 
characterized  cDNA  clones  from  olfactory  tissue 
that  encode  ion  channels  with  properties  similar 
to  those  found  in  the  visual  system. 

The  olfactory  system  is  also  interesting  as  a 
model  for  neuron  differentiation  and  develop- 
ment. The  olfactory  neuroepithelium  is  the  only 
neuronal  tissue  in  adult  mammals  that  undergoes 
continual  regeneration.  The  lifetime  of  sensory 
neurons  is  approximately  40  days,  after  which 
they  are  shed  from  the  epithelium  and  replaced 


from  a  population  of  neuroblast-like  precursor 
cells.  Moreover,  if  the  nerve  leading  from  the  sen- 
sory neurons  to  the  olfactory  bulb  is  severed,  all 
1 0  million  receptor  cells  are  rapidly  lost  and  sub- 
sequently replaced  in  a  relatively  synchronous 
fashion.  My  laboratory  is  beginning  to  address  the 
mechanism  of  regulation  of  olfactory  neuron- 
specific  genes. 

We  have  identified  a  specific  DNA  sequence 
upstream  of  all  the  genes  known  to  be  expressed 
specifically  by  the  olfactory  neurons  and  have  ob- 
served in  olfactory  homogenates  a  binding  activ- 
ity specific  for  that  sequence.  This  putative  tran- 
scriptional regulator  might  direct  the  expression 
of  the  entire  repertoire  of  genes  involved  in  olfac- 
tory signal  transduction  and  neuronal  maturation. 

We  have  recently  identified  a  number  of  cDNA 
clones  that  encode  proteins  expressed  only  in  the 
mature  sensory  neurons  and  are  attempting  to 
elucidate  their  role  in  the  cell.  A  novel  group  of 
proteins,  those  expressed  transiently  during  neu- 
ron maturation,  may  include  receptors  for  neuro- 
genic as  well  as  neurotrophic  factors.  Several  of 
the  genes  we  have  identified  appear  to  encode 
membrane-bound  or  cell  surface  proteins. 

We  are  continuing  to  use  several  techniques  to 
elucidate  the  mechanism  of  signal  transduction. 
Likewise,  the  identification  of  proteins  asso- 
ciated with  the  replacement  of  olfactory  neurons 
provides  the  tools  to  study  neural  development, 
not  just  in  the  olfactory  system  but  also  in  other 
areas  of  the  brain.  In  the  future  we  will  focus  on 
the  molecular  components  that  underlie  the 
complex  mechanisms  of  signal  transduction,  sig- 
nal processing,  and  the  formation  of  neural 
connections. 


332 


The  Molecular  Basis  of  Hereditary  Diseases 
of  the  Kidney 


Stephen  T.  Reeders,  M.D. — Associate  Investigator 

Dr.  Reeders  is  also  Associate  Professor  of  Internal  Medicine  and  Genetics  at  Yale  University  School  of 
Medicine.  He  attended  Cambridge  University  with  the  intention  of  majoring  in  physics,  but,  realizing  that 
developments  in  molecular  biology  were  providing  the  basis  for  new  approaches  to  the  study  of  human 
disease,  he  switched  to  medicine  and  continued  to  study  at  Oxford  University.  After  qualifying  in 
medicine,  he  sought  clinical  training  in  intensive  care,  cardiology,  nephrology,  and  neurology.  Then,  with 
Sir  David  Weatherall  at  Oxford,  he  began  to  use  molecular  genetic  techniques  to  study  human  disease, 
with  emphasis  on  hereditary  diseases  of  the  kidney,  diseases  that  heretofore  had  received  little  attention 
from  geneticists. 


CRITICAL  for  normal  functioning  of  the  kid- 
ney is  the  integrity  of  the  glomerular  base- 
ment membrane  (GBM) ,  a  complex  extracellular 
structure  that  forms  one  of  the  main  barriers  be- 
tween the  blood  and  the  urine.  The  GBM  is  com- 
posed of  several  proteins,  including  five  related 
but  subtly  different  collagens  that  interact  to 
form  a  chicken-wire  mesh  holding  the  membrane 
together.  One  of  the  interests  of  our  laboratory  is 
Goodpasture  syndrome,  an  autoimmune  disorder 
in  which,  for  unknown  reasons,  autoantibodies 
are  suddenly  targeted  at  the  collagen  components 
of  basement  membrane  in  the  lungs  and  kidneys. 
In  the  kidney,  these  autoantibodies  produce  a 
sudden  and  devastating  severe  inflammation, 
which  frequently  leads  to  acute  renal  failure,  irre- 
versible unless  treated.  The  nephritis  is  often  ac- 
companied by  autoimmune  lung  damage,  mani- 
fested by  bleeding  into  the  alveoli. 

Previous  studies  have  shown  that  the  probable 
target  of  Goodpasture  autoantibodies  is  the  a  3 
chain  of  basement  membrane  collagen.  To  under- 
stand the  pathogenesis  more  clearly,  we  under- 
took to  isolate  and  purify  the  collagen  chain  to 
study  its  structure.  Because  the  protein  is  present 
in  very  small  amounts  and  is  accompanied  by  four 
similar  proteins,  purifying  it  has  proved  difficult. 
We  have  therefore  isolated,  cloned,  and  se- 
quenced the  gene  for  the  a3  chain  of  basement 
membrane  collagen  and  have  used  the  sequence 
information  to  predict  the  primary  structure  and 
compare  this  protein  with  other  basement  mem- 
brane collagens. 

In  collaboration  with  Billy  Hudson  (Kansas 
City),  we  used  knowledge  of  the  primary  struc- 
ture to  identify  several  potential  antibody- 
binding  sites  (epitopes)  in  the  a3  molecule.  We 
synthesized  short  peptides  and  used  them  to  test 
the  binding  of  some  of  these  sites,  which  we  local- 
ized to  within  a  small  region  at  the  carboxyl  ter- 
minus. At  least  one  of  the  peptides  has  very  high 
affinity  for  Goodpasture  antibodies  and  adsorbs 
them  from  patients'  serum.  Knowledge  of  the 
epitope  structure  should  enable  us  to  develop  a 


means  of  selectively  adsorbing  Goodpasture  anti- 
bodies, opening  possibilities  for  a  new  treatment 
modality.  In  addition,  this  information  may  pro- 
vide clues  to  the  development  of  autoimmunity 
in  this  disorder. 

Mariko  Mariyama,  a  Howard  Hughes  associate 
in  our  laboratory,  has  recently  isolated  a  novel 
basement  membrane  collagen,  a4.  The  compari- 
son of  the  structure  of  this  molecule  with  the 
known  basement  membrane  collagen  allows  the 
evolution  of  these  molecules  to  be  inferred.  Two 
classes  of  the  molecule  exist  in  all  species  from 
roundworm  to  humans,  suggesting  a  functional 
divergence.  The  expression  of  a3  and  a4  occurs 
in  a  limited  number  of  tissues,  and  they  are  re- 
gionally localized  within  these  tissues.  In  kidney, 
for  example,  they  are  found  in  the  glomerulus 
but  not  in  the  tubular  or  vascular  endothelial 
basement  membranes.  Our  gene  mapping  data 
suggest  that  the  expression  of  a3  and  aA  is  coordi- 
nated by  transcription  of  the  genes  from  opposite 
DNA  strands.  A  similar  arrangement  has  been 
found  for  the  al  and  a2  genes,  homologues  of  al 
and  a2. 

One  of  the  major  projects  in  our  laboratory  is  a 
study  of  the  molecular  and  cellular  pathology  of 
autosomal  dominant  polycystic  kidney  disease 
(ADPKD) ,  one  of  the  commonest  causes  of  kid- 
ney failure  in  humans,  affecting  at  least  1  in 
1,000  of  the  population.  The  disease  is  an  enor- 
mous burden  to  families  and  the  community, 
since  the  majority  of  patients  develop  irreversible 
kidney  failure  in  middle  life  and  require  dialysis 
or  transplantation  for  survival. 

Having  previously  ascertained  that  the  majority 
of  the  inherited  mutations  in  ADPKD  lie  close  to 
the  tip  of  the  short  arm  of  chromosome  16,  we 
have  isolated  and  cloned  a  small  segment  of  DNA 
(550,000  base  pairs)  that  includes  the  mutated 
gene.  This  region  turns  out  to  be  extremely  gene 
rich,  and  we  have  already  isolated  22  genes  from 
within  it.  These  include  novel  cyclin  A-like  and 
r«s-like  genes,  a  gene  encoding  a  zinc  finger  pro- 
tein, and  a  gene  having  homology  to  the  (8- 


335 


The  Molecular  Basis  of  Hereditary  Diseases  of  the  Kidney 


subunit  of  the  G  protein  family.  Since  we  have 
not  been  able  to  detect  any  large-scale  deletions 
or  rearrangements  affecting  any  of  the  22,  we 
have  begun  to  examine  the  sequence  of  these 
genes  in  detail  and  to  look  for  mutations  that  may 
affect  only  one  or  two  nucleotides.  This  work  is 
supported  by  a  grant  from  the  National  Institutes 
of  Health. 

Our  laboratory  has  been  interested  in  the  struc- 
ture of  telomeres,  the  ends  of  chromosomes.  We 
have  shown  that  banks  of  repetitive  sequence 
reminiscent  of  the  sequence  of  human  telomeres 


(TTAGGG)  are  also  present  at  other  sites  within 
the  human  genome.  One  of  the  most  interesting 
arrays  of  telomere-like  sequence  is  embedded  in 
the  middle  of  the  long  arm  of  chromosome  2. 
Comparison  of  the  chromosome  banding  pattern 
of  humans  with  that  of  several  closely  related 
apes  suggests  that  this  region  of  the  chromosome 
contains  a  point  at  which  two  ancestral  ape  chro- 
mosomes fused.  Jaap  IJdo,  a  Howard  Hughes  asso- 
ciate, has  cloned  this  fusion  point  and  shown  that 
it  consists  of  a  head-to-head  telomere-telomere 
fusion. 

In  situ  hybridization  of  a  cloned  DNA  segment 
containing  subtelomeric  sequences  to  a  human 
chromosome  2  (left).  The  fluorescent  probe  rec- 
ognizes the  sequences  at  the  tips  of  several 
chromosomes,  including  those  of  the  long  arm 
of  chromosome  2  shown  here.  In  addition,  the 
probe  recognizes  another  sequence  (left 
arrow)  that  was  buried  in  the  middle  of  chro- 
mosome 2  when  chromosomes  of  ancestral 
apes  fused  to  create  the  human  chromosome. 
(To  this  day  the  great  apes  have  one  more  chro- 
mosome pair  than  humans.)  The  point  of  fu- 
sion is  very  close  to  a  rare  fragile  site  that  was 
observed  in  the  chromosome  2  used  in  this 
study.  The  chromosome  on  the  right  is  shown 
with  the  hybridization  signal  removed  so  that 
the  fragile  site,  which  appears  as  a  gap  ( right 
arrow ),  can  be  discerned. 

From  If  do,  f.W.,  Baldini,  A.,  Wells,  R.A., 
Ward,  D.C.,  and  Reeders,  S.T.  1992.  Genomics 
12:835-835. 


354 


Extracellular  Factors  Affecting 
Neuron  Development 


Louis  F.  Reichardt,  Ph.D. — Investigator 

Dr.  Reichardt  is  also  Professor  of  Physiology  and  of  Biochemistry  and  Biophysics  at  the  University  of 
California,  San  Francisco.  He  received  his  Ph.D.  degree  in  biochemistry  from  Stanford  University  for  work 
on  control  of  gene  expression  by  a  bacterial  virus,  the  bacteriophage  X.  Dr.  Reichardt  entered  the  field  of 
neurobiology  as  a  postdoctoral  fellow  in  Paul  Patterson 's  laboratory  at  Harvard  University,  where  he 
studied  factors  that  regulate  the  transmitter  phenotype  of  individual  neurons.  Among  his  honors  are  a 
McKnight  Scholars  Award,  a  Sloan  Award,  and  a  Guggenheim  Fellowship. 


MY  laboratory  is  investigating  molecules  in 
the  extracellular  environment  of  neurons 
(conducting  nerve  cells)  that  direct  their  devel- 
opment in  vivo.  These  include  trophic  (nutri- 
tive) factors,  exemplified  by  nerve  growth  factor 
(NGF),  and  molecules  in  the  extracellular  matrix 
or  on  the  surface  of  cells  that  serve  as  substrates 
for  the  growth  of  axons  (long  nerve  fibers) .  Such 
molecules  help  to  regulate  neuronal  survival, 
axon  growth,  and  synapse  (nerve  junction) 
formation. 

Neurons  require  contact  with  targets  to  survive 
during  development.  Experimentally  increasing 
or  decreasing  the  volume  of  target  tissue  corre- 
spondingly increases  or  decreases  neuronal  sur- 
vival. To  explain  these  target  influences,  it  has 
been  proposed  that  target  organs  synthesize  tro- 
phic factors  required  for  the  survival  of  the  inner- 
vating neurons.  Defects  in  the  synthesis  of  these 
factors  or  in  the  neuron's  ability  to  respond  to 
them  may  explain  some  neurodegenerative 
disorders. 

The  key  to  understanding  trophic  factors  is  to 
identify  and  understand  the  actions  of  their  re- 
ceptors. One  of  the  proteins  that  functions  as  a 
receptor  for  the  family  of  NGF-related  trophic 
factors  is  called  the  low-affinity  NGF  receptor. 
Antibodies  to  this  protein  have  been  prepared 
and  used  to  show  that  it  is  distinct  from  a  second 
protein  that  constitutes  a  distinct  high-affinity  re- 
ceptor for  NGF.  Work  in  the  laboratories  of  David 
Kaplan,  Mariano  Barbacid,  and  their  colleagues 
has  recently  shown  that  this  second  protein  is  the 
trk  proto-oncogene  product,  an  NGF-tyrosine  ki- 
nase. The  same  antibodies  have  been  used  to 
show  that  NGF-dependent  survival  and  axon 
growth  by  response  neurons  do  not  require  the 
low-affinity  receptor.  These  antibodies  are  being 
used  to  examine  the  role  of  this  class  of  receptor 
in  mediating  other  responses  to  NGF. 

Our  laboratory  has  devoted  considerable  effort 
to  identifying  molecules  that  promote  the  groMT:h 
of  neuronal  processes.  We  have  tried  to  identify 
both  the  molecules  that  axons  recognize  in  their 
environment  and  the  receptors  that  neurons  use 
for  binding  to  these  molecules.  Our  results  have 


shown  that  laminin,  an  adhesive  protein,  is  by  far 
the  most  active  of  the  glycoproteins  that  cells  se- 
crete into  the  extracellular  matrix.  Other  pro- 
teins that  have  similar  activities  include  fibronec- 
tin,  thrombospondin,  and  vitronectin. 

We  have  also  shown  that  neurons  utilize  a  fam- 
ily of  receptors,  the  integrins,  to  bind  to  laminin 
and  other  matrix  glycoproteins.  Distinct  recep- 
tors appear  to  mediate  neuronal  adhesion  and 
growth  cone  motility  on  laminin,  fibronectin, 
and  collagen.  In  the  past  two  years  we  have  puri- 
fied several  of  these  proteins  and  isolated  clones 
encoding  subunits  of  the  receptors.  Specific  anti- 
bodies to  these  subunits  have  been  prepared  and 
are  being  used  to  study  their  regulation .  Of  partic- 
ular interest,  we  have  identified  the  receptors 
used  by  both  peripheral  neurons  and  retinal  neu- 
rons to  interact  with  laminin,  a  heterotrimer  as- 
sembled from  three  different  gene  products — A, 
Bl,  and  B2.  Josh  Sanes  and  Eva  Engvall  have 
shown  that  at  least  two  different  A-like  genes  and 
two  different  Bl  -like  genes  exist  and  are  differen- 
tially expressed,  making  it  possible  to  assemble 
four  different  isoforms  of  laminin,  which  are  dif- 
ferentially distributed  in  embryos.  We  have 
shown  that  individual  integrin  heterodimers  dis- 
tinguish between  these  isoforms,  making  it  possi- 
ble for  cells  to  exhibit  different  responses  to  indi- 
vidual isoforms.  Work  in  the  past  year  has  also 
identified  candidate  receptors  for  thrombospon- 
din and  tenascin. 

Evidence  suggests  that  the  activity  of  integrin 
receptors  may  modulate  the  behavior  of  axons  in 
vivo.  In  studies  on  regulation  of  a^jSi,  the  major 
laminin  receptor  in  the  neuroretina,  we  have 
shown  that  receptor  function  can  be  regulated  on 
several  levels.  First,  expression  of  the  genes  en- 
coding the  two  subunits  is  regulated.  Retinal  gan- 
glion cells  lose  responsiveness  to  laminin  when 
they  contact  their  synaptic  partners  in  the  optic 
tectum,  and  this  reflects  down-regulation  of  ex- 
pression of  the  gene.  Second,  other  neurons  in 
the  retina  modulate  responses  to  laminin  by  ex- 
hibiting changes  in  the  activity  of  integrin  recep- 
tors on  their  surfaces.  These  changes  occur  rap- 
idly and  can  be  modulated  by  external  and 
internal  agents.  Yet  a  third  level  of  regulation  ap- 


335 


Extracellular  Factors  Affecting  Neuron  Development 


pears  to  modulate  the  signals  conveyed  by  inte- 
grin  receptor  binding  to  laminin  and  other  pro- 
teins. These  signals  can  be  modulated  by  trophic 
factors,  such  as  NGF. 

In  addition  to  proteins  secreted  into  the  extra- 
cellular environment  of  cells,  neurons  also  recog- 
nize and  use  for  axon  extension  integral  mem- 
brane proteins  on  the  surfaces  of  different  cell 
types.  Recent  work  in  our  laboratory  has  identi- 
fied some  of  the  major  proteins  that  neurons  use 
for  axonal  growth  in  the  central  and  peripheral 
nervous  systems.  Thus  neurons  have  been  shown 
to  use  both  extracellular  matrix  and  cell-cell  ad- 
hesion molecules  for  growth  of  axons  on 
Schwann  cells,  the  major  cell  type  with  which 
they  interact  in  peripheral  nerves.  A  similar  com- 
bination of  adhesive  interactions  promotes 
growth  of  retinal  axons  on  astroglia.  The  move- 
ments of  growth  cones  of  retinal  ganglion  cells 
have  been  directly  monitored  in  vivo  by  time- 
lapse  microscopy.  Injection  of  antibodies  to  indi- 
vidual cell  adhesion  molecules  has  been  shown 
to  alter  dramatically  the  behavior  of  these  growth 
cones. 

In  studies  on  interactions  of  neurons  with  other 


cells  in  the  brain,  it  became  clear  that  additional 
adhesion  molecules,  not  yet  identified  or  charac- 
terized, must  be  important.  We  have  used  molec- 
ular biological  methods  to  identify  two  of  these 
with  localized  expression  patterns  in  the  brain. 
One  of  these,  the  integrin  ag/^n  is  localized  on 
axons  in  many  of  the  major  axon  tracts  of  the 
brain.  Its  concentration  on  axons  in  these  tracts 
suggests  that  it  mediates  interactions  between 
these  axons.  A  potential  ligand  for  this  integrin 
that  is  also  localized  in  these  tracts  has  been  iden- 
tified. The  second  novel  adhesion-promoting 
molecule  is  B-cadherin,  a  Ca^'^-dependent  adhe- 
sion molecule  that  is  also  expressed  in  the  ner- 
vous system.  It  appears  to  be  concentrated  in  spe- 
cific cells  in  several  areas  of  the  brain.  Some  but 
not  all  neurons  are  able  to  interact  with  it.  Both 
molecules  are  also  expressed  in  some  areas  out- 
side the  nervous  system. 

Future  work  will  focus  on  determining  how 
these  neuronal  receptors  act  to  promote  growth 
cone  motility,  how  they  are  regulated  during  de- 
velopment, and  what  their  importance  is  in  regu- 
lating the  grov^T:h  of  axons  during  development 
and  regeneration. 


356 


Molecular  Genetics  of  RNA  Processing 
and  Behavior 


Michael  Rosbash,  Ph.D. — Investigator 

Dr.  Rosbash  is  also  Professor  of  Biology  at  Brandeis  University  and  Adjunct  Professor  of  Molecular  Biology 
at  Massachusetts  General  Hospital,  Boston.  He  received  his  Ph.D.  degree  in  biophysics  from  the 
Massachusetts  Institute  of  Technology  and  was  a  postdoctoral  fellow  at  the  University  of  Edinburgh, 
where  he  studied  with  J.  O.  Bishop.  Dr.  Rosbash  was  a  Guggenheim  Fellow  in  Paris,  France. 


MY  laboratory  is  interested  in  two  fundamen- 
tal problems.  Our  earliest  and  foremost  in- 
terest is  the  molecular  genetics  of  RNA  process- 
ing. For  this  subject  our  principal  experimental 
system  is  the  budding  yeast  Saccharomyces  cere- 
visiae,  which  is  amenable  to  both  genetic  and 
biochemical  attack.  Our  more  recent  interest  is 
the  molecular  genetics  of  behavior — in  particu- 
lar, circadian  rhythms.  This  problem  is  addressed 
in  the  fruit  fly  Drosophila  melanogaster,  be- 
cause the  organism  is  amenable  to  behavioral  as 
well  as  biochemical  and  genetic  approaches. 

Within  the  area  of  RNA  processing,  we  are  most 
interested  in  understanding  certain  aspects  of 
pre-messenger  RNA  splicing,  the  process  by 
which  the  undesirable  sections  of  a  pre-mRNA 
molecule  are  removed  and  the  remaining 
"sense"  sections  sewn  together.  Our  interests  are 
primarily  focused  on  the  more  biological  aspects 
of  the  problem.  These  include  how  the  places  in 
the  molecule  to  be  cut — the  two  splice  sites  in 
the  case  of  a  pre-mRNA  with  a  single  intron — are 
defined.  They  also  include  how  splice  site 
partners,  in  the  case  of  a  pre-mRNA  with  multiple 
introns,  are  specified. 

The  latter  question  is  particularly  puzzling,  be- 
cause most  of  these  splice  sites  appear  similar. 
Yet  there  is  clearly  an  order  to  the  process,  al- 
though the  basis  for  this  order  is  not  apparent. 
The  adjective  "biological"  is  used  to  distinguish 
these  issues  from  more  "chemical"  consider- 
ations, such  as  how  the  active  site  of  the  splicing 
enzyme  is  formed  and  how  the  efficiency  and 
specificity  of  the  splicing  reaction  are  dictated. 

We  are  addressing  these  issues  of  splice  site 
definition  and  partner  assignment  by  examining 
the  interactions  between  a  pre-mRNA  substrate 
and  splicing  factors.  Although  some  of  the  work  is 
done  in  vivo — that  is,  in  intact  cells  where  the 
interactions  are  inferred  from  their  conse- 
quences— most  of  our  efforts  have  concentrated 
on  interactions  that  take  place  during  in  vitro 
pre-mRNA  splicing  in  a  whole-cell  yeast  extract. 
We  have  focused  particularly  on  the  earliest  in- 
teractions, those  that  apparently  reflect  recogni- 
tion of  the  pre-mRNA  substrate  by  the  splicing 
machinery. 


Our  studies  indicate  that  the  factor  Ul  small 
nuclear  ribonucleoprotein  (snRNP)  plays  a  prom- 
inent role  in  these  early  interactions.  Conse- 
quently we  have  expended  considerable  effort  in 
characterizing  this  snRNP  and  its  constituents,  as 
well  as  the  pre-mRNA-Ul  snRNP  interaction.  Sur- 
prisingly, both  ends  of  the  intron  interact  with  Ul 
snRNP,  suggesting  that  certain  aspects  of  splice 
site  recognition,  if  not  splice  site  partner  assign- 
ment, are  already  defined  early  during  the 
spliceosome  assembly  process,  well  before  the 
cleavage  and  ligation  steps  of  the  actual  splicing 
process  take  place.  This  work  is  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

We  are  also  interested  in  another  biological 
aspect  of  RNA  processing — namely,  how  mRNA 
is  transported  from  the  nucleus,  where  it  is  syn- 
thesized, to  the  cytoplasm,  where  it  is  translated 
into  protein.  This  transport  problem  interfaces 
with  the  splicing  process,  since  RNA  needs  to  be 
transported  to  the  cytoplasm  but  usually  not  be- 
fore the  splicing  is  completed.  Otherwise,  incom- 
pletely spliced  molecules  would  be  prematurely 
transported,  which  would  give  rise  to  untranslat- 
able pre-mRNAs  in  the  cytoplasm. 

The  problem  of  RNA  transport  is  poorly  under- 
stood, and  even  less  well  understood  in  yeast  than 
in  mammalian  cells.  In  yeast,  however,  there  is 
the  possibility  of  addressing  the  problem  with  ge- 
netic tools.  At  present  we  are  localizing  pre- 
mRNA  and  splicing  factors  within  the  yeast  nu- 
cleus, in  an  attempt  to  define  a  cytological  path 
that  the  RNA  may  follow  in  leaving  the  nucleus. 
The  goal  is  to  use  existing  temperature-sensitive 
mutants  and  to  uncover  new  ones,  both  to  study 
RNA  transport  and  to  define  some  of  the  gene 
products  important  for  this  process. 

Rhythms 

Our  goals  in  this  project  are  to  define  the  bio- 
chemical machinery  that  underlies  the  mysteri- 
ous yet  ubiquitous  process  of  circadian  rhythmic- 
ity.  We  are  using  genetics  and  biochemistry  to 
define  candidate  genes  and  gene  products  that 
may  participate  in  fundamental  aspects  of  these 
rhythms.  Our  entree  into  the  process  is  the  pe- 
riod gene  (per)  of  Drosophila  melanogaster. 


337 


Molecular  Genetics  of  RNA  Processing  and  Behavior 


Mutants  in  this  gene,  originally  identified  more 
than  20  years  ago,  have  profound  effects  on  circa- 
dian  rhythms  of  locomotor  activity  and  of  eclo- 
sion  (emergence  of  adults  from  the  pupal  case) . 

Recently  we  discovered  that  the  per  gene  prod- 
ucts, mRNA  as  well  as  protein,  undergo  circadian 
fluctuations  in  level  during  the  circadian  cycle. 
These  observations  and  others  indicated  that  there 
is  a  feedback  loop  in  which  the  per  protein  nega- 
tively affects  the  level  of  its  own  mRNA.  Since  the 
per  mRNA  also  gives  rise  to  the  per  protein  during 
translation,  this  putative  feedback  loop  contains 
all  of  the  elements  required  to  define  a  circadian 
clock,  including  a  substantial  (and  mysterious) 
delay  between  the  mRNA  and  protein  accumula- 
tion curves.  We  are  studying  several  aspects  of  this 
loop  in  an  attempt  to  confirm  (or  refute)  its  im- 
portance to  the  circadian  clock. 


Because  no  close  relatives  of  the  per  protein 
with  a  known  biochemical  function  are  found  in 
the  database,  we  cannot  say  for  certain  what  gene 
family  it  belongs  to  or  what  biochemical  function 
it  serves.  We  suspect,  however,  that  it  is  a  tran- 
scription factor,  or  serves  to  modulate  transcrip- 
tion, and  that  its  effect  on  its  own  mRNA  levels  is 
quite  direct.  A  current  goal  is  to  test  this  hypothe- 
sis and  to  define  the  biochemical  function  of  the 
per  gene  product,  especially  insofar  as  rhythms 
are  concerned. 

With  the  support  of  a  grant  from  the  National 
Institutes  of  Health,  we  are  also  in  the  process  of 
defining  and  studying  several  other  genes  that  af- 
fect rhythms.  Although  these  genes  are  in  a  less 
well  developed  state  of  examination  than  the  per 
gene,  some  appear  interesting  and  may  provide 
additional  insight  into  clock  mechanisms. 


338 


Molecular  Mechanisms  of  Transcription, 
Regulation,  and  Development 
of  the  Neuroendocrine  System 


Michael  G.  Rosenfeld,  M.D. — Investigator 

Dr.  Rosenfeld  is  also  Professor  of  Medicine  in  the  Eukaryotic  Regulatory  Biology  Program  at  the  University 
of  California,  San  Diego,  School  of  Medicine.  He  received  his  undergraduate  degree  from  the  Johns  Hopkins 
University  and  his  medical  degree  from  the  University  of  Rochester.  His  internship  and  medical  residency 
were  completed  at  Barnes  Hospital,  St.  Louis.  Before  accepting  his  current  position,  he  received 
postdoctoral  training  at  NIH.  Dr.  Rosenfeld  also  holds  an  adjunct  position  at  the  Salk  Institute. 


OVER  the  past  year  our  central  research  focus 
has  been  the  determination  of  molecular 
mechanisms  that  induce  specific  neuroendocrine 
phenotypes  and  the  further  definition  of  signal 
transduction  pathways  that  lead  to  regulated  pat- 
terns of  gene  expression. 

The  neuroendocrine  system  coordinates  the 
complex  pattern  of  regulation  necessary  to 
achieve  the  precise  temporal,  spatial,  and  homeo- 
static  patterns  of  gene  expression  required  by 
complex  organisms.  Development  of  the  central 
nervous  system  and  endocrine  organs  involves 
precise  patterns  of  responses  to  morphogens  and 
other  regulatory  signals  that  ultimately  establish 
the  intricate  patterns  of  neural  and  endocrine 
phenotypes.  The  cloning  and  analysis  of  genes 
encoding  receptors  and  cell-specific  transcrip- 
tion factors  have  permitted  an  initial  definition  of 
developmental  and  regulatory  strategies. 

The  anterior  pituitary  gland  has  provided  a  suit- 
able model  to  investigate  the  molecular  basis  for 
generating  specific  cell  phenotypes  in  an  organ. 
The  rat  genes  for  growth  hormone  and  prolactin 
(hormone  that  stimulates  and  sustains  lactation) 
exhibit  precisely  restricted  expression  in  the 
cells  of  origin,  somatotrophs  and  lactotrophs,  re- 
spectively. We  found  that  prolactin  gene  expres- 
sion is  dictated  by  a  distal  enhancer  and  a  proxi- 
mal region,  each  containing  at  least  four  critical 
cell-specific  elements.  These  two  domains,  each 
capable  of  targeting  tissue-specific  gene  expres- 
sion, act  synergistically  to  generate  high  levels  of 
prolactin  gene  expression  in  transgenic  mice. 
Similarly,  grov^h  hormone  gene  expression  in- 
volves the  action  of  related  cell-specific  cis- 
active  elements.  A  33-kDa  cell-specific  transcrip- 
tion factor,  referred  to  as  Pit- 1 ,  was  characterized 
and  its  encoding  cDNA  defined. 

Bacterially  expressed  Pit-1  specifically  and 
with  high  affinity  binds  to  prolactin  and  growth 
hormone  promoters.  Additional  elements  and 
factors  are  required  to  achieve  the  full  physiologi- 
cal levels  and  restricted  patterns  of  expression  of 
the  prolactin  and  growth  hormone  genes.  For  ex- 
ample, the  estrogen  receptor  synergistically  acts 
with  Pit- 1  in  activation  of  the  rat  prolactin  gene's 
distal  enhancer. 


Pit-1  is  a  member  of  a  family  of  regulators  that 
contain  a  POU  domain  consisting  of  a  variant  ho- 
meodomain  and  a  second,  76-amino  acid  se- 
quence of  striking  homology.  We  have  found  that 
the  76-amino  acid  POU-specific  domain  func- 
tions in  high-affinity  DNA  binding,  in  conferring 
site-specificity,  and  in  protein-protein  interac- 
tions critical  for  transcriptional  activation  by 
Pit-1.  The  major  transcription-activating  domain 
of  Pit-1  is  a  70-amino  acid,  N'-terminal,  serine, 
threonine-rich  sequence,  distinct  from  recog- 
nized motifs.  A  detailed  structure-function  analy- 
sis has  suggested  that  the  determinants  of  DNA 
binding  by  the  Pit-1  POU-domain  protein  are  dis- 
tinct from  those  of  the  classical  homeodomain 
proteins,  and  that  Pit- 1  DNA  binding  is  regulated 
by  phosphorylation  of  a  specific,  conserved  resi- 
due in  the  POU  homeodomain. 

A  genetic  approach  was  utilized  to  determine 
the  functional  role  of  Pit-1  during  organogenesis. 
Based  on  the  demonstration  of  disrupted  patterns 
of  the  Pit-1  gene  in  genetic  dwarf  mice,  it  has 
been  established  that  this  POU-domain  protein 
acts  as  a  developmental  regulator  to  determine 
patterns  of  commitment,  progression,  and  prolif- 
eration of  three  specific  cell  types  in  the  anterior 
pituitary  gland.  In  the  case  of  lactotrophs  and  so- 
matotrophs, proliferation  is  restricted  in  the 
Pit- 1 -defective  animal  cell  type,  while  the  thyro- 
troph provides  an  initial  example  of  an  estab- 
lished cell  phenotype  that  disappears  at  the  time 
the  initial  Pit-1  protein  is  expressed.  These  data 
indicate  that  one  role  of  a  developmental  tran- 
scriptional regulator  can  be  survival  of  an  estab- 
lished cell  type. 

A  strategy  was  devised  to  isolate  new  members 
of  the  POU-domain  gene  family.  Ten  new 
members  were  identified  in  neural  tissues.  Two, 
referred  to  as  Brn-1  and  Brn-2,  exhibit  virtually 
identical  patterns  of  expression  in  the  central 
nervous  system,  though  Brn-1  is  clearly  ex- 
pressed in  the  medullary  zone  of  the  kidney 
while  Brn-2  is  not.  A  third  member,  Brn-3,  is  pre- 
dominantly expressed  in  the  peripheral  nervous 
system.  Tst  l  transcripts  are  present  in  mamma- 
lian brain  cells  and  in  testis.  Subsequently  other 


339 


Molecular  Mechanisms  of  Transcription,  Regulation,  and  Development 
of  the  Neuroendocrine  System 


POU  domains  have  been  identified,  each  ex- 
pressed in  a  unique  pattern  early  in  development 
and  during  organogenesis. 

Most  POU-domain  genes  were  widely  ex- 
pressed in  all  levels  of  the  neural  tube  (including 
the  retina)  during  early  development,  and  hybrid- 
ization in  the  ventricular  (proliferative)  zone  of 
the  neuroepithelium  is  evident  for  all  four  tran- 
scripts at  all  levels.  The  time  course  of  anatomic 
restriction  in  the  developing  neural  tube  is 
distinct,  and  the  patterns  for  each  gene  product 
tend  to  reflect  the  adult  loci  of  expression.  In 
addition,  reactive  transcripts  for  some  of  these 
genes  are  also  expressed  during  mammalian 
neurogenesis. 

We  have  recently  identified  many  additional 
family  members  in  the  brain  and  have  obtained 
initial  evidence  that  at  least  some  members  of  the 
family  can  bind  to  specific  elements  in  distinct 
classes  of  neuronally  expressed  genes.  We  find 
that  neurally  expressed  POU-domain  proteins 
can  be  considered  to  represent  distinct  related 
families,  each  of  which  binds  to  related,  but  dis- 
tinct, DNA  response  elements.  Putative  target 
genes  have  been  identified  for  several  of  these 
factors,  and  analysis  of  their  function  has  revealed 
both  inhibitory  and  stimulatory  domains. 

Interactions  between  diverse  transcription  fac- 
tors generate  heterodimers  that  exert  distinct  pat- 
terns of  gene  activation.  Thus,  in  the  case  of  the 
retinoic  acid  receptor,  we  find  that  a  series  of  cell 
type-specific  coregulators  impose  novel  hierar- 
chies of  binding  site  preferences.  Unique  posi- 


tive and  negative  transcriptional  regulators 
impose  variable  patterns  of  gene  activation,  po- 
tentially contributing  to  the  refinement  of  pheno- 
typic  variance  required  in  the  central  nervous 
system.  One  class  of  coregulators  has  been  char- 
acterized by  expression  cloning  techniques. 

Development  of  the  neuroendocrine  system  is 
also  initially  regulated  by  post-transcriptional 
strategies.  We  have  provided  evidence  that  a  gene 
of  the  neuroendocrine  system,  the  calcitonin/ 
CGRP  (calcitonin  gene-related  peptide)  gene, 
contains  genomic  regions  that  represent  discrete 
hormone-encoding  domains.  The  ultimate  ex- 
pression of  these  domains  is  dependent  on  alter- 
native RNA-processing  events  that  differentially 
include  or  exclude  specific  exons  encoding  cer- 
tain components  of  polypeptide  regulators  in  the 
mature  mRNA  products.  The  rat  and  human  calci- 
tonin/CGRP  genes  contain  six  exons.  More  than 
95  percent  of  the  mature  transcripts  in  thyroid  C 
cells,  encoding  calcitonin,  are  produced  by 
splicing  of  the  first  three  to  four  exons.  CGRP 
mRNA  is  the  only  detectable  mature  transcript  in 
rat  neuronal  tissue,  which  appears  to  reflect  the 
actions  of  a  specific  regulatory  machinery  con- 
trolling post-transcriptional  RNA  splice  acceptor 
choice.  CGRP  appears  to  be  an  important  regula- 
tor of  blood  pressure. 

Current  investigations  continue  to  explore 
these  novel  aspects  of  transcriptional  and 
post-transcriptional  regulatory  strategies  in  neu- 
roendocrine gene  expression.  Results  may  pro- 
vide new  ways  of  studying  the  problem  of 
organogenesis. 


340 


Development  of  the  Drosophila  Visual  System 


Gerald  M.  Rubin,  Ph.D. — Investigator 

Dr.  Rubin  is  also  John  D.  MacArthur  Professor  of  Genetics  at  the  University  of  California,  Berkeley,  and 
Adjunct  Professor  of  Biochemistry  and  Biophysics  at  the  University  of  California,  San  Francisco,  School 
of  Medicine.  He  received  his  B.S.  degree  in  biology  from  the  Massachusetts  Institute  of  Technology  and  his 
Ph.D.  degree  in  molecular  biology  from  the  University  of  Cambridge.  Dr.  Rubin's  postdoctoral  work  was 
done  at  Stanford  University  with  David  Hogness.  He  has  held  faculty  positions  at  Harvard  Medical  School 
and  the  Carnegie  Institution  of  Washington.  Dr.  Rubin  is  a  member  of  the  National  Academy  of  Sciences 
and  counts  among  his  other  honors  the  American  Chemical  Society  Eli  lilly  Award  in  Biological  Chemistry. 


RESEARCH  in  our  laboratory  is  directed  to- 
ward studies  of  differentiation  and  gene  regu- 
lation in  the  developing  nervous  system.  Our  ex- 
perimental approach  involves  studying  genes 
whose  mutations  disrupt  neural  development. 
During  the  past  year,  we  have  focused  our  work 
on  several  genes  important  for  the  determination 
of  cell  fates  in  the  developing  retina  of  the  fruit 
fly  Drosophila. 

Two  very  different  but  not  exclusive  mecha- 
nisms can  account  for  the  selection  of  distinct 
developmental  pathways.  First,  cells  may  be  pro- 
grammed in  a  lineage-dependent  manner  by  the 
asymmetric  partitioning  of  determinants  during 
cell  division.  Different  developmental  pathways 
are  then  selected  in  the  daughter  cells  in  re- 
sponse to  the  different  localized  determinants.  Al- 
ternatively, cellular  differentiation  may  occur  in 
a  lineage-independent  manner,  where  the  posi- 
tion that  a  cell  occupies  in  a  developing  field 
determines  its  fate.  In  this  case,  diffusible  sub- 
stances, such  as  hormones,  or  interactions  with 
adjacent  cells  are  the  primary  determinants  of 
cellular  differentiation.  Although  the  mecha- 
nisms used  to  read  and  interpret  such  positional 
information  are  largely  unknown,  short-range 
cellular  interactions  are  thought  to  be  of  princi- 
pal importance  in  a  wide  variety  of  developmen- 
tal phenomena. 

The  compound  eye  of  Drosophila  melanogas- 
ter  is  an  attractive  system  to  study  the  mecha- 
nisms underlying  lineage-independent  develop- 
mental decisions,  since  it  consists  of  a  small 
number  of  different  cell  types  that  develop  in  a 
lineage-independent  manner.  The  compound  eye 
is  a  two-dimensional  array  of  800  repeating  units, 
or  ommatidia.  Each  ommatidium  contains  8  pho- 
toreceptor cells  as  well  as  1 2  nonneuronal  acces- 
sory cells.  Each  photoreceptor  cell  has  a  distinct 
cellular  identity,  based  on  both  its  position 
within  the  ommatidium  and  its  projection  pattern 
to  the  optic  lobes  of  the  brain.  The  stereotyped 
arrangement  of  this  small  number  of  nerve  cells, 
together  with  the  dispensability  of  the  visual  sys- 
tem under  laboratory  conditions,  makes  the  com- 


pound eye  an  attractive  model  system  to  study 
genes  involved  in  the  specification  of  nerve  cells. 

Assembly  of  ommatidia  begins  in  an  initially 
unpatterned  monolayer  of  epithelial  cells,  the 
eye  imaginal  disc.  Ommatidial  assembly  does  not 
occur  synchronously  throughout  the  disc  but  in- 
stead begins  at  the  posterior  edge  and  progresses 
anteriorly.  Eye  discs  removed  from  larvae  just 
prior  to  pupariation  show  a  smoothly  graded  se- 
ries of  ommatidia  at  different  stages  of  develop- 
ment, covering  just  over  half  of  the  disc.  Examina- 
tion of  individual  cells  in  the  forming  ommatidia 
has  shown  that  the  photoreceptors  differentiate 
in  a  fixed  sequence,  beginning  with  the  central 
R8  photoreceptor  and  proceeding  pairwise  with 
R2  and  R5,  R3  and  R4,  Rl  and  R6,  and  finally  R7. 

The  fate  of  a  cell  within  a  developing  ommatid- 
ium appears  to  be  governed  by  the  specific  com- 
bination of  signals  received  by  that  cell  from  its 
immediate  neighbors.  We  would  like  to  under- 
stand how  such  signals  are  generated,  received, 
and  interpreted.  Our  approach  has  been  to  study 
mutations  that  specifically  disrupt  these  pro- 
cesses, as  illustrated  by  our  studies  of  the  seven- 
less  gene. 

The  sevenless  gene  is  essential  for  the  develop- 
ment of  a  single  type  of  photoreceptor  cell.  In  the 
absence  of  proper  sevenless  function,  the  cells 
that  would  normally  become  the  R7  photorecep- 
tors become  instead  nonneuronal  cells.  Previous 
morphological  and  genetic  analysis  has  indicated 
that  the  product  of  the  sevenless  gene  is  involved 
in  reading  or  interpreting  the  positional  informa- 
tion that  specifies  this  particular  developmental 
pathway.  We  have  isolated  and  characterized  the 
sevenless  gene.  Our  data  indicate  that  sevenless 
encodes  a  transmembrane  protein  with  a  tyrosine 
kinase  domain.  The  structural  analogies  between 
the  sevenless  protein  and  certain  hormone  recep- 
tors suggest  that  developmental  pathway  selec- 
tions dependent  on  cell-cell  interactions  may 
involve  molecular  mechanisms  similar  to  physio- 
logical or  developmental  changes  induced  by 
long-range  diffusible  factors. 

To  investigate  the  role  of  the  sevenless  protein 


341 


Development  of  the  Drosophila  Visual  System 


in  R7  development,  we  have  examined  the  pat- 
tern of  sevenless  expression  in  the  developing 
retina  and  have  studied  the  effects  of  experimen- 
tally altering  this  pattern.  By  transiently  express- 
ing the  sevenless  protein,  we  have  shown  that 
there  is  only  a  brief  period  during  eye  develop- 
ment when  the  sevenless  protein  is  required  for 
t-he  formation  of  the  R7  photoreceptor.  Our  re- 
sults are  consistent  with  the  proposal  that  seven- 
less directly  reads  positional  information  re- 
quired to  specify  the  R7  developmental  pathway. 

A  major  current  goal  is  to  elucidate  the  intra- 
cellular signal  transduction  pathway  that  is  acti- 
vated by  stimulation  of  the  sevenless  protein. 
That  is,  how  does  activation  of  the  sevenless  tyro- 
sine kinase  instruct  a  cell  to  become  an  R7  pho- 
toreceptor? Defining  the  biologically  relevant 
substrates  of  tyrosine  kinase  receptors  has  been  a 
long-standing  and  difficult  problem.  We  have 
taken  a  genetic  approach  toward  identifying 
genes  whose  products  act  downstream  of  seven- 
less, including  those  that  might  be  direct  sub- 
strates for  the  sevenless  kinase. 

We  have  utilized  two  strategies.  First,  we  have 
looked  for  other  mutations  that  give  the  same  phe- 
notype  as  sevenless — transformation  of  the  R7 
cell  into  a  nonneuronal  cell  type.  In  this  way  we 
isolated  the  seven-in-absentia  (sina)  gene. 
Function  of  the  sina  gene  is  required  only  in  R7 
for  correct  R7  cell  development.  The  sina  pro- 
tein, which  has  a  potential  metal-binding  do- 
main, is  localized  in  the  nuclei  of  several  omma- 
tidial  precursor  cells,  including  R7,  and  sina 
expression  in  R7  appears  before  R7  overtly  be- 
gins to  differentiate.  These  data  indicate  that  the 
sina  gene  product  is  necessary  at  a  stage  in  the 


determination  of  R7  cell  fate  when  R7  receives 
and  interprets  developmental  signals  from  neigh- 
boring cells  and  possibly  acts  by  regulating  gene 
expression. 

Second,  we  utilized  a  crippled  sevenless  pro- 
tein, whose  activity  is  just  barely  adequate  to 
specify  R7  cell  development,  to  establish  a  highly 
sensitive  assay  for  other  components  of  this  signal 
transduction  pathway.  Using  this  assay  we  looked 
for  other  genes  in  which  a  50  percent  reduction 
of  the  level  of  their  protein  products  resulted  in  a 
failure  to  specify  R7  cells.  In  this  way  we  have 
identified  seven  genes  that  appear  to  encode 
products  that  act  to  interpret  the  signal  mediated 
by  the  sevenless  receptor. 

The  putative  products  of  two  of  these  seven 
genes  have  been  identified.  One  encodes  a  ras 
protein.  The  ras  oncogene  is  implicated  in  as 
many  as  30  percent  of  human  tumors.  The  ras 
proteins  exist  in  two  different  states:  an  inactive 
GDP-bound  state  and  an  active  GTP-bound  state. 
The  active  ras  protein  transmits  a  signal  by  inter- 
action with  unidentified  cellular  targets.  The 
other  locus  whose  product  we  have  identified  en- 
codes a  protein  that  is  homologous  to  the  Sac- 
charomyces  cerevisiae  CDC25  protein,  an  acti- 
vator of  GDP-GTP  exchange  by  ras  proteins. 
These  results  suggest  that  the  stimulation  of  ras 
protein  activity  is  a  key  element  in  the  signaling 
by  sevenless  and  that  this  stimulation  may  be 
achieved  by  activating  the  exchange  of  GTP  for 
bound  GDP  by  the  ras  protein.  The  evolutionary 
conservation  of  the  ras  signaling  pathway  sug- 
gests that  studies  in  Drosophila  could  provide 
clues  to  the  role  of  ras  in  oncogenesis  and  devel- 
opmental abnormalities  in  humans. 


342 


lebrand  Factor  Precursor 


GPIb 
Botrocetin 
Heparin  Heparin 
Collagen  Factor  VIZI  Sulfatide  Collagen  GPIIb-IIIa 


488 

474   471     4G4  463 


Top:  Structure-function  relationships  of  von  Willebrand factor. 
The  factor  precursor  comprises  four  types  of  homologous  do- 
mains (A-D),  repeated  two  to  five  times  each.  The  mature  sub- 
unit  consists  of 2, 050  amino  acids.  Binding  sites  are  indicated 
for  collagen,  heparin,  factor  VIII,  platelet  glycoprotein  lb,  bo- 
trocetin, sulfatide,  and  platelet  glycoprotein  Ilb-IIIa.  The  loca- 
tions of  two  Arg-Gly- Asp- containing  sequences,  RGD  and 
RGBS,  are  indicated. 

Bottom:  Mutations,  indicated  by  brackets,  in  exon  28  of  the 
gene  encoding  von  Willebrand  factor  cause  von  Willebrand 
disease  types  IIA  and  IIB.  The  segment  of  the  factor  shown  in- 
cludes amino  acid  residues  463-921,  designated  by  single- 
letter  code.  The  positions  of  repeated  domains  D3  and  A 1-3  are 
indicated.  The  green  zigzag  segments  from  Cys474  to  Pro488 
and  Cys695  to  Pro708  indicate  regions  proposed  to  interact 
directly  with  platelet  glycoprotein  lb.  One  proposed  type  IIA 
mutation  (Val551  Phe,  orange  circle)  occurs  in  the  region 
of  the  type  IIB  mutations. 

From  Sadler,  f.E.  1991.  J  Biol  Chem  266:22777-22780. 


The  Regulation  of  Blood  Coagulation 

J:) 


J.  Evan  Sadler,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Sadler  is  also  Associate  Professor  of  Medicine  and  of  Biochemistry  and  Molecular  Biophysics  at 
Washington  University  School  of  Medicine,  St.  Louis.  He  obtained  his  undergraduate  degree  in  chemistry 
from  Princeton  University.  He  then  attended  Duke  University,  where  he  received  first  his  Ph.D.  degree  in 
biochemistry  with  Robert  Hill  and  then  his  M.D.  degree.  Following  his  internship  and  residency  in 
medicine  at  Duke  University  Medical  Center,  Dr.  Sadler  was  a  Hematology  Fellow  in  the  laboratory  of  Earl 
Davie  at  the  University  of  Washington,  Seattle. 


UNDER  normal  circumstances,  blood  clots  oc- 
cur only  at  sites  of  vascular  injury,  and  un- 
necessary clots  are  dissolved  promptly.  Inappro- 
priate blood  clots  can  cause  devastating  illness, 
such  as  stroke  and  myocardial  infarction.  Abnor- 
mal thrombosis  also  complicates  many  com- 
mon diseases,  including  certain  cancers  and 
infections. 

In  the  blood,  proteins  and  small  cells  called 
platelets  are  required  for  clot  formation.  The  en- 
dothelial cells  that  line  all  blood  vessels  and  cir- 
culating white  blood  cells  are  not,  however,  pas- 
sive bystanders  in  these  reactions,  but  actively 
promote  or  inhibit  clotting.  Compounds  that  are 
produced  during  inflammation  modulate  these 
cellular  activities. 

We  are  investigating  the  structure,  function, 
regulation,  and  evolution  of  proteins  that  control 
blood  coagulation.  Our  goal  is  to  understand  how 
these  opposing  tendencies — to  stimulate  or  to  in- 
hibit clotting — are  balanced  to  achieve  normal 
hemostasis  and  prevent  dangerous  thrombosis. 
These  studies  will  increase  our  knowledge  of  the 
interaction  between  blood  coagulation  and  in- 
flammation and  may  provide  a  foundation  for  the 
design  of  new  therapies  for  thrombotic  disorders. 
Studies  of  thrombomodulin,  thrombin,  and  tissue 
factor  are  supported  by  a  grant  from  the  National 
Institutes  of  Health. 

von  Willebrand  Factor 
and  von  Willebrand  Disease 

The  von  Willebrand  factor  (vWF)  is  a  blood 
protein  that  is  made  by  endothelial  cells  and  is 
required  for  normal  platelet  function.  vWF  also 
binds  to  and  stabilizes  blood  coagulation  factor 
VIII,  the  factor  that  is  deficient  in  classical  hemo- 
philia. The  structure  of  vWF  was  determined  indi- 
rectly by  cDNA  cloning:  vWF  contains  12  re- 
peated domains  that  belong  to  four  families  of 
ancestral  sequences.  Hereditary  deficiency  of 
vWF,  or  von  Willebrand  disease,  is  the  most  com- 
mon genetic  bleeding  disorder  of  humans.  Mild 
abnormalities  of  vWF  function  can  be  detected  in 
nearly  1  percent  of  the  population. 


We  determined  the  structure  of  the  human  vWF 
gene  and  also  of  a  related  pseudogene  that  has 
diverged  recently  from  the  authentic  vWF  gene. 
This  allowed  us  to  investigate  von  Willebrand 
disease  at  the  level  of  gene  sequence.  We  charac- 
terized deletions  of  the  vWF  gene  that  cause  se- 
vere von  Willebrand  disease  in  five  unrelated  pa- 
tients. These  particular  patients  treat  transfused 
vWF  as  a  foreign  protein  and  make  inhibitory  anti- 
bodies to  it.  Deletions  in  the  vWF  gene  predis- 
pose to  the  formation  of  such  antibodies. 

These  studies  were  extended  to  include  pa- 
tients with  variants  of  von  Willebrand  disease 
who  make  a  defective  vWF  molecule.  Among 
more  than  30  unrelated  patients,  1 1  difi'erent 
mutations  were  characterized  within  a  single 
exon  of  the  vWF  gene.  Six  of  these  mutations  are 
within  a  small  vWF  domain  that  appears  to  modu- 
late the  affinity  of  vWF  for  platelets;  of  these  six 
mutations,  five  cause  a  paradoxical  increase  in 
binding  and  the  sixth  causes  a  decrease  in  bind- 
ing. The  remaining  five  mutations  are  within  an 
adjacent  domain  of  the  protein  and  cause  loss  of 
function,  either  by  impairing  vWF  biosynthesis  or 
increasing  vWF  degradation.  The  association  of 
severe  bleeding  with  both  increased  and  de- 
creased function  illustrates  the  importance  of  bal- 
anced vWF  function  for  normal  hemostasis. 

A  recently  described  variant  of  von  Willebrand 
disease,  recognized  in  a  patient  from  Normandy, 
France,  is  characterized  by  defective  binding  of 
vWF  to  blood  coagulation  factor  VIII.  In  such  pa- 
tients factor  VIII  is  unstable,  and  this  results  in  a 
secondary  factor  VIII  deficiency  that  mimics  clas- 
sical hemophilia.  Among  several  unrelated  af- 
fected families,  three  diff^erent  mutations  were 
identified  in  the  factor  Vlll-binding  site  of  vWF. 
The  corresponding  recombinant  vWF  proteins  ex- 
hibited the  same  defect  in  factor  VIII  binding  as 
natural  vWF  Normandy,  confirming  that  these 
mutations  cause  the  disease.  The  genetic  defects 
of  these  and  other  such  patients  provide  insight 
into  structure-function  relationships  of  vWF  and 
may  suggest  new  therapeutic  strategies  to  inhibit 
or  augment  vWF  function. 


345 


The  Regulation  of  Blood  Coagulation 


Thrombomodulin  and  Thrombin 

Thrombomodulin  is  a  protein  of  the  endothe- 
lial cell  surface  that  binds  thrombin,  a  blood  clot- 
ting en2yme.  Because  of  its  effects  on  thrombin 
activity,  thrombomodulin  is  an  essential  natural 
anticoagulant.  Several  inflammatory  mediators 
decrease  the  expression  of  thrombomodulin  by 
endothelial  cells.  Understanding  this  process 
may  help  us  understand  the  abnormal  blood  coag- 
ulation that  accompanies  much  human  disease. 
We  have  cloned  and  expressed  variants  of  recom- 
binant human  thrombomodulin  in  a  variety  of 
cultured  cell  lines.  These  cells  have  been  used  to 
define  the  structural  requirements  for  thrombo- 
modulin cofactor  activity. 

Thrombin  is  a  protease  enzyme  that  is  required 
to  form  blood  clots  from  fibrinogen.  Thrombin 
also  can  inhibit  clotting  by  first  binding  to  throm- 
bomodulin and  then  digesting  certain  other 
blood  coagulation  factors.  Disruption  of  throm- 
bin's normal  balance  between  promoting  and  in- 
hibiting clotting  can,  in  principle,  cause  either 
bleeding  or  thrombosis.  We  have  constructed 
mutant  forms  of  thrombin  w^ith  predominantly  an- 
ticoagulant or  procoagulant  activities.  Such  mu- 
tant thrombins  help  to  define  the  structural  basis 
for  the  different  activities  of  thrombin.  In  addi- 
tion, they  provide  reagents  to  test  the  physiologi- 
cal importance  of  specific  thrombin  activities, 
and  mutant  thrombins  may  also  be  found  to 
have  therapeutic  procoagulant  or  anticoagulant 
properties. 

Tissue  Factor 

Tissue  factor,  a  cell  surface  protein  that  is 
found  on  many  cells  that  do  not  normally  contact 
the  blood,  is  the  most  important  physiological 
initiator  of  blood  coagulation.  When  blood  ves- 
sels are  damaged,  tissue  factor  is  exposed  to  and 


binds  to  blood  coagulation  factor  VII.  The  factor 
Vll-tissue  factor  complex  then  initiates  a  cascade 
of  reactions  that  cause  blood  to  clot.  Both  mono- 
cytes and  endothelial  cells  express  tissue  factor 
activity  in  response  to  many  stimuli,  and  this  con- 
tributes to  the  abnormal  thrombosis  that  accom- 
panies systemic  infections. 

We  have  isolated  cDNA  clones  for  human  tissue 
factor  and  localized  the  gene  to  chromosome  1 . 
These  clones  have  been  used  to  study  the  regula- 
tion of  tissue  factor  in  endothelial  cells.  Tumor 
necrosis  factor,  a  protein  made  during  inflamma- 
tion, causes  a  dramatic  but  transient  increase  in 
tissue  factor  activity.  This  appears  to  be  the  result 
of  activating  the  gene,  which  is  normally  silent  in 
endothelium.  A  second  level  of  control  can  be 
employed  to  amplify  this  response.  Tissue  factor 
mRNA  normally  is  degraded  very  rapidly,  but  it  is 
stabilized  by  some  agents  that  stimulate  endothe- 
lial cells,  and  this  may  contribute  to  the  induc- 
tion of  tissue  factor  activity  during  inflammation. 
We  are  currently  studying  the  structural  ba- 
sis for  this  regulation  of  tissue  factor  mRNA 
degradation. 

The  systems  we  are  investigating  provide  abun- 
dant opportunities  to  answer  biological  ques- 
tions concerning  the  regulation  of  blood  coagula- 
tion and  to  approach  fundamental  questions 
related  to  signal  transduction,  gene  expression, 
and  protein  structure-function  relationships. 
These  studies  may  illustrate  how  several  proteins 
can  be  coordinately  regulated  to  promote  blood 
clotting  reactions  on  the  vascular  endothelium 
during  inflammation.  We  will  continue  to  ex- 
plore the  mechanisms  by  which  vWF,  thrombo- 
modulin, thrombin,  and  tissue  factor  are  regu- 
lated and  will  extend  this  work  to  other 
endothelial  cell  proteins  that  can  promote  or  in- 
hibit thrombosis. 


346 


Molecular  Mechanism  of  Transmembrane  Signal 
Transduction  by  G  Protein-coupled  Receptors 


Thomas  p.  Sakmar,  M.D. — Assistant  Investigator 

Dr.  Sakmar  is  also  Assistant  Professor  at  the  Rockefeller  University.  He  received  his  A.B.  degree  in 
chemistry  and  his  M.D.  degree  from  the  University  of  Chicago.  He  completed  a  medical  residency  at 
Massachusetts  General  Hospital,  Boston,  and  conducted  postdoctoral  research  in  the  laboratory 
of  H.  Gobind  Khorana  at  the  Massachusetts  Institute  of  Technology. 


IN  our  laboratory  the  vertebrate  visual  proteins 
rhodopsin  and  transducin  serve  as  a  model  sys- 
tem for  structure-function  studies  on  the  molecu- 
lar mechanism  of  transmembrane  signaling. 
These  visual  proteins  are  members  of  a  superfam- 
ily  of  related  G  proteins  (guanine-binding  regula- 
tory proteins)  and  G  protein-coupled  receptors. 
Light-activated  rhodopsin  catalyzes  guanine  nu- 
cleotide exchange  by  transducin,  v^^hich  ulti- 
mately leads  to  a  change  in  membrane  cation 
conductance  and  a  neural  signal.  Our  approach  is 
to  reconstitute  heterologously  expressed  rho- 
dopsin and  transducin  in  defined  in  vitro  systems 
and  to  use  biochemical  and  biophysical  methods 
to  probe  site-directed  mutants. 

Our  current  interests  include  structure- 
function  relationships  in  rhodopsin.  For  exam- 
ple, we  are  studying  the  ground  state  structure  of 
the  receptor,  the  interactions  between  specific 
amino  acid  residues  and  the  1  l-c?s-retinal  chro- 
mophore  that  control  spectral  properties  and 
photochemistry,  the  mechanism  by  which  a  pho- 
tochemical signal  is  transmitted  from  the  core  of 
the  receptor  to  the  surface,  and  the  specific  do- 
mains on  the  cytoplasmic  surface  that  bind  and 
activate  transducin. 

We  employ  a  multifaceted  approach,  including 
the  use  of  a  variety  of  complementary  spectro- 
scopic techniques.  For  example,  the  structure 
and  environment  of  the  retinal  chromophore  in 
rhodopsin  and  its  photointermediates  can  be  stud- 
ied with  resonance  Raman  spectroscopy.  In  a  con- 
tinuation of  collaborative  work  with  Steven  Lin 
and  Richard  Mathies,  we  obtained  microprobe 
resonance  Raman  spectra  of  solutions  containing 


only  microgram  quantities  of  mutant  pigments. 

The  results  confirmed  and  supplemented  our 
earlier  observations  concerning  the  role  of  a  spe- 
cific carboxylate  group  in  rhodopsin  that  acts  to 
stabilize  the  positive  charge  of  the  protonated 
Schiff  base  chromophore  linkage.  A  model  of  the 
chromophore  binding  pocket  of  rhodopsin  was 
proposed  and  is  being  used  to  direct  further  stud- 
ies into  the  mechanism  of  wavelength  regulation 
by  visual  pigments. 

We  are  also  interested  in  identifying  specific 
domains  of  rhodopsin  and  transducin  involved  in 
binding  and  activation.  Flash  photolysis  studies 
of  site-directed  rhodopsin  mutants  had  previ- 
ously shown  that  at  least  the  second  and  third 
cytoplasmic  loops  of  rhodopsin  are  involved  in 
activation  of  bound  transducin.  Some  cytoplas- 
mic mutations  prevent  transducin  binding  as 
well.  Recently  we  have  developed  a  spectrofluo- 
rimetric  method  designed  to  allow  simultaneous 
illumination  and  measurements  of  rhodopsin- 
transducin  interactions  by  intrinsic  fluorescence. 

Rhodopsin-catalyzed  binding  of  GTP,  or  a  GTP 
analogue  to  transducin,  results  in  a  large  increase 
in  its  intrinsic  fluorescence.  Mixtures  of  transdu- 
cin and  rhodopsin  can  be  assayed  by  this  method 
to  determine  the  kinetic  rate  constants  of  their 
interaction  and  to  evaluate  the  specific  effects  of 
mutations.  A  series  of  site-directed  mutants  of 
rhodopsin  with  alterations  in  their  cytoplasmic 
domains  have  been  studied.  The  results  may  be 
relevant  to  other  seven-transmembrane  helix  re- 
ceptors that  couple  to  G  proteins  and  play  roles 
in  cellular  physiology,  growth,  development, 
and  differentiation  in  the  nervous  system. 


347 


i 

I 

1 


Molecular  Genetics  of  Development 
in  Drosophila 


Shigeru  Sakonju,  Ph.D. — Assistant  Investigator 

Dr.  Sakonju  is  also  Assistant  Professor  of  Human  Genetics  at  the  University  of  Utah  School  of  Medicine.  He 
received  a  B.A.  degree  from  Columbia  Union  College  and  a  Ph.D.  degree  in  biology  from  the  Johns  Hopkins 
University,  having  developed  his  doctoral  thesis  in  the  Department  of  Embryology  at  the  Carnegie 
Institution  of  Washington,  Baltimore,  with  Donald  Brown.  He  was  a  Helen  Hay  Whitney  Postdoctoral 
Fellow  with  E.  B.  Lewis  at  the  California  Institute  of  Technology  and  at  Stanford  University 
with  David  Hogness. 


DURING  the  development  of  organisms,  the 
fertihzed  egg  undergoes  many  divisions  to 
produce  a  mukicellular  body.  In  the  fruit  fly  Dro- 
sophila melanogaster,  the  body  is  made  up  of 
several  fused  segments  in  the  head,  3  thoracic 
segments  with  wings  and  legs,  and  10  abdominal 
segments,  each  showing  unique  characteristics. 
This  basic  pattern  of  body  segments,  invariant 
from  generation  to  generation,  is  dictated  by  a 
genetic  blueprint  within  the  organism's  own 
genome. 

The  characteristics,  or  identity,  of  each  body 
segment  are  determined  by  the  activities  of  the 
so-called  homeotic  genes.  When  these  do  not 
function  properly,  a  body  segment  or  group  of 
segments  transforms  to  take  on  the  characteristics 
of  another  segment.  Thus  homeotic  genes  can  be 
thought  of  as  master  regulatory  switches  that  trig- 
ger the  genetic  circuits  necessary  to  form  normal 
body  patterns.  Genes  similar  to  the  homeotic 
genes  of  the  fruit  fly  are  found  in  other  organisms, 
including  humans.  The  goal  in  my  laboratory  is  to 
understand  how  the  homeotic  genes  accomplish 
their  task  at  the  molecular  level. 

In  Drosophila,  three  homeotic  genes — Ultra- 
bithorax  (JJbx),  abdominal- A  (abd-A),  and 
Abdominal-B  (Abd-E) — are  responsible  for  de- 
termining the  characteristics  of  two  thoracic  and 
nine  abdominal  segments.  These  genes  are  lo- 
cated in  a  chromosomal  region  called  the  bitho- 
rax  complex  (BX-C) .  Flies  carrying  mutations  in 
any  one  of  the  three  BX-C  genes  show  characteris- 
tic transformations  of  body  segments.  By  observ- 
ing which  segments  are  transformed  in  these  mu- 
tants, we  know  that  Ubx  is  required  in  two 
thoracic  and  eight  abdominal  segments,  abd-A  in 
the  second  through  eighth  abdominal  segments, 
and  Abd-B  in  the  fifth  through  ninth  abdominal 
segments. 

To  learn  what  homeotic  proteins  do  in  the  cell, 
we  have  focused  on  the  proteins  encoded  by  Ubx 
and  abd-A.  Many  homeotic  proteins,  including 
these,  have  been  shown  to  bind  DNA  in  vitro, 
suggesting  that  they  act  by  binding  to  transcrip- 
tional signals  of  other  genes  to  regulate  expres- 
sion. Whether  this  is  how  they  act  in  living  organ- 


isms has  not  been  directly  shown.  Therefore  we 
have  chosen  to  study  the  regulation  of  a  potential 
target  gene  of  Ubx  and  abd-A  proteins,  called  An- 
tennapedia  (Antp),  as  a  paradigm  for  the  mecha- 
nism of  homeotic  protein  actions. 

We  have  shown  that  both  Ubx  and  abd-A  pro- 
teins bind  to  a  number  of  sites  on  the  DNA  seg- 
ment that  contains  the  signals  necessary  for  tran- 
scription of  the  Antp  gene.  We  have  further 
shown,  by  creating  mutations  in  the  binding  sites 
and  assaying  their  effects,  that  this  binding  is  es- 
sential for  repressing  the  Antp  gene  expression  in 
the  embryo.  Our  study  therefore  provides  direct 
evidence  that  homeotic  proteins  do  turn  on  or  oflF 
transcription  of  their  target  genes. 

What  are  the  target,  or  downstream,  genes  regu- 
lated by  homeotic  proteins?  Little  is  known  about 
potential  target  genes,  even  though  many  are 
thought  to  exist.  To  identify  such  targets,  we  have 
utilized  a  method,  called  the  enhancer  trap,  of 
detecting  genes  with  expression  patterns  that 
suggest  homeotic  gene  regulation.  A  number  of 
candidates  have  been  isolated,  and  we  are  testing 
them  for  further  evidence  of  being  the  down- 
stream genes.  We  have  shown  that  the  expression 
of  several  candidate  genes  is  abnormal  in  the  mu- 
tant embryos  that  do  not  produce  Ubx  protein, 
suggesting  that  the  protein  does  regulate  this 
gene. 

We  are  also  interested  in  determining  how  the 
homeotic  genes  with  a  relatively  small  number  of 
encoded  proteins  can  specify  many  unique  body 
segments.  One  answer  lies  in  the  fact  that  these 
genes  are  expressed  in  different  but  overlapping 
sets  of  segments.  For  example,  of  the  three  ho- 
meotic proteins  from  the  BX-C,  only  Ubx  protein 
is  detected  in  the  second  and  third  thoracic  seg- 
ments; both  Ubx  and  abd-A  proteins  are  present  in 
the  second  through  fourth  abdominal  segments; 
and  all  three  BX-C  proteins  are  found  in  the  fifth 
through  eighth  abdominal  segments. 

These  three  combinations  would  of  course  de- 
fine only  three  segment  identities  if  all  cells 
within  segments  were  expressing  the  same  combi- 
nation of  the  homeotic  genes.  In  fact,  cells  within 
a  segment  do  not  express  the  same  combination. 


349 


Molecular  Genetics  of  Development  in  Drosophila 


We  find  that  some  cells  within  a  given  abdominal 
segment  express  abd-A  protein  exclusively  or  pre- 
dominantly; other  cells,  Ubx  protein;  and  still 
other  cells,  Abd-B  protein.  This  kind  of  "mosaic" 
expression  can,  in  theory,  specify  an  unlimited 
number  of  segmental  identities.  We  have  there- 
fore asked  the  following  question:  Is  the  mosaic 
-expression  necessary  for  correct  specification  of 
these  identities? 

To  answer  this  question,  we  altered  flies  geneti- 
cally so  that  homeotic  proteins  could  be  ex- 
pressed at  will.  When  Abd-B  protein  is  expressed 


in  all  cells,  thoracic  and  abdominal  segments  ex- 
hibit some  characteristics  of  the  eighth  abdomi- 
nal segment.  When  both  Ubx  and  Abd-B  proteins 
are  expressed  simultaneously,  thoracic  segments 
transform  to  the  first  abdominal  segment,  which 
is  specified  by  Ubx,  but  abdominal  segments  re- 
main largely  unaltered.  Therefore,  surprisingly, 
we  do  not  observe  completely  nonsensical  iden- 
tities, as  might  have  been  expected  from  this  ab- 
normal situation,  suggesting  that  mosaic  expres- 
sion is  not  required,  at  least  at  a  gross  level,  for 
correct  specification  of  segments. 


350 


Generating  a  Repertoire  of  Antigen-Specific 
Receptors 


W9 


David  G.  Schatz,  Ph.D. — Assistant  Investigator 

Dr.  Schatz  is  also  Assistant  Professor  of  Immunobiology  at  Yale  University  School  of  Medicine.  He  received 
undergraduate  degrees  in  molecular  biophysics  and  biochemistry  from  Yale  University  and  in  philosophy 
and  politics  from  Oxford  University.  His  Ph.D.  degree  and  postdoctoral  training  were  done  with  David 
Baltimore  at  the  Massachusetts  Institute  of  Technology  and  the  Whitehead  Institute. 


CELLS  of  the  immune  system  act  in  concert  to 
protect  against  infectious  agents  and  trans- 
formed (malignant)  cells.  At  the  heart  of  this  pro- 
tective system  are  the  antigen  receptor  molecules 
found  on  B  and  T  lymphocytes:  the  immunoglob- 
ulin (Ig)  and  the  T  cell  receptor  (TCR).  Each 
lymphocyte  expresses  a  distinct  receptor  mole- 
cule that  confers  on  the  cell  a  unique  antigen 
specificity.  The  millions  of  different  genes 
needed  to  encode  these  receptors  are  assembled 
from  component  gene  segments  by  a  site-specific 
process  known  as  V(D)J  recombination — so 
named  for  the  V  (variable) ,  D  (diversity) ,  and  J 
(joining)  gene  segments  used  in  the  reaction. 
V(D)J  recombination  is  critical  for  the  develop- 
ment of  B  and  T  lymphocytes  and  is  the  only  site- 
specific  recombination  process  know^n  to  occur 
in  vertebrates.  We  are  interested  in  two  funda- 
mental questions  concerning  such  recombina- 
tion: What  is  the  biochemical  mechanism  of  the 
reaction,  and  what  molecular  mechanisms  regu- 
late the  reaction  during  lymphoid  development? 

This  recombination  reaction  has  been  inten- 
sively studied  since  its  discovery  in  1976,  yet  lit- 
tle was  known  by  the  late  1980s  about  the  enzy- 
matic machinery  (recombinase)  that  carried  it 
out.  Particularly  frustrating  was  the  inability  to 
identify  the  gene  or  genes  encoding  the  V(D)J 
recombinase,  despite  a  detailed  understanding  of 
the  substrates  and  products.  While  working  with 
David  Baltimore,  I  developed  a  novel  genetic  ap- 
proach to  the  identification  of  these  genes.  Using 
a  highly  sensitive  assay  for  V(D)J  recombinase  ac- 
tivity, I  was  able  to  demonstrate  that  gene  transfer 
(transfection  of  genomic  DNA)  could  activate  the 
V(D)J  recombinase  in  nonlymphoid  cells,  a  sur- 
prising result  implying  that  a  single  genetic  locus 
was  sufficient  for  the  activation. 

Marjorie  Oettinger  and  I  then  isolated  this  ge- 
netic locus  and  quickly  encountered  a  second 
surprise.  The  locus  contained  not  one  but  rwo 
genes,  which  functioned  together  to  activate  the 
V(D)J  recombinase.  Indeed,  a  mixture  of  these 
recombination-activating  genes,  called  RAG-1 
and  RAG- 2,  was  thousands  of  times  more  potent 
than  was  either  gene  alone.  We  then  went  on  to 


demonstrate  that  the  two  genes  are  only  coex- 
pressed  in  developing  lymphocytes — in  exactly 
those  cells  that  are  assembling  Ig  and  TCR  genes. 
This  and  a  variety  of  other  data  suggest  that  RAG- 1 
and  RAG- 2  encode  the  critical,  lymphoid-specific 
components  of  the  V(D)J  recombinase.  Interest- 
ingly, we  found  that  RAG- 1 ,  but  apparently  not 
RAG- 2,  is  transcribed  in  neurons  in  the  central 
nervous  system,  raising  the  possibility  that  RAG- 1 
has  important  roles  in  processes  other  than  clas- 
sic V(D)J  recombination. 

A  central  goal  in  our  current  research  is  to  un- 
derstand the  enzymatic  mechanism  of  V(D)J  re- 
combination, an  elusive  goal  thus  far,  largely  be- 
cause efforts  to  reconstitute  the  reaction  in  a 
cell-free  system  have  been  unsuccessful.  As  a  first 
step  toward  this  goal,  we  are  studying  the  bio- 
chemical and  enzymatic  properties  of  the  RAG- 1 
and  RAG-2  proteins.  Our  initial  focus  is  on  devel- 
oping the  necessary  reagents — in  particular, 
highly  purified  preparations  of  the  proteins  and 
antibodies  that  specifically  recognize  them. 

We  will  use  these  tools  to  examine  the  bio- 
chemical properties  of  the  RAG  proteins,  asking 
whether  they  exhibit  the  activities  expected  of 
proteins  involved  in  recombination  (e.g.,  topo- 
isomerase,  endonuclease,  exonuclease,  or  ligase 
activities) .  We  are  also  interested  in  determining 
if  the  RAG  proteins  bind  to  DNA,  either  nonspecif- 
ically  or  by  interacting  with  elements  of  the 
V(D)J  "recombination  signal  sequence"  [the 
DNA  element  required  for  V(D)J  recombination]. 
In  addition,  we  hope  to  ascertain  whether  the 
RAG-1  and  RAG-2  proteins  interact  with  one  an- 
other or  with  other  proteins  and  how  these  inter- 
actions vary  during  lymphoid  development  and 
among  different  cell  types. 

Our  second  major  focus  is  to  understand  the 
molecular  mechanisms  that  regulate  V(D)J  re- 
combination during  lymphoid  development.  The 
assembly  of  Ig  and  TCR  genes  is  a  complex  and 
highly  ordered  process  regulated  in  part  at  the 
level  of  expression  of  the  V(D)J  recombinase.  Us- 
ing RAG-1  and  RAG-2  as  indicators,  we  are  study- 
ing when  and  how  the  recombinase  is  turned  on 
and  then  off  again  as  B  and  T  cells  develop.  Ex- 
periments performed  in  collaboration  with  Craig 


351 


Generating  a  Repertoire  of  Antigen-Specific  Receptors 


Thompson  (HHMI,  University  of  Michigan)  and 
Larry  Turka  suggest  that  signals  transduced 
through  membrane-bound  TCR  molecules  may 
play  a  role  in  mediating  the  termination  of 
V(D)J  recombinase  expression  during  T  cell 
development. 

V(D)J  recombination  is  also  regulated  at  the 
ievel  of  the  availability,  or  accessibility,  of  the 
gene  segments  to  the  V(D)J  recombinase  .  Accessi- 
bility in  turn  appears  to  be  mediated  by,  or  paral- 
leled by,  transcription  of  the  unrearranged 
("germline")  gene  segments.  Analysis  of  germ- 
line  transcription  during  B  cell  development  has 
yielded  important  insights  into  the  relationship 
between  transcription  and  accessibility  but  has 


left  unaddressed  important  questions  concerning 
T  cell  development.  We  are  interested  both  in 
how  a  stem  cell  (a  cell  with  the  potential  to  de- 
velop into  multiple  cell  types)  becomes  commit- 
ted to  the  T  cell  lineage  and  how  the  different 
sublineages  of  T  cells  are  established.  Since  the 
regulation  of  V(D)J  recombination  is  interwoven 
with  these  developmental  decisions,  we  are  ex- 
amining the  structure  and  developmental  profile 
of  germline  TCR  gene  transcripts.  We  hope  to  de- 
termine the  sequence  of  molecular  events  that 
lead  to  the  assembly  of  TCR  genes  and  to  ask 
whether  defects  in  this  process  contribute  to  the 
pathogenesis  of  immunological  disease,  particu- 
larly autoimmunity. 


352 


Intracellular  Protein  Transport 


Randy  W.  Schekman,  Ph.D. — Investigator 

Dr.  Schekman  is  also  Professor  of  Biochemistry  and  Molecular  Biology  at  the  University  of  California, 
Berkeley,  and  Adjunct  Professor  of  Biochemistry  and  Biophysics  at  the  University  of  California,  San 
Francisco.  As  a  graduate  student,  he  studied  the  enzymology  ofDNA  replication  with  Arthur  Kornberg  at 
Stanford  University.  His  current  interest  in  cellular  membranes  developed  during  a  postdoctoral  period 
with  S.  J.  Singer  at  the  University  of  California,  San  Diego.  At  Berkeley,  he  developed  a  genetic  approach 
to  the  study  of  eukaryotic  membrane  trafftc.  Among  his  awards  is  the  American  Society  for  Microbiology 
Eli  Lilly  Award  in  Microbiology  and  Immunology.  Dr.  Schekman  was  recently  elected  to  the  National 
Academy  of  Sciences. 


RESEARCH  in  our  laboratory  is  devoted  to  mo- 
lecular description  of  the  processes  of  poly- 
peptide translocation  from  the  cytosol  into  the 
endoplasmic  reticulum  (ER)  and  of  vesicular 
transport  among  organelles  of  the  secretory 
pathway. 

Genetic  and  Biochemical  Dissection 
of  the  Secretory  Process 

A  genetic  approach  to  the  study  of  eukaryotic 
protein  transport  involved  the  isolation  of  condi- 
tional mutants.  We  isolated  a  series  of  secretory 
{sec)  mutants  in  the  yeast  Saccharomyces  cerevi- 
siae  that  are  temperature-sensitive  for  cell  sur- 
face growth,  division,  and  secretion.  Most  of  the 
mutants  accumulate  secretory  proteins  in  an  in- 
tracellular pool  that  can  be  released  when  cells 
are  returned  to  a  permissive  temperature.  More 
than  30  gene  products  have  been  implicated  in 
the  process  of  delivering  membrane  and  secre- 
tory proteins  to  the  cell  surface. 

A  combined  genetic  and  cytologic  evaluation 
of  the  sec  mutants  has  allowed  a  description  of 
the  secretory  pathway.  Protein  transport  in  yeast 
appears  to  be  mediated  by  the  same  organelles 
and  proteins  that  operate  in  mammalian  cells.  Mo- 
lecular cloning  analysis  of  SEC  genes  has  re- 
vealed striking  structural  and  functional  homol- 
ogy with  corresponding  mammalian  genes. 

We  have  developed  biochemical  assays  that 
measure  the  early  events  of  polypeptide  translo- 
cation into  the  ER  and  of  vesicle-mediated  pro- 
tein transport  from  the  ER  to  the  Golgi  apparatus. 
The  cell-free  reactions  represent  physiologically 
meaningful  processes.  Extracts  prepared  from 
mutant  cells  reproduce  the  temperature-sensitive 
defects  observed  in  vivo.  In  favorable  circum- 
stances the  mutant  defects  are  repaired  by  addi- 
tion of  a  protein  fraction  obtained  from  wild-type 
yeast,  and  such  restoration  of  transport  activity 
may  be  used  to  purify  functional  forms  of  SEC 
gene  products. 


Protein  Translocation  Across  Membranes 

Protein  translocation  into  the  lumen  of  the  en- 
doplasmic reticulum  represents  the  initial  step  in 
assembly  of  the  eukaryotic  cell  surface.  This  pro- 
cess has  been  reconstituted  with  detergent-solubi- 
lized  membrane  proteins  and  purified  cytosolic 
proteins,  yet  the  mechanism  of  polypeptide  pen- 
etration is  unclear.  We  have  isolated  mutants  that 
are  defective  in  translocation,  using  a  genetic  se- 
lection that  requires  secretory  polypeptides  to  be 
retained  in  the  cytosol.  The  work  on  these  mu- 
tants is  supported  by  a  grant  from  the  National 
Institutes  of  Health  and  will  not  be  described 
here. 

An  independent  line  of  investigation  concerns 
the  mechanism  of  protein  translocation  from  the 
cytosol  into  the  vacuole.  The  vacuole  contains  an 
array  of  hydrolytic  enzymes  and  is  believed  to 
play  an  important  role  in  the  degradation  of  cyto- 
solic proteins  and  intracellular  membranes.  The 
mechanism  for  importing  substrates  into  the  vacu- 
ole has  not  been  evaluated. 

Last  year  we  reported  a  novel  pathway  for  the 
localization  and  degradation  of  fructose  1,6-bis- 
phosphatase  (FBPase),  a  key  regulatory  enzyme 
of  gluconeogenesis.  FBPase  is  localized  to  the  cy- 
tosol when  cells  are  grown  on  a  poor  carbon 
source,  such  as  ethanol.  When  cells  are  trans- 
ferred to  glucose,  FBPase  is  degraded  in  a  process 
called  catabolite  inactivation,  which  depends  on 
active  vacuolar  proteases.  In  a  protease-deficient 
strain,  FBPase  enters  the  vacuole  and  remains  in- 
tact. Import  into  the  vacuole  was  shown  to  de- 
pend on  protein  synthesis  during  the  period  of 
transfer  to  glucose  medium.  In  addition,  vacuolar 
import  requires  the  transfer  of  a  protein,  possibly 
an  import  receptor,  via  the  secretory  pathway. 

The  mechanism  of  this  new  import  pathway  is 
being  pursued  by  the  isolation  of  mutants  defec- 
tive in  the  degradation  of  FBPase.  Thus  far  a  large 
number  of  genes  have  been  identified  that  are 
required  for  import  of  FBPase  into  the  vacuole. 
Preliminary  evidence  suggests  that  the  import 


353 


Intracellular  Protein  Transport 


may  be  mediated  by  an  independent  organelle, 
the  peroxisome.  Mutations  that  block  peroxiso- 
mal assembly  also  block  FBPase  degradation.  Fur- 
thermore, a  peroxisomal  enzyme,  acyl-CoA  thio- 
lase,  is  subject  to  the  same  glucose-stimulated, 
vacuolar-dependent  degradation  process.  One 
possible  explanation  of  these  results  is  that 
FBPase  may  be  imported  into  or  somehow  asso- 
ciated with  peroxisomes  and  then  localized  to 
the  vacuole  by  autophagic  uptake  of  the 
peroxisome. 

Vesicle  Transport  Early  in  the  Secretory 
Pathway 

Subsequent  stages  in  the  secretory  pathway  in- 
volve protein  sorting  and  transport  from  the  en- 
doplasmic reticulum  to  the  Golgi  apparatus  and 
from  there  to  the  cell  surface.  Genes  required  for 
each  of  these  steps  are  being  evaluated  by  molec- 
ular cloning  and  by  development  of  cell-free  reac- 
tions that  measure  individual  steps  in  the  trans- 
port process.  An  assay  that  depends  on  Sec 
proteins  has  been  reconstituted  in  vitro.  Yeast 
a-factor  precursor  is  translocated  into  the  ER  lu- 
men of  gently  lysed  yeast  spheroplasts.  In  the 
presence  of  soluble  proteins  and  ATP,  the  precur- 
sor is  transferred  to  the  Golgi  apparatus.  This  sys- 
tem allows  the  purification  and  functional  charac- 
terization of  Sec  proteins. 

Transfer  of  secretory  proteins  from  the  ER  to 
the  Golgi  apparatus  is  mediated  by  small  vesicle 
carriers.  In  this  limb  of  the  pathway,  defective  sec 
mutants  fall  into  two  categories:  class  I,  mutant 
cells  that  accumulate  ER  tubules  at  a  restrictive 
temperature  {SEC  12,  -13,  -16,  and  -23);  and 
class  II,  mutant  cells  that  also  accumulate  several 
thousand  60-nm  vesicles  (SEC17,  -18,  and  -22). 

Genetic  epistasis  tests  indicate  that  class  I 
genes  must  execute  their  function  prior  to  class  II 
genes.  This  implies  that  class  I  products  partici- 
pate in  the  production  of  the  60-nm  vesicles  that 
are  consumed,  by  fusion  with  the  Golgi  appara- 
tus, through  the  action  of  class  II  gene  products. 
Genetic  interactions  among  members  of  class  I 
and  II  genes  suggest  that  the  Sec  proteins  in  each 


group  act  in  a  complex,  or  at  least  in  a  concerted 
manner,  to  perform  their  respective  roles  in  vesi- 
cle budding  or  fusion. 

Transport  of  the  a-factor  precursor  in  vitro  is 
mediated  by  diffusible  vesicles.  Transport  vesi- 
cles contain  a  core-glycosylated  precursor  and 
are  physically  separable  from  donor  ER  and  target 
Golgi  membranes.  Budding  of  vesicles  from  the 
ER  requires  a  crude  cytosol  fraction,  ATP, 
Secl2p,  Secl3p,  Sec23p,  GTP,  and  a  ras-like 
GTP-binding  protein,  Sarlp.  Fusion  of  the  vesi- 
cles to  the  Golgi  compartment  is  measured  by 
conversion  of  the  precursor  to  a  more  highly  gly- 
cosylated form.  Enriched  transport  vesicles  target 
to  the  Golgi  compartment  and  then  fuse  in  dis- 
tinct subreactions  that  require  cytosol,  Ca^"^,  ATP, 
and  only  a  subset  of  Sec  proteins. 

To  allow  the  purification  of  functional  Sec23p, 
we  have  developed  an  assay  based  on  restoration 
of  transport  in  sec23  lysates  by  wild-type  protein 
fractions.  The  purified  protein  has  been  isolated 
in  two  forms:  a  Sec23p  monomer,  and  a  hetero- 
oligomer  that  consists  of  Sec23p  and  a  105-kDa 
protein  (Sec24p)  that  is  also  required  for  vesicle 
budding.  The  SEC 2 3  and  -24  genes  have  both 
been  cloned,  and  though  the  sequences  confirm 
the  observed  molecular  weights  of  the  isolated 
polypeptides,  no  homology  to  known  proteins 
was  found. 

Localization  of  Sec  proteins  in  yeast  has  met 
with  only  limited  success.  Yeast  morphologic 
analysis  is  limited  by  the  small  cell  size  and  the 
difficulty  of  specimen  preservation.  Fortunately 
the  functional  and  structural  conservation  of  Sec 
proteins  allows  their  localization  in  mammalian 
cells.  The  first  and  most  favorable  example  is 
Sec23p,  where  antibodies  directed  against  the 
yeast  protein  cross-react  with  the  mammalian  ho- 
mologue.  Immunolocalization  studies  reveal  a 
striking  enrichment  of  Sec23p  in  the  cytoplasmic 
pocket  that  separates  the  transitional  ER  and  the 
cis  face  of  the  Golgi  complex.  This  location  is 
completely  consistent  with  the  role  proposed  for 
yeast  Sec23p  and  further  confirms  that  the  secre- 
tory pathway  is  fundamentally  conserved  across 
the  broad  spectrum  of  the  eukaryotic  kingdom. 


354 


Development  and  Function  of  the  Synapse 


Richard  H.  Scheller,  Ph.D. — Associate  Investigator 

Dr.  Scheller  is  also  Associate  Professor  of  Molecular  and  Cellular  Physiology  and  Associate  Professor  of 
Biological  Sciences  (by  courtesy)  at  Stanford  University.  He  received  his  B.S.  degree  from  the  University 
of  Wisconsin- Madison  and  his  Ph.D.  degree  in  chemistry  from  the  California  Institute  of  Technology. 
Dr.  Scheller  was  a  postdoctoral  student  with  Richard  Axel  and  Eric  Kandel  at  Columbia  University. 


THE  nervous  system  is  composed  of  large  num- 
bers of  unique  cells  that  communicate  with 
each  other  via  the  regulated  release  of  chemical 
neurotransmitters.  These  synaptic  interactions 
govern  animal  behavior,  and  modulation  of  the 
efficacy  of  synaptic  communication  is  thought  to 
underlie  learning  and  memory.  We  are  interested 
in  understanding  the  molecular  mechanisms  of 
synaptic  formation  during  development  and  re- 
generation in  the  peripheral  nervous  system  after 
nerve  injury.  It  is  also  our  goal  to  contribute  to  an 
understanding  of  how  the  nerve  terminal  func- 
tions in  the  regulation  of  neurotransmitter 
release. 

Processing  and  Packaging  of  Neuropeptides 

Many  synapses  release  two  types  of  chemical 
messengers:  fast-acting,  or  classical  transmitters, 
and  slower-acting  messengers,  or  neuromodula- 
tors. Most  of  the  various  chemicals  used  as  mes- 
sengers in  the  brain  are  neuropeptides.  These 
molecules  are  synthesized  as  larger  precursors 
that  are  processed  to  smaller  active  peptides.  One 
interesting  neuropeptide  precursor  is  expressed 
in  an  identified  set  of  neurons,  the  bag  cells,  in 
the  marine  snail  Aplysia.  When  these  neurons 
fire,  they  release  a  set  of  neuropeptides  derived 
from  a  single  precursor.  These  peptides  act  on 
neurons  and  peripheral  tissues  to  regulate  egg 
laying,  a  stereotyped  behavior. 

Interestingly,  the  peptides  produced  on  the 
egg-laying  hormone  (ELH)  precursor  are  pack- 
aged in  two  types  of  vesicles.  These  vesicles  con- 
tain different  sets  of  peptides  and  are  differen- 
tially localized  within  the  neurons.  We  are 
interested  in  understanding  how  the  peptides  ini- 
tially synthesized  on  a  single  precursor  are  sorted 
into  different  vesicles.  We  are  also  interested  in 
understanding  the  physiological  significance  of 
the  differential  packaging  and  localization. 

When  the  ELH  precursor  is  transfected  into 
mammalian  pituitary  tumor  cells  (AtT-20  cells), 
ELH  is  packaged  with  the  endogenous  hormone. 
The  amino-terminal  region  of  the  precursor  is  de- 
graded within  the  secretory  cell,  probably  in  the 
lysosomes.  Thus  the  AtT-20  cells,  like  the  bag 


cells,  differentially  route  the  two  regions  of  the 
ELH  prohormone.  Mutating  the  first  cleavage  site 
from  a  set  of  four  basic  residues  to  two  basic  resi- 
dues results  in  constitutive  secretion  of  the 
amino-terminal  region  of  the  precursor,  not  intra- 
cellular degradation.  These  results  suggest  that 
the  subcellular  location  of  the  first  endoproteoly- 
tic  processing  event  is  critical  in  determining  the 
routing  of  the  processing  intermediates. 

Mechanisms  of  Synaptic  Transmission 

When  the  action  potential  travels  down  the 
nerve  and  enters  a  release  zone,  changes  in  the 
membrane  potential  open  channels  that  allow  cal- 
cium to  enter  the  cell.  The  calcium  promotes 
transmitter  release  and  membrane  fusion.  The 
membrane  then  recycles,  forming  new  vesicles, 
which  are  then  replenished  with  chemical  trans- 
mitter. This  cycle  might  be  considered  the  funda- 
mental process  that  underlies  nervous  system 
function,  yet  little  is  known  about  the  molecular 
mechanisms  involved.  In  an  attempt  to  define  the 
molecular  mechanisms  that  regulate  membrane 
flow  in  the  nerve,  our  laboratory  and  others  have 
begun  to  characterize  the  proteins  associated 
with  the  critical  organelle  in  the  process,  the  syn- 
aptic vesicle.  For  these  studies,  we  use  mamma- 
lian brain  and  the  electric  organs  of  marine  rays. 
These  electric  organs  have  a  concentration  of  syn- 
apses approximately  100-fold  higher  than  that  of 
skeletal  muscle.  In  addition,  these  synapses  are 
homogeneous;  they  all  use  the  neurotransmitter 
acetylcholine. 

Purified  synaptic  vesicles  contain  about  20-50 
protein  bands  when  fractionated  on  acrylamide 
gels.  Genes  encoding  many  of  these  proteins  have 
been  characterized  and  the  primary  sequence  of 
the  molecules  determined.  Some  of  the  proteins 
show  interesting  homologies  to  other  molecules, 
and  others  are  turning  out  to  have  counterparts  in 
yeast  where  genetic  studies  of  membrane  traffick- 
ing have  provided  insight  into  the  secretory  pro- 
cess. It  has  also  become  apparent  that  many  of  the 
synaptic  vesicle  proteins  are  members  of  small 
gene  families.  Individual  members  of  these  gene 
families  are  differentially  expressed  through  the 


355 


Development  and  Function  of  the  Synapse 


brain,  resulting  in  a  variety  of  combinations  of 
these  molecules  on  different  vesicles. 

Molecular  fractionation  and  immunoprecipita- 
tion  techniques  suggest  that  several  of  the  synap- 
tic vesicle  proteins  interact  to  form  a  large  multi- 
meric  complex.  This  complex  contains  a 
previously  uncharacterized  35-kDa  protein  that 
is  a  major  substrate  for  casein  kinase.  We  have 
purified  this  protein  and  isolated  cDNAs  encod- 
ing the  molecule.  The  protein  is  unique  in  the 
database  and  is  predicted  to  be  anchored  in  the 
membrane  by  a  hydrophobic  carboxyl  terminus 
and  to  have  an  amino-terminal  region  oriented 
toward  the  cytoplasm.  Like  the  synaptic  vesicle 
proteins,  this  molecule  is  a  member  of  a  small 
gene  family.  Experiments  are  directed  tow^ard  un- 
derstanding the  localization  and  function  of  this 
protein  and  the  other  synaptic  vesicle  proteins. 

Synapse  Development 

Motor  neurons  in  the  spinal  cord  send  axons  to 
muscle  fibers  throughout  the  body.  When  axons 
contact  muscle  fibers,  a  highly  ordered  structure 
consisting  of  a  presynaptic  nerve  terminal  and  a 
postsynaptic  site  develops.  The  presynaptic  ter- 
minal comprises  an  active  zone  rich  in  synaptic 
vesicles  containing  neurotransmitter.  The  post- 
synaptic element  is  made  up  of  a  membrane  rich 
in  receptors  for  the  neurotransmitter  and  an  in- 
dentation in  the  membrane  called  the  junctional 
fold.  An  extracellular  matrix,  or  basal  lamina, 
surrounds  the  muscle  fiber,  including  the  space 
between  the  nerve  and  muscle. 

One  of  the  key  events  in  the  development  of 
the  neuromuscular  junction  is  the  redistribution 
of  neurotransmitter  receptors  that  occurs  when 
nerve  contacts  muscle.  Initially  receptors  for  the 
neurotransmitter,  in  this  case  acetylcholine,  are 
randomly  distributed  on  the  muscle  fiber.  When 
the  nerve  contacts  muscle,  neurotransmitter  re- 
ceptors aggregate  under  the  nerve  terminal  in  an 
appropriate  position  to  detect  the  chemicals  re- 
leased during  synaptic  transmission. 


Agrin,  a  component  of  the  extracellular  matrix, 
causes  acetylcholine  receptors  to  cluster  when 
added  to  muscle  fibers  growing  in  culture.  We 
have  isolated  recombinant  DNA  clones  encoding 
agrin  molecules  and,  through  an  analysis  of  the 
nucleotide  sequence,  have  defined  the  primary 
amino  acid  sequence  of  the  molecule.  When  we 
compare  the  predicted  agrin  sequence  with  the 
proteins  in  the  data  bank,  two  types  of  similarities 
are  revealed.  The  first  is  to  a  class  of  molecules 
that  inhibit  proteases  and  the  second  to  a  protein 
motif  called  EGF  (epidermal  growth  factor)  re- 
peats. The  gene  is  expressed  in  embryonic  motor 
neurons  at  the  time  they  are  first  contacting  mus- 
cle fibers.  Two  regions  of  the  agrin  gene  are  alter- 
nately spliced  and  may  produce  up  to  eight  forms 
of  the  molecule. 

Expression  of  agrin  encoding  cDNAs  in  CHO 
and  COS  cell  lines  results  in  the  association  of  the 
protein  with  the  surface  of  the  transfected  cells, 
probably  through  assembly  into  an  extracellular 
matrix.  Coculture  of  agrin-expressing  cells  with 
primary  muscle  fibers  or  a  C2  myoblast  cell  line 
results  in  aggregation  of  acetylcholine  receptors 
at  sites  of  contact  between  the  transfected  cell 
and  the  muscle  fibers.  Only  the  forms  of  the  pro- 
tein containing  an  eight-amino  acid  sequence, 
which  is  the  product  of  alternate  RNA  splicing, 
are  capable  of  causing  clusters  on  S27  cells,  a 
mutant  C2  line  lacking  proteoglycans.  These  data 
suggest  that  agrin  may  cluster  receptors  via  two 
mechanisms,  one  of  which  is  proteoglycan  de- 
pendent. Another  possibility  is  that  the  eight 
amino  acids  provide  a  binding  site  that  results  in  a 
high-affinity  interaction,  overriding  the  need  for 
the  proteoglycan  component. 

Since  agrin  is  stably  maintained  in  the  synaptic 
basal  lamina  after  nerve  or  muscle  damage,  it  may 
also  play  a  role  in  regeneration  events.  Under- 
standing the  mechanisms  of  peripheral  synapse 
regeneration  may  lead  to  procedures  that  could 
aid  in  central  nervous  system  regeneration. 


356 


Molecular  Pathogenicity  Studies 
of  Enteric  Bacteria 


Gary  K.  Schoolnik,  M.D. — Associate  Investigator 

Dr.  Schoolnik  is  also  Associate  Professor  of  Medicine  and  of  Microbiology  and  Immunology  at  Stanford 
University  School  of  Medicine.  He  received  his  M.D.  degree  from  the  University  of  Washington.  He  was  an 
intern,  resident,  and  chief  resident  in  internal  medicine  at  Massachusetts  General  Hospital,  a  fellow  in 
infectious  diseases  with  King  Holmes  and  Thomas  Buchanan  at  the  University  of  Washington,  and  a 
postdoctoral  fellow  with  Emil  Gotschlich  at  the  Rockefeller  University.  He  founded  the  Division  of 
Geographic  Medicine  at  Stanford  University  and  has  established  a  research  center  for  the  study  of 
infectious  diseases  in  southern  Mexico. 


BACTERIAL,  viral,  and  parasitic  infections  of 
the  gastrointestinal  tract  cause  an  estimated 
500  million  illnesses  and  5  million  deaths  each 
year  among  children  living  in  the  developing 
countries.  The  principal  mission  of  our  labora- 
tory is  to  discover  how  these  infectious  agents 
cause  disease,  how  they  are  spread,  and  how  this 
information  can  lead  to  new  tactics  for  preven- 
tion and  control.  This  effort  has  entailed  work  in 
two  settings:  molecular  studies  in  our  laboratory 
at  Stanford  University,  and  epidemiological  in- 
vestigations at  a  field  laboratory  in  southeastern 
Mexico,  where  infections  of  this  kind  are 
common. 

In  the  first  setting,  the  unit  of  analysis  is  the 
organism  itself.  We  try  to  determine  how  it  at- 
taches to,  invades,  and  damages  human  cells.  This 
anthropomorphic  orientation  views  the  disease- 
causing  capacity  of  an  infectious  agent  as  the  cen- 
tral research  issue.  In  the  second  setting,  the  unit 
of  analysis  is  a  household  or  a  village.  In  this  con- 
text we  seek  to  understand  how  the  organism  is 
transmitted  within  the  community,  what  its  reser- 
voirs are,  and  how  it  manages  to  survive  as  a  via- 
ble entity  in  the  real  world. 

This  ecological  orientation  seeks  to  understand 
how  the  organism  adapts  to  different  environmen- 
tal habitats.  Within  the  context  of  a  Third  World 
village,  these  habitats  include  contaminated  food 
and  well  water,  sewage,  and  the  gastrointestinal 
tracts  of  people  and  animals.  By  using  biochemi- 
cal and  genetic  tools  to  study  infectious  agents  as 
they  inhabit  and  move  among  these  different  envi- 
ronmental niches,  we  have  begun  to  understand 
the  underlying  molecular  mechanisms  for  this  re- 
markable capacity.  This  in  turn  is  beginning  to 
lead  to  new  strategies  for  the  control  of  these  dis- 
eases through  vaccination  and  epidemiological 
interventions.  Examples  of  work  in  progress  are 
described  below. 

Enteropathogenic  Escherichia  coli 

Enteropathogenic  E.  coli  (EPEC)  are  a  common 
cause  of  infantile  diarrhea  in  Third  World  chil- 
dren. When  biopsies  of  the  small  intestine  are 


performed,  colonies  of  EPEC  are  found  attached 
to  the  underlying  epithelia.  It  is  evident  that  the 
bacteria  interact  not  only  with  the  host  cells  to 
which  they  are  bound  but  also  with  each  other. 
Beneath  these  adherent  colonies,  structural 
changes  in  the  host  cell  also  occur,  indicating 
that  EPEC  have  altered  the  absorptive  surface  of 
the  intestinal  cell.  It  is  now  clear  that  structural 
changes  of  this  kind  are  directly  responsible  for 
the  diarrheal  syndrome. 

From  these  studies  we  have  learned  that  EPEC's 
pathogenic  strategy  consists  of  at  least  three  dis- 
tinct steps,  which  probably  occur  in  the  follow- 
ing order:  the  coalescence  of  individual  bacteria 
into  infectious  units  composed  of  several  to 
hundreds  of  organisms  (an  event  that  probably 
occurs  in  the  small  intestine,  soon  after  ingestion 
of  the  bacteria);  attachment  of  these  infectious 
units  to  the  surface  of  intestinal  epithelial  cells; 
and,  following  close  contact  between  the  bacte- 
ria and  the  epithelial  cells,  the  organism's  partial 
penetration  of  the  cell,  in  association  with  con- 
comitant rearrangement  of  its  cytoskeleton. 

Each  of  these  steps  is  performed  by  a  distinct 
surface  structure  of  the  bacteria.  First,  the  bacte- 
ria are  organized  into  infectious  units  through  the 
production  of  rope-like  appendages  that  emanate 
from  the  organism's  surface.  Termed  "bundle- 
forming  pili,"  these  appendages  create  a  network 
within  which  the  bacteria  become  enmeshed. 
Then  the  bacteria  bind  to  human  intestinal  epithe- 
lial cells  through  the  activity  of  rod-like  filaments 
that  project  from  the  organism  like  spines  from  a 
porcupine.  These  filaments  bind  fibronectin  mol- 
ecules that  are  located  around  the  periphery  of 
each  intestinal  epithelial  cell.  Finally,  an  outer 
membrane  protein  of  the  organism  interacts  with 
integrin-like  molecules  of  the  epithelial  cell,  an 
event  that  triggers  changes  in  the  architecture  of 
the  cell's  cytoskeleton. 

The  production  of  at  least  one  of  these  surface 
structures  is  controlled  by  physiochemical  sig- 
nals that  operate  in  the  intestinal  lumen,  where 
they  serve  as  unique  signatures  of  that  habitat. 
Thus,  when  under  the  influence  of  these  signals, 


357 


Molecular  Pathogenicity  Studies  of  Enteric  Bacteria 


EPEC  produce  bundle-forming  pili  and  coalesce 
as  infectious  units.  In  contrast,  when  in  inani- 
mate environmental  reservoirs  such  as  water  or 
sewage,  their  production  is  repressed  and  they 
exist  as  single,  well-separated  organisms.  This 
phenomenon,  together  with  the  findings  de- 
scribed above,  shows  that  EPEC  exemplify  two 
features  of  bacterial  pathogenesis:  first,  that 
pathogenicity  is  often  the  consequence  of  several 
distinct  virulence  determinants  of  the  organism 
acting  together  and,  second,  that  the  expression 
of  these  determinants  may  be  regulated  by  local 
features  of  the  organism's  environment. 

We  are  now  attempting  to  use  these  findings  to 
design  an  EPEC  vaccine  for  the  prevention  of  diar- 
rheal illnesses  in  children.  We  hope  to  delete 
from  the  EPEC  chromosome  the  gene  that  causes 
functional  abnormalities  of  the  host  cell  while 
retaining  the  genes  that  direct  the  cell  attachment 
capacity  and  the  production  of  bundle-forming 
pili.  A  genetically  modified  strain  of  this  kind 
should  be  attenuated  with  respect  to  its  viru- 
lence, yet  fully  capable  of  colonizing  the  small 
intestine,  where  it  would  be  expected  to  stimu- 
late a  protective  immune  response. 

Using  Attenuated  Strains  of  the  Typhoid 
Fever  Bacillus  as  Vaccines 

Fully  virulent  strains  of  Salmonella  typhi 
cause  typhoid  fever,  whereas  an  attenuated  strain 
of  the  same  species,  administered  as  a  living  oral 
vaccine,  is  now  FDA  approved  and  widely  used 
for  the  prevention  of  typhoid  fever.  Attenuated 
strains  of  this  kind  have  been  proposed  as  vehi- 
cles that  might  be  able  to  carry  other  vaccine  sub- 
stances— termed  "passenger  antigens" — de- 
rived from  a  variety  of  microorganisms.  Such  a 
vaccine  would  stimulate  immunity  not  only  to  S. 
typhi,  but  to  other  infectious  agents  as  well.  We 
have  been  studying  this  system  for  the  delivery  of 
T  cell  epitopes. 

For  the  purposes  of  this  study,  T  cell  epitopes 
are  considered  to  be  localized  regions  of  a  pro- 
tein that  stimulate  a  cellular  immune  response 
and  are  required  for  the  elimination  of  intracellu- 
lar pathogens,  including  some  bacteria  and  many 


viruses  and  protozoan  parasites.  We  have  found 
that  T  cell  epitopes  can  be  effectively  processed 
and  presented  to  the  immune  system  when  ex- 
pressed in  the  center  of  the  flagellin  protein  of  S. 
typhi.  Flagellin  is  the  protein  building  block  of  a 
whip-like  extracellular  filament  that  acts  like  a 
motor  to  propel  the  bacterium  through  liquids. 
Because  these  vaccine  strains  would  express  a  fla- 
gellin protein  that  would  also  contain  a  foreign  T 
cell  epitope,  they  are  referred  to  as  "chimeric" 
flagellins. 

We  have  taken  advantage  of  recent  information 
about  how  epitopes  of  this  kind  are  processed  by 
cellular  components  of  the  immune  system.  It  is 
now  clear  that  one  common  pathway  would  en- 
tail the  presence  of  the  chimeric  flagellin  in  the 
endosome  of  a  macrophage.  In  that  specialized 
compartment  of  the  cell,  it  would  be  digested  by 
endosomal  proteolytic  enzymes,  resulting  in  the 
release  of  the  epitope  as  a  small  peptide  prior  to 
its  presentation  to  other  components  of  the  im- 
mune system.  A  T  cell  epitope  was  placed  in  the 
flagellin  protein  flanked  at  either  end  by  amino 
acids  known  to  be  sites  of  cleavage  by  endosomal 
proteolytic  enzymes.  This  has  yielded  a  five-  to 
eight-fold  increase  in  the  magnitude  of  the  result- 
ing immune  response. 

Further  modifications  have  entailed  placing 
the  expression  of  these  chimeric  flagellins  under 
the  control  of  the  heat-shock  protein  promoter, 
which  is  known  to  be  activated  in  Salmonella 
growing  in  macrophages.  Other  modifications  in- 
clude the  use  of  multiple  tandem  copies  of  these 
epitopes  and  the  use  of  flagellins  that  are  secreted 
as  soluble  proteins  into  the  macrophage  endo- 
some. Taken  together,  these  studies  have  demon- 
strated that  it  is  possible  to  use  attenuated  strains 
of  S.  typhi  to  deliver  T  cell  epitopes  and  that  the 
effectiveness  of  this  delivery  system  can  be  en- 
hanced by  the  design  features  described  above. 

We  are  now  working  in  collaboration  with  sci- 
entists at  the  National  Institutes  of  Health  to  place 
T  cell  epitopes  of  a  human  immunodeficiency 
virus  (HIV)  protein  in  Salmonella  flagellins, 
with  a  view  to  preparing  useful  vaccines  for  the 
prevention  of  AIDS. 


358 


HEp-2  cells  infected  with  Escherichia  coli  express  the  inv  gene  from  the  invasive  bacterial  pathogen 
Yersinia  enterocolitica.  Data  were  recorded from  a  computer-controlled  confocal  laser  microscope 
and  processed  on  a  graphics  display  terminal.  The  HEp-2  cells  (green)  were  stained  with  the 
actin-speciflc  stain  FITC-phallicidin,  while  the  invasin- bearing  bacteria  (red)  were  visualized  by 
indirect  immunofluorescence  using  antisera  against  E.  coli  outer-membrane  proteins.  Areas  of 
extensive  overlap  appear  yellow.  Results  indicate  that  actin,  a  major  component  of  the  cytoskele- 
ton  that  controls  cell  shape  and  movement,  accumulates  in  the  region  of  entering  bacteria. 

From  Young,  V.B.,  Falkow,  S.,  and  Schoolnik,  G.K.  75>i)2.  J  Cell  Biol  116:197-207,  by  copyright 
permission  of  the  Rockefeller  University  Press. 


359 


Stereo  pairs  showing  a  three-dimensional  view  of  in  situ  hybridization  signals  to  two  neighboring 
genes.  DNA  from  the  white  gene  (seen  here  in  green )  and  from  the  Notch  gene  ( red )  were  hybrid- 
ized to  the  giant  chromosomes  from  Drosophila  salivary  glands,  fluorescently  labeled,  and  visual- 
ized by  wide-field  three-dimensional  optical  sectioning  microscopy.  Top  picture  shows  these  sig- 
nals overlaid  on  the  chromosome  (blue );  bottom,  the  signals  alone.  The  structure  of  the  two  genes 
is  seen  to  be  distinctly  different.  Scale  bar  =  1  fim. 

Research  and  photograph  by  Susan  Parmelee  in  the  laboratories  of  fohn  Sedat  and  David 
Agard. 


360 


Three-Dimensional  Structure  of  Eukaryotic 
Chromosomes 


John  W.  Sedat,  Ph.D. — Investigator 

Dr.  Sedat  is  also  Professor  of  Biochemistry  and  Biophysics  at  the  University  of  California,  San  Francisco. 
He  received  his  Ph.D.  degree  in  biology  from  the  California  Institute  of  Technology.  His  postdoctoral  work 
with  Fred  Sanger  was  done  at  the  Medical  Research  Council  in  Cambridge,  England.  Before  joining  the 
faculty  at  UCSF,  Dr.  Sedat  was  a  research  associate  at  Yale  University. 


THE  three-dimensional  structure  of  chromo- 
somes, both  in  the  nucleus  and  during  cell 
division,  remains  a  major  unsolved  problem  in 
biology.  Our  laboratory,  in  collaboration  with 
that  of  David  Agard  (HHMI,  University  of  Califor- 
nia, San  Francisco) ,  is  investigating  chromosome 
structure  from  the  perspective  of  several  inter- 
locking questions:  1)  What  is  the  architecture  of 
the  chromosome  in  the  intact  diploid  nucleus? 
How  does  the  three-dimensional  structure 
change  as  a  function  of  development,  or  progres- 
sion through  the  cell  cycle?  2)  What  is  the  archi- 
tecture of  a  given  gene  in  the  nucleus?  Do  the 
structural  attributes  reflect  the  detailed  molecu- 
lar information?  3)  How  do  interphase  chromo- 
somes condense  to  form  the  intricate  mitotic 
structure  at  cell  division? 

The  fruit  fly  Drosophila  melanogaster,  well- 
known  for  its  genetics,  development,  and  bio- 
chemistry, was  chosen  as  a  model  biological  sys- 
tem. Although  the  initial  emphasis  is  structural, 
molecular  genetics  and  biochemistry  provide 
functional  correlations. 

The  UCSF  three-dimensional  optical  micro- 
scope has  been  developed  to  the  point  that  data  at 
several  wavelengths  can  be  routinely  collected, 
even  as  a  function  of  time  (four-dimensional  mi- 
croscopy) ,  and  can  be  used  without  computer  ex- 
perience. Still,  we  continue  to  perfect  and  en- 
hance the  instrumentation.  We  increased  the 
time  resolution  for  data  collection  and  greatly 
improved  the  image  quality  of  the  four- 
dimensional  data,  permitting  analysis  of  much  in- 
formation on  biological  structures.  We  continue 
to  write  software,  with  extensive  mathematical 
analysis,  to  correct  systematic  image  acquisition 
problems,  to  display  results  in  a  variety  of  for- 
mats, and  to  model  and  analyze,  often  quantita- 
tively, the  complex  three-dimensional  data.  We 
have  started  to  develop  a  computer-based  meth- 
odology to  extract  and  analyze  quantitatively  the 
large-  and  small-scale  motion  of  chromosomes  or 
structure  within  the  nucleus. 

Four-Dimensional  Optical  Microscopy 

We  have  continued  to  study  the  structure  of  the 


cellular  nucleus  in  living  Drosophila  embryos. 
Nuclei  were  labeled  by  microinjection  of  fluores- 
cent histones,  or  other  chromosomal  proteins. 
Nuclear  and  chromosomal  structures  were  fol- 
lowed throughout  the  cell  cycle  during  embry- 
onic development.  In  addition  to  discerning 
structural  changes,  we  can  now  infer  function. 

Topoisomerase  II — A  Key  Nuclear  Protein 

Our  studies  include  an  effort  to  understand  the 
role  of  various  proteins  in  the  organization  and 
dynamics  of  chromosomes.  We  have  therefore 
studied  the  distribution  and  dynamics  of  the  DNA 
strand-passing  (unknotting)  enzyme  topoisomer- 
ase II.  High-resolution  three-dimensional  imag- 
ing of  Drosophila  embryonic  chromosomes 
shows  a  heterogeneous  distribution  of  topoiso- 
merase II  along  the  chromosome.  At  metaphase 
and  anaphase,  the  enzyme  can  be  clearly  seen  to 
be  situated  adjacent  to  the  chromosome.  This 
suggests  that  its  localization  may  be  linked  to  its 
activity:  the  enzyme  may  concentrate  at  sites  of 
chromosome  condensation  and/or  segregation. 
These  data  argue  against  a  purely  structural  role 
for  the  enzyme. 

We  have  studied  the  dynamics  of  localization 
by  injecting  fluorescently  labeled  antibodies 
against  the  enzyme,  or  the  labeled  enzyme  itself, 
into  live  embryos  and  then  imaging  them  by  our 
three-dimensional  microscopy  as  a  function  of 
time.  The  resulting  time-lapse  movies  have 
shown  that  the  concentration  of  nuclear  topoiso- 
merase II  changes  dramatically  throughout  the 
cell  cycle.  The  highest  levels  occur  in  late  inter- 
phase, the  lowest  levels  in  telophase.  A  very  dy- 
namic fibrillar  complex  is  evident  during  inter- 
phase. Experiments  that  will  disclose  the 
functional  relevance  of  this  structure  are  in 
progress. 

A  Molecular  Dissection 
of  the  Nuclear  Periphery 

Recently  we  showed  that  the  lamin  proteins  of 
the  nuclear  envelope  (NE)  form  a  highly  discon- 
tinuous network  in  somatic  interphase  nuclei. 
Several  obvious  questions  arise.  First,  where  are 


361 


Three-Dimensional  Structure  of  Eukaryotic  Chromosomes 


the  other  known  components  of  the  nuclear  pe- 
riphery (pore  complexes,  chromatin)  relative  to 
this  network?  Second,  what,  if  anything,  occupies 
these  large,  lamin-empty  regions?  Third,  how  are 
these  structures  assembled  as  the  NE  re-forms 
during  telophase? 

Chromatin  in  the  nuclear  periphery  displays  an 
^mteresting  structural  paradox  in  that  a  large  frac- 
tion appears  to  be  aligned  beneath  the  lamin  net- 
work, but  with  very  little  contacting  lamins  di- 
rectly. The  majority  of  it  seems  to  be  at  a  distance 
of  about  0.2  /xm.  This  result  is  consistent  with 
much  indirect  evidence  for  a  strong  interaction 
between  chromatin  and  the  nuclear  lamina,  but 
strongly  suggests  that  a  direct  physical  contact  is 
not  involved. 

We  injected  lamins  and  lamin-specific  mono- 
clonal antibody  Fab  fragments,  both  fluores- 
cently  labeled,  into  early  Drosophila  embryos  to 
study  four-dimensional  lamin-NE  dynamics.  In 
these  experiments  the  embryos  develop  normally 
and  hatch  on  time.  We  observe  a  highly  discon- 
tinuous lamin  network  in  vivo,  with  interlamin 
fiber  spacings  at  least  as  large  as  those  observed  in 
fixed  samples.  From  these  experiments,  new 
four-dimensional  data,  spanning  prophase  to 
metaphase  in  the  cell  cycle,  show  a  surprisingly 
complex  series  of  lamin  structural  rearrange- 
ments. Lamins  do  not  completely  disassemble 
and  disperse  at  the  onset  of  mitosis,  but  remain 
well  localized  with  complex  structural  dynamics 
until  well  into  the  mitotic  process.  Further  lamin 
structural  changes  take  place  at  anaphase  and 
telophase. 

These  studies  emphasize  that  lamins  appear  es- 
sential for  the  nuclear  structural  reorganizations 
that  take  place  at  all  points  of  the  cell  cycle.  If, 
however,  we  inject  fluorescently  labeled  inter- 
phase lamins,  a  very  different  picture  results. 
Arrested  nuclear  structures  leading  to  chromo- 
somal/nuclear aggregates  are  seen.  These  studies 
suggest  that  structural/functional  assays  will  be 
required  for  proper  interpretation  of  the 
biochemistry. 

Three-Dimensional  in  situ  Hybridization 

We  are  continuing  our  study  of  nuclear  organi- 
zation, using  three-dimensional  fluorescence  in 


situ  hybridization.  The  studies,  described  in  our 
previous  report,  were  performed  in  Drosophila 
embryos,  primarily  at  the  histone  gene  locus,  a 
500-kb,  tandemly  repeated  gene  cluster.  We  have 
extended  our  techniques  to  other  whole-mount 
tissues  from  Drosophila,  including  developing 
imaginal  tissues  (which  will  later  form  the  adult 
fly).  We  have  also  improved  our  hybridization 
protocols  and  can  now  detect  chromosomal 
probes  as  small  as  1 2  kb  with  high  signal-to-noise 
resolution.  Using  these  techniques,  we  are  in- 
vestigating the  arrangement  of  chromosomes 
throughout  the  cell  cycle  and  as  a  function  of 
development. 

A  focus  of  our  studies  is  the  question  of  homolo- 
gous chromosome  pairing  (one  chromosome 
from  the  male  parent,  the  other  from  the  female) . 
This  is  of  particular  interest  in  Drosophila  biol- 
ogy because  genetic  evidence  has  indicated  that 
different  alleles  at  certain  homologous  loci  can 
influence  one  another,  implying  that  the  loci 
communicate  in  some  fashion.  Such  phenomena 
have  been  termed  transvections,  or  more  gener- 
ally, "trans-sensing  effects."  By  determining  the 
nuclear  positions  of  the  histone  gene  cluster  in 
developing  embryos,  we  have  shown  that  the  ho- 
mologous chromosomes  bearing  this  locus  are  for 
the  most  part  spatially  distinct,  or  unpaired, 
throughout  most  of  early  embryonic  develop- 
ment, but  that  a  transition  occurs  just  prior  to 
cellularization,  a  distinct  time  point  in  develop- 
ment, after  which  the  locus  is  seen  to  be  paired  at 
high  frequency. 

We  are  currently  extending  this  analysis  to 
other  genetic  loci,  to  determine  whether  this  un- 
paired/paired transition  occurs  simultaneously  at 
all  chromosomal  positions.  We  have  preliminary 
evidence  that  loci  more  distant  from  the  centro- 
mere than  the  histone  locus  may  show  different 
pairing  behavior  in  early  embryos.  We  are  particu- 
larly interested  in  carrying  out  this  type  of  analy- 
sis for  loci  that  are  known  to  have  transvection  or 
trans-sensing  effects. 

This  general  methodology  has  a  number  of  po- 
tential applications  to  problems  of  cell  lineage, 
neural  architecture,  and  pattern  formation  in  de- 
velopment. We  are  pursuing  some  of  these  inter- 
ests in  collaboration  with  other  laboratories. 


362 


A  Molecular  Basis  of  Familial  Hypertrophic 
Cardiomyopathy 


Jonathan  G.  Seidman,  Ph.D. — Investigator 

Dr.  Seidman  is  also  Professor  of  Genetics  at  Harvard  Medical  School.  He  received  his  undergraduate  degree 
from  Harvard  University  and  his  Ph.D.  degree  from  the  University  of  Wisconsin-Madison,  where  he 
studied  with  William  McClain.  His  postdoctoral  studies  were  carried  out  in  Philip  Leder's  laboratory  at 
the  National  Institute  of  Child  Health  and  Human  Development. 


FAMILIAL  hypertrophic  cardiomyopathy  (FHC) 
is  a  heart  muscle  disorder  with  an  autosomal 
dominant  pattern  of  inheritance.  The  disease  is 
characterized  clinically  by  unexplained  myocar- 
dial hypertrophy  and  variable  symptomatology 
that  can  include  syncope,  arrhythmias,  conges- 
tive heart  failure,  and  sudden  death.  Diagnosis  in 
young  people  is  particularly  important.  The  inci- 
dence of  sudden  death  appears  higher  in  this 
group  and  can  occur  without  warning. 

Indeed,  hypertrophic  cardiomyopathy  is  one  of 
the  most  common  autopsy  findings  among  young 
athletes  who  die  suddenly.  The  large  majority  of 
these  were  undiagnosed  previously.  Diagnosis  in 
this  age  group  may  be  particularly  difficult,  since 
the  diagnostic  clinical  and  echocardiographic  cri- 
teria may  not  be  manifest  until  adulthood. 

Because  mutations  in  the  cardiac  myosin 
heavy-chain  (MHC)  genes  were  implicated  as  the 
cause  of  FHC  in  two  families,  we  decided  to  un- 
dertake a  direct  analysis  of  these  genes  in  affected 
individuals  from  other  families.  During  the  past 
year  we  spent  considerable  effort  attempting  to 
find  in  24  unrelated  families  the  cardiac  MHC 
mutations  that  cause  FHC. 

We  had  previously  demonstrated  that  most 
FHC  mutations  are  missense  or  point  mutations  in 
the  MHC  genes.  We  have  successfully  employed  a 
variety  of  techniques  in  the  detection  of  missense 
mutations  within  genes.  Most  of  these  are  based 
upon  amplification  of  genomic  DNA  sequences 
and  analyses  of  individual  exons.  Application  of 
these  approaches  to  the  study  of  FHC  mutations 
was  more  difficult  because  the  ^  MHC  polypep- 
tide is  encoded  in  40  exons,  and  hence  40  inde- 
pendent analyses  are  required  to  examine  the  en- 
tire gene.  Furthermore,  FHC  is  an  autosomal 
dominant  disorder,  and  affected  individuals  are 
heterozygous,  bearing  one  mutated  and  one  nor- 
mal gene.  Genomic  analyses  may  fail  to  detect 
deletions  of  entire  exons  or  mutations  that  al- 
tered gene  splicing  because  of  the  presence  of 
one  normal  gene. 

Access  to  messenger  RNA  in  which  intronic  se- 
quences have  been  excised  would  overcome 
these  limitations  and  allow  analysis  of  coding  re- 


gions in  a  more  rapid  and  convenient  manner. 
Although  MHC  mRNAs  are  abundant  in  the  heart, 
expression  elsewhere  is  low  and  restricted  to  se- 
lected fibers  in  slow-twitch  skeletal  muscle.  Nor- 
mal and  mutant  MHC  sequences  were  detected 
in  RNA  transcripts  from  peripheral  lympho- 
cytes and  lymphocyte  cell  lines  transformed  by 
Epstein-Barr  virus  (EBV) .  This  finding  permitted 
examination  of  (8  MHC  mRNA,  even  though  car- 
diac tissue  was  not  available. 

Seven  different  ^  cardiac  MHC  mutations  were 
found  among  24  unrelated  FHC  probands.  Four 
mutations  were  identified  in  two  or  more  fami- 
lies. One  of  these,  the  Arg453Cys  mutation,  prob- 
ably occurred  independently  in  families  B  and  E, 
because  only  family  B  also  contains  a  hybrid  a/jS 
cardiac  MHC  gene  on  the  same  chromosome. 
Whether  other  mutations  that  are  shared  by  appar- 
ently unrelated  individuals  arose  independently 
within  mutational  hot  spots  or  represent  a 
founder  mutation  is  uncertain.  Characterization 
of  other  families  should  also  elucidate  whether 
the  mutations  that  can  cause  the  FHC  phenotype 
are  restricted  in  number. 

The  identification  of  seven  different  mutations 
in  a  disease  with  significant  morbidity  and  pre- 
mature death  suggests  that  many  of  these  are  of 
relatively  recent  origin  in  human  evolution. 
Since  FHC  mutations  are  not  likely  to  provide  a 
selective  advantage,  but  have  not  been  lost  in  the 
population,  they  probably  reflect  a  high  inci- 
dence of  new  mutational  events  in  the  (8  cardiac 
MHC  gene. 

The  natural  history  of  FHC  is  quite  variable, 
and  diagnostic  tests  have  been  unable  to  identify 
those  with  a  more  serious  prognosis  or  those  at 
risk  for  sudden,  unexpected  death.  To  determine 
whether  particular  ft  cardiac  MHC  gene  muta- 
tions correlate  with  clinical  outcome,  we  com- 
pared several  indices  with  genotype.  Data  from 
families  with  the  same  mutation  are  pooled. 
Disease-related  deaths  were  infrequent  in  fami- 
lies with  the  Val606Met  mutations  as  compared 
with  the  Arg249Gln,  Arg403Gln,  or  Arg453Cys 
mutations.  While  the  incidence  of  disease-related 
deaths  in  individuals  with  the  Arg249Gln  muta- 


363 


A  Molecular  Basis  of  Familial  Hypertrophic  Cardiomyopathy 


tion  is  similar  to  that  associated  witli  other  muta- 
tions, the  average  age  at  death  is  significantly 
older  for  affected  individuals  within  this  family. 

To  assess  cumulative  survival  of  affected  indi- 
viduals with  respect  to  age,  we  compared  sur- 
vival curves  of  FHC  families  with  five  different 
mutations  for  which  sufficient  numbers  of  af- 
fected individuals  (alive  or  deceased)  were  avail- 
able. These  analyses  confirmed  that  persons  with 
the  Val606Met  mutation  survive  longer  than 
those  with  the  Arg453Cys  or  Arg403Glu 
mutations. 

The  Arg249Glu  mutation  appears  to  produce 
an  intermediate  phenotype.  Survival  in  these  indi- 
viduals is  better  than  in  those  with  the  Arg453Cys 
or  the  Arg403Glu  mutation.  While  survival  ap- 
pears shorter  in  individuals  with  the  Arg249Glu 
mutation  than  in  those  with  the  Val606Met  muta- 
tion, this  difference  is  not  statistically  significant. 
Individuals  with  the  Arg453Cys  mutation  (with 
or  without  the  hybrid  gene)  and  those  with  the 
Arg403Cys  mutation  have  similar  life  expectan- 
cies, dying  prematurely. 

The  seven  different  mutations  are  clustered  in 
the  globular  head  of  the  polypeptide,  and  we 
postulate  that  the  defective  myosins  made  by 
these  genes  poison  myosin  function  by  impairing 
physiologic  interactions  with  other  contractile  el- 
ements. Six  of  seven  missense  mutations  affect 
the  charge  of  the  altered  residue.  Perhaps  the  fact 


that  the  Val606Met  mutation  does  not  alter  the 
net  charge  of  the  polypeptide  accounts  for  the 
better  survival  of  affected  individuals. 

Identification  of  multiple  disease-causing  mu- 
tations implies  that  mutational  events  within  the 
iS  cardiac  MHC  gene  are  not  uncommon  and  that  a 
number  of  FHC-causing  cardiac  MHC  mutations 
have  occurred  during  the  course  of  human  evolu- 
tion. A  high  frequency  of  myosin  mutation  may 
explain  the  relatively  high  incidence  of  sporadic 
(10-20  percent)  hypertrophic  cardiomyopathy. 

The  value  of  identifying  FHC  mutations  was  fur- 
ther demonstrated  by  analysis  of  a  large  family 
affected  by  mutation  Arg249Glu.  Related  adult 
family  members  were  clinically  evaluated  for 
FHC,  and  blood  samples  were  obtained  for  inde- 
pendent genetic  diagnosis.  The  clinical  and  ge- 
netic diagnoses  were  in  complete  concordance. 

Thirteen  children  (ages  2-20)  were  also  evalu- 
ated. Statistically,  half  should  have  been  affected, 
but  only  one  child  had  clinically  demonstrable 
FHC.  Genotype  analysis  of  these  children  re- 
vealed six  who  inherited  the  mutant  MHC  gene. 

These  data  underscored  the  insensitivity  of 
clinical  diagnostic  criteria  for  FHC  in  children 
and  young  adults.  Genetically  based  diagnoses  of 
FHC  permit  preclinical  diagnosis  and  should  fa- 
cilitate prenatal  diagnosis.  Furthermore,  the  abil- 
ity to  make  a  preclinical  diagnosis  in  families 
makes  possible  longitudinal  studies  of  disease  de- 
velopment and  interventional  trials. 


364 


I 

Time  course  of  the  calcium  concentration  in  \ 
the  dendrite  of  a  CAl  hippocampal  pyramidal 
neuron  in  response  to  synaptic  stimulation  as  a 
function  of  time,  running  horizontally.  The 
concentration  along  the  dendrite,  running  ver- 
tically, is  shown  in  false  color,  with  the  resting 
level  in  blue  and  the  highest  concentration  in 
red. 

Research  of  Richard  Adams  and  Terrence 
Sejnowski. 


366 


Computational  Neurobiology  of  Sensory 
Representations 


TerrenceJ.  Sejnowski,  Ph.D. — Investigator 

Dr.  Sejnowski  is  also  Professor  at  the  Salk  Institute  for  Biological  Studies  and  Professor  of  Biology  and 
Neuroscience  at  the  University  of  California,  San  Diego.  He  received  his  B.S.  degree  in  physics  from  Case 
Western  University  and  his  M.A.  and  Ph.D.  degrees  in  physics  from  Princeton  University.  He  was  a 
postdoctoral  fellow  with  Alan  Gelperin  in  the  Biology  Department  at  Princeton  and  with  Stephen  Kuffler 
at  Harvard  Medical  School,  where  he  studied  mechanisms  of  synaptic  transmission.  Dr.  Sejnowski  was  a 
member  of  the  faculty  of  the  Biophysics  Department  at  the  Johns  Hopkins  University  before  moving  to 
San  Diego.  He  and  Patricia  Churchland  have  recently  written  The  Computational  Brain,  a  book  on 
computational  neuroscience. 


WE  do  not  yet  understand  how  the  nervous 
system  enables  us  to  recognize  objects,  to 
learn  new  skills,  and  to  plan  actions.  The  discov- 
ery that  single  neurons  in  the  visual  system  can  be 
highly  selective  in  responding  to  visual  stimuli 
led  to  the  view  that  the  perception  of  complex 
objects  could  be  directly  linked  to  the  activity  of 
individual  neurons.  This  possibility  raises  a  num- 
ber of  questions,  such  as  what  degree  of  influ- 
ence a  single  neuron  can  have  on  behavior  and 
whether  there  are  enough  neurons  in  the  brain  to 
account  for  the  large  number  of  objects  that  can 
be  perceived. 

An  alternative  possibility  relies  on  populations 
of  neurons  to  represent  perceptual  states.  On  this 
account,  the  information  essential  to  the  repre- 
sentation of  an  object  is  distributed  over  a  large 
population  of  neurons.  It  is  difficult  to  imagine 
how  a  pattern  of  activity  in  a  large  number  of 
neurons  distributed  widely  throughout  the  brain 
could  be  used  to  recognize  an  object  and  serve  as 
the  input  for  motor  actions.  Computer  models 
incorporating  cellular  information  from  single- 
cell  recordings  and  constrained  by  psychological 
measurements  on  performance  can  help  to  orga- 
nize these  data  and  provide  a  conceptual  frame- 
work for  understanding  distributed  represen- 
tations. Such  models  are  being  used  to  explore 
how  the  visual  cortex  represents  the  three- 
dimensional  world,  how  this  representation  may 
arise  during  development,  and  how  the  informa- 
tion coded  by  these  neurons  might  be  used  to 
coordinate  actions  such  as  eye  movements. 

The  perception  of  depth  depends  upon  a  num- 
ber of  visual  cues,  but  only  one  of  them  relies  on 
the  slight  positional  shift  that  occurs  between  the 
diff^erent  viewpoints  of  the  two  eyes,  called  the 
image  disparity.  Neurons  in  the  first  cortical  stage 
of  vision  are  sensitive  to  disparity,  and  the  devel- 
opment of  this  sensitivity  is  dependent  on  binocu- 
lar vision  during  a  critical  period. 

In  the  adult  visual  cortex,  neurons  are  ob- 
served to  be  either  dominated  by  input  from  one 
eye  (monocular  cells)  or  relatively  balanced  with 


input  from  both  eyes  (binocular  cells) .  Further- 
more, binocular  cells  tend  to  be  stimulated  maxi- 
mally when  images  are  in  exact  correspondence 
in  both  eyes,  thus  preferring  zero  disparity,  and 
relatively  monocular  cells  tend  to  prefer  nonzero 
disparities.  We  have  simulated  the  development 
of  a  layer  of  cortical  cells  receiving  inputs  from 
both  eyes  and  show  how  such  a  relationship  be- 
tween ocularity  and  disparity  might  arise. 

The  key  feature  of  our  model  is  the  use  of 
correlations  of  activity  both  within  each  eye  and 
between  the  eyes.  We  assume  that  two  retinal 
cells  close  to  each  other  will  have  more  corre- 
lated activity  than  two  cells  far  apart.  Corre- 
sponding points  in  the  two  eyes  will  also  tend  to 
be  correlated,  since  they  will  look,  on  average,  at 
the  same  point  in  space.  However,  the  correla- 
tion between  the  eyes  will  also  be  spread  out  by 
convergent  and  divergent  eye  movement.  We  can 
simulate  visual  development  in  two  stages:  prena- 
tal, when  the  two  retinae  have  essentially  inde- 
pendent activities,  and  postnatal,  when  the  eyes 
are  open  and  have  correlated  activities.  By  vary- 
ing the  amount  of  development  that  occurs  in  the 
model  before  eye  opening,  we  can  show  that  a 
mixture  of  monocular  and  binocular  cells  arises 
with  the  observed  relationship  to  disparity. 

Disparity  provides  information  about  the  rela- 
tive positions  of  objects  in  space,  but  this  cue  is 
insufficient  to  recover  the  absolute  distance  of 
objects  from  the  viewer.  However,  the  distance  of 
an  object  from  the  viewer  can  be  computed  by 
combining  relative  depth  cues  with  other  infor- 
mation, such  as  eye  position.  We  have  developed 
a  network  model  to  explore  how  the  vergence  of 
the  two  eyes  (angle  between  the  two  lines  of 
sight)  and  the  binocular  disparity  could  be  com- 
bined to  represent  the  distance  to  an  object. 

Single  neurons  have  a  wide  range  of  disparity 
tuning  curves  that  are  broad  and  overlapping. 
Such  a  distributed  representation  of  disparity  was 
used  in  a  network  model  to  encode  the  inputs. 
The  network  was  trained  to  transform  disparity 
and  vergence  input  information  by  projections 


367 


Computational  Neurobiology  of  Sensory  Representations 


through  a  layer  of  hidden  units  to  an  output  layer 
that  represented  the  perceived  egocentric  depth 
of  the  object,  as  determined  by  psychophysical 
measurements.  The  disparity  tuning  curves  of  the 
hidden  units  were  similar  to  those  of  the  input 
units,  and  varying  the  vergence  did  not  change 
the  shape  of  the  tuning  curves;  however,  the  ver- 
-^'gence  did  modulate  their  amplitude. 

Similar  "gain  fields"  for  conjugate  eye  move- 
ments have  been  observed  in  the  posterior  pari- 
etal cortex,  a  region  of  the  brain  that  is  essential 
for  our  internal  representation  of  external  space. 
The  predictions  of  this  model  can  be  tested  by 
recording  single-unit  activity  in  the  cerebral  cor- 
tex of  awake  and  behaving  monkeys,  and  several 
laboratories  are  pursuing  these  experiments.  Pre- 
liminary results  support  the  model. 

Neurons  in  the  early  stages  of  visual  processing 
in  the  cerebral  cortex  are  organized  in  retinoto- 
pic  maps.  Thus  visual  features  are  arranged  in  a 
system  of  coordinates  that  is  based  on  the  posi- 
tion of  features  in  the  visual  field  of  the  retina 
rather  than  on  the  absolute  position  of  features  in 
space.  Psychological  experiments  provide  fur- 
ther evidence  that  simple  visual  features  such  as 
orientation  and  direction  of  motion  are  organized 
according  to  retinal  coordinates.  At  later  stages  of 
visual  processing,  the  receptive  fields  of  neurons 
become  very  large;  and  in  the  posterior  parietal 
cortex,  containing  areas  important  for  sensory- 
motor  coordination,  the  visual  responses  of  neu- 
rons are  modulated  by  both  eye  and  head  posi- 
tion. A  previous  model  of  the  parietal  cortex 
showed  that  the  modulation  of  the  neurons  ob- 
served there  is  consistent  with  a  distributed  spa- 
tial transformation  from  retinal  to  spatial  coordi- 
nates. Our  model  of  the  transformation  from 
disparity  to  distance  by  vergence  modulation  can 
be  considered  a  generalization  of  this  model  to 
include  the  third  dimension  of  space. 

All  these  models  assume  that  the  responses  of 
neurons  in  the  early  stages  of  visual  processing  in 
cerebral  cortex  depend  only  on  retinal  informa- 
tion and  not  on  the  direction  of  gaze.  Several  labo- 
ratories have  now  reported  that  eye  position  does 
in  fact  modulate  the  visual  response  of  many  neu- 
rons in  early  stages  of  visual  processing.  Further- 
more, this  modulation  appears  to  be  qualitatively 
similar  to  that  previously  reported  for  neurons  in 
the  parietal  cortex.  These  new  findings  suggest 
that  transformations  from  retinal  to  spatial  repre- 
sentations could  be  initiated  much  earlier  than 
previously  thought. 


We  have  used  network  models  to  study  the 
consequences  of  incremental  spatial  transforma- 
tions in  a  feedforward  hierarchy  of  cortical  maps. 
Our  model  shows  that  it  is  possible  for  visual  fea- 
tures to  be  encoded  in  spatial  coordinates  already 
at  very  early  stages  of  visual  processing.  We  call 
this  new  type  of  spatial  map  a  retinospatiotopic 
representation  and  are  exploring  its  counterin- 
tuitive properties.  The  model  makes  several  sur- 
prising predictions  that  we  are  testing  with  per- 
ceptual experiments  on  human  observers. 

The  primate  visual  system  is  very  good  at  com- 
plex motion-processing  tasks  such  as  tracking  a 
moving  object  against  a  textured  background 
under  a  variety  of  luminance  conditions.  In  order 
to  track  a  moving  object,  the  visual  system  must 
integrate  many  local  motion  estimates  from  many 
neurons,  each  with  limited  spatial  receptive 
fields.  No  single  neuron  has  the  information 
needed  to  estimate  the  velocity  of  the  object. 

We  have  developed  a  simple  model  for  motion 
processing  in  the  visual  areas  of  cortex  that  spe- 
cialize in  representing  motion.  The  model  as- 
sumes two  pools  of  filters  at  each  location  on  the 
visual  field:  one  pool  computes  estimates  of  mo- 
tion in  a  local  region  of  the  visual  field,  while  the 
other  estimates  the  relevance  or  reliability  of 
each  local  motion  estimate,  based  on  the  estimate 
itself  and  on  additional  information  from  the 
visual  scene.  Outputs  from  the  second  pool  can 
"gate"  the  outputs  from  the  first  pool  through  a 
gain-control  mechanism,  before  the  local  motion 
estimates  are  integrated  to  form  more-global  esti- 
mates. The  proposed  mechanism  of  gain  control 
is  consistent  with  measured  responses  of  cortical 
cells  under  conditions  of  interfering  motion  of 
transparent  stimuli. 

These  models  provide  representations  of  ob- 
jects in  space  that  are  highly  distributed.  We  also 
want  to  understand  how  these  distributed  repre- 
sentations can  be  used  to  direct  the  motor  system 
to  orient  toward  these  objects.  For  example,  mo- 
tion estimates  can  be  used  to  direct  the  eyes  to 
track  moving  objects,  and  distance  estimates  can 
be  used  to  guide  hand  movements  to  reach  out  for 
objects.  We  are  developing  models  of  motor  sys- 
tems in  the  brain  that  will  complement  these 
models  of  sensory  processing.  The  models  of  mo- 
tor control  are  based  on  networks  of  neurons  that 
include  feedback  connections,  which  makes 
them  highly  dynamic.  New  principles  of  neural 
processing  may  emerge  as  more-detailed  dynami- 
cal properties  of  neurons  are  incorporated  into 
these  models. 


368 


Adenovirus  as  a  Model  for  Control 
of  Gene  Expression 


Thomas  E.  Shenk,  Ph.D. — Investigator 

Dr.  Shenk  is  also  Elkins  Professor  of  Molecular  Biology  at  Princeton  University  and  Adjunct  Professor  of 
Biochemistry  at  the  Robert  Wood  Johnson  Medical  School,  University  of  Medicine  and  Dentistry  of  New 
Jersey.  He  received  his  Ph.D.  degree  in  microbiology  from  Rutgers  University  for  studies  with  Victor  Stollar, 
and  his  postdoctoral  training  with  Paul  Berg  at  Stanford  University.  Before  coming  to  Princeton,  he  was 
Assistant  Professor  of  Microbiology  at  the  University  of  Connecticut  Health  Center  and  then  Professor  of 
Microbiology  at  the  State  University  of  New  York  School  of  Medicine  at  Stony  Brook.  Dr.  Shenk  counts 
among  his  honors  the  Eli  Lilly  Award  in  Microbiology  from  the  American  Society  for  Microbiology  and  an 
American  Cancer  Society  Professorship. 


ADENOVIRUSES  are  widespread,  and  humans 
are  first  infected  when  quite  young.  Gener- 
ally the  infection  results  in  cold-like  symptoms 
and  resolves  without  complication.  Some  human 
adenoviruses,  however,  induce  a  variety  of  be- 
nign and  malignant  tumors  if  injected  into  a  rat  or 
hamster.  Since  these  viruses  contain  DNAand  are 
tumorigenic  under  certain  conditions,  they  are 
classified  as  DNA  tumor  viruses. 

Adenoviruses  can  be  propagated  easily  in  cul- 
tured cells.  When  human  cells  are  infected,  the 
approximately  30  viral  genes  are  expressed,  the 
viral  chromosome  is  replicated,  and  individual 
DNA  molecules  are  packaged  into  protein  shells 
to  produce  virus  progeny.  Since  viral  genes  are 
expressed  at  high  levels  compared  with  most  cel- 
lular genes,  and  since  this  expression  is  tightly 
regulated,  the  adenovirus  is  a  useful  probe  for 
studying  the  control  of  gene  expression. 

During  the  past  year,  much  of  our  effort  has 
focused  on  transcriptional  control  of  viral  gene 
expression.  The  first  viral  gene  to  be  expressed 
after  infection  of  a  cell  is  the  ElA  gene,  which 
encodes  a  protein  that  activates  expression  of  ad- 
ditional viral  genes.  The  ElA  protein  appears  to 
activate  transcription  (copying  of  genetic  infor- 
mation) through  several  physiologically  distinct 
mechanisms.  One  of  these  involves  a  cellular 
transcription  factor  that  we  have  termed  YY- 1 

We  first  identified  the  binding  site  for  YY- 1  in 
the  P5  transcriptional  control  region  of  adeno- 
associated  virus,  a  defective  virus  that  depends  on 
a  variety  of  adenovirus  gene  products  for  its  repli- 
cation. The  ElA  protein  activates  expression  of 
the  P5  control  region,  and  the  critical  sequence 
element  required  for  activation  is  a  DNA  segment 
constituting  the  binding  site  for  YY-1 .  To  investi- 
gate its  function,  this  site  was  inserted  upstream 
of  several  heterologous  promoters,  and  it  re- 
pressed their  activity.  The  repression  was  re- 
lieved in  the  presence  of  ElA  protein.  In  fact,  the 
protein  also  activated  transcription  through  the 
YY-1  binding  site.  The  combination  of  these  two 
effects,  relief  of  repression  and  activation,  in- 


duced transcription  by  a  factor  of  1,000  in  some 
test  genes.  Thus  the  combination  of  the  YY-1 
binding  site  and  ElA  protein  formed  a  powerful 
biological  on/off  switch. 

In  order  to  study  the  YY- 1  factor,  we  prepared 
some  from  cultured  human  cells  and  determined 
a  short  amino  acid  sequence  from  the  purified 
protein.  This  sequence  was  used  to  design  a  short 
probe  DNA,  which  enabled  us  to  identify  and  iso- 
late a  cDNA  clone  encoding  the  protein.  Se- 
quence analysis  of  the  clone  revealed  that  YY- 1  is 
a  4l4-amino  acid  protein  with  a  zinc  finger 
DNA-binding  motif.  Protein  was  expressed  from 
the  clone  and  shown  to  bind  specifically  to  the 
YY-1  recognition  site. 

The  binding  activity  of  YY- 1  was  altered  by  fus- 
ing it  to  the  DNA-binding  domain  of  the  yeast 
GAL4  protein.  This  approach  is  widely  used  to 
study  transcription  factors,  since  it  provides  the 
opportunity  to  direct  binding  of  the  factor  under 
study  in  a  highly  specific  fashion  to  a  test  gene 
construct  that  contains  the  yeast  GAL4  DNA- 
binding  site.  By  redirecting  the  factor  to  bind  to  a 
novel  site,  it  was  possible  to  study  the  activity  of 
the  fusion  protein  in  cells  that  contain  high  levels 
of  endogenous  YY-1 .  As  anticipated,  the  YY-1  fu- 
sion protein  repressed  transcription  of  the  test 
gene  in  the  absence  of  the  ElA  protein,  and  the 
repression  was  relieved  by  the  ElA  protein.  Muta- 
tional analysis  of  the  fusion  protein  has  demon- 
strated that  the  YY- 1  zinc  finger  domain  is  respon- 
sible for  its  ability  to  repress  transcription. 

Since  the  ElA  protein  would  not  be  expected 
to  cause  the  YY-1  fusion  protein  to  detach  from 
the  GAL4  DNA-binding  site,  it  appears  likely  that 
YY- 1  remains  bound  to  the  control  region  but  is 
somehow  altered  in  the  presence  of  the  ElA  pro- 
tein so  that  transcription  is  not  repressed.  Several 
experiments  indicate  that  the  ElA  protein  can 
bind  directly  to  YY-1.  For  example,  if  the  two 
proteins  are  mixed,  they  sediment  as  a  complex 
in  a  sucrose  gradient.  Furthermore,  radioactively 
labeled  ElA  can  bind  to  YY-1  that  has  been  sepa- 
rated from  a  complex  mixture  of  proteins  by  elec- 
trophoresis and  bound  to  a  membrane  filter. 


369 


Adenovirus  as  a  Model  for  Control  of  Gene  Expression 


We  are  currently  mapping  the  domains  on  ElA 
and  YY-1  through  which  they  interact.  The  loca- 
tion of  these  domains  might  provide  insight  to 
the  mechanism  by  which  the  viral  activator  alters 
YY-1  function.  We  are  also  searching  for  cellular 
proteins  that  might  interact  with  the  domain  of 
YY-1  responsible  for  repression. 
^  The  P5  promoter  of  the  adeno-associated  virus 
contains  two  binding  sites  for  YY- 1 .  The  site  that 
was  originally  studied  is  centered  about  60  base 
pairs  upstream  of  the  transcriptional  initiation 
site,  and  it  is  responsible  for  the  repression  and 
ElA-mediated  activation  discussed  above.  The 
second  site  is  centered  at  the  P5  transcriptional 
control  region.  Its  location  suggested  that  YY-1 
might  serve  an  initiator  function  in  addition  to  its 
repression  function.  Short  DNA  sequences 
surrounding  several  transcriptional  start  sites 
have  been  shown  to  be  capable  of  starting  tran- 
scription and  have  been  termed  initiator  ele- 
ments. These  sequences  can  direct  RNA  polymer- 
ase to  initiate  transcription  at  the  correct  start  site 
in  the  absence  of  any  other  binding  sites  for 
known  transcription  factors.  When  binding  sites 
for  additional  factors  are  added,  the  initiator  se- 
quences become  much  more  efficient. 

The  binding  site  for  YY- 1  proved  to  behave  as 
an  initiator  element.  Furthermore,  by  studying  its 
activity  in  cell-free  extracts  and  using  antibodies 


specific  for  YY- 1 ,  we  were  able  to  show  that  YY- 1 
protein  is  required  for  the  initiator  activity  dis- 
played by  its  binding  site.  Work  in  progress  to 
determine  whether  YY- 1  interacts  with  other  pro- 
teins in  the  initiation  complex  should  elucidate 
its  role  in  transcription. 

It  is  intriguing  that  YY- 1  (Yin  and  Yang  factor 
1 )  is  able  to  exert  opposite  effects  when  it  binds 
to  different  locations  within  a  promoter.  In  the 
adeno-associated  virus  promoter,  it  represses 
transcription  when  bound  upstream  of  the  start 
site,  and  it  contributes  in  a  positive  sense  to  the 
initiation  event  when  bound  at  the  start  site.  Our 
longer  term  goal  will  be  to  understand  how  the 
factor  can  mediate  these  two  different  activities. 

Finally,  it  is  important  to  note  that  in  addition 
to  activating  transcription  through  YY-1  and 
other  cellular  transcription  factors,  the  ElA  pro- 
tein can  oncogenically  transform  cells.  Almost 
certainly,  at  least  part  of  its  oncogenic  activity 
results  from  its  ability  to  bind  to  proteins  such  as 
the  cellular  retinoblastoma  protein,  a  recessive 
oncogene  product  whose  proper  function  is  re- 
quired for  appropriate  growth  regulation  of  cells. 
It  is  possible,  however,  that  interactions  of  ElA 
protein  with  transcriptional  regulatory  proteins 
also  contribute  to  oncogenesis.  Work  is  in 
progress  to  determine  whether  alteration  of 
YY-1  function  can  play  a  role  in  cellular 
transformation. 


370 


Growth  Control  of  Myeloid  Cells 


Charles  J.  Sherr,  M.D.,  Ph.D. — Investigator 

Dr.  Sherr  is  also  a  member  of  the  Department  of  Tumor  Cell  Biology  at  St.  Jude  Children 's  Research 
Hospital  and  Adjunct  Professor  of  Biochemistry  at  the  University  of  Tennessee  College  of  Medicine, 
Memphis.  He  received  his  medical  degree  and  his  Ph.D.  degree  in  immunology  from  New  York  University 
School  of  Medicine,  where  he  studied  with  Jonathan  Uhr.  After  a  pathology  residency  at  Bellevue  Hospital 
Center,  New  York,  he  joined  George  Todaro's  laboratory  at  the  National  Cancer  Institute,  where  he  began 
studies  on  retroviral  oncogenes.  After  10  years  on  the  staff  of  the  NCI,  Dr.  Sherr  relocated  to  St.  Jude 
Children 's  Research  Hospital. 


EACH  day  humans  produce  billions  of  blood 
cells,  which  enter  the  circulation  from  their 
sites  of  origin  in  the  bone  marrow.  The  majority 
are  red  cells  (erythrocytes) ,  which  transport  oxy- 
gen, and  the  remainder  are  white  cells  (leuko- 
cytes), which  play  a  vital  role  in  preventing  in- 
fection by  bacteria,  viruses,  and  other  parasites. 
Different  classes  of  white  cells  carry  out  special- 
ized functions:  macrophages  and  granulocytes  in- 
gest and  kill  microorganisms,  and  lymphocytes 
recognize  foreign  antigens  and  produce  antibod- 
ies to  combat  them. 

The  process  of  blood  cell  production  (hema- 
topoiesis)  is  regulated  by  a  group  of  protein 
growth  factors,  termed  colony-stimulating  fac- 
tors (CSFs)  or  interleukins.  These  factors  stimu- 
late the  precursors  of  mature  white  cells  to  form 
colonies  in  agar  composed  of  differentiated 
blood  cell  elements  and  v/ere  named  for  the  types 
of  colonies  they  produced.  For  example,  M-CSF 
(or  CSF-1)  specifically  induces  macrophage  colo- 
nies, G-CSF  promotes  granulocyte  development, 
and  GM-CSF  stimulates  the  growth  and  differen- 
tiation of  both  types  of  cells.  CSFs,  now  produced 
in  quantity  through  genetic  engineering  tech- 
niques, have  become  part  of  the  clinical  arma- 
mentarium and  are  efficacious  in  extrinsically 
regulating  blood  cell  production  and  in  height- 
ening host  defense  against  infection. 

Signal  Transduction  by  the  CSF-1  Receptor 

The  actions  of  CSFs  in  supporting  cell  prolifera- 
tion and  survival  are  mediated  through  their  bind- 
ing to  specific  receptors  expressed  on  the  sur- 
faces of  their  target  cells.  The  macrophage  CSF-1 
receptor  (CSF-1  R)  consists  of  an  extracellular 
growth  factor-binding  portion,  joined  through  a 
single  membrane-spanning  segment  to  an  intra- 
cellular enzymatic  domain.  Binding  of  CSF-1  to 
its  receptor  on  the  outside  of  the  cell  triggers  the 
activity  of  the  intracellular  enzymatic  moiety — a 
kinase — inducing  it  to  add  phosphate  molecules 
to  other  proteins.  These  phosphorylation  events 
modify  the  biochemical  behavior  of  multiple  tar- 
get proteins,  some  of  which  relay  signals  to  the 


cell  nucleus  that  alter  gene  expression,  DNA  syn- 
thesis, and  cell  division. 

The  ability  of  many  cell  types  to  respond  to 
CSF- 1  is  simply  determined  by  whether  they  ex- 
press its  receptor.  By  introducing  the  gene  en- 
coding CSF-IR  into  naive  cells,  we  can  sensitize 
them  to  the  stimulatory  effects  of  the  growth  fac- 
tor. Such  manipulations  have  allowed  us  to  study 
genetically  engineered  receptor  variants  for  their 
capacity  to  transduce  signals  for  cell  growth  or 
differentiation. 

One  strategy  is  to  alter  different  portions  of  the 
intracellular  domain  of  CSF-IR  in  order  to  pin- 
point structural  motifs  that  determine  its  interac- 
tion with  its  "downstream"  targets.  A  conse- 
quence has  been  the  development  of  receptors 
that  are  impaired  in  transducing  signals  through 
certain  pathways  but  not  others.  In  particular  in- 
stances the  introduction  of  other  complementing 
genes  into  cells  that  express  partially  defective 
receptor  mutants  has  reconstituted  full  receptor 
activity,  thereby  providing  genetic  evidence  for 
functional  relationships  between  different  gene 
products  in  signaling  pathways. 

Such  experiments  have  revealed  that  the  com- 
binatorial actions  of  target  proteins  that  bind  to, 
or  are  phosphorylated  by,  CSF- 1 R  can  in  part  de- 
termine the  specificity  of  the  biological  response 
in  different  cell  types,  thereby  influencing  deci- 
sions governing  cell  proliferation,  survival,  and 
fate. 

Oncogenic  Potential  of  CSF-IR 

CSF- 1 R  is  encoded  by  the  FMS  proto-oncogene 
and  can  be  converted  by  mutations  to  an  "onco- 
protein" capable  of  inducing  tumors.  Certain 
mutations  in  the  extracellular  domain  of  CSF-IR 
can  mimic  the  action  of  CSF-1  and  activate  the 
receptor  kinase  in  the  complete  absence  of  the 
growth  factor.  The  sustained  and  unregulated  sig- 
nals for  cell  growth  that  arise  from  this  class  of 
mutant  receptors  might  naturally  contribute  to 
malignancies  involving  macrophages  or  their 
bone  marrow  progenitor  cells. 

We  have  recently  used  a  prospective  genetic 


371 


Growth  Control  of  Myeloid  Cells 


approach  to  identify  sites  within  CSF-IR  that, 
when  mutated,  can  endow  the  receptor  with  on- 
cogenic activity.  By  randomly  mutagenizing  seg- 
ments of  FMS  and  screening  "libraries"  of  mu- 
tated genes  for  their  ability  to  induce  cell 
transformation,  we  identified  several  sites  in  the 
receptor  where  "activating  mutations"  occur. 
-With  such  information  in  hand,  it  is  now  possible 
to  search  for  the  presence  of  similar  genetic  le- 
sions in  the  FMS  genes  of  myeloid  leukemia  cells 
and  so  determine  whether  FMS  mutations  play  an 
etiologic  role  in  such  diseases. 

Role  of  CSF-1  in  Cell  Cycle  Progression 

After  proliferating  macrophages  complete  cell 
division  (mitosis,  or  M  phase),  they  enter  an  8-  to 
10-hour  gap  phase  (Gi),  during  which  they  pre- 
pare to  replicate  their  chromosomal  DNA.  The 
ensuing  period  of  DNA  synthesis  (S  phase)  lasts 
for  seven  hours,  and  once  DNA  replication  is 
complete,  the  cells  enter  a  second,  shorter  gap 
phase  (G2)  before  dividing  again  and  redistribut- 
ing copies  of  duplicated  chromosomes  to  each 
daughter  cell.  CSF-1  is  only  required  throughout 
Gi  for  cells  to  enter  S  phase,  and  once  DNA  syn- 


thesis begins,  macrophages  can  complete  cell  di- 
vision in  the  absence  of  the  growth  factor.  On  the 
other  hand,  the  requirement  for  persistent  CSF- 
IR-mediated  signals  throughout  the  entire  Gj 
interval  implies  that  the  expression  of  growth 
factor-responsive  genes  must  be  temporally  regu- 
lated over  an  8-  to  10-hour  period. 

Genetic  data  accumulated  through  studies  of 
yeasts  indicate  that  cell  division-cycle  genes 
called  Gi  cyclins  act  to  prepare  cells  for  DNA  syn- 
thesis. We  recently  isolated  a  novel  class  of  "D- 
type"  cyclins  from  mammalian  cells,  at  least  two 
of  which  are  differentially  regulated  by  CSF-1 
during  the  Gj  interval  of  the  macrophage  cell  cy- 
cle. Related  genes  are  expressed  in  other  cell  lin- 
eages, where  their  expression  is  governed  by  dif- 
ferent growth  factors.  Our  idea  is  that  these 
cyclins  control  progression  through  the  Gj  inter- 
val in  mammalian  cells  by  mechanistically  link- 
ing early  steps  in  growth  factor-mediated  signal 
transduction  with  the  timing  of  the  cell  cycle 
clock.  As  might  be  expected,  perturbations  in  the 
regulation  of  these  cyclins  occur  in  specific  types 
of  tumor  cells  and  thus  appear  to  contribute  to 
malignancy. 


372 


The  Role  of  Second  Messengers 
in  Ion  Channel  Regulation 


Steven  A.  Siegelbaum,  Ph.D. — Associate  Investigator 

Dr.  Siegelbaum  is  also  Associate  Professor  of  Pharmacology  in  the  Center  for  Neurobiology  and  Behavior, 
Columbia  University  College  of  Physicians  and  Surgeons.  He  received  his  A.B.  degree  in  biochemical 
sciences  from  Harvard  College  and  his  Ph.D.  degree  in  pharmacology  from  Yale  University,  studying  the 
role  of  calcium  in  cardiac  electrical  activity.  He  then  did  postdoctoral  research  with  David  Colquhoun  at 
University  College,  London,  and  with  Philippe  Ascher  at  the  Ecole  Normale  Superieure  in  Paris,  where  he 
studied  the  nicotinic  acetylcholine  receptor  ion  channel,  before  joining  the  faculty  of  Columbia  University. 
He  has  received  the  Herbert  J.  Kayden  Award  in  Biomedical  Science  of  the  New  York  Academy  of  Sciences. 


THE  electrical  activity  of  nerve  and  muscle 
cells  is  regulated  by  the  actions  of  hormones, 
neurotransmitters,  and  sensory  stimuli  such  as 
light,  odors,  and  pressure.  Regulation  of  neuro- 
nal activity  often  depends  on  the  production  of 
intracellular  second  messengers — small  metabo- 
lites such  as  cyclic  AMP,  cyclic  GMP,  and  various 
products  of  phospholipid  metabolism.  These  sec- 
ond messengers  then  act  to  alter  the  function  of 
ion  channels,  the  membrane  proteins  that  govern 
the  electrical  signaling  of  cells.  Previous  research 
in  our  laboratory  focused  on  the  role  that  regula- 
tion of  ion  channel  function  by  second  messen- 
gers plays  in  learning  and  memory.  Recently  we 
have  become  interested  in  the  role  of  second  mes- 
sengers in  olfactory  signal  transduction,  the  sub- 
ject summarized  below. 

The  second  messenger  that  was  first  found  to 
play  a  role  in  regulating  electrical  activity  was 
cyclic  AMP.  From  initial  studies  by  Earl  Suther- 
land and  his  colleagues,  a  number  of  hormones 
and  neurotransmitters  have  been  shown  to  act  by 
elevating  cAMP  concentrations  in  cells.  Later 
studies  by  Edwin  Krebs  and  his  colleagues 
showed  that  most  of  the  effects  of  cAMP  were  due 
to  the  activation  of  a  cAMP-dependent  protein  ki- 
nase (cAMP-PK),  which  phosphorylates  many 
types  of  proteins.  Over  the  past  several  years  it 
has  become  clear  that  neurotransmitters  can  alter 
the  activity  of  ion  channels  by  causing  the  produc- 
tion of  cAMP,  leading  to  the  activation  of  cAMP- 
PK,  which  can  then  directly  phosphorylate  ion 
channels.  In  general,  cAMP-dependent  actions 
are  relatively  slow;  they  require  several  seconds 
to  produce  changes  in  electrical  activity  because 
of  the  relatively  slow  rates  of  protein  phosphory- 
lation and  dephosphorylation.  Thus  phosphory- 
lation-dependent  second  messenger  actions  are 
generally  not  well  suited  to  mediating  rapid  neu- 
ronal signaling  that  occurs  during  fast  synaptic 
transmission  or  sensory  processing. 

Role  of  Second  Messengers  in  Olfactory 
Signal  Transduction 

Olfactory  signal  transduction  has  provided  neu- 


robiologists  with  an  intriguing  puzzle  on  several 
levels.  First,  how  does  the  olfactory  system  recog- 
nize and  discriminate  among  thousands  of  differ- 
ent odors?  Are  there  a  limited  number  of  recep- 
tors that  each  bind  many  hundreds  of  odorants,  or 
are  there  hundreds  of  receptors  that  are  each  spe- 
cific for  a  single  or  a  few  different  odorants?  Sec- 
ond, how  does  the  binding  of  an  odorant  to  its 
receptor  generate  an  electrical  signal  in  the  olfac- 
tory neuron?  Third,  how  does  our  olfactory  sys- 
tem enable  us  initially  to  detect  odors  at  very  low 
concentrations  and  yet  become  insensitive  to  the 
same  stimuli  after  several  minutes?  In  one  sense 
this  form  of  adaptation  is  the  simplest  form  of 
learning:  our  olfactory  system  "learns"  to  ignore 
a  certain  stimulus. 

Recently  some  of  the  puzzles  associated  with 
olfaction  began  to  be  solved.  Linda  Buck  and 
Richard  Axel  (HHMI,  Columbia  University  Col- 
lege of  Physicians  and  Surgeons)  identified  a  sur- 
prisingly large  gene  family  that  may  code  for 
hundreds  of  distinct  odorant  receptors  in  rat  ol- 
factory neurons,  providing  for  the  requisite  speci- 
ficity. The  discrepancy  between  the  slow  time 
course  of  most  cAMP-mediated  responses  and  the 
more  rapid  olfactory  signaling  was  resolved  when 
Tadashi  Nakamura  and  Geoffrey  Gold  reported  an 
ion  channel  in  toad  olfactory  neurons  that  was 
directly  activated  by  cAMP.  Thus  the  activation  or 
gating  of  the  channel  did  not  depend  on  the  rela- 
tively slow  processes  of  phosphorylation  and  de- 
phosphorylation, but  rather  seemed  to  be  due  to 
the  direct  binding  of  cAMP  to  the  channel.  This 
channel  was  activated  equally  well  by  cAMP  or 
cGMP. 

Properties  of  a  Cyclic  Nucleotide-gated 
Channel 

The  first  gene  for  a  cyclic  nucleotide-gated 
(CNG)  channel  to  be  cloned  came  from  photore- 
ceptors, where  the  channel  participates  in  photo- 
transduction  and  is  selectively  activated  by 
cGMP.  More  recently  several  laboratories  have 
cloned  the  genes  for  olfactory  neuron  CNG  chan- 
nels from  several  species.  In  collaboration  with 


373 


The  Role  of  Second  Messengers  in  Ion  Channel  Regulation 


Richard  Axel's  group,  we  have  been  studying  the 
properties  of  a  CNG  channel  cloned  from  olfac- 
tory neurons  of  catfish.  Our  goal  is  to  understand 
how  the  binding  of  cyclic  nucleotides  leads  to 
channel  activation  and  how  the  channel  itself 
may  participate  in  adaptation. 

The  gene  for  the  catfish  olfactory  channel  is 
"highly  homologous  to  olfactory  channel  genes 
from  other  species  and  to  the  gene  for  the  photo- 
receptor channel.  The  channel  is  expressed  selec- 
tively in  olfactory  neurons,  supporting  its  role  in 
olfactory  signal  transduction.  Like  the  rat  olfac- 
tory CNG  channel,  the  catfish  channel  is  also  acti- 
vated directly  by  both  cAMP  and  cGMP.  Unlike 
the  rat  channel,  however,  the  catfish  channel 
does  not  discriminate  between  cAMP  and  cGMP, 
suggesting  a  structural  difference  in  the  cyclic 
nucleotide-binding  sites.  Using  genetic  engineer- 
ing, we  are  trying  to  define  the  structural  bases 
for  these  differences. 

We  have  also  used  single-channel  recording  to 
measure  the  unitary  currents  that  flow  through  an 
open  CNG  channel.  We  find  that  the  probability 
of  a  channel  being  open  increases  with  rising  cy- 
clic nucleotide  concentration.  When  the  cloned 
channel  is  expressed  in  frog  oocytes,  it  requires 
relatively  high  concentrations  of  cAMP  or  cGMP 
(around  50  micromolar)  to  become  activated. 
Surprisingly,  the  same  channel  studied  in  its  na- 
tive environment,  the  catfish  olfactory  neuron, 
requires  20-fold  lower  cyclic  nucleotide  concen- 


trations for  activation.  This  difference  in  cyclic 
nucleotide  sensitivity  could  mean  that  the  oo- 
cytes fail  to  process  the  channel  correctly  (for 
example,  by  not  phosphorylating  it  properly)  or 
that  they  lack  some  important  channel-regulating 
protein.  Defining  the  factors  responsible  for  the 
discrepancy  between  the  cloned  and  native  chan- 
nels is  an  important  goal. 

In  other  work,  we  are  focusing  on  the  role  of 
the  channel  in  odor  adaptation.  When  the  olfac- 
tory CNG  channel  is  activated,  it  allows  calcium 
ions  to  enter  the  olfactory  neuron  from  the  out- 
side medium.  In  photoreceptors,  a  similar  influx 
of  calcium  underlies  visual  adaptation.  We  find 
that  intracellular  calcium  greatly  reduces  the  re- 
sponse of  the  olfactory  channel  to  cyclic  nucleo- 
tides. This  effect  appears  to  result  from  a  shift  in 
the  dose-response  curve  for  channel  activation  to 
higher  concentrations  of  cyclic  nucleotides.  The 
inhibitory  effect  of  calcium  occurs  at  physiologi- 
cal calcium  levels.  Moreover,  the  effect  is  not  due 
to  a  direct  action  of  calcium  on  the  channel  but 
rather  appears  to  involve  an  intermediate  regula- 
tory protein  that  is  loosely  associated  with  the 
channel.  Thus  calcium  acts  as  a  negative  feedback 
regulator  of  olfactory  responses. 

Thus  the  olfactory  system  provides  a  useful 
model  for  studying  neuronal  signal  transduction 
and  neuronal  plasticity.  Studies  on  the  molecular 
bases  of  these  phenomena  should  provide  us  with 
insight  into  many  of  the  basic  mechanisms  con- 
trolling nerve  cell  behavior. 


374 


Chemistry  of  Cellular  Regulation 


Paul  B.  Sigler,  M.D.,  Ph.D. — Investigator 

Dr.  Sigler  is  also  Professor  of  Molecular  Biophysics  and  Biochemistry  at  Yale  University.  He  studied 
chemistry  at  Princeton  University  and  received  his  M.D.  degree  from  Columbia  University.  He  then  spent 
two  years  as  a  house  officer  in  the  Department  of  Medicine  at  Columbia-Presbyterian  Medical  Center, 
New  York.  He  began  his  work  on  crystallography  with  David  Davies  at  NIH.  He  studied  as  a  Helen  Hay 
Whitney  Fellow  at  the  MRC  Laboratory  of  Molecular  Biology  in  Cambridge,  England,  where  he  received 
his  Ph.D.  degree  in  biochemistry.  Before  accepting  his  present  position.  Dr.  Sigler  was  Professor  of 
Biochemistry  and  Molecular  Biology  at  the  University  of  Chicago.  Dr.  Sigler  was  recently  elected  to  the 
National  Academy  of  Sciences. 


MY  colleagues  and  I  continue  to  pursue  our 
interest  in  the  molecular  mechanism  of  reg- 
ulation at  the  level  of  transcription  and  trans- 
membrane signaling.  Our  approach  is  to  use 
high-resolution  x-ray  crystallography  to  deter- 
mine the  structure  of  the  relevant  macromole- 
cules  and  their  assemblies.  We  infer  chemical 
mechanisms  from  these  structures  and  test  them 
with  biochemical  and  physicochemical  experi- 
ments and  with  directed  mutagenesis. 

Transcriptional  Regulation 

In  the  area  of  transcriptional  control,  our  pri- 
mary focus  remains  the  mechanism  by  which 
DNA-binding  regulatory  proteins  are  targeted  to 
their  responsive  genes.  Our  earliest  and  best- 
studied  experimental  system  has  been  the  alloste- 
ric  regulation  of  the  trp  repressor  and  the  forma- 
tion of  the  trp  repressor-operator  complex. 
Having  defined  the  stereochemistry  of  the 
protein-DNA  interface,  we  are  now  extending  the 
study  (partly  in  collaboration  with  Julian  Sturte- 
vant,  Yale  University)  to  include  an  analysis  of 
the  thermodynamic  parameters  underlying  the 
trp  repressor's  ability  to  bind  selectively  to  the 
trp  operator. 

Crystallographic  structural  analyses  have  been 
extended  to  the  mechanism  by  which  steroid  re- 
ceptors bind  selectively  to  their  DNA  targets.  The 
first  complex  in  this  series  (in  collaboration  with 
Len  Freedman  and  Keith  Yamamoto,  University 
of  California,  San  Francisco)  shows  the  DNA- 
binding  domain  in  complex  with  its  idealized  tar- 
get, the  symmetrical  glucocorticoid  response  ele- 
ment. It  shows  how  the  two  zinc  fingers  of  the 
domain  interact  with  each  other  to  form  a  unified 
globular  domain  and  how  the  domain  makes 
chemical  contacts  with  the  bases  that  identify  the 
"half  sites"  of  the  symmetrical  response  element. 
It  also  shows  a  surprisingly  novel  mechanism  by 
which  the  receptor  recognizes  the  invariant 
three-base  pair  spacing  between  the  half  sites. 

Interactions  with  the  DNA  phosphates  stabilize 
a  change  in  the  protein's  structure  that  causes  it 


to  form  a  firm  dimer.  In  so  doing,  the  DNA-bind- 
ing surfaces  of  each  subunit  are  placed  in  perfect 
registry  with  the  complementary  surfaces  of  the 
DNA  target's  half  sites.  Thus  the  DNA  itself  helps 
form  a  dimer  that  discriminates  between  DNA  se- 
quences that  have  the  correct  spacings  between 
their  half  sites  and  those  that  do  not.  These  same 
DNA-induced  conformational  changes  are  also 
likely  to  potentiate  functional  contacts  between 
the  receptor  and  other  components  of  the  tran- 
scription initiation  assembly. 

Work  is  under  way  to  examine  the  arrangement 
by  which  the  glucocorticoid  receptor  contacts 
other  transcription  factors  on  "composite"  regu- 
latory sites.  We  are  also  examining  the  way  other 
members  of  this  steroid  receptor  superfamily  in- 
teract with  their  respective  response  elements. 
Only  through  a  comparative  study  can  we  build  a 
reliable  picture  of  the  targeting  mechanism. 

Most  recently  we  have  solved  (in  collaboration 
with  Laimonis  Laimins,  HHMI,  University  of  Chi- 
cago) the  structure  of  the  complex  between  the 
E2  transcription  factor  of  bovine  papilloma  virus 
and  the  DNA  sequence  (enhancer)  to  which  it 
binds.  E2  is  involved  in  replication  of  the  viral 
genome  and  controls  expression  of  the  genes  re- 
sponsible for  transformation  of  cells  infected 
with  this  oncogenic  virus.  The  structure  has  been 
refined  at  an  unprecedented  level  of  detail  (1.7 
A)  and  exhibits  a  barrel-like  dimeric  architecture 
that  has  never  been  seen  before  in  any  protein 
structure,  let  alone  in  a  DNA-binding  domain. 
The  DNA  is  severely  bent  over  this  barrel  as  it 
forms  specific  contacts  with  the  protein.  These 
results  are  now  being  correlated  with  genetic  in- 
formation produced  by  others. 

Several  other  specific  DNA  complexes  of  tran- 
scriptional regulatory  proteins  are  in  earlier 
stages  of  crystallographic  analysis.  A  particularly 
interesting  one  is  the  arg  repressor,  which  regu- 
lates the  expression  of  arginine  biosynthetic 
genes.  It  is  unique  because  it  is  a  hexamer  of 
identical  subunits.  Its  target  is  also  unusual,  in 
that  it  is  two  symmetrical  1 8-base  pair  sequences 
whose  symmetry  axes  are  separated  by  2 1  base 


375 


Chemistry  of  Cellular  Regulation 


pairs,  or  exactly  two  helical  turns  of  DNA.  Our 
studies  (in  collaboration  with  Werner  Maas,  New 
York  University)  support  the  inference  that  the 
operator  must  be  sharply  bent  as  it  wraps  around 
the  hexamer.  In  view  of  the  distortion  that  this 
protein  can  introduce  into  DNA,  it  is  interesting 
that  the  arg  repressor  has  been  implicated  as  a 
-binding  "scaffold"  in  the  site-specific  recombina- 
tion process  that  resolves  fused  ColEl  plasmids. 
The  architecture  of  this  protein-DNA  complex 
should  give  insight  into  the  DNA  topology  in- 
volved in  site-specific  recombination. 

Transmembrane  Signaling 

In  the  area  of  transmembrane  signaling,  we 
have  recently  solved  (in  collaboration  with  Jeff 
Browning  of  Biogen)  the  structure  of  the  human 
secretory  phospholipase  A2  that  is  associated  with 
both  acute  and  chronic  inflammation.  This  study 
contrasts  the  free  enzyme  with  the  enzyme  in  a 
complex  with  a  transition-state  analogue  and 
shows  that  the  mechanism  of  this  enzyme  con- 
forms to  what  we  have  proposed  earlier  for  inter- 
facial  catalysis.  This  structure  could  provide  a 
firm  stereochemical  platform  for  anti-inflamma- 
tory drug  design. 


In  order  to  function,  this  class  of  enzymes  must 
attach  itself  firmly  to  the  surface  of  the  mem- 
brane. Others  have  shown  that  the  attachment  of 
the  enzyme  to  the  membrane  surface  is  primarily 
due  to  electrostatic  forces.  Our  calculations  (in 
collaboration  with  Barry  Honig,  Columbia  Uni- 
versity) of  the  charge-potential  distribution  for  a 
large  series  of  phospholipase  A2  structures  ac- 
count nicely  for  this  effect. 

Recently  our  attention  has  turned  to  the  trans- 
membrane signal  cascade  that  employs  seven- 
helical  transmembrane  receptors,  G  proteins, 
and  target  enzymes  involved  in  either  the  synthe- 
sis or  cleavage  of  phosphodiester  bonds  (e.g., 
adenyl cyclase,  phospholipase  C,  cGMP  phospho- 
diesterase) .  Because  of  the  immediate  availability 
of  large  amounts  of  pure  protein,  we  have  chosen 
to  work  first  on  the  bovine  visual  receptor  sys- 
tem. Our  first  goal  is  to  solve  a  recently  obtained 
well-ordered  crystal  form  of  T^^  •  OTP,  the  solu- 
ble transducer  of  the  activation  signal.  We  hope 
to  extend  this  work  to  include  complexes  of 
Tg„  with  the  activated  receptor  (rhodopsin) 
and  its  target  molecule,  the  7-subunit  cGMP 
phosphodiesterase . 


376 


The  Mitochondrial  Genome  of  Trypanosomes 


Larry  Simpson,  Ph.D. — Investigator 

Dr.  Simpson  is  also  Professor  of  Cell  and  Molecular  Biology  at  the  University  of  California,  Los  Angeles. 
He  received  his  B.A.  degree  in  biology  from  Princeton  University  and  his  Ph.D.  degree  in  molecular 
parasitology  with  William  Trager  at  the  Rockefeller  University.  His  postdoctoral  training  was  with  Maurice 
Steinert  at  the  Free  University  of  Bruxelles. 


THE  kinetoplastids  represent  a  large  group  of 
parasitic  protozoa  that  are  tiie  causal  agents 
for  a  variety  of  human  and  animal  diseases,  in- 
cluding African  sleeping  sickness,  Chagas'  dis- 
ease, and  leishmaniasis.  There  are  no  vaccines  for 
any  of  these,  and  chemotherapies  are  either  non- 
existent or  inadequate.  As  cells,  the  kinetoplast- 
ids represent  one  of  the  most  ancient  lineages  of 
the  eukaryotic  kingdom  and  thereby  have  many 
novel  physical  and  biochemical  features.  Some 
species,  such  as  Crithidia,  are  parasitic  in  only  a 
single  invertebrate  host.  Others,  such  as  Leish- 
mania,  Trypanosoma,  and  Pfoytomonas,  are  par- 
asitic in  both  an  invertebrate  and  a  vertebrate  (or 
plant)  host. 

Kinetoplast  DNA 

The  mitochondrial  genome  of  these  cells, 
which  is  known  as  kinetoplast  DNA,  consists  of  a 
single  giant  network  containing  approximately 
10,000  minicircles  and  20-50  maxicircles,  all 
linked  together  by  catenation.  The  maxicircles 
comprise  a  subset  of  mitochondrial  genes  that  en- 
codes two  small  rRNAs,  three  subunits  of  cy- 
tochrome oxidase,  cytochrome  b,  four  subunits 
of  NADH  dehydrogenase,  and  three  yet  unidenti- 
fied proteins.  No  tRNAs  appear  to  be  encoded  by 
the  mitochondrial  genome  and  therefore  must  be 
imported  into  the  organelle  by  some  yet  to  be 
defined  mechanism.  The  function  of  the  minicir- 
cle  was  unknown  until  recently. 

RNA  Editing 

RNA  editing  is  a  post-transcriptional  process  in 
which  uridine  (U)  residues  are  inserted  and  de- 
leted from  coding  regions  of  the  primary  tran- 
scripts of  several  maxicircle  "cryptogenes."  The 
extent  of  editing  varies  from  a  few  U  residues  to 
hundreds,  at  hundreds  of  sites  throughout  the  en- 
tire mRNA  (pan-editing) .  The  function  of  editing 
is  to  create  translatable  mRNAs  encoding  mito- 
chondrial proteins. 

The  extent  of  editing  of  specific  genes  varies 
from  species  to  species.  For  example,  the  NADH 
dehydrogenase  subunit  7  mRNA  is  internal  and 
5'-edited  in  Leishmania  tarentolae  but  pan- 


edited  in  Trypanosoma  brucei.  We  have  re- 
cently shown  that  at  least  one  G-rich  intergenic 
region  in  both  species  encodes  a  transcript  that  is 
pan-edited  to  produce  ribosomal  protein  SI  2  for 
the  mitochondrial  ribosome.  It  is  likely  that  five 
other  G-rich  regions  represent  additional  pan- 
edited  cryptogenes,  which  would  bring  the  total 
mitochondrial  structural  gene  content  in  these 
cells  to  17,  of  which  12  are  cryptogenes. 

Mechanism  of  RNA  Editing 

We  discovered  in  1990  that  maxicircles  also 
encode  another  class  of  RNAs — the  guide  RNAs 
(gRNAs) — which  contain  the  necessary  se- 
quence information  for  the  edited  genes.  These 
are  small  RNAs,  which  can  form  perfect  duplex 
hybrids  with  edited  mRNAs,  both  within  the 
edited  region  and  3'  of  the  edited  region,  pro- 
vided G-U  base  pairs  are  allowed.  The  gRNAs  also 
have  nonencoded  3'  oligo-[U]  tails  ranging  in 
length  from  5  to  28  nucleotides.  We  then  discov- 
ered that  gRNAs  are  also  encoded  in  the  minicir- 
cles, finally  providing  a  genetic  role  for  these 
enigmatic  molecules. 

We  have  proposed  two  models  for  the  involve- 
ment of  gRNAs  in  editing.  In  both  models  the  ini- 
tial interaction  of  the  gRNA  and  the  mRNA  is  the 
formation  of  an  anchor  duplex  just  downstream 
of  the  pre-edited  region.  The  "enzyme  cascade" 
model  invokes  an  endonuclease  that  cleaves  at 
the  first  mismatch,  a  terminal  uridylyl  transferase 
that  adds  a  U  to  the  3'-hydroxyl,  and  an  RNA  ligase 
joining  the  two  mRNA  fragments.  In  the  "transes- 
terification"  model,  the  added  U's  are  derived 
from  the  oligo-[U]  tail  of  the  gRNA  by  means  of 
two  successive  transesterifications.  In  both  mod- 
els the  gRNA  provides  an  internal  guide  sequence 
to  specify  the  precise  addition  and  deletion  of  U's 
at  specific  sites. 

A  major  goal  is  to  obtain  an  in  vitro  system  in 
which  the  entire  editing  process  occurs  in  an  ac- 
curate manner,  thereby  allowing  a  biochemical 
dissection  and  reconstitution  of  the  underlying 
enzymatic  machinery.  We  have  recently  shown 
that  at  least  the  initial  step  of  RNA  editing — the 
formation  of  gRNA-mRNA  chimeric  molecules — 


577 


The  Mitochondrial  Genome  of  Trypanosomes 


can  be  performed  in  vitro  by  incubation  of  syn- 
thetic RNAs  with  a  mitochondrial  extract  and  that 
this  process  is  anchor-dependent. 

The  work  on  RNA  editing  was  supported  by  a 
grant  from  the  National  Institutes  of  Health. 

Evolutionary  Considerations 

'  The  existence  of  split  genes  is  not  novel,  but 
the  existence  of  genes  in  which  the  RNA  product 
of  one  gene  contains  information  for  the  correc- 
tion of  coding  sequences  within  transcripts  of  the 
other  gene  is  novel.  If  the  transesterification 
model  proves  to  be  correct,  this  would  suggest 
that  RNA  editing  is  on  the  same  evolutionary 
pathway  as  RNA  splicing  and  may  in  fact  repre- 
sent a  primitive  type  of  trans-splicing  with  partial 
integration.  The  trypanosome  type  of  RNA  editing 
has  not  yet  been  reported  in  other  organisms,  but 
other  types  of  modifications  of  the  sequences  of 
coding  RNAs  have  been  observed  in  organisms  as 
diverse  as  plants  and  humans.  C  to  U  changes  at 
specific  sites  occur  in  transcripts  of  several  hu- 
man genes  and  also  in  transcripts  from  many  plant 
mitochondrial  and  chloroplast  genes.  The  deter- 
mination of  site  specificity  for  these  multiple 
transitions  is  completely  unknown. 

The  evolution  of  RNA  editing  in  the  trypano- 
somes is  interesting  in  itself.  To  investigate  it,  we 
plan  to  examine  representatives  of  more-primi- 
tive kinetoplastid  lineages. 

The  Kinetoplast  Genome  as  a  Target 
for  Disease  Diagnosis 

We  have  shown  that  the  kinetoplast  DNA  mini- 
circle  molecule  of  Trypanosoma  cruzi,  the 
causal  agent  of  Chagas'  disease,  is  an  appropriate 
multicopy  target  for  detection  and  strain  classifi- 
cation of  the  parasite  in  patients,  animals,  or  in- 
sects. The  minicircle  of  T.  cruzi  consists  of  four 
conserved  regions  and  four  variable  regions. 


There  are  multiple  minicircle  sequence  classes  in 
a  network,  and  minicircles  in  different  strains  are 
very  polymorphic.  Primers  to  the  conserved  re- 
gion were  used  for  polymerase  chain  reaction 
(PGR)  amplification  of  minicircle  fragments 
from  either  the  conserved  region  or  the  variable 
region.  This  method  is  being  developed  into  a 
diagnostic  procedure  to  detect  small  numbers  of 
parasites  in  blood  of  chronically  ill  patients  and 
to  classify  the  strain  of  the  parasite. 

We  recently  developed  a  method  to  recover 
blood  from  patients  and  preserve  total  DNA  with- 
out refrigeration.  The  DNA  is  then  cleaved  with  a 
chemical  nuclease  to  release  linearized  minicir- 
cles, and  the  fragments  are  amplified  with  spe- 
cific primer  sets.  We  are  attempting  to  expand 
this  diagnostic  procedure  by  developing  a  multi- 
plex PCR-based  assay  for  the  detection  of  multi- 
ple blood-borne  viral  and  parasitic  disease 
agents. 

The  kinetoplast  DNA  minicircle  has  also 
proved  to  be  an  appropriate  target  for  the  diagno- 
sis of  other  pathogenic  kinetoplastid  infections, 
such  as  those  caused  by  Leishmania  species. 

Grants  from  the  World  Health  Organization 
and  the  Rockefeller  Foundation  supported  this 
diagnostic  work. 

RNA  Editing  as  Possible  Target 
for  Intervention 

Whenever  a  parasite  has  a  biochemical  path- 
way that  is  unique  to  the  parasite  and  not  found  in 
the  human  host,  there  is  a  potential  for  selective 
chemotherapy.  As  RNA  editing  is  dissected  bio- 
chemically, we  plan  to  examine  the  possibility  of 
inhibiting  components  of  the  editing  machinery 
in  the  parasites  without  affecting  the  host.  This 
may  open  up  a  new  direction  for  selective  che- 
motherapy of  the  many  trypanosome-caused 
diseases. 


378 


Regulation  of  Gene  Activity  During 
B  Cell  Development 


Harinder  Singh,  Ph.D. — Assistant  Investigator 

Dr.  Singh  is  also  Assistant  Professor  in  the  Department  of  Molecular  Genetics  and  Cell  Biology  at  the 
University  of  Chicago.  He  received  his  Ph.D.  degree  in  biochemistry,  molecular  biology,  and  cell  biology 
with  Lawrence  Dumas  at  Northwestern  University.  His  postdoctoral  research  was  done  with  Phillip  Sharp 
at  the  Massachusetts  Institute  of  Technology,  as  a  Jane  Coffin  Childs  fellow.  He  remained  at  MIT  as  a 
research  associate  until  his  move  to  Chicago. 


MY  research  interests  are  focused  on  the 
analysis  of  transcriptional  regulatory  cir- 
cuits that  turn  genes  on  or  off  during  the  growth 
and  differentiation  of  B  lymphocytes,  cells  that 
produce  antibodies.  The  B  cell  lineage  is  a  very 
useful  model  for  exploring  the  molecular  basis  of 
differential  gene  activity  in  mammalian  develop- 
ment. We  are  seeking  answers  to  the  following 
questions.  What  is  the  nature  of  the  genetic  ele- 
ment (s)  linked  to  a  target  gene  that  controls  its 
transcriptional  activity?  What  is  the  nature  of  the 
regulatory  protein (s)  that  recognizes  this  genetic 
element?  How  upon  binding  near  its  target  gene 
does  the  regulatory  protein  modulate  the  activity 
of  the  enzyme  complex  that  transcribes  the  gene? 
What  is  the  mechanism  by  which  a  growth  or  de- 
velopmental signal  is  transduced  by  the  regula- 
tory protein (s)  to  effect  gene  activity?  How  does 
the  structure  of  the  chromatin  within  which  the 
target  gene  is  packaged  influence  the  function  of 
the  regulatory  protein (s)? 

The  heavy-  and  light-chain  genes  encoding  the 
immunoglobulin  (Ig)  molecule  are  selectively 
transcribed  in  B  cells.  These  genes  are  assembled 
from  gene  segments  through  an  ordered  series  of 
somatic  recombination  events  that  occur  in  a  de- 
veloping B  cell.  The  heavy-chain  gene  locus  is  the 
first  to  undergo  recombination  and  transcrip- 
tional activation,  thereby  defining  the  pre-B  cell 
developmental  state.  Subsequently  one  of  two 
light-chain  gene  loci  is  recombined  and  ex- 
pressed, resulting  in  the  development  of  a  mature 
B  cell.  Ig  genes  contain  multiple  cis-acting  tran- 
scriptional regulatory  elements  that  restrict  their 
expression  to  appropriate  stages  in  the  B  lineage. 

Previous  work  by  various  research  groups  has 
resulted  in  the  identification,  characterization, 
and  cloning  of  a  B  cell-specific  regulatory  pro- 
tein, Oct-2.  This  protein  recognizes  the  octanu- 
cleotide  sequence  ATTTGCAT,  which  confers  B 
cell  specificity  to  Ig  gene  promoters.  The  same 
sequence  motif  is  also  a  functional  component  of 
the  heavy-chain  and  K-light-chain  gene  en- 
hancers. Oct-2  can  activate  transcription  of  a  re- 
porter gene  linked  to  an  Ig  promoter  in  a  non-B 
cell.  Thus  Oct-2  appears  to  be  both  necessary  and 


sufficient  for  regulating  the  activity  of  Ig  pro- 
moters. The  deduced  amino  acid  sequence  of 
Oct-2  reveals  a  region  of  similarity  that  is  shared 
with  three  other  regulatory  proteins:  Pit- 1 ,  Oct- 1 , 
and  unc-86.  This  region  is  termed  the  POU  box 
and  includes  a  subdomain  related  to  the 
homeobox. 

The  levels  of  the  Oct-2  protein  are  regulated 
during  B  cell  differentiation.  The  Oct-2  protein  is 
expressed  at  low  levels  in  pre-B  cells.  Oct-2  lev- 
els are  elevated  5-  to  10-fold  upon  pre-B  cell 
differentiation,  and  higher  Oct-2  protein  levels 
correlate  with  activation  of  the  Ig  /c-light-chain 
gene  locus.  Increased  expression  of  Oct-2  in 
pre-B  cells  is  induced  by  signaling  with  the  B  cell 
mitogen,  bacterial  lipopolysaccharide,  as  well  as 
the  lymphokine  interleukin-1  (IL-1).  Transform- 
ing growth  factor-/8,  an  inhibitor  of  k  gene  induc- 
tion in  pre-B  cells,  blocks  the  up-regulation  of 
Oct-2  but  not  the  activation  of  NF-kB.  The  latter  is 
another  regulatory  protein  that  has  been  previ- 
ously implicated  in  controlling  the  activity  of  the 
K  locus.  We  have  proposed  a  model  in  which  the 
concerted  action  of  increased  levels  of  Oct-2  and 
activated  NF-kB  controls  the  proper  stage-specific 
expression  of  the  k  locus.  To  test  genetically  the 
function  of  Oct-2  in  regulating  immunoglobulin 
gene  transcription,  we  have  used  gene  targeting 
to  disrupt  sequentially  the  two  copies  of  the 
Oct-2  gene  in  a  B  cell.  The  analysis  of  the  mutant 
and  wild-type  cells  is  currently  under  way. 

Expression  of  the  Oct-2  gene  has  been  shown 
to  be  regulated  at  the  level  of  transcription  dur- 
ing pre-B  cell  differentiation.  By  studying  the 
control  of  Oct-2  expression,  regulatory  proteins 
interacting  with  Oct-2  in  the  genetic  hierarchy 
underlying  B  cell  difi'erentiation  can  be  identi- 
fied and  isolated.  A  promoter  controlling  tran- 
scription of  the  Oct-2  gene  has  been  identified. 
This  region  is  being  analyzed  and  will  enable  the 
characterization  of  transcription  factors  that 
regulate  Oct-2  expression  as  well  as  B  cell 
development. 

B  cells  need  to  activate  the  expression  of  two 
other  genes,  mb- 1  and  B29,  to  function  as  anti- 
gen-recognizing cells.  These  genes  encode  mem- 
brane proteins  that  associate  with  the  antibody 


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Regulation  of  Gene  Activity  During  B  Cell  Development 


molecule  (antigen  receptor)  and  are  required  for 
the  expression  of  the  receptor  on  the  cell  surface. 
The  mb-l  gene  is  activated  early  in  B  cell  ontog- 
eny, continues  to  be  expressed  in  mature  B  cells, 
but  is  turned  olF  in  plasma  cells.  We  have  identi- 
fied the  mb-l  promoter  and  shown  that  it  con- 
tains a  regulatory  domain  that  functions  in  a  cell- 
=-«ype  and  stage-specific  manner.  A  25-base  pair 


element  within  this  domain  is  necessary  and  suf- 
ficient for  activity.  This  element  is  recognized  by 
a  novel  transcriptional  activator  termed  BLyF, 
whose  distribution  correlates  with  the  pattern  of 
expression  of  mb-l  in  B  lineage  cells.  Thus  BLyF 
may  play  an  important  role  in  B  cell  develop- 
ment, in  part  by  regulating  the  activity  of  the 
mb-l  gene. 


380 


Regulation  of  Gene  Expression 
in  Developing  Lymphocytes 


Stephen  T.  Smale,  Ph.D. — Assistant  Investigator 

Dr.  Smale  is  also  Assistant  Professor  of  Microbiology  and  Immunology  and  a  member  of  the  Molecular 
Biology  Institute  at  the  University  of  California  School  of  Medicine,  los  Angeles.  He  received  his  Ph.D. 
degree  in  biochemistry  from  the  University  of  California,  Berkeley,  where  he  studied  with  Robert  Tjian. 
Dr.  Smale 's  postdoctoral  research  was  done  with  David  Baltimore  at  the  Whitehead  Institute, 
Massachusetts  Institute  of  Technology. 


.1 


HEMATOPOIESIS  refers  to  a  complex  develop- 
mental process  through  which  pluripotent 
stem  cells  in  mammalian  fetal  liver  or  adult  bone 
marrow  give  rise  to  several  types  of  terminally 
differentiated  blood  cells.  The  immune  system  in- 
corporates many  of  these  cell  types,  which  in- 
clude B  and  T  lymphocytes,  granulocytes,  mono- 
cytes, and  killer  cells,  to  protect  the  host  from 
infection  by  a  variety  of  means.  When  defects 
arise  in  the  hematopoietic  pathway,  the  effects 
are  often  severe.  In  some  cases,  precursor  cells 
cannot  develop  to  maturity,  resulting  in  immuno- 
deficiency. In  other  cases,  uncontrolled  prolifera- 
tion of  developing  cells  results  in  leukemias  and 
lymphomas.  To  understand  the  basis  of  these  de- 
fects, we  must  explain  the  regulation  of  hema- 
topoiesis  at  the  molecular  level. 

The  approach  used  in  our  laboratory  to  study 
the  regulation  of  the  hematopoietic  pathway  is  to 
identify  proteins  that  directly  activate  or  inacti- 
vate genes  expressed  at  specific  stages  of  B  and  T 
lymphocyte  development.  Lymphocytes  play  a 
central  role  in  the  immune  response  by  mediat- 
ing the  recognition  of  foreign  and  infectious  mat- 
ter. As  B  and  T  cells  mature,  a  wide  variety  of 
genes  are  turned  on  and  off  in  a  specific  temporal 
pattern.  An  analysis  of  the  mechanisms  by  which 
these  genes  are  precisely  controlled  is  a  good 
starting  point  for  studying  the  regulation  of 
hematopoiesis. 

Our  laboratory  currently  focuses  on  the  regula- 
tion of  the  gene  encoding  terminal  deoxynucleo- 
tidyltransferase  (TdT) .  This  gene  is  turned  on  for 
only  a  short  time  during  both  B  and  T  cell  devel- 
opment. The  TdT  protein  is  a  template-indepen- 
dent DNA  polymerase  that  appears  to  play  a  role 
in  generating  diversity  within  the  antibody  and  T 
cell  receptor  molecules. 

We  chose  to  analyze  the  TdT  gene  because  of 
its  expression  patterns  in  normal  and  leukemic 
cells.  In  normal  cells  TdT  expression  is  unusual 
because  it  is  found  in  both  early  B  and  T  cells, 
suggesting  that  it  may  be  regulated  by  transcrip- 
tion factors  common  to  these  two  related  but  dis- 
tinct lineages.  Moreover,  TdT  is  expressed  at  high 
levels  in  acute  lymphocytic  leukemias  (ALLs)  and 


at  lower  levels  in  many  acute  myeloid  leukemias 
(AMLs).  TdT  expression  in  AMLs  is  particularly 
intriguing  because  it  does  not  occur  in  normal 
myeloid  cells  or  myeloid  precursors.  Therefore 
the  abnormal  expression  of  TdT  in  AMLs  suggests 
that  the  protein  or  proteins  that  deregulate  TdT 
may  also  play  a  role  in  leukemogenesis. 

To  study  the  mechanisms  of  regulating  TdT  ex- 
pression, we  have  analyzed  the  promoter,  the 
transcriptional  control  region  surrounding  the 
start  site  for  TdT  RNA  synthesis.  Our  analysis  fo- 
cuses on  two  aspects  of  promoter  function:  1)  the 
DNA  sequence  elements  and  proteins  responsible 
for  directing  TdT  expression  specifically  in  lym- 
phoid cells  and  2)  the  unusual  architecture  of  the 
TdT  promoter  region,  which  is  fundamentally 
different  from  that  of  most  other  mammalian  pro- 
moters that  have  been  studied  in  detail.  Our  pro- 
gress in  these  two  areas  is  described  below. 

Regulated  Expression  of  the  TdT  Gene 

To  understand  the  specific  activation  of  the 
TdT  gene  in  B  and  T  lymphocytes,  we  are 
currently  focusing  on  a  DNA  sequence  element 
located  60  nucleotides  upstream  from  the  TdT 
transcription  start  site.  We  have  shown  that  this 
element  of  approximately  15  base  pairs,  called 
D',  is  essential  for  efficient  promoter  activity  in 
lymphocytes.  We  have  identified  two  different 
classes  of  proteins  that  interact  with  the  D'  re- 
gion, one  or  both  of  which  may  be  important  for 
TdT  transcription.  We  identified  the  first  protein, 
called  LyF- 1 ,  by  looking  for  protein-DNA  interac- 
tions at  the  D'  site  in  protein  extracts  derived 
from  lymphocytes.  We  have  characterized  this 
novel  protein  after  purifying  it  from  a  murine 
thymoma  cell  line.  LyF-1  is  a  50-kDa  protein  and 
was  found  to  be  highly  enriched  in  lymphoid 
cells  relative  to  a  variety  of  nonlymphoid  cells. 
Moreover,  LyF- 1  was  found  to  bind  to  two  differ- 
ent sites  in  the  TdT  promoter  as  well  as  to  the 
promoters  for  four  other  lymphocyte-specific 
genes. 

The  second  class  of  proteins  that  bind  to  the 
TdT  D'  site  are  those  related  to  the  ets  family  of 
DNA-binding  proteins.  Mammalian  ets-1  was  first 


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Regulation  of  Gene  Expression  in  Developing  Lymphocytes 


identified  as  the  cellular  homologue  to  a  retro- 
viral oncogene  and  was  shown  only  recently  to 
bind  to  specific  DNA  sequence  elements.  As  with 
most  genes  expressing  DNA-binding  proteins,  the 
ets- 1  gene  is  related  to  several  other  genes  found 
in  the  mammalian  genome,  all  of  which  encode 
proteins  that  bind  to  similar  DNA  sequence  ele- 
ments. We  have  expressed  in  bacteria  three  dif- 
ferent members  of  the  ets  family — ets-1,  fli-1, 
and  PU.l — and  have  found  that  all  three  bind 
tightly  to  the  TdT  D'  site.  Interestingly,  all  three 
of  these  proteins  are  expressed  at  high  levels  in 
certain  subsets  of  hematopoietic  cells,  and  both 
ets-1  and  fli-1  are  expressed  in  most,  if  not  all, 
cell  types  that  express  the  TdT  gene. 

We  have  determined  that  LyF-1  is  not  ets-1  or 
fli-1  and  is  unlikely  to  be  a  member  of  the  ets 
family  of  proteins.  Therefore  our  current  goal  is 
to  determine  which  of  these  proteins — LyF-1, 
ets- 1 ,  fli-1 ,  or  a  combination  of  LyF- 1  and  an  ets 
member — is  required  to  activate  TdT  transcrip- 
tion by  interacting  with  the  D'  element.  Those 
proteins  required  for  TdT  activation  are  likely  to 
be  important  for  regulating  lymphocyte  develop- 
ment and  will  become  candidates  for  involve- 
ment in  the  deregulation  of  TdT  expression  in 
ALL  and  AML.  This  work  is  supported  by  a  grant 
from  the  National  Institutes  of  Health. 

Unusual  Design  of  the  TdT  Promoter 

The  TdT  promoter  is  unusual  in  that  it  does  not 
contain  a  TATA  box,  which  is  a  common  DNA  se- 
quence element  found  in  most  promoters  that 
have  been  characterized  in  detail.  The  TATA  ele- 
ment is  known  to  carry  out  several  important 
functions  in  promoters,  including  1)  recruitment 
of  the  RNA  polymerase  to  the  transcription  start 
site  region,  2)  determination  of  the  site's  loca- 
tion, and  3)  determination  of  the  direction  of 
transcription  from  the  promoter.  To  understand 
the  regulation  of  the  TdT  promoter,  we  are  inquir- 
ing into  how  these  functions  are  carried  out  in  the 
absence  of  a  TATA  box.  This  question  is  espe- 


cially important  for  an  understanding  of  tran- 
scriptional control  during  lymphocyte  develop- 
ment, since  it  appears  that  the  promoters  of  most 
genes  expressed  during  this  process  do  not  con- 
tain TATA  boxes. 

Previously  we  found  that  in  place  of  the  TATA 
element,  which  is  typically  located  30  nucleo- 
tides upstream  of  the  transcription  start  site,  the 
TdT  promoter  contains  a  distinct  element  that  in- 
stead overlaps  the  start  site.  This  element,  which 
we  call  an  initiator  (Inr) ,  is  like  the  TATA  element 
in  its  importance  for  promoter  function  and  also 
in  pinpointing  the  RNA  start  site  to  a  specific 
nucleotide. 

We  are  now  comparing  further  the  activities 
and  mechanisms  of  action  of  TATA  boxes  and  Inr 
elements.  Currently  our  data  suggest  that  the 
mechanisms  for  TATA  and  Inr-mediated  tran- 
scription follow  very  similar  steps  prior  to  the 
actual  onset  of  RNA  synthesis. 

We  would  also  like  to  understand  the  contribu- 
tions of  TATA  and  Inr  elements  toward  determin- 
ing the  start  site  location  and  the  direction  of 
transcription  within  a  promoter.  Some  promoters 
contain  both  TATA  and  Inr  elements.  Although 
both  elements  can  influence  start  site  localiza- 
tion and  transcriptional  directionality,  the  TATA 
element  appears  to  be  much  more  powerful  than 
an  Inr.  Our  results,  however,  also  have  led  us  to 
challenge  the  belief  that  the  orientation  of  a  TATA 
box  within  a  promoter  alone  determines  the  di- 
rection of  transcription.  Instead,  our  data  suggest 
that  the  direction  of  transcription  within  a  simple 
promoter  is  determined  by  the  location  of  a  TATA 
box  or  Inr  element  relative  to  a  specific  activator 
element. 

We  are  continuing  to  define  the  rules  by  which 
a  promoter  determines  the  direction  of  RNA  syn- 
thesis in  order  to  understand  further  the  design  of 
more-complex  promoters,  including  those  that 
contain,  like  the  TdT  promoter,  important  se- 
quence elements  both  upstream  and  downstream 
of  the  transcription  start  site. 


382 


Developmental  Genetics 


Philippe  M.  Soriano,  Ph.D.,  D.Sc. — Assistant  Investigator 

Dr.  Soriano  is  also  Assistant  Professor  at  the  Institute  for  Molecular  Genetics  and  Department  of  Cell 
Biology,  Baylor  College  of  Medicine.  He  obtained  his  Ph.D.  degree  in  biochemistry  and  his  D.Sc.  degree 
from  the  University  of  Paris.  He  did  postdoctoral  research  in  France,  and  then  with  Rudolf  Jaenisch  in 
Germany  and  at  the  Whitehead  Institute  for  Biomedical  Research  of  the  Massachusetts  Institute  of 
Technology,  before  joining  the  faculty  at  Baylor.  Dr.  Soriano  is  a  Pew  Scholar  in  the  biomedical  sciences. 


THE  major  aim  of  our  research  is  to  extend  the 
understanding  of  early  development  of  the 
mouse.  Our  approach  is  to  create  mice  that  are 
mutant  in  genes  critical  for  this  process.  Two 
techniques  are  being  used:  random  mutagenesis, 
which  should  discover  unknown  genes,  and  tar- 
geted mutagenesis  in  previously  characterized 
genes  for  which  no  mutation  is  known. 

The  random  mutagenesis  project  is  designed  to 
identify  developmentally  regulated  and  critical 
genes  in  the  embryo.  Identification  of  the  mu- 
tated gene  in  many  of  the  classical  mouse  mu- 
tations has  been  difficult  because  there  is  no 
convenient  tag  by  which  to  clone  the  muta- 
tion. Insertion  mutagenesis  in  transgenic  mice, 
wherein  a  gene  is  disrupted  by  introducing  a  for- 
eign fragment  of  DNA  into  the  germline,  is  attrac- 
tive, since  the  transgene  can  serve  as  a  tag.  How- 
ever, only  1  out  of  20  transgenic  strains  displays 
an  overt  phenotype,  so  the  approach  is  laborious 
and  time  consuming.  We  have  circumvented  this 
problem  by  preselecting  for  mutations  in  embry- 
onic stem  (ES)  cells,  which  are  then  introduced 
into  embryos  to  colonize  the  germline. 

The  selection  procedure  we  have  used  is 
termed  "promoter  trapping."  In  this  method,  a 
reporter  gene  is  placed  downstream  of  a  splice 
acceptor,  and  the  construct  is  then  introduced 
into  ES  cells.  Expression  of  the  reporter  gene  can 
only  originate  from  a  flanking  cellular  promoter. 
Therefore  transgenic  mice  derived  from  ES  cells 
selected  for  such  events  can  be  used  both  to  trace 
the  activity  of  the  tagged  gene,  by  expression  of 
the  reporter  gene,  and  to  mutate  the  gene.  The 
reporter  gene  we  have  used  encodes  a  fusion  pro- 
tein with  both  (8-galactosidase  (;8-gal)  and  neo- 
mycin phosphotransferase  (neo)  activity.  This  al- 
lows one  to  select  directly  for  promoter  trap 
events,  since  neo  confers  resistance  to  the  drug 
G418,  and  to  follow  the  activity  of  the  trapped 
gene  by  incubating  the  embryos  in  a  dye  that 
turns  cells  blue  if  |8-gal  is  present. 

Thirty-one  transgenic  lines  have  now  been  gen- 
erated using  a  retroviral  promoter  trap  vector  and 
have  been  examined  both  for  patterns  of  expres- 
sion and  for  phenotype.  We  are  particularly  inter- 
ested in  strains  that  exhibit  restricted  patterns  of 


expression  at  gastrulation,  a  critical  stage  of  de- 
velopment in  the  early  mouse  embryo.  Twelve 
strains  carry  a  recessive  lethal  mutation  due  to  the 
promoter  trap  insertion,  and  one  induces  steril- 
ity. This  demonstrates  that  the  method  is  very 
valuable  for  isolating  developmental  mutants. 

Further  analysis  of  one  of  the  embryonic  lethal 
strains  suggests  a  mutation  in  a  transcription  fac- 
tor. The  other  strains,  which  do  not  display  a  phe- 
notype associated  with  the  promoter  trap  event, 
may  reflect  mutations  in  nonessential  genes.  This 
first  aspect  of  our  research  is  supported  by  the 
National  Institutes  of  Health. 

It  is  also  possible  to  make  specific  mutations  by 
targeting  genes  believed  to  play  critical  roles  in 
development,  based  on  their  previous  character- 
ization. This  approach  relies  on  the  ability  to  in- 
troduce into  ES  cells  mutant  gene  constructs  that 
will  recombine  homologously  with  the  normal 
gene,  at  the  correct  chromosomal  location,  there- 
fore creating  a  mutation  in  the  gene  of  interest. 
Our  initial  efforts  focused  on  the  gene  encoding 
c-src,  the  first  proto-oncogene  identified. 

Src  is  a  protein-tyrosine  kinase,  broadly  ex- 
pressed but  at  particularly  high  levels  in  neurons 
and  in  platelets.  The  src  gene  was  knocked  out  in 
ES  cells,  and  the  mutation  was  transmitted 
through  the  germline.  Surprisingly,  animals  ho- 
mozygous for  the  mutation  do  not  die  at  an  early 
stage,  but  develop  osteopetrosis,  a  disease  char- 
acterized by  an  impaired  function  of  the  osteo- 
clasts, the  cells  that  normally  resorb  bone.  As  a 
result  of  the  mutation,  the  mutant  mice  fail  to 
undergo  normal  tooth  eruption  and  have  to  be 
maintained  on  a  soft  food  diet.  We  have  been  able 
to  show  that  the  defect  lies  in  the  osteoclasts 
proper,  rather  than  in  accessory  cells  such  as  os- 
teoblasts that  condition  osteoclast  activity.  How- 
ever, no  defect  is  found  in  cells  or  tissues  where 
src  is  most  highly  expressed. 

To  explain  the  lack  of  a  more  severe  pheno- 
type, we  have  tested  the  hypothesis  that  another 
5rc-related  kinase  may  be  compensating  for  loss 
of  src  in  the  mutant  mice.  Mice  mutant  in  the 
closely  related  c-yes  and  fyn  genes  have  therefore 
been  generated  by  homologous  recombination  in 
ES  cells.  Neither  of  these  mutations  results  in  an 


383 


Developmental  Genetics 


overt  phenotype.  However,  further  analysis  of  the 
fyn  mutation  has  revealed  that  thymocytes  are  un- 
able to  proliferate  in  response  to  stimulation 
through  the  T  cell  receptor,  demonstrating  that 
this  kinase  is  essential  for  normal  thymocyte 
function.  Surprisingly,  peripheral  T  cells  can  re- 
spond through  the  T  cell  receptor,  implying  that 
-Other  kinase (s)  may  be  involved.  This  mutation 
may  have  further  effects  on  the  repertoire  of  T 
cells,  and  further  studies  are  under  way. 


To  test  the  concept  that  5rc-related  kinases 
compensate  for  one  another,  we  have  crossbred 
the  mutant  mice  and  examined  progeny  for  dou- 
ble mutant  genotypes.  The  vast  majority  of  src- 
yes  and  src-fyn  double  mutants  die  at  birth — 
supporting  the  compensation  hypothesis.  The 
nature  of  the  defect  in  the  double  mutants  is 
under  further  investigation.  This  second  aspect  of 
our  research  has  been  supported  in  part  by  the 
National  Institutes  of  Health. 


Expression  pattern  of  the  ^galactosidase  (^-gal)  gene  in  blastocysts  of  a  'promoter  trap"  strain 
of  transgenic  mice,  showing  labeling  (blue)  in  both  the  inner  cell  mass  and  the  outlying  trophec- 
toderm.  By  this  technique,  mice  derived  from  selected  embryonic  stem  cells  can  be  used  both  to 
trace  the  activity  of  a  ^-gal-tagged  gene  and  to  mutate  the  strain. 
Research  of  Glenn  Friedrich  and  Philippe  Soriano. 


384 


Understanding  How  Eggs  Work 


Allan  C.  Spradling,  Ph.D. — Investigator 

Dr.  Spradling  is  also  a  staff  member  of  the  Department  of  Embryology  at  the  Carnegie  Institution  of 
Washington  in  Baltimore  and  Adjunct  Professor  of  Biology  and  Microbiology  at  the  fohns  Hopkins 
University.  He  earned  his  B.A.  degree  in  physics  from  the  University  of  Chicago  and  his  Ph.D.  in  cell  biology 
from  the  Massachusetts  Institute  of  Technology.  His  postdoctoral  study  was  done  at  Indiana  University 
with  Anthony  Mahowald.  Dr.  Spradling  is  a  member  of  the  National  Academy  of  Sciences  and  has  received 
many  honors  for  his  work. 


ALTHOUGH  the  union  of  egg  and  sperm  initi- 
ates the  complex  processes  that  ultimately 
result  in  a  new  animal,  the  roles  played  by  the 
two  cells  in  embryonic  development  are  by  no 
means  equivalent.  Even  nonspecific  treatments, 
such  as  pricking  with  a  needle,  will  stimulate 
many  types  of  eggs  to  develop  in  the  absence  of 
sperm.  Without  an  egg,  however,  neither  a  sperm 
cell  nor  any  other  can  even  begin  the  processes 
that  lead  to  an  embryo  and  ultimately  an  adult.  In 
fact,  eggs  have  undergone  extensive  and  intricate 
preparations  that  allow  them  to  support  and  di- 
rect embryogenesis.  If  the  mystery  of  a  new  life 
could  be  condensed  into  a  single  cell,  that  cell 
would  surely  be  an  egg. 

Eggs  Pose  Exceptional  Biological  Problems 

Egg  cells  are  very  different  in  structure  and  in 
biological  capacity  from  other  cells.  Most 
chicken  cells  weigh  less  than  one  millionth  of  an 
ounce,  whereas  a  single  chicken  egg  makes  a  nice 
breakfast.  Eggs  not  only  differ  in  size  but  in  many 
other  important  ways.  For  example,  the  egg's 
genes  function  differently  from  those  of  other 
cells;  so  extensive  are  these  differences  that  the 
chromosomes  appear  in  the  microscope  quite 
unlike  those  of  other  tissues.  In  eggs  the  products 
of  many  genes  accumulate  and  are  stored  in  spe- 
cial forms  so  that  they  can  be  utilized  at  precisely 
the  right  times  during  embryonic  development. 
Indeed,  storage  of  material  is  one  of  the  reasons 
many  eggs  are  enormous. 

A  great  deal  remains  to  be  learned  about  egg 
structure.  How  are  gene  products  specially 
stored,  and  what  are  their  specific  functions  later 
in  development?  Is  each  product  located  in  a  par- 
ticular place?  What  are  the  mechanisms  that  al- 
low these  materials  to  be  produced  and  stock- 
piled in  the  appropriate  manner?  The  sheer 
complexity  of  an  egg,  with  its  tens  of  thousands 
of  specific,  highly  organized  components,  has 
until  recently  blocked  all  attempts  to  describe 
egg  structure  in  molecular  detail,  much  less  to 
understand  the  logic  that  allows  this  structure  to 
develop  into  an  even  more  intricate  organism. 
However,  over  the  past  10  years  or  so,  powerful 


new  methods  in  molecular  biology,  such  as  gene 
cloning,  gene  transfer,  and  fruit  fly  genetics,  have 
begun  to  unravel  the  fascinating  secrets  stored 
within  an  eggshell. 

Using  Genetics  to  Study  Eggs 

It  is  now  possible  to  study  structures  as  com- 
plex as  eggs  because  techniques  of  both  genetics 
and  molecular  biology  allow  us  to  take  eggs  apart 
gene  by  gene.  Each  component  of  an  egg  is  speci- 
fied by  a  gene  carried  on  the  chromosomes.  Much 
research  on  eggs  utilizes  the  fruit  fly  Drosophila, 
since  far  more  has  been  revealed  about  its  genes 
during  70  years  of  genetic  studies  than  about 
those  of  any  other  complex  organism,  including 
humans.  A  genetic  mutation  in  the  fly  inactivates 
one  gene  among  the  20,000  to  50,000,  and  if  that 
gene  specifies  an  egg  component,  then  the  mu- 
tant fly  will  produce  defective  eggs. 

Frequently,  however,  the  eggs  can  still  par- 
tially function.  For  example,  in  a  particular  mu- 
tant, development  might  begin  normally,  but 
then  stop  because  of  a  lack  of  stored  food.  By 
studying  what  goes  wrong  with  the  mutant  eggs, 
biologists  can  deduce  the  normal  function  of  a 
particular  gene's  product.  In  this  case,  one  might 
conclude  that  the  gene  was  involved  in  produc- 
ing the  stored  food.  To  understand  eggs,  it  will  be 
necessary,  at  a  minimum,  to  create  mutations  and 
carry  out  such  studies  on  all  the  fruit  fly  genes, 
one  by  one. 

If  researchers  were  limited  to  looking  at  egg 
defects  in  a  microscope,  progress  would  still  be 
slow.  Many  of  the  defective  eggs  would  not  con- 
tain any  detectable  problems;  they  just  wouldn't 
work.  It  is  at  this  point  that  the  ability  to  clone  the 
gene  molecularly  becomes  essential  to  further  ad- 
vancement. Cloning  is  simply  a  way  of  purifying 
an  individual  gene  so  that  its  DNA  structure  can 
be  determined  in  the  laboratory. 

There  are  two  major  benefits.  First,  the  struc- 
ture of  the  gene's  product,  a  specific  protein  that 
actually  becomes  part  of  the  egg,  can  now  be  de- 
termined by  the  process  of  DNA  sequencing.  Sec- 
ond, specific  antibodies  can  be  prepared  that 
only  bind  to  the  product  of  that  particular  gene. 


385 


Understanding  How  Eggs  Work 


When  these  antibodies  are  used,  eggs  that  previ- 
ously appeared  normal  in  the  microscope  can 
suddenly  be  seen  to  have  specific  defects.  Such 
molecular  biological  studies  allow  us  to  under- 
stand in  much  greater  detail  vv^here  in  an  egg  par- 
ticular gene  products  are  located,  where  they  go 
as  development  begins,  and  what  role  they  are 
4ikely  to  play  in  the  process. 

Transporting  Materials  Into  Eggs 

One  project  of  our  research  group  concerns 
how  the  massive  amount  of  material  found  in  an 
egg  actually  gets  there.  In  the  fruit  fly  most  of  the 
egg  contents  are  piped  in  from  15  nearby  cells 
called  nurse  cells.  Four  of  these  cells  are  attached 
directly  to  the  egg  by  special  "pipes"  made  of  the 
same  material  as  the  outer  surface  of  cells,  and 
the  remaining  1 1  are  attached  to  other  nurse  cells 
by  similar  pipes.  Thus  some  of  the  material  in  an 
egg  has  been  shipped  through  as  many  as  four 
nurse  cells  on  the  way  to  its  destination.  Nurse 
cells  not  only  control  somehow  the  rate  and  direc- 
tion of  transport,  but  even  select  certain  products 
for  priority  delivery,  moving  them  well  ahead  of 
the  majority  of  the  egg's  contents. 

To  learn  more  about  the  equipment  flies  use  to 
make  their  eggs,  we  identified  some  mutant 
strains  in  which  either  the  connections  between 
nurse  cells  and  egg  did  not  form  properly,  or  ma- 
terial did  not  flow  very  well.  We  have  now  stud- 
ied one  mutant  in  some  detail.  When  mother  flies 
lack  this  gene,  they  are  able  to  attach  on  average 
only  four  nurse  cells  to  their  eggs.  As  a  result  the 
eggs  usually  do  not  grow  properly  and  are  unable 
to  function.  Rarely,  a  developing  egg  becomes 
connected  to  as  many  as  11  nurse  cells,  and  a  few 
of  these  eggs  can  develop,  so  the  affected  females 
are  not  completely  sterile. 

We  determined  the  structure  of  the  protein  that 
was  missing  in  the  mutant,  and  were  surprised  to 
learn  that  it  was  very  similar  to  one  found  in  large 
amounts  just  under  the  surface  of  human  and 
other  mammalian  red  blood  cells.  This  protein, 
called  "adducin,"  has  been  studied  for  some  time 


by  Vann  Bennett  (HHMI,  Duke  University).  It  has 
been  found  at  various  levels  in  many  types  of  cell, 
where  it  is  thought  to  strengthen  cell  membranes 
by  acting  as  a  kind  of  skeleton  supporting  the 
floppy  lipid  bilayer.  Red  cell  adducin  is  thought 
to  act  with  other  membrane  skeleton  proteins  to 
mold  red  cells  into  their  characteristic  concave 
disk  shape  and  to  lend  the  physical  properties 
that  let  them  pass  through  small  capillaries.  As 
with  many  human  proteins,  however,  the  actual 
function  is  uncertain  because  the  effects  of  re- 
moving a  red  cell's  adducin  are  unknown. 

At  present  we  are  attempting  to  learn  in  detail 
what  adducin  does  to  help  nurse  cells  and  eggs 
hook  up  properly.  The  pipes  form  during  cell  di- 
vision. Normal  daughter  cells  separate  when  a  re- 
gion of  membrane  clamps  down  as  though  con- 
stricted by  a  knot  from  outside.  Eventually  the 
two  cells  are  choked  off  completely.  During  the 
last  few  divisions  that  give  rise  to  the  nurse  cells 
and  egg,  these  constrictions  simply  stop  short, 
leaving  a  small  interconnecting  region  of  cell 
membrane.  This  junction  subsequently  forms 
into  a  pipe.  In  the  adducin  mutants,  many  of  the 
constrictions  are  unable  to  stop,  so  cells  that 
should  have  built  a  connecting  pipe  become  com- 
pletely separated.  The  pipes  that  do  form  in  the 
mutant  are  much  weaker  looking  than  normal, 
may  not  transport  materials  very  well,  and  appear 
susceptible  to  breakage. 

Understanding  how  adducin  contributes  to 
fruit  fly  egg  production  may  provide  insight  into 
the  details  of  how  it  works  in  humans.  The  mu- 
tant flies  also  have  several  other  abnormalities 
besides  their  inability  to  connect  nurse  cells  and 
the  egg  properly,  indicating  that  the  function  of 
adducin  can  be  studied  in  a  variety  of  different 
situations.  Experimenters  can  now  make  what- 
ever changes  in  the  protein's  structure  they  wish, 
introduce  a  modified  gene  encoding  the  altered 
protein  back  into  the  mutant  strain  of  fruit  fly, 
and  see  if  the  modified  protein  will  "rescue" 
some  or  all  of  the  fly's  problems  or  create  new 
ones.  This  approach  can  be  applied  to  the  many 
different  gene  products  that  make  up  a  fruit  fly  egg. 


586 


Structural  Studies  of  Regulatory  Proteins 


Stephen  R.  Sprang,  Ph.D. — Associate  Investigator 

Dr.  Sprang  is  also  Associate  Professor  of  Biochemistry  at  the  University  of  Texas  Southwestern  Medical 
Center  at  Dallas.  He  received  a  B.S.  degree  from  California  State  University  at  Los  Angeles,  and  a  Ph.D. 
degree  from  the  University  of  Wisconsin-Madison,  where  Muttaiya  Sundaralingam  was  his  advisor.  His 
postdoctoral  training  was  at  the  University  of  Alberta,  Edmonton,  with  Robert  Fletterick.  Again  with  Dr. 
Fletterick,  he  was  an  assistant  research  biochemist  at  the  University  of  California,  San  Francisco, 
before  assuming  his  present  position. 


CELLS  communicate  with  each  other  by  se- 
creting growth  factors,  cytokines,  or  hor- 
mones into  the  extracellular  space,  and  by  re- 
sponding to  factors  produced  by  other  cells. 
These  chemical  messengers  act  by  changing  pat- 
terns of  gene  expression  within  target  cells, 
thereby  altering  the  cells'  metabolic  or  develop- 
mental program.  Among  the  major  unsolved  ques- 
tions in  biology  is  how  cells  specifically  recog- 
nize these  chemical  messengers  and  how  the 
messages  are  transduced  within  cells. 

We  are  using  x-ray  crystallographic  techniques 
to  define  the  molecular  nature  of  the  interaction 
between  specific  growth  factors  and  their  cog- 
nate receptors.  Receptors  are  themselves  protein 
molecules  distributed  on  the  outer  surface  of  the 
plasma  membrane  surrounding  the  target  cell. 
The  goal  of  our  research  is  to  learn  how  receptor 
molecules  specifically  recognize  growth  factors, 
and  how,  if  at  all,  the  receptors  alter  their  struc- 
ture and  chemical  properties  as  a  consequence  of 
their  interaction  with  the  factors. 

Tumor  Necrosis  Factors  and  Fibroblast 
Growth  Factors 

Tumor  necrosis  factors  (TNF)  are  produced  by 
cells  of  the  immune  system.  Macrophages — the 
white  blood  cells  responsible  for  engulfing  cel- 
lular debris — produce  large  quantities  of  TNF-a 
(also  known  as  cachectin)  when  stimulated  by 
toxins  carried  on  the  surface  of  bacteria,  as  oc- 
curs in  an  infection.  The  avid  attachment  of  this 
cytokine  to  receptors  present  on  a  variety  of  cells 
in  the  body  triggers  a  series  of  events  that  mediate 
inflammation,  endotoxic  shock,  and  the  wasting 
phenomenon,  cachexia,  from  which  the  cytokine 
takes  one  of  its  names. 

A  related  cytokine,  TNF-|8,  is  produced  by  T 
lymphocytes  and  has  many  properties  in  common 
with  TNF-a,  including  the  ability  to  bind  to  the 
same  receptors.  Both  molecules  are  toxic  to  many 
types  of  tumor  cells. 

Two  different  types  of  TNF  receptor  have  been 
found  which,  surprisingly,  bear  little  amino  acid 
sequence  identity  to  each  other.  Both  receptors, 
however,  contain  a  structural  "motif"  composed 


of  four  inexact  copies  of  a  repeat  rich  in  the  sul- 
fur-containing amino  acid  cysteine.  Preliminary 
data  from  our  own  and  other  laboratories  suggest 
that  each  trimer  of  TNF-a  interacts  with  three  re- 
ceptor molecules.  We  hope  to  learn  how  these 
distantly  related  molecules  interact  with  the 
same  receptors  and  how  this  interaction  might 
mediate  a  cellular  response. 

The  three-dimensional  atomic  structures  of 
TNF-a,  and  most  recently  of  TNF-/?,  have  been 
determined  by  Michael  Eck,  a  graduate  student  in 
the  laboratory  (and  now  a  Research  Associate 
with  Don  Wiley  and  Stephen  Harrison  at  HHMI, 
Harvard  University).  Despite  the  differences  in 
amino  acid  composition  and  sequence,  TNF-a 
and  TNF-/3  have  the  same  three-dimensional 
structure  and  assemble  to  form  trimers  composed 
of  three  identical  protein  subunits  packed  about 
a  threefold  axis  of  symmetry.  However,  because 
of  the  differences  in  the  amino  acid  composition 
of  the  two  cytokines,  the  surface  of  the  TNF-a 
trimer  is  chemically  quite  different  from  that  of 
the  TNF-/3  trimer.  We  hope  to  determine  how  the 
same  receptor  can  recognize  these  different  mo- 
lecular surfaces  with  equal  affinity. 

More  importantly  from  a  pharmacological  per- 
spective, we  would  like  to  be  able  to  design  TNF 
molecules  that  interact  with  one,  but  not  the 
other  receptor,  or  receptor  antagonists  that  inter- 
act with  only  one  of  the  two  TNF  species.  Toward 
this  goal,  we  are  now  attempting  to  determine  the 
structure  of  theTNF-receptor  complexes.  The  lab- 
oratory has  been  successful  in  growing  crystals  of 
the  TNF-binding  fragment  of  one  of  the  two  re- 
ceptors, and  crystallographic  studies  are  now 
under  way.  We  are  also  attempting  to  produce 
crystals  of  the  complex  formed  between  TNF-a 
and  one  of  the  receptors  in  order  to  study  directly 
the  interaction  between  the  two  molecules. 

Last  year  we  reported  the  three-dimensional 
structure  of  basic  fibroblast  growth  factor 
(bFGF),  one  of  a  group  of  seven  structurally  re- 
lated proteins  that  promote  cell  division.  This 
factor  is  present  in  the  space  between  cells,  par- 
ticularly in  endothelial  tissue,  where  it  stimulates 
the  movements  of  cells  to  sites  of  tissue  genera- 


587 


Structural  Studies  of  Regulatory  Proteins 


tion  or  repair.  Our  work  has  lately  focused  on  the 
interaction  between  bFGF  and  heparin,  a  com- 
plex, negatively  charged  sugar  polymer  that  coats 
endothelial  cells  and  is  required  to  induce  the 
mitogenic  response  that  bFGF  elicits.  In  a  collabo- 
ration with  Phillip  Barr  of  Chiron,  Inc.,  the  labo- 
ratory is  also  attempting  to  determine  the  struc- 
ture of  the  receptor  for  bFGF. 

The  signals  generated  by  the  engagement  of  ex- 
tracellular messenger  molecules  with  their  re- 
ceptors are,  in  many  cases,  transmitted  across  the 
plasma  membrane  to  members  of  the  G  protein 
family.  For  example,  when  epinephrine  binds  to 
the  /3-adrenergic  receptor,  an  intracellular  pro- 
tein called  Gsa  is  induced  to  bind  the  nucleotide 
GTP  and  discard  a  pair  of  regulatory  subunits. 
The  GTP-bound  Gsa  is  then  an  activator  of  a 
membrane-bound  enzyme  that  catalyzes  the  syn- 
thesis of  the  intracellular  messenger  molecule 
cyclic  AMP.  In  collaboration  with  Alfred  Gilman 
(University  of  Texas  Southwestern  Medical 
Center  at  Dallas),  we  are  undertaking  crystallo- 
graphic  studies  of  Gsa  and  the  related  protein 
Gia  to  learn  how  these  proteins  may  interact  with 
other  components  of  the  signal  transduction  sys- 
tem. Research  Associate  David  Coleman  has  ob- 
tained small  crystals  of  the  complex  between  Gia 
and  a  nonhydrolyzable  GTP  analogue,  and  we 
hope  soon  to  resolve  the  structure  of  this  fascinat- 
ing molecule. 

Glycogen  Phosphorylase 

A  second  focus  of  the  laboratory  has  been  to 


understand  how  a  certain  class  of  complex  biolog- 
ical catalysts,  called  allosteric  enzymes,  regulate 
their  own  activity.  An  example  of  such  an  en- 
zyme, studied  in  our  laboratory,  is  glycogen 
phosphorylase.  This  molecule  catalyzes  the 
breakdown  of  a  storage  carbohydrate  called  gly- 
cogen into  sugar  units  that  can  be  used  directly  by 
the  body  to  fuel  muscle  contraction  or  to  main- 
tain constant  levels  of  glucose  in  the  blood.  The 
activity  of  this  enzyme  increases  when  it  binds  to 
"cellular  messenger"  molecules  such  as  adeno- 
sine monophosphate,  which  signals  an  energy 
deficit  in  the  cell,  and  decreases  when  it  binds 
glucose,  which  signals  an  energy  surplus.  Glyco- 
gen phosphorylase  can  be  chemically  modified 
(phosphorylated)  by  other  enzymes  in  response 
to  hormonal  signals  (epinephrine),  which  also 
increases  the  catalytic  activity  of  this  enzyme. 
Our  goal  is  to  understand  the  molecular  mechan- 
ics of  the  process  by  which  catalytic  machines 
such  as  phosphorylase  can  alter  their  activity  in 
response  to  the  cellular  messengers. 

We  have  recently  determined  the  three-dimen- 
sional structure  of  active  glycogen  phosphorylase 
with  the  activator  AMP  bound  to  a  regulatory  site 
in  the  molecule.  In  comparing  this  structure  with 
the  inactive  conformation  of  the  molecule  deter- 
mined in  Robert  Fletterick's  laboratory  (Univer- 
sity of  California,  San  Francisco) ,  we  have  learned 
how  the  three-dimensional  structure  of  the  mole- 
cule is  altered  in  such  a  way  as  to  increase  the 
affinity  of  the  enzyme  for  glycogen. 


388 


Insulin  and  the  Islets  of  Langerhans 


Donald  F.  Steiner,  M.D. — Senior  Investigator 

Dr.  Steiner  is  also  A.  N.  Pritzker  Distinguished  Service  Professor  of  Biochemistry  and  Molecular  Biology 
and  of  Medicine  at  the  University  of  Chicago  Pritzker  School  of  Medicine.  He  received  his  M.D.  degree  at 
the  University  of  Chicago.  His  interest  in  insulin  developed  during  postdoctoral  training  with  Robert 
Williams  at  the  University  of  Washington  School  of  Medicine.  After  joining  the  faculty  at  the  University  of 
Chicago,  he  studied  insulin  action  in  the  liver  and,  later,  insulin  biosynthesis.  This  work  led  to  his 
discovery  of  proinsulin  and preproinsulin.  Dr.  Steiner  has  received  many  honors,  including  the  Lilly  and 
Gairdner  Awards,  the  Wolf  Prize  in  Medicine,  and  several  honorary  degrees. 


INSULIN  is  essential  for  normal  growth  and  uti- 
lization of  food.  Diabetes,  a  disease  due  to 
insulin  deficiency  or  defects  in  its  action,  is  char- 
acterized by  high  blood  sugar  and  such  complica- 
tions as  blindness,  heart  disease,  stroke,  and  in- 
creased susceptibility  to  infections.  It  affects  2-3 
percent  of  people  in  developed  countries. 

Diabetes  can  be  controlled  through  various 
combinations  of  diet,  oral  hypoglycemic  agents, 
and/or  insulin  injections,  depending  on  the  type 
and  severity  of  the  disease.  Such  therapies,  how- 
ever, are  often  less  than  fully  satisfactory  because 
they  may  only  retard  the  development  of  compli- 
cations. A  better  understanding  of  how  insulin  is 
formed  and  secreted  in  a  regulated  manner  into 
the  bloodstream  and  how  it  acts  on  tissue  recep- 
tors to  control  metabolism  and  growth  are  vitally 
important  to  the  development  of  new  therapeutic 
approaches. 

Insulin  is  only  made  in  the  islets  of  Langerhans 
— small  clusters  of  cells  dispersed  throughout 
the  pancreas.  Specialized  islet  cells  also  secrete 
other  hormones  that  influence  metabolism,  in- 
cluding glucagon,  somatostatin,  amylin  (or  islet 
amyloid  polypeptide),  and  pancreatic  polypep- 
tide. The  islet  hormones,  like  other  regulatory 
peptides  in  the  body,  are  derived  from  larger  pro- 
teins called  prepro hormones. 

These  precursors  contain  additional  portions 
that  may  guide  them  along  special  intracellular 
pathways,  where  they  are  concentrated  into  stor- 
age vesicles  and  processed  into  their  biologically 
active  forms.  The  contents  of  these  vesicles  are 
then  released  into  the  bloodstream  in  varying 
proportions  to  meet  physiological  requirements. 
One  goal  of  our  research  is  to  learn  more  about 
how  newly  formed  prohormones  are  separated 
from  other  proteins  in  the  cell,  concentrated  into 
secretory  granules,  and  processed  into  active  hor- 
mones by  highly  specialized  enzymes  before  be- 
ing secreted. 

Insulin  secretion  from  |8-cells  in  response  to 
elevated  plasma  glucose  is  a  complex  electro- 
chemical process  resembling  the  transmission  of 
nerve  impulses  or  the  contractions  of  the  heart. 


In  the  insulin-producing  |8-cells,  a  specialized 
sensor  mechanism  couples  the  metabolism  of 
glucose  to  ion  channels  in  the  plasma  membrane. 
These  channels,  upon  membrane  depolarization, 
allow  the  selective  entry  of  calcium  into  the  cell, 
triggering  the  release  of  secretory  granule  con- 
tents. Certain  oral  hypoglycemic  agents  used  to 
treat  diabetes — the  sulfonylurea  drugs — appear 
to  stimulate  insulin  secretion  by  inhibiting  a  spe- 
cialized potassium  channel  in  the  ;8-cell  mem- 
brane, which  then  initiates  electrical  depolariza- 
tion of  the  cell.  We  are  trying  to  learn  more  about 
the  structure  of  this  and  other  important  ion 
channels  in  insulin-producing  cells  to  under- 
stand both  their  normal  functions  and  their  possi- 
ble malfunction  in  some  forms  of  diabetes. 

We  also  are  studying  mutations  that  affect  insu- 
lin or  proinsulin  structure.  Some  of  these  occur 
in  families  and  are  associated  with  mild  diabetes 
as  a  result  of  the  synthesis  of  abnormal  insulin 
molecules  with  greatly  reduced  biological  activ- 
ity. Other  mutations  in  the  insulin  gene  primarily 
affect  the  conversion  of  proinsulin  into  insulin, 
leading  to  elevated  proinsulin  in  the  circulation. 

Insulin  acts  on  tissues  by  binding  to  a  large  and 
complex  protein  receptor  that  is  present  on  the 
surface  of  most  cells,  activating  a  tyrosine  kinase 
that  alters  many  intracellular  processes  through  a 
cascade  of  intracellular  phosphorylations.  Insu- 
lin binding  to  the  receptor  also  leads  to  the  up- 
take and  degradation  of  the  hormone  in  the  liver 
and  other  tissues.  This  process,  known  as  recep- 
tor-mediated endocytosis,  plays  an  important 
role  by  rapidly  removing  insulin  from  the  circu- 
lation. By  studying  inherited  defects  in  proinsu- 
lin and  insulin  receptor  molecules,  we  are  learn- 
ing more  about  the  normal  processes  of  islet 
hormone  production  and  action  and  how  their 
derangement  can  lead  to  disease. 

Prohormone-converting  Enzymes 

We  have  recently  identified  two  cDNAs,  which 
we  call  PC2  and  PC3,  in  neuroendocrine  cells. 
These  encode  proteases  having  catalytic  domains 
similar  to  that  of  Kex2,  a  yeast  prohormone- 


589 


Insulin  and  the  Islets  of  Langerhans 


processing  enzyme.  PC2  is  more  abundant  in  islet 
(S-cells,  while  PC  3  predominates  in  the  anterior 
pituitary.  Expression  of  PC2  in  Xenopus  oocytes 
gives  rise  to  a  68-kDa  protein  that  is  active  on 
substrates  having  characteristic  sequences  for 
prohormone  cleavage  recognition  (i.e.,  Lys-Arg 
or  Arg-Arg) .  The  proteolytic  activity  requires  cal- 
cium ions  and  is  highest  at  pH  5.5.  Other  expres- 
sion studies  carried  out  in  collaboration  with 
Gary  Thomas  and  his  co-workers  at  the  Vollum 
Institute  indicate  that  PC  2  and  PC  3  separately  or 
together  can  appropriately  process  both  proopio- 
melanocortin or  proinsulin.  Studies  are  now  in 
progress  on  the  genes  encoding  these  proteases 
and  their  evolutionary  origins,  as  well  as  on  the 
synthesis,  sorting,  and  activation  of  PC2  and/or 
PC3  in  various  neuroendocrine  cells. 

Regulation  of  Insulin  Secretion 

We  have  recently  isolated  and  characterized 
cDNA  and  genomic  clones  encoding  several  volt- 
age-dependent K"^  channel  isoforms  expressed  in 
human  islets  or  insulin-producing  tumors.  These 
channels  are  related  to  the  Shaker  family  of  Dro- 
sophila  K"^  channels.  Their  electrophysiological 
characteristics  have  been  examined  by  voltage 
clamp  recordings  of  oocytes  injected  with  syn- 
thetic mRNA.  Efforts  are  also  under  way  to  iden- 
tify and  characterize  an  ATP-dependent  potas- 
sium channel  believed  to  play  a  key  role  in 
initiating  /S-cell  depolarization  in  response  to 
glucose  or  sulfonylureas. 

Insulin  Receptor  Studies 

Studies  nearing  completion  have  revealed  an 
interesting  and  potentially  important  aspect  of 
insulin  proreceptor  processing,  namely  that  un- 
cleaved  proreceptors  having  exon  1 1  (which  en- 
codes 1 2  amino  acids  near  the  carboxyl  terminus 
of  the  a-subunit)  are  fully  functional,  while  those 
lacking  this  exon  (due  to  alternative  splicing  of 


the  mRNA)  are  markedly  reduced  in  their  afi&nity 
for  insulin.  Since  most  cells  express  receptors 
without  exon  1 1 ,  failure  to  cleave  the  prorecep- 
tor (as  occurs  in  a  family  we  have  studied  that  has 
mutant  proreceptors  that  cannot  be  processed) 
leads  to  severe  insulin-resistant  diabetes.  The 
liver,  kidney,  islet  ;8-cells,  and  placenta  are  the 
only  tissues  that  express  insulin  receptors  with 
exon  1 1 ,  suggesting  that  alternative  splicing  may 
be  physiologically  relevant. 

Biosynthesis  of  Islet  Amyloid 
Polypeptide  (lAPP) 

LAPP,  or  amylin,  a  recently  discovered  product 
of  the  (8-cell,  is  a  peptide  related  to  CGRP  (calci- 
tonin gene-related  peptide)  that  is  found  in  amy- 
loid deposits  in  the  islets  of  elderly  diabetics  and 
may  play  a  role  in  impairing  |8-cell  function.  We 
have  characterized  the  gene  and  cDNAs  encoding 
the  precursor  of  lAPP  in  humans  and  several  other 
mammalian  species.  We  are  currently  studying  a 
transgenic  mouse  model  to  identify  factors  that 
may  contribute  to  amyloid  deposition  (in  collabo- 
ration with  Niles  Fox  of  the  Lilly  Research 
Laboratories) . 

Evolution  of  Insulin  and  Insulin-like 
Growth  Factors 

We  have  used  the  polymerase  chain  reaction  to 
identify  genes  encoding  insulin  or  the  closely  re- 
lated insulin-like  growth  factors  IGF-I  and  -II  in 
primitive  vertebrates.  The  identification  of  a  hy- 
brid insulin/IGF  molecule  in  amphioxus,  a  pro- 
tochordate,  suggests  that  the  insulin-like  growth 
factors  diverged  from  an  ancestral  preproinsulin- 
like  protein  in  the  very  earliest  stages  of  verte- 
brate evolution  (about  600  million  years  ago). 
Studies  nearing  completion  on  the  identification 
and  structure  of  insulin  and  IGF  receptors  in 
these  lower  forms  also  lend  support  to  this  model 
for  the  origin  of  the  insulin-like  growth  factors  as 
unique  vertebrate  growth  regulators. 


390 


Autoantibody  Probes  for  Mammalian 
Gene  Expression 


Joan  A.  Steitz,  Ph.D. — Investigator 

Dr.  Steitz  is  also  Professor  of  Molecular  Biophysics  and  Biochemistry  at  Yale  University  School  of  Medicine. 
She  received  her  Ph.D.  degree  in  biochemistry  and  molecular  biology  ( with  James  Watson )  from  Harvard 
University  and  did  postdoctoral  work  at  the  Medical  Research  Council  Laboratory  of  Molecular  Biology 
(with  Frances  Crick)  in  Cambridge,  England.  Her  many  honors  include  the  Passano  Foundation  Young 
Scientist  Award,  the  Eli  Lilly  Award  in  Biological  Chemistry,  the  U.S.  Steel  Award  in  Molecular  Biology, 
the  National  Medal  of  Science,  the  Dickson  Prize  for  Science,  the  Warren  Triennial  Prize  (shared  with 
Thomas  Cech),  and  the  Christopher  Columbus  Discovery  Award.  Dr.  Steitz  is  a  member  of  the  National 
Academy  of  Sciences. 


KNOWLEDGE  gained  from  basic  research  in 
the  biomedical  sciences  sometimes  pro- 
vides answers  to  key  questions  in  clinical  medi- 
cine. Occasionally,  however,  the  path  is  reversed 
and  clinical  studies  provide  information  or  mate- 
rials that  help  to  unravel  basic  biological  pro- 
cesses. An  example  of  this  is  our  use  of  sera  from 
human  patients  to  determine  the  roles  of  previ- 
ously mysterious  small  particles  in  normal  cells. 
Particles  called  small  nuclear  ribonucleoproteins 
(snRNPs:  pronounced  "snurps")  are  found  in  the 
nucleus  of  the  cells  of  humans  and  other  higher 
organisms.  Each  snRNP  is  a  tight  cluster  of  one  or 
more  proteins  with  a  small  RNA  molecule. 
SnRNPs  are  abundant  in  virtually  all  human  cells 
and  are  remarkably  similar  among  various  mam- 
malian species,  suggesting  that  the  particles  must 
play  important  cellular  roles. 

Systemic  lupus  erythematosus  (SLE)  is  one  of  a 
number  of  diseases  in  which  the  immune  system 
mistakenly  makes  antibodies  against  the  body's 
own  molecules.  Curiously,  molecules  that  are 
very  abundant  in  cells  and  highly  conserved  in 
evolution,  such  as  DNA,  are  the  most  common 
targets  of  autoimmunity.  Thus  SLE  patients  often 
make  autoantibodies  against  snRNPs. 

Using  SLE  patients'  antibodies  to  probe  both 
the  structures  and  functions  of  snRNPs,  we  have 
investigated  the  roles  of  various  kinds  of  snRNPs 
in  gene  expression.  These  investigations  began  in 
1979  when  studies  by  Michael  Lerner  (then  an 
M.D./Ph.D.  student;  now  HHMI,  Yale  University 
School  of  Medicine)  led  to  the  hypothesis  that  the 
most  abundant  snRNP  in  mammalian  cells,  called 
the  Ul  snRNP,  might  be  involved  in  RNA  splic- 
ing, an  early  step  in  gene  expression.  In  the  mak- 
ing of  a  gene's  product,  its  information  coded  in 
DNA  is  transcribed  into  an  RNA  copy  called  pre- 
messenger  RNA,  which  is  then  "processed"  into 
mRNA  to  direct  the  synthesis  of  a  protein.  The 
DNA  and  the  pre-mRNA  contain  segments  called 
exons,  which  code  for  the  gene's  product,  and 
segments  called  introns,  which  are  intermittent 
noncoding  regions.  Before  leaving  the  cell  nu- 


cleus as  mRNA,  the  pre-mRNA  is  cut,  the  exons 
are  spliced  together,  and  the  introns  discarded. 
The  individual  exons  must  be  precisely  joined  in 
the  order  they  originally  had  in  the  gene.  Some- 
times differences  in  the  way  exons  are  spliced 
can  lead  to  anomalous  protein  products  in 
various  tissues. 

Evidence  that  snRNPs  play  central  roles  in  pre- 
mRNA  splicing  has  been  obtained  in  several  types 
of  experiments,  including  use  of  autoantibodies 
from  SLE  patients  to  inhibit  splicing  in  active  cell 
extracts.  We  now  know  that  participation  of  the 
most  abundant  snRNPs  in  mammalian  cells  (the 
Ul ,  U2,  U5,  and  U4/U6  particles)  is  essential  and 
that  splicing  requires  assistance  from  the  snRNP 
proteins  as  well  as  their  RNA  molecules.  SnRNPs 
recognize  the  splice  junctions  and  the  so-called 
intron  branch  point  (where  an  unusual  RNA 
structure  is  formed  as  an  intermediate  in  splic- 
ing) and  then  assemble  to  align  the  exon  ends  so 
that  precise  splicing  can  occur.  In  this  sense, 
snRNPs  are  much  like  the  ribosomal  subunits 
(also  containing  both  RNA  and  protein)  that  as- 
semble onto  an  mRNA  to  translate  it  into  protein. 

Current  efforts  in  splicing  are  directed  at  un- 
derstanding exactly  how  RNA-RNA  interactions  in 
the  active  splicing  body  (called  the  spliceosome) 
contribute  to  catalysis.  Here,  we  are  using  two 
types  of  crosslinking  approaches  to  identify  con- 
tacts between  the  pre-mRNA  molecule  and  either 
proteins  or  snRNAs  in  the  assembled  splice- 
osome. Novel  crosslinks  are  now  being  analyzed 
that  suggest  how  a  cut-off  exon  may  be  held  in  the 
spliceosome  for  subsequent  ligation.  Also  evolv- 
ing is  an  increased  understanding  of  how  the 
spliceosome  is  related  to  some  "self-splicing"  in- 
trons, which  are  removed  without  proteins  or 
other  factors. 

Mammalian  cells  also  contain  many  minor 
snRNPs  that  are  closely  related  to  the  splicing 
snRNPs.  One  is  the  U7,  which  is  only  1/1 ,000  as 
abundant  as  the  splicing  snRNPs.  We  have  re- 
cently demonstrated  that  it  participates  in  form- 
ing the  3'  ends  of  histone  mRNAs  by  using  base- 
pairing  to  recognize  a  specific  sequence  in  the 


391 


Autoantibody  Probes  for  Mammalian  Gene  Expression 


pre-mRNA  just  downstream  of  the  cut  site.  Other 
related  low- abundance  snRNPs  containing  Ull 
and  Ul  2  RNA  interact  to  form  a  two-snRNP  com- 
plex, but  their  function  is  unknown. 

A  new  autoantibody  directed  against  the  Ull 
snRNP  has  recently  been  found  in  the  serum  of  a 
patient  with  scleroderma.  Also  in  this  group  of 
-related  snRNPs,  which  probably  all  function  in 
some  aspect  of  mRNA  maturation,  are  viral 
snRNPs.  For  instance,  in  marmoset  cells  infected 
by  Herpesvirus  saimiri,  which  causes  malignant 
transformation  of  the  T  cells,  the  viral  genome 
encodes  five  small  RNAs.  Current  results  suggest 
that  these  viral  snRNPs  may  act  to  slow  the  degra- 
dation of  mRNAs  coding  for  oncoproteins  or 
growth  factors,  thereby  enhancing  the  process  of 
cellular  transformation. 

Another  type  of  patient  autoantibody  is  di- 
rected against  a  different  class  of  small  RNPs  lo- 
calized in  the  nucleolus,  where  ribosomal  RNA 
processing  and  assembly  occur.  The  most  abun- 
dant nucleolar  snRNP  (containing  U3  RNA)  is  es- 
sential for  the  first  step  of  ribosomal  RNA  process- 
ing. Recent  analyses  have  revealed  how  U3  binds 
to  the  ribosomal  pre-RNA,  making  specific  base 


pairs.  Separate  studies  are  dissecting  the  signals 
that  dictate  the  delivery  of  the  U3  and  related 
snRNPs  to  their  nucleolar  site  of  action. 

Yet  another  autoantibody  type  precipitates 
EBERs,  two  small  RNAs  specified  by  Epstein-Barr 
virus  (EBV),  the  causative  agent  of  infectious 
mononucleosis,  also  implicated  in  several  human 
cancers.  Since  EBERs  are  among  the  few  viral 
products  that  are  expressed  in  EBV-transformed 
cells,  they  must  be  important  to  the  induction  or 
maintenance  of  the  transformed  state.  A  highly 
abundant,  highly  conserved  cell  protein  that 
binds  the  EBERs  appears  to  reside  in  the  cell's 
nucleolus.  We  hope  that  its  further  characteriza- 
tion will  lead  to  an  elucidation  of  EBER  function. 

Thus  autoantibodies  are  potent  probes  for  de- 
ciphering some  of  the  fundamental  reactions  oc- 
curring in  all  mammalian  cells,  those  involved  in 
gene  expression.  Characterization  of  new  cellu- 
lar particles  like  snRNPs  is  significant  for  future 
studies  of  basic  cellular  processes  and  their  alter- 
ation by  disease.  Furthermore,  our  research  has 
provided  new  ways  of  diagnosing  patient  autoan- 
tibodies, which  are  helpful  in  the  diagnosis  and 
treatment  of  diseases  like  SLE. 


592 


Structural  Studies  of  Protein-Nucleic  Acid 
Interactions 


Thomas  A.  Steitz,  Ph.D. — Investigator 

Dr.  Steitz  is  also  Professor  of  Molecular  Biophysics  and  Biochemistry  and  of  Chemistry  at  Yale  University. 
He  received  a  B.A.  degree  in  chemistry  from  Lawrence  College  in  Appleton,  Wisconsin,  and  a  Ph.D.  degree 
in  molecular  biology  and  biochemistry  from  Harvard  University,  with  W.  N.  Lipscomb.  After  a 
postdoctoral  year  at  Harvard,  he  moved  to  the  MRC  Laboratory  of  Molecular  Biology  in  Cambridge, 
England,  as  a  fane  Coffin  Childs  fellow,  with  D.  M.  Blow.  He  next  Joined  the  Yale  faculty,  where  he  has 
remained,  except  for  sabbatical  work  with  K.  Weber  in  Gottingen,  West  Germany,  with  A.  Klug  at 
Cambridge,  and  with  f.  Abelson  at  the  California  Institute  of  Technology.  He  has  received  the  Pfizer  Prize 
of  the  American  Chemical  Society  and  is  a  member  of  the  National  Academy  of  Sciences. 


OUR  general  long-term  goal  is  to  determine 
the  detailed  molecular  mechanisms  by 
which  those  proteins  and  nucleic  acids  that  are 
involved  in  the  central  dogma  of  molecular  biol- 
ogy (DNA  replication,  transcription,  and  transla- 
tion) achieve  their  biological  functions.  Virtually 
all  aspects  of  the  maintenance  and  expression  of 
information  stored  in  the  genome  involve  inter- 
actions between  proteins  and  nucleic  acids.  We 
are  seeking  to  provide  a  structural  and  chemical 
basis  for  these  fundamental  processes. 

Synthetase-tRNA  Complex 

Enzymes  called  aminoacyl-tRNA  synthetases 
translate  the  genetic  code  by  attaching  the 
correct  amino  acid  to  a  tRNA  containing  the  ap- 
propriate anticodon.  Of  significant  current  inter- 
est is  how  these  synthetases  can  accurately  distin- 
guish among  the  60  or  so  similar  tRNA  molecules. 
Furthermore,  how  does  RNA  recognition  by  a 
protein  differ  from  DNA  recognition?  Finally,  can 
the  structure  of  the  present-day  synthetase-tRNA 
complex  provide  any  insights  into  the  evolution 
of  this  central  process  and  the  evolution  of  the 
genetic  code  itself? 

We  have  determined  the  crystal  structure  of 
glutaminyl-tRNA  synthetase  (GlnRS),  a  64,000- 
molecular-weight  monomeric  protein,  com- 
plexed  with  tRNA^'"  and  ATP.  The  GlnRS  consists 
of  four  domains  arranged  to  give  an  elongated 
molecule  that  interacts  with  the  inside  of  the  L- 
shaped  tRNA  from  its  anticodon  to  its  acceptor 
end.  GlnRS  specifically  recognizes  the  correct 
tRNA  by  interactions  with  the  three  bases  of  the 
anticodon  and  with  base  pairs  of  the  amino  acid 
acceptor  stem  of  the  tRNA.  The  three  bases  of  the 
anticodon  are  unstacked  and  splayed  out;  each 
base  binds  into  a  separate  recognition  pocket  on 
the  enzyme.  The  extensive  hydrogen-bonding  in- 
teractions between  the  protein  and  the  anticodon 
bases  make  the  enzyme  specific  for  the  two  gluta- 
mine  anticodons  (UUG  and  CUG)  but  none  of  the 
other  62  possible  anticodons.  The  structural 


bases  of  Gln-tRNA  synthetase  recognition  are 
currently  being  pursued  by  determining  the 
structures  of  mutant  tRNAs  complexed  with  the 
enzyme. 

The  protein  domain  that  contains  the  active 
site  has  a  structure  similar  to  that  of  the  homolo- 
gous domains  of  the  tyrosyl-  and  methionyl-tRNA 
synthetases  and  by  amino  acid  sequence  similar- 
ity is  homologous  to  another  10  of  the  20  synthe- 
tases. Synthetases  for  10  amino  acids  belong  to  a 
second  unrelated  class  of  synthetases.  This  work 
is  supported  by  a  grant  from  the  National  Insti- 
tutes of  Health. 

Regulation  of  Gene  Expression 

In  Escherichia  colt  a  reduction  in  glucose  con- 
centration results  in  a  rise  in  the  levels  of  a  sec- 
ond messenger  molecule,  cAMP,  and  subse- 
quently to  an  increase  in  the  proteins  that 
metabolize  other  sugars.  This  is  achieved  because 
cAMP  binds  to  the  catabolite  gene  activator  pro- 
tein (CAP),  which  in  turn  binds  to  specific  se- 
quences at  transcription  start  sites,  activating  the 
transcription  of  the  catabolite  genes.  We  wish  to 
know  how  the  binding  of  cAMP  promotes  the  se- 
quence-specific DNA  binding  of  CAP  and  how 
this  binding  then  activates  the  transcribing  en- 
zyme RNA  polymerase. 

We  have  now  determined  the  structure  of  CAP 
cocrystallized  with  both  a  30-bp  DNA  fragment 
and  cAMP.  The  earlier  CAP  •  cAMP  structure  had 
shown  each  subunit  of  this  dimer  to  consist  of 
two  domains,  the  larger  of  which  binds  cAMP. 
The  two  small  domains  are  seen  to  bind  DNA, 
with  the  helix-turn-helix  interacting  in  the  major 
groove  as  anticipated.  Strikingly,  in  the  first  com- 
plex the  DNA  is  bent,  with  an  overall  bend  of 
about  90° .  In  both  complexes  most  of  the  bend  is 
achieved  by  two  large  kinks  of  about  43°  each. 
The  relationships  between  this  CAP-induced  DNA 
bend  and  transcription  activation  are  presently 
being  pursued  by  attempts  to  crystallize  CAP  with 
polymerase  and  DNA. 


393 


Structural  Studies  of  Protein-Nucleic  Acid  Interactions 


Replication  of  DNA 

E.  co// DNA  polymerase  I  functions  primarily  in 
the  repair  of  DNA  but  is  homologous  to  polymer- 
ases involved  in  replication.  We  have  determined 
the  structure  of  the  Klenow  fragment,  a  portion 
of  Pol  I  that  retains  the  polymerase  and  a  3'-  to 
5'-editing  exonuclease  activity.  We  have  shown 
that  a  larger  structural  domain,  which  has  a  cleft 
sufficient  in  size  to  bind  duplex  DNA,  contains 
the  active  site  for  the  polymerase  reaction, 
whereas  a  smaller  domain  has  the  active  site  for 
the  exonuclease  activity.  Using  site-directed  mu- 
tagenesis, we  have  made  an  enzyme  devoid  of  the 
editing  exonuclease  activity  and  determined  its 
structure.  We  have  grown  two  crystal  forms  of 
this  protein  complexed  with  a  small  DNA  sub- 
strate. A  high-resolution  structure  of  one  crystal 
form  shows  a  single-stranded  tetranucleotide 
bound  to  the  exonuclease  active  site  and  1 1  base 
pairs  of  duplex  DNA  bound  to  a  cleft  that  runs  at 
right  angles  to  the  major  cleft.  There  are  changes 
in  the  structure  of  the  polymerase  domain,  in- 
cluding the  movement  of  a  thumb-like  structure. 
To  access  the  polymerase  active  site  in  the  cleft,  it 
appears  that  the  DNA  will  have  to  bend  by  90°. 
These  structures  begin  to  address  the  issues  of 
how  these  two  active  sites  work  together  on  the 
same  DNA  substrate  and  how  they  both  function 
to  enhance  the  DNA-copying  fidelity  of  this  and 
other  polymerases.  This  work  is  supported  by  a 
grant  from  the  American  Cancer  Society. 

Genetic  Recombination 

We  have  recently  determined  the  crystal  struc- 
tures of  two  proteins  that  are  involved  in  genetic 
recombination.  One  protein,  called  resolvase, 
catalyzes  a  site-specific  recombination  between 
two  duplex  DNAs  of  identical  sequence.  Resolv- 
ase is  the  product  of  a  transposable  element  (a 
jumping  gene)  that  can  move  throughout  the  E. 
coli  population  spreading  drug  resistance  genes. 
This  protein  can  bind  to  a  specific  duplex  DNA 
sequence,  align  two  DNA  segments  having  the 
same  sequence,  cleave  the  two  DNA  duplexes, 
rearrange  the  duplexes,  and  re-ligate  them,  re- 
sulting in  a  recombinational  event.  We  have  de- 
termined the  structure  of  the  catalytic  domain  of 
this  enzyme  at  2. 5 -A  resolution.  This  structure 
helps  to  explain  the  phenotypes  of  many  resolv- 
ase mutant  proteins.  This  structure  and  that  of  the 
intact  protein  determined  at  3-A  resolution  pro- 
vide ideas  for  a  possible  recombination  mecha- 


nism. We  have  recently  cocrystallized  this  pro- 
tein with  a  31 -bp  fragment  containing  the 
recombination  site,  whose  structure  should  pro- 
vide further  clues  to  the  mechanism  of  this 
reaction. 

E.  coli  recK  protein  is  essential  for  general  re- 
combination in  E.  coli.  Using  the  energy  of  ATP 
hydrolysis,  recK  protein  promotes  the  pairing  of 
homologous  duplex  DNAs  in  preparation  for  re- 
combination. The  structure  of  rech.  protein  has 
been  refined  at  2. 3 -A  resolution.  The  subunit 
forms  a  helical  filament  in  the  crystal  very  similar 
to  that  formed  on  DNA  and  thus  enables  us  to 
understand  the  many  mutant  recK  proteins  made 
during  the  past  decade  and  relate  its  structure  to 
its  functions  in  nucleotide  binding,  DNA  binding, 
and  the  SOS  response.  Our  goal  is  to  understand 
how  ATP  hydrolysis  and  the  homologous  pairing 
of  DNA  are  coupled.  We  have  now  produced 
good  crystals  of  the  next  enzyme  in  the  pathway 
of  recombination,  ruvC,  which  cleaves  the  DNA 
recombination  intermediate  called  the  Holliday 
structure.  The  work  on  resolvase  and  the  recK 
protein  is  supported  by  a  grant  from  the  National 
Institutes  of  Health. 

HIV  Proteins 

During  the  past  year  we  have  determined  the 
structure  of  human  immunodeficiency  virus 
(HIV)  reverse  transcriptase  complexed  with  a 
nonnucleotide  inhibitor  that  shows  promise  as  an 
anti-AIDS  drug.  The  enzyme  is  a  heterodimer  of  a 
66-kDa  polypeptide  containing  a  polymerase  and 
RNase  H  domain  and  a  5 1  -kDa  polypeptide  con- 
taining only  the  polymerase  domain.  This  di- 
meric  polymerase  shows  an  asymmetric  structure 
with  one  active-site  cleft  running  from  the  p66 
domain  to  the  RNase  H  domain.  The  p5 1  subunit 
has  no  cleft.  The  similarities  between  the  cata- 
lytic domains  of  HIV  reverse  transcriptase  and 
Klenow  fragment  suggest  that  all  polymerases 
have  the  same  catalytic  site.  We  have  been  suc- 
cessful in  diffusing  other  nonnucleotide  inhibi- 
tors into  the  crystal  and  find  that  they  bind  into  a 
deep  pocket  near  to,  but  not  overlapping  with, 
the  polymerase  catalytic  site.  We  expect  that  de- 
sign of  additional  inhibitors  based  on  the  struc- 
ture will  be  possible. 

Recently  a  peptide  fragment  of  the  transactiva- 
tor  protein  Tat  has  been  cocrystallized  with  a 
fragment  of  the  RNA  site  to  which  it  binds,  TAR. 
This  work  is  supported  in  part  by  a  grant  from  the 
National  Institutes  of  Health. 


394 


A  representation  of  the  crystal  structure  o/ Escherichia  coli  tecA  protein.  This 
protein  catalyzes  DMA  strand  exchange  in  general  genetic  recombination. 
Shown  are  12  subunits  of  the  crystal  in  a-carbon  backbone  representation, 
with  adjacent  subunits  alternately  light  and  dark  blue.  A  bound  ADP  molecule 
is  represented  in  orange. 

Figure  by  Randall  Story  and  Thomas  Steitz  from  research  reported  in  Story, 
R.M.,  Weber,  I.T.,  and  Steitz,  T.A.  1992.  Nature  555:318-525. 


395 


Visualization  of  photoreceptor  axons  in  the  developing  visual  system  o/Drosophila.  The  axon 
bundles  emerge  from  each  ommatidium  of  the  developing  retina  and  project  through  an  epithelial 
tube,  the  optic  stalk,  to  specific  retinotopic  positions  in  the  brain.  Guidance  of  these  axons  relies 
largely  on  positional  information. 
Research  of  Hermann  S  teller. 


396 


Pattern  Formation  and  Neuronal  Cell  Recognition 
in  the  Drosophila  Visual  System 


Hermann  Steller,  Ph.D. — Assistant  Investigator 

Dr.  Steller  is  also  Associate  Professor  of  Neurobiology  at  the  Massachusetts  Institute  of  Technology  and 
Adjunct  Assistant  Neurobiologist  at  Massachusetts  General  Hospital,  Boston.  He  was  born  in  the  Federal 
Republic  of  Germany  and  received  a  Diplom  in  biology  from  the  Johann- Wolf  gang- Goethe  University, 
Frankfurt.  His  graduate  studies  were  done  with  Vincenzo  Pirrotta  at  the  European  Molecular  Biology 
Laboratory  and  with  Ekkehard  Bautz  at  Heidelberg  University.  His  postdoctoral  work  was  done  with 
Gerald  Rubin  in  the  Department  of  Biochemistry  at  the  University  of  California,  Berkeley.  Dr.  Steller  is 
currently  also  a  Searle  Scholar  and  a  Pew  Fellow  in  the  Biomedical  Sciences. 


THE  overall  objective  of  our  research  is  to  un- 
derstand how  functional  neuronal  circuits 
are  established  and  maintained  during  develop- 
ment. Our  current  work  is  focused  on  three  major 
areas. 

Axon  Guidance  and  Neuronal 
Cell  Recognition 

We  are  studying  two  different  stages  of  visual 
system  development  to  investigate  the  cellular 
and  molecular  mechanisms  by  which  axons  find 
and  recognize  their  proper  synaptic  partners.  The 
optic  nerve  of  the  Drosophila  larva  is  a  simple, 
well-described  model  system.  Connectivity  de- 
fects of  the  larval  optic  nerve  can  be  rapidly  and 
reliably  detected  in  mutant  embryos  by  staining 
with  neuron-specific  antibodies.  In  addition,  a 
simple  behavioral  test,  larval  phototaxis,  pro- 
vides an  assay  for  functional  connections  of  the 
larval  optic  nerve.  This  permits  systematic 
screening  for  mutants  with  abnormal  axonal  pro- 
jections, which  can  be  subsequently  analyzed  in 
detail  with  respect  to  defects  in  axonal  guidance, 
target  recognition,  and  synapse  formation. 

We  have  previously  identified  a  gene,  discon- 
nected {disco),  which  is  required  for  establish- 
ing stable  connections  between  the  larval  optic 
nerve  and  its  target  cells  in  the  developing  brain. 
We  have  cloned  the  disco  gene  and  determined 
its  structure,  nucleotide  sequence,  and  pattern  of 
expression.  These  studies  suggest  that  disco  en- 
codes a  transcription  factor  with  autoregulatory 
properties.  Consistent  with  such  a  function  we 
have  recently  found  that  disco  protein  has  se- 
quence-specific DNA-binding  activity  in  vitro 
and  that  two  high-affinity  binding  sites  are  lo- 
cated very  close  to  the  disco  transcription  unit. 
Ectopic  expression  of  disco  protein  under  an  in- 
ducible promoter  in  transgenic  flies  results  in  se- 
vere developmental  defects  and  embryonic  lethal- 
ity. These  defects  include  a  drastic  reduction  of 
the  axon  scaffold  and  connectivity  defects  in  both 
the  peripheral  and  central  nervous  systems.  We 
are  now  testing  the  idea  that  disco  regulates  the 
expression  of  cell  adhesion  and/or  cell  recogni- 


tion molecules  that  are  required  for  the  establish- 
ment of  stable  connections  between  the  larval 
optic  nerve  and  its  target  cells  in  the  brain. 

More  recently  we  have  begun  to  study  axon 
guidance  and  neuronal  cell  recognition  in  the 
adult  visual  system.  The  compound  eye  of  Dro- 
sophila consists  of  approximately  800  repeating 
units  called  ommatidia.  Each  ommatidium  con- 
tains eight  photoreceptor  neurons,  which  repre- 
sent three  major  cell  types  that  project  to  differ- 
ent target  cells  in  the  optic  ganglia.  The  major 
class  of  photoreceptors,  Rl-6,  establishes  synap- 
tic connections  with  neurons  in  the  first  optic 
ganglion,  the  lamina.  Photoreceptor  axons  from 
R7  and  R8  project  deeper  into  the  brain  to  differ- 
ent regions  of  the  second  ganglion,  the  medulla. 
Early  during  visual  system  development,  all  eight 
photoreceptor  axons  from  each  ommatidium 
grow  as  a  bundle  to  specific  retinotopic  positions 
in  the  developing  brain.  The  growth  cones  of 
these  axons  have  to  navigate  over  a  long  distance 
and  make  a  number  of  highly  specific  choices. 

We  would  like  to  understand  what  signals 
guide  axons  to  their  proper  destinations  and  how 
these  signals  are  generated,  received,  and  inter- 
preted. To  address  these  questions,  we  have 
screened  for  mutations  that  perturb  the  projec- 
tion pattern  of  photoreceptor  cells  at  very  early 
developmental  stages,  when  axons  enter  the 
brain.  We  have  found  a  number  of  mutants  with 
severely  abnormal  patterns  of  axon  ingrowth.  The 
developmental  and  genetic  characterization  of 
this  material  is  in  progress. 

Role  of  Innervation  for  Neurogenesis 
and  Survival  of  Target  Cells 

It  has  been  noticed  for  many  years  that  synaptic 
input  can  have  a  profound  influence  on  the  fate 
and  differentiation  of  target  cells.  Cell  death  in 
the  absence  of  incoming  projections  is  a  dramatic 
example  of  how  innervation  can  affect  develop- 
mental decisions,  and  many  neurological  dis- 
orders are  thought  to  arise  from  defective  interac- 
tions between  neurons  and  their  targets.  In 


397 


Pattern  Formation  and  Neuronal  Cell  Recognition  in  the  Drosophila 
Visual  System 


Drosophila  the  proper  development  of  the  aduh 
optic  gangha,  the  central  nervous  system  portion 
of  the  visual  system,  depends  on  innervation  from 
the  eye.  In  the  absence  of  retinal  innervation, 
adult  flies  entirely  lack  the  first  optic  ganglion, 
the  lamina,  which  receives  direct  synaptic  input 
from  the  outer  photoreceptor  cells  Rl-6. 
^'  We  have  found  that  the  birth  of  lamina  neurons 
is  controlled  by  innervation  from  the  developing 
eye.  The  arrival  of  photoreceptor  axons  in  the 
brain  induces  a  wave  of  mitotic  activity  that  pro- 
duces the  lamina  neurons.  These  results  suggest  a 
novel  mechanism  for  matching  the  number  of  tar- 
get neurons  in  the  first  optic  ganglion  to  the  num- 
ber of  incoming  photoreceptor  axons,  and  they 
explain  how  developmental  synchrony  between 
the  Drosophila  retina  and  first  optic  ganglion  is 
achieved.  We  are  now  using  several  different  ap- 
proaches to  elucidate  the  detailed  cellular  and 
molecular  mechanisms  underlying  this  process. 

While  the  importance  of  retinal  innervation  on 
the  development  of  the  adult  optic  ganglia  of 
Drosophila  is  well  documented,  little  is  known 
about  retrograde  effects  of  the  brain  on  photore- 
ceptor cells  in  the  compound  eye.  We  have  re- 
cently discovered  the  first  evidence  for  the  exis- 
tence of  such  retrograde  effects  in  the 
Drosophila  visual  system.  Although  photorecep- 
tor cells  develop  normally  in  the  absence  of  con- 
nections to  the  optic  ganglia,  we  have  found  that 
their  continued  survival  requires  these  connec- 
tions. This  situation  is  reminiscent  of  trophic  in- 


teractions that  are  commonly  found  in 
vertebrates. 

Genetic  Control  of  Cell  Death 

Apoptosis,  the  deliberate  and  orderly  removal 
of  cells  by  natural  death,  is  a  prominent  feature  of 
normal  development  throughout  the  animal 
kingdom.  In  many  organisms,  a  large  number  of 
cells  die  in  the  absence  of  obvious  external  in- 
sults. For  example,  in  vertebrates  neurogenesis 
produces  about  twice  as  many  neurons  as  are 
needed  in  the  mature  nervous  system,  and  approx- 
imately half  of  these  neurons  are  eliminated  by 
cell  death.  We  are  interested  in  isolating  genes 
that  are  required  for  the  initiation  or  execution  of 
cell  death  in  Drosophila.  We  have  found  that  the 
ultrastructural  characteristics  of  cell  deaths  seen 
in  the  Drosophila  embryo  are  strikingly  similar 
to  apoptotic  deaths  described  in  other  systems. 
We  have  developed  techniques  utilizing  the  vital 
dyes  acridine  orange  and  nile  blue  that  permit  the 
rapid  and  reliable  visualization  of  apoptotic  cells 
in  live  embryos,  and  we  have  used  these  methods 
to  screen  for  cell  death-defective  mutants.  We 
have  identified  a  complex  genetic  locus  on  the 
third  chromosome  that  is  required  for  either  the 
commitment  to  or  the  execution  of  a  cell  death 
program.  We  have  cloned  the  DNA  encompassing 
this  locus,  and  we  expect  that  its  molecular  char- 
acterization will  provide  insight  into  the  bio- 
chemical mechanisms  underlying  cell  death  in 
Drosophila,  and  possibly  other  organisms  as 
well. 


398 


Molecular  Genetics  of  Nematode  Development 
and  Behavior 


Paul  W.  Sternberg,  Ph.D. — Associate  Investigator 

Dr.  Sternberg  is  also  Associate  Professor  of  Biology  at  the  California  Institute  of  Technology  and  Adjunct 
Assistant  Professor  of  Anatomy  and  Cell  Biology  at  the  University  of  Southern  California  School  of 
Medicine,  los  Angeles.  He  received  a  B.A.  degree  in  biology  and  mathematics  from  Hampshire  College  and 
a  Ph.D.  degree  in  biology  from  the  Massachusetts  Institute  of  Technology  for  work  with  Robert  Horvitz. 
He  did  postdoctoral  research  in  yeast  molecular  genetics  with  Ira  Herskowitz  at  the  University  of 
California,  San  Francisco.  Dr.  Sternberg  is  also  a  Presidential  Young  Investigator. 


USING  the  nematode  Caenorhabditis  ele- 
gans,  our  laboratory  takes  a  molecular  genet- 
ics approach  to  basic  questions  in  developmental 
biology  and  neurogenetics:  What  are  the  molecu- 
lar mechanisms  by  which  cells  interact  to  estab- 
lish a  spatial  pattern  of  cell  types?  What  is  the 
genetic  and  cellular  basis  for  morphogenesis? 
What  establishes  the  asymmetry  of  individual 
cells?  How  are  the  instructions  for  innate  behav- 
ior encoded  in  the  genome?  Our  major  strategy  is 
to  identify  mutations  that  make  cells  or  animals 
misbehave  and  then  to  study  the  functions  of  the 
genes  defined  by  these  mutations,  using  a  combi- 
nation of  molecular  cloning  and  genetic  analysis. 
A  second  strategy  is  to  clone  nematode  homo- 
logues  of  genes  identified  in  mammals  and  then 
to  elucidate  the  functions  of  those  genes  in 
nematodes. 

In  this  past  year  we  focused  on  the  develop- 
ment and  function  of  the  C.  elegans  male  spic- 
ules— innervated  structures  crucial  to  successful 
mating.  Each  of  the  two  spicules  comprises  nine 
cells:  two  sensory  neurons,  one  motoneuron,  and 
six  supporting  cells.  By  studying  spicule  develop- 
ment, we  have  identified  a  new  example  of  in- 
duction during  nematode  development.  In  the  de- 
veloping male,  either  of  two  cells  signals  spicule 
precursor  cells  to  generate  particular  sets  of  spic- 
ule cells.  This  inductive  signaling  process  re- 
quires the  tin- 3  growth  factor,  the  let- 2 3  tyrosine 
kinase,  and  the  let-60  ras  genes  that  we  cloned 
over  the  last  two  years,  tin- 3  encodes  an  induc- 
tive signal  for  the  hermaphrodite  vulva,  and  it  is 
likely  that  it  acts  as  an  inductive  signal  for  proper 
spicule  development  as  well.  Thus  we  have 
found  that  a  cascade  of  proto-oncogenes  specifies 
cell  fates  in  several  aspects  of  nematode  develop- 
ment. In  addition  to  this  inductive  signal,  at  least 
three  other  signals  are  also  necessary  for  the 
correct  specification  of  spicule  precursor  cells. 

Because  C.  elegans  hermaphrodites  are  inter- 
nally self-fertilizing — each  animal  producing 
both  sperm  and  ova — male  mating  and  thus  spic- 
ule function  is  dispensable.  Thus  mutant  strains 
defective  in  male  mating  can  be  easily  propa- 
gated and  the  mating  process  studied.  We  have 


used  a  simple  behavioral  test — the  ability  of 
males  to  sire  progeny — to  isolate  mutants  that  are 
unable  to  mate.  Some  mutant  males  have  obvious 
defects  in  the  development  of  male-specific 
structures.  Others,  called  Cod  (for  copulation 
defective) ,  are  anatomically  normal  yet  defective 
in  mating  behavior. 

By  studying  the  Cod  mutants,  we  hope  to  eluci- 
date how  genes  control  each  step  in  male  mating 
behavior.  We  have  isolated  a  set  of  mutants,  have 
characterized  the  mating  defect  of  each  strain, 
and  have  begun  placing  these  mutations  on  the 
genetic  map.  Most  of  the  mutants  analyzed  are 
defective  at  only  a  single  step  in  the  mating  pro- 
cess. These  steps  include  1)  attraction  to  her- 
maphrodites, 2)  maintaining  contact  with  her- 
maphrodites, 3)  location  of  the  vulva,  4) 
insertion  of  spicules,  and  5)  transfer  of  sperm. 
For  example,  a  mutant  male  defective  in  step  4 
will  locate  the  hermaphrodite  vulva  but  fail  to 
insert  his  spicule.  Having  mutants  blocked  at  de- 
fined steps  will  allow  us  to  identify  genes  neces- 
sary to  specify  this  innate  behavior. 

To  identify  the  cells  responsible  for  each  step 
in  mating  behavior,  we  kill  individual  cells  with  a 
laser  microbeam  and  observe  the  consequences. 
For  example,  the  spicule  motoneuron  and  both 
spicule  sensory  neurons  are  necessary  for  spicule 
insertion.  One  of  our  G  protein  a-subunit  genes  is 
expressed  in  one  of  these  neurons,  suggesting 
that  it  might  serve  to  regulate  spicule  insertion. 
Another  G  protein,  homologous  to  human  G,,,  is 
expressed  in  male  diagonal  muscles,  required  for 
initial  steps  in  mating.  We  have  begun  to  test 
whether  any  of  the  Cod  mutants  are  defective  in 
these  G  protein  genes. 

The  establishment  of  cellular  asymmetry  is  a 
fundamental  aspect  of  cell  regulation.  We  have 
begun  to  study  this  problem  in  the  context  of  the 
2°  vulval  precursor  cell.  The  lin-18  gene  (cell 
lineage  gene  number  18)  is  necessary  for  the 
asymmetry  of  the  2°  vulval  precursor  cell.  We 
have  mapped  the  lin-18  gene  to  a  manageable 
region  of  the  X  chromosome.  We  have  found  that 
the  2°  cells  will  orient  posteriorly  in  the  animal, 
unless  they  receive  a  signal  from  the  developing 
gonad  to  orient  anteriorly.  This  signal  is  distinct 


399 


Molecular  Genetics  of  Nematode  Development  and  Behavior 


from  the  inductive  signal  that  instructs  the  2° 
cells  to  be  2°  and  not  other  types  of  cells.  The 
lin-18  gene  must  be  involved  in  the  response  to 
this  signal. 

In  another  project,  we  have  been  analyzing  the 
function  of  the  nematode  homologues  of  signal 
transduction  molecules  identified  in  mammals. 
-We  have  cloned  a  nematode  gene  similar  to  the 
raf-1  protein  kinase  as  well  as  four  nematode 
genes  similar  to  mammalian  G  protein  a-subunit 
genes.  We  have  placed  these  on  the  C.  elegans 


genomic  map.  To  identify  the  cells  expressing 
these  genes,  we  have  constructed  chimeric  genes 
consisting  of  their  transcriptional  control  regions 
linked  to  a  reporter  gene.  We  then  introduced  the 
engineered  genes  back  into  the  nematode  by  mi- 
croinjection of  DNA  to  create  transgenic  nema- 
todes. All  four  G  protein  genes  are  expressed  pri- 
marily in  neurons,  including  neurons  in  the  male 
tail.  We  plan  to  engineer  poisonous  forms  of 
these  genes  to  disrupt  their  function  in  intact 
animals. 


A  G  protein  a-subunit  gene  expressed  in  a  sensory  neuron  of  a  male  spicule  o/ Caenorhabditis 
elegans.  The  cell  body  is  seen  as  a  large  round  blue  spot,  and  the  dendrite  as  blue  inside  the  yellow 
spicule. 

Research  of  Paul  Sternberg;  photograph  by  Jane  Mendel. 


400 


Why  Do  We  Drink  Coffee  and  Tea? 


Charles  F.  Stevens,  M.D.,  Ph.D. — Investigator 

Dr.  Stevens  is  also  Professor  of  Molecular  Neurobiology  at  the  Salk  Institute  for  Biological  Studies  and 
Adjunct  Professor  of  Pharmacology  and  of  Neuroscience  at  the  University  of  California  School  of  Medicine, 
San  Diego.  He  received  his  B.A.  degree  in  psychology  at  Harvard  University,  his  M.D.  degree  at  Yale 
University,  and  his  Ph.D.  degree  in  biophysics  at  the  Rockefeller  University  for  studies  with  Keffer  Hartline. 
He  was  a  member  of  the  faculties  at  the  University  of  Washington  Medical  School  and  at  Yale  Medical 
School  before  joining  the  Salk  Institute.  Dr.  Stevens  is  a  member  of  the  National  Academy  of  Sciences. 


COFFEE  and  tea  are  widely  used  in  many  soci- 
eties as  mild  stimulants.  How  do  they  work? 
Recent  research  in  our  laboratory  provides  an 
answer. 

One  of  the  more  remarkable  features  of  the 
brain  is  its  ability  to  modify  its  own  neuronal 
characteristics,  a  phenomenon  known  as  neuro- 
modulation.  Neurons  can  cause  other  neurons  to 
alter  their  properties,  and  thus  can  change  the 
computations  carried  out  by  the  modified  neuro- 
nal circuits. 

Neuromodulation  occurs  in  many  ways.  One  of 
the  most  important  is  the  regulation  of  synaptic 
strength — the  force  and  effectiveness  with  which 
one  neuron  transmits  signals  to  another.  Some 
changes  in  synaptic  strength  are  very  long  lasting 
and  are  thought  to  underlie  the  storage  of  memo- 
ries. Other  modifications  of  synaptic  strength  oc- 
cur more  rapidly  and  constitute  a  moment-to- 
moment  tuning  up  of  circuit  function.  A  major 
goal  of  our  laboratory  has  been  to  elucidate  the 
mechanisms  through  which  synaptic  strength  is 
neuromodulated.  In  the  course  of  this  work,  we 
have  gained  insights  into  the  actions  of  caffeine 
and  theophylline,  the  stimulants  in  coffee  and 
tea,  respectively. 

The  chemical  adenosine  is  released  at  synapses 
together  with  neurotransmitters  and  is  present  in 
the  fluids  bathing  neurons.  Neurons  display  on 
their  surface  several  different  types  of  receptors 
for  adenosine,  which  couple  adenosine  binding 
to  second  messenger  cascades  (such  as  the  one 
involving  cAMP) .  Adenosine  is  a  very  potent  regu- 
lator of  synaptic  strength:  concentrations  above 
the  usual  levels  result  in  large  decreases  in 
strength.  Here  is  the  interesting  part:  methylxan- 
thines,  a  family  of  chemicals  that  includes  caf- 
feine and  theophylline,  bind  to  adenosine  recep- 
tors and  block  their  uptake  of  adenosine. 

Some  "fake"  agonists  that  bind  to  a  receptor 
will  activate  it  as  if  they  were  natural  agonists,  but 
the  methylxanthines  do  not  do  this.  They  occupy 
the  receptor's  adenosine  binding  site  and  thereby 
prevent  adenosine  from  doing  so.  Rather  than 
mimic  the  action  of  adenosine,  the  methylxan- 
thines act  as  if  taking  it  away.  Thus  they  increase 


synaptic  strength,  indicating  that  the  normal 
brain  levels  of  adenosine  are  sufficient  to  pro- 
duce and  maintain  a  partial  decrease  in  synaptic 
strength. 

We  examined  the  effect  of  adenosine  itself,  and 
of  adenosine  receptor  blockers,  on  synaptic 
transmission  in  brain  slices  from  the  hippocam- 
pal  region  (specifically  from  dentate)  of  rats.  Us- 
ing the  whole-cell  recording  method,  a  tech- 
nique that  permits  high-resolution  detection  of 
currents  that  flow  as  a  result  of  synaptic  activa- 
tion, we  were  able  to  determine  the  specific  con- 
sequences of  adenosine  and  methylxanthine 
action. 

To  interpret  the  effects  of  these  drugs,  one 
needs  to  know  certain  features  of  normal  synaptic 
transmission.  A  synapse  contains  a  number  of  mi- 
croscopic membrane-bounded  spheres,  known  as 
synaptic  vesicles,  whose  contents  are  released 
from  the  cell  by  a  membrane  fusion  event  (exo- 
cytosis).  Each  of  these  vesicles  contains  a  unit 
amount  of  neurotransmitter,  such  as  glutamate, 
that  binds  to  special  receptors  in  the  target  neu- 
ron surface  membrane  and  produces  a  signal.  The 
strength  of  a  synapse  therefore  depends  jointly  on 
the  number  of  vesicles  whose  contents  are  re- 
leased and  on  the  size  of  the  response  produced 
by  one  vesicleful  of  the  neurotransmitter.  Be- 
cause the  size  of  the  synaptic  response  must  be 
integral — a  multiple  of  that  produced  by  a  single 
vesicle — this  single-vesicle  response  is  called  a 
quantum  or  quantal  response.  The  quantal  size 
depends,  in  turn,  on  the  number  of  transmitter 
molecules  contained  in  a  vesicle  and  on  the  num- 
ber and  responsiveness  of  the  receptors  displayed 
by  the  target  neuron. 

Only  a  certain  number  of  vesicles  are  available, 
of  course,  to  release  their  contained  neurotrans- 
mitter. This  number  is  usually  denoted  by  N. 
What  is  interesting  and  significant  about  the  neu- 
rotransmitter release  process  is  that  it  is  probabi- 
listic. That  is,  only  a  fraction  of  the  N  vesicles 
available  to  release  their  contents  do  so,  and  that 
number  is  determined  the  same  way  one  would 
calculate  the  number  of  heads  in  A''  coin  flips, 
with  a  probability  p  for  heads  on  any  flip.  Thus 


401 


Why  Do  We  Drink  Coffee  and  Tea? 


the  strength  of  a  synapse  is  determined  by  TV,  p, 
and  the  quantai  size.  There  are  techniques  for 
measuring  the  quantai  size  directly,  and  a  kind  of 
statistical  computation,  known  as  quantai  analy- 
sis, can  estimate  TV  and  p. 

We  have  carried  out  this  procedure  in  the  dentate 
neurons  with  either  added  adenosine  or  adenosine 
receptor  antagonists.  The  quantai  responded  size 
was  unaffected  by  any  of  the  drugs.  Quantai  analysis, 
however,  reveals  a  pronounced  effect  on  the  release 
probability:  adenosine  decreases  and  methylxan- 
thines  increase  the  chances  that  a  vesicle  will  un- 
dergo exocytosis  and  release  its  neurotransmitter 
when  a  nerve  impulse  arrives  at  the  synapse. 

Thus  methylxanthines  antagonize  baseline 
adenosine  effects  and  strengthen  synapses.  Why 
does  this  act  as  a  stimulant?  About  two-thirds  of 
the  brain's  synapses  are  excitatory  and  the  other 
third  are  inhibitory.  Many  drugs  shift  the  balance 
between  excitation  and  inhibition  and  thereby 


have  a  net  stimulatory  or  depressive  effect  on  the 
brain's  activity.  Barbiturates,  alcohol,  and  benzo- 
diazipines  (e.g.,  Valium)  all  selectively  increase 
the  strength  of  inhibitory  synapses  and  shift  the 
balance  from  excitation  toward  inhibition.  We 
find  that  the  effects  of  adenosine  and  adenosine 
antagonists  on  synaptic  transmission  are  limited 
to  excitatory  synapses.  Apparently  the  inhibitory 
synapses  lack  adenosine  receptors  or  the  second 
messenger  cascades  that  are  necessary  for  the  ex- 
pression of  their  action. 

In  summary,  coffee  and  tea  act  (as  many  have 
discovered  independently)  as  antialcohol  and  an- 
tibarbiturate  agents,  in  that  they  shift  the  brain's 
excitation/inhibition  balance  in  the  excitatory  di- 
rection. They  do  this  by  antagonizing  the  effects 
of  background  levels  of  adenosine  on  synaptic 
strength.  Their  precise  mechanism  of  action  is  to 
increase  the  probability  of  release  of  neurotrans- 
mitter molecules  from  synaptic  vesicles. 


402 


Morphogen  Gradients  and  the  Control  of  Body 
Pattern  in  Drosophila 


Gary  Struhl,  Ph.D. — Associate  Investigator 

Dr.  Struhl  is  also  Associate  Professor  of  Genetics  and  Development  at  Columbia  University  College  of 
Physicians  and  Surgeons.  He  received  B.S.  and  M.S.  degrees  in  biology  from  the  Massachusetts  Institute  of 
Technology  and  his  Ph.D.  degree  in  genetics  from  the  University  of  Cambridge,  England.  His  graduate 
studies  were  carried  out  with  Peter  Lawrence  at  Cambridge.  Before  joining  the  faculty  at  Columbia,  he 
conducted  molecular  and  genetic  studies  on  Drosophila  in  the  laboratory  of  Tom  Maniatis  at  Harvard 
University.  In  addition  to  several  fellowships,  he  received  the  McKnight  Neuroscience  Development  Award. 


SINCE  the  birth  of  embryology  as  an  experi- 
mental discipline,  it  has  been  apparent  that 
the  development  of  cell  and  body  patterns  de- 
pends on  robust  and  complex  systems  of  spatial 
information.  Yet,  until  recently,  we  had  little 
idea  of  the  physical  nature  of  such  systems  or  the 
mechanisms  by  which  they  are  generated  or 
interpreted.  Considerable  attention  has  been 
given  to  the  possibility  that  gradients  of  "form- 
generating"  substances,  or  morphogens,  might 
provide  this  information.  For  example,  a  local- 
ized diffusible  morphogen  could  generate  a  gra- 
dient that  would  provide  a  series  of  concentra- 
tion thresholds,  each  determining  distinct 
cellular  behaviors  (e.g.,  the  development  of  par- 
ticular pattern  elements)  as  a  function  of  distance 
from  the  source. 

Current  research  in  this  laboratory  is  directed 
toward  identifying  potential  morphogen  gra- 
dients and  determining  both  how  they  arise  and 
how  they  control  pattern.  Over  the  past  20-30 
years,  a  variety  of  embryologic  and  genetic  exper- 
iments have  suggested  that  such  gradients  play  a 
key  role  in  controlling  cell  and  body  patterns  in 
insects.  Indeed,  Christiane  Niisslein-Volhard  and 
her  colleagues  have  identified  several  such  mor- 
phogens involved  in  specifying  the  basic  body 
plan  of  the  fruit  fly  Drosophila. 

One  of  these  determinants,  the  protein  product 
of  the  gene  bicoid  (bed) ,  is  clearly  expressed  as  a 
gradient  that  peaks  at  the  anterior  pole  of  the 
early  embryo  and  controls  anterior  body  pattern 
(head  and  thorax).  Another,  nanos  (nos),  is  re- 
quired for  generating  posterior  body  pattern  (ab- 
domen), though  its  properties  are  less  well  un- 
derstood. Our  immediate  goal  during  the  past 
year  or  so  has  been  to  determine  how  these  two 
systems  work.  As  we  describe  below,  the  con- 
trolled expression  of  a  single  regulatory  protein, 
hunchback  (hb),  has  proved  to  be  the  key  to  un- 
derstanding both  molecular  mechanisms. 


rior  pole  to  a  boundary  halfway  down  the  body. 
This  event  depends  critically  on  the  bed  morpho- 
gen. Moreover,  it  is  essential  for  the  generation  of 
a  large  portion  of  anterior  pattern,  including  all 
three  thoracic  segments. 

In  an  initial  series  of  experiments,  we  found 
that  bed  behaves  as  a  transcriptional  regulator, 
capable  of  directly  binding  the  hb  gene  and  acti- 
vating transcription  in  a  remarkably  sensitive, 
concentration-dependent  fashion.  Hence,  when 
the  concentration  of  bed  protein  exceeds  a  criti- 
cal threshold,  it  binds  a  series  of  adjacent  sites  in 
the  DNA  immediately  upstream  of  the  hb  pro- 
moter and  activates  transcription;  however,  when 
the  concentration  of  bed  protein  falls  beneath 
this  threshold,  binding  does  not  occur  and  the  hb 
gene  remains  silent.  The  concentration  gradient 
of  bed  protein  therefore  determines  where  the  hb 
gene  is  transcribed  by  positioning  this  threshold 
along  the  anteroposterior  axis. 

In  the  process  of  examining  how  the  bed  gra- 
dient controls  hb  gene  expression,  we  created  a 
series  of  hybrid  genes  that  contain  only  some  of 
the  binding  sites  for  bed  protein  normally  present 
upstream  of  the  hb  promoter.  These  hybrid 
genes,  like  hb  itself,  are  activated  in  anterior  por- 
tions of  the  body  under  the  control  of  the  bed 
protein  gradient.  However,  their  domains  of  ex- 
pression are  abbreviated  relative  to  that  of  hb, 
extending  only  a  quarter  or  a  third  of  the  way 
down  the  body.  These  and  other  experiments 
suggest  that  the  bed  gradient  can  trigger  several 
spatially  distinct  responses,  the  posterior  bound- 
ary of  each  depending  on  the  affinity  of  bed  pro- 
tein for  a  given  target  gene. 

Recently  several  new  genes  have  been  identi- 
fied that  appear  to  be  expressed  in  a  series  of 
overlapping  anterior  domains.  These  genes  are 
required  for  generating  particular  portions  of 
head  and  thoracic  pattern  and  may  constitute 
other  direct  targets  of  the  bed  gradient. 


Anterior  Pattern:  Transcriptional 
Activation  of  hb  by  bed 

Early  in  embryogenesis,  the  hb  gene  is  acti- 
vated in  a  broad  domain  extending  from  the  ante- 


Posterior  Pattern:  Translation  Repression 
of  hb  by  nos 

Transcriptional  activation  of  the  hb  gene  by 
bed  is  only  one  of  two  sources  of  hb  mRNA  in  the 


403 


Morphogen  Gradients  and  the  Control  of  Body  Pattern  in  Drosophila 


early  embryo.  The  second  source  is  maternal  hb 
mRNA  synthesized  during  oogenesis  and  depos- 
ited uniformly  throughout  the  egg.  These  tran- 
scripts are  translated  only  after  fertilization,  and 
their  translation  is  repressed  in  more-posterior 
portions  of  the  egg,  owing  to  nos  activity  emanat- 
ing from  the  posterior  pole. 

We  recently  found  that  the  ability  of  nos  to 
repress  hb  translation  depends  on  the  presence  of 
two  copies  of  a  short  cis-acting  regulatory  se- 
quence in  the  3'  noncoding  portion  of  the  hb 
mRNA.  As  in  the  case  of  the  DNA  sites  mediating 
the  binding  and  regulation  of  hb  by  bed  protein, 
the  number  and  quality  of  the  target  sites  in  hb 
mRNA  can  determine  the  pattern  of  expression  of 
hb  protein. 

However,  in  contrast  to  bed,  which  has  many 
other  signaling  roles,  nos  appears  to  have  no  role 
other  than  to  block  posterior  expression  of  hb 
protein.  Indeed,  we  have  been  able  to  show  that 
nos  activity  is  completely  dispensable  if  the 
translation  of  maternally  derived  hb  mRNAs  is 
blocked  by  other  means  (such  as  mutation) .  Thus 
the  control  of  posterior  body  pattern  may  depend 
solely  on  the  repression  of  maternal  hb  tran- 
scripts by  nos. 

Posterior  Body  Pattern  Is  Controlled 

by  hb  Protein  Acting 

as  a  Classical  Gradient  Morphogen 

The  bed  and  nos  determinants  have  opposite 
effects  on  hb  protein  expression.  The  bed  gene 
triggers  the  synthesis  of  hb  transcripts,  and  hence 
protein,  anteriorly;  nos  blocks  the  translation  of 


both  maternal  and  zygotic  hb  transcripts  posteri- 
orly. Together  the  actions  of  bed  and  nos  gener- 
ate a  graded  pattern  of  hb  protein  in  which  the 
concentration  declines  from  uniformly  high  lev- 
els in  the  anterior  half  of  the  body  to  undetect- 
able levels  in  the  posterior  half. 

As  described  above,  our  analysis  of  the  interac- 
tion between  nos  and  hb  indicates  that  nos  itself 
can  be  rendered  dispensable,  provided  that  ma- 
ternal hb  transcripts  can  be  inactivated  by  other 
means.  In  these  unusual  embryos,  hb  protein  is 
still  expressed  differentially  along  the  anteropos- 
terior axis,  owing  to  zygotic  activation  of  the  hb 
gene  by  bed.  This  finding,  taken  together  with  the 
critical  role  nos  normally  plays  in  repressing  the 
translation  of  hb  mRNAs,  suggests  that  the  distri- 
bution of  hb  protein  may  be  the  critical  determi- 
nant of  posterior  body  pattern. 

We  tested  this  possibility  by  generating  em- 
bryos in  which  the  differential  expression  of  hb 
protein  along  the  anteroposterior  axis  has  been 
systematically  altered  while  all  other  known  sig- 
naling systems  are  eliminated  or  held  constant.  By 
examining  the  expression  of  the  subordinate  reg- 
ulatory genes  Kriippel,  knirps,  and  giant,  each  of 
which  is  normally  responsible  for  controlling  a 
particular  subdomain  of  thoracic  and  abdominal 
pattern,  we  have  been  able  to  show  that  the  gra- 
dient of  hb  protein  provides  a  series  of  distinct 
concentration  thresholds  that  govern  where  these 
genes  are  expressed. 

Thus  hb  appears  to  control  posterior  body  pat- 
tern by  acting  as  a  classical  gradient  morphogen. 
In  this  regard,  it  functions  as  the  posterior  coun- 
terpart to  the  anterior  morphogen  bed. 


404 


Secretory  Pathways  in  Neurons 


Thomas  C.  Siidhof,  M.D. — Investigator 

Dr.  Siidhof  is  also  Professor  of  Molecular  Genetics  at  the  University  of  Texas  Southwestern  Medical  Center 
at  Dallas.  He  received  his  M.D.  degree  and  his  doctorate  from  the  Georgia  Augusta  University  of  Gottingen, 
FRG.  He  obtained  postdoctoral  training  first  with  Victor  Whittaker  at  the  Max  Planck  Institute  for 
Biophysical  Chemistry,  Gottingen,  and  then  with  Michael  Brown  and  Joseph  Goldstein  in  Dallas. 


NEURONS  communicate  with  one  another  by 
means  of  chemical  signals.  The  known  com- 
munication pathways  between  neurons  are  of  sev- 
eral kinds: 

•  Fast,  point-to-point  transmission  of  signals 
between  neurons  occurs  at  the  synapse. 

•  Long-lasting  modulatory  signals  that  often 
reach  many  cells  are  transmitted  outside  of  syn- 
apses by  neuropeptides  and  other  mediators. 

•  Short-range  diffuse  signals  are  probably 
spread  by  lipophilic  messengers,  such  as  nitric 
oxide  or  arachidonic  acid. 

Of  these  pathways,  signaling  between  neurons 
at  the  synapse  is  quantitatively  the  major  form  of 
cell-to-cell  communication  in  the  central  ner- 
vous system.  Synapses  are  abundant  in  the  ner- 
vous system,  and  their  activity  provides  the  basis 
of  brain  function.  However,  the  slow  communica- 
tion pathways  are  clearly  an  essential  counterpart 
to  the  fast  point-to-point  signals.  The  coexistence 
of  different  signaling  pathways  in  the  same  neu- 
ron increases  the  complexity  of  the  neuronal 
networks.  Brain  function  will  clearly  not  be 
understood  until  we  gain  insight  into  the  molecu- 
lar mechanisms  that  govern  these  signaling 
pathways. 

Work  in  our  laboratory  addresses  the  question 
of  how  nerve  cells  send  out  chemical  signals.  We 
are  concentrating  on  the  synapse  as  the  most 
abundant  signaling  pathway.  Here  the  chemical 
signals,  the  neurotransmitters,  are  prepackaged 
in  unique  cellular  organelles  called  synaptic  vesi- 
cles and  released  from  the  presynaptic  neuron  by 
secretion.  This  secretion  is  achieved  by  exocyto- 
sis,  the  fusion  of  synaptic  vesicles  with  the  synap- 
tic cell  membrane.  After  fusion  the  empty  synap- 
tic vesicles  are  quickly  re-endocytosed  and 
refilled  with  neurotransmitter.  They  become 
competent  for  secretion  again  in  a  short  time,  al- 
lowing the  neurons  to  fire  rapidly. 

We  have  taken  two  avenues  to  the  exploration 
of  the  molecular  basis  of  signal  transmission  at 
the  synapse.  The  first  approach  has  been  to  study 
synaptic  vesicles  and  their  components  as  the 
central  organelle  in  neurotransmitter  release. 
The  second  approach  consists  of  a  characteriza- 


tion of  the  presynaptic  plasma  membrane  as  the 
point  of  signal  release. 

Characterizing  the  molecular  components  of 
synaptic  vesicles  constitutes  a  long-term  project 
that  we  are  largely  carrying  out  in  collaboration 
with  Reinhard  Jahn  (HHMI,  Yale  University). 
This  project  has  led  to  the  molecular  characteriza- 
tion of  more  than  10  synaptic  vesicle  proteins, 
which  together  account  for  approximately  one- 
third  to  one-half  of  the  total  vesicle  protein  by 
mass.  The  goal  of  this  project  is  twofold. 

First,  we  would  like  to  achieve  a  complete  de- 
scription of  the  synaptic  vesicle  in  molecular 
terms.  This  is  not  only  a  precondition  to  any  even- 
tual understanding  of  how  the  synaptic  vesicle 
pathway  works,  but  would  also  provide  the  first 
molecular  anatomy  of  an  organelle. 

Second,  we  would  like  to  explore  the  functions 
of  each  vesicle  protein  in  neurotransmitter  re- 
lease, using  biochemical  and  genetic  techniques. 
This  part  of  the  project  has  progressed  to  the 
point  that  interesting  biochemical  properties  of 
several  vesicle  proteins  have  been  elucidated. 
Moreover,  the  feasibility  of  mouse  genetics  to 
probe  the  functions  of  these  proteins  has  been 
demonstrated.  For  example,  we  have  recently 
shown  that  a  synaptic  vesicle  protein  named  syn- 
aptotagmin  binds  Ca^"^  and  phospholipids  in  a 
ternary  complex  at  physiologic  Ca^"^  concentra- 
tions. This  result  suggests  a  function  for  synapto- 
tagmin  in  synaptic  vesicle  fusion,  a  possibility 
now  being  explored  in  transgenic  mice. 

Another  approach  we  are  pursuing  to  elucidate 
synapse  function  is  the  study  of  the  presynaptic 
plasma  membrane.  The  membrane  serves  two  ba- 
sic functions.  It  binds  synaptic  vesicles  and  fuses 
with  them  in  a  Ca^"^-dependent  manner,  thereby 
releasing  neurotransmitter;  and  it  contacts  the 
postsynaptic  site  and  aligns  pre-  and  postsynaptic 
membranes  with  each  other.  Both  functions 
are  probably  performed  by  specific  protein 
components. 

To  identify  a  component  of  the  presynaptic 
plasma  membrane  that  may  be  involved  in  its 
functions,  we  have  studied  the  receptor  for  a  neu- 
rotoxin called  a-latrotoxin.  This  protein,  derived 
from  venom  of  the  black  widow  spider,  binds  spe- 


405 


Secretory  Pathways  in  Neurons 


cifically  to  the  presynaptic  membrane  and  causes 
massive  neurotransmitter  release.  Purification 
and  amino  acid  sequencing  of  the  receptor  for 
a-latrotoxin  led  to  the  discovery  of  a  new  family 
of  neuron-specific  cell  surface  proteins  that  we 
named  neurexins.  Their  structure  and  localiza- 
tion suggest  that  they  may  be  synaptic  recogni- 
tion molecules. 

Surprisingly,  we  also  found  that  the  a-latrotoxin 
receptor  interacts  with  a  synaptic  vesicle  protein, 


synaptotagmin,  whose  Ca  -binding  properties 
have  already  implicated  it  in  exocytosis.  These 
results  raise  the  possibility  that  the  neurexins 
may  have  a  role  in  aligning  the  synapse  both 
extra-  and  intracellularly.  Further  studies  of  the 
neurexins  should  allow  us  to  gain  new  insights 
into  how  synapses  are  formed  and  neurotransmit- 
ters released.  Results  should  be  relevant  for  un- 
derstanding brain  function  under  normal  and 
pathologic  conditions. 


406 


Transcription  Factors  in  Cell  Growth 
and  Kidney  Differentiation 


Vikas  P.  Sukhatme,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Sukhatme  is  also  Associate  Professor  of  Medicine  and  of  Molecular  Genetics  and  Cell  Biology  at  the 
University  of  Chicago.  He  received  his  Ph.D.  degree  in  theoretical  physics  from  the  Massachusetts  Institute 
of  Technology.  He  then  received  his  M.D.  degree  from  Harvard  Medical  School.  After  residency  and  clinical 
fellowship  training  at  the  Massachusetts  General  Hospital,  Boston,  he  completed  his  postdoctoral  work  at 
Stanford  University. 


MY  laboratory  has  been  engaged  in  cloning 
and  characterizing  mammalian  genes  that 
code  for  transcription  factors.  One  focus  is  on 
signal  transduction,  specifically  on  mitogenic  sig- 
naling, and  another  aims  at  identifying  transcrip- 
tion factors  that  control  kidney  development. 
These  two  interests  have  recently  intersected,  as 
delineated  below. 

The  Egr  Family  of  Immediate-Early 
Transcription  Factors 

Extracellular  "signals"  in  the  form  of  neuro- 
transmitters, growth  factors,  hormones,  and  ma- 
trix are  known  to  control  cellular  phenotype. 
These  agents  lead  to  the  generation  of  second 
messenger  signals  in  the  plasma  membrane  and 
cytosol.  In  turn,  these  biochemical  events  modu- 
late the  expression  of  so-called  immediate-early 
genes  (lEGs) ,  whose  induction  does  not  require 
de  novo  protein  synthesis.  Several  years  ago,  we 
and  others  identified  several  lEGs  in  the  context 
of  a  mitogenic  response,  and  more  specifically  in 
the  transition  of  a  cell  out  of  a  quiescent  state 
(Go)  into  Gi .  Of  particular  interest  to  us  has  been 
a  subset  of  lEGs  that  encode  transcription  factors 
(proteins  that  bind  DNA  and  regulate  gene  tran- 
scription), since  they  might  couple  short-term 
responses  in  the  form  of  second  messenger  events 
to  long-term  changes  in  gene  expression  instru- 
mental in  altering  phenotype. 

The  best  characterized  of  these  immediate- 
early  transcription  factor  genes  include  members 
of  the  Fos  family.  c-Fos,  identified  as  the  cellular 
homologue  of  the  \-Fos  oncogene  present  in  two 
viruses  that  cause  osteosarcomas,  was  discovered 
in  1984  to  be  an  lEG  in  serum-stimulated  fibro- 
blasts. However,  it  was  not  until  four  years  later 
that  Fos  was  shown  to  be  part  of  the  AP- 1  tran- 
scription factor  complex,  composed  of  Fos-Jun 
heterodimers  and  other  less  well  characterized 
proteins.  c-Jun  was  likewise  first  identified  as  the 
cellular  homologue  of  the  \-Jun  transforming 
gene.  It  was  suspected  to  be  a  transcription  factor 
through  homology  to  part  of  the  yeast  GCN4 
protein. 

In  1 987  we  discovered  (concurrently  with  sev- 


eral other  laboratories)  the  Egr  family  of  lEGs. 
The  best-characterized  gene  in  this  family  is 
Egr-1  (early  growth  response  gene-1).  Egr-1 
(also  known  as  Zif-268,  Tis-8,  NGFI-A,  and 
Krox-24)  was  isolated  as  a  serum-inducible  lEG 
in  quiescent  fibroblasts  (Gq-Gj  transition),  utiliz- 
ing a  differential  screening  protocol.  The  gene  is 
induced  by  mitogen  stimulation  in  every  mam- 
malian cell  type  tested,  including  B  cells;  T  cells; 
kidney  mesangial,  glomerular,  and  tubular  epi- 
thelial cells;  hepatocytes;  and  vascular  smooth 
muscle  and  endothelial  cells.  It  is  also  induced  by 
nerve  growth  factor  in  PCI  2  pheochromocytoma 
cells,  a  physiological  context  in  which  mitotic 
cells  convert  to  a  nonmitotic  state. 

The  cDNA  structure  predicts  a  protein  whose 
carboxyl  terminus  contains  three  zinc  fingers  of 
the  Cys2-HiS2  type,  first  identified  in  the  Xenopus 
transcription  factor  TFIIIA.  This  prediction  has 
recently  been  verified  by  Carl  Pabo  (HHMI,  Mas- 
sachusetts Institute  of  Technology)  and  his  col- 
leagues through  analysis  of  the  crystal  structure 
of  the  zinc  finger  domain  cocrystallized  with  its 
target  DNA  sequence  GCGGGGGCG. 

We  have  been  interested  in  identifying  the 
events,  from  cell  surface  to  nucleus,  that  modu- 
late Egr  l  expression.  Although  identification  of 
such  "upstream"  or  "proximal"  events  can  ei- 
ther be  attempted  in  the  context  of  mitogenesis 
or  in  other  situations  in  which  Egr- 1  mRNA  levels 
change,  most  of  our  work  has  been  restricted  to 
cell  proliferation  studies  in  fibroblasts.  It  has 
been  found  that  multiple  kinases  regulate  Egr- 1 
expression.  For  example,  activation  of  the  PDGF 
(platelet-derived  growth  factor)  receptor  or  the 
EGF  (epidermal  growth  factor)  receptor  by  their 
cognate  ligands  leads  to  Egr-1  induction. 

v-Src  has  been  shown  to  regulate  Egr-1  inde- 
pendent of  protein  kinase  C.  More  recently,  we 
have  found  that  v-Raf,  a  serine-threonine  kinase, 
whose  activation  results  from  the  convergence  of 
diverse  cell  surface  signals,  leads  to  Egr-1  induc- 
tion. Furthermore,  a  dominant  negative  mutant  of 
v-Raf  will  ablate  the  v-Src  induction  of  Egr-1 ,  sug- 
gesting that  v-Src  stimulates  Egr- 1  via  v-Raf.  Even 
if  Egr-1  served  as  nothing  more  than  a  target 


407 


Transcription  Factors  in  Cell  Growth  and  Kidney  Differentiation 


marker,  studies  like  these  are  helping  to  define 
the  circuitry  among  second  messenger  cascades. 

Additional  studies  currently  under  way  are 
aimed  at  defining  structure-function  relation- 
ships of  the  Egr-1  protein.  The  protein  contains 
several  activation  domains  and  a  small  modular 
repressor  region  whose  sequence  has  been  highly 
conserved  in  evolution.  Nuclear  localization  is 
dependent  upon  a  bipartite  sequence,  and  DNA 
binding  is  determined  by  the  three  zinc  fingers. 

A  major  challenge  ahead  is  to  define  a  func- 
tional role  for  the  Egr  l  protein.  Attempts  at 
abrogating  Egr- 1  activity  may  prove  useful  in  de- 
fining a  phenotype.  In  this  regard,  the  use  of  anti- 
sense  methodology — either  as  oligonucleotides 
or  in  the  form  of  stable  inducible  vectors — is 
under  investigation.  The  latter  technique,  when 
applied  to  c-Fos,  has  resulted  in  a  remarkable  and 
reversible  inhibition  of  cell  growth.  Another  ap- 
proach is  also  available  to  inhibit  Egr- 1  activity: 
nature  has  designed  its  own  inhibitor  of  Egr-1 
function  in  the  form  of  a  tumor-suppressor  gene, 
one  whose  absence  is  likely  to  lead  to  Wilms'  tu- 
mor (see  below). 

Transcription  Factors  in  Kidney 
Development 

A  second  recent  focus  of  our  laboratory  is  on 
trying  to  define  molecular  events  that  character- 
ize the  development  of  the  kidney.  It  is  well 
known  that  metanephric  mesenchymal  (blaste- 
mal)  cells  convert  into  epithelial  cells  over  a  4-  to 
5 -day  period  (in  the  rat  or  mouse)  in  response  to 
invasion  of  the  ureteric  bud.  Little  is  known  at  the 
molecular  and  cellular  level  of  the  events  that 
transpire  during  this  conversion  process.  Our  aim 
is  to  define  a  hierarchy  of  transcriptional  regula- 
tors whose  expression  is  modulated  during 
nephrogenesis.  Toward  this  end,  several  studies 
are  under  way. 

One  strategy  is  to  identify  zinc  finger  transcrip- 
tional regulators  expressed  in  the  kidney  during 
differentiation.  Using  a  so-called  H/C-link  probe 
directed  against  a  region  often  conserved  in  zinc 
finger  proteins,  we  have  isolated  from  a  kidney 


library  a  dozen  cDNA  clones  encoding  zinc  finger 
proteins.  These  are  now  being  characterized  by 
their  developmental  profile  (Northern  blots  and 
in  situ  hybridization)  and  by  limited  sequence 
analysis.  One  of  these  clones  hybridizes  at  high 
stringency  to  the  candidate  Wilms'  tumor  anti- 
oncogene  (WTl)  identified  recently  by  reverse 
genetic  means  in  the  laboratories  of  David  Hous- 
man  (Massachusetts  Institute  of  Technology) 
and  Gail  Bruns  (Harvard  University).  Another 
clone  has  identified  a  zinc  finger  protein  that 
is  expressed  very  early  in  human  kidney 
development. 

In  a  second  approach,  work  is  in  progress  to  set 
up  stem  cell  cultures  from  embryonic  kidney  for 
the  development  of  cell  lines  arrested  at  different 
stages  of  differentiation.  If  these  cells  can  be 
maintained  and  then  made  to  differentiate  in  cul- 
ture under  appropriate  conditions,  they  will  be 
invaluable  for  studying  the  early  molecular  and 
cellular  events  in  nephrogenesis. 

Third,  we  are  pursuing  an  interesting  connec- 
tion between  Egr-1  and  WTl .  WTl  is  a  zinc  finger 
protein;  three  of  its  four  zinc  fingers  show  a  60- 
70  percent  similarity  to  the  three  zinc  fingers  of 
Egr  l.  Indeed,  WTl  and  Egr-l  bind  to  a  common 
sequence,  as  shown  by  the  work  of  Frank 
Rauscher  (Wistar  Institute).  We  have  recently 
shown  (in  collaboration  with  Dr.  Rauscher)  that 
WTl  acts  as  a  transcriptional  repressor,  whereas 
Egr- 1  is  a  transcriptional  activator.  These  findings 
may  have  exciting  biological  implications,  be- 
cause several  binding  sites  exist  for  the  Egr-1/ 
Wilms'  tumor  proteins  in  the  promoter  sequence 
of  the  insulin-like  growth  factor  II  (IGF-II). 

IGF-II  levels  are  known  to  be  high  in  Wilms' 
tumor,  and  these  levels  fall  during  development. 
Thus  the  possibility  exists  that  IGF-II  is  a  target 
for  the  repressive  action  of  the  Wilms'  tumor  pro- 
tein and  for  positive  regulation  by  Egr- 1 .  If  this 
model  holds,  it  will  be  a  way  to  explain  the  find- 
ings of  aberrant  regulation  of  IGF-II  in  Wilms' 
tumor  and  will  also  provide  major  insight  into  the 
mesenchymal  cell-to-epithelial  cell  conversion 
in  the  kidney. 


408 


Structure  and  Function  of  Voltage-Dependent 
Calcium  Channels 


Tsutomu  Tanabe,  Ph.D. — Assistant  Investigator 

Dr.  Tanabe  is  also  Assistant  Professor  of  Cellular  and  Molecular  Physiology  at  Yale  University  School  of 
Medicine.  He  received  his  B.A.,  M.A.,  and  Ph.D.  degrees  from  Kyoto  University  in  Kyoto,  Japan.  He  began 
studies  on  the  structure  and  function  of  the  receptors  and  ion  channels  in  excitable  membrane  when  he 
was  a  graduate  student  in  Shosaku  Numa's  laboratory.  After  he  received  his  doctorate,  he  continued  to 
conduct  research  in  Dr.  Numa's  laboratory  as  a  faculty  member  before  coming  to  Yale. 


VOLTAGE-dependent  calcium  channels  play 
important  roles  in  the  regulation  of  a  variety 
of  cellular  functions,  including  membrane  excit- 
ability, muscle  contraction,  synaptic  transmis- 
sion, and  secretion.  At  least  four  types  of  calcium 
channels  have  been  distinguished  by  their  elec- 
trophysiological and  pharmacological  properties. 

Recently,  molecular  biological  studies,  com- 
bined with  electrophysiology,  have  provided  evi- 
dence that  this  diversity  of  calcium  channels 
derives  largely  from  differences  in  their  pore- 
forming  ttj-subunit  and  that  the  other  subunits 
associated  with  can  modify  channel  function. 
Furthermore,  the  diversity  of  the  several  subunits 
was  shown  to  originate  not  only  from  differences 
in  the  genes  encoding  them  but  also  from  alterna- 
tive splicing  of  their  RNAs. 

Several  types  of  calcium  channels  are  known  to 
be  coexpressed  in  single  cells,  and  the  cells  ap- 
parently use  them  for  different  purposes.  We  are 
interested  in  the  structure-function  relationships 
of  calcium  channels  and  the  molecular  basis  of 
one  type's  specialization. 

Muscular  dysgenesis  (mdg)  is  a  fatal  autosomal 
recessive  mutation  of  mice.  It  eliminates  excita- 
tion-contraction (E-C)  coupling  and  dihydropyri- 
dine  (DHP) -sensitive  calcium  current  of  the  slow 
L-type  from  skeletal  muscle.  Analysis  of  genomic 
DNA  and  skeletal  muscle  RNA  indicates  that  the 
mdg  mutation  is  associated  with  alterations  of  the 
structural  gene  for  the  skeletal  muscle  DHP  re- 
ceptor. Injection  of  an  expression  plasmid  carry- 


ing the  cDNA  of  the  receptor  restores  both  E-C 
coupling  and  skeletal  L-type  calcium  current, 
suggesting  a  dual  role  of  this  molecule. 

The  restored  coupling  resembles  that  of  nor- 
mal skeletal  muscle,  which  does  not  require 
entry  of  extracellular  calcium  ions.  By  contrast, 
injection  into  dysgenic  myotubes  of  an  expres- 
sion plasmid  carrying  cDNA  of  the  cardiac  DHP 
receptor  produces  rapid,  cardiac-like  L-type 
current  and  cardiac-type  E-C  coupling,  which 
does  require  calcium  ion  entry. 

To  investigate  the  molecular  basis  for  these  dif- 
ferences in  calcium  currents  and  E-C  coupling, 
we  expressed  various  cDNAs  of  the  chimeric  DHP 
receptor  in  dysgenic  myotubes,  with  the  follow- 
ing results.  Expression  of  cDNAs  encoding  chi- 
meras with  regions  of  the  skeletal  muscle  DHP 
receptor  replacing  one  or  more  of  the  corre- 
sponding large,  putative  cytoplasmic  regions  of 
the  cardiac  DHP  receptor  showed  that  the  region 
linking  repeats  II  and  III  is  a  major  determinant  of 
skeletal  muscle-type  E-C  coupling. 

Expression  of  cDNAs  encoding  chimeras  in 
which  repeats  of  the  skeletal  muscle  DHP  recep- 
tor are  replaced  by  corresponding  repeats  from 
the  cardiac  receptor  showed  that  repeat  I  deter- 
mines whether  the  chimeric  calcium  channel  ac- 
tivation will  be  slow  (skeletal  muscle-like)  or 
rapid  (cardiac-like). 

We  are  also  focusing  on  the  drug-binding  sites  of 
calcium  channel  molecules,  including  those  of 
channel  antagonist  (L-type  channel  blocker),  and 
the  mechanism  of  calcium-dependent  inactivation. 


409 


The  Molecular  Biology  of  Liver  Regeneration 


Rebecca  A.  Taub,  M.D. — Associate  Investigator 

Dr.  Taub  is  also  Assistant  Professor  of  Human  Genetics  at  the  University  of  Pennsylvania  School  of 
Medicine.  She  received  her  B.A.  degree  in  biochemistry  from  Yale  University,  attended  Yale  University 
School  of  Medicine,  and  completed  residency  training  in  internal  medicine  at  Yale.  She  was  a  postdoctoral 
fellow  with  Philip  Leder  at  Harvard  Medical  School,  where  she  later  joined  the  Department  of  Internal 
Medicine  as  Assistant  Professor  before  moving  to  Philadelphia. 


THE  liver  has  unusual  properties  of  regenera- 
tion. When  the  two  largest  lobes  of  the  liver 
are  removed,  leaving  the  smaller  lobes  com- 
pletely intact,  the  remaining  cells  can  grow  and 
divide  until  the  liver  regains  its  former  size, 
whereupon  growth  ceases.  Many  growth  factors 
have  been  implicated  in  regulating  this  process, 
but  the  mechanisms  remain  poorly  understood. 
After  many  years  of  study  the  same  interesting 
questions  about  liver  regeneration  remain:  What 
makes  the  liver  start  regenerating?  What  regulates 
liver  growth  during  regeneration,  allowing  the 
liver  to  maintain  its  normal  architecture?  What 
makes  the  liver  stop  regenerating  when  it  has  re- 
gained its  initial  size? 

Understanding  liver  regeneration  will  help  ex- 
plain how  the  liver  responds  to  toxic  damage  or 
infections  like  hepatitis.  Additionally,  because 
increasing  numbers  of  liver  transplants  are  being 
performed  and  successful  transplants  require 
liver  regeneration,  it  is  essential  to  understand 
the  biological  and  molecular  bases  for  liver  cell 
growth. 

My  colleagues  and  I  are  interested  in  determin- 
ing what  genes  are  expressed  during  liver  regen- 
eration and  how  their  protein  products  are  in- 
volved in  regulating  the  process.  It  is  important 
to  determine  if  these  genes  are  identical  to  those 
that  regulate  the  growth  of  all  cells.  We  are  study- 
ing liver  cell  growth  in  two  systems:  a  continu- 
ously growing  liver  cell  line  that  responds  to  in- 
sulin as  a  growth  factor  and  regenerating  liver 
tissue  from  rats. 

In  our  early  studies,  we  identified  more  than 
40  novel  genes  that  are  rapidly  expressed  in  re- 
sponse to  growth  factors  in  liver  cells.  Although 
many  of  the  genes  are  expressed  in  other  growing 
cells  and  seem  to  be  part  of  the  general  growth 
response,  some  of  the  genes  are  specific  to  grow- 
ing liver.  Many  of  these  genes  encode  proteins 
that  function  in  the  cell  nucleus,  possibly  regu- 
lating the  cascade  of  gene  expression  occurring 
when  cells  grow.  Some  of  these  genes  have  al- 
tered expression  in  cancer  cells,  contributing  to 
the  aberrant  proliferation. 

Our  studies  have  focused  on  understanding  the 


actions  of  the  proteins  encoded  by  some  of  these 
genes  and  their  potential  roles  in  regulation  of 
liver  regeneration.  Because  we  isolated  so  many 
novel  genes,  it  was  important  to  establish  criteria 
for  determining  which  of  the  encoded  proteins 
are  likely  to  have  important  regulatory  roles  in 
liver  regeneration.  We  decided  to  explore  further 
the  exact  roles  of  novel  proteins  falling  into  four 
functional  categories. 

The  first  category  includes  proteins  that  regu- 
late the  expression  of  genes.  Because  so  many 
genes  need  to  be  turned  on  for  liver  regeneration 
to  proceed,  proteins  that  regulate  gene  expres- 
sion are  likely  to  be  important. 

The  second  category  includes  proteins  that  are 
secreted  from  cells  and  may  function  to  regulate 
the  growth  of  surrounding  cells.  The  liver  must 
maintain  its  cellular  architecture  during  regener- 
ation, and  because  it  is  made  up  of  many  different 
cell  types,  intercellular  communications  must 
exist  during  regeneration.  Secreted  proteins 
could  be  involved  in  mediating  such  intercellular 
signals. 

The  third  category  includes  proteins  whose  ex- 
pression is  specific  to  regenerating  liver  and  not 
other  growing  cells.  We  found  several  in  this  cate- 
gory, a  few  of  which  are  highly  expressed  in  re- 
generating liver.  These  proteins  could  have  im- 
portant functions  in  liver-specific  growth. 

The  fourth  category  includes  several  genes 
whose  expression  is  nicely  induced  in  regenerat- 
ing liver  but  abnormally  high  in  the  liver  tumor 
cell  line  that  grows  in  response  to  insulin.  These 
genes  could  be  functioning  as  oncogenes  in  the 
liver  tumor  cell  line. 

One  of  the  novel  genes  that  we  isolated,  RL/ 
IF-1  (regenerating  liver  inhibitory  factor),  en- 
codes a  protein  that  inhibits  the  activity  of  certain 
gene-transactivating  proteins  in  the  NF-kB/RcI 
family.  We  examined  the  relative  activity  of 
NF-/cB/Rel  proteins  during  liver  regeneration  and 
found  to  our  surprise  that  although  most  of  the 
proteins  in  this  family  remain  in  an  inhibited  or 
inactive  form,  one  protein,  PHF-1,  becomes  dra- 
matically active  in  its  ability  to  interact  with  gene 
sequences  within  minutes  after  hepatectomy. 
The  activation  of  PHF-1  is  the  earliest  change  we 


411 


The  Molecular  Biology  of  Liver  Regeneration 


have  observed  in  the  liver  posthepatectomy,  and 
our  findings  suggest  that  PHF- 1  could  have  a  role 
as  an  initiating  signal  in  liver  regeneration. 

LRF- 1  (liver  regeneration  factor)  is  one  of  the 
proteins  that  we  have  studied  that  is  highly  ex- 
pressed in  regenerating  liver  and  functions  as  a 
DNA-binding  protein,  controlling  the  expression 
of  target  genes.  It  falls  into  the  category  of  so- 
called  leucine  zipper  proteins,  in  which  a  stretch 
of  amino  acids  containing  evenly  spaced  leucine 
residues  allows  one  molecule  of  LRF- 1  to  interact 
with  another  molecule  of  LRF-1  or  of  a  related 
protein.  LRF-1  activates  or  inhibits  the  expression 
of  target  genes  in  a  singular  manner,  implying 
that  LRP-1  has  a  unique  role  in  regulating  events 
in  the  regenerative  process.  As  the  relative  level 
of  LRF- 1  complexes  increases  posthepatectomy, 
activation  of  the  many  liver-specific  genes  oc- 
curs, resulting  in  maintenance  of  normal  liver 
function  and  metabolic  homeostasis  during 
regeneration. 


Another  goal  of  our  research  (supported  in  part 
by  research  funds  from  other  agencies)  is  to  un- 
derstand the  involvement  of  insulin  in  the  regula- 
tion of  hepatic  growth.  It  is  well  known  that  dia- 
betics have  poor  healing  capacity,  and  early 
studies  have  shown  that  the  livers  of  diabetic  ani- 
mals show  poor  regenerative  capacity.  Because 
we  are  studying  a  liver  cell  line  that  is  growth- 
regulated  by  insulin,  we  have  been  able  to  com- 
pare the  insulin-regulated  growth  response  in 
these  cells  with  the  response  during  liver  regen- 
eration. Differences  in  expression  of  more  than 
1 0  of  the  novel  genes  we  have  identified  suggest 
that  insulin,  if  it  is  an  important  growth  factor 
during  liver  regeneration,  must  act  several  hours 
after  the  hepatectomy  and  is  not  an  initiating  fac- 
tor. Additionally,  in  examining  the  encoded  func- 
tion of  several  genes  that  are  aberrantly  regulated 
in  the  hepatic  cell  line,  we  are  learning  more 
about  the  specific  signaling  pathways  involved  in 
hepatic  growth. 


412 


Protein-Tyrosine  Phosphatases  and  the  Control 
of  Lymphocyte  Activation 


Matthew  L.  Thomas,  Ph.D. — Assistant  Investigator 

Dr.  Thomas  is  also  Associate  Professor  of  Pathology  and  Assistant  Professor  of  Molecular  Microbiology  at 
Washington  University  School  of  Medicine,  St.  Louis.  He  received  his  Ph.D.  degree  from  the  University  of 
Utah  after  completing  his  thesis  research  at  Harvard  Medical  School.  His  postdoctoral  training  was  done 
with  Alan  Williams  at  Oxford  University  and  with  Ian  Trowbridge  at  the  Salk  Institute.  Prior  to  his  present 
appointment.  Dr.  Thomas  was  an  Established  Investigator  of  the  American  Heart  Association. 


THE  molecular  mechanisms  by  which  cells  re- 
spond to  their  environment  are  a  central 
theme  in  many  areas  of  medical  research.  Our 
interests  are  in  understanding  how  signals  re- 
ceived by  proteins  of  leukocyte  surface  mem- 
branes result  in  changes  in  a  wide  variety  of  cel- 
lular processes.  In  particular,  we  are  interested  in 
lymphocyte  activation. 

Lymphocytes,  by  virtue  of  their  ability  to  recog- 
nize an  infinite  variety  of  foreign  antigens,  play  a 
key  role  in  effecting  and  regulating  an  immune 
response.  Upon  binding  antigen,  lymphocytes 
undergo  profound  biochemical  changes  to  en- 
gage the  cellular  machinery  required  for  clonal 
expansion  and  to  produce  molecules  needed  to 
fight  infection.  A  key  regulatory  mechanism  by 
which  many  different  cell  types  control  the  signal 
transduction  process  is  phosphorylation  and/or 
dephosphorylation  of  distinct  protein-tyrosine 
residues  of  specific  substrates.  This  process  is 
controlled  by  families  of  enzymes  that  either  add 
phosphate  (protein-tyrosine  kinases)  or  remove 
phosphate  (protein-tyrosine  phosphatases).  The 
research  interests  of  my  laboratory  are  centered 
on  how  protein-tyrosine  phosphatases  control 
lymphocyte  activation. 

The  protein-tyrosine  phosphatase  family  can 
be  divided  into  two  main  branches:  transmem- 
brane and  intracellular.  The  leukocyte-common 
antigen  CD45  is  a  major  transmembrane  protein- 
tyrosine  phosphatase  of  lymphocytes  and  is  ex- 
pressed by  all  nucleated  cells  of  hematopoietic 
origin.  To  study  the  function  of  this  molecule,  we 
generated  nontransformed  T  cell  clones  deficient 
in  the  expression  of  CD45.  The  deficiency  re- 
sulted in  the  cells'  inability  to  respond  to  antigen. 
CD45-deficient  cells  still  respond  to  other  prolif- 
erative signals,  such  as  stimulation  by  growth  fac- 
tors or  plant  lectin  mitogens  or  direct  activation 
of  protein  kinase  C.  However,  these  cells  cannot 
proliferate  or  produce  cytokines  in  response  to  T 
cell  antigen  receptor  stimulus,  and  their  ability  to 
cytolyze  target  cells  is  also  impaired.  Therefore 
the  protein-tyrosine  phosphatase  CD45  is  re- 
quired for  antigen-induced  T  cell  activation. 
We  are  investigating  the  molecular  mechanism 


of  antigen-induced  CD45-controlled  activation 
by  examining  the  differences  in  tyrosine  phos- 
phorylation among  the  CD4  5 -expressing  and 
-nonexpressing  cell  lines.  We  have  observed  in- 
creased tyrosine  phosphorylation  of  members  of 
the  Src-tyrosine  kinase  family  of  proteins  in  the 
CD45-deficient  cells,  indicating  that  they  are  po- 
tential substrates  for  CD45.  Three  members  of 
the  Src  family,  p56'^\  p59'^,  and  ip62^^\  are  ex- 
pressed by  T  cells,  and  recent  studies  have  indi- 
cated that  kinase  activity  for  all  three  is  decreased 
in  the  CD45-deficient  T  cells.  Src  family 
members  are  negatively  regulated  by  phosphory- 
lation of  a  carboxyl-terminal  tyrosine  residue. 
CD45  may  serve  to  dephosphorylate  the  car- 
boxyl-terminal tyrosine  site  and  thus  function  to 
activate  members  of  the  Src  family.  The  inability 
of  antigen  to  activate  CD45-deficient  T  cells  im- 
plies that  activation  of  Src  family  members  is  criti- 
cal to  antigen-induced  T  cell  activation  and  that 
CD45  is  the  phosphatase  important  in  initiating 
this  process.  This  work  is  supported  in  part  by  a 
grant  from  the  National  Institutes  of  Health. 

Immortalization  of  CD45-deficient  T  cells  by 
fusion  with  a  transformed  thymoma  cell  line  has 
permitted  the  development  of  a  model  system  in 
which  function  can  be  reconstituted  by  cDNA 
transfection.  We  are  currently  analyzing  regions 
of  functional  importance  in  the  CD45  molecule 
by  reconstituting  the  cells  with  altered  CD45 
cDNAs.  These  experiments  should  allow  precise 
definition  of  the  regions  of  the  molecule  impor- 
tant in  regulating  T  cell  activation. 

To  analyze  further  how  protein-tyrosine  phos- 
phatases control  lymphocyte  activation,  we  have 
isolated  multiple  cDNAs  that  encode  protein- 
tyrosine  phosphatases  expressed  by  leukocytes. 
LRP  is  a  transmembrane  phosphatase  whose  exte- 
rior domain  is  predicted  to  be  a  highly  glycosy- 
lated, elongated  rod.  This  type  of  structure  is  also 
found  at  the  amino  terminus  of  CD45's  exterior 
domain  and  may  provide  a  novel  means  of  regu- 
lating the  phosphatase  activity.  To  understand  its 
function  and  regulation,  we  have  developed  a 
monoclonal  antibody  that  recognizes  the  native 
protein.  Use  of  this  antibody  has  allowed  us  to 


413 


Protein-Tyrosine  Phosphatases  and  the  Control  of  Lymphocyte  Activation 


examine  the  expression  of  the  protein  by  var- 
ious leukocyte  populations,  the  extent  of  post- 
translational  modification,  and  the  ability  of  the 
antibody  to  induce  changes  in  lymphocyte  activa- 
tion and  proliferation. 

PEP  and  SHP  are  both  intracellular  protein- 
tyrosine  phosphatases  expressed  primarily  by 
leukocytes.  PEP  contains  a  large  carboxyl-termi- 
nal  extension  following  the  phosphatase  domain. 
This  region  is  unusual  when  compared  with  other 
intracellular  phosphatases,  in  that  it  contains  se- 
quences indicative  of  nuclear  localization  and 
rapid  protein  turnover.  By  tagging  the  protein 
with  a  sequence  that  can  be  traced,  we  have  ob- 
tained preliminary  results  indicating  that  PEP 
does  indeed  localize  to  the  nucleus.  It  is  possible, 
therefore,  that  PEP  is  involved  in  regulating  gene 
transcription  or  other  nuclear  functions. 

SHP  contains  two  src-homology  region-2 
(SH2)  domains  linked  in  tandem  immediately 
amino  terminal  to  the  PTPase  catalytic  domain. 
SH2  domains  are  found  in  proteins  involved  in 
transducing  mitogenic  signals,  such  as  all  the 
nonreceptor  protein-tyrosine  kinases,  the  p21"* 


GTPase-activating  protein,  and  the  7-isoform  of 
the  phosphatidylinositol-specific  phospholipase 
C.  Functionally,  SH2  domains  bind  phosphory- 
lated  tyrosine  residues.  Thus  a  protein  that  con- 
tains SH2  domains  serves  to  amplify  and  direct 
the  biochemical  pathway  of  a  response  that  is  in- 
duced by  an  initial  increase  in  tyrosine  phosphor- 
ylation. Recent  results  indicate  that  the  SHP  SH2 
domains  are  capable  of  binding  a  low-molecular- 
weight  phosphotyrosine  protein.  The  existence 
of  a  phosphatase  that  contains  SH2  domains  sug- 
gests that  SHP  may  serve  to  modulate  kinase-in- 
duced  signals.  We  are  examining  the  plausibility 
of  this  model  by  identifying  the  specific  proteins 
with  which  SHP  interacts. 

Through  an  understanding  of  how  protein- 
tyrosine  phosphatases  effect  lymphocyte  activa- 
tion, we  hope  to  gain  a  more  thorough  knowledge 
of  the  biochemical  steps  involved  in  control- 
ling and  regulating  an  immune  response.  The 
functional  characterization  of  multiple  protein- 
tyrosine  phosphatases  expressed  by  lymphocytes 
has  allowed  us  to  define  steps  in  which  members 
of  this  family  function  in  lymphocyte  activation. 


414 


Molecular  Regulation  of  Lymphoid  Cell  Growth 
and  Development 


Craig  B.  Thompson,  M.D. — Associate  Investigator 

Dr.  Thompson  is  also  Associate  Professor  of  Internal  Medicine  and  of  Microbiology  and  Immunology  at  the 
University  of  Michigan  Medical  School.  He  received  his  undergraduate  degree  from  Dartmouth  College 
and  his  medical  degree  from  the  University  of  Pennsylvania.  Following  an  internship  and  residency  at  the 
Peter  Bent  Brigham  Hospital,  Boston,  he  spent  eight  years  as  a  research  medical  officer  in  the  United  States 
Navy.  During  this  time,  he  conducted  his  research  at  Boston  University,  the  Uniformed  Services  University 
of  the  Health  Sciences,  the  Fred  Hutchinson  Cancer  Research  Center,  and  the  Naval  Medical  Research 
Institute. 


THE  lymphoid  immune  system  comprises  two 
major  cell  types:  the  T  cell,  which  identifies 
and  destroys  cells  expressing  foreign  proteins; 
and  the  B  cell,  which  secretes  antibodies  that 
bind  to  foreign  substances,  targeting  them  for 
elimination.  The  central  role  of  the  system  in  the 
natural  resistance  to  infectious  diseases  is  demon- 
strated by  the  infections  encountered  by  patients 
with  immunologic  deficiencies.  Many  of  the  seri- 
ous infectious  and  neoplastic  complications  asso- 
ciated with  the  acquired  immune  deficiency  syn- 
drome (AIDS)  are  the  result  of  depletion  of  the 
helper  T  cells. 

A  better  understanding  of  the  molecular 
mechanisms  associated  with  generation  of  both  B 
and  T  cells  during  development  would  aid  in  our 
ability  to  understand  and  treat  various  immunode- 
ficiencies. The  goals  of  our  laboratory  are  to  un- 
derstand the  molecular  events  associated  with 
the  development  of  the  lymphoid  immune  sys- 
tem and  to  define  the  mechanisms  by  which  the 
functions  of  these  cells  are  controlled. 

T  cells  are  divided  into  two  major  subsets: 
helper  T  cells,  which  produce  the  lymphokines 
that  regulate  immune  responses,  and  cytotoxic  T 
cells,  which  can  kill  cells  expressing  foreign  pro- 
teins. The  cooperation  of  these  two  cell  types  is 
needed  for  the  immune  system  to  reject  a  foreign 
cell  or  a  cell  bearing  foreign  proteins.  Initiation 
of  a  helper  T  cell  response  requires  activation  of 
the  T  cell  receptor  by  foreign  histocompatibility 
genes.  Recent  evidence  suggests,  however,  that  T 
cell  receptor  activation  alone  does  not  lead  the 
helper  cell  to  produce  sufficient  amounts  of  lym- 
phokines to  initiate  an  immune  response.  Addi- 
tional co-stimulatory  signals  are  required. 

Previously  we  showed  that  the  CD  2  8  receptor 
expressed  on  helper  T  cells  serves  as  a  surface 
component  of  a  signal  transduction  pathway  that 
can  enhance  T  cell  lymphokine  production.  In 
vitro,  interaction  of  CD28  with  its  natural  ligand 
B7,  expressed  on  activated  B  cells  or  macro- 
phages, can  act  as  a  co-stimulus  to  such  enhance- 
ment. Now  we  have  evidence  that  CD28  activa- 


tion of  primary  T  cells  is  also  a  required 
co-stimulatory  event  in  the  initiation  of  a  cell- 
mediated  immune  response. 

We  have  investigated  the  role  of  the  CD  2  8 
pathway  in  allogenic  responses  by  using  a  soluble 
CD28  receptor  homologue  termed  CTLA-4-Ig, 
produced  by  our  collaborator  Peter  Linsley.  This 
protein  is  a  recombinant  that  displays  an  affinity 
for  the  CD28  ligand,  B7,  approximately  20-fold 
higher  than  does  cell-bound  CD28.  Therefore  it 
acts  as  a  competitive  inhibitor  of  CD28  engage- 
ment. In  vitro,  CTLA-4-Ig  was  found  to  be  able  to 
inhibit  completely  the  ability  of  cells  from  one 
rat  strain  to  respond  against  cells  from  another. 

Based  on  these  data,  the  ability  of  CTLA-4-Ig  to 
prevent  the  induction  of  organ  graft  rejection  was 
tested  in  a  cardiac  transplant  model.  Trans- 
planted hearts  in  control  animals  receiving  no  im- 
munotherapy are  rejected  within  one  week  of 
transplantation.  In  contrast,  when  activation  of 
the  CD28  pathway  is  blocked  by  daily  administra- 
tion of  CTLA-4-Ig,  rejection  of  the  cardiac  trans- 
plant is  prevented. 

These  data  support  the  hypothesis  that  helper  T 
cell  co-stimulation  by  the  CD28-ligand,  B7,  is  a 
required  event  in  the  initiation  of  a  T  cell- 
mediated  immune  response.  Furthermore,  the 
work  suggests  that  preventing  B7  activation  of  a  T 
cell  may  not  only  help  to  prevent  transplant  re- 
jection but  may  also  help  to  decrease  the  severity 
of  autoimmune  diseases  such  as  rheumatoid  ar- 
thritis. Work  to  address  this  issue  is  being 
planned. 

The  major  role  of  the  B  cell  immune  system  is 
to  generate  the  approximately  10  million  diff'er- 
ent  antibody  molecules  needed  to  protect  the 
body  from  foreign  substances.  B  cells  derive  their 
name  from  the  bursa  of  Fabricius,  a  developmen- 
tal organ  in  birds  that  is  required  for  B  cell  matura- 
tion. Mammals  lack  this  organ  but  are  still  able  to 
generate  a  B  cell  immune  system.  Over  the  past 
several  years,  our  laboratory  has  investigated  the 
role  of  the  bursa  in  B  cell  development  in  the 
chicken.  We  are  attempting  to  characterize  dif- 


415 


Molecular  Regulation  of  Lymphoid  Cell  Growth  and  Development 


ferences  in  the  generation  of  B  cells  in  mammals 
and  birds  to  shed  light  on  mammalian  B  cell 
production. 

Our  studies  have  helped  to  demonstrate  that 
the  primary  molecular  mechanism  by  which 
chickens  generate  a  wide  variety  of  antibody  mol- 
ecules is  different  from  that  used  by  mammals. 
Antibody  diversity  in  the  chicken  is  achieved  by  a 
process  known  as  gene  conversion,  which  occurs 


during  B  cell  development  in  the  bursa  of  Fabri- 
cius.  Gene  conversion  requires  that  the  immature 
B  cell  migrate  to  the  bursa,  and  that  the  mature  B 
cell,  having  undergone  conversion,  emigrate  to 
the  peripheral  lymphoid  organs.  Understanding 
how  this  migration  is  controlled  in  a  regulated 
fashion  should  lead  to  insights  into  how  cells 
in  a  multicellular  organism  migrate  during 
development. 


416 


The  Molecular  Basis  of  Metamorphosis 


Carls.  Thummel,  Ph.D. — Assistant  Investigator 

Dr.  Thummel  is  also  Assistant  Professor  of  Human  Genetics  at  the  University  of  Utah  School  of  Medicine. 
He  obtained  his  undergraduate  degree  in  biology  from  Colgate  University  and  his  Ph.D.  degree  in 
biochemistry,  working  with  Robert  Tjian,  at  the  University  of  California,  Berkeley.  He  received 
postdoctoral  training  in  the  laboratory  of  David  Hogness  at  Stanford  University. 


THE  fruit  fly  Drosophila  melanogaster  pro- 
vides an  ideal  model  system  for  studying  the 
development  of  eukaryotes.  Three-quarters  of  a 
century  of  biological,  physiological,  and  genetic 
experiments,  combined  v^^ith  recent  intensive  mo- 
lecular studies,  has  led  to  a  greater  understanding 
of  its  development  than  that  of  any  other  higher 
organism. 

Halfway  through  the  fly's  life  cycle,  a  pulse  of 
the  steroid  hormone  ecdysone  triggers  a  dramatic 
morphological  transformation,  from  the  rela- 
tively immobile  feeding  larva  to  a  highly  motile, 
reproductively  active  adult  fly.  We  are  studying 
the  molecular  basis  of  the  ecdysone-induced  reg- 
ulatory mechanisms  that  allow  metamorphosis  to 
proceed. 

When  the  larva  begins  to  undergo  metamorpho- 
sis, its  salivary  glands  contain  giant  polytene 
chromosomes  that  can  be  visualized  by  light  mi- 
croscopy. These  500-fold  overreplicated,  inter- 
phase chromosomes  lie  in  register  beside  one  an- 
other. A  characteristic  banding  pattern  along  the 
length  of  the  polytene  chromosomes  allows  any 
gene  of  interest  to  be  located  precisely.  Regions 
of  the  genome  that  are  undergoing  transcription 
are  often  represented  by  large  areas  of  decon- 
densed  chromatin  that  can  be  seen  as  puffs.  Thus 
the  transcriptional  activity  of  specific  genes  at 
specific  times  can  be  followed  in  these  chromo- 
somes by  observing  the  appearance  and  disap- 
pearance of  puff^s  during  development. 

Approximately  10  puffs  can  be  distinguished 
when  the  salivary  gland  chromosomes  first  be- 
come large  enough  to  see.  These  puff's  remain 
until  the  end  of  the  larval  phase,  when  the  burst 
of  ecdysone  triggers  a  dramatic  change  in  the 
puffing  pattern.  Approximately  six  puffs  are  in- 
duced directly  by  the  steroid  hormone.  These 
early  ecdysone-inducible  puffs  appear  to  encode 
regulatory  proteins  that  repress  their  own  expres- 
sion and  induce  the  formation  of  over  100  late 
puffs.  This  second  wave  of  puffs  is  believed  to 
encode  the  proteins  responsible  for  initiating 
metamorphosis. 

By  isolating  and  characterizing  the  ecdysone- 
inducible  genes  that  lie  within  the  early  puffs,  we 


hope  to  learn  how  these  genes  are  induced  by  the 
hormone  and  how  their  encoded  proteins  might 
function  in  a  regulatory  capacity.  In  a  broader 
sense,  this  project  provides  a  model  system  for 
characterizing  the  role  of  steroid  hormones  in  reg- 
ulating gene  expression  and  for  addressing  the 
question  of  how  gene  hierarchies  are  controlled 
during  development. 

Our  studies  have  focused  on  E74,  an  ecdysone- 
inducible  gene  that  is  located  within  the  large 
early  puff  at  position  74EF  in  the  polytene  chro- 
mosomes. This  unusually  complex  gene  encodes 
three  nested  mRNAs  that  derive  from  unique  start 
sites  but  share  a  common  3'  end.  The  distal  pro- 
moter directs  the  synthesis  of  a  60-kb  primary 
transcript  that  is  spliced  to  form  the  6-kb  E74A 
mRNA.  Two  other  promoters,  located  40  kb 
downstream  from  the  E74A  promoter,  direct  the 
synthesis  of  4.8-  and  5.1-kb  E74B  mRNAs.  Al- 
though the  E74A  and  E74B  mRNAs  are  distinct 
from  one  another  by  virtue  of  their  unique  5' 
exons,  the  majority  of  these  mRNAs  are  identical, 
derived  from  a  common  set  of  three  3'  exons.  This 
nested  arrangement  of  the  E74  transcripts  leads 
to  the  synthesis  of  two  related  E74  proteins  that 
have  unique  amino-terminal  domains  joined  to  a 
common  carboxyl-terminal  domain. 

The  sequence  of  the  carboxyl  terminus  of  the  E74 
proteins  is  very  similar  to  a  portion  of  the  protein 
encoded  by  the  ets  oncogene.  This  85-amino  acid 
ETS  domain  defines  a  family  of  proteins  and  has  been 
shown  to  function  as  a  site-specific  DNA-binding  do- 
main that  recognizes  a  purine-rich  DNA  sequence. 
Studies  of  proteins  related  to  oncogenes,  such  as 
E74,  may  help  us  learn  more  about  how  the  normal 
counterparts  of  these  disease  genes  function  during 
development. 

By  using  antibody  detection  techniques  to  lo- 
calize the  E74A  protein  bound  to  the  giant  poly- 
tene chromosomes,  we  have  identified  approxi- 
mately 70  binding  sites,  most  of  which 
correspond  to  late  ecdysone-inducible  puffs. 
Based  on  this  observation,  we  predict  that  at  least 
one  function  for  the  E74A  protein  is  to  activate 
late  gene  expression.  In  support  of  this  predic- 
tion, many  late  puffs  are  either  reduced  or  absent 


417 


The  Molecular  Basis  of  Metamorphosis 


in  the  polytene  chromosomes  of  mutant  animals 
that  do  not  express  E74A  protein.  Our  long-term 
goal  is  to  identify  some  of  these  late  genes  in 
order  to  determine  what  role  they  might  play 
during  metamorphosis  and  whether  they  are  di- 
rectly regulated  by  E74A. 

The  precise  timing  of  the  genetic  response  to 
efcdysone  can  be  clearly  seen  in  the  pattern  of 
puffs  that  arise  at  the  onset  of  metamorphosis. 
Our  characterization  of  E74  transcription  has 
provided  insights  into  how  timing  can  be  built 
into  a  genetic  regulatory  hierarchy.  Ecdysone  di- 
rectly activates  the  E74A  promoter,  resulting  in  a 
dramatic  induction  of  the  6-kb  mRNA.  This  tran- 
script, however,  does  not  appear  in  the  cyto- 
plasm until  one  hour  after  promoter  activation. 
The  delay  corresponds  quite  closely  to  the  time  it 
takes  for  RNA  polymerase  to  traverse  the  60-kb 
transcription  unit,  indicating  that  the  length  of 
the  .£"74^1  unit  functions  as  a  timer  to  delay  signifi- 
cantly the  appearance  of  the  encoded  mRNA.  The 
unusual  length  of  the  E74A  primary  transcript 
sets  it  apart  from  most  transcription  units  in  Dro- 
sopfoila,  which  are  only  slightly  longer  than  the 
final  processed  mRNA. 

Ecdysone  also  directly  activates  the  20-kb 
E74B  transcription  unit.  In  agreement  with  its 
primary  transcript  length,  mature  E74B  mRNA 
appears  between  1 5  and  30  minutes  after  ecdy- 
sone addition.  Thus  the  structure  of  the  E74  gene 
dictates  an  invariant  order  of  appearance  of  its 
transcripts  in  response  to  ecdysone. 

The  earlier  appearance  of  E74B  mRNA  is  en- 
hanced by  its  activation  at  an  approximately  25- 
fold  lower  ecdysone  concentration  than  E74A. 
E74B  is  further  distinguished  from  E74A  by  its 
repression  at  a  significantly  higher  ecdysone  con- 
centration than  that  required  for  its  induction, 
close  to  the  concentration  required  for  E74A  ac- 
tivation. These  regulatory  properties  lead  to  an 


ecdysone-induced  switch  in  E74  expression, 
with  an  initial  burst  of  E74B  transcription  fol- 
lowed by  a  burst  of  E74A  transcription.  These 
studies  provide  a  means  to  translate  the  profile  of 
a  steroid  hormone  pulse  into  different  amounts 
and  times  of  regulatory  gene  expression  that,  in 
turn,  could  direct  different  developmental  re- 
sponses in  a  temporally  and  spatially  regulated 
manner. 

To  date,  E74  has  provided  a  valuable  paradigm 
for  our  molecular  characterization  of  the  ecdy- 
sone regulatory  hierarchy.  We  have  recently  be- 
gun to  extend  our  studies  to  include  three  other 
early  genes,  all  of  which,  like  E74,  are  unusually 
long  and  encode  multiple  DNA-binding  proteins. 
Characterization  of  the  temporal  regulation  of 
these  early  genes,  like  that  described  above  for 
E74,  has  confirmed  that  both  promoter  sensitiv- 
ity to  ecdysone  and  primary  transcript  length  con- 
tribute to  the  timing  of  early  gene  activation. 

In  addition,  these  studies  have  allowed  us  to 
divide  the  early  transcription  units  into  two 
classes.  One  class  of  transcripts  is  activated  by  a 
low  ecdysone  concentration,  like  E74B,  and  ap- 
pears to  play  earlier  roles  in  the  regulatory  hierar- 
chy. This  class  includes  transcripts  encoding  the 
ecdysone  receptor,  needed  for  initiating  the  ge- 
netic response  to  the  hormone.  The  second  class, 
typified  by  E74A,  is  activated  by  a  higher  hor- 
mone concentration  and  appears  to  play  a  later 
role  in  the  hierarchy. 

In  addition,  analysis  of  the  effects  of  early  gene 
mutations  on  both  early  and  late  gene  expression 
should  provide  valuable  clues  regarding  func- 
tional regulatory  interactions  within  the  hierar- 
chy. The  predictions  that  arise  from  these  genetic 
studies  can  then  be  tested  at  the  molecular  level. 
An  additional  long-term  goal  v/ill  be  to  isolate 
more  early  and  late  ecdysone-inducible  genes  to 
extend  our  understanding  of  this  complex  devel- 
opmental process. 


418 


The  Regulation  of  Mammalian  Development 


Shirley  M.  Tilghman,  Ph.D. — Investigator 

Dr.  Tilghman  is  also  Howard  A.  Prior  Professor  of  the  Life  Sciences  in  the  Molecular  Biology  Department  at 
Princeton  University  and  Adjunct  Professor  of  Biochemistry  at  the  University  of  Medicine  and  Dentistry 
of  New  Jersey,  Robert  Wood  Johnson  Medical  School.  She  obtained  a  B.Sc.  degree  at  Queen's  University  in 
Kingston,  Ontario,  Canada.  Following  two  years  in  Sierra  Leone,  West  Africa,  where  she  taught  secondary 
school,  she  attended  graduate  school  at  Temple  University  in  Philadelphia,  where  she  received  her  Ph.D. 
degree  in  biochemistry.  Her  postdoctoral  work  was  done  with  Philip  Leder  at  NLH  Before  joining  the 
faculty  at  Princeton,  Dr.  Tilghman  held  positions  at  Temple  University  and  the  Institute  for  Cancer 
Research,  Philadelphia. 


ORDERLY  development  of  the  mammalian 
embryo  requires  the  appropriate  activation 
and  subsequent  modulation  of  genes  in  a  spatial 
and  temporal  manner.  For  the  vast  majority  of 
genes,  both  the  mother's  and  father's  copies  are 
activated  and  modulated  identically,  but  for  a 
small  class  of  genes,  only  the  mother's  or  father's 
copy  is  expressed.  Such  genes  are  parentally  im- 
printed. That  is,  during  the  process  that  generates 
eggs  or  sperm,  these  genes  are  marked  in  such  a 
way  that  the  resulting  embryo  can  distinguish  the 
parental  origin  and  express  it  accordingly.  Our 
laboratory  is  studying  a  locus  that  encodes  at  least 
two  imprinted  genes  on  the  distal  end  of  mouse 
chromosome  7. 

One,  the  insulin-like  growth  factor  II  gene 
(Igf2)  encodes  a  fetal-specific  growth  factor  that 
is  exclusively  expressed  from  the  paternal  chro- 
mosome. The  other  gene  is  HI 9,  which  encodes 
an  RNA,  evolutionarily  conserved,  that  is  only 
found  in  high  abundance  during  fetal  develop- 
ment in  tissues  originating  in  endoderm  and  me- 
soderm. Unlike  Igf2,  HI 9  is  exclusively  ex- 
pressed from  the  maternal  chromosome.  These 
genes  lie  in  tandem  about  75  kilobases  (kb)  of 
DNA  apart  and  are  expressed  in  a  very  similar 
manner  during  mouse  embryogenesis. 

We  are  investigating  the  activation  and  role  of 
these  two  differentially  imprinted  genes.  The 
function  of  the  H19  RNA  is  unknown.  Its  pattern 
of  sequence  conservation  in  mammals  is  reminis- 
cent of  other  functional  RNAs,  such  as  those  asso- 
ciated with  telomerases  and  RNase  P.  We  are  us- 
ing both  genetic  and  biochemical  approaches  to 
understand  its  role  during  development. 

Over  the  past  50  years  a  large  number  of  muta- 
tions have  been  described  that  affect  all  aspects  of 
mouse  physiology.  However,  molecular  access  to 
the  genes,  which  would  allow  us  to  identify  those 
of  developmental  importance,  has  been  difficult 
because  the  mouse  genome  is  so  large.  Advances 
in  DNA  analysis  and  cloning  methods  have  effec- 
tively reduced  the  barriers  to  studying  these 


genes  at  the  molecular  level.  We  have  generated  a 
yeast  artificial  chromosome  library  that  now  con- 
tains 2.5  copies  of  the  mouse  genome  in  over 
28,000  yeast  strains,  each  of  which  harbors  a  seg- 
ment of  mouse  genomic  DNA  averaging  275  kb. 
The  library  has  been  constructed  to  serve  the 
mouse  genome  community,  and  over  60  labo- 
ratories worldwide  have  so  far  screened  it  suc- 
cessfully. This  work  is  supported  by  a  grant  from 
the  National  Institutes  of  Health. 

We  have  used  the  mouse  Fused  (Fu)  locus  to 
test  the  utility  of  the  library  for  isolating  large 
chromosomal  DNA  segments.  Fu,  a  dominant  mu- 
tation on  mouse  chromosome  17,  generates 
kinky  tails  in  heterozygotes  and  early  embryonic 
lethality  in  homozygotes.  The  lethality  is  asso- 
ciated with  overgrowth  of  neuroectoderm  and 
duplications  of  the  body  axis.  To  localize  Fu  pre- 
cisely, 1,000  progeny  were  generated  by  back- 
crossing  mice  carrying  the  Kinky  allele  of  Fu 
with  a  distantly  related  wild  mouse,  Mus  spretus. 
Because  these  mice  are  genetically  very  distinct, 
it  is  easy  to  follow  the  segregation  of  genes  in 
their  progeny. 

The  offspring  were  scored  for  seven  molecular 
markers  that  map  in  a  small  interval  around  Fu, 
and  a  high-density  genetic  map  gives  us  molecu- 
lar landmarks  every  100-200  kb  of  DNA.  One 
marker,  a  pseudogene  of  the  a-globin  gene  fam- 
ily, cannot  be  separated  from  Fu  in  this  cross, 
suggesting  that  it  is  very  close  to  the  gene — 
within  100-200  kb.  We  have  used  this  close 
proximity  to  isolate  approximately  650  kb  of 
DNA  around  the  marker.  By  comparing  the  segre- 
gation of  this  DNA  with  that  of  Fu,  we  should  be 
able  to  pinpoint  Fu's  location. 

A  similar  approach  has  been  adopted  for  the 
piebald  (5)  locus  on  mouse  chromosome  14. 
Mice  carrying  the  original  5  mutation  have  spot- 
ted coats,  a  result  of  the  absence  of  melanocytes 
in  genetically  specified  regions  of  the  midsec- 
tion. Mice  carrying  more-severe  s  mutations  are 
almost  entirely  white.  In  addition,  they  develop 


419 


The  Regulation  of  Mammalian  Development 


megacolon,  as  a  result  of  the  absence  of  enteric 
ganglia  in  the  gut.  Both  melanocytes  and  enteric 
ganglia  develop  from  the  neural  crest,  a  migrat- 
ing cell  population  that  contributes  to  many  cell 
types.  By  studying  the  s  gene,  we  hope  to  gain 
insight  into  the  genes  that  control  the  birth,  mi- 
gration, and  maturation  of  these  cells. 

A  high-density  map  around  s  is  under  way,  us- 
ing mice  carrying  the  original  5  mutation  and  an- 
other wild  strain,  Mus  castaneus.  In  addition, 
the  molecular  studies  are  greatly  aided  by  the 
many  different  mutant  alleles  of  5  created  over 
the  past  40  years  at  the  Oak  Ridge  National  Labo- 
ratory by  William  and  Leane  Russell.  These  al- 


leles, many  of  which  are  deletions,  will  be  invalu- 
able in  mapping  the  region  on  chromosome  14 
that  contains  the  5  gene. 

To  gain  insight  into  the  nature  of  the  5  mutation, 
we  are  also  comparing  the  behavior  of  neural  crest 
cells  in  s-bearing  and  normal  mice.  For  this  pur- 
pose we  have  exploited  a  transgenic  mouse  strain 
carrying  a  (S-galactosidase  gene  that  is  expressed  in 
early  neural  crest  cells  and  their  derivatives.  To 
study  later  stages  in  melanocyte  development,  we 
are  utilizing  an  antibody  to  the  cell  surface  protein 
c-kit,  which  is  expressed  in  these  cells.  We  will 
thus  identify  the  stage  in  neural  crest  development 
that  the  s  mutation  affects. 


420 


Mechanisms  of  Gene  Regulation  in  Animal  Cells 


Robert  Tjian,  Ph.D. — Investigator 

Dr.  Tjian  is  also  Professor  of  Molecular  and  Cell  Biology  at  the  University  of  California,  Berkeley,  and 
Adjunct  Professor  of  Biochemistry  and  Biophysics  at  the  University  of  California,  San  Francisco.  He 
received  the  Ph.D.  degree  in  biochemistry  and  molecular  biology  from  Harvard  University.  Following  this 
he  was  a  Junior  Fellow  of  the  Harvard  Society  of  Fellows  while  a  resident  at  Cold  Spring  Harbor  Laboratory 
and  later  a  staff  investigator  there  before  moving  to  Berkeley.  His  honors  include  the  Monsanto  Molecular 
Biology  Award  of  the  National  Academy  of  Sciences  and  the  Pfizer  Award  for  Enzymology.  Dr.  Tjian  was 
recently  elected  to  the  National  Academy  of  Sciences. 


THE  main  research  interest  of  our  laboratory  is 
the  mechanism  by  which  the  genetic  infor- 
mation stored  in  DNA  molecules  is  retrieved  in  a 
controlled  and  orderly  fashion  during  the  process 
called  transcription,  which  leads  to  the  produc- 
tion of  specific  proteins  in  animal  cells.  We  have 
taken  a  biochemical  approach  to  the  problem  of 
gene  control  and  have  devised  various  means  of 
isolating  the  cellular  components  responsible  for 
transcription  and  of  reconstructing  this  complex 
reaction  in  the  test  tube.  In  this  way  we  are  study- 
ing how  specific  genes  are  turned  on  and  off  dur- 
ing cell  growth  and  development  of  eukaryotic 
organisms.  The  mechanisms  that  govern  the  acti- 
vation of  genes  are  of  fundamental  importance  in 
understanding  the  normal  metabolic  processes 
that  maintain  and  perpetuate  living  cells,  as  well 
as  in  deciphering  cellular  and  genetic  disorders. 

Biochemical  Analysis  of  Cancer  Genes 

A  living  cell  contains  hundreds  of  thousands  of 
protein  molecules,  each  carrying  out  its  allotted 
function.  However,  if  either  the  production  or 
action  of  these  molecules  is  altered,  severe  mal- 
function can  result,  such  as  uncontrolled  growth 
leading  to  cancer.  Thus  certain  key  molecules  in 
the  milieu  of  a  normal  cell's  constituents  have 
the  potential  to  cause  tumors  when  their  func- 
tion is  disrupted.  Such  molecules  are  called 
oncoproteins. 

Our  group  previously  isolated  from  human 
cells  a  family  of  rare  proteins  that  have  subse- 
quently been  shown  to  be  oncogenic,  encoded  by 
thejun  and  fos  genes.  These  regulatory  proteins 
are  normally  involved  in  controlling  the  action  of 
many  other  genes,  but  when  their  activities  are 
perverted — for  example,  by  viruses — they  can 
lead  to  the  production  of  cancer-causing  cells. 

Recent  advances  in  the  study  of  the  nuclear  on- 
cogene ^wn  reveal  that  the  ability  of  its  protein  to 
activate  transcription  is  regulated  by  a  cell-rype- 
specific  inhibitor  that  interacts  with  a  unique 
portion  of  the  molecule,  rendering  it  less  potent. 
It  is  anticipated  that  the  isolation  and  character- 


ization of  this  specific  negative  regulator  of  jun 
will  help  unravel  the  molecular  signaling  path- 
ways responsible  for  transducing  information 
from  the  outside  of  the  cell  into  the  nucleus, 
where  gene  expression  is  controlled.  Moreover, 
such  inhibitors  of  jun  activity  may  also  be  found 
to  represent  new  members  of  the  anti-oncogenic 
or  tumor-suppressor  family  of  biological  regula- 
tory molecules. 

Studies  of  Trans-activating  Proteins 
That  Regulate  Gene  Expression 

A  major  hurdle  has  been  the  development  of 
biochemical  techniques  that  allow  the  purifica- 
tion of  certain  rare  and  fragile  regulatory  pro- 
teins. Through  use  of  DNA-affiniry  chromatogra- 
phy procedures  pioneered  in  this  laboratory,  it  is 
now  possible  to  isolate  such  transcription  pro- 
teins and,  in  turn,  to  clone  molecularly  the  genes 
that  encode  them.  The  ability  to  proliferate  this 
biologically  important  class  of  genes  provides  a 
powerful  approach  toward  understanding  their 
structure  and  function.  In  the  past  two  years  the 
laboratory  has  isolated  and  characterized  some 
10  different  genes  that  are  directly  responsible 
for  the  tissue-selective,  temporally  programmed, 
and  basal-level  control  of  gene  expression  in  ani- 
mal cells. 

In  addition,  these  recent  studies  are  beginning 
to  reveal  new  concepts  regarding  the  surprisingly 
modular  construction  of  the  derivative  proteins 
as  well  as  their  unusual  plasticity  and  functional 
flexibility.  Most  importantly,  specific  structural 
motifs  that  lie  within  these  proteins  have  been 
recognized  as  carrying  out  distinct  functions. 
These  findings  provide  the  theoretical  basis  for 
analysis  of  other  as  yet  undiscovered  regulatory 
factors  and  will  greatly  aid  our  ability  to  decipher 
their  mechanisms  of  action. 

How  Promoter-Specific  Regulators 
Trigger  Transcription 

One  of  the  remaining  fundamental  mysteries  is 
the  mode  of  action  by  which  sequence-specific 


421 


Mechanisms  of  Gene  Regulation  in  Animal  Cells 


DNA-binding  proteins,  such  as  the  prototype  hu- 
man factor  Spl,  direct  transcriptional  interac- 
tions. To  address  this  critical  issue,  our  group  re- 
cently fractionated  and  isolated  the  multiple 
components  necessary  to  reconstitute  transcrip- 
tion. In  the  process  of  dissecting  the  general  tran- 
scriptional apparatus,  we  discovered  two  previ- 
ously undetected  components  that  serve  as  the 
functional  bridge  between  upstream  trans-activa- 
tors and  the  initiation  complex.  These  novel  fac- 
tors appear  to  be  part  of  the  missing  link  that  di- 
rects promoter-selective  transcription  in  animal 
cells,  and  it  is  likely  that  they  will  be  members  of 
a  diverse  and  essential  class  of  regulatory 
proteins. 

Indeed,  this  past  year  has  seen  significant  pro- 
gress in  the  purification  and  characterization  of 
transcriptional  "coactivators"  and  TATA-binding 
protein  (TBP) -associated  factors  (TAFs).  In  partic- 
ular, several  TAFs  and  coactivators  have  been  pu- 
rified from  both  Drosophila  and  human  cells.  In 
addition,  the  genes  encoding  this  novel  class  of 
transcription  factors  have  recently  been  cloned. 
The  structure  and  function  of  these  multisubunit 
complexes  should  prove  to  be  very  revealing. 

One  of  the  most  exciting  and  unexpected  find- 
ings this  year  was  the  discovery  that  the  TBP,  a 
general  transcription  factor  thought  to  be  only 
responsible  for  RNA  polymerase  II  transcription 
of  mRNA,  is  also  an  integral  subunit  of  a  TBP-TAF 
complex  responsible  for  recognition  of  the  RNA 
polymerase  I  promoter.  Most  interestingly,  the 
RNA  polymerase  I  complex  carries  out  all  the 
functions  ascribed  to  the  species-specific  tran- 
scription factor  SLl ,  and  the  subunit  composition 
reveals  the  presence  of  TBP  and  three  novel  TAFs, 
apparently  adapted  uniquely  to  direct  RNA  poly- 
merase I  transcription.  This  surprising  finding 
provides  a  unifying  mechanism  of  transcription 
initiation. 

Transcription  of  Developmentally 
Regulated  Genes 

One  of  our  long-term  interests  is  the  mecha- 
nisms underlying  regulation  and  expression  in 


the  development  of  higher  organisms.  We  have 
begun  to  address  this  issue  in  two  ways:  first,  by 
initiating  a  series  of  in  vitro  experiments  aimed 
at  dissecting  the  transcriptional  regulation  of 
Drosophila  genes,  including  the  alcohol  dehy- 
drogenase, Ultrabithorax,  Antennapedia,  dopa 
decarboxylase,  and  hunchback  genes.  A  major 
advance  was  the  development  of  in  vitro  tran- 
scription reactions  from  staged  Drosophila  em- 
bryos that  accurately  initiate  RNA  synthesis  and 
recapitulate  the  temporal  program  of  transcrip- 
tion displayed  by  these  tissue-specific  and  devel- 
opmentally regulated  genes. 

A  second  approach  has  been  to  investigate  the 
regulatory  mechanism  of  RNA  polymerase  initia- 
tion factors  in  vivo.  Various  systems  have  been 
adapted  to  introduce  altered  genes  back  into  cells 
or  whole  organisms  to  study  their  patterns  of 
expression. 

These  in  vitro  and  in  vivo  studies  have  re- 
cently led  to  two  exciting  results.  First,  a  negative 
regulator  of  a  developmentally  important  gene 
has  been  identified  by  direct  biochemical  means, 
and  its  mode  of  operation  can  now  be  dissected. 
The  finding  of  specific  transcriptional  repressors 
is  of  particular  importance  because  it  is  thought 
that  an  interplay  of  positive  activators  and  nega- 
tive regulators  is  seminal  to  the  spatially  re- 
stricted patterns  of  expression  observed  during 
embryogenesis. 

Also  arising  from  Drosophila  studies  are  the 
identification  and  subsequent  biochemical  char- 
acterization of  a  transcription  factor  that  appears 
to  govern  the  expression  of  genes  in  cells  of  the 
central  nervous  system.  The  gene  encoding  this 
neurogenic-specific  activator  has  recently  been 
isolated,  and  its  structure  is  expected  to  reveal 
interesting  information.  Advantages  in  the  use  of 
fruit  flies  include  the  ability  to  probe  the  develop- 
mental and  tissue-specific  function  of  this  neuro- 
genic regulator  in  a  rapid  and  highly  informative 
manner  not  readily  applicable  to  mammalian 
cells.  These  studies  are  expected  to  yield  new 
insights  concerning  the  tissue-specific  distribu- 
tion and  temporal  timing  of  expression  during 
development. 


422 


Studies  on  T  Lymphocytes  and  Mammalian  Memory 


Susumu  Tonegawa,  Ph.D. — Investigator 

Dr.  Tonegawa  is  also  Professor  of  Biology  at  the  Massachusetts  Institute  of  Technology.  He  received  a  B.S. 
degree  in  chemistry  from  Kyoto  University  in  Kyoto,  Japan,  and  a  Ph.D.  degree  in  biology  from  the 
University  of  California,  San  Diego.  His  postgraduate  training  and  research  were  at  UCSD  in  the 
laboratory  of  Masaki  Hayashi  and  at  the  Salk  Institute  with  Renato  Dulbecco.  Dr.  Tonegawa  was  a 
member  of  the  Basel  Institute  for  Immunology  in  Basel,  Switzerland,  before  joining  the  Department  of 
Biology  and  Center  for  Cancer  Research  at  MIT.  He  was  awarded  the  Nobel  Prize  for  physiology  or 
medicine  in  1987. 


T lymphocytes  play  a  pivotal  role  in  the  body's 
defense  against  a  variety  of  infectious  agents 
and  malignant  tumors  as  well  as  in  the  rejection 
of  grafted  foreign  tissues.  Our  laboratory  contin- 
ues to  study  the  development  and  functions  of  T 
lymphocytes,  with  a  particular  focus  on  more  re- 
cently discovered  and  less-characterized  yb  T 
cells.  In  addition,  outwork  on  mammalian  mem- 
ory has  progressed  significantly  during  the  past 
year. 

Our  study  on  yb  T  cells  could  be  divided  into 
three  categories:  development  and  selection, 
specificities,  and  function.  As  for  the  early  devel- 
opment of  these  cells,  one  major  issue  that  we 
continued  to  pursue  is  the  mechanism  by  which 
the  yb  lineage  segregates  from  the  a.0  lineage 
from  common  progenitor  cells.  Following  our 
earlier  observations  that  differential  activation  of 
the  transcriptional  silencer  associated  with  the  y 
genes  of  the  T  cell  receptor  (TCR)  plays  a  pivotal 
role  in  the  cell  lineage  segregation,  we  character- 
ized the  silencer  DNA  elements  in  detail  and 
identified  proteins  that  bind  to  these  elements. 
We  intend  to  clone  the  genes  encoding  the 
silencer-binding  proteins  and  produce  mice  with 
mutations  in  these  genes.  Analysis  of  the  mice 
should  be  highly  informative  in  the  dissection  of 
the  lineage  segregation  mechanism. 

a/?  T  cells  are  known  to  undergo  in  the  thymus 
a  critical  maturation  step  called  positive  selec- 
tion. This  depends  on  an  appropriate  interaction 
between  the  TCRs  and  a  self  determinant  en- 
coded in  part  by  the  genes  of  the  major  histocom- 
patibility complex  (MHC).  Following  our  earlier 
study  indicating  that  epithelium-associated  yb  T 
cells  (called  s-IEL  and  vut-IEL)  undergo  a  similar 
positive  selection,  we  have  now  obtained  evi- 
dence of  positive  selection  for  circulating  yb  T 
cells.  Thus  the  development  of  a  transgenic  yb  T 
cell  clone  (KN6)  specific  for  an  MHC  class  I  prod- 
uct (encoded  by  the  T2^  gene)  was  shown  to  be 
blocked  at  an  immature  state  in  a  mouse  geneti- 
cally deficient  in  the  expression  of  MHC  class  I 
gene  products. 

We  also  studied  T  cell  development  by  exploit- 


ing the  embryonic  stem  (ES)  cell  gene-targeting 
method.  We  produced  four  types  of  mutant  mice 
relevant  for  the  analysis  of  T  cell  development 
and  functions.  The  first  and  second  types  of  mice 
have  mutations  in  the  TCRa  and  TCRjS  genes,  re- 
spectively, and  are  deficient  in  mature  T  cells 
but  not  in  mature  yb  T  cells.  The  third  type  of 
mouse  has  a  TCR6  gene  mutation  and  is  deficient 
in  yb  T  cells  but  not  in  afi  T  cells.  The  fourth  type 
was  produced  by  mutating  the  RAG-1  gene, 
whose  product  is  required  for  somatic  rearrange- 
ment of  immunoglobulin  and  TCR  genes.  Neither 
mature  T  cells  nor  B  cells  are  present  in  the 
RAG-1  mutant  mice. 

The  initial  analysis  of  these  mice  indicated  that 
there  is  no  major  interaction  between  the  devel- 
opment of  and  yb  T  cells  and  that  the  muta- 
tions in  the  various  genes  block  lymphocyte  de- 
velopment at  distinct  stages.  Detailed  analysis  of 
the  immature  lymphocytes  accumulating  in  these 
mutant  mice  is  expected  to  augment  our  under- 
standing of  lymphocyte  development. 

Specificities  of  yb  T  cells  were  studied  by  char- 
acterizing the  self-reactive  yb  T  cell  subset 
(V4C1)  on  the  one  hand,  and  by  testing  our  hy- 
pothesis that  TL  class  I  molecules  have  evolved  to 
present  peptides  to  yb  TCRs.  In  the  V4C1  subset 
study,  we  produced  three  types  of  monoclonal 
antibody  (mAb),  each  of  which  blocks  self- 
reactivity  of  these  TCRs  in  vitro.  These  mAbs  rec- 
ognize 1)  a  determinant  specifically  present  on 
the  75  TCR  utilized  as  the  immunogen  (called 
clonotypic),  2)  determinants  present  on  all 
V74C71 -expressing  TCR,  or  3)  determinants  pres- 
ent on  the  receptor  for  vitronectin,  a  family  of 
cell  adhesion  molecules  previously  implicated  as 
coreceptors  for  this  T  cell  subset.  These  mAbs 
will  be  extremely  useful  in  the  elucidation  of  the 
ligand  for  the  self-reactive  yb  T  cell  subset,  as 
well  as  for  the  analysis  of  its  development  and 
tissue  localization. 

We  are  studying  the  general  peptide-present- 
ing  role  of  TL  class  I  molecules  by  producing 
mouse  mutants  lacking  a  cluster  of  these  mole- 
cules. We  have  thus  far  identified  ES  cell  clones 


423 


Studies  on  T  Lymphocytes  and  Mammalian  Memory 


devoid  of  a  large  number  of  TL  class  I  genes.  Also 
being  pursued  is  biochemical  characterization  of 
a  TL  class  I  molecule  encoded  by  the  gene  73'', 
whose  product  has  been  shown  to  be  expressed 
specifically  on  the  surface  of  gut  epithelial  cells 
with  which  a  76  T  cell  subset,  i-IEL,  is  associated. 
We  intend  to  purify  the  putative  peptides  bound 
to  the  T3^  molecule  and  subject  them  to  microse- 
quencing.  The  sequences  may  lead  to  the  identi- 
fication of  the  antigenic  proteins. 

We  have  pursued  functions  of  76  T  cells  by  im- 
munizing KN6  transgenic  mice  with  the  ligand- 
bearing  C57BL/6J  spleen  cells  and  following  the 
proliferation  of  the  transgenic  cells  and  the  disap- 
pearance of  the  injected  spleen  cells  from  the 
host  spleen.  These  studies  indicate  that  KN6  76  T 
cells  are  capable  of  responding  to  the  allogenic 
cells  by  cytotoxicity. 

Functions  of  76  T  cells  have  also  been  studied 
by  following  the  antibody  response  of  afi  T-defi- 
cient  mutant  mice  to  thymus-dependent  and 
-independent  antigens.  So  far  these  mice  respond 
only  to  the  thymus-independent  antigen.  A  possi- 
ble role  of  76  T  cells  in  this  type  of  antibody  re- 
sponse is  being  studied  using  76  T-deficient  mu- 
tant mice.  The  T-  or  76  T-deficient  mice  have 
also  been  infected  with  listeria,  mycobacteria,  or 
malaria.  Resistance  and/or  immunity  against 
these  infectious  agents  will  be  determined.  We 
are  also  studying  the  response  of  the  mutant  mice 
to  skin  grafts  and  injected  tumor  cells. 

In  addition  to  the  studies  on  76  T  cells,  we 
found,  using  the  multigene  transfection  tech- 
nique, that  the  afi  TCR-CD3  complex  can  be  ex- 
pressed on  the  surface  of  nonlymphoid  cells 
without  either  the  CD37  or  CD35  subunit.  This 
suggests  the  intriguing  possibility  that  these  mul- 
tiple forms  of  TCR-CD3  complex  are  utilized  by 
normal  T  cells  for  difi'erential  purposes.  The  hy- 
pothesis is  being  tested  by  producing  mouse  mu- 
tants that  are  defective  either  in  the  CD37  or 
CD36  gene. 

We  are  also  interested  in  studying  how  infor- 
mation is  stored  and  retrieved  in  the  brain.  The 
approach  that  we  have  taken  is  to  investigate  the 
biochemistry,  physiology,  and  behavior  of  mice 
mutant  for  genes  thought  to  be  involved  in  synap- 
tic plasticity.  Using  homologous  recombination, 
we  have  disrupted  the  a-subunit  of  the  calcium 
calmodulin  kinase  II  (CaMKII)  gene  in  embry- 
onic stem  cells  and  have  used  these  cells  to  gener- 
ate a  mouse  strain  lacking  the  gene.  The  a-sub- 


unit  of  CaMKII  is  neural  specific  and  comprises 
most  of  the  CaMKII  holoenzyme  in  postnatal  hip- 
pocampus and  forebrain.  Peptides  that  emulate 
either  the  calmodulin-binding  domain  or  the  in- 
hibitory domain  of  this  kinase  seem  to  block  the 
induction  of  long-term  potentiation  (LTP) .  Mice 
lacking  the  a-subunit  develop  normally  and  are 
viable. 

Gross  neuroanatomical  studies  did  not  detect 
any  abnormalities.  We  studied  the  electrophysiol- 
ogy  of  the  CAl  fields  of  the  mutant  hippocampus 
in  collaboration  with  the  laboratory  of  Charles 
Stevens  (HHMI,  Salk  Institute).  In  normal  animals 
we  were  able  to  induce  LTP  in  more  than  90  per- 
cent (n  =  12;  3  animals)  of  all  slices.  However, 
we  could  stably  potentiate  less  than  1 0  percent  of 
slices  from  mutant  animals  («  =  17;  5  animals). 
Furthermore,  the  unpotentiated  excitatory  post- 
synaptic potentials  of  normal  and  mutant  animals 
were  identical,  suggesting  that  synaptic  transmis- 
sion was  not  impaired  in  the  mutant  animals. 

We  also  studied  the  mutant  mice  in  difl'erent 
versions  of  the  Morris  water  maze,  in  collabora- 
tion with  Jeanne  Wehner's  laboratory.  The  results 
indicate  that  the  mutant  mice,  but  not  their  nor- 
mal litter  mates,  are  specifically  impaired  in  tasks 
that  demand  the  use  of  configured  spatial  infor- 
mation. Our  analysis  of  the  mice  mutant  for  the  a 
CaMKII  demonstrates  the  involvement  of  this  ki- 
nase with  LTP  and  with  configural  learning. 

Past  evaluations  of  the  involvement  of  LTP  on 
learning  and  memory  have  mainly  depended  on 
the  potential  role  that  the  NMDA  (7V-methyl-D- 
aspartate)  receptor  plays  on  both  phenomena.  Un- 
fortunately, NMDA  blockers  seem  to  impair  both 
learning  and  performance,  confounding  the  in- 
terpretation of  those  experiments.  However,  our 
results  suggest  that  performance  measures  unre- 
lated to  learning  were  not  responsible  for  the 
learning  impairment  observed  in  our  mutant 
animals. 

A  key  aspect  of  our  studies  has  been  the  appar- 
ent specificity  of  the  phenotype:  the  develop- 
ment and  neuroanatomy  of  the  mutant  mice  are 
apparently  normal,  as  well  as  nonpotentiated  syn- 
aptic transmission.  Furthermore,  behavioral  anal- 
ysis of  the  mutant  animals  argues  for  a  selective 
impairment  on  hippocampal-dependent  learn- 
ing. We  are  continuing  to  test  the  Hebbian  hy- 
pothesis by  making  mutants  for  other  compo- 
nents of  LTP  and  for  genes  that  might  affect 
the  physiology  of  specific  regions  involved  in 
learning. 


424 


dIV  in  ftplysia  sensory  neuron:  processes  vs«  cell  bodii  29.2 


Before  5~HT       19»  after  50  mM  5-HT    49s  after  B-HT 


Concentrations  of  the  intracellular  messenger  cAMP  (cyclic  adenosine  3' ,5' monophosphate) 
within  a  single  neuron  from  the  sea  snail  Aplysia  californica.  77?^  cAMP  is  detected  by  its  ability  to 
affect  an  enzyme,  cAMP- dependent  protein  kinase.  This  enzyme  was  produced  by  recombinant 
DNA  technology,  labeled  with  fluorescent  dyes,  and  injected  into  the  neuron,  which  was  then 
grown  in  tissue  culture.  The  cAMP-sensitive  signal  from  the  enzyme  was  imaged  by  confocal 
fluorescence  microscopy,  a  technique  that  can  isolate  one  plane  of  focus  within  a  live  specimen. 
The  concentrations  of  cAMP  are  denoted  by  a  rainbow  of  colors,  with  blues  and  reds  representing 
the  lowest  and  highest  levels,  respectively. 

The  upper  left  panel  shows  the  neuron  before  stimulation.  The  upper  middle  panel  shows  the 
same  cell  shortly  after  addition  of  5  hydroxytryptamine  ( 5-HT,  also  known  as  serotonin )  to  the 
medium.  Although  applied  to  the  entire  cell,  5  HT,  an  important  neurotransmitter,  elevates  cAMP 
to  a  much  greater  extent  in  the  fine  dendrites.  Over  the  next  100  seconds  (upper  right,  lower  left 
panels ),  the  cAMP  shows  some  spread  to  the  main  cell  body,  but  the  dendrites  continue  to  have  the 
highest  cAMP  levels.  The  cAMP  response  begins  to  decay  even  while  the  5-HT  is  still  present  (lower 
middle  panel )  and  returns  to  baseline  when  it  is  removed  (lower  right).  These  images  are  the  first 
direct  visualization  of  local  generation  of  c AMP  in  the  fine  outgrowths  and  its  diffusion  toward 
the  cell  body  and  nucleus.  It  is  known  to  be  a  key  controller  of  both  short-  and  long-term  plasticity 
in  many  neurons. 

Research  of  Brian  Bacskai,  Benny  Hochner,  Martyn  Mahaut-Smith,  Stephen  Adams,  Bong-kiun 
Kaang,  Eric  Kandel,  and  Roger  Tsien. 


426 


Molecular  Engineering  Applied  to  Cell  Biology 
and  Neurobiology 


Roger  Y.  Tsien,  Ph.D. — Investigator 

Dr.  Tsien  is  also  Professor  of  Pharmacology  and  of  Chemistry  at  the  University  of  California  School  of 
Medicine,  San  Diego.  His  undergraduate  degree  was  from  Harvard  College,  in  chemistry  and  physics,  but 
it  was  at  the  University  of  Cambridge,  England,  while  obtaining  a  Ph.D.  degree  in  physiology,  that  he 
was  "introduced  to  the  potential  synergism  between  organic  chemistry  and  cell  biology.  "  After  a 
postdoctoral  fellowship  at  Gonville  and  Caius  College,  Cambridge,  Dr.  Tsien  became  a  faculty  member  at 
the  University  of  California,  Berkeley.  Seven  years  later  his  laboratory  moved  to  the  University  of 
California,  San  Diego.  His  recent  honors  include  the  Passano  Foundation  Young  Scientist  Award,  the 
Spencer  Award  in  Neurobiology  from  Columbia  University,  and  the  Bowditch  Lectureship  of  the  American 
Physiological  Society. 


THE  overall  goal  of  my  laboratory  is  to  gain  a 
better  understanding  of  information  process- 
ing both  inside  individual  living  cells  and  in  net- 
works of  neurons.  Our  preferred  approach  is 
through  the  rational  design,  synthesis,  and  use  of 
new  molecules  to  detect  and  manipulate  intra- 
cellular biochemical  signals,  usually  by  optical 
means  such  as  fluorescence  readout  or  photo- 
chemical release  of  messenger  substances.  For  ex- 
ample, we  have  created  fluorescent  dye  mole- 
cules that  detect  calcium  ions  (Ca^"^)  with  great 
specificity  and  sensitivity,  so  that  while  the  cells 
are  living  and  performing  their  normal  functions, 
we  can  image  Ca^"*^  levels  inside  cells  with  a  spa- 
tial resolution  of  a  micron  or  so  and  a  temporal 
resolution  of  a  fraction  of  a  second.  These  dyes 
have  found  wide  application  in  cell  biology, 
since  a  rise  in  intracellular  Ca^"*^  levels  is  one  of 
the  commoner  mechanisms  by  which  cell  mem- 
branes control  biochemical  events  inside  the 
ceil,  such  as  muscle  contraction,  synaptic  trans- 
mission, glandular  secretion,  enzyme  activation, 
embryonic  fertilization,  and  growth  stimulation. 

The  detection  of  intracellular  signals  such  as 
Ca^"^  is  doubly  important.  It  should  help  in  trac- 
ing the  complex  biochemistries  involved  in  such 
signaling,  and  it  affords  a  nondestructive  way  to 
watch  the  activity  of  many  individual  cells  simul- 
taneously. The  latter  ability  is  particularly  rele- 
vant to  understanding  how  neural  networks  pro- 
cess information  by  harnessing  many  individual 
but  interconnected  neurons  in  parallel.  The  domi- 
nant established  techniques  for  monitoring 
neural  activity  either  listen  intensively  to  a  single 
neuron  at  a  time  or  record  some  smeared-out 
average  of  what  thousands,  millions,  or  billions 
of  cells  are  doing.  If  we  can  continue  to  improve 
the  spatial  and  temporal  resolution  of  present 
Ca^"^  imaging,  we  may  succeed  in  eavesdropping 
on  conversations  within  small  groups  of  individu- 
ally identified  neurons  or  in  taking  snapshots  of 
the  instantaneous  state  of  activity  of  yet  larger  en- 
sembles. Because  imaging  is  inherently  good  for 


following  multiple  events  in  parallel,  it  would  be 
a  major  help  in  analyzing  the  workings  of  the 
brain,  which  is  still  the  most  awesome  and  com- 
plex molecular  assembly  known.  We  recognize 
that  optical  methods,  although  unsurpassed  in 
their  combination  of  spatial  and  temporal  resolu- 
tion, are  best  applied  to  small  regions  of  thin 
transparent  tissues.  For  larger  volumes  of  opaque 
organs,  especially  in  intact  organisms,  other 
forms  of  visualization,  such  as  magnetic  reso- 
nance imaging,  are  more  appropriate,  so  we  are 
also  seeking  to  extend  our  molecular  designs  to 
create  suitable  non-optical  indicators. 

A  recent  example  of  molecular  engineering  is 
our  development  of  a  fluorescent  sensor  for 
cAMP.  This  important  intracellular  messenger 
plays  a  crucial  role  in  the  actions  of  a  great  many 
hormones,  in  the  detection  of  odors  and  tastes, 
and  in  the  mechanisms  of  learning  and  memory. 
In  this  case  we  did  not  design  the  sensing  mole- 
cules from  scratch  but  rather  modified  the  natu- 
ral protein  that  cells  normally  use  to  respond  to 
cAMP.  In  collaboration  with  Susan  Taylor  and  her 
laboratory,  Stephen  Adams  attached  fluorescent 
labels  on  cAMP-dependent  protein  kinase  in  such 
a  way  that  cAMP  not  only  activates  the  normal 
activity  of  this  enzyme  but  produces  an  immedi- 
ate optical  signal  that  we  can  image  microscopi- 
cally. This  labeled  protein  enables  us  to  visualize 
cAMP  levels,  to  show  that  diff'erent  regions  of  a 
single  cell  can  have  differing  responses  to  neuro- 
transmitter and  drug  stimulation,  and  to  see  that  a 
subunit  of  the  enzyme  can  move  in  and  out  of  the 
nucleus  as  the  cAMP  level  rises  and  falls.  While  it 
is  in  the  nucleus,  it  is  ideally  placed  to  modify 
gene  expression. 

A  particularly  dramatic  example  of  the  dy- 
namics of  cAMP  signaling  comes  from  sensory 
neurons  of  the  marine  mollusk  Aplysia  califor- 
nica.  These  neurons  have  been  extensively  stud- 
ied by  Eric  Kandel  (HHMI,  Columbia  University) , 
James  Schwartz,  and  their  collaborators  as  models 
for  both  short-  and  long-term  neuronal  plasticity. 


427 


Molecular  Engineering  Applied  to  Cell  Biology  and  Neurobiology 


In  collaboration  with  Dr.  Kandel's  group,  Brian 
Bacskai  and  Martyn  Mahaut-Smith  have  injected 
the  labeled  protein  kinase  into  the  neurons,  ei- 
ther in  culture  or  in  intact  ganglia,  and  imaged 
the  nucleus,  the  surrounding  cytoplasm  of  the 
cell  body,  and  the  peripheral  outgrowths  of  the 
cell.  Bath  application  of  the  relevant  neurotrans- 
mitter, 5-hydroxytryptamine,  produces  rapid  in- 
creases in  cAMP  with  remarkable  spatial  gra- 
dients— high  in  the  peripheral  outgrovvths  yet 
only  slightly  elevated  in  the  central  cell  body. 

Optical  sections  through  the  nucleus  show  that 
it  tends  to  exclude  the  holoenzyme  (injected  into 
the  cytoplasm)  as  long  as  cAMP  concentrations 
remain  at  basal  levels.  Prolonged  elevation  of 
cAMP  and  dissociation  of  the  holoenzyme  causes 
gradual  translocation  of  the  catalytic  subunit  into 
the  nucleus  over  tens  of  minutes.  The  observed 
gradient  puts  high  cAMP  where  it  is  most  needed 
for  short-term  plasticit}',  at  the  distal  processes 
where  the  presynaptic  terminals  would  be  in 
vivo.  Only  strong  or  repeated  stimulations  would 
be  able  to  raise  the  cAMP  concentration  in  the 
cell  body  sufficiently  to  release  the  catalytic  sub- 
unit  to  diffuse  into  the  nucleus,  phosphorylate 
transcription  factors,  and  cause  longer-term 
changes  in  gene  expression.  There  is  some  evi- 
dence that  cAMP  changes  in  the  mammalian  brain 
may  also  be  important  in  comparable  forms  of 
plasticity,  so  we  are  trying  to  extend  our  studies 
to  the  appropriate  mammalian  neurons. 

Eventually  we  hope  to  extend  optical  methods 
to  detect  macromolecular  biochemical  signals 
such  as  protein  phosphorylation  or  gene  tran- 
scription. These  events  currently  are  assayed  by 
grinding  up  millions  of  cells,  so  that  time  resolu- 
tion is  limited  and  differences  between  individ- 
ual cells  or  subregions  are  impossible  to  discern. 
Our  experience  with  imaging  ionic  messengers 
and  cAMP  suggests  that  cells  have  considerable 
individuality  and  complex  behavior  patterns. 
These  were  missed  with  destructive  population 
assays,  which  might  be  somewhat  analogous  to 


studying  human  psychology  on  the  basis  only  of 
anonymous  nationwide  averages  in  which  the 
respondents  are  executed  after  each  poll.  We 
therefore  seek  continuous,  nondestructive  read- 
out from  single  cells.  Approaches  currently 
under  development  (by  Julie  Matheson  and  Gre- 
gor  Zlokamik,  respectively)  include  microinjec- 
tion of  peptides  whose  fluorescence  is  altered  by 
phosphorylation  and  development  of  membrane- 
permeant  fluorogenic  substrates  for  reporter  en- 
zymes whose  nucleotide  sequences  can  be  fused 
to  genes  or  promoter  sequences  of  interest. 

A  complementary  approach  is  to  perturb  intra- 
cellular signals  in  a  controlled  manner  to  see  how 
the  cell  or  tissue  responds.  Many  important  intra- 
cellular messengers  such  as  cAMP  or  inositol 
phosphates  contain  one  or  more  phosphate 
groups  that  prevent  permeability  through  mem- 
branes. A  general  method  for  making  membrane- 
permeant  analogues  has  not  been  available  but 
would  be  highly  useful  for  artificial  stimulation 
of  the  putative  transduction  pathways  to  see  what 
physiological  functions  result,  especially  re- 
sponses that  cannot  be  assayed  in  microinjected 
or  permeabilized  cells.  Recently  Carsten  Schultz 
has  discovered  such  a  method,  esterification  of 
the  organophosphate  anions  with  acetoxymethyl 
groups,  which  increases  the  potency  of  extracel- 
lularly  applied  cAMP  analogues  by  about  100- 
fold  and  yields  the  first  membrane-permeant  de- 
rivatives of  inositol  phosphates  yet  reported.  This 
methodology  should  help  us  to  find  new  func- 
tions for  these  ubiquitous  messengers. 

Our  projects  encompass  a  wide  range  of  disci- 
plines, including  organic  synthesis,  theoretical 
and  experimental  optical  spectroscopy  and  pho- 
tochemistry, protein  chemistry,  computerized 
microscopy  and  image  processing,  cell  biology, 
and  neurobiology. 

The  work  of  Stephen  Adams,  Martyn  Mahaut- 
Smith,  Julie  Matheson,  Carsten  Schultz,  and  Gre- 
gor  Zlokarnik  in  my  laboratory  was  supported  by 
grants  from  the  National  Institutes  of  Health. 


428 


Genetic  Defects  in  the  Metabolic  Pathways 
Interconnecting  the  Urea 
and  Tricarboxylic  Acid  Cycles 


David  L.  Valle,  M.D. — Investigator 

Dr.  Valle  is  also  Professor  of  Pediatrics,  Medicine,  Molecular  Biology  and  Genetics,  and  Biology  at  the 
Johns  Hopkins  University  School  of  Medicine.  He  received  both  his  undergraduate  degree  in  zoology  and 
his  medical  degree  from  Duke  University.  His  internship  and  residency  in  pediatrics  were  completed  at 
the  Johns  Hopkins  Hospital.  His  postdoctoral  research  in  metabolism  was  done  at  NIH. 


KJMAN  biochemical  genetics  has  been  a 
ruitful  area  of  study  since  its  beginning 
with  the  work  of  Sir  Archibald  Garrod  early  in  this 
century.  Inherited  defects  in  our  body's  chemis- 
try or,  as  Garrod  called  them,  inborn  errors  of 
metabolism,  are  intrinsically  interesting  and 
serve  as  important  models  for  all  genetic  diseases. 
My  colleagues  and  I  have  been  involved  in  the 
study  of  several  aspects  of  these  disorders, 
including  clinical  diagnosis,  biochemical  charac- 
terization, delineation  of  pathophysiologic  mech- 
anisms, development  of  new  therapeutic  ap- 
proaches, and  molecular  studies  of  the  involved 
genes. 

We  have  focused  on  disorders  of  amino  acid 
metabolism,  particularly  those  involving  two 
fundamentally  important  areas  of  metabolism: 
the  urea  cycle,  which  is  involved  in  the  conver- 
sion of  excess  nitrogen  from  a  toxic  to  a  nontoxic, 
readily  excreted  form;  and  the  tricarboxylic  acid 
cycle,  an  essential  component  of  energy  metabo- 
lism. Recently  we  have  extended  these  interests 
to  include  inborn  errors  in  the  biogenesis  of  the 
peroxisome,  a  ubiquitous,  subcellular  organelle 
that  contains  about  40  enzymes  important  in  a 
variety  of  anabolic  and  catabolic  processes. 

One  of  the  amino  acid  disorders  that  we  are 
studying  intensively  is  an  inborn  error  of  orni- 
thine metabolism  known  as  gyrate  atrophy  of  the 
choroid  and  retina  (GA).  This  progressive,  blind- 
ing chorioretinal  degeneration  with  associated 
cataract  formation  is  inherited  as  an  autosomal 
recessive  trait.  The  primary  biochemical  defect  is 
deficiency  of  the  enzyme  ornithine-6-aminotrans- 
ferase  (OAT),  which  results  in  an  approximate 
10-fold  accumulation  of  ornithine  in  all  bodily 
fluids. 

Despite  the  systemic  nature  of  the  metabolic 
abnormality  in  GA,  the  clinical  phenotype  is  lim- 
ited to  the  eye.  Thus  GA  is  one  of  a  very  few  iso- 
lated, inherited  retinal  degenerations  for  which  a 
primary  biochemical  defect  is  known.  In  an  ex- 
tensive molecular  analysis  of  the  OAT  genes  of  85 
probands  from  GA  families  around  the  world,  we 
have  detected  34  OAT  mutations.  Other  investi- 
gators have  added  another  20,  and  together  these 


54  OAT  mutations  account  for  128  (75  percent) 
of  the  possible  170  mutant  alleles  in  our  patient 
population. 

This  compilation  of  OAT  mutations  allows  one 
to  determine  their  consequences  on  the  steady- 
state  levels  of  OAT  mRNA  and  on  the  structure 
and  function  of  OAT  protein  in  the  patients'  cul- 
tured skin  fibroblasts  or  when  expressed  in  a  het- 
erologous system,  Chinese  hamster  ovary  cells, 
which  lack  endogenous  OAT  mRNA  and  protein. 
We  find  that  more  than  80  percent  of  the  mutant 
alleles  produce  normal  amounts  of  normally 
sized  OAT  mRNA.  A  small  fraction  (approxi- 
mately 10  percent)  of  mutant  alleles,  all  with 
point  mutations  that  truncate  the  open  reading 
frame  in  the  penultimate  exon  or  earlier,  have 
markedly  reduced  levels  of  OAT  mRNA.  In  con- 
trast to  their  mRNA  phenotype,  approximately  80 
percent  of  the  OAT  mutant  alleles,  including  at 
least  13  missense  mutations,  yield  little  or  no  de- 
tectable OAT  antigen.  Thus  destabilization  of  the 
protein  is  the  most  common  consequence  of 
these  mutations.  However,  two  missense  alleles, 
Rl  80T  and  Rl  54L,  inactivate  OAT  function  with- 
out reducing  OAT  antigen.  We  speculate  that  the 
involved  residues  may  play  a  role  in  the  active 
site  of  OAT.  Studies  are  now  in  progress  to  pro- 
duce the  large  quantities  of  OAT  necessary  for 
x-ray  crystallography  to  determine  directly  the 
consequences  of  these  mutations  in  OAT  struc- 
ture and  function.  A  portion  of  this  work  on  gy- 
rate atrophy  is  supported  by  a  grant  from  the  Na- 
tional Institutes  of  Health. 

The  OAT-catalyzed  reaction  is  an  essential  step 
in  the  metabolic  pathway  that  interconnects  the 
urea  and  tricarboxylic  acid  cycles  and,  as  might 
be  predicted,  is  subject  to  complex  regulation.  In 
liver,  the  regulation  of  OAT  expression  is  coordi- 
nated with  other  urea  cycle-related  enzymes.  We 
have  identified  a  sequence  motif  in  the  5'-flank- 
ing  region  of  OAT  that  is  also  present  in  the  pro- 
moters of  several  other  urea  cycle  enzymes  and 
have  obtained  evidence  that  this  motif  is  a  cis- 
acting  element  involved  in  the  regulation  of  these 
genes.  Surprisingly,  localization  of  OAT  expres- 
sion by  in  situ  hybridization  and  immunohisto- 


429 


Genetic  Defects  in  the  Metabolic  Pathways  Interconnecting  the  Urea 
and  Tricarboxylic  Acid  Cycles 


chemistry  reveals  an  additional  complication: 
OAT  expression  is  limited  to  one  zone  of  the  he- 
patic lobule,  namely  a  small  population  of  hepa- 
tocytes  surrounding  the  central  vein,  whereas 
most  other  urea  cycle-related  enzymes  are  in  the 
periportal  region.  This  zonal  expression  persists 
even  when  OAT  activity  is  induced  40 -fold  by 
alterations  in  dietary  protein.  We  hope  to  identify 
cis-  and  trans-acting  elements  that  mediate  this 
aspect  of  OAT  expression.  Furthermore,  to  un- 
derstand coordinated  aspects  of  OAT  regulation 
better,  we  are  cloning  the  genes  for  other  en- 
zymes that  are  metabolically  related  to  OAT.  We 
used  complementation  in  Saccharomyces  cere- 
visiae  mutants  to  clone  the  human  cDNA  for 
pyrroline-5-carboxylate  reductase,  the  enzyme 
that  catalyzes  the  conversion  of  the  product  of 
the  OAT  reaction  to  proline.  We  have  now  cloned 
and  mapped  the  structural  gene  for  this  enzyme 
and  are  beginning  a  comparison  of  the  promoter 
regions  of  the  reductase  to  that  of  OAT. 

Our  interest  in  GA  has  stimulated  us  to  identify 
other  genes  that  may  be  involved  in  inherited  reti- 
nal degenerations.  We  have  proceeded  along  two 
lines  of  investigation:  1)  cloning  genes  important 
for  photoreceptor  function  and  2)  using  posi- 
tional cloning  to  identify  candidate  genes  from  a 
region  of  the  genome  known  to  harbor  genes  for 
several  retinal  degenerations  (Xpl  1 .2-Xpll.3). 
As  part  of  the  former  strategy,  we  have  cloned  the 
cDNA  and  cloned  and  mapped  the  structural  gene 
for  recoverin.  Recoverin  is  a  calcium-binding 
protein  whose  expression  is  limited  to  the  pho- 
toreceptor. When  intraphotoreceptor  calcium 
falls,  recoverin  stimulates  retinal  guanylate  cy- 
clase, so  that  photoreceptor  cGMP  concentra- 
tions return  to  high,  dark-adapted  levels.  We  are 
beginning  to  examine  the  possible  role  of  re- 
coverin in  a  variety  of  retinal  degenerations.  In 


our  positional  cloning  studies  of  the  Xpl  1.2  re- 
gion of  the  human  genome,  we  have  assembled 
yeast  artificial  chromosome  (YAC)  contigs  cover- 
ing most  of  this  region  and  have  utilized  one  of 
the  YACs  as  a  probe  to  screen  a  human  retinal 
cDNA  library.  We  have  cloned  at  least  five  cDNAs, 
all  of  which  map  back  to  the  correct  Xpl  1.2  re- 
gion. These  are  being  sequenced  and  will  be  used 
as  probes  in  Northern  blots  of  patient  samples  to 
test  for  their  possible  involvement  in  these 
disorders. 

We  have  also  begun  an  investigation  of  inborn 
errors  of  peroxisome  biogenesis  and  function. 
Zellweger  syndrome,  a  neurodevelopmental  dis- 
order fatal  in  infancy,  is  the  disease  paradigm. 
Cells  and  tissues  from  these  patients  exhibit  defi- 
ciency of  virtually  all  peroxisomal  enzymes  and 
lack  normal-appearing  peroxisomes.  We  have 
cloned  the  genes  for  two  peroxisomal  membrane 
proteins,  the  70-kDa  peroxisomal  membrane 
protein  (PMP70)  and  the  35-kDa  protein 
(PMP35).  PMP70  isamemberof  theATP-binding 
cassette  (ABC)  transporter  protein  family  that 
also  includes  the  mammalian  multiple-drug  resis- 
tance protein  (MDR)  and  the  CFTR  protein  in- 
volved in  cystic  fibrosis.  We  have  cloned  the  en- 
tire PMP70  cDNA,  determined  its  sequence,  and 
used  it  to  clone,  map,  and  characterize  the 
PMP70  gene.  In  collaboration  with  Hugo  Moser, 
we  are  analyzing  the  possible  role  of  PMP70  in 
Zellweger  syndrome.  We  have  identified  three 
PMP70  mutant  alleles,  and  our  results  suggest 
that  PMP70  mutations  account  for  one  of  the 
Zellweger  complementation  groups.  We  also 
have  determined  the  complete  sequence  of  hu- 
man PMP35  and  identified  one  mutant  allele  in 
another  Zellweger  complementation  group.  We 
now  are  focusing  on  expression  systems  to  test 
directly  the  functional  consequences  of  these 
mutations  on  peroxisomal  biogenesis. 


450 


Human  Molecular  Genetics  in  Two  X-linked  Diseases 


Stephen  T.  Warren,  Ph.D. — Associate  Investigator 

Dr.  Warren  is  also  Associate  Professor  of  Biochemistry  and  of  Pediatrics  at  Emory  University  School  of 
Medicine.  He  received  his  Ph.D.  degree  in  genetics  from  Michigan  State  University.  Prior  to  joining  the 
faculty  at  Emory,  he  did  postdoctoral  research  with  Richard  Davidson  at  the  University  of  Illinois  School 
of  Medicine  in  Chicago. 


MY  laboratory  is  involved  in  human  molecu- 
lar genetics  and  is  especially  interested  in 
the  identification  of  genes  responsible  for  ge- 
netic disease.  Our  efforts  currently  focus  on  X- 
linked  disease,  particularly  the  fragile  X  syn- 
drome and  Emery-Dreifuss  muscular  dystrophy. 

Fragile  X  Syndrome 

Fragile  X  syndrome  is  the  commonest  form  of 
inherited  mental  retardation  and  one  of  the  most 
prevalent  genetic  diseases  known,  affecting  ap- 
proximately 1  per  1,000  persons  worldwide.  As 
the  name  implies,  fragile  X  syndrome  is  asso- 
ciated with  a  fragile  chromosomal  site,  which  has 
been  localized  to  band  position  Xq27.3.  Fragile 
sites  are  heritable  loci  that  form  cytologically  evi- 
dent gaps  within  chromosomes  under  specific 
biochemical  induction.  Although  such  sites  are 
numerous  throughout  the  human  genome,  the 
fragile  X  site  is  the  only  one  associated  with  a 
disease. 

Fragile  X  syndrome  is  unusual  among  mamma- 
lian genetic  disorders  in  that  20  percent  of  the 
males  with  a  fragile  X  chromosome  are  not  af- 
fected while  approximately  30  percent  of  carrier 
females  show  some  degree  of  mental  impairment. 
The  fragile  X  mutation  is  less  frequently  pene- 
trant (i.e.,  resulting  in  mental  retardation)  among 
the  siblings  of  these  normal  carrier  males  (called 
transmitting  males),  and  penetrance  increases 
with  each  generation  from  a  transmitting  male 
until  it  reaches  so-called  Mendelian  ratios,  where 
half  of  the  male  children  of  a  carrier  female  are 
affected,  typical  of  an  X-linked  gene.  One  excep- 
tion is  that  daughters  of  transmitting  males  are 
never  affected,  but  their  children  may  be.  This 
confusing  hereditary  pattern,  unique  among  ge- 
netically studied  organisms,  has  been  referred  to 
as  the  Sherman  paradox,  in  reference  to  Steph- 
anie Sherman's  description  of  it. 

Our  work  over  the  past  year  has  not  only  identi- 
fied the  gene  responsible  for  fragile  X  syndrome 
but  has  also  uncovered  an  unusual  mutation 
whose  behavior  explains  the  Sherman  paradox. 
Working  with  an  international  group  of  collabora- 
tors, including  Thomas  Caskey  (HHMI,  Baylor 


College  of  Medicine),  David  Nelson  (Baylor), 
and  Ben  Oostra  (Erasmus  University,  the  Nether- 
lands) ,  we  identified  yeast  artificial  chromosome 
(YAC)  clones,  previously  developed  in  my  labora- 
tory, that  mapped  near  chromosome  breakpoints 
involving  the  fragile  X  site.  Using  cloned  DNA 
derived  from  one  of  these  YACs,  we  identified  a 
cDNA  encoded  by  a  gene  that  the  translocation 
breakpoints  had  interrupted.  This  gene,  termed 
fragile  X  mental  retardation  1  {FMR-1),  pro- 
duces a  4.4-kb  message  expressed  at  high  levels 
in  the  brain  and  testes.  Male  fragile  X  patients 
have  macro-orchidism,  or  enlarged  testes,  in  ad- 
dition to  mental  retardation. 

Within  the  FMR-1  mRNA  is  an  unusual  repeat 
of  the  trinucleotide  CGG.  In  normal  individuals, 
there  are  most  frequently  29  repeats,  though  this 
can  vary  between  6  and  52.  Among  transmitting 
males  and  most  normal  carrier  females,  there  are 
between  52  and  200  CGG  repeats.  Among  men- 
tally retarded  patients,  the  codon  repeats  up  to 
1,300  times  and  is  markedly  unstable  in  mitotic 
cells.  Importantly,  when  the  repeat  expands 
beyond  250,  it  spontaneously  methylates  the 
nearby  DNA,  turning  off  the  FMR-1  gene.  Re- 
moval of  the  gene  product  by  hypermethylation 
in  response  to  the  massive  augmentation  of  CGG 
repeats  is  believed  to  be  the  mechanism  of  fragile 
X  syndrome. 

Work  with  our  colleagues  in  Houston  revealed 
an  apparent  relationship  between  the  number  of 
repeats  in  a  normal  carrier  female  and  the  proba- 
bility of  having  a  mentally  retarded  son.  In  gen- 
eral, the  smaller  the  abnormal  repeat,  the  lower 
the  risk  of  expansion  to  the  full  fragile  X  muta- 
tion in  an  offspring.  Above  a  threshold  of  approxi- 
mately 200  repeats,  the  fragile  X  chromosome 
when  passed  down  always  undergoes  expansion 
to  the  full  mutation.  Hence  carrier  mothers  with 
200  repeats  have  a  50  percent  risk  of  having  a 
retarded  son,  while  those  with  only  70  repeats 
have  a  9  percent  risk.  This  explains  the  paradox 
of  penetrance  in  fragile  X  syndrome:  the  sequen- 
tial increase  in  the  CGG  repeat  with  each  genera- 
tion imparts  a  concomitant  increase  in  risk  of 
having  an  affected  child. 


451 


Human  Molecular  Genetics  in  Two  X-linked  Diseases 


We  are  focusing  on  three  major  efforts  related 
to  the  FMR-1  gene.  The  first  is  to  determine  the 
mechanism  of  expansion  of  the  CGG  repeat  by 
both  the  direct  sequence  analysis  of  the  fragile  X 
mutation  and  the  introduction  of  long  synthetic 
CGG  repeats  into  mammalian  cells  and,  subse- 
quently, into  mice.  Second,  we  are  attempting  to 
understand  the  normal  function  of  the  FMR-1 
gene  product.  Toward  this  goal,  we  have  recently 
cloned  and  sequenced  the  homologous  gene 
from  the  mouse  and  are  in  the  process  of  cloning 
the  yeast  and  the  nematode  genes.  Finally,  we  are 
excited  about  finding  a  number  of  other  human 
genes,  distinct  from  FMR-1,  that  similarly  contain 
long  CGG  repeats.  These  genes,  whose  functions 
are  presently  not  understood,  may  share  a  similar 
functional  utilization  of  the  CGG  repeat;  may  un- 
dergo similar  mutational  changes,  perhaps  lead- 
ing to  diseases  exhibiting  unusual  genetic  pat- 
terns; and  may  represent  other,  autosomal  fragile 
sites. 

Emery-Dreifuss  Muscular  Dystrophy 

Emery-Dreifuss  muscular  dystrophy  (EDMD)  is 
the  most  frequent  X-linked  muscular  dystrophy 
following  the  Duchenne  and  Becker  types.  It  is  a 
slowly  progressive  disease  that  usually  leaves  af- 


fected males  ambulatory  until  middle  age.  Heart 
muscle  involvement  is  frequent,  sometimes  re- 
sulting in  early  sudden  death  due  to  heart  block. 
If  the  disease  is  identified  early,  such  a  death  can 
be  prevented  by  pacemaker  implantation. 

We  have  performed  genetic  linkage  studies  in 
two  large  families  and  have  localized  the  EDMD 
gene  to  the  terminal  band  of  the  X  chromosome 
(band  Xq28)  just  distal  from  the  fragile  X  site. 
We  can  now  place  the  gene  within  an  approxi- 
mate 2,000  kb  of  DNA.  Using  selective  cDNA  li- 
braries and  DNA  of  this  region  cloned  into  cos- 
mids  or  YACs,  we  are  mapping  muscle-expressed 
genes  within  Xq28.  This  entails  use  of  a  somatic 
cell  hybrid  mapping  panel  containing  six  distinct 
Xq28  fragments.  Any  genes  that  map  within  the 
region  believed  to  contain  the  EDMD  gene  will  be 
used  to  search  for  mutations  in  patients. 

Another  outcome  of  our  genetic  mapping  stud- 
ies has  been  the  identification  of  young,  asymp- 
tomatic males  who  will  eventually  suffer  from 
EDMD.  Cardiac  function  of  these  males  is  being 
carefully  followed,  and  pacemaker  implantation 
is  performed  when  warranted  prior  to  full  heart 
block.  Thus  we  have  virtually  eliminated  sudden 
death  due  to  EDMD  in  these  two  families. 


In  situ  hybridization  detecting  human  DNA 
within  the  metaphase  chromosomes  of  a  so- 
matic cell  hybrid  ( micro21D),  which  contains 
a  human-rodent  translocation  between  the 
centric  fragile  X  chromosome  (yellow)  and  a 
rodent  chromosome  arm  ( red).  The  hybrid  was 
constructed  under  conditions  favoring  re- 
arrangements specific  for  the  fragile  X  site.  In- 
deed, the  human  translocation  breakpoint  in 
this  hybrid  is  within  the  CGG  repeat  of  the  frag- 
ile X  mutation.  Somatic  cell  hybrids  with  trans- 
locations of  this  nature  proved  instrumental  in 
cloning  the  fragile  X  site  and  its  associated  gene 
(FMR-1). 
Research  of  Stephen  Warren. 


432 


The  MyoD  Gene  Family:  A  Nodal  Point  During 
Specification  of  Muscle  Cell  Lineage 


Harold  M.  Weintraub,  M.D.,  Ph.D. — Investigator 

Dr.  Weintraub  is  also  a  Full  Member  in  the  Division  of  Basic  Sciences  at  the  Fred  Hutchinson  Cancer 
Research  Center  and  Affiliate  Professor  of  Pathology  and  Zoology  at  the  University  of  Washington,  Seattle. 
He  received  his  M.D.-Ph.D.  degree  from  the  University  of  Pennsylvania  School  of  Medicine  and  completed 
his  postdoctoral  studies  at  the  Medical  Research  Council  Laboratory  of  Molecular  Biology  in  Cambridge, 
England.  Before  joining  the  staff  at  the  Hutchinson  Center,  Dr.  Weintraub  was  in  the  Department  of 
Biochemical  Sciences  at  Princeton.  He  is  a  member  of  the  National  Academy  of  Sciences  and  the  American 
Academy  of  Arts  and  Sciences.  Among  his  many  honors  are  the  Eli  Lilly  Award  and  the  Richard  Lounsbery 


Award  from  the  National  Academy  of  Sciences. 


THE  MyoD  gene  converts  many  differentiated 
cell  types  into  muscle.  MyoD  is  a  member  of 
the  protein  family  characterized  by  the  basic 
helix-loop-helix,  a  68-amino  acid  domain  in 
MyoD  that  is  necessary  and  sufficient  for  myo- 
genesis.  MyoD  binds  cooperatively  to  muscle- 
specific  enhancers  and  activates  transcription. 
The  helix-loop-helix  motif  is  responsible  for  di- 
merization,  and,  depending  on  its  dimerization 
partner,  MyoD  activity  can  be  controlled. 

MyoD  senses  and  integrates  many  facets  of  the 
cell  state.  The  gene  is  expressed  only  in  skeletal 
muscle  cells  and  their  precursors;  in  nonmuscle 
cells  it  is  repressed  by  specific  genes.  MyoD  acti- 
vates its  own  transcription,  perhaps  stabilizing 
commitment  to  myogenesis.  Despite  this  seem- 
ingly overwhelming  evidence  that  MyoD  is  cru- 
cial for  myogenesis  in  vertebrates,  recent  experi- 
ments with  Michael  Krause  and  with  Andrew  Fire 
show  that  zygotic  deletions  of  MyoD  in  worms 
result  in  embryos  that  retain  the  capacity  to  acti- 
vate muscle  cell  differentiation. 

Muscle-Specific  Transcriptional 
Activation  by  MyoD 

Our  laboratory  has  focused  on  the  mechanism 
by  which  MyoD  activates  transcription.  Previous 
experiments  showed  that  when  the  13-amino 
acid  basic  region  of  the  ubiquitously  expressed 
basic  helix-loop-helix  gene  E12  replaces  the 
corresponding  basic  region  of  MyoD,  the  result- 
ing MyoD-E12Basic  chimeric  protein  can  bind 
specifically  to  muscle-specific  enhancers  in  vitro 
and  form  dimers  with  El 2,  but  cannot  activate  a 
cotransfected  reporter  gene  or  convert  lOTVi 
cells  to  muscle.  Back  mutation  of  this  chimeric 
protein  (with  the  corresponding  residues  in 
MyoD)  reestablishes  activation.  A  specific  ala- 
nine is  involved  in  increasing  DNA  binding,  and  a 
specific  threonine  is  required  for  activation. 

A  reporter  gene  containing  MyoD-binding  sites 
located  downstream  from  the  transcription  start 


site  was  used  to  show  that  MyoD-E12Basic  can 
bind  in  vivo  and  thereby  inhibit  expression  of  the 
reporter.  In  vivo  binding  is  also  supported  by  the 
fact  that  the  addition  of  the  "constitutive"  VPl6 
activation  domain  to  MyoD-El  2Basic  restores  full 
trans-activation  potential.  The  normal  MyoD  acti- 
vation domain  maps  within  the  amino-terminal 
53  residues  and  can  be  replaced  functionally  by 
the  activation  domain  of  VP16. 

The  activity  of  the  MyoD  activation  domain  is 
dramatically  elevated  when  deletions  are  made 
almost  anywhere  in  the  rest  of  the  MyoD  mole- 
cule, suggesting  that  the  activation  domain  in 
MyoD  is  usually  masked.  Surprisingly,  MyoD- 
E12Basic  can  activate  transcription  in  CVl  and 
B78  cells  (but  not  in  IOT1/2  or  3T3  cells),  sug- 
gesting that  the  activation  function  of  the  basic 
domain  requires  a  specific  factor  present  in  CVl 
and  B78  cells.  We  propose  that  the  masked  MyoD 
activation  domain  requires,  in  order  to  function, 
the  participation  of  another  factor  that  recog- 
nizes the  basic  region. 

By  replacing  the  MyoD  basic  region  and  the 
adjacent  four-residue  junction  region  with  helix 
1  into  the  corresponding  region  of  El 2,  we  have 
recently  shown  that  this  small  section  of  MyoD  is 
sufficient  for  myogenesis.  Our  work  suggests  that 
only  three  residues,  A114,  Tn,,  and  K124,  are 
uniquely  critical  for  "recognition  factor"  func- 
tion and  subsequent  activation  of  myogenic  gene 
transcription. 


Control  of  MyoD  Activity 

A  variety  of  transforming  agents,  including  a 
variety  of  growth  factors,  the  oncogenes  src,  ras, 
fos,jun,fps,  erbA,  myc,  and  El  A,  and  such  chem- 
ical agents  as  butyrate  and  phorbol  esters,  inhibit 
myogenic  differentiation.  Most  of  these  reagents 
can  inactivate  the  expressed  MyoD  protein;  in  ad- 
dition, several  (such  as  ras  and  fos)  also  inhibit 
MyoD  transcription.  Whether  this  is  a  secondary 


433 


The  MyoD  Gene  Family:  A  Nodal  Point  During  Specification 
of  Muscle  Cell  Lineage 


effect  due  to  an  inhibition  of  the  autoactivation 
function  of  MyoD  protein  or  a  more  direct  inhibi- 
tion of  MyoD  transcription  remains  to  be  deter- 
mined. Rhabdomyosarcoma  cells  (derived  from 
tumors  of  patients  who  harbor  a  genetic  predis- 
position to  myogenic  tumors)  differentiate 
poorly  but  express  MyoD,  suggesting  that  loss  of 
anti-oncogene  activity  at  the  rhabdomyosarcoma 
locus  can  also  impinge  on  MyoD  action.  The  spe- 
cific pathway  by  which  each  of  these  oncogenes, 
anti-oncogenes,  and  growth  factors  inhibits  myo- 
genesis  provides  a  potential  clue  to  how  MyoD 
might  integrate  information  coming  from  many 
aspects  of  cellular  function. 

Recently,  in  collaboration  with  the  laboratory  of 
Inder  Verma,  we  found  that  the  leucine  zipper  re- 
gion of  the  jun  oncogene  actually  binds  to  the  he- 
lix-loop-helix region  of  MyoD,  both  in  vivo  and  in 
vitro.  Similarly,  assays  for  a  recognition  factor  for 
MyoD  activation  show  that  such  a  factor,  which  is 
missing  in  rhabdomyosarcoma  cell  lines,  can  be 
provided  in  trans  by  fusion  with  1  OTVi  cells.  Possi- 
bly failure  to  activate  myogenesis  leads  to  increased 
proliferation  and  then  secondary  effects  that  give 
rise  to  rhabdomyosarcomas. 


Activation  of  MyoD  During  Development 

We  are  studying  developmental  activation  of 
MyoD  in  mice,  worms,  and  frogs.  In  both  mice 
and  worms,  deletional  analysis  has  identified 
regulatory  sequences  upstream  of  the  MyoD 
gene  that  are  important  for  correct  developmen- 
tal activation  of  MyoD.  Current  efforts  focus  on 
identifying  trans-acting  elements  that  integrate 
with  these  sequences.  In  worms,  several  mater- 
nal-effect mutants  have  been  isolated  by  Jim 
Priess  and  his  colleagues.  These  mutant  em- 
bryos produce  excess  muscle  from  the  wrong 
lineage.  It  is  possible  that  these  mutants  define 
elements  involved  in  the  segregation  of  myo- 
genic potential  to  specific  cells  during  early 
cleavage  stages.  In  apparent  contrast  to  worms, 
frogs  seem  to  activate  MyoD  in  all  cells  of  the 
blastoderm;  however,  expression  is  stabilized 
only  in  those  presumptive  mesodermal  cells 
that  become  induced  by  vegetal  inducing  fac- 
tors such  as  activin.  Frogs  also  contain  maternal 
MyoD  mRNA,  which,  however,  seems  not  to  be 
crucial  for  subsequent  myogenesis,  as  its  de- 
struction with  anti-sense  DNA  results  in  normal 
muscle  gene  activation. 


454 


Structural  and  Functional  Studies  of  the  T  Cell 
Antigen  Receptor 


Arthur  Weiss,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Weiss  is  also  Ephraim  P.  Engleman  Distinguished  Professor  of  Rheumatology  and  Associate  Professor 
of  Medicine  and  of  Microbiology  and  Immunology  at  the  University  of  California,  San  Francisco.  He 
received  his  undergraduate  education  at  the  Johns  Hopkins  University  and  was  an  M.D./Ph.D.  student  at 
the  University  of  Chicago,  where  he  studied  immunology  in  the  laboratory  of  Frank  Fitch.  He  did 
postdoctoral  work  with  Jean- Charles  Cerottini  and  K.  Theodore  Brunner  at  the  Swiss  Institute  for 
Experimental  Research,  Lausanne.  After  an  internship  and  residency  in  internal  medicine  at  UCSF,  he 
became  a  postdoctoral  fellow  in  rheumatology  with  John  Stobo. 


THE  immune  system  has  evolved  to  provide  an 
organism  with  a  flexible  and  dynamic  mecha- 
nism to  respond  specifically  to  a  wide  variety  of 
antigens.  During  the  initiation  of  an  immune  re- 
sponse, antigen  must  not  only  be  recognized  by 
antigen-specific  lymphocytes,  but  this  recogni- 
tion event  must  lead  to  cellular  activation.  T  and 
B  lymphocytes  comprise  the  antigen-specific 
components  of  the  cellular  immune  system.  The 
activation  of  T  lymphocytes  is  critical  to  most  im- 
mune responses,  since  it  permits  these  cells  to 
exert  their  potent  regulatory  or  effector  activi- 
ties. During  activation,  relatively  quiescent  cells 
undergo  complex  changes  involving  cell  differ- 
entiation and  proliferation. 

Following  exposure  to  antigen,  activation  of  T 
lymphocytes  is  limited  to  only  those  cells  ex- 
pressing antigen-specific  receptors.  Activation  is 
a  consequence  of  ligand-receptor  interactions 
that  occur  at  the  interface  of  the  T  cell  and  an 
antigen-presenting  cell.  These  interactions  initi- 
ate intracellular  biochemical  events  within  the  T 
cell  that  culminate  in  cellular  responses.  Our 
goal  is  to  understand  how  cell  surface  molecules 
on  the  T  cell,  and  in  particular  the  T  cell  antigen 
receptor  (TCR),  initiate  T  cell  activation. 

Although  it  is  clear  that  a  number  of  different 
cell  surface  molecules  on  the  T  lymphocyte  and 
the  antigen-presenting  cell  may  participate  in  the 
complex  cell-cell  interaction  that  occurs  during 
antigen  presentation,  the  TCR  must  play  a  promi- 
nent role.  Here  the  familiar  lock  and  key  analogy 
is  appropriate.  Antigen  is  the  ligand  (key)  for  a 
particular  set  of  clonally  distributed  receptors 
(locks)  on  T  lymphocytes.  Antigen  often  repre- 
sents a  protein  fragment  that  is  physically  asso- 
ciated with  a  molecule  of  the  major  histocompati- 
bility complex  (MHC). 

The  TCR  is  an  extraordinarily  complex  struc- 
ture. It  consists  of  an  a//3-chain  disulfide-linked 
heterodimer  (Ti)  derived  from  immunoglobulin- 
like  genes  that  is  noncovalently  associated  with 
six  invariant  chains  of  the  CD3  complex  and  a 
f-chain  dimer.  CD3  consists  of  four  chains  (5-,  7-, 
and  two  e-chains)  derived  from  three  closely 


linked  homologous  genes  located  on  chromo- 
some 1 1 .  The  f  dimer,  derived  from  the  products 
of  two  homologous  genes  on  chromosome  1 ,  may 
represent  a  homodimer  (f^  or  heterodimer  con- 
sisting of  fry  (77  is  an  alternatively  spiced  form  of 
f)  or  (7,  a  homologous  protein,  is  also  a  com- 
ponent of  the  IgfFc  receptor  on  mast  cells  and 
basophils) .  Ti  is  the  ligand-binding  subunit  of  the 
TCR,  since  it  contains  all  the  information  needed 
to  recognize  antigen  and  MHC  specificities.  CD3 
and  f  have  been  thought  to  play  some  role  in 
transducing  the  ligand  occupancy  state  of  Ti 
across  the  plasma  membrane.  Hence  the  struc- 
tural basis  for  the  association  of  Ti,  CD3,  and  f  is 
of  interest. 

Previous  studies  from  our  laboratory  have  dem- 
onstrated that  coexpression  of  all  of  the  chains  of 
the  oligomeric  TCR — Ti,  CD3,  and  f — on  the 
plasma  membrane  is  obligatory  for  efficient  TCR 
expression.  Studies  from  this  laboratory  demon- 
strated that  the  structural  and  functional  basis  for 
the  interaction  between  Ti,  CD 3,  and  f  is  con- 
tained within  regions  including  the  transmem- 
brane domains  of  these  proteins.  Further  muta- 
tional studies  are  in  progress  to  understand  more 
precisely  how  Ti,  CD3,  and  f  interact  function- 
ally within  these  domains. 

Since  the  transmembrane  regions  are  responsi- 
ble for  the  Ti-CD3-r  association,  we  have  taken 
advantage  of  this  information  to  separate  regions 
or  domains  of  CD3  and  f  from  Ti.  This  has  been 
accomplished  by  constructing  chimeric  mole- 
cules between  other  cell  surface  molecules 
linked  to  the  cytoplasmic  domain  of  TCR  chains. 
A  chimeric  molecule  consisting  of  the  CDS  extra- 
cellular and  transmembrane  domains  fused  to 
the  f  cytoplasmic  domain  acquired  the  signal- 
transducing  capacity  of  the  entire  TCR.  This  find- 
ing demonstrates  that  the  cytoplasmic  domain  of 
CD3  f  can  link  the  TCR  to  intracellular  signaling 
machinery. 

These  studies  are  being  extended  to  define  the 
region  of  f  that  interacts  with  such  intracellular 
molecules  and  to  identify  these  molecules.  Re- 
cently we  identified  a  70-kDa  tyrosine  phospho- 


435 


Structural  and  Functional  Studies  of  the  T  Cell  Antigen  Receptor 


protein  that  associates  with  the  f-chain;  we  are  in 
the  process  of  characterizing  this  protein.  We 
would  also  like  to  understand  the  function  of  the 
associated  CD3  complex  within  the  complex 
oligomeric  TCR.  Moreover  the  ability  to  create 
such  functional  chimeric  receptors  may  permit 
the  creation  of  novel  antiviral  or  antitumor  TCRs 
that  may  be  of  value  in  gene  therapy. 

Stimulation  of  the  TCR  initiates  cellular  activa- 
tion by  inducing  a  transmembrane  signal  that  is 
manifested  as  the  formation  of  intracellular  bio- 
chemical mediators  called  second  messengers, 
which  can  initiate  or  influence  cellular  response 
pathways.  Recent  studies  demonstrate  that  the 
TCR  activates  an  uncharacterized  protein-tyro- 
sine  kinase  (PTK)  as  the  initial  event  leading  to 
cellular  activation.  This  PTK  appears  to  be  asso- 
ciated with  the  TCR  f-chain.  The  activation  of 
this  PTK  results  in  the  tyrosine  phosphorylation 
of  many  cellular  proteins,  one  of  which  is  phos- 
pholipase  C-7I  (PLC-7I).  The  phosphorylation 
of  PLC-7I  activates  this  enzyme  to  hydrolyze  a 
rare  membrane  lipid,  PIP2  (phosphatidylinositol 
4,5-bisphosphate).  This  yields  two  potent  intra- 
cellular second  messengers  (inositol  1,4,5-tris- 
phosphate  and  diacylglycerol)  that  regulate  the 
mobilization  of  intracellular  calcium  and  activa- 
tion of  the  enzyme  protein  kinase  C,  respectively. 
These  latter  two  events  are  physiologically  im- 
portant to  subsequent  cellular  responses. 

The  mechanism  by  which  the  TCR  couples  to 
intracellular  signaling  pathways  is  largely  unde- 
fined, as  are  many  of  the  components  of  the  sig- 
naling pathways  themselves.  To  define  and  char- 
acterize the  molecular  basis  by  which  the  TCR 
regulates  these  pathways,  we  are  using  a  somatic 
cell  genetic  approach.  In  work  supported  by  a 
grant  from  the  National  Institutes  of  Health,  we 
have  isolated  a  number  of  mutants  derived  from  T 
cell  leukemic  lines  that  are  defective  in  TCR- 
mediated  activation  of  the  inositol  phospholipid 


pathway.  Unlike  the  parental  cells,  none  of  these 
mutants  produce  lymphokines  in  response  to 
TCR  stimulation.  These  mutants  define  four  dis- 
tinct gene  products  other  than  the  TCR  chains 
that  are  required  for  the  functional  activation  of 
the  inositol  phospholipid  pathway. 

In  one  mutant,  the  activation  of  the  tyrosine 
kinase  pathway  is  still  intact;  however,  some  of 
the  substrates  of  the  tyrosine  kinase  pathway  are 
not  phosphorylated,  including  PLC-7I.  The  phe- 
notype  of  this  mutant  suggests  that  there  may  be  a 
coupling  protein  or  second  PTK  that  is  deficient. 

The  defect  in  another  of  these  mutants  can  be 
attributed  to  the  absence  of  a  cell  surface  protein, 
CD45,  with  tyrosine  phosphatase  activity.  The 
absence  of  CD45  prevents  the  TCR  from  activat- 
ing the  tyrosine  kinase  or  phosphatidylinositol 
inositol  pathway.  At  least  one  target  of  the  CD45 
tyrosine  phosphatase  is  a  regulatory  phospho- 
tyrosine  residue  in  the  PTK  Ick.  Thus  a  protein- 
tyrosine  phosphatase  regulates  the  activity  of  a 
PTK.  This  suggests  a  complex  autoregulatory  sys- 
tem that  we  are  intensively  studying.  In  the  re- 
maining two  mutants,  biochemical  studies  have 
complemented  our  genetic  approach.  In  neither 
of  these  mutants  is  the  PTK  pathway  activated. 
Preliminary  studies  suggest  that  one  of  these  mu- 
tants is  deficient  in  a  previously  identified  PTK. 
Thus  these  mutants  are  proving  to  be  valuable 
tools  with  which  to  dissect  the  complexities  of 
the  signal  transduction  pathways  and  their  rela- 
tionships to  cellular  responses. 

T  cell  activation  is  a  complex  process  that  is 
regulated  by  cell  surface  molecules.  Investiga- 
tion of  the  molecules  and  events  involved  in  the 
activation  of  T  cells  should  lead  to  a  more  com- 
plete understanding  of  T  cell  biology  and  a  more 
rational  approach  to  the  manipulation  of  the  im- 
mune system.  Moreover,  through  the  study  of  the 
activation  of  T  cells,  it  is  likely  that  insight  into 
other  biological  systems  involving  cell  prolifera- 
tion and  differentiation  will  emerge. 


456 


Following  the  Life  History  of  Lymphocytes 


Irving  L.  Weissman,  M.D. — Investigator 

Dr.  Weissman  is  also  the  Karel  and  Avice  Beekhuis  Professor  of  Cancer  Biology  and  Professor  of  Pathology, 
Developmental  Biology,  and  (by  courtesy)  Biology  at  Stanford  University  School  of  Medicine.  He  directs 
the  Program  for  Molecular  and  Genetic  Medicine  and  the  Immunology  Program.  He  received  his  M.D. 
degree  from  Stanford  and  remained  to  do  postdoctoral  studies  in  the  Department  of  Radiology.  He  also 
studied  at  Oxford  with  Jim  Gowans  in  1964  and  returned  in  1975  for  part  of  a  sabbatical  year,  which  he 
then  completed  with  Melvin  Cohn  at  the  Salk  Institute.  Dr.  Weissman  is  a  member  of  the  National 
Academy  of  Sciences  and  the  American  Academy  of  Arts  and  Sciences. 


LIKE  all  other  blood  cells,  lymphocytes — the 
principal  players  in  immune  recognition  of 
self  from  nonself — are  derived  ultimately  from 
stem  cells  in  the  bone  marrow.  It  is  both  biologi- 
cally and  clinically  important  to  delineate  the  de- 
cisions these  bone  marrow  precursors  make  as 
they  pass  through  microenvironments  that  define 
the  type  of  lymphocyte  (or  other  blood  cell)  they 
shall  become.  We  have  focused  on  identifying  the 
earliest  cells  in  mouse  and  human  bone  marrow 
that  have  multipotent  capacity,  the  so-called  he- 
matopoietic (blood-forming)  stem  cells. 

Several  years  ago  we  were  able  to  isolate  the 
hematopoietic  stem  cell  of  the  mouse.  This  year 
we  showed  that  no  other  cell  type  in  the  bone 
marrow  has  stem  cell  activity  or  potential.  We 
have  also  demonstrated  its  full  developmental 
potential  by  transferring  a  single  stem  cell  from 
one  mouse  strain  mixed  with  1 00  stem  cells  from 
another  strain  into  lethally  irradiated  mice  of  the 
second  strain.  Progeny  from  the  single  marked 
stem  cell  regularly  gave  rise  to  over  100  million 
blood  cells  of  all  types,  including  hundreds  to 
thousands  of  stem  cells. 

These  thousands  of  stem  cells,  derived  from  the 
initially  injected  single  cell,  could  be  retrieved 
and  transferred  to  a  second  generation  of  irra- 
diated animals,  all  of  whom  were  fully  reconsti- 
tuted. Thus  this  stem  cell  has  a  remarkable  pro- 
file of  activities,  including  that  of  massive 
self-renewal. 

In  the  past  year  we  also  found  that  stem  cells  in 
mouse  fetuses  have  the  capability  of  giving  rise  to 
a  broader  variety  of  T  cells  than  do  stem  cells  in 
the  adult  bone  marrow.*  The  additional  types  of 
T  cells  derived  from  fetal  stem  cells  are  those 
cells  that  move  from  the  fetal  thymus  to  the  skin 
and  other  epithelial  coverings  of  the  body,  pre- 
sumably to  act  as  sentinels  to  protect  against  in- 
coming infectious  microorganisms. 

Most  remarkably,  in  preparation  for  this  added 
capacity  in  fetal  life,  stem  cells  apparently  start  a 
T  cell  developmental  "clock"  on  one  T  cell  re- 
ceptor chromosome.  We  propose  that  after  sev- 
eral stem  cell  divisions,  the  clock  moves  past  that 


part  of  the  chromosome  that  will  be  expressed  as 
receptors  for  antigens  expressed  on  epithelial  T 
cells,  and  concentrates  only  on  other  T  cell  re- 
ceptors to  fight  off  infection  in  other  sites  of  the 
body.  We  believe  that  the  genetic  events  that  set 
developmental  clocks  and  then  shut  them  down 
at  the  level  of  hematopoietic  stem  cells  lie  at  the 
heart  of  understanding  the  determination  of 
choices  that  cells  can  make  in  general,  and  hope 
to  develop  in  the  next  few  years  new  methods  to 
investigate  the  regulators  that  set  the  clock  and 
how  they  do  so  in  the  developmental  history  of 
the  mouse  embryo. 

Several  years  ago  our  laboratory  developed  a 
mouse  model  of  human  organ  function,  wherein 
human  hematopoietic  and  lymphoid  organs,  such 
as  fetal  liver,  thymus,  and  bone  marrow,  could  be 
implanted  in  the  immunodeficient  SCID  mouse.* 
We  found  that  the  human  lymphoid  microenvi- 
ronments implanted  in  the  SCID  mouse  could 
provide  the  right  soil  for  human  T  cell  lymphoma 
growth,  while  the  same  primary  tumors  from  pa- 
tients will  not  grow  in  the  SCID  mouse  in  any 
other  microenvironment.  This  presents  the  op- 
portunity to  study  the  earliest  stages  of  growth 
and  malignant  progression  of  human  cancers, 
lymphomas,  and  leukemias,  if  the  general  princi- 
ple holds  that  their  early  growth  is  dependent  on 
the  organ  in  which  they  find  themselves.  This  im- 
plies that  there  might  exist  factors  within  human 
organs  that  are  responsible  for  the  early  neoplas- 
tic grov^h  of  cancer  cells,  some  of  which  may  be 
tissue  specific. 

In  the  past  year,  we  also  identified  the  genes 
that  encode  a  Peyer's  patch  homing  receptor — 
the  molecule  that  is  involved  in  the  traffic  of  lym- 
phocytes from  the  bloodstream  to  intestinal  lym- 
phoid organs  such  as  Peyer's  patches,  appendix, 
and  mesenteric  lymph  nodes.  The  molecule  is  a 
member  of  the  integrin  family  of  adhesion  pro- 
teins and  uses  the  combination  a^^-j.  Lymphomas 
that  express  a^^^  were  found  to  metastasize  to 


*  This  work  is  supported  by  a  grant  from  the  National  Insti- 
tutes of  Health. 


457 


Following  the  Life  History  of  Lymphocytes 


Peyer's  patches  as  well  as  to  bind  to  the  blood 
vessels  in  the  traffic  zones  of  Peyer's  patches, 
whether  the  a^fi-j  molecules  were  found  to  be  ex- 
pressed on  these  lymphomas  or  were  transfected 
to  them  by  our  cDNA  clones.  Antibodies  to  either 
or  jSy  could  block  this  selective  adhesive  event. 
We  have  also  developed  a  new  mouse  strain  by 
transgenic  technology,  deleting  all  cells  that  ex- 
press the  activated  killer  cell  gene  granzyme  A 
(first  identified  and  cloned  in  this  laboratory)  by 
attaching  to  that  gene  a  "suicide"  gene  obtained 
from  Richard  Palmiter  (HHMI,  University  of 


Washington).  When  killer  cells  are  activated  in 
this  mouse  strain  they  begin  to  express  the  diph- 
theria toxin  A  chain  suicide  protein,  and  die.  Un- 
expectedly, we  have  uncovered  in  this  transgenic 
mouse  strain  a  profound  effect  on  the  life  span  of 
all  CDS  T  cells,  and  not  just  killer  T  cells  and 
natural  killer  cells.  In  the  next  year,  we  plan  to 
utilize  these  mice  to  delineate  the  role  of  killer 
cells  in  normal  and  pathological  immune  reac- 
tions in  vivo  and  to  define  the  mechanism  by 
which  non-killer  CDS  T  cells  are  deleted  after 
their  emigration  from  the  thymus. 


438 


Function  and  Regulation  of  the  Cystic  Fibrosis 
Transmembrane  Conductance  Regulator 


Michael  J.  Welsh,  M.D. — Investigator 

Dr.  Welsh  is  also  Professor  of  Internal  Medicine  and  of  Physiology  and  Biophysics  at  the  University  of  Iowa 
College  of  Medicine,  Iowa  City.  He  earned  his  M.D.  degree  from  the  University  of  Iowa.  He  completed  his 
residency  at  the  University  of  Iowa  College  of  Medicine;  held  clinical  and  research  fellowships  in 
pulmonary  diseases  and  cardiovascular  research  at  the  University  of  California,  San  Francisco;  and  did 
postgraduate  research  in  physiology  and  cell  biology  at  the  University  of  Texas,  Houston.  He  then  returned 
to  the  University  of  Iowa  as  a  faculty  member. 


CYSTIC  fibrosis  (CF)  is  a  common  lethal  ge- 
netic disease  involving  defective  electrolyte 
transport  by  several  epithelia.  In  normal  epithelia 
of  the  airways,  the  intracellular  second  messen- 
ger cAMP  regulates  chloride  (Cl~)  channels  in 
the  apical  membrane.  These  channels  provide 
both  a  pathway  through  which  Cl~  flows  and  a 
key  point  for  regulation  of  its  movement.  When 
cAMP  increases,  the  channels  open  and  Cl~  flows 
from  the  cell  into  the  airway  lumen,  drawing 
water  with  it.  Secretion  of  salt  and  water  is  impor- 
tant in  generating  the  respiratory  tract  fluid,  a  crit- 
ical component  of  the  mucocilliary  defense 
mechanism.  In  CF  airway  epithelia,  cAMP  fails  to 
open  the  Cl~  channels.  As  a  result  secretion  is 
defective  and  the  respiratory  tract  fluid  is  abnor- 
mal. This  defect  is  believed  to  be  the  major  cause 
of  morbidity  and  mortality  in  CF  lung  disease. 

CF  is  caused  by  mutations  in  the  gene  encoding 
the  cystic  fibrosis  transmembrane  conductance 
regulator  (CFTR).  When  we  expressed  the  nor- 
mal CFTR  gene  in  CF  airway  epithelial  cells,  the 
channel  defect  was  corrected.  That  result  indi- 
cated that  CFTR  was  somehow  intimately  asso- 
ciated with  cr  channels,  but  did  not  reveal  its 
function.  Toward  further  understanding,  we  ex- 
pressed CFTR  in  a  number  of  nonepithelial  mam- 
malian cells  lacking  both  endogenous  CFTR  and 
cAMP-activated  CP  channels.  In  every  case  in 
which  CFTR  was  expressed,  the  cell  generated  a 
unique  CI"  channel  that  was  activated  by  cAMP. 
Such  channels  were  not  observed  in  cells  lacking 
the  CFTR  gene. 

The  cAMP-regulated  CP  channels  displayed 
regulatory  and  biophysical  properties  that  were 
identical  to  those  observed  in  cells  expressing  en- 
dogenous CFTR,  as  well  as  those  in  the  apical 
membrane  of  airway  epithelia.  We  stress  apical 
membrane  because  that  is  where  the  CF  defect  is 
observed.  These  results  suggested  that  CFTR  itself 
might  be  a  CP  channel.  This  conclusion  was 
quite  controversial  because  CFTR  did  not  resem- 
ble any  channels  previously  described.  It  seemed 
instead  to  resemble  a  family  of  proteins  that  in- 
clude membrane  pumps. 

To  test  the  hypothesis  that  CFTR  is  a  CP  chan 
nel,  we  changed  specific  amino  acids  within  the 


CFTR  sequence.  When  an  ion  crosses  a  cell  mem- 
brane through  a  channel,  it  must  interact  with  the 
amino  acids  in  that  channel.  In  changing  some  of 
the  positively  charged  amino  acids  to  negatively 
charged  ones,  we  changed  the  anion  selectivity 
sequence.  The  channels  normally  favor  CP  over 
iodide  (P),  but  after  two  of  the  amino  acids  were 
mutated  in  CFTR,  the  channel  favored  P  over 
CP.  The  ability  to  change  the  properties  of  the 
conduction  mechanism  by  altering  specific 
amino  acids  provided  the  most  compelling  evi- 
dence that  CFTR  is  itself  a  cAMP-regulated  CP 
channel. 

The  evidence  that  CFTR  forms  a  CP  channel 
and  that  the  electrolyte  transport  defect  in  CF  is 
in  the  apical  membrane  suggested  that  CFTR 
would  be  located  in  the  apical  membrane  of  se- 
cretory epithelia.  To  test  that  hypothesis,  we  de- 
veloped antibodies  to  CFTR  and  localized  it  with 
immunofluorescence  confocal  microscopy.  In 
several  lines  of  intestinal  epithelial  cells  that  se- 
crete CP,  we  found  that  CFTR  was  located  in  the 
apical  membrane.  That  result  indicates  that  CFTR 
is  in  a  position  where  it  can  directly  mediate  CP 
movement  across  the  membrane. 

An  increase  in  the  cellular  concentration  of 
cAMP  opens  the  CFTR  CP  channel.  Several  of  our 
studies  have  shown  that  CFTR  is  phosphorylated 
and  thus  regulated  by  a  cAMP-dependent  protein 
kinase  (PKA).  (Kinases  regulate  cell  proteins  by 
attaching  a  phosphate  group.)  When  we  used 
cell-free  patches  of  membrane  containing  CFTR, 
we  found  that  addition  of  the  catalytic  subunit  of 
PKA  opened  the  CFTR  CP  channel. 

Moreover,  in  vitro  biochemical  studies  dis- 
closed that  PKA  phosphorylates  CFTR  on  seven 
residues.  In  vivo  studies  further  showed  that  PKA 
phosphorylates  four  serine  residues  located 
within  a  portion  of  the  protein  called  the  R  do- 
main. Evidence  that  these  reactions  are  important 
for  opening  the  channel  came  from  the  observa- 
tion that  PKA  failed  to  open  the  channel  when  the 
four  serines  had  been  changed  to  alanines.  Fur- 
thermore, when  the  R  domain  was  deleted  from 
CFTR,  the  channel  no  longer  required  PKA  to 
open:  it  was  open  even  without  phosphorylation. 

The  CFFR  CP  channel  also  contains  stretches 


439 


Function  and  Regulation  of  the  Cystic  Fibrosis  Transmembrane 
Conductance  Regulator 


of  amino  acids  similar  to  those  found  in  a  number 
of  proteins  that  utilize  ATP.  To  determine  if  the 
CFTR  Cl~  channel  utilizes  ATP,  we  first  phos- 
phory'lated  it  with  PKA  and  then  examined  the 
effect  of  ATP  on  cell-free  membrane  patches  con- 
taining CFTR.  We  found  that  ATP  is  required  for 
the  channel  to  open.  The  channel  appears  to  re- 
quire the  hydrolysis  of  ATP;  nonhydrolyzable  ana- 
logues would  not  open  it.  This  observation  pro- 
vides the  first  example  of  an  ion  channel 
regulated  by  ATP  hydrolysis.  We  were  surprised 
because  an  ion  channel,  by  its  nature,  is  a  passive 
structure.  The  results  suggest  that  the  energy 
from  ATP  hydrolysis  may  be  required  to  open  the 
channel  and  that,  once  open,  Cl~  flows  through 
passively. 

These  studies  have  begun  to  define  the  func- 
tion of  CFTR,  and  in  so  doing,  they  begin  to  ex- 
plain why  the  apical  membrane  of  CF  epithelia 


are  impermeable  to  CP.  They  also  suggest  that 
correction  of  the  underlying  defect  would  be  a 
feasible  therapeutic  strategy. 

In  addition  to  the  studies  of  the  CFTR  Cl~  chan- 
nel, the  laboratory  is  investigating  other  channels 
in  epithelial  cells.  Other  CI"  channels  are  not  as 
well  explored  as  the  CFTR  channel;  but  if  their 
regulation  were  understood,  they  might  possibly 
be  utilized  to  bypass  the  CFTR  CP  channel  de- 
fect. Potassium  channels,  too,  are  important,  be- 
cause they  generate  the  intracellular  voltage  that 
drives  Cl~  out  of  the  cell  across  the  apical  mem- 
brane into  the  airway  lumen.  Finally,  the  labora- 
tory has  a  major  focus  on  the  regulation  of  intra- 
cellular calcium.  The  calcium  ion  is  known  to 
regulate  some  CP  channels  in  the  airway  epithe- 
lial cells,  and  again,  a  better  understanding  of  this 
process  might  open  an  approach  by  which  the  CF 
defect  could  be  circumvented. 


CI 


ATP 


ATP 


Model  showing  the  proposed  domains  of  the  cystic  fibrosis  transmem- 
brane conductance  regulator  ( CFTR).  Membrane  is  cross-hatched  area. 
MSD  refers  to  membrane- spanning  domain,  NBD  to  nucleotide-binding 
domain,  and  PKA  to  cAMP-dependent  protein  kinase.  Mutation  of  the 
CFTR  gene  is  responsible  for  the  defective  function  of  chloride  channels  in 
CF  patients. 

Adapted  from  Anderson,  M.P.,  Berger,  H.A.,  Rich,  D.P.,  Gregory,  R.J., 
Smith,  A.E.,  and  Welsh,  M.f.  1991.  Cell  67:775-784.  Copyright©  1991  by 
Cell  Press. 


440 


Identification  of  the  Gene  Responsible  for 
Adenomatous  Polyposis 


Raymond  L.  White,  Ph.D. — Investigator 

Dr.  White  is  also  Professor  of  Human  Genetics  and  Adjunct  Professor  of  Cellular,  Viral,  and  Molecular 
Biology  at  the  University  of  Utah  School  of  Medicine.  He  received  a  B.S.  degree  in  microbiology  from  the 
University  of  Oregon,  Eugene,  and  a  Ph.D.  degree  in  microbiology  from  the  Massachusetts  Institute  of 
Technology.  He  did  postdoctoral  research  with  David  Hogness  at  Stanford  University.  Dr.  White  has  held 
various  academic  appointments  at  the  University  of  Massachusetts,  Worcester,  and  the  University  of  Utah. 
He  was  recently  named  the  Thomas  D.  Dee  II  Professor  of  Human  Genetics  at  the  University  of  Utah.  Dr. 
White  is  a  corecipient  of  the  National  Health  Council's  National  Medical  Research  Award  and  was  recently 
elected  to  the  National  Academy  of  Sciences. 


FORMATION  of  a  polyp  in  colonic  epithelium 
is  an  early  event  in  the  development  of  colon 
cancer.  In  some  families  a  dominantly  inherited 
mutation  causes  familial  adenomatous  polyposis 
coli  (APC),  a  condition  characterized  by  large 
numbers  of  polyps  and  a  consequently  high  risk 
for  colon  cancer  among  those  who  inherit  the 
mutant  allele.  Because  colon  carcinoma  is  the 
leading  cause  of  cancer  death  in  the  United 
States,  identification  of  the  gene  that  causes  poly- 
posis in  APC  families  has  been  the  goal  of  investi- 
gators seeking  to  understand  cellular  mecha- 
nisms that  can  lead  to  malignancy.  This  goal  was 
achieved  when  the  gene  APC  was  isolated  in 
1991. 

Genetic  linkage  studies,  combined  with  evi- 
dence from  microscopic  analysis  of  altered  chro- 
mosomes, had  localized  the  polyposis-causing 
mutation  in  APC  families  to  a  specific  region  on 
the  long  arm  of  chromosome  5 .  Molecular  tech- 
niques, including  physical  mapping  of  large  DNA 
fragments,  were  brought  into  play  to  define  the 
region  more  clearly,  because  numerous  genes  be- 
sides APC  were  likely  to  reside  in  that  portion  of 
the  chromosome. 

With  molecular  probes  derived  from  the  large 
DNA  fragments,  small  deletions  were  found  in  the 
DNA  of  two  unrelated  patients  with  APC.  These 
deletions  proved  to  be  the  key  to  isolation  of 
APC,  for  they  defined  a  very  small  region  in 
which  to  search.  A  nearby  gene,  MCC,  which  is 
often  mutated  in  sporadic  colonic  tumors,  had 
been  a  candidate;  but  MCC  was  not  interrupted 
by  either  of  the  small  deletions,  and  the  search 
continued. 

Three  other  genes  were  found  that  did  fall 
within  the  100-kilobase  region  deleted  in  both 
patients,  and  each  of  those  was  investigated  for 
the  presence  of  very  small  ("point")  mutations 
in  the  DNA  of  other  persons  with  APC.  One  of 
these  genes  was  subsequently  identified  as  APC 
on  the  basis  of  point  mutations  in  several 
patients. 

Although  each  of  these  APC-specific  genetic  al- 


terations had  occurred  in  a  different  coding  re- 
gion of  the  gene,  all  were  of  a  kind  that  could  be 
expected  to  prevent  the  generation  of  a  normal 
protein  product.  In  one  patient  the  mutation  was 
new  in  his  family.  His  parents'  chromosomes 
carried  normal  copies  of  APC,  but  two  of  his  chil- 
dren had  inherited  his  mutant  allele. 

APC,  like  NFl  (neurofibromatosis  type  1 ) ,  an- 
other tumor-associated  gene  cloned  recently,  ap- 
pears to  fall  into  the  growing  category  of  tumor- 
suppressor  genes.  There  were  no  clues,  however, 
to  its  physiological  function,  as  had  come  to  light 
for  NFl  with  the  discovery  of  similarities  in 
amino  acid  sequence  between  its  predicted  prod- 
uct and  proteins  known  to  participate  in  trans- 
duction of  growth  signals  within  the  cell.  The 
only  structural  motif  that  could  be  identified  was 
the  presence  of  "heptad  repeats,"  or  series  of 
seven  amino  acids  in  tandem  arrays.  Heptad  re- 
peats often  suggest  that  a  protein  can  interact 
with  similar  proteins  to  form  coiled  helical 
structures. 

The  putative  product  of  MCC  also  contains 
heptad  repeats.  It  is  intriguing  to  speculate  upon 
the  possibility  of  interaction  between  this  pro- 
tein and  the  APC  product,  in  view  of  the  fact  that 
alterations  in  both  genes  are  known  to  play  im- 
portant roles  in  the  pathway  to  colon  cancer. 

Some  families  exhibit  an  unusually  high  inci- 
dence of  colon  cancer  without  showing  a  pattern 
of  classical  polyposis;  that  is,  affected  members 
may  develop  very  few  polyps.  Inherited  muta- 
tions in  the  APC  gene  are  being  sought  in  these 
families,  and  colonic  tumors  in  the  general  popu- 
lation are  being  tested  for  mutant  APC  alleles. 
Characterization  of  a  variety  of  APC  mutations 
may  help  to  explain  differences  in  clinical  pro- 
files among  people  with  a  genetic  predisposition 
to  colon  cancer.  It  may  also  lead  to  new  informa- 
tion about  the  role  of  the  gene  in  normal  cellular 
processes. 

For  many  families  with  APC,  discovery  of  the 
gene  has  immediate  value  for  presymptomatic, 
even  prenatal,  diagnosis  of  carrier  status.  When  a 


441 


Identification  of  the  Gene  Responsible  for  Adenomatous  Polyposis 


particular  mutation  has  been  characterized  in  a 
family,  members  who  carry  the  disease  allele  can 
be  identified  directly.  Families  for  whom  the  mu- 
tant allele  is  as  yet  unidentified  may  still  benefit 
from  analysis  of  genetic  linkage  between  the  pu- 
tative disease  allele  and  a  highly  polymorphic 


DNA  marker  system  that  has  been  detected  within 
one  of  the  other  genes  lying  in  the  APC  deletion 
region.  Early  diagnosis  at  the  molecular  level  will 
free  unaffected  members  of  these  families  from 
some  anxiety  and  spare  them  the  discomfort  and 
expense  of  frequent  colonoscopic  examinations. 


442 


Mechanisms  of  the  Biological  Activities  of 
Membrane  Glycoproteins 


Don  C.  Wiley,  Ph.D. — Investigator 

Dr.  Wiley  is  also  Professor  of  Biochemistry  and  Biophysics  at  Harvard  University  and  Research  Associate  in 
Medicine  at  the  Laboratory  of  Molecular  Medicine  at  the  Children's  Hospital,  Boston.  He  received  his  Ph.D. 
degree  in  biophysics  from  Harvard  University.  He  then  joined  the  faculty  at  Harvard  and  served  as 
Assistant  and  Associate  Professor  of  Biochemistry  and  Molecular  Biology  before  attaining  his  present 
position.  Dr.  Wiley  is  Chairman  of  the  Department  of  Biochemistry  and  Molecular  Biology.  He  is  a  member 
of  the  National  Academy  of  Sciences  and  a  Fellow  of  the  American  Academy  of  Arts  and  Sciences.  Among 
his  honors  is  the  Louisa  Gross  Horwitz  Prize  from  Columbia  University. 


Tcell  recognition  occurs  when  cell  surface  his- 
tocompatibility glycoproteins  present  anti- 
gens, processed  to  small  peptides,  to  an  antibody- 
like molecule  on  the  T  cell  receptor.  An  individ- 
ual organism  has  only  a  small  number  of  different 
histocompatibility  molecules  (probably  less  than 
a  dozen),  so  that  each  histocompatibility  gly- 
coprotein must  be  able  to  "present"  many, 
possibly  thousands,  of  different  antigenic  pep- 
tides to  thousands  or  more  distinct  T  cell  re- 
ceptors throughout  the  immunological  life  of 
the  individual. 

In  the  past  year  we  have  been  able  to  visualize 
the  conformation  of  peptides  bound  to  a  histo- 
compatibility glycoprotein,  HIA-B27.  The  ex- 
tended conformation  of  the  peptide  appears  to  be 
specified  by  the  HLA-binding  site,  so  that  the  two 
ends  of  the  peptide  are  bound  to  specialized  re- 
gions at  the  two  ends  of  the  binding  groove.  A  few 
of  the  side  chains  of  a  9-mer  peptide  interact  with 
pockets  in  the  surface  of  the  HLA  molecule, 
whose  size  and  chemical  composition  vary  from 
allele  to  allele  in  the  population.  We  also  eluted, 
sequenced,  and  identified  1 1  self-peptides  from 
HLA-B27.  All  11  had  arginine,  a  positively 
charged  amino  acid,  at  peptide  position  2,  which 
correlates  with  the  x-ray  crystallographic  finding 
that  position  2  fits  into  a  deep  pocket  with  a  nega- 
tively charged  glutamic  acid  at  the  bottom.  (That 
residue  is  polymorphic  and  changes  the  specific- 
ity of  that  pocket  in  other  alleles.) 

We  are  also  now  able  to  reconstitute  class  I  mol- 
ecules from  polypeptide  chains  produced  in 
Escherichia  coli  with  single  peptides  and  have 
crystallized  HLA-A2  with  a  series  of  peptide  anti- 
gens from  influenza  virus  and  HIV-l  (human  im- 
munodeficiency virus  type  1 ) .  One  complex  dif- 
fracts beyond  1 .5-A  resolution  when  the  crystal  is 
frozen  at  -  160°C.  (All  of  the  crystals  in  our  labo- 
ratory are  now  frozen  to  this  temperature  to  pre- 
serve crystallographic  order  and  eliminate  radia- 
tion damage.) 


In  our  studies  of  class  II  histocompatibility  an- 
tigens (a  collaboration  with  Joan  Gorga  and  Jack 
Strominger),  we  now  have  three  crystals  under 
study:  human  DRl,  human  DRl  plus  a  superanti- 
gen,  and  DRl  expressed  in  insect  cells  and  com- 
plexed  with  a  single  influenza  virus  peptide.  The 
complex  of  a  single  peptide  with  a  class  II  mole- 
cule was  generated  by  expressing  a  soluble  class 
II  molecule  in  cells  from  insects,  which  lack  an 
immune  system.  Empty  DRl  molecules  were  pro- 
duced that  rapidly  and  stoichiometrically  bound 
peptide.  The  empty  molecules  were  stabilized 
against  aggregation  and  sodium  dodecyl  sulfate- 
induced  denaturation  by  addition  of  peptide,  ar- 
guing that  peptide  binding  is  accompanied  by  a 
conformational  change. 

Our  laboratory  is  also  studying  how  influenza 
virus  infects  cells.  About  10  years  ago  we  deter- 
mined the  three-dimensional  structure  of  the  in- 
fluenza virus  hemagglutinin  (HA),  the  viral  gly- 
coprotein responsible  for  binding  the  virus  to 
cells  and  for  fusing  the  viral  membrane  to  a  cellu- 
lar membrane  to  effect  infectious  entry.  Recently 
we  determined  the  structure  of  a  series  of  com- 
plexes between  the  HA  and  derivatives  of  sialic 
acid,  the  cellular  receptor  for  influenza  virus.  We 
have  synthesized  a  number  of  these  new  ligands 
and  determined  the  crystal  structure  of  the  com- 
plexes to  confirm  an  atomic  model  for  virus-cell 
binding  that  we  proposed.  In  the  process  a  sec- 
ond binding  site  has  been  located  on  the  HA  at  an 
interface  between  domains  of  the  molecule, 
which,  although  probably  not  physiological,  may 
offer  opportunities  for  the  design  of  a  ligand  to 
stabilize  the  interface  against  the  conformational 
change  required  for  the  HA's  membrane  fusion 
activity. 

A  number  of  other  crystallographic  and  bio- 
chemical studies  are  under  way  on  influenza  C 
virus,  on  a  low-pH  fusion-active  conformation  of 
the  influenza  HA,  on  trypanosome  surface  anti- 
gens, and  on  the  glycoprotein  of  HIV- 1  in  com- 
plex with  its  cellular  receptor,  CD4. 


443 


Studies  of  Blood  Cell  Formation 


David  A.  Williams,  M.D. — Assistant  Investigator 

Dr.  Williams  is  also  Associate  Professor  and  Kipp  Investigator  of  Pediatrics  at  Indiana  University  School  of 
Medicine,  Indianapolis.  It  was  here  that  he  received  his  medical  degree,  after  graduating  in  biology  from 
Indiana  State  University.  His  postdoctoral  training  includes  a  pediatric  residency  at  Children's  Hospital 
Medical  Center,  Cincinnati;  research  fellowships  with  Richard  Mulligan  at  the  Massachusetts  Institute  of 
Technology  Center  for  Cancer  Research  and  the  Whitehead  Institute  for  Biomedical  Research;  and  clinical 
fellowships  in  pediatric  hematology /oncology  at  Harvard  Medical  School,  the  Children 's  Hospital,  and 
'%t,     Dana-Farber  Cancer  Institute,  Boston.  He  was  Assistant  Professor  of  Pediatrics  at  Harvard  Medical  School 
before  assuming  his  present  position. 


THE  goal  of  work  in  our  laboratory  is  to  under- 
stand the  relationship  between  supporting 
cells  in  the  bone  marrow  cavity  and  the  develop- 
ment of  blood  cells.  The  production  of  blood 
cells  from  primitive  "stem  cells"  in  the  marrow 
is  dependent  on  interactions  between  the  latter 
cells  and  certain  supporting  cells.  Understanding 
the  process  of  blood  cell  formation  is  important 
for  treatments  that  utilize  bone  marrow  trans- 
plantation, including  gene  transfer  methods  to 
correct  genetic  diseases  affecting  the  blood- 
forming  system. 

Studies  of  the  Bone  Marrow  Environment 

The  production  of  enormous  numbers  of  blood 
cells  in  the  bone  marrow  environment  and  their 
delivery  to  the  blood  circulation  constitute  a 
highly  regulated  and  complex  process.  Either  ex- 
cessive or  insufficient  production  of  the  primi- 
tive stem  cells  or  the  more  mature  daughter  cells 
is  associated  with  human  diseases.  Little  is 
known,  however,  about  how  this  process  is  regu- 
lated. Our  approach  to  the  problem  has  been  to 
simplify  interactions  between  stem  cells  and  sup- 
porting cells  by  immortalizing  single  supporting 
cells  from  the  bone  marrow  and  studying  these 
interactions  in  detail. 

In  collaboration  with  Vikrum  Patel  at  North- 
western University,  we  have  defined  a  protein 
and  its  receptor  that  appear  to  be  important  in 
anchoring  stem  cells  to  supporting  cells.  This  in- 
teraction can  be  blocked  in  mice  and  in  vitro 
using  antibodies  to  the  receptor.  The  stem  cells 
that  adhere  to  supporting  cells  (stromal  cells)  us- 
ing this  receptor  appear  to  have  a  high  capacity  to 
form  new  stem  and  daughter  cells.  The  adherence 
of  stem  cells  to  supporting  cells  is  important  in 
the  survival  of  stem  cells  both  in  vitro  and  in  the 
bone  marrow  cavity.  A  better  understanding  of 
this  interaction  may  lead  to  new  methods  for 
growing  blood  cells  and  expanding  the  number 
of  stem  cells  in  the  laboratory. 

An  important  protein  in  the  growth  of  blood 
cells  was  recently  identified  in  our  laboratory  as 


"Steel  factor."  This  protein  is  defective  in  a 
mouse  mutant  (Steel  mouse) ,  which  has  a  form  of 
aplastic  anemia,  as  well  as  infertility  and  skin  ab- 
normalities. The  protein  is  normally  made  in  two 
forms  that  differ  with  respect  to  how  it  is  pre- 
sented to  blood  stem  cells  in  the  bone  marrow 
environment.  We  are  investigating  the  role  of 
these  two  different  types  of  Steel  factor 
presentation. 

Deniz  Toksoz,  a  former  HHMI  associate,  has  re- 
cently shown  that  presentation  of  Steel  factor  in  a 
localized  fashion  leads  to  survival  of  immature 
human  blood  cells  (termed  progenitor  cells) 
longer  in  culture  than  when  the  presentation  is 
nonlocalized.  This  observation,  in  combination 
with  the  identification  of  proteins  involved  in 
blood  cell  adhesion  (described  above) ,  may  have 
important  implications  in  developing  a  system 
for  growing  bone  marrow  cells  in  vitro.  Manas 
Majumdar,  an  HHMI  associate,  is  currently  study- 
ing the  role  of  this  protein  on  blood  formation 
during  development  of  the  mouse. 

Studies  on  the  role  of  the  supporting  cells  in 
the  bone  marrow  have  also  led  us  to  the  identifi- 
cation and  production  (by  recombinant  meth- 
ods) of  another  protein  involved  in  blood  forma- 
tion, termed  IL-11  (interleukin- 11 ) .  In  recent 
work  utilizing  IL-11,  Xunxiang  Du  has  demon- 
strated that  it  has  the  capacity  to  help  bone 
marrow  stem  cells  reestablish  blood  formation 
after  intravenous  injection  (i.e.,  bone  marrow 
transplantation)  in  mice.  This  protein  affects  the 
growth  of  several  different  types  of  blood  cells, 
including  platelets  and  neutrophils.  We  are  in- 
vestigating the  use  of  this  growth  factor  to  pre- 
vent some  of  the  severe  complications  of  chemo- 
therapy, such  as  infection  and  bleeding.  This 
work  is  also  supported  by  grants  from  the  Na- 
tional Institutes  of  Health. 

Effects  of  Growth-regulating  Proteins 
During  Blood  Development  in  the 
Early  Embryo 

In  the  developing  mammalian  embryo,  blood 


445 


Studies  of  Blood  Cell  Formation 


formation  begins  in  the  yolk  sac.  Little  is  known 
about  the  interaction  between  supporting  cells  in 
this  environment  and  the  earliest  primitive  stem 
cells,  a  very  few  of  which  are  thought  to  give  rise 
to  all  blood  cells  in  the  adult.  Our  laboratory  has 
derived  cell  lines  from  each  part  of  the  yolk  sac, 
and  Merv  Yoder,  a  visiting  scientist,  is  character- 
izing the  interactions  of  supporting  cells  from  the 
yolk  sac  with  blood  stem  cells  derived  from 
mouse  adult  bone  marrow,  yolk  sac,  and  the  em- 
bryonic liver. 

In  addition,  Pamela  Kooh,  an  HHMI  associate, 
is  examining  the  effects  of  expression  of  various 
growth  proteins  during  fetal  development.  She  is 
utilizing  embryonic  stem  cell  technology  to  ex- 
amine mouse  fetal  blood  development  in  vivo 
and  in  vitro. 

Gene  Transfer  and  Somatic  Gene  Therapy 

A  long-standing  focus  of  research  in  our  labora- 
tory has  been  the  use  of  viral  vehicles,  or  vectors, 
to  introduce  new  genetic  material  into  bone 
marrow  stem  cells.  These  manipulated  cells  can 
then  be  introduced  into  a  recipient  by  bone 
marrow  transplantation,  so  that  the  daughter 
blood  cells  contain  the  new  gene.  In  the  future 
these  methods,  called  somatic  gene  therapy,  may 
provide  a  means  of  curing  severe  genetic 
diseases. 


Our  work  has  utilized  information  we  have 
gained  about  blood  formation  to  improve  the  de- 
livery of  genes  into  mouse  stem  cells.  Barry  Lus- 
key,  an  HHMI  associate  in  our  laboratory,  has 
shown  that  use  of  the  Steel  factor  ensures  that 
100  percent  of  mice  transplanted  with  manipu- 
lated bone  marrow  can  be  made  to  express  hu- 
man protein.  We  hope  to  apply  this  knowledge 
clinically.  Children  with  adenosine  deaminase 
(ADA)  deficiency,  a  genetic  disease  involving 
bone  marrow-derived  cells,  exhibit  severe  com- 
bined immunodeficiency  (SCID) .  With  a  view  to 
treating  this  fatal  condition,  we  are  collaborating 
with  David  Bodine  and  Arthur  Nienhuis  at  the  Na- 
tional Institutes  of  Health  in  testing  the  ap- 
proaches outlined  above  in  monkeys,  but  so  far 
without  success. 

Tom  Moritz,  a  postdoctoral  fellow  in  our  labo- 
ratory, is  collaborating  with  Ronald  Hoffman  at 
Indiana  University  in  investigating  the  transfer  of 
the  ADA  gene  in  human  bone  marrow  cells  that 
have  been  highly  enriched  for  stem  cells.  At  this 
point  it  is  unclear  whether  the  use  of  such  stem 
cell  enrichment  will  improve  the  efficiency  of 
gene  transfer  into  blood  stem  cells.  Gene  transfer 
efficiency  is  a  critical  issue  in  the  successful  ex- 
tension to  humans  of  gene  transfer  methods  devel- 
oped in  mice  by  this  and  other  laboratories. 


446 


Growth  Factor-stimulated  Cell  Proliferation 


Lewis  T.  Williams,  M.D.,  Ph.D. — Investigator 

Dr.  Williams  is  also  Professor  of  Medicine  at  the  University  of  California,  San  Francisco.  He  received  his 
undergraduate  degree  from  Rice  University  and  his  M.D.  and  Ph.D.  degrees  from  Duke  University,  where 
he  studied  with  Robert  Lefkowitz.  He  then  completed  a  clinical  residency  in  internal  medicine  and 
specialty  training  in  cardiology  at  Massachusetts  General  Hospital,  Boston.  Before  joining  the  faculty  at 
UCSF,  he  was  Assistant  Professor  of  Medicine  at  Harvard  Medical  School.  Among  his  honors  is  the 
Outstanding  Young  Investigator  Award  of  the  American  Federation  for  Clinical  Research. 


POLYPEPTIDE  groAvth  factors  regulate  the  pro- 
liferation and  migration  of  cells  in  the  devel- 
oping tissues  of  embryonic  animals.  The  actions 
of  these  factors  appear  to  be  recapitulated  in 
adults  when  damaged  or  senescent  tissues  are  re- 
paired. Our  research  group  is  investigating  the 
action  of  platelet-derived  growth  factor  (PDGF) . 
This  potent  growth  factor  for  fibroblast  and 
smooth  muscle  cells  is  found  in  platelets  and  is 
released  at  sites  of  tissue  injury.  PDGF  is  also  pro- 
duced by  other  tissues,  including  endothelial 
cells  that  line  the  inner  surfaces  of  blood  vessels. 
In  this  context  PDGF  is  likely  to  play  a  major  role 
in  stimulating  the  proliferation  of  smooth  muscle 
cells  that  constitute  atherosclerotic  plaques.  Its 
role  in  vascular  proliferation  appears  to  be  espe- 
cially prominent  in  the  recurrent  blockage  of  cor- 
onary arteries  that  occurs  after  clinical  interven- 
tions such  as  angioplasty  or  atherectomy,  which 
are  undertaken  in  an  attempt  to  restore  blood 
flow  through  vessels  narrowed  by  atherosclero- 
sis. PDGF  also  plays  a  role  in  the  growth  of  some 
tumors.  At  least  one  monkey  sarcoma  is  caused  by 
the  aberrant  production  of  PDGF,  which  stimu- 
lates the  tumor  cells  to  grow  in  an  uncontrolled 
fashion. 

Like  other  growth  factors,  PDGF  acts  on  cells 
by  first  binding  to  specific  receptor  sites  located 
on  the  cell  surface.  This  interaction  of  PDGF  with 
its  receptor  is  transmitted  as  a  signal  across  the 
cell  membrane  and  triggers  a  series  of  complex 
reactions  inside  the  cell  that  culminate  in  DNA 
synthesis  and  cell  division.  To  study  the  mecha- 
nism of  signal  transmission  by  the  PDGF  receptor, 
we  purified  the  receptor  from  mouse  cells, 
cloned  the  gene  that  encodes  the  mouse  receptor 
protein,  expressed  this  receptor  in  cells  that  nor- 
mally lack  PDGF  receptors,  and  demonstrated 
that  this  expressed  receptor  mimics  the  actions  of 
native  PDGF  receptors  and  mediates  all  of  the 
known  responses  to  PDGF. 

The  PDGF  receptor  is  anchored  at  the  surface 
of  the  cell  and  is  oriented  so  that  approximately 
half  of  the  receptor,  the  PDGF-binding  domain,  is 
located  outside  the  cell,  and  the  other  half  of  the 
receptor  is  located  inside  the  cell.  The  receptor 


appears  to  consist  of  several  distant  regions, 
termed  domains,  that  have  distinct  functions.  Us- 
ing the  cloned  gene  for  the  receptor,  we  can  pro- 
duce individual  domains  of  the  receptor  protein 
and  study  the  functions  of  these  domains.  For  ex- 
ample, we  have  produced  receptor  fragments 
that  contain  the  PDGF-binding  domain  but  lack 
the  other  portions  of  the  molecule.  To  localize 
more  precisely  the  portion  of  the  receptor  that  is 
essential  for  binding  PDGF,  we  are  now  prepar- 
ing even  smaller  versions  of  the  binding  domain 
by  deleting  portions  of  the  molecule.  When  a 
minimal  domain  for  binding  is  defined,  we  will 
study  the  three-dimensional  structure  of  this  sim- 
plified molecule  and  use  this  information  to  de- 
sign agents  that  can  block  the  binding  of  PDGF  to 
its  receptor.  These  agents  should  function  as 
blockers  of  the  actions  of  PDGF  and  will  facilitate 
the  study  of  the  role  of  PDGF  in  atherosclerosis 
and  cancer. 

One  of  the  major  problems  in  growth  factor 
research  has  been  to  determine  how  the  portion 
of  the  receptor  inside  the  cell  senses  that  the  bind- 
ing domain  on  the  outside  of  the  cell  has  inter- 
acted with  PDGF.  We  have  recently  found  that 
the  transmission  of  the  signal  from  the  outside 
domain  to  the  inside  of  the  cell  involves  two  ma- 
jor steps.  First,  when  a  receptor  molecule  binds 
PDGF,  the  receptor  pairs  with  another  identical 
receptor  molecule  to  form  a  receptor  dimer.  Each 
of  the  two  receptor  molecules  in  the  dimeric 
complex  phosphorylates  its  partner,  thereby 
modifying  the  partner.  The  phosphorylation  reac- 
tion results  in  the  addition  of  a  phosphate  group 
to  the  partner  and  is  accomplished  by  a  region  of 
the  receptor  termed  the  kinase  domain.  We  have 
designed  and  produced  mutant  receptors  that 
have  normal  PDGF-binding  domains  but  have  de- 
fective kinase  domains.  When  these  mutants  are 
introduced  into  cells  that  have  normal  receptors, 
a  dimer  (heterodimer)  is  formed  between  the 
normal  and  kinase-defective  receptors.  The  nor- 
mal and  mutant  receptors  in  the  heterodimer 
complex  cannot  phosphorylate  each  other  and 
cannot  transmit  the  signals  required  to  initiate 
cell  growth.  These  experiments  prove  that  forma- 
tion of  a  dimer  consisting  of  two  normal  recep- 


447 


Growth  Factor-stimulated  Cell  Proliferation 


tors  is  required  for  proper  functioning  and  signal 
transduction  of  the  receptor.  By  introducing  mu- 
tant receptors  into  specific  tissues  of  animals  we 
hope  to  be  able  to  block  the  function  of  the  nor- 
mal receptors  and  assess  the  role  of  PDGF  in  phys- 
iological processes  and  in  disease  states. 

When  the  dimerized  receptor  is  phosphory- 
lated,  the  second  major  step  in  signal  transduc- 
tion occurs.  The  phosphorylated  receptor  physi- 
cally binds  to  signaling  molecules  that  are  inside 
the  cell.  We  have  recently  found  that  the  interac- 
tion between  the  receptor  and  signaling  mole- 
cules occurs  at  the  phosphorylation  sites  on  the 
receptor.  Using  information  about  the  structures 
of  these  sites,  we  are  designing  ways  to  disrupt 
the  interaction  between  the  receptors  and  the  sig- 
naling molecules.  We  are  also  using  the  receptors 
as  a  tool  for  discovering  previously  unidentified 
signaling  molecules  that  mediate  that  prolifera- 
tive response  to  PDGF.  Recently  we  have  identi- 
fied one  of  these  molecules,  phosphatidylinositol 
3-kinase  (PI  3-kinase),  that  appears  to  play  an  im- 
portant role  in  PDGF-stimulated  proliferation. 
Other  investigators  have  found  that  PI  3-kinase  is 
also  important  in  the  cell  transformation  caused 
by  some  viruses  that  cause  cancer  in  animals.  Us- 
ing the  receptor  as  a  probe,  we  recently  purified 
the  PI  3-kinase  and  cloned  the  gene  for  this  signal- 
ing molecule.  We  hope  that  by  studying  the  inter- 
action of  the  receptor  and  PI  3-kinase  we  can  un- 
derstand an  important  set  of  reactions  that  are 
involved  in  regulating  cell  growth. 


We  recently  have  begun  to  study  the  fibroblast 
grovvT:h  factors  (FGF).  These  factors  appear  to 
play  important  roles  in  the  earliest  stages  of  em- 
bryogenesis  and  in  angiogenesis  (the  formation 
of  new  blood  vessels) .  The  development  of  new 
vessels  can  be  beneficial  (e.g.,  in  offsetting  ath- 
erosclerotic narrowing  of  blood  vessels  in  the 
heart)  or  deleterious  (e.g.,  in  the  formation  of 
new  vessels  that  supply  nutrients  to  tumors  or  in 
the  vascular  proliferation  that  occurs  in  the  eyes 
of  diabetic  patients) .  We  have  identified  some  of 
the  FGF  receptors  and  are  now  examining  their 
mechanisms  of  action.  This  work  on  FGF  recep- 
tors is  supported  by  a  grant  from  the  National  In- 
stitutes of  Health. 

The  long-range  goal  of  these  studies  is  to  probe 
the  role  of  grov^h  factors  in  normal  embryonic 
development,  in  tissue  repair,  and  in  prolifera- 
tive diseases.  Using  the  tools  of  molecular  biol- 
ogy, cell  biology,  and  protein  chemistry,  we  and 
other  research  groups  are  identifying  the  factors, 
receptors,  and  regulatory  molecules  involved  in 
these  processes.  Studies  of  the  spatial  and  tem- 
poral distribution  of  the  growth  factors  and  re- 
ceptors in  normal  and  diseased  tissues  will  pro- 
vide insight  into  the  function  of  these  molecules. 
By  understanding  the  molecular  details  of  the 
protein-protein  interactions  involved  in  growth 
factor  action,  it  may  be  possible  to  devise  new 
therapeutic  strategies  to  treat  proliferative 
diseases. 


448 


Somatic  Cell  Gene  Transfer 


James  M.  Wilson,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Wilson  is  also  Associate  Professor  of  Internal  Medicine  and  Biological  Chemistry  at  the  University  of 
Michigan  Medical  School.  He  received  his  undergraduate  degree  in  chemistry  from  Albion  College  and  his 
Ph.D.  (biological  chemistry)  and  M.D.  degrees  from  the  University  of  Michigan.  He  completed  a  residency 
and  clinical  fellowship  at  Massachusetts  General  Hospital,  Boston,  and  conducted  postdoctoral  research 
with  Richard  Mulligan  at  the  Whitehead  Institute,  Massachusetts  Institute  of  Technology.  Dr.  Wilson  is 
investigating  ways  to  treat  genetic  diseases  by  correcting  the  basic  defects. 


GENE  replacement  therapies  are  being  consid- 
ered for  the  treatment  of  a  variety  of  ac- 
quired and  inherited  diseases.  These  novel  thera- 
peutic modalities  involve  the  transfer  of  genetic 
material  into  somatic  tissues  of  affected  individ- 
uals. The  development  of  new  therapies  for  car- 
diopulmonary diseases,  based  on  gene  transfer 
into  hepatocytes  and  airway  epithelial  cells,  is  a 
major  focus  of  my  laboratory. 

Familial  hypercholesterolemia  (FH)  is  an  auto- 
somal dominant  disorder  in  humans  that  is  an  ex- 
cellent model  for  developing  gene  replacement 
therapies  of  hyperlipidemic  states.  Patients  with 
the  homozygous  deficient  form  of  FH  have  severe 
hypercholesterolemia  and  suffer  life-threatening 
coronary  artery  disease  in  childhood  that  is  refrac- 
tory to  conventional  medical  therapies.  The  mo- 
lecular basis  of  this  disorder  is  a  systemic  defi- 
ciency in  the  receptor  that  degrades  low-density 
lipoprotein  (LDL) ,  the  primary  carrier  of  choles- 
terol in  the  blood.  Hepatocytes  are  the  appro- 
priate target  for  gene  transfer  in  this  disease, 
since  the  liver  is  the  organ  primarily  responsible 
for  LDL  metabolism  and  cholesterol  excretion. 

We  have  used  an  animal  model  for  FH — the 
Watanabe  heritable  hyperlipidemic  (WHHL)  rab- 
bit— to  develop  different  types  of  liver-directed 
gene  replacement  therapies.  One  approach  is  sim- 
ilar in  concept  to  the  well-described  bone 
marrow-directed  gene  therapies.  This  ex  vivo 
method  involves  isolating  hepatocytes  from  a  ge- 
netically deficient  animal,  transferring  functional 
genetic  material  into  the  hepatocytes  in  vitro, 
and  returning  the  corrected  cells  to  the  affected 
recipient.  We  have  used  recombinant  retrovi- 
ruses to  transfer  a  functional  gene  for  the  human 
LDL  receptor  stably  into  hepatocytes  derived 
from  WHHL  rabbits.  Transplantation  of  these 
cells  into  WHHL  rabbits  leads  to  substantial  de- 
creases in  serum  cholesterol  for  over  four 
months.  A  similar  approach  may  be  therapeutic  in 
patients  with  homozygous  FH. 

An  alternative  and  less  invasive  approach  is  to 
deliver  a  functional  LDL  receptor  gene  into  the 
hepatocytes  in  vivo.  We  have  developed  an  in 
vivo  gene  delivery  system  that  is  based  on  inter- 


actions with  hepatocyte-specific  receptors.  Using 
this  approach  we  can  deliver  reporter  genes  spe- 
cifically to  hepatocytes  in  vivo  and  obtain  ex- 
pression of  the  recombinant  gene  in  liver  for  at 
least  four  months.  Administration  of  a  gene 
transfer  substrate  containing  the  LDL  receptor 
gene  into  the  circulation  of  WHHL  rabbits  led  to 
significant  reductions  in  the  level  of  serum  cho- 
lesterol. Hepatic  overexpression  of  LDL  receptor 
by  gene  transfer  can  potentially  prevent  the  ath- 
erosclerotic disease  in  FH  and  other  hyperlipid- 
emic states.  This  work  is  also  supported  by  a  grant 
from  the  National  Institutes  of  Health. 

Cystic  fibrosis  (CF)  is  an  inherited  disease 
characterized  by  abnormal  salt  and  water  trans- 
port that  leads  to  pathology  within  the  pancreas 
and  lung.  CF  is  the  most  common  lethal  congeni- 
tal disease  among  Caucasians,  with  a  prevalence 
of  1  in  2,000  births.  The  primary  defect  in  the 
lung  appears  to  be  the  production  of  thick  abnor- 
mal mucus  that  plugs  the  airways  and  leads  to 
infections.  The  recent  discovery  of  the  gene  that 
causes  CF  suggests  a  new  therapeutic  strategy  in 
which  normal  functioning  CF  genes  are  directly 
introduced  into  pancreatic  or  respiratory  cells  of 
affected  patients. 

As  a  first  step  in  the  development  of  a  genetic 
cure  for  CF,  we  have  attempted  to  correct  the 
physiological  abnormality  in  cells  from  a  CF  pa- 
tient by  introducing  into  them  a  CF  gene  that 
encodes  CFTR  (cystic  fibrosis  transmembrane  reg- 
ulator), a  normal  functioning  product.  Replica- 
tion-defective viruses  were  used  to  shuttle  a  nor- 
mal CF  gene  into  pancreatic  cells  from  a  patient 
with  CF.  Prior  to  gene  transfer,  the  cells  mani- 
fested the  typical  abnormalities  associated  with 
this  disease  (i.e.,  decreased  transport  of  salt 
across  the  membrane).  Following  gene  transfer, 
the  cells  regained  normal  regulation  of  salt  trans- 
port. This  demonstrates  the  feasibility  of  gene 
therapy  in  CF  at  a  cellular  level. 

Rational  development  of  approaches  for  recon- 
stituting CFTR  expression  in  vivo  requires  a  defi- 
nition of  endogenous  CFTR  expression  in  the 
normal  human  airway  as  well  as  an  understanding 
of  the  biology  of  the  epithelial  cells  that  line  the 


449 


Somatic  Cell  Gene  Transfer 


airway.  Using  antibody  and  RNA  probes,  we  have 
characterized  the  distribution  of  CFTR  expres- 
sion in  the  human  bronchus.  The  airway  epithe- 
lial cells  express  low  levels  of  CFTR.  The  predom- 
inant site  of  expression,  however,  is  in  the 
submucosal  glands  that  are  responsible  for  the 
production  and  delivery  of  mucus  to  the  airway 
lumen.  These  findings  suggest  that  effective  gene 
therapies  may  require  genetic  reconstitution  in 
both  the  epithelium  and  the  submucosal  glands. 


This  work  is  also  supported  by  the  Cystic  Fibrosis 
Foundation. 

We  have  entered  a  new  phase  of  the  develop- 
ment of  CF  gene  therapies  in  which  approaches 
for  delivering  functional  CF  genes  into  airway 
cells  in  vivo  are  being  designed  and  tested.  Di- 
rect inhalation  of  the  gene  transfer  substrates 
could  provide  a  noninvasive  way  of  delivering 
the  genes  to  the  appropriate  cells.  Several  viral 
and  nonviral  approaches  are  being  considered. 


450 


Model  for  gene  therapy  of  the  cystic  fibrosis  airway:  human  bronchial 
xenograft  reconstituted  with  cells  from  a  CF  patient  following  retro- 
viral infection  with  the  ^-galactosidase  reporter  gene.  Darkfield photo- 
micrograph shows  blue  cells  following  a  histochemical  stain  for 
0-galactosidase. 

Research  and  photograph  by  fohn  Engelhardt  in  the  laboratory  of 
fames  Wilson. 


Differentiation  of  human  myeloid  leukemia  cells  to  eosinophils  in  the  bone  marrow  of  a  mouse 
with  severe  combined  immunodeficiency  disease  (SCID). 

From  Sawyers,  C.L.,  Gishizky,  M.L.,  Quan,  S.,  Golde,  D.W.,  and  Witte,  O.N.  1992.  Blood 
79:2089-2098. 


452 


Normal  and  Abnormal  Lymphocyte 
Growth  Regulation 


Owen  N.  Witte,  M.D. — Investigator 

Dr.  Witte  is  also  Professor  of  Microbiology  and  Molecular  Genetics  at  the  University  of  California,  Los 
Angeles,  holding  the  President's  Chair  in  Developmental  Immunology.  He  received  his  B.S.  degree  in 
microbiology  from  Cornell  University  and  his  M.D.  degree  from  Stanford  University,  where  he  trained  with 
Irving  Weissman  in  the  Medical  Scientist  Training  Program.  Dr.  Witte  completed  postdoctoral  training 
with  David  Baltimore  at  the  Massachusetts  Institute  of  Technology  before  joining  the  UCLA  faculty.  His 
honors  include  the  Milken  Family  Medical  Foundation  Award  in  Basic  Cancer  Research  and  the  Rosenthal 
Foundation  Award  of  the  American  Association  for  Cancer  Research. 


OUR  ability  to  resist  a  wide  range  of  infectious 
agents  depends  on  the  normal  function  of 
the  immune  system.  The  humoral  portion  of  this 
system  is  responsible  for  the  production  of  spe- 
cific antibody  molecules  from  B  lymphocytes. 
Too  low  a  growth  rate  can  result  in  immunodefi- 
ciency; too  high  a  growth  rate,  in  various  types  of 
leukemia  or  lymphoma.  Our  laboratory  has 
concentrated  on  defining  the  growth  control 
mechanisms  that  regulate  the  production  of  B 
lymphocytes. 

The  ABL  Oncogene  in  Murine  and 
Human  Leukemias 

The  ABL  oncogene  was  first  isolated  as  the  ac- 
tive genetic  element  of  the  Abelson  murine  leu- 
kemia virus.  This  agent  is  capable  of  causing  a 
wide  range  of  leukemias  in  mice.  The  biological 
properties  of  the  ABL  gene  product  depend  on  its 
activity  as  a  tyrosine-specific  kinase. 

The  human  homologue  of  the  ABL  gene  has 
now  been  strongly  implicated  in  the  pathogene- 
sis of  a  family  of  human  leukemias  that  harbor  a 
specific  cytogenetic  abnormality.  This  is  a  chro- 
mosome translocation  that  uses  mRNA  splicing  to 
join  part  of  the  BCR  gene  (of  unknown  function) 
from  chromosome  22  to  part  of  the  ABL  gene 
from  chromosome  9,  forming  the  so-called  Phila- 
delphia chromosome,  or  Ph  1 .  The  tyrosine  kinase 
activity  of  the  chimeric  BCR/ABL  gene  product 
is  evoked  and  strongly  correlates  with  the  trans- 
formation activity  of  the  protein. 

Two  different  forms  of  BCR/ABL  protein  can 
occur,  depending  on  the  precise  position  of  the 
chromosomal  breakpoints.  In  human  chronic  my- 
elogenous leukemia,  a  larger  protein  product 
called  P210  BCR/ABL  is  produced,  and  in  the 
case  of  Phi -positive  acute  lymphocytic  leuke- 
mia, a  PI 85  BCR/ABL  protein  product  is  com- 
monly found.  We  have  undertaken  to  determine 
the  precise  contribution  of  BCR  sequences  to  the 
tyrosine  kinase  activity  of  the  ABL  segment  and  to 
the  malignant  potential  of  the  gene  product. 

A  variety  of  studies  have  now  documented  the 
specific  role  of  BCR  in  the  activation  of  the  ABL 


tyrosine  kinase.  Site-directed  mutagenesis  has  es- 
tablished that  the  BCR  segment  is  essential  for 
transformation  by  the  chimeric  oncogene  and 
acts  through  the  tyrosine  kinase  domain  of  ABL. 
We  have  recently  described  a  new  class  of  pro- 
tein-protein interactions  that  regulate  this  activa- 
tion. Within  the  first  exon  segment  of  BCR,  there 
are  two  strong  SH2  (SRC  homologous  domain  2) 
binding  regions  that  can  tightly  bind  to  ABL.  All 
previous  SH2  interactions  have  been  mediated  by 
proteins  containing  phosphotyrosine.  Interest- 
ingly, the  BCR  protein  requires  phosphoserine 
and  phosphothreonine  for  strong  binding,  but 
not  phosphotyrosine.  BCR  represents  a  new  class 
of  protein-protein  interaction  domains  important 
in  intracellular  signaling. 

Further  analysis  of  BCR  has  shown  that  it  also 
represents  a  new  class  of  protein  kinases.  The  first 
exon  region  of  BCR  contains  a  serine/threonine 
kinase  activity  that  can  be  distinguished  from  the 
traditional  protein  kinase  family  by  several  crite- 
ria, including  its  containment  of  catalytic  activity 
within  a  single  exon,  its  reactivity  with  nucleo- 
tide analogues,  and  the  role  of  essential  cysteines 
within  the  nucleotide-binding  cleft.  Work  from 
other  laboratories  shows  that  BCR  also  has  a 
GTPase-activating  protein  domain  at  its  carboxyl 
terminus  that  regulates  the  action  of  small  gua- 
nine nucleotide-binding  proteins.  BCR  appears 
to  be  a  multifunctional  protein.  Its  normal  func- 
tion remains  to  be  determined. 

The  normal  ABL  oncogene  products  are  ex- 
pressed in  many  cell  types,  but  their  role  in  mam- 
malian cell  physiology  is  unknown.  Gross  struc- 
tural changes  can  activate  their  oncogenic 
potential.  It  has  been  difficult  to  identify  more- 
subtle  mutations  that  might  activate  ABL,  be- 
cause the  normal  gene  is  toxic  to  most  cell  types 
when  highly  expressed.  The  precise  mechanism 
of  toxicity  is  not  established,  but  probably  relates 
to  a  cell  cycle-blocking  effect. 

Full-length  cDNA  copies  of  the  cellular  ABL 
genes  are  cloned  into  a  retroviral  vector  that  has 
been  modified  to  allow  amplification  in  an  acute 
transfection  system.  Retroviral  particles  are  pro- 


453 


Normal  and  Abnormal  Lymphocyte  Growth  Regulation 


duced  that  can  transmit  the  cellular  ABL  gene  at 
high  efficiency  to  a  wide  variety  of  cell  types. 
Using  this  system,  we  have  been  able  to  select  for 
transformed  clones  that  harbor  new  classes  of  ac- 
tivating mutations  in  ABL. 

One  new  class  of  mutants  shows  the  loss  of  se- 
quences downstream  of  the  kinase  domain.  These 
narturally  selected  deletion  mutants  and  molecu- 
lar constructs  created  to  mimic  their  structure  are 
weak  transforming  alleles  that  require  a  comple- 
menting oncogene  to  induce  fibroblast  cells  to 
grow  in  agar.  Their  most  striking  phenotypic 
characteristic  is  the  very  low  activation  of  the  ty- 
rosine kinase  activity  of  ABL.  This  is  very  different 
than  all  previous  transformation  alleles  of  ABL. 

It  has  previously  been  shown  that  all  forms  of 
the  ABL  oncogene  can  synergize  for  their  biologi- 
cal effects  with  members  of  the  MYC  gene  family. 
We  have  recently  tested  the  hypothesis  that  MYC 
is  not  only  complementary  to  ABL  transformation, 
but  essential.  Using  a  series  of  dominant  negative 
mutants  to  inactivate  the  effective  dosage  of  MYC 
in  both  fibroblast  and  hematopoietic  cells,  we 
have  observed  a  dramatic  decrease  in  the  ability 
of  BCR/ABL  and  viral  forms  of  ABL  to  transform. 
The  dominant  negative  mutants  have  no  suppres- 
sive effect  on  other  types  of  oncogenes,  including 
MOS.  Such  essential  combinations  could  be  im- 
portant considerations  in  devising  strategies  for 
the  treatment  of  certain  cancers. 

Regulation  of  Stem  and  Progenitor 
Cell  Growth 

In  collaboration  with  Naomi  Rosenberg  of 
Tufts  Medical  School,  we  have  developed  a  sys- 
tem for  retroviral  infection  of  murine  bone 
marrow  stem  cells  with  the  BCR/ABL  oncogene 
and  reimplantation  into  syngeneic  hosts.  This 
procedure  leads  to  tumors  with  the  characteris- 
tics of  human  chronic  myelogenous  leukemia.  In- 
terestingly, animals  infected  with  the  PI  85  BCR/ 
ABL  forms  show  more-aggressive  tumors  that 
invade  nonhematopoietic  organs  and  show 
shorter  latency  than  animals  infected  with  the 
P210  form.  This  system  should  be  valuable  for 
analyzing  new  therapies  directed  at  the  BCR/ABL 
oncogene  and  for  defining  the  grov^h  regulation 
of  primitive  hematopoietic  stem  cells. 

To  elucidate  the  mechanisms  that  lead  to  dereg- 
ulated growth  of  stem  cells,  we  have  developed 
an  in  vitro  system  to  monitor  the  effects  of  BCR/ 
ABL   on   multipotential   stem   cells.  Marrow 


enriched  for  such  cells  was  infected  with  P2 1 0 
BCR/ABL  expressing  retrovirus  and  plated  in  agar 
in  very  low  concentrations  of  growth  factors  like 
Steel  factor  and  interleukin-3  (IL-3).  These  low 
concentrations  are  incapable  of  stimulating  col- 
ony growth  but  could  synergize  with  BCR/ABL. 

A  variety  of  colony  types  grew  in  this  assay  sys- 
tem, including  multipotential  cells  capable  of 
differentiating  to  form  mast  cells,  macrophages, 
granulocytes,  and  pre-B  cells.  Cell  lines  of  these 
different  sublineages  established  from  such  mul- 
tipotent  colonies  express  a  high  level  of  the 
BCR/ABL  gene  product  but  are  only  subtly  trans- 
formed in  their  growth  factor  requirements  and 
nonmalignant  when  transferred  to  syngeneic 
animals. 

Reliable  test  models  for  the  growth  of  human 
myeloid  leukemias  are  limited  to  a  small  subset  of 
these  leukemias  that  can  be  grown  into  continu- 
ous cell  lines  in  vitro.  We  have  recently  estab- 
lished a  reproducible  procedure  for  the  growth 
of  freshly  explanted  human  acute  myelogenous 
leukemia  and  blast  crisis  specimens  from  chronic 
myelogenous  leukemia  patients.  Cells  from  the 
bone  marrow  of  such  patients  are  implanted 
under  the  kidney  capsule  of  the  severe  combined 
immunodeficient  (SCID)  mouse.  At  this  site  the 
human  cells  grow  and  then  migrate  to  the  bone 
marrow  and  peripheral  blood  in  a  pattern  quite 
similar  to  that  of  the  original  human  disease. 

One  long-range  goal  of  our  group  is  to  develop 
effective  in  vitro  culture  techniques  for  the  prop- 
agation and  enrichment  of  stem  and  progenitor 
cells  for  different  lineages.  Previously  we  used 
the  growth  stimulatory  properties  of  the  BCR/ 
ABL  oncogene  in  concert  with  selected  bone 
marrow-adherent  stromal  lines  to  grow  clonal 
lines  of  B  lymphoid  progenitor  cells  that  could 
repopulate  the  B  cell  lineage  of  immunodefec- 
tive  (SCID)  mice.  By  modifying  the  culture  con- 
ditions, we  have  now  been  able  to  cultivate  such 
progenitor  cells  without  the  need  for  co-stimula- 
tion by  the  oncogene.  These  populations  are  very 
effective  in  reconstituting  the  B  cell  lineage  in 
vivo. 

We  have  used  these  progenitor  cells  to  prepare 
several  cDNA  libraries  in  order  to  search  for  new 
members  of  the  tyrosine  kinase  gene  family  and 
other  regulatory  genes  that  may  be  important  in  B 
cell  development.  A  new  member  of  the  SRC  fam- 
ily of  tyrosine  kinases  has  been  identified  that  is 
specific  to  the  B  cell  lineage. 


454 


Translational  Regulation 


Sandra  L.  Wolin,  M.D.,  Ph.D. — Assistant  Investigator 

Dr.  Wolin  is  also  Assistant  Professor  of  Cell  Biology  at  Yale  University  School  of  Medicine.  She  received  her 
undergraduate  degree  in  biochemistry  from  Princeton  University  and  her  M.D.  and  Ph.D.  degrees  from 
Yale  University,  where  she  worked  with  Joan  Steitz.  Her  postdoctoral  research  was  done  with  Marc 
Kirschner  and  Peter  Walter  at  the  University  of  California,  San  Francisco,  where  she  was  a  fellow  of  the 
Helen  Hay  Whitney  Foundation  and  a  Lucille  P.  Markey  Scholar. 


MY  laboratory  is  particularly  interested  in  the 
mechanisms  that  regulate  the  translation  of 
messenger  RNA  (mRNA)  into  proteins.  The  infor- 
mation in  mRNA  is  translated  by  a  large  RNA- 
protein  complex,  the  ribosome.  It  has  been 
known  for  some  time  that  the  ribosomes  do  not 
move  along  the  mRNA  at  an  even  pace — that  they 
pause  at  discrete  places  along  the  way.  Why  ribo- 
somes pause  is  not  well  understood  but  is 
thought  to  be  due  either  to  rare  codons  or  to  sec- 
ondary mRNA  structures. 

In  certain  mRNAs  the  pausing  of  ribosomes  is 
thought  to  cause  them  to  slip  and  lose  their  place, 
such  that  a  different  protein  is  now  translated. 
This  slipping  is  known  as  ribosome  frameshifting. 
We  have  been  investigating  why  ribosomes  pause 
during  translation,  and  we  would  like  to  under- 
stand how  cells  can  use  ribosome  pausing  to  regu- 
late the  synthesis  of  particular  proteins. 

To  address  these  questions,  we  use  a  method 
that  allows  us  to  determine  the  distribution  of 
ribosomes  on  an  mRNA  with  single-nucleotide 
precision.  In  this  way  we  can  obtain  a  detailed 
picture  of  the  translation  process.  Using  this  as- 
say, we  have  found  that  the  ribosomes  often 
pause  directly  over  the  initiation  codon  of  the 
mRNA.  This  pausing  by  fully  assembled  ribo- 
somes appears  to  represent  a  slow  step  in  eukar- 
yotic  protein  initiation  that  had  not  been  previ- 
ously detected. 

By  performing  translation  reactions  in  the  pres- 
ence of  inhibitors  that  block  distinct  steps  in  poly- 
peptide initiation  and  then  examining  the  posi- 
tion of  the  resulting  paused  ribosomes,  we  have 
narrowed  down  this  major  slow  step  to  one  of  two 
points  in  the  initiation  pathway.  We  are  continu- 
ing to  characterize  this  intermediate  in  transla- 
tion initiation  and  are  also  investigating  the  possi- 
bility that  this  slow  step  in  protein  synthesis  may 
be  a  point  at  which  cells  regulate  translation  of 
particular  mRNAs. 

We  are  also  interested  in  understanding  how 
the  availability  of  particular  tRNA  molecules  (in- 
termediates in  protein  synthesis)  affects  ribo- 
some pausing  during  elongation  of  the  nascent 
protein.  To  study  this  question,  we  have  prepared 


translation  extracts  in  which  the  tRNA  molecules 
have  been  removed.  By  adding  back  different 
tRNA  populations,  we  can  now  manipulate  the 
levels  of  individual  tRNAs.  In  this  way  we  hope  to 
determine  how  different  tRNA  species  contribute 
to  ribosome  pausing  and  frameshifting. 

We  are  also  studying  how  the  attachment  of 
ribosomes  to  the  endoplasmic  reticulum  mem- 
brane affects  their  movement  along  mRNAs  en- 
coding secreted  proteins.  The  synthesis  of  these 
proteins  designed  for  export  outside  the  cell  be- 
gins when  ribosomes,  free  in  the  cytoplasm,  initi- 
ate translation  on  the  mRNAs.  A  common  feature 
of  these  secretory  proteins  is  the  presence  of  a 
signal  peptide,  usually  an  amino-terminal  exten- 
sion of  15-30  amino  acids.  A  signal-recognition 
particle  (SRP),  a  small  cytoplasmic  ribonucleo- 
protein,  binds  to  the  signal  sequence  emerging 
from  the  ribosome  and  arrests  elongation  tran- 
siently. This  translational  arrest  is  released  after 
the  ribosome-SRP  complex  interacts  with  a  spe- 
cific component  of  the  endoplasmic  reticulum 
membrane,  the  SRP  receptor. 

Concomitant  with  the  resumption  of  protein 
synthesis,  translocation  of  the  nascent  polypep- 
tide begins.  All  subsequent  translation  is  carried 
out  by  ribosomes  that  are  attached  to  the  endo- 
plasmic reticulum  membrane. 

We  have  found  that  the  point  at  which  mem- 
brane insertion  of  the  nascent  polypeptide  begins 
is  distinct  for  different  proteins,  which  may  re- 
flect differences  in  the  size  and  structures  of  indi- 
vidual signal  peptides.  It  also  appears  that  certain 
features  of  mRNA  structure  that  cause  ribosomes 
to  pause  during  translation  in  solution  do  not  al- 
ways result  in  their  pausing  when  attached  to 
microsomal  membranes.  Thus  it  appears  likely 
that  mRNA  secondary  structure  can  be  altered 
by  attachment  through  ribosomes  to  these 
membranes. 

We  are  currently  determining  whether  ribo- 
somes, following  termination,  are  able  to  reini- 
tiate translation  on  microsomal  membranes.  If  so, 
it  would  provide  a  molecular  explanation  for  the 
"circular  polysomes"  that  have  long  been  ob- 
served attached  to  the  endoplasmic  reticulum. 


455 


Molecular  Genetics  and  Studies  Toward  Gene 
Therapy  for  Metabolic  Disorders 


Savio  L.  C.  Woo,  Ph.D. — Investigator 

Dr.  Woo  is  also  Professor  in  the  Department  of  Cell  Biology  and  Institute  for  Molecular  Genetics  at  Baylor 
College  of  Medicine.  He  obtained  his  undergraduate  degree  in  chemistry  from  Loyola  College,  Montreal, 
and  his  Ph.D.  degree  in  biochemistry  from  the  University  of  Washington,  where  he  worked  with  Earl  Davie. 
His  postdoctoral  research  was  done  at  the  University  of  British  Columbia,  Vancouver,  in  the  Division  of 
Neurological  Sciences. 


A major  focus  in  my  laboratory  has  been  the 
analysis  of  human  metabolic  disorders  at  the 
molecular  level.  Phenylketonuria  (PKU),  the  dis- 
ease under  investigation,  is  an  inborn  error  in 
amino  acid  metabolism  that  causes  severe  and 
permanent  mental  retardation  in  untreated  chil- 
dren. The  condition  is  caused  by  defects  in  the 
liver  enzyme  phenylalanine  hydroxylase  (PAH) 
and  is  transmitted  from  the  parents  to  both  boys 
and  girls.  In  the  United  States  it  affects  300-400 
newborns  a  year,  and  1  of  every  50  individuals  is 
an  asymptomatic  carrier  of  the  disease  trait.  A  sec- 
ond area  under  intense  investigation  is  technol- 
ogy development  for  the  cure  of  these  genetic 
disorders  by  somatic  gene  therapy.  The  diseases 
being  studied  here  include  PKU  and  hemophilia 
B,  which  is  transmitted  from  asymptomatic  car- 
rier mothers  to  their  sons. 

Prenatal  Diagnosis  for  Phenylketonuria 

Our  laboratory  has  isolated  the  human  PAH 
gene  by  molecular  cloning  and  used  the  cloned 
gene  to  analyze  cellular  DNA  of  normal  and  af- 
fected individuals.  Extensive  benign  variations  in 
this  gene  were  discovered  and  used  to  trace  the 
transmission  of  individual  PAH  genes  from  the 
parents  to  the  children  in  PKU  families.  A  fetus 
inheriting  the  same  PAH  genes  as  an  affected  sib- 
ling will  have  PKU.  This  has  led  to  a  prenatal 
diagnosis  procedure  for  PKU  in  families  with  pre- 
viously affected  children,  and  the  procedure  has 
been  adopted  in  the  United  States  and  other  west- 
ern European  countries. 

Prognosis  by  Gene  Analysis 

There  is  a  wide  range  of  severity  of  clinical 
symptoms  among  PKU  patients,  and  their  treat- 
ment depends  on  measurement  of  the  phenylala- 
nine level  in  blood,  which  is  often  highly  vari- 
able. The  development  of  an  independent 
method  for  the  determination  of  prognosis  is  im- 
portant for  proper  management.  We  have  ana- 
lyzed the  mutations  in  the  PAH  gene  of  a  large 
number  of  PKU  patients  who  have  been  closely 
supervised  for  the  past  20  years.  The  severity  of 
their  clinical  conditions  is  primarily  dependent 


on  the  inheritance  of  mutations  in  their  PAH 
genes  that  are  either  totally  or  partially  defective. 
In  the  future  this  correlation  will  allow  physi- 
cians to  prescribe  proper  medical  treatment  after 
simple  gene  analysis. 

Population  Dynamics 

A  number  of  these  PKU  genes  have  distinctive 
distributions  in  the  European  continent.  One  is 
very  prevalent  in  eastern  Europe,  and  its  fre- 
quency decreases  in  a  gradual  fashion  from  east  to 
west.  These  results  suggested  that  the  mutation 
occurred  in  eastern  Europe  some  time  ago  and 
was  then  spread  throughout  the  European  conti- 
nent by  migration  of  people  in  prehistoric  times. 
Two  other  prevalent  PKU  genes  are  very  frequent 
in  either  northern  or  southern  Europe  but  less 
frequent  in  the  neighboring  countries.  When  sim- 
ilar analysis  was  performed  in  Israel  and  China, 
independent  centers  of  major  PKU  mutations 
were  also  discovered.  These  results  strongly  sug- 
gest that  multiple  PKU  mutations  occurred  inde- 
pendently in  various  regions  of  the  world  and 
then  spread  into  neighboring  areas  in  Europe  and 
Asia. 

Somatic  Gene  Therapy 

The  other  major  goal  of  our  laboratory  is  to  ex- 
plore the  potential  for  somatic  gene  therapy  of 
genetic  disorders.  The  PAH  gene  has  been  in- 
serted into  the  genome  of  an  incapacitated  virus. 
The  recombinant  viruses  are  able  to  transduce 
mammalian  cells  and  incorporate  themselves 
into  the  genome  of  the  host  cells,  but  they  are  no 
longer  able  to  produce  new  virus  to  continue  the 
infection  process.  The  recombinant  viruses  were 
used  to  transduce  cultured  rodent  hepatoma  cells 
and  normal  liver  cells,  thereby  conferring  upon 
them  the  ability  to  synthesize  the  corresponding 
human  enzyme. 

These  results  have  led  to  the  development  of 
hepatocyte  transplantation  technologies  in  labo- 
ratory animals.  A  variety  of  inert  substances  were 
used  as  support  for  mouse  liver  cells  prior  to 
transplantation,  but  the  transplanted  hepatocytes 


457 


Molecular  Genetics  and  Studies  Toward  Gene  Therapy 
for  Metabolic  Disorders 


lived  for  only  a  few  weeks  in  the  recipient  ani- 
mals. When  the  hepatocytes  were  returned  to  the 
liver  by  direct  injection  into  the  portal  vein  or  the 
spleen  of  recipient  mice,  however,  the  cells  mi- 
grated to  the  liver  and  incorporated  themselves 
into  the  parenchyma.  They  not  only  lived  for  as 
long  as  the  mice  did  but  also  continued  to  func- 
tion as  liver  cells  in  the  transplanted  animals.  Us- 
ing liver  cells  isolated  from  a  mouse  strain  that  is 
deficient  in  hepatic  PAH  activity,  we  demon- 
strated that  enzymatic  activity  was  reconstituted 
in  these  cells  after  retroviral-mediated  gene 
transfer.  The  PKU  mouse  model  will  be  critically 
important  to  test  the  efficacy  of  our  hepatic  gene 
transfer  and  hepatocyte  transplantation  protocols 
for  the  correction  of  PKU  in  the  future. 

To  develop  a  larger  animal  model  for  somatic 
gene  therapy,  with  technologies  that  may  be  di- 
rectly applied  to  human  patients  in  the  future,  we 
selected  a  colony  of  hemophilic  dogs.  Methods 
were  developed  to  obtain  a  liver  lobe  from  nor- 
mal dogs  by  partial  hepatectomy  and  to  disperse 
the  hepatocytes  into  single  cells  in  culture.  In- 
stead of  a  few  million  cells  (which  we  obtained 


from  the  mouse),  several  billion  cells  were  ob- 
tained from  a  single  canine  liver  lobe.  The  iso- 
lated hepatocytes  were  transplanted  back  into  the 
same  animal  by  direct  injection  into  the  spleen. 
Alternatively,  a  catheter  was  inserted  into  the 
splenic  vein  with  a  subcutaneous  port  to  permit 
direct  external  injection  of  hepatocytes  into  the 
port.  It  was  observed  that  a  billion  hepatocytes 
can  be  easily  transplanted,  and  greater  than  half 
of  these  cells  migrate  to  the  liver  and  survive  in 
the  animal  for  a  minimum  of  four  months.  Using  a 
recombinant  retroviral  vector  containing  the  hu- 
man coagulation  factor  IX  gene  to  transduce  nor- 
mal canine  hepatocytes,  we  also  observed  high 
levels  of  human  factor  IX  protein  in  the  culture 
media.  With  these  technological  developments, 
we  shall  attempt  to  correct  the  genetic  deficiency 
in  hemophilia  B  dogs  by  somatic  gene  therapy. 

Prenatal  diagnosis  of  PKU  and  disease  progno- 
sis by  gene  analysis  were  supported  by  a  grant 
from  the  National  Institutes  of  Health.  Somatic 
gene  therapy  research  on  the  PKU  mouse  and  he- 
mophilia B  dog  models  is  also  supported  in  part 
by  a  grant  from  the  National  Institutes  of  Health. 


458 


Paracrine  Control  of  Blood  Vessel  Function 
Role  of  the  Endothelins 


Masasbi  Yanagisawa,  M.D.,  Ph.D. — Associate  Investigator 

Dr.  Yanagisawa  is  also  Associate  Professor  of  Molecular  Genetics  at  the  University  of  Texas  Southwestern 
Medical  Center  at  Dallas.  He  received  his  M.D.  and  Ph.D.  degrees  from  the  University  of  Tsukuba,  Japan, 
where  he  worked  with  Tomoh  Masaki.  Before  moving  to  Dallas,  he  was  Assistant  Professor  of 
Pharmacology  at  Kyoto  University,  Japan. 


DISORDERS  of  blood  vessels,  including  all 
forms  of  heart  attack  and  stroke,  represent 
the  most  frequent  disease  cause  of  death  in  devel- 
oped countries.  The  inner  surface  of  blood  vessel 
walls  is  lined  with  a  thin  monolayer  of  flat  cells 
called  vascular  endothelium.  These  cells  cover  a 
total  surface  area  of  nearly  700  m^  throughout 
the  human  body.  The  endothelium  is  also  unique 
in  that  it  is  the  only  tissue  that  has  direct  physical 
contact  with  circulating  blood  under  healthy 
conditions.  Nevertheless,  until  a  little  over  10 
years  ago,  the  endothelial  cells  were  considered 
to  be  merely  a  "bio-inert  dialysis  bag."  In  other 
words,  they  just  kept  blood  flowing  smoothly 
without  unwanted  clotting  and  allowed  nutri- 
tional components  and  metabolites  to  pass  freely 
between  the  blood  and  interstitial  space. 

Evidence  accumulated  recently,  however,  indi- 
cates that  the  endothelium  plays  much  more 
complex  roles  in  many  different  facets  of  physiol- 
ogy and  pathology.  The  endothelial  cells  can  re- 
spond to  both  chemical  (circulating  and  local 
hormones)  and  mechanical  (local  blood  flow  and 
pressure)  information  carried  in  the  circulating 
blood. 

In  response  to  these  stimuli,  these  cells  trigger 
remodeling  and  de  novo  formation  of  blood  ves- 
sels by  secreting  growth  factors  and  migrating 
into  adjacent  tissues.  They  also  regulate  leuko- 
cyte infiltration  and  lymphocyte  homing  by  ex- 
pressing a  variety  of  cell  adhesion  molecules. 
They  integrate  blood  coagulation,  fibrinolysis, 
and  platelet  function  by  producing  various  pro- 
and  anticoagulant  factors.  They  control  vascular 
permeability  by  actively  transcytosing  and  metab- 
olizing plasma  components.  Finally,  they  regu- 
late blood  pressure  and  local  blood  flow  by  both 
activating  and  inactivating  circulating  vasoactive 
factors  and,  more  importantly,  by  generating  an 
array  of  vasoactive  molecules.  Thus  we  now  rec- 
ognize the  vascular  endothelium  as  an  active  and 
dynamic  transducer  that  senses  and  interprets 
blood-borne  signals. 

Central  to  these  functions  of  the  endothelium 
is  local  communication  of  the  endothelial  cells 
with  vascular  smooth  muscle  cells  underneath. 


As  mentioned  above,  the  endothelial  cells  send 
out  an  array  of  physiological  and  pathological 
signals  toward  the  smooth  muscle  cells  by  means 
of  the  endothelium-derived  vasoactive  factors. 
Among  those  are  prostacyclin  and  nitric  oxide 
(also  called  endothelin-derived  relaxing  factor), 
both  strong  vasodilators. 

This  background  led  us  in  1988  to  identify  en- 
dothelin-1  (ET-1),  a  novel  vasoconstrictor  pro- 
duced by  vascular  endothelial  cells.  ET-1  is  a 
small  peptide  consisting  of  21  amino  acid  resi- 
dues wound  into  a  rigid  structure  by  two  sets  of 
intrachain  disulfide  bridges.  It  is  the  most  potent 
vasoactive  molecule  known,  causing  a  strong  and 
extremely  sustained  constriction  of  all  blood  ves- 
sels both  in  vitro  and  in  vivo. 

Our  subsequent  studies  demonstrated  in  hu- 
mans and  other  mammals  three  endothelin- 
related  genes  that  encode,  besides  ET-1,  two  ad- 
ditional isopeptides  of  the  endothelin  family 
called  ET-2  and  -3.  Endothelin  isopeptides  are 
expressed  with  distinct  distribution  patterns  in 
many  mammalian  tissues.  While  the  endothelial 
cells  are  the  most  abundant  source  of  ET- 1 ,  one  or 
more  of  the  three  isopeptides  are  expressed 
widely  in  other  tissues  such  as  brain,  lung,  and 
kidney. 

The  production  of  endothelins  is  regulated  in 
both  directions.  In  endothelial  cells,  it  is  up- 
regulated  by  various  chemical  stimuli,  including 
classical  vasoactive  hormones  (epinephrine,  an- 
giotensin II,  and  vasopressin) ,  products  from  co- 
agulation/platelet activation  (thrombin  and 
transforming  growth  factor-jS) ,  factors  implicated 
in  septic  shock  (bacterial  endotoxin,  interleukin- 
1,  and  tumor  necrosis  factor),  oxidized  low- 
density  lipoprotein,  etc.  ET-1  production  is  also 
augmented  by  mechanical  stimuli  such  as  stretch 
and  fluid-dynamical  shear  stress.  In  contrast,  cer- 
tain vasodilators  (nitric  oxide  and  atrial  natri- 
uretic peptides)  attenuate  the  ET-1  production 
via  increase  of  intracellular  cGMP  levels. 

Like  many  other  peptide  hormones  and  neuro- 
peptides, endothelins  are  processed  from  the 
corresponding  larger  precursor  proteins  (pre- 
pro-proteins).  However,  biologically  active  21- 


459 


Paracrine  Control  of  Blood  Vessel  Function:  Role  of  the  Endothelins 


residue  endothelins  are  produced  via  a  formerly 
unknown  type  of  proteolytic  processing.  Biologi- 
cally inactive  intermediates  of  approximately  40 
amino  acids,  called  big  endothelins,  are  first  cut 
out  from  the  prepro-proteins.  Their  carboxyl- 
terminal  half  is  then  sheared  off  to  produce  the 
amino-terminal  active  peptides. 

'This  unusual  endoproteolytic  activation  is  cata- 
lyzed by  a  novel  membrane-bound  metallopro- 
tease  called  endothelin-converting  enzyme.  This 
unique  enzyme  is  sensitive  to  the  metallopro- 
tease  inhibitor  phosphoramidon  and  is  distinct 
from  any  other  protease  know^n.  Since  big  ET-1  is 
at  least  100-200  times  less  active  than  mature 
ET-1  in  constricting  vascular  smooth  muscle 
strips,  the  converting  enzyme  is  essential  in  pro- 
ducing the  potent  vasoconstrictive  agent.  There- 
fore the  enzyme  could  also  be  an  important  target 
for  pharmacological  intervention  in  the  endothe- 
lin  system.  If  one  could  develop  an  inhibitor  of 
endothelin-converting  enzyme,  it  could  possibly 
be  used  as  a  novel  class  of  vasodilatory  drug. 

Apart  from  their  potent  and  long-lasting  vaso- 
constrictor/pressor  activities,  endothelins  pos- 
sess a  wide  spectrum  of  nonvascular  actions  in 
various  tissues.  Furthermore,  two  distinct  sub- 
types of  endothelin  receptors  (called  ET^  and 
ETg)  with  different  selectivities  to  the  three  iso- 
peptides  have  been  pharmacologically  demon- 
strated and  molecularly  cloned  by  us  and  other 
researchers.  In  keeping  with  the  wide  spectrum 
of  endothelin  action,  the  two  receptors  are  ex- 
pressed in  a  variety  of  vascular  and  nonvascular 
tissues. 

The  observation  that  many  endothelin-produc- 
ing  tissues  also  express  one  or  more  subtypes  of 


endothelin  receptors  suggests  the  importance  of 
the  peptide  family  as  locally  acting  mediators 
(paracrine  and/or  autocrine)  rather  than  circu- 
lating agents.  The  idea  is  further  supported  by 
several  lines  of  evidence.  For  example,  clearance 
of  endothelins  from  the  circulation  is  extremely 
rapid,  with  an  initial  half-life  of  less  than  2-3 
min;  plasma  concentration  of  immunoreactive 
ET-1  is  well  below  the  threshold  concentration 
for  pharmacological  actions;  and  circulating  en- 
dothelins are  capable  of  inducing  the  release  of 
vasodilator  substances  such  as  nitric  oxide  and 
prostacyclin  via  the  receptors  on  the  endothelial 
cells,  thereby  limiting  their  own  vasoconstrictor 
actions. 

Increased  plasma  concentrations  of  FT- 1  have 
been  reported  in  patients  with  various  disease 
states,  including  vasospasm  and  hypertension, 
where  abnormal  vasomotor  function  is  impli- 
cated. Moreover,  in  animal  models  of  certain  vas- 
cular disorders,  such  as  acute  myocardial  infarc- 
tion, cerebral  vasospasm,  and  acute  ischemic 
renal  failure,  treatment  with  antiendothelin 
neutralizing  antibody  significantly  ameliorates 
the  abnormal  vasoconstriction  seen  in  these 
conditions. 

It  is  hoped  that  further  insight  into  the  physio- 
logical and  pathobiological  roles  of  this  complex 
system  of  peptidic  mediators  will  be  gained  in 
the  near  future  with  the  development  of  endothe- 
lin receptor  antagonists,  inhibitors  of  endothelin- 
converting  enzyme,  and  mice  deficient  in  en- 
dothelin/endothelin  receptors.  This  may  lead  to 
a  new  level  of  understanding  of  how  cardiovascu- 
lar homeostasis  is  maintained  via  local  communi- 
cation between  cells  of  blood  vessel  walls  in 
health  and  disease. 


460 


Mechanism  of  Phototransduction  in  Retinal  Rods 
and  Cones 


King- Wat  Yau,  Ph.D. — Investigator 

Dr.  Yau  is  also  Professor  of  Neuroscience  at  the  Johns  Hopkins  University  School  of  Medicine.  He  received 
an  A.B.  degree  in  physics  from  Princeton  University  and  a  Ph.D.  degree  in  neurobiology  from  Harvard 
University.  He  did  postdoctoral  research  at  Stanford  University  with  Denis  Baylor  and  at  Cambridge 
University,  England,  with  Alan  Hodgkin.  For  six  years  thereafter,  he  was  on  the  faculty  at  the  Department 
of  Physiology  and  Biophysics  of  the  University  of  Texas  Medical  Branch  at  Galveston.  He  has  received  the 
Rank  Prize  in  Optoelectronics  from  the  Rank  Prize  Funds,  England. 


VISION  begins  in  the  rods  and  cones  of  the 
retina,  where  light  is  absorbed  and  trans- 
duced into  a  neural  signal  consisting  of  an  elec- 
trical hyperpolarization  at  the  photoreceptor 
membrane.  This  signal  is  relayed  to  second-order 
neurons  in  the  retina  through  a  modulation  of  the 
release  of  synaptic  transmitter  at  the  photorecep- 
tor's terminal.  In  darkness  the  transmitter  is  re- 
leased at  a  high  rate,  and  in  light  the  membrane 
hyperpolarization  reduces  the  release  in  a  graded 
fashion.  This  modulation  of  synaptic  transmitter 
release  can  lead  to  a  hyperpolarizing  or  depolar- 
izing response  to  light  in  a  second-order  neuron, 
depending  on  the  polarity  of  a  given  synapse. 

The  phototransduction  process — the  way  the 
hyperpolarizing  response  to  light  is  generated  in 
the  receptors — is  as  follows.  In  darkness  an  ionic 
conductance  in  the  plasma  membrane  of  the  re- 
ceptor's outer  segment  (the  part  of  the  cell  that 
contains  the  visual  pigment)  is  kept  open  by  the 
cyclic  nucleotide  guanosine  3':5'-cyclic  mono- 
phosphate (cGMP),  letting  both  Na^  and  Ca^"^ 
into  the  cell.  This  "dark"  current  depolarizes  the 
cell  and  causes  the  steady  release  of  synaptic 
transmitter  described  above. 

Light  activates  the  following  reaction  cascade: 
light  photoisomerization  of  visual  pigment  -»• 
G  protein  activation  ->  cGMP  phosphodiesterase 
stimulation  cGMP  hydrolysis.  As  a  result,  the 
cGMP  level  falls  in  the  outer  segment,  causing 
the  ionic  conductance  to  close  and  leading  se- 
quentially to  membrane  hyperpolarization  and 
the  reduction  of  synaptic  transmitter  release.  This 
phototransduction  scheme  applies  to  both  rods 
and  cones,  with  only  quantitative  differences  be- 
tween the  two  types  of  receptors. 

One  consequence  of  the  conductance  closure 
in  the  light  is  that  the  Ca^^  influx  stops.  The  re- 
sulting imbalance  between  influx  and  efflux 
leads  to  a  decrease  in  the  intracellular  free  Ca^* 
concentration.  This  Ca^"*^  decrease  reduces  a  tonic 
inhibition  exerted  by  Ca^+  on  the  cGMP-synthe- 
sizing  enzyme  guanylate  cyclase  and  causes  an 
increase  in  the  synthesis  of  cGMP  in  the  light. 
Thus  Ca^"^  mediates  a  negative  feedback  control 
on  the  light-activated  cGMP  hydrolysis,  and  this 


feedback  should  be  a  candidate  mechanism  un- 
derlying the  well-known  phenomenon  of  back- 
ground light  adaptation  in  photoreceptors.  In- 
deed, we  have  found  that  this  adaptation 
essentially  disappears  upon  removing  the  feed- 
back experimentally  by  eliminating  the  Ca^"^  in- 
flux and  efflux. 

The  effect  of  Ca^"^  on  rod  guanylate  cyclase  has 
been  studied  by  others  in  biochemical  experi- 
ments in  vitro.  This  effect  is  now  known  to  in- 
volve recoverin,  a  novel  Ca^^-binding  protein 
that  activates  guanylate  cyclase  at  low  Ca^^  con- 
centrations but  loses  this  ability  when  Ca^"^  is 
bound  to  it.  One  drawback  of  the  biochemical 
experiments  is  that  unphysiological  ionic  con- 
centrations (e.g.,  very  high  Mg^"^)  were  used  to 
measure  the  cyclase  activity.  We  have  now  stud- 
ied this  Ca^^  modulation  of  the  cyclase  in  more 
physiological  conditions,  by  recording  the 
cGMP-activated  current  from  an  isolated,  open- 
ended  rod  outer  segment  with  a  suction  pipette 
while  dialyzing  the  interior  of  the  outer  segment 
with  different  Ca'^^  concentrations. 

The  Ca^^  effect  on  the  guanylate  cyclase  could 
be  derived  from  the  magnitude  of  the  cGMP- 
activated  current.  We  have  found  that  the  cyclase 
activity  is  very  sensitive  to  the  free  Ca^^  concen- 
tration, with  its  maximum  activity  being  approxi- 
mately halved  at  100  nM  Ca^^.  This  is  similar  to 
biochemical  measurements.  The  cyclase  depen- 
dence on  Ca^^  shows  a  Hill  coefficient  of  approxi- 
mately 1.5,  which  is  lower  than  the  value  of 
around  4  in  biochemical  measurements.  The  cy- 
clase activity  becomes  relatively  insensitive  to 
Ca^^  at  a  GTP  concentration  of  greater  than  1  mM, 
suggesting  that  the  effect  of  Ca^"^,  acting  through 
recoverin,  may  primarily  be  to  reduce  the  affinity 
of  the  enzyme  for  its  substrate.  Further  experi- 
ments on  this  problem  are  in  progress. 

Another  problem  we  are  working  on  is  a  molec- 
ular characterization  of  the  cGMP-activated  con- 
ductance mediating  phototransduction.  The  con- 
ductance now  appears  to  belong  to  a  family  of 
cyclic  nucleotide-gated  channels  that  includes 
the  cGMP-gated  channels  in  retinal  rods  and 
cones,  as  well  as  the  cGMP/cAMP-gated  channel 


461 


Mechanism  of  PhototransdiicHon  in  Retinal  Rods  and  Cones 


mediating  olfactory  transduction  in  olfactory 
cilia.  These  channels  show  both  similarities  and 
differences.  In  particular,  the  rod  and  olfactory 
channels  have  similar  ion-permeation  character- 
istics, but  the  olfactory  channel  shows  a  30-fold 
higher  affinity  for  cyclic  nucleotides.  To  under- 
stand the  molecular  determinant  for  this  differ- 
ence, we,  in  collaboration  with  Randall  Reed 
(HHMI,  Johns  Hopkins  University),  have  con- 
structed chimeras  between  the  rod  and  the  olfac- 
tory channels  and  tested  for  modifiications  in 
function.  The  difference  in  amino  acid  residues  at 
the  putative  cyclic  nucleotide-binding  site  on 
the  two  channel  molecules  has  little  to  do  with 
their  difference  in  affinity  for  cyclic  nucleotides. 
Other  regions  of  the  molecule  must  therefore  be 
involved,  possibly  through  steric  or  allosteric  in- 
teractions in  the  folded  molecule.  Identification 
of  these  regions  is  still  in  progress. 

Separately,  several  independent  clones  have 


been  isolated  from  a  human  retinal  cDNA  library 
based  on  structural  homology  to  the  published 
sequence  of  the  bovine  rod  channel.  One  of  these 
has  been  successfully  expressed  in  human  293 
cells  and  found  to  have  properties  identical  to  the 
bovine  rod  channel.  Thus  this  clone  most  proba- 
bly encodes  for  the  human  rod  channel.  The 
other  clones  have  yet  to  be  expressed  function- 
ally in  a  cell  line.  Based  on  the  presence  of  a  puta- 
tive cyclic  nucleotide-binding  domain  as  well  as 
other  features  in  their  nucleotide  sequences, 
however,  they  probably  also  encode  for  cyclic 
nucleotide-gated  channels.  Antipeptide  antibod- 
ies are  being  made  to  identify  the  locations  of  the 
encoded  proteins  in  the  retina.  Continuing  ef- 
forts are  also  being  made  to  express  these  clones 
functionally.  Cloning  at  the  genomic  level  is  be- 
ing carried  out  to  provide  further  clarification. 

Part  of  the  above  work  is  supported  by  a  grant 
from  the  National  Institutes  of  Health. 


462 


Molecular  Mechanisms  of  Ion  Channel  Function 


Gary  Yellen,  Ph.D. — Assistant  Investigator 

Dr.  Yellen  is  also  Assistant  Professor  of  Neuroscience  and  Biophysics  at  the  Johns  Hopkins  University 
School  of  Medicine.  He  received  his  undergraduate  degree  in  biochemical  sciences  from  Harvard  College 
and  his  Ph.D.  degree  in  physiology  from  Yale  University,  where  he  studied  with  Charles  Stevens.  Dr.  Yellen 
did  his  postdoctoral  research  on  ion  channel  physiology  as  a  Life  Sciences  Research  Foundation 
postdoctoral  fellow  at  Brandeis  University,  where  he  worked  with  Christopher  Miller. 


ALL  electrical  signaling  in  the  nervous  system 
is  controlled  by  ion  channels,  a  class  of 
membrane  proteins  that  form  pores  through  the 
membrane.  Charged  ions  such  as  sodium,  potas- 
sium, and  calcium  pass  through  ion  channels  and 
carry  an  electrical  current.  The  channels  them- 
selves are  regulated,  so  that  the  pores  are  only 
open  when  the  proper  chemical  or  electrical  sig- 
nal is  present,  and  only  certain  ions  can  pass 
through  a  particular  kind  of  channel.  By  under- 
standing how  channels  open  and  close  and  how 
they  are  regulated,  we  define  the  repertoire  of 
molecular  changes  used  by  neurons  when  they 
signal,  sense,  and  learn. 

Ion  channels,  like  other  membrane  proteins, 
have  resisted  standard  biochemical  and  structural 
analysis.  Their  structure  has  only  recently  begun 
to  be  elucidated  by  a  combination  of  protein 
chemistry  and  molecular  biology.  On  the  other 
hand,  we  have  detailed  knowledge  of  the  func- 
tioning of  ion  channels.  Because  each  ion  chan- 
nel catalyzes  the  transport  of  millions  of  ions  per 
second,  we  can  measure  electrically  the  current 
carried  by  just  a  single-channel  protein  molecule. 
This  technique  of  single-channel  recording  has 
allowed  us  to  make  a  detailed  model  for  the  con- 
formational changes  between  open  and  closed 
states  induced  by  chemical  ligands  and  changes 
in  voltage,  but  we  still  have  no  knowledge  of  the 
protein  structures  that  underlie  these  conforma- 
tional changes. 

My  laboratory  uses  a  combination  of  high- 
resolution  functional  analysis  (by  single-channel 
recording)  and  direct  manipulation  of  the  struc- 
ture of  the  channel  protein.  Site-directed  muta- 
genesis allows  us  to  modify  any  amino  acid  in  a 
protein  for  which  we  have  the  cloned  genetic  ma- 
terial. Rather  than  modifying  the  protein  directly, 
we  change  the  DNA  sequence  and  then  inject  the 
modified  messenger  RNA  into  immature  frog  eggs 
(oocytes) ,  which  manufacture  the  modified  pro- 
tein. This  method  allows  us  to  test  specific  the- 
ories about  which  parts  of  the  channel  protein  are 
important  for  specific  functional  features. 

We  have  applied  this  combination  of  strategies 
to  voltage-activated  potassium  channels,  which 


participate  in  electrical  signaling.  By  systematic 
mutagenesis,  we  have  identified  the  region  of  the 
potassium  channel  protein  that  lines  the  pore 
through  which  ions  cross  the  membrane.  We 
found  specific  amino  acid  residues  in  the  protein 
sequence  that  control  the  sensitivity  of  the  chan- 
nel to  tetraethylammonium,  an  organic  ion  that 
can  block  current  through  the  channel.  Natural 
variation  of  one  of  these  amino  acids  explains  the 
differences  in  drug  sensitivity  between  potassium 
channels  in  different  organs  or  species.  Amino 
acids  in  this  region  of  the  protein  can  also  alFect 
the  rate  at  which  ions  are  conducted  through  the 
pore.  These  discoveries  put  us  in  a  position  to 
discover  the  basic  mechanisms  of  ion  selectivity 
and  channel  gating  at  the  level  of  individual 
amino  acids. 

We  have  also  used  recording  from  single  potas- 
sium channels  to  demonstrate  that  one  of  the 
mechanisms  by  which  these  channels  open  and 
close  is  a  simple  occlusion  of  the  pore  by  part  of 
the  channel  protein.  Earlier  work  established  that 
the  pore  could  be  directly  blocked  or  occluded 
by  internal  organic  ions;  we  have  established  that 
the  natural  gating  occurs  by  a  very  similar  mecha- 
nism involving  a  tethered  blocking  particle.  The 
most  direct  demonstration  of  this  is  that  potas- 
sium ions  passing  through  the  pore  from  one  side 
can  clear  the  tethered  blocking  particle  from  the 
opposite  side. 

Further  work  in  progress  to  determine  the 
structural  basis  for  potassium  channel  gating  in- 
cludes introducing  chemically  reactive  cysteine 
residues  at  specific  locations  in  the  protein  se- 
quence. Channel  proteins  with  cysteines  inserted 
at  critical  locations  should  show  a  specific 
change  in  function  when  treated  with  reagents 
that  modify  the  cysteine  side  chain.  The  reactivity 
of  these  side  chains  will  depend  on  both  their 
location  in  the  channel  protein  and  the  specific 
conformational  state  of  the  protein  at  the  time  of 
reaction. 

We  have  also  used  the  site-directed  strategy  to 
study  acetylcholine-activated  cation  channels, 
which  convert  neurochemical  signals  into  elec- 
trical signals  at  synapses.  We  have  changed  amino 


465 


Molecular  Mechanisms  of  Ion  Channel  Function 


acids  in  the  region  of  the  protein  that  binds  ace- 
tylcholine and  identified  specific  residues  that 
play  a  critical  role  in  binding  and  signal  transduc- 
tion by  acetylcholine.  These  studies  are  teaching 
us  more  about  the  molecular  basis  of  drug  rec- 


ognition and  of  signal  transduction  in  this 
protein. 

Dr.  Yellen  is  now  Associate  Professor  of  Neu- 
robiology at  Harvard  Medical  School. 


464 


Drosophila  Behavior  and  Neuromuscular 
Development 


Michael  W.  Young,  Ph.D. — Investigator 

Dr.  Young  is  also  Professor  of  Genetics  at  the  Rockefeller  University.  He  received  his  B.A.  degree  in  biology 
from  the  University  of  Texas,  Austin.  Staying  on  to  work  at  the  university  with  Burke  Judd,  he  earned  his 
Ph.D  degree  for  genetic  and  cytological  studies  o/Drosophila  chromosome  structure.  Dr.  Young  did 
postdoctoral  work  in  biochemistry  at  Stanford  University  Medical  School  with  David  Hogness.  He  moved 
to  Rockefeller  as  a  fellow  of  the  Andre  and  Bella  Meyer  Foundation. 


A biological  clock,  composed  of  a  few  thou- 
sand cells  within  the  mammalian  brain,  con- 
trols timing  of  daily  behaviors  such  as  sleep  with 
an  accuracy  of  minutes.  Chemical  and  electrical 
rhythms  have  been  detected  in  these  mammalian 
pacemaker  tissues.  Still,  little  is  known  about  the 
underlying  biochemistry  used  to  calculate  time. 

The  genes  and  proteins  central  to  biological 
timing  are  beginning  to  be  recovered  and  charac- 
terized in  a  simpler  model  organism,  the  fruit  fly 
Drosophila.  The  best-studied  gene  in  the  Dro- 
sophila clock  system  has  been  named  per  {pe- 
riod^. Several  mutant  forms  of  the  gene  have 
been  recovered  that  affect  the  pace  of  the  insect's 
clock  and  certain  aspects  of  cell  physiology. 

In  the  per^  mutant,  circadian  locomotor  activ- 
ity rhythms  have  a  long  period  of  30  rather  than 
24  hours.  For  the  mutant per^,  daily  cycles  have  a 
shortened,  1 9-hour  period.  Mutants  with  no  daily 
rhythms  are  designated  per^ .  Corresponding 
changes  in  cycle  time  are  found  for  a  high- 
frequency  rhythm — a  courtship  song  produced 
in  males  by  pulses  of  wing  beating:  an  80-second 
song  (instead  of  the  normal  55  seconds)  for  per\ 
40  seconds  for  per^,  and  song  arrhythmicity  for 
per^.  Also,  the  mutations  change  the  period  of  a 
daily  oscillation  in  per  transcription,  which  may 
be  important  for  establishing  rhythmic  behavior. 
Finally,  for  at  least  some  tissues,  the  mutants  ap- 
pear to  alter  conductance  of  specialized  channels 
(gap  junctions)  between  cells. 

The  molecular  changes  associated  with  the 
mutations  have  been  established.  The  per^  mu- 
tant cannot  express  a  full-length  protein.  On  the 
other  hand,  per^  and  per^  make  per  proteins,  but 
these  are  altered  by  substitution  of  a  single,  dif- 
ferent amino  acid.  Comparable  changes  in  cycle 
time  can  also  be  effected  by  altering  the  amount 
of  per  protein  the  fly  produces.  For  example,  mi- 
croinjection of  a  gene  that  underproduces  the  per 
protein  20-fold  induces  40-hour  daily  rhythms. 
From  these  results  it  has  been  suggested  that  the 
per^  and  per^  mutations  generate,  respectively, 
hyper-  and  hypoactive  proteins. 

In  a  recent  effort  to  understand  how  changes  in 
protein  structure  can  affect  per  activity,  genes 


carrying  new  mutations,  produced  in  vitro,  were 
reintroduced  into  the  fly  by  microinjection.  It 
was  found  that  mutations  changing  the  structure 
of  a  certain  segment  of  about  20  amino  acids  pre- 
dominantly confer  short-period  (per'-like)  rhyth- 
micity.  Apparently  the  mutations  identify  a  re- 
gion of  the  per  protein  that  regulates  the  activity 
level  in  normal  flies. 

A  variety  of  experiments  have  demonstrated 
that  the  per  protein  acts  in  the  nervous  system  to 
control  daily  and  circaminuten  rhythms.  We  have 
become  interested  in  tracking  the  development 
of  the  fly's  clock  in  an  effort  to  determine  when  it 
begins  to  run,  whether  it  requires  signals  from 
outside  the  organism  to  start,  and  where  the  first 
cells  expressing  per  arise  and  develop.  We  have 
learned  that  Drosophila  reared  in  constant  envi- 
ronmental conditions  spontaneously  start  their 
clocks  only  hours  after  formation  of  the  embryo. 
Evidence  of  a  running  clock  is  first  seen  after 
completion  of  the  embryonic  nervous  system  and 
just  following  cessation  of  high  levels  of  per  ex- 
pression in  certain  neural  cells. 

Until  recently,  only  the  per  locus  was  known  to 
be  essential  for  production  of  biological  rhythms 
in  the  fruit  fly.  Genetic  screens  for  rhythm  muta- 
tions have  led  to  the  discovery  of  additional,  in- 
dispensable genes.  Of  special  interest  is  a  new 
mutation  found  on  the  second  chromosome  {per 
maps  to  the  X  chromosome).  The  new  mutation, 
like per^,  renders  flies  arrhythmic,  and  in  molecu- 
lar studies  appears  to  block  the  circadian  rhythm 
in  per  transcription.  Thus  the  newly  recognized 
gene  may  be  required  for  per  to  function. 

Development  of  Skin,  Muscle,  and  Nerve 

In  the  embryo  the  nervous  system  and  skin  are 
derived  from  a  common  set  of  cells,  the  ecto- 
derm. Each  of  these  cells  must  choose  a  fate,  and 
in  certain  Drosophila  mutants  the  choice  goes 
awry.  For  one  set  of  mutants  known  as  "neuro- 
genic," the  capacity  to  choose  skin  has  been  lost 
and  only  nerve  is  formed.  We  believe  the  mutants 
have  lost  the  ability  to  form  a  set  of  signals  that  act 
early  in  development,  so  we  are  using  the  muta- 


465 


Drosophila  Behavior  and  Neuromuscular  Development 


tions  to  mark,  isolate,  and  characterize  the  prod- 
ucts of  these  genes  and  putative  developmental 
signals.  Neurogenic  mutations  have  been  recov- 
ered at  seven  genetic  loci:  Notch,  big  brain, 
mastermind,  neuralized.  Delta,  almondex,  and 
Enhancer  of  split. 

Notch  has  been  characterized  more  completely 
than  any  other  gene  in  the  neurogenic  group.  It 
produces  a  very  large,  2 ,700-amino  acid  protein, 
predominantly  composed  of  an  uninterrupted 
array  of  36  copies  of  a  hormone-like  molecule,  a 
relative  of  epidermal  growth  factor.  The  Notch 
protein  spans  the  cellular  membrane,  with  the  36 
hormone  copies  exposed  to  its  neighbors.  We 
surmise  that  the  neighbors  read  that  signal  and,  in 
return,  tell  the  A'^orc^-bearing  cell  to  come  up 
with  the  correct  allocations  of  skin  and  nerve. 
Since  the  entire  string  of  hormone  copies  is  teth- 
ered to  the  cell's  surface,  signaling  between  cells 
must  be  intimate.  Only  cells  that  can  touch  each 
other  could  communicate  through  such  a 
protein. 

From  work  with  temperature-sensitive  muta- 
tions, we  know  that  Notch  proteins  are  used  to 
instruct  development  throughout  embryonic, 
larval,  and  pupal  life.  Mutations  altering  the 
structures  of  individual  hormone-like  elements 
of  the  Notch  protein  have  been  examined  to  de- 
termine the  role  each  plays  in  early  and  late  devel- 
opment. Of  the  36  hormone  repeats,  no  two  are 
identical,  and  changes  in  different  hormone  ele- 
ments produce  different  developmental  abnor- 
malities. This  must  mean  that  alternate  parts  of 
the  Notch  hormone  string  are  read  as  develop- 
ment unfolds.  In  part  these  specificities  could  al- 
low a  cell  to  talk  to  changing  neighbors  from  the 


time  of  cell  birth  to  differentiation  into  adult 
tissue. 

What  do  signals  from  these  genes  tell  a  cell  to 
do?  For  several  years  gene  action  at  Notch,  Delta, 
big  brain,  almondex,  neuralized,  mastermind, 
and  Enhancer  of  split  have  been  assumed  to  stim- 
ulate an  undifferentiated  ectodermal  cell  to  de- 
velop as  an  epidermal  cell.  The  genes  are  thought 
to  provide  a  series  of  epidermalizing  signals  dur- 
ing cell  differentiation,  with  loss  of  function  gen- 
erating a  nerve  cell.  New  work  in  our  laboratory 
shows  this  simple  picture  to  be  inaccurate.  Notch 
proteins  have  now  been  found  in  cells  giving  rise 
to  embryonic  muscle.  In  Notch  mutants,  strong 
effects  on  muscle  development  are  seen,  with  in- 
creased numbers  of  some  muscle  cell  types  gen- 
erated, probably  at  the  expense  of  others.  Thus 
parallel  changes  in  muscle,  skin,  and  nerve  devel- 
opment take  place  in  Notch  mutants.  Of  most  sig- 
nificance, comparable  effects  on  muscle  develop- 
ment are  seen  with  mutations  of  Delta,  big  brain, 
mastermind,  almondex.  Enhancer  of  split,  and 
neuralized. 

We  have  learned  three  things  from  these  stud- 
ies: 1 )  the  developmental  fates  of  many  cell  types 
are  switched  in  neurogenic  mutants;  2)  the  genes 
must  provide  differentiation  signals  that  cells 
composing  different  germ  layers  can  read,  with 
no  apparent  overlap  in  the  final  developmental 
fates;  and  3)  the  genes  defined  by  the  neurogenic 
mutations  probably  work  together  to  form  a  sin- 
gle developmental  pathway,  which  generates  a 
common  differentiation  signal  in  all  cells  af- 
fected in  the  mutants,  since  any  developmental 
anomaly  caused  by  loss  of  one  gene  in  the  group 
predicts  a  comparable  developmental  change 
upon  loss  of  any  other  gene  in  the  series. 


466 


los/jun 


NGF 


r 


Fos/Jun 
binding  site 

TH 

inactii^e 


Fos  and  Jun 
protein 
levels  low 


Fos/Jun 
binding  site 


TH 

active 

Fos/Jun 
synthesis 
induced 
by  NGF 


lra?/jun? 


Fos/Jun 
binding  site 


TH 

repressed 

Fra/Jun 

synthesis 

induced 


Model  for  a  mechanism  of  control  over  a  gene  that  may  play  a  role  in  Parkinson's  disease.  The 
polypeptide  hormone  NGF  (nerve  growth  factor)  binds  to  receptors  on  the  surface  of  nerve  cells 
( neurons)  and  induces  expression  of  the  gene  encoding  tyrosine  hydroxylase  (TH).  This  enzyme 
carries  out  a  critical  step  in  the  synthesis  of  the  neurotransmitter  dopamine,  whose  release  by 
neurons  transmits  signals  to  neighboring  cells.  Thus  NGF  may  indirectly  control  the  neuron's 
signaling  capacity.  In  the  model,  NGF  induces  synthesis  of  members  of  the  Fos  family  of  proteins, 
which  form  complexes  with  fun  family  proteins.  These  complexes  bind  to  a  regulatory  element  of 
the  TH  gene  and  stimulate  TH  expression.  Later,  proteins  related  to  Fos — Fra's — are  expressed 
and  repress  TH  gene  activity.  A  deficiency  in  dopamine  production  can  lead  to  Parkinson's  dis- 
ease and  other  neurological  disorders. 
Research  of  Elena  Gizang- Ginsberg  and  Edward  Ziff. 


468 


Control  of  Transcription  by  Transmembrane 
Signals 


Edward  B.  Ziff,  Ph.D — Investigator 

Dr.  Ziff  is  also  Professor  of  Biochemistry  at  New  York  University  Medical  Center.  He  received  his  B.A. 
degree  in  chemistry  from  Columbia  University  and  the  Ph.D  degree  in  biochemistry  from  Princeton 
University.  He  then  studied  DNA  structure  with  Fred  Sanger  at  the  MRC  Laboratory  of  Molecular  Biology 
in  Cambridge.  He  later  conducted  research  on  DNA  tumor  viruses  at  the  Imperial  Cancer  Research  Fund 
Laboratory,  London,  and  in  the  Department  of  Molecular  Cell  Biology  at  the  Rockefeller  University.  He 
later  joined  New  York  University  Medical  Center  and  began  the  study  of  cellular  mechanisms  that  control 
proliferation  and  differentiation. 


THE  remarkable  process  of  development  re- 
quires that  a  fertilized  egg  undergo  many 
rounds  of  cell  division  with  accompanying  differ- 
entiation to  generate  specialized  cell  types  that 
ultimately  form  the  mature  organism.  For  devel- 
opment to  proceed  normally,  just  the  right  num- 
ber and  types  of  cells  must  be  available  at  each 
stage.  It  follows  that  any  cell's  decision  to  divide 
and/or  differentiate  must  be  carefully  regulated. 
These  two  critical  processes,  proliferation  and 
differentiation,  are  also  controlled  in  the  adult — 
for  example,  during  the  maintenance  of  tissues 
and  in  wound  healing. 

The  decision  of  a  cell  to  divide  or  to  express  a 
specialized  function  is  often  determined  by  sig- 
nals from  its  environment.  Prominent  among  the 
agents  that  convey  such  signals  are  the  growth 
factors,  which  are  polypeptides  synthesized  and 
secreted  by  cells.  Our  laboratory  studies  the  mo- 
lecular mechanisms  by  which  growth  factors  and 
other  transmembrane  signaling  agents  exert  their 
effects  on  cell  proliferation  and  differentiation. 
The  transforming  genes  of  DNA  tumor  viruses, 
such  as  adenovirus,  often  modify  these  programs 
and  are  therefore  useful  for  dissecting  the  growth 
regulatory  pathways. 

Growth  factors  transmit  signals  to  cells  by  bind- 
ing to  specific  receptor  proteins,  which  span  the 
cell's  plasma  membrane  and  induce  second  mes- 
senger signals  in  the  cytoplasm.  The  latter  signals 
have  a  multitude  of  targets,  some  in  the  cyto- 
plasm and  some  in  the  nucleus.  Although  individ- 
ual growth  factors  may  exert  profound  changes 
on  cells,  some  effects,  such  as  the  induction  of 
cell  proliferation,  may  require  the  combined  ac- 
tions of  more  than  one  growth  factor.  When  the 
signal  pathways  are  inappropriately  activated, 
cells  may  lose  control  of  growth  and  form  a  tu- 
mor. When  the  pathways  are  blocked,  essential 
cell  types  may  degenerate  and  die.  It  follows  that 
errors  in  signaling  can  result  in  diverse  diseases. 

In  the  case  of  the  nervous  system,  transmem- 
brane signals  induced  by  neurotransmitters  regu- 
late the  properties  of  neurons.  These  small  mole- 
cules, released  by  neurons  at  synapses,  bind  to 


receptors  on  postsynaptic  target  cells.  Neuro- 
transmitter stimulation  of  target  neurons  is  a  criti- 
cal step  in  the  rapid  transmission  of  nerve  im- 
pulses, but  it  can  also  more  slowly  regulate  the 
activities  of  specific  genes,  allowing  the  nervous 
system  to  modify  its  properties  in  response  to  its 
environment.  Such  modification  may  underlie 
the  processes  of  neural  adaptation  and  memory. 

Our  laboratory  has  identified  a  group  of  imme- 
diate early-response  genes  that  are  rapidly  in- 
duced by  growth  factor  stimulation  and  appear  to 
be  primary  targets  in  the  nucleus  for  the  growth 
factor-induced  signals.  Our  work  focuses  on  the 
c-fos  gene,  which  is  very  rapidly  induced  by  a 
wide  range  of  transmembrane  signals.  This  gene 
encodes  a  protein,  c-Fos,  which  is  a  member  of  a 
family  of  transcription  factors  that  bind  to  spe- 
cific sites  in  the  regulatory  regions  of  other  genes 
and  thereby  control  their  activity.  In  this  manner 
c-Fos  acts  as  an  intermediary  for  the  conversion  of 
short-term  transmembrane  signals  into  longer- 
term  changes  in  the  cell.  These  studies  are  sup- 
ported by  the  American  Cancer  Society. 

We  are  particularly  concerned  with  the  role  of 
c-fos  in  programs  of  neuronal  differentiation  in- 
duced by  nerve  growth  factor  (NGF).  In  vivo, 
NGF  is  required  for  the  differentiation  and  main- 
tenance of  peripheral  neurons.  Expression  of  c- 
fos  appears  to  be  a  first  step  in  the  activation  of  a 
multistage  gene  expression  program  induced  by 
NGF  that  can  culminate  in  cell  division  or  the 
induction  of  terminally  differentiated  functions. 

In  our  model  neuronal  cells,  we  have  shown 
that  neurotransmitters  as  well  as  growth  factors 
can  also  induce  the  expression  of  c-Fos.  It  is  ap- 
parent from  this  and  other  studies  that  c-Fos  has  a 
critical  role  in  the  adult  nervous  system,  not  just 
in  neural  development. 

We  have  shown  that  c-Fos  induced  by  NGF  can 
cooperate  with  a  second  protein  factor,  c-Jun,  to 
induce  the  gene  for  tyrosine  hydroxylase  (TH), 
an  enzyme  that  catalyzes  a  critical  step  in  the  pro- 
duction of  neurotransmitters  in  the  catechol- 
amine family.  The  combined  c-Fos  and  c-Jun  form 
a  heterodimer  that  binds  to  a  TH  gene  regulatory 


469 


Control  of  Transcription  by  Transmembrane  Signals 


element  and  induces  expression.  This  participa- 
tion of  c-Fos  in  TH  control  may  allow  a  neuron  to 
coordinate  the  production  of  catecholamine  neu- 
rotransmitters with  the  activity  of  the  neuron 
during  its  function  in  the  nervous  system.  Indeed, 
stimuli  provided  by  light  or  smell  may  use  this 
pathway  to  control  neuronal  activity. 

The  c-Fos  protein  belongs  to  a  family  of  pro- 
teins including  FosB  and  Fral  and  Fra2,  all  of 
which  may  bind  to  c-Jun  or  to  members  of  the  Jun 
protein  family.  The  resulting  complexes  in  turn 
may  all  bind  to  the  same  DNA  element.  The  dif- 
ferent c-Fos  family  members  differ  in  their  pat- 
terns of  expression  following  cell  stimulation, 
as  well  as  in  their  structures  outside  the  DNA- 
binding  domain.  This  suggests  that  they  may  regu- 
late gene  activity  differentially  when  complexed 
with  a  DNA  element  such  as  the  TH  gene  regula- 
tory element. 

Recent  studies  in  our  laboratory  indicate  that 
the  TH  gene  is  repressed  by  a  mechanism  in 
which  the  c-Fos  protein,  an  activator,  is  replaced 
by  a  different  c-Fos  family  member,  which  serves 
as  a  repressor.  Indeed,  other  Fos  family  members, 
including  FosB,  become  the  predominant  species 
as  c-Fos  levels  dwindle  and  TH  transcription  is 
shut  off. 

We  have  gone  on  to  show  that  NGF  induces 
other  genes  as  well.  One  of  these  encodes  periph- 
erin,  a  neuron-specific  intermediate  filament 
protein  that  is  present  in  the  axons  of  peripheral 
neurons  as  a  component  of  the  neuronal  cytoskel- 
eton.  Our  studies  of  the  developing  rat  nervous 
system  indicate  that  peripherin  expression  coin- 
cides with  the  final  steps  of  neuronal  maturation 
and  acquisition  of  function. 

The  mechanisms  that  control  peripherin  ex- 
pression appear  to  be  quite  distinct  from  those 
that  control  c-Fos  or  TH.  We  detect  no  binding 
site  for  the  Fos-Jun  complex  in  the  peripherin 
gene.  Instead,  a  negative  regulatory  element  ap- 


pears to  release  an  inhibitory  factor,  thus  activat- 
ing the  gene. 

Study  of  these  mechanisms  may  give  a  clue  to 
how  cells  permanently  exit  from  the  cell  cycle 
and  induce  the  expression  of  genes  that  they  em- 
ploy after  losing  the  capacity  to  proliferate.  One 
event  that  may  block  exit  from  the  cell  cycle  is 
the  loss  of  control  of  expression  of  c-myc,  an- 
other growth  factor-induced  gene.  It  encodes  a 
protein,  c-Myc,  that  is  distantly  related  to  c-Fos 
and  has  specialized,  but  poorly  understood,  func- 
tion in  inducing  cell  proliferation.  It  is  expressed 
at  abnormally  high  levels  in  many  tumors. 

We  have  identified  a  DNA  nucleotide  sequence 
to  which  c-Myc  can  bind  and  a  protein  partner  of 
c-Myc  called  Myn  that  can  stimulate  its  DNA  bind- 
ing. Our  studies  of  c-Myc  and  Myn  and  their  com- 
plexes with  DNA  indicate  they  are  controlled  at 
many  levels,  including  modification  of  the  ma- 
ture proteins  by  phosphorylation.  Our  studies 
seek  to  reveal  the  role  of  c-Myc  in  normal  cell 
proliferation  and  in  tumorigenesis.  These  studies 
are  supported  by  the  National  Institutes  of 
Health. 

We  find  that  c-Myc  expression  is  elevated  in  the 
naturally  occurring  childhood  brain  tumor  me- 
dulloblastoma.  The  tumor  cells  have  been 
blocked  to  differentiation  and  have  proliferated 
abnormally.  We  also  find  that  the  transforming 
gene  of  adenovirus.  El  a,  like  c-myc,  can  block 
the  differentiation  of  our  model  neurons,  mimick- 
ing the  state  of  the  tumor. 

We  are  introducing  various  sorts  of  mutants  of 
the  c-Myc  protein  into  cells  to  disrupt  pathways 
stimulated  by  the  normal,  wild-type  c-Myc.  These 
experiments  may  reveal  other  protein  factors  that 
cooperate  with  c-Myc  in  controlling  cell  prolifer- 
ation and  differentiation.  We  seek  to  understand 
how  cells  coordinate  their  functional  maturation 
with  a  loss  of  ability  to  proliferate  as  they  un- 
dergo the  final  stages  of  differentiation. 


470 


Cell-Cell  Interactions  Determine  Cell  Fate  in  the 
Drosophila  Retina 


S.  Lawrence  Zipursky,  Ph.D. — Associate  Investigator 

Dr.  Zipursky  is  also  Associate  Professor  of  Biological  Chemistry  at  the  University  of  California  School  of 
Medicine,  Los  Angeles.  He  received  his  Ph.D.  degree  from  Albert  Einstein  College  of  Medicine,  where  he 
studied  mechanisms  ofDNA  replication  in  bacteria  in  the  laboratory  of  ferard  Hurwitz.  He  moved  to  the 
California  Institute  of  Technology  to  pursue  postdoctoral  studies  in  Drosophila  neurogenetics  with 
Seymour  Benzer.  He  has  been  on  the  faculty  of  UCLA  for  seven  years. 


WE  are  interested  in  two  questions  in  devel- 
opmental biology.  First,  How  are  specific 
cell  fates  established  through  cell-cell  interac- 
tions? And  second,  What  are  the  mechanisms  un- 
derlying the  specificity  of  neuronal  connectivity? 
To  address  these  issues,  we  have  focused  our  stud- 
ies on  the  development  of  the  Drosophila  visual 
system.  I  will  briefly  describe  progress  we  have 
made  in  understanding  the  cellular  and  molecu- 
lar mechanisms  underlying  the  specification  of  a 
unique  cell  fate  in  the  developing  retina.  This 
work  is  supported  in  part  by  a  grant  from  the  Na- 
tional Institutes  of  Health.  We  are  using  similar 
genetic  and  molecular  approaches  toward  under- 
standing how  different  classes  of  photoreceptor 
neurons  identify  their  unique  postsynaptic  tar- 
gets in  the  developing  brain. 

Development  of  the  Drosophila  Retina 

The  Drosophila  retina  has  a  near-crystalline 
structure  of  some  800  identical  units  called  om- 
matidia.  Each  ommatidium  contains  a  group  of 
cells  organized  in  a  stereotyped  fashion,  with 
eight  photoreceptor  neurons,  designated  R1-R8, 
forming  its  core,  surrounded  by  accessory  cells 
that  produce  screening  pigments  and  the  lens. 
Genetic  studies  in  the  1970s  established  that 
there  were  no  strict  cell  lineage  relationships  be- 
tween the  cells  in  the  fly's  eye.  Observations  have 
supported  the  view  that  the  developmental  mech- 
anisms regulating  the  acquisition  of  cell  identity 
are  dependent  not  on  a  cell's  ancestry  but  rather 
on  its  interactions  with  other  developing  cells. 

The  R  cells  are  the  first  to  develop  within  the 
ommatidial  unit.  Their  stereotyped  and  sequen- 
tial pattern  of  diff'erentiation  suggested  to  early 
workers  that  cell  fates  were  established  as  a  con- 
sequence of  a  cascade  of  inductive  interactions. 
Cell  fates  were  proposed  to  be  a  result  of  unique 
inductive  cues  provided  by  differentiating  neigh- 
boring cells.  The  first  cell  to  differentiate  is  R8, 
followed  by  the  cells  surrounding  it.  The  last  cell 
to  differentiate  is  R7.  It  contacts  the  differentiat- 
ing R8,  Rl,  and  R6  cells  as  well  as  a  number  of 
other  unpatterned  and  undifferentiated  cells.  It 
was  proposed  that  the  unique  R7  cell  fate  was 


induced  by  specific  signals  requiring  direct  cell 
contacts  between  the  R7  precursor  and  the  Rl, 
R6,  and  R8  cells. 

An  Inductive  Event  Specifying 
R7  Development 

The  first  step  toward  a  molecular  description 
of  the  developmental  mechanisms  regulating  R7 
development  was  the  discovery  of  the  sevenless 
{sev)  mutation.  In  flies  carrying  this  mutation, 
the  R7  cell  fails  to  assume  its  normal  fate  and 
becomes  a  nonneuronal  lens-secreting  cone  cell. 
The  sev  gene  was  shown  to  encode  a  receptor 
tyrosine  kinase,  which  is  expressed  in  the  R7  pre- 
cursor cell  and  many  other  cells  of  the  develop- 
ing eye.  The  molecular  structure  of  the  sev  pro- 
tein, its  expression  pattern,  and  its  genetic 
requirement  in  the  R7  precursor  cell  led  to  the 
proposal  that  it  is  a  receptor  for  an  inductive  cue 
specifying  an  R7  cell  fate. 

Several  years  ago  we  identified  a  mutation  in 
another  gene,  bride  of  sevenless  (boss),  which 
resulted  in  a  phenotype  identical  to  sev.  Genetic 
studies  indicated  that  boss  is  required  in  R8,  not 
for  its  own  development  but  for  that  of  the  R7 
cell.  This  raised  the  intriguing  notion  that  the 
boss  protein  is  an  inductive  ligand  to  which  the 
sev  receptor  binds.  Molecular  analysis  of  the  boss 
protein  revealed  that  it  is  an  integral  membrane 
protein  with  a  large  extracellular  domain,  multi- 
ple transmembrane  segments,  and  a  short  cyto- 
plasmic tail.  Using  antibodies,  we  showed  that 
boss  is  specifically  expressed  in  the  R8  neuron. 

Three  observations  argue  that  the  boss  protein 
is  a  ligand  for  the  sev  receptor.  First,  mixtures  of 
sev  and  boss-expressing  tissue  culture  cells  bind 
specifically  to  one  another.  Second,  membranes 
containing  the  boss  protein  rapidly  and  specifi- 
cally activate  the  sev  tyrosine  kinase  activity.  And 
finally,  boss  was  shown  to  be  transferred  from  the 
surface  of  the  R8  cell  to  an  organelle  in  R7  re- 
ferred to  as  a  multivesicular  body.  This  internal- 
ization is  strictly  dependent  upon  the  presence  of 
the  sev  protein  on  the  surface  of  the  R7  cell. 

Earlier  workers  had  demonstrated  that  the  sev 


471 


Cell-Cell  Interactions  Determine  Cell  Fate  in  the  Drosophila  Retina 


protein  is  expressed  on  many  cells  within  an  om- 
matidium  in  addition  to  the  R7  precursor  cell. 
Nevertheless,  only  one  cell  assumes  an  R7  cell 
fate.  Four  of  these  additional  cells  contact  the 
boss-expressing  R8  cell,  and  the  others  are  one  or 
more  cell  diameters  away.  We  have  shown  that 
multiple  mechanisms  limit  the  induction  to  only 
one  cell,  through  studies  in  which  we  examined 
R7  development  in  various  mutant  backgrounds 
and  in  response  to  ectopically  expressed  boss 
protein.  These  studies  revealed  that  the  cells  that 
do  not  contact  the  R8  cells  are  competent  to  re- 
spond to  the  inductive  cue  and  do  so  if  presented 


with  a  surface-bound  form  of  boss.  Conversely, 
the  sev-expressing  cells  that  normally  contact  R8 
are  restricted  from  responding  to  the  cue  as  a  re- 
sult of  a  very  early  developmental  event  that 
commits  them  to  alternative  fates. 

We  believe  that  similar  molecular  and  develop- 
mental strategies  involving  the  carefully  con- 
trolled expression  of  surface-bound  ligands  and 
competing  alternative  developmental  pathways 
will  be  important  in  establishing  the  remarkable 
cellular  diversity  and  organization  of  more- 
complex  nervous  systems,  such  as  the  vertebrate 
brain. 


472 


Molecular  Genetics  of  Sensory  Transduction 


Charles  S.  Zuker,  Ph.D. — Associate  Investigator 

Dr.  Zuker  is  also  Associate  Professor  of  Biology  and  of  Neurosciences  at  the  University  of  California  School 
of  Medicine,  San  Diego.  He  received  his  Ph.D.  degree  from  the  Massachusetts  Institute  of  Technology  for 
studies  with  Harvey  Lodish.  He  carried  out  postdoctoral  research  with  Gerald  Rubin  in  the  Department  of 
Biochemistry  at  the  University  of  California,  Berkeley,  before  joining  the  Department  of  Biology  at  UCSD. 
Dr.  Zuker  is  currently  a  Pew  Fellow  in  the  Biomedical  Sciences  and  a  Fellow  of  the  March  of  Dimes 
Foundation. 


AN  understanding  of  signal  transduction  is  es- 
sential to  elucidating  the  cellular  and  molec- 
ular basis  of  information  processing  in  biological 
systems.  The  primary  event  in  the  processing  of 
visual  stimuli  is  phototransduction,  the  conver- 
sion of  light  energy  into  a  change  in  the  ionic 
permeabilities  of  the  photoreceptor  cell  mem- 
brane. The  aim  of  our  research  is  to  clarify  mecha- 
nisms used  for  signal  transduction  in  the  visual 
system,  using  a  combined  molecular,  genetic, 
and  physiological  approach.  The  study  of  this  pro- 
cess in  Drosophila  applies  powerful  molecular 
genetic  techniques  to  identify  novel  transduc- 
tion molecules  and  to  examine  their  function  in 
vivo,  in  their  normal  cellular  and  organismal 
environment. 

Experimental  Strategy 

Over  the  past  few  years  my  colleagues  and  I 
have  been  working  on  the  isolation  and  character- 
ization of  genes  important  for  photoreceptor  cell 
function.  Most  recently  we  have  focused  on  genes 
encoding  proteins  involved  in  the  regulation  of 
the  visual  transduction  cascade.  We  have  identi- 
fied a  protein  kinase  C  (PKC)  that  is  expressed 
exclusively  in  the  Drosopbilavisual  system.  Anal- 
ysis of  the  light  response  from  mutants  defective 
in  this  PKC  showed  it  to  be  required  for  the  deac- 
tivation and  rapid  desensitization  of  the  visual 
cascade.  The  availability  of  a  PKC  mutant  in  Dro 
sophila  provides  the  basis  for  genetic  and  bio- 
chemical studies  to  identify  biologically  relevant 
substrates  and  regulators  of  this  enzyme. 

Rhodopsin,  like  other  G  protein-coupled  re- 
ceptors, is  phosphorylated  by  a  specific  kinase 
upon  activation  (rhodopsin  kinase).  This  reac- 
tion is  thought  to  be  involved  in  the  termination 
of  rhodopsin's  active  state.  Phosphorylation,  how- 
ever, is  not  sufficient  for  receptor  inactivation, 


since  the  phosphorylated  form  can  still  activate 
the  G  protein.  Complete  inactivation  has  been 
shown  {in  vitro)  to  require  the  stoichiometric 
interaction  of  rhodopsin  with  a  protein  called 
arrestin,  or  S  antigen.  This  protein  has  also  been 
implicated  in  a  number  of  autoimmune  retinal 
disorders  in  mammals.  We  have  isolated  the 
genes  encoding  two  photoreceptor  cell-specific 
arrestin  molecules  in  Drosophila  and  have  gen- 
erated mutants  of  these  genes.  Our  results  should 
help  us  understand  the  molecular  basis  of  G  pro- 
tein-coupled receptor  regulation  and  of  relevant 
abnormalities  in  the  human  visual  and  nervous 
systems. 

Mechanotransduction 

We  have  recently  begun  to  study  mechano- 
transduction, the  process  by  which  specialized 
sensory  cells  convert  mechanical  stimuli — for 
instance,  sound,  touch,  gravity,  or  movement — 
into  electrical  (neuronal)  signals.  In  contrast  to 
phototransduction,  nothing  is  known  about  the 
molecular  basis  of  mechanosensitivity.  Our  aim  is 
to  identify  genes  and  proteins  involved  in  mech- 
anotransduction by  isolating  mutations  that  affect 
mechanosensory  behavior. 

When  Drosophila  larvae  are  touched  gently, 
they  contract  and  retreat.  We  have  developed  a 
screen  for  mutant  larvae  defective  in  this  behav- 
ior and  have  isolated  several  mutant  lines.  Muta- 
tions that  specifically  affect  the  working  of  the 
sensory  organs  are  of  greatest  interest.  Mapping 
the  mutations — in  prelude  to  isolating  and  clon- 
ing the  affected  genes — is  in  progress.  In  addi- 
tion, we  have  developed  a  molecular  approach  to 
isolate  genes  expressed  specifically  in  mechano- 
sensory organs.  We  are  using  developmental  mu- 
tants that  overproduce  or  lack  mechanosensory 
bristles  to  isolate  such  genes  by  subtractive 
hybridization. 


475 


J 


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i 

I 

i 


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i 


1 


Investigators  by  Location 


ALABAMA 
Birmingham 

University  of  Alabama  at  Birmingham 
and  associated  hospitals 

Cooper,  Max  D.,  M.D. 

CALIFORNIA 
Berkeley 

University  of  California,  Berkeley, 
and  associated  hospitals 

Goodman,  Corey  S.,  Ph.D. 
Rubin,  Gerald  M.,  Ph.D. 
Schekman,  Randy  W.,  Ph.D. 
Tjian,  Robert,  Ph.D. 

Los  Angeles 

University  of  California,  Los  Angeles,  and 
associated  hospitals 

Kaback,  H.  Ronald,  M.D. 
Simpson,  Larry,  Ph.D. 
Smale,  Stephen  T.,  Ph.D. 
Witte,  Owen  N.,  M.D. 
Zipursky,  S.  Lawrence,  Ph.D. 

University  of  Southern  California 
and  associated  hospitals 

Lai,  Michael  M.-C,  M.D.,  Ph.D. 
Palo  Alto 

Stanford  University  and  the 
Stanford  University  Hospital 

Aldrich,  Richard  W.,  Ph.D. 
Barsh,  Gregory  S.,  M.D.,  Ph.D. 
Brown,  Patrick  O.,  M.D.,  Ph.D. 
Crabtree,  Gerald  R.,  M.D. 
Davis,  Mark  M.,  Ph.D. 
Francke,  Uta,  M.D. 

Goodnow,  Christopher  C,  B.V.Sc,  Ph.D. 
Kobilka,  Brian  K.,  M.D. 
Nusse,  Roel,  Ph.D. 
Scheller,  Richard  H.,  Ph.D. 
Schoolnik,  Gary  K.,  M.D. 
Weissman,  Irving  L.,  M.D. 

Pasadena 

California  Institute  of  Technology 
and  associated  hospitals 

Anderson,  David  J.,  Ph.D. 
Bjorkman,  Pamela  J.,  Ph.D. 
Sternberg,  Paul  W.,  Ph.D. 

San  Diego 

The  Salk  Institute  for  Biological  Studies 
Evans,  Ronald  M.,  Ph.D. 


Sejnowski,  TerrenceJ.,  Ph.D. 
Stevens,  Charles  F.,  M.D.,  Ph.D. 

University  of  California,  San  Diego, 
and  the  UCSD  Medical  Center 

Bevilacqua,  Michael  P.,  M.D.,  Ph.D. 
Emr,  Scott  D.,  Ph.D. 
Rosenfeld,  Michael  G.,  M.D. 
Tsien,  Roger  Y.,  Ph.D. 
Zuker,  Charles  S.,  Ph.D. 

San  Francisco 

University  of  California,  San  Francisco, 
and  associated  hospitals 

Agard,  David  A.,  Ph.D. 
Ganem,  Donald  E.,  M.D. 
Gitschier,  Jane  M.,  Ph.D. 
Grosschedl,  Rudolf,  Ph.D. 
Jan,  Lily  Y.,  Ph.D. 
Jan,  Yuh  Nung,  Ph.D. 
Kan,  Yuet  Wai,  M.D.,  D.Sc. 
Littman,  Dan  R.,  M.D.,  Ph.D. 
Payan,  Donald  G.,  M.D. 
Peterlin,  B.  Matija,  M.D. 
Reichardt,  Louis  F.,  Ph.D. 
Sedat,  John  W  ,  Ph.D. 
Weiss,  Arthur,  M.D.,  Ph.D. 
Williams,  Lewis  T.,  M.D.,  Ph.D. 

COLORADO 
Boulder 

University  of  Colorado  at  Boulder  and  the 
University's  Health  Sciences  Center 

Cech,  Thomas  R.,  Ph.D. 
Kirkegaard,  Karia  A.,  Ph.D. 

Denver 

National  Jewish  Center  for  Immunology 
and  Respiratory  Medicine 

Kappler,  John  W.,  Ph.D. 
Marrack,  Philippa,  Ph.D. 

University  of  Colorado  Health  Sciences  Center 
and  associated  hospitals 

Mailer,  James  L.,  Ph.D. 

CONNECTICUT 
New  Haven 

Yale  University  and  associated  hospitals 

Artavanis-Tsakonas,  Spyridon,  Ph.D. 
Bottomly,  H.  Kim,  Ph.D. 
Briinger,  Axel  T.,  Ph.D. 
Cresswell,  Peter,  Ph.D. 


475 


Investigators  by  Location 


De  Camilli,  Pietro,  M.D. 
Flavell,  Richard  A.,  Ph.D. 
Fox,  Roben  O.,  Ph.D. 
Ghosh,  Sankar,  Ph.D. 
Horwich,  Arthur  L.,  M.D. 
Jahn,  Reinhard,  Ph.D. 
Janeway,  Charles  A  ,  Jr.,  M.D. 
Lerner,  Michael  R.,  M.D.,  Ph.D. 
Reeders,  Stephen  T.,  M.D. 
Schatz,  David  G  ,  Ph.D. 
Sigler,  Paul  B.,  M.D.,  Ph.D. 
Steitz,  Joan  A.,  Ph.D. 
Steitz,  Thomas  A.,  Ph.D. 
Tanabe,  Tsutomu,  Ph.D. 
Wolin,  Sandra  L.,  M.D.,  Ph.D. 

GEORGIA 
Atlanta 

Emory  University  School  of  Medicine 
and  associated  hospitals 

Warren,  Stephen  T.,  Ph.D. 

ILLINOIS 

Chicago 

The  University  of  Chicago  and 
The  University  of  Chicago  Hospitals 

Bell,  Graeme  I.,  Ph.D. 
Fuchs,  Elaine,  Ph.D. 
Laimins,  Laimonis  A.,  Ph.D. 
Lindquist,  Susan  L.,  Ph.D. 
Singh,  Harinder,  Ph.D. 
Steiner,  Donald  F.,  M.D. 
Sukhatme,  Vikas  P.,  M.D.,  Ph.D. 

Evanston 

Northwestern  University  and  associated  hospitals 
Lamb,  Robert  A.,  Ph.D. 


Donelson,  John  E.,  Ph.D. 
Welsh,  Michael  J.,  M.D. 

MARYLAND 
Baltimore 

The  Carnegie  Institution  of  Washington 
and  The  Johns  Hopkins  Hospital 

McKnight,  Steven  Lanier,  Ph.D. 
Spradling,  Allan  C,  Ph.D. 

The  Johns  Hopkins  University 
and  Hospital 

Beachy,  Philip  A.,  Ph.D. 

Corden,  Jeffry  L  ,  Ph.D. 

Craig,  Nancy  L.,  Ph.D. 

Desiderio,  Stephen  V.,  M.D.,  Ph.D. 

Huganir,  Richard  L.,  Ph.D. 

Nathans,  Daniel,  M.D. 

Nathans,  Jeremy,  M.D.,  Ph.D. 

Reed,  Randall  R.,  Ph.D. 

Valle,  David  L.,  M.D. 

Yau,  King-Wai,  Ph.D. 

Yellen,  Gary,  Ph.D. 

MASSACHUSETTS 
Boston 

Brigham  and  Women's  Hospital 

Chin,  William  W.,  M.D. 
Cunningham,  James  M.,  M.D. 
Maas,  Richard  L.,  M.D.,  Ph.D. 

The  Children 's  Hospital 


Alt,  Frederick  W.,  Ph.D. 
Kunkel,  Louis  M.,  Ph.D. 
Nadal-Ginard,  Bernardo,  M.D. 
Orkin,  Stuart  H.,  M.D. 


Ph.D. 


INDIANA 
Bloomington 

Indiana  University  and  associated  hospitals 

Kaufman,  Thomas  C,  Ph.D. 

Indianapolis 

Indiana  University  School  of  Medicine 
and  associated  hospitals 

Williams,  David  A.,  M.D. 

IOWA 
Iowa  City 

University  of  Iowa  and  associated  hospitals 


Harvard  Medical  School  and 
Brigham  and  Women 's  Hospital 

Church,  George  M.,  Ph.D. 
Duyk,  Geofifrey  M.,  M.D.,  Ph.D. 
Leder,  Philip,  M.D. 
Perrimon,  Norbert,  Ph.D. 
Seidman,  Jonathan  G.,  Ph.D. 

Massachusetts  General  Hospital 

Alexander-Bridges,  Maria  C,  M.D., 
Corey,  David  P.,  Ph.D. 
Habener,  Joel  F.,  M.D. 


Ph.D. 


Campbell,  Kevin  P.,  Ph.D. 


476 


Investigators  by  Location 


Tufts  University  School  of  Medicine 
and  associated  hospitals 

Isberg,  Ralph  R.,  Ph.D. 
Cambridge 

Harvard  College,  Arts  and  Sciences, 
and  The  Children 's  Hospital 

Harrison,  Stephen  C,  Ph.D. 
Wiley,  Don  C,  Ph.D. 

Massachusetts  Institute  of  Technology 
and  associated  hospitals 

Horvitz,  H.  Robert,  Ph.D. 
Hynes,  Richard  O.,  Ph.D. 
Kim,  Peter  S.,  Ph.D. 
Lehmann,  Ruth,  Ph.D. 
Pabo,  Carl  O.,  Ph.D. 
Page,  David  C,  M.D. 
Steller,  Hermann,  Ph.D. 
Tonegawa,  Susumu,  Ph.D. 

Waltham 

Brandeis  University  and  associated  hospitals 

Miller,  Christopher,  Ph.D. 
Rosbash,  Michael,  Ph.D. 

Worcester 

University  of  Massachusetts 
and  associated  hospitals 

Davis,  Roger  J.,  Ph.D. 

MICfflGAN 
Ann  Arbor 

University  of  Michigan  and  associated  hospitals 

Bonadio,  Jeffrey  F.,  M.D. 
Collins,  Francis  S.,  M.D.,  Ph.D. 
Feinberg,  Andrev^^  P.,  M.D.,  M.P.H. 
Ginsburg,  David,  M.D. 
Kurnit,  David  M.,  M.D.,  Ph.D. 
Leiden,  Jeffrey  M.,  M.D.,  Ph.D. 
Lowe,  John  B.,  M.D. 
Nabel,  GaryJ.,  M.D.,  Ph.D. 
Thompson,  Craig  B.,  M.D. 
Wilson,  James  M.,  M.D.,  Ph.D. 

MISSOURI 
St.  Louis 

Washington  University  and  associated  hospitals 

Atkinson,  John  P.,  M.D. 
Chaplin,  David  D.,  M.D.,  Ph.D. 
Holers,  V.  Michael,  M.D. 


Korsmeyer,  Stanley  J.,  M.D. 
Loh,  Dennis  Y.-D.,  M.D. 
Olson,  Maynard  V.,  Ph.D. 
Pike,  Linda  J.,  Ph.D. 
Sadler,  J.  Evan,  M.D.,  Ph.D. 
Thomas,  Matthevi^  L.,  Ph.D. 

NEW  JERSEY 
Princeton 

Princeton  University  and  associated  medical  centers 

Shenk,  Thomas  E.,  Ph.D. 
Tilghman,  Shirley  M.,  Ph.D. 

NEW  YORK 
Bronx 

Albert  Einstein  College  of  Medicine 
of  Yeshiva  University  and 
associated  hospitals 

Bloom,  Barry  R.,  Ph.D. 
Jacobs,  William  R.,  Jr.,  Ph.D. 

Cold  Spring  Harbor 

Cold  Spring  Harbor  Laboratory 
and  associated  hospitals 

Beach,  David  H.,  Ph.D. 
New  York 

Columbia  University  and  associated  hospitals 

Axel,  Richard,  M.D. 
Hendrickson,  Wayne  A.,  Ph.D. 
Jessell,  Thomas  M.,  Ph.D. 
Kandel,  Eric  R.,  M.D. 
Siegelbaum,  Steven  A.,  Ph.D. 
Struhl,  Gary,  Ph.D. 

Cornell  University  Medical  College 

O'Donnell,  Michael  E.,  Ph.D. 

Memorial  Sloan-Kettering  Cancer  Center 

Massague,  Joan,  Ph.D. 

New  York  University  (Medical  Center  and 
Washington  Square )  and  associated  hospitals 

Movshon,  J.  Anthony,  Ph.D. 
Ziff,  Edward  B  ,  Ph.D. 

The  Rockefeller  University  and 
Rockefeller  University  Hospital 

Blobel,  Gunter,  M.D.,  Ph.D. 
Burley,  Stephen  K.,  Ph.D. 


477 


Investigators  by  Location 


Desplan,  Claude,  Ph.D. 
Friedman,  Jefifrey  M.,  M.D.,  Ph.D. 
Geliebter,  Jan,  Ph.D. 
Heintz,  Nathaniel,  Ph.D. 
Kuriyan,  John,  Ph.D. 
Nussenzweig,  Michel  C,  M.D.,  Ph.D. 
Sakmar,  Thomas  P.,  M.D. 
Young,  Michael  W.,  Ph.D. 

Stony  Brook 

State  University  of  New  York  at  Stony  Brook  and 
University  Hospital  at  Stony  Brook 

Adams,  Paul  R.,  Ph.D. 

NORTH  CAROLINA 
Durham 

Duke  University,  including  Duke  University 
Medical  Center 

Bennett,  G.  Vann,  M.D.,  Ph.D. 
Blackshear,  Perry  J.,  M.D.,  D.Phil. 
Caron,  Marc  G.,  Ph.D. 
Cullen,  Bryan  R.,  Ph.D. 
Davis,  Laura  I.,  Ph.D. 
Lefkowitz,  Robert  J.,  M.D. 
Nevins,  Joseph  R.,  Ph.D. 
Parker,  Keith  L.,  M.D.,  Ph.D. 

OKLAHOMA 
Oklahoma  City 

Oklahoma  Medical  Research  Foundation 
and  associated  hospitals 

Esmon,  Charles  T.,  Ph.D. 

OREGON 
Eugene 

University  of  Oregon  and  associated  hospitals 
Matthews,  Brian  W.,  Ph.D. 

PENNSYLVANIA 
Philadelphia 

University  of  Pennsylvania 
and  associated  hospitals 

Brugge,  Joan  S.,  Ph.D. 
Dreyfuss,  Gideon,  Ph.D. 
Kadesch,  Thomas  R.,  Ph.D. 
Liebhaber,  Stephen  A.,  M.D. 
Malim,  Michael  H.,  Ph.D. 
Nussbaum,  Robert  L.,  M.D. 
Taub,  Rebecca  A.,  M.D. 


TENNESSEE 
Memphis 

St.  Jude  Children 's  Research  Hospital 
Sherr,  Charles  J.,  M.D.,  Ph.D. 

Nashville 

Vanderbilt  University,  including  Vanderbilt 
University  Hospital 

Exton,  John  H.,  M.D.,  Ph.D. 

TEXAS 
Dallas 

University  of  Texas  Southwestern  Medical  Center 
at  Dallas  and  associated  hospitals 

Beutler,  Bruce  A.,  M.D. 
Deisenhofer,  Johann,  Ph.D. 
Fischer  Lindahl,  Kirsten,  Ph.D. 
Garbers,  David  L.,  Ph.D. 
Gething,  Mary-Jane  H.,  Ph.D. 
Sprang,  Stephen  R.,  Ph.D. 
Siidhof,  Thomas  C,  M.D. 
Yanagisawa,  Masashi,  M.D.,  Ph.D. 

Houston 

Baylor  College  of  Medicine  and  associated  hospitals 

Beaudet,  Arthur  L.,  M.D. 
Bellen,  Hugo  J.,  D.V.M.,  Ph.D. 
Belmont,  John  W.,  M.D.,  Ph.D. 
Caskey,  C.  Thomas,  M.D. 
Cohen,  Stephen  M.,  Ph.D. 
Ledley,  Fred  D.,  M.D. 
Overbeek,  Paul  A  ,  Ph.D. 
Quiocho,  Florante  A.,  Ph.D. 
Soriano,  Philippe  M.,  Ph.D.,  D.Sc. 
Woo,  Savio  L.  C,  Ph.D. 

Rice  University  and  associated  hospitals 

Gomer,  Richard  H  ,  Ph.D. 

UTAH 

Salt  Lake  City 

University  of  Utah,  including  University  of  Utah 
Medical  Center 

Capecchi,  Mario  R.,  Ph.D. 
Gesteland,  Raymond  F.,  Ph.D. 
Lalouel,  Jean-Marc,  M.D.,  D.Sc. 
Sakonju,  Shigeru,  Ph.D. 
Thummel,  Carl  S.,  Ph.D. 
White,  Raymond  L.,  Ph.D. 


478 


Investigators  by  Location 


WASfflNGTON 
Seattle 

Fred  Hutchinson  Cancer  Research  Center 

Henikofr,  Steven,  Ph.D. 
Weintraub,  Harold  M.,  M.D.,  Ph.D. 

University  of  Washington 
and  associated  hospitals 

Bevan,  Michael  J.,  Ph.D. 
Glomset,  John  A.,  M.D. 
Hurley,  James  B.,  Ph.D. 
Palmiter,  Richard  D  ,  Ph.D. 
Perlmutter,  Roger  M.,  M.D.,  Ph.D. 

WISCONSIN 
Madison 

University  of  Wisconsin 
and  associated  hospitals 

Carroll,  Sean  B.,  Ph.D. 


479 


International  Research  Scholars 


In  recognition  of  the  contributions  of  scientists  outside  of  the  United  States  to  advances  in  biomedi- 
cal science,  the  Institute  initiated  in  1991  its  International  Research  Scholars  Program.  This  is  a 
limited  experimental  effort  that  provides  research  support  through  five-year  grants  to  promising 
scientists  working  in  areas  of  fundamental  biomedical  research  related  to  the  Institute's  ongoing 
medical  research  programs. 

The  investigators  invited  to  compete  for  the  initial  grant  awards  were  located  in  Canada  and  Mexico. 
From  those  invited  to  compete,  24  were  selected  (14  in  Canada,  10  in  Mexico).  Grants  also  were 
awarded  to  the  Mexican  Academia  de  la  Investigacion  Cientifica  and  to  the  United  States  National 
Academy  of  Sciences  for  joint  activities  over  four  years  to  promote  the  exchange  of  scientific  informa- 
tion and  encourage  cooperation  between  the  scientific  communities  in  each  country,  particularly  in 
the  life  sciences. 

The  next  phase  of  the  international  program  will  focus  on  biomedical  scientists  in  the  United 
Kingdom,  Australia,  and  New  Zealand.  The  emphasis  remains  on  the  support  of  outstanding  scientists 
whose  research  careers  are  still  developing,  rather  than  those  in  the  later  phases  of  a  distinguished 
career.  Grant  awards  for  this  second  group  will  be  announced  late  in  1992. 


Canada 


Mexico 


Bernstein,  Alan,  Ph.D. 
Finlay,  B.  Brett,  Ph.D. 
Greenblatt,  Jack,  Ph.D. 
Grinstein,  Sergio,  Ph.D 
Gros,  Philippe,  Ph.D. 


Arias,  Carlos  F.,  Ph.D. 

Calva,  Edmundo,  Ph.D. 

Cota,  Gabriel,  Ph.D. 

Darszon,  Alberto,  Ph.D. 

Guarneros  Pena,  Gabriel,  Ph.D. 

Herrera-Estrella,  Luis  R.,  Ph.D. 

Lizardi,  Paul  M.,  Ph.D. 

Orozco,  Esther,  Ph.D. 

Possani,  Lourival  Domingos,  Ph.D. 

Romo,  Ranulfo,  M.D.,  Ph.D. 


Joyner,  Alexandra  L.,  Ph.D. 
McGhee,  James  D.,  Ph.D. 
Mosmann,  Tim  R..,  Ph.D. 


Pawson,  Tony,  Ph.D. 
Read,  Randy  J.,  Ph.D. 
Rossant,  Janet,  Ph.D. 
Roy,  Jean-Pierre,  M.D. 
Snutch,  Terry  P.,  Ph.D 
Tsui,  Lap-Chee,  Ph.D. 


481 


Molecular  Biology  and  Epidemiology 
for  Control  of  Rotavirus  Diarrhea 


Carlos  F.  Arias,  Ph.D. — International  Research  Scholar 

Dr.  Arias  is  Investigador  Titular  B,  Department  of  Molecular  Biology,  at  the  Biotechnology  Institute, 
National  Autonomous  University  of  Mexico,  Cuernavaca.  He  received  his  undergraduate  degree  in 
biochemistry  and  his  M.S.  and  Ph.D.  degrees  in  biomedical  research  from  the  National  University  of 
Mexico,  Mexico  City.  He  recently  conducted  sabbatical  research  with  James  Strauss  at  the  California 
Institute  of  Technology. 


ACUTE,  infectious  diarrhea  is  the  commonest 
Lcause  of  morbidity  and  mortality  among 
young  children  living  in  developing  countries, 
accounting  for  as  many  as  one  billion  illnesses 
and  between  four  and  five  million  deaths  an- 
nually. Rotaviruses  are  the  leading  cause  of  se- 
vere diarrheal  disease  in  children  under  three 
years  of  age,  and  it  is  estimated  that  an  effective 
vaccine  would  save  about  800,000  children's 
lives  per  year. 

Because  rotavirus  plays  such  an  important  role 
in  severe  infantile  gastroenteritis,  and  because 
even  advanced  levels  of  hygiene  seem  unable  to 
control  the  spread  of  rotavirus  infections,  there 
has  been  considerable  interest  in  developing  ef- 
fective vaccination  strategies. 

Our  laboratory  is  interested  in  studying  how 
rotaviruses  attach  and  enter  their  host  cell  and 
how  they  replicate  there  to  produce  the  viral 
progeny.  Also  among  our  interests  are  the  host 
immune  response  to  rotavirus  infection  and  the 
epidemiology  of  these  viruses  in  Mexico. 

As  in  other  infectious  agents,  proteins  located 
at  the  surface  of  the  rotavirus  particles  are  in- 
volved in  the  early  interactions  (attachment  and 
penetration)  of  the  virions  with  the  host  cell. 
These  proteins  contain  antigenic  determinants 
that  represent  major  immunological  targets.  The 
structural  and  functional  characterization  of  the 
viral  surface  proteins,  as  well  as  the  study  of  im- 
mune response  in  the  infected  host,  should  con- 
tribute to  the  development  of  rationally  designed 
therapeutic  agents  and  new  prevention  strate- 
gies. Moreover,  the  success  of  a  vaccine  may  de- 
pend on  knowledge  of  the  epidemiology  of  the 
pathogen  in  the  particular  geographic  area  where 
the  vaccine  is  to  be  used. 

The  surface  of  the  rotavirus  contains  two  pro- 
teins, VP4  and  VP7.  VP4  forms  spikes  that  extend 
from  the  viral  surface.  This  protein  has  been  asso- 
ciated with  a  variety  of  viral  functions,  including 
the  determination  of  virulence  in  vivo  and  the 
ability  to  agglutinate  red  blood  cells  (hemagglu- 
tination). VP4  is  also  important  in  the  penetra- 
tion of  the  virion  into  the  cell.  On  the  other  hand. 


the  protein  responsible  for  the  initial  attachment 
of  rotavirus  to  its  host  cell  remains  controversial; 
both  VP4  and  VP7  have  been  proposed. 

The  attachment  of  animal  rotaviruses  to  cells  in 
culture  is  mediated  by  compounds  containing 
sialic  acid,  since  treatment  of  cells  with  sialidases 
greatly  reduces  the  binding  of  virus  particles  to 
the  cell  surface.  In  addition,  the  hemagglutinat- 
ing  activity  of  rotaviruses  and  their  binding  to 
cultured  epithelial  cells  can  also  be  inhibited  by 
incubation  of  the  virus  with  sialoglycoproteins 
such  as  glycophorin. 

We  have  isolated  rotavirus  mutants  whose  bind- 
ing is  no  longer  inhibited  by  treatment  of  cells 
with  sialidases  or  incubation  with  glycophorin. 
The  detailed  analysis  of  these  mutants  should 
give  us  information  about  the  protein  (s)  in- 
volved in  the  attachment  of  the  virions  to  the  cell. 
Furthermore,  we  are  constructing  deletion  mu- 
tants and  chimeras  between  VP4  genes  of  hemag- 
glutinating  and  nonhemagglutinating  rotavi- 
ruses, to  define  further  the  protein  domains 
involved  in  the  binding  of  the  virus  to  epithelial 
and  red  blood  cells. 

After  the  virus's  initial  attachment  to  the  cell 
surface,  the  next  step  in  its  infection  cycle  is  to 
enter  the  cell.  The  entry  of  the  virus  particle  can 
be  augmented  by  treating  it  with  trypsin,  and 
probably  is  trypsin  dependent.  Experiments  in 
progress  have  shown  that  this  proteolytic  treat- 
ment induces  three  specific  cleavages  of  VP4.  We 
are  interested  in  identifying  the  cleavage (s)  di- 
rectly associated  with  enhancement  of  viral  infec- 
tivity  and  in  learning  about  the  mechanism 
through  which  the  cleaved  VP4  protein  mediates 
the  virion's  penetration  of  the  cell. 

Both  surface  proteins  have  also  been  character- 
ized immunologically.  It  has  been  found  that  anti- 
bodies to  either  protein  neutralize  the  virus  in 
vitro  and  are  capable  of  passively  protecting  ani- 
mals from  rotavirus  challenge.  In  addition,  oral 
infection  with  live  rotavirus  stimulates  protec- 
tive immunity,  which  can  be  mediated  by  VP4 
and/or  VP7.  These  observations  make  the  two 
proteins  attractive  candidates  for  recombinant 
subunit  vaccines. 


483 


Molecular  Biology  and  Epidemiology  for  Control  of  Rotavirus  Diarrhea 


Rotavirus  infections  are  limited  to  the  gut,  and 
it  is  believed  that  a  vaccine  will  have  to  be  given 
orally  to  stimulate  a  local  (mucosal)  immune  re- 
sponse in  order  to  induce  protection.  An  attrac- 
tive approach  to  this,  alternative  to  the  use  of  live 
attenuated  virus,  is  the  use  of  live  oral  vaccines 
containing  nonpathogenic  strains  of  enteric  bac- 
teria carrying  the  genes  that  code  for  the  protec- 
tive rotavirus  antigens.  We  are  currently  con- 
structing expression  vectors  to  direct  the 
synthesis  of  the  surface  proteins  of  rotavirus  in 
the  enteric  bacteria  Salmonella  and  Lactobacil- 
lus, and  will  test  the  potential  of  these  recombi- 


nant strains  to  induce  a  protective  immune  re- 
sponse against  rotavirus  infection. 

Finally,  our  laboratory  is  also  interested  in  the 
study  of  some  aspects  of  the  epidemiology  of  ro- 
tavirus. Six  different  serotypes  of  human  rotavir- 
uses have  been  identified,  four  of  which  appear 
to  account  for  the  majority  of  isolates.  We  have 
found  that  the  four  major  serotypes  circulate  in 
Mexico.  We  are  currently  studying  the  molecular 
determinants  of  virulence  of  rotavirus  and  inves- 
tigating how  the  frequency  of  the  different  sero- 
types changes  over  time  and  in  relation  to 
virulence. 


In  situ  localization  o/c-kit  and  Steel  ex- 
pression in  adult  mouse  cerebellum.  The 
protein  products  of  the  proto- oncogene 
c-kit  and  its  ligand,  the  protein  product 
of  the  Steel  gene,  interact  to  activate  Kit 
tyrosine  kinase  activity  vital  to  the  func- 
tion of  melanocytes,  blood  cells,  and 
germ  cells.  The  abbreviations  identify 
the  molecular  layer  (mol),  granular 
layer  (gran),  and  Purkinje  cells  (Pur). 

From  Motro,  B.,  van  der  Kooy,  D., 
Rossant,  f.,  Reith,  A.,  and  Bernstein,  A. 
1991.  Development  115:1207-1221. 


484 


Molecular  Genetics  of  Normal  and  Leukemic 
Hematopoiesis 


Alan  Bernstein,  Ph.D. — International  Research  Scholar 

Dr.  Bernstein  is  Head  of  the  Division  of  Molecular  and  Developmental  Biology  and  Associate  Director  of 
the  Samuel  Lunenfeld  Research  Institute  at  Mount  Sinai  Hospital,  Toronto.  He  received  his  Ph.D.  degree 
in  medical  biophysics  from  the  University  of  Toronto  and  trained  as  a  postdoctoral  fellow  at  the  Imperial 
Cancer  Research  Fund  Laboratories,  London.  Before  moving  to  Mount  Sinai  he  was  Senior  Scientist  in 
the  Division  of  Biological  Research  at  the  Ontario  Cancer  Institute.  He  is  the  first  incumbent  of  the  Anne 
Tanenbaum  Chair  in  Molecular  and  Developmental  Biology  and  was  recently  elected  a  Fellow  of  the  Royal 
Society  of  Canada. 


MY  laboratory  is  interested  in  the  molecular 
mechanisms  that  govern  the  orderly  produc- 
tion of  hematopoietic  cells  in  the  adult  mammal. 
It  is  clear  from  earlier  work  involving  the  genera- 
tion of  unique  clonal  markers  (either  visible  cyto- 
genetic markers  or  the  chromosomal  integration 
sites  of  retroviral  vectors)  that  hematopoiesis  in- 
volves the  proliferation  and  differentiation  of 
pluripotent  stem  cells.  These  cells  have  the  devel- 
opmental and  proliferative  capacity  to  produce 
the  millions  of  myeloid  and  lymphoid  cells  that 
are  continuously  required  in  adult  life.  Thus  the 
hematopoietic  system  has  served  as  an  attractive 
and  important  experimental  model  of  develop- 
mental processes.  Defects  in  hematopoiesis  can 
also  lead  to  disease,  including  anemias,  leuke- 
mias,  and  such  genetic  disorders  as  sickle  cell 
anemia  and  thalassemia,  making  the  understand- 
ing of  this  system  important  to  both  medicine  and 
biology. 

We  are  taking  two  main  approaches  to  eluci- 
date the  mechanisms  that  control  the  regulation 
of  hematopoietic  stem  cells.  The  first  approach 
involves  the  analysis  of  mouse  mutations  that 
disrupt  hematopoiesis,  while  the  second  involves 
the  study  of  the  multistage  erythroleukemias  in- 
duced by  the  various  forms  of  Friend  murine  leu- 
kemia virus,  a  murine  retrovirus. 

The  white-spotting  (IT)  and  Steel  (^Sl)  Loci 

Mutations  at  either  the  dominant  white- 
spotting  {W}  or  Steel  (SI)  loci  can  lead  to  coat 
color  defects,  severe  macrocytic  anemia,  and  ste- 
rility. This  similarity  in  pleiotropic  phenotype  of 
W^and  SI  mutants  is  striking,  particularly  as  there 
is  no  common  developmental  origin  of  cells  that 
give  rise  to  melanocytes,  blood  cells,  and  germ 
cells.  Furthermore,  the  Wand  SI  loci  are  clearly 
distinct  genes,  as  they  map  on  mouse  chromo- 
somes 5  and  10,  respectively. 

Initial  insights  into  their  mechanism  of  action 
came  from  in  vivo  cell-mixing  experiments,  in- 
volving either  bone  marrow  transplantation  or 
aggregation  chimeras  with  animals  of  different  ge- 
notypes. These  early  studies  established  that  the 


developmental  defects  in  W  mutant  mice  result 
from  a  cell-autonomous,  intrinsic  stem  cell  de- 
fect, whereas  the  cellular  defect  in  5/  mutant 
mice  is  in  the  microenvironment  in  which  these 
cells  develop  in  the  embryo  and  function  in  the 
adult  animal. 

Several  years  ago,  we  showed  that  H^was  allelic 
with  the  proto-oncogene  c-kit,  a  member  of  the 
family  of  genes  that  includes  c-fms  and  pdgfr, 
which  encode  transmembrane  receptors  with  ty- 
rosine kinase  activity.  Different  W  alleles  are  ei- 
ther deletions  or  point  mutations  in  the  cytoplas- 
mic tyrosine  kinase  domain  that  result  in  partial 
or  complete  loss  of  tyrosine  kinase  activity  and  a 
consequent  reduction  in  the  signaling  capability 
of  the  Kit  receptor.  Thus  the  W  mutant  mouse 
provided  the  first  example  of  a  germline  muta- 
tion in  a  mammalian  proto-oncogene.  This  work 
is  supported  by  a  grant  from  the  National  Insti- 
tutes of  Health. 

These  findings  predicted  that  SI  might  encode 
the  ligand  for  the  Kit  receptor  and  that  this  ligand 
might  be  a  potent  growth  or  survival  factor  for 
melanoblasts,  germ  cells,  and  hematopoietic 
stem  cells.  The  microenvironmental  nature  of  the 
defects  in  SI  mutant  mice  also  predicted  that  the 
5/  protein  product  might  be  membrane  bound. 
All  three  predictions  have  turned  out  to  be 
correct.  Thus  the  W-Sl  gene  pair  has  provided 
strong  molecular  genetic  evidence  for  the  impor- 
tant role  that  cell-cell  interactions  play  in  the  de- 
velopment and  function  of  hematopoietic  and 
other  stem  cells.  Their  protein  products  are  strik- 
ingly similar  in  design  and  function  to  those  of 
the  sev  and  boss  genes,  which  control  the  devel- 
opment of  the  R7  photoreceptor  cell  in  the  com- 
pound eye  of  the  fruit  fly  Drosophila. 

Friend  Leukemia  Virus 

In  the  second  approach,  we  are  identifying  and 
analyzing  cellular  genes  important  in  the  induc- 
tion of  the  multistage  erythroleukemias  induced 
by  Friend  leukemia  virus.  Several  years  ago,  we 
showed  that  the  p53  gene  was  inactivated  by  de- 
letion or  retroviral  insertion  in  approximately  30 


485 


Molecular  Genetics  of  Normal  and  Leukemic  Hematopoiesis 


percent  of  independent  leukemic  clones  isolated 
from  the  spleens  of  mice  infected  with  Friend 
virus.  These  experiments  suggested  that  />53  is  a 
tumor-suppressor  gene,  a  conclusion  supported 
by  recent  experiments  demonstrating  allelic  loss 
and  mutation  in  the  p53  gene  in  a  broad  spectrum 
and  high  proportion  of  human  cancers. 

Tlie  importance  of  p53  in  the  evolution  of 
Friend  leukemia  also  became  evident  from  stud- 
ies on  transgenic  mice  that  express  high  levels  of 
mutant  forms  of  the  p55  protein.  In  addition  to 
displaying  an  increased  spontaneous  predisposi- 
tion to  a  variety  of  malignancies,  these  p53  trans- 
genic mice  are  more  susceptible  to  the  late  stages 
of  Friend  leukemia.  Thus  inactivation  of  the  p53 
gene,  either  somatically  after  Friend  virus  infec- 
tion or  as  the  result  of  inheritance  of  dominant 
negative  alleles  of  p53,  appears  to  be  a  central 
event  in  the  disease.  These  studies  also  suggested 
that  it  is  the  accumulation  of  a  specific  set  of  mu- 
tational events,  rather  than  the  order  in  which 
they  normally  occur,  that  is  critical  for  leukemia 
induction. 

In  addition  to  inactivation  of  the  p53  gene,  ac- 
tivation of  one  of  two  novel  members  of  the  ets 
gene  family  of  DNA-binding  transcriptional  acti- 
vators occurs  during  the  evolution  of  Friend  leu- 
kemia. Transcription  of  the  Spi-\  gene  is  acti- 
vated as  a  result  of  the  integration  of  Friend 
spleen  focus-forming  virus  (SFFV)  in  95  percent 
of  erythroleukemic  clones  induced  in  adult  mice 
by  SFFV.  In  contrast,  we  showed  last  year  that 
transcription  of  another  ets  family  member,  Fli- 1 , 
is  activated  by  insertion  of  the  replication- 
competent  Friend  MuLV  in  75  percent  of  erythro- 
leukemia  clones  induced  after  infection  of  new- 


born mice.  The  members  of  the  ets  gene  family, 
which  include  c-ets- 1  and  c-ets-2,  elk- 1  and  elf-1, 
erg,  E74,  5p/-l/PU.l,  and  Fli-\,  contain  a  con- 
served domain  of  80-90  amino  acids,  the  ETS  do- 
main, which  is  involved  in  specific  DNA  binding, 
as  well  as  a  less-conserved  transcriptional  activa- 
tion domain. 

The  strict  specificity  of  the  integration  sites  for 
SFFV  and  Friend  MuLV  is  intriguing,  as  Spi-\  and 
Fli-\  are  both  members  of  the  same  gene  family 
and  both  SFFV  and  Friend  MuLV  induce  erythro- 
leukemias  involving  activation  of  the  receptor  for 
erythropoietin  and  inactivation  of  the  p53  gene. 
We  are  currently  pursuing  the  hypothesis  that 
Fli- 1  and  Spi- 1  are  functionally  distinct  genes,  en- 
coding proteins  that  transactivate  a  distinct  set  of 
genes  downstream  in  the  leukemogenic  pathway. 
These  studies  involve  the  analysis  of  the  DNA- 
binding  specificity  of  the  Fli-\  and  Spi-  \  proteins 
as  well  as  the  generation  of  mutant  mice  carrying 
either  gain-  or  loss-of-function  mutations  in  these 
genes.  This  work  is  supported  by  a  grant  from  the 
National  Cancer  Institute  of  Canada. 

The  ets  gene  family  was  first  discovered  by  ana- 
lyzing the  genome  of  the  avian  erythroblastosis 
virus  E26,  which  contains  a  part  of  a  myb  fusion 
protein,  the  \-ets  oncogene.  Thus  at  least  three 
members  of  the  ets  family,  \-ets,  Spi-1,  and  Fli-1, 
are  involved  in  the  leukemic  transformation  of 
erythroid  cells.  This  specificity  of  ets  genes  for 
the  erythroid  lineage  suggests  that  members  of 
this  gene  family  are  involved  in  the  regulation  of 
one  or  more  genes  critical  to  erythropoiesis.  We 
are  interested  in  identifying  these  genes  to 
gain  further  insights  into  both  the  regulation 
of  normal  hematopoiesis  and  the  induction  of 
leukemia. 


486 


Molecular  Biology  of  Two 
Enteropathogenic  Bacteria 


Edmundo  Calva,  Ph.D. — International  Research  Scholar 

Dr.  Calva  is  Associate  Professor  and  Chairman  of  Molecular  Biology  at  the  Biotechnology  Institute, 
National  Autonomous  University  of  Mexico  (UNAM),  Cuernavaca.  He  received  his  Ph.D.  degree  in 
molecular  biology  from  the  University  of  Wisconsin-Madison  under  Richard  Burgess  and  did  postdoctoral 
research  at  UNAM.  He  is  currently  President  of  the  Mexican  Biochemical  Society. 


THE  study  of  infectious  diseases  provides  op- 
portunities to  explore  various  basic  biolog- 
ical phenomena.  During  an  infection,  entero- 
pathogenic bacteria  participate  in  numerous 
biological  events  that  may  lead  to  intestinal  ill- 
ness. Upon  oral  ingestion,  the  bacteria  must  un- 
dergo a  series  of  interactions  with  the  host  that 
involve  specific  molecules,  either  on  the  surface 
or  in  the  interior  of  the  bacterial  and  host  cells. 
For  instance,  bacteria  adhere  to  epithelial  gut 
cells  and  produce  enterotoxins  that  result  in  diar- 
rhea. They  can  invade  host  tissue  with  the  same 
effect.  Invasion  sometimes  results  in  a  systemic 
infection,  in  which  the  bacteria  have  means  of 
protection  against  the  immune  system,  seriously 
jeopardizing  health. 

Current  research  questions  in  the  area  of  bacte- 
rial pathogenesis  address  the  molecular  mecha- 
nisms of  the  bacteria-host  interaction.  Our  knowl- 
edge of  adherence,  invasion,  resistance  to  the 
immune  system,  and  enterotoxin  production, 
among  other  processes,  is  just  being  unveiled.  We 
know  only  scant  details  of  some  of  these  pro- 
cesses in  a  few  bacteria,  and  even  then  are  not 
completely  aware  of  the  genetic  variation  within 
the  species  studied — variation  that  may  result  in 
different  strains  with  varying  capabilities  for 
causing  disease.  Furthermore,  some  bacterial  and 
parasite  antigens  share  structures  with  stress  pro- 
teins, like  those  expressed  during  heat  shock.  An 
open  area  of  study  involves  defining  the  global 
genetic  circuits  that  regulate  virulence  factors 
and  determining  whether  these  "regulons"  share 
common  features  with  those  encountered  in  the 
response  to  stresses  other  than  infection. 

Health  biotechnology  should  benefit  from  the 
definition  of  bacterial  antigens  that  have  a  role  in 
infection  or  against  which  a  specific  immune  re- 
sponse is  mounted,  thus  permitting  the  develop- 
ment of  better  vaccines  and  diagnostic  proce- 
dures as  well  as  furthering  our  understanding  of 
the  structure  and  function  of  the  immune  system. 
Benefits  should  also  emerge  from  the  isolation 
and  characterization  of  specific  bacterial  genes, 
together  with  knowledge  about  their  distribution 
and  polymorphism  in  bacterial  populations.  Such 


information  should  permit  the  rapid  and  specific 
detection  of  bacteria  through  nucleic  acid  ampli- 
fication procedures.  These  can  not  only  be  useful 
for  monitoring  infections  in  animals,  plants,  or 
humans,  or  the  contamination  of  foodstuff,  but 
should  also  be  valuable  in  the  definition  of  modes 
of  transmission  and  environmental  reservoirs  for 
the  bacteria.  In  this  manner,  molecular  epidemi- 
ology will  very  likely  shed  light  on  yet  other  bio- 
logical phenomena. 

Salmonella  typhi 

S.  typhi  is  the  causal  agent  of  typhoid  fever 
(TF)  in  humans,  a  disease  estimated  to  afflict  an- 
nually more  than  1 2  million  persons  worldwide. 
TF  is  the  result  of  a  systemic  infection,  in  which  S. 
typhi  can  be  isolated  from  blood  cultures.  As  a 
gram-negative  bacterium,  5.  typhi  has  an  outer 
membrane  that  surrounds  an  inner  one  and  the 
cell  wall.  Thus  exposed  to  the  cell's  exterior  envi- 
ronment, outer  membrane  proteins  (OMPs)  have 
been  shown  to  be  important  immunogens  for 
protection  against  various  bacterial  infections  in 
laboratory  models.  We  have  demonstrated  the 
utility  of  OMPs  for  the  rapid  immunodiagnosis  of 
TF  in  patients  from  different  parts  of  the  world, 
most  of  whom  raise  specific  antibodies  to  OMP 
preparations. 

Our  laboratory  has  reported  the  isolation  and 
characterization  of  an  S.  typhi  gene  coding  for 
OmpC,  a  major  OMP.  Amino  acid  sequence  align- 
ment with  other  OMPs  that  form  pores  (porins) 
has  allowed  structure  prediction  in  the  porin  su- 
perfamily.  Using  site-directed  mutagenesis,  we 
have  constructed  a  gene  that  codes  for  a  chimeric 
protein  containing  a  foreign  epitope  inserted  in  a 
region  predicted  to  be  exposed  on  the  cell  sur- 
face. This  epitope  is  a  segment  of  a  capsid  protein 
from  rotavirus,  a  causal  agent  of  diarrhea,  against 
which  neutralizing  antibodies  are  made.  The  chi- 
meric protein  does  indeed  contain  this  epitope 
on  a  segment  exposed  at  the  bacterial  cell  sur- 
face, supporting  the  notion  that  the  native  region 
is  located  toward  the  exterior  and  indicating  that 
OmpC  could  be  used  as  a  carrier  of  heterologous 
epitopes.  This  information  on  protein  topology 


487 


Molecular  Biology  of  Two  Enteropathogenic  Bacteria 


can  be  useful  in  the  design  of  multivalent  vac- 
cines against  several  infectious  agents. 

We  have  used  the  S.  typhi  ompC  gene  to  probe 
the  genetic  variability  of  the  salmonella  genus. 
In  this  manner  we  have  been  able  to  establish 
some  phylogenetic  relationships  among  several 
species. 

We  have  also  determined  that  expression  of  5. 
typhi  OmpC  is  influenced  differently  by  medium 
osmolarity  than  its  well-studied  counterpart  in 
Escherichia  coli.  Nevertheless,  expression  of  5. 
typhi  ompC  is  also  dependent  on  the  E.  coli 
OmpR  transcriptional  activator.  Apparently  the 
two  bacteria  differ  in  the  mechanisms  of  gene  ex- 
pression in  response  to  osmotic  stress,  although 
common  effectors  appear  to  be  shared.  We  are 
presently  studying  this  phenomenon  in  detail. 

In  addition,  we  have  isolated  other  S.  typhi 
OMP  genes,  namely  ompF  and  phoE,  and  are 
characterizing  these  to  gain  a  better  understand- 
ing of  structure-function  relations  in  porins  and 
their  genes. 

Campylobacter  jejuni 

C.  jejuni  is  one  of  the  major  causal  agents  of 
diarrhea  throughout  the  world.  Molecular  biol- 


ogy of  this  organism  has  developed  slowly, 
mostly  because  of  the  difficulty  of  maintaining 
stably  cloned  C.  jejuni  DNA  segments  in  E.  coli. 
Consequently,  only  a  handful  of  C.  jejuni  genes 
have  been  isolated.  The  mechanisms  underlying 
this  DNA  instability  are  not  understood,  and  re- 
search in  this  area  might  well  yield  valuable 
results. 

The  isolation  of  C.  jejuni,  producing  a  cholera- 
like  enterotoxin,  has  been  associated  with  clini- 
cal symptoms  of  a  watery  secretory  type  of  diar- 
rhea. We  have  shown  that  the  C.  jejuni 
chromosome  contains  sequences  similar  to  the 
enterotoxin  genes  of  Vibrio  cholerae  (CT  entero- 
toxin) and  E.  coli  (LT,  heat-labile  enterotoxin). 
In  cloning  and  characterizing  these  sequences, 
we  have  explored  different  host  cells  and  differ- 
ent-size C.  jejuni  DNA  fragments  in  order  to  find  a 
successful  cloning  procedure.  In  the  process,  we 
have  isolated  two  cryptic  fragments,  one  highly 
specific  for  C.  jejuni  and  C.  coli  (another 
diarrhea-producing  Campylobacter)  and  the 
other  revealing  genetic  variability  between  the 
two  bacteria.  We  are  testing  the  usefulness  of 
both  DNA  probes  in  field  epidemiology  and  char- 
acterizing them  at  the  nucleotide  level. 


488 


Functional  Heterogeneity 
in  Prolactin-secreting  Cells 


Gabriel  Cota,  Ph.D. — International  Research  Scholar 

Dr.  Cota  is  Professor  of  Physiology,  Biophysics,  and  Neurosciences  at  the  Center  for  Research  and 
Advanced  Studies,  National  Polytechnic  Institute,  Mexico  City.  He  received  his  Ph.D.  degree  in  physiology 
and  biophysics  from  the  Center  and  subsequently  carried  out  postdoctoral  research  with  Clay  M. 
Armstrong  at  the  University  of  Pennsylvania,  as  a  Pogarty  International  Research  Fellow. 


OUR  laboratory  is  interested  in  the  cellular 
mechanisms  involved  in  the  control  of  pro- 
lactin secretion.  Prolactin  is  a  vertebrate  hor- 
mone that  participates  in  the  regulation  of  a  di- 
versity of  physiological  processes,  including 
lactation.  This  versatile  chemical  messenger  is 
produced  in  the  pituitary  gland  by  endocrine 
cells  called  lactotropes  or  mammotropes.  Basic 
information  about  lactotrope  function  and  its 
control  offers  insight  into  the  pathogenesis  of  hy- 
perprolactinemia,  a  frequent  hypothalamic  pitu- 
itary disorder  in  humans. 

Until  recently,  lactotropes  were  commonly 
thought  to  comprise  a  homogeneous  cell  popula- 
tion in  the  normal  pituitary  gland.  However,  stud- 
ies performed  on  cultured  pituitary  cells  indicate 
the  existence  of  a  considerable  lactotrope  hetero- 
geneity. In  these  accessible  model  systems,  sub- 
sets of  lactotropes  that  differ  in  basal  secretory 
activity  or  responsiveness  to  extracellular  regula- 
tory factors  have  been  distinguished.  Our  work 
over  the  last  three  years  has  focused  on  the  origin 
of  such  functional  differences. 

Lactotrope  Subtypes 

We  have  identified  two  subpopulations  of  lac- 
totropes in  pituitary  cultures  derived  from  adult 
male  rats.  We  used  the  reverse  hemolytic  plaque 
assay,  an  immunological  technique  that  permits 
the  microscopic  visualization  of  single-cell  se- 
cretions. In  this  assay,  cells  releasing  the  appro- 
priate hormone  induce  lysis  of  indicator  erythro- 
cytes. The  size  of  the  zone  of  hemolysis,  or 
plaque,  around  an  individual  secretor  provides 
an  index  of  the  cumulative  amount  of  hormone 
released  by  that  cell.  In  keeping  with  previous 
observations  by  Jimmy  Neill  (University  of  Ala- 
bama) on  the  secretory  behavior  of  female  rat 
lactotropes,  we  found  that  under  basal  con- 
ditions some  prolactin-secreting  cells  form  small 
plaques  (SP  lactotropes)  and  others  induce  large 
plaques  (LP  lactotropes).  Thus  SP  and  LP  lacto- 
tropes secrete  distinct  amounts  of  prolactin  per 
unit  of  time  in  the  basal  state. 

Calcium  Channel  Activity  as  a  Determinant 
of  Lactotrope  Heterogeneity 

There  is  much  evidence  that  calcium  ion  plays 


a  major  role  in  pituitary  cells  as  an  intracellular 
messenger  in  hormone  secretion.  In  particular, 
basal  prolactin  secretion  is  thought  to  be  sus- 
tained by  calcium  influx  through  plasma  mem- 
brane calcium  channels,  which  transiently  open 
during  spontaneous  action  potentials.  This  sug- 
gests that  differences  in  basal  secretory  rate 
among  lactotrope  subtypes  might  arise,  at  least  in 
part,  from  a  differential  expression  of  calcium 
channels.  To  test  this  possibility,  we  investigated 
the  calcium  channel  activity  of  lactotrope  sub- 
types, using  electrophysiological  techniques. 

Our  analysis  indicates  that  both  SP  and  LP 
lactotropes  express  two  classes  of  voltage-gated 
calcium  channels  in  the  plasma  membrane: 
low-voltage-activated  (LVA)  and  high-voltage- 
activated  (HVA)  channels.  The  activity  of  LVA 
channels  does  not  significantly  differ  between 
the  two  lactotrope  subtypes.  By  contrast,  the  sur- 
face density  of  HVA  channels  is  markedly  higher 
in  LP  cells  than  in  SP  cells. 

In  addition,  we  tested  the  effect  of  nifedipine 
on  prolactin  secretion.  Nifedipine  is  a  dihydro- 
pyridine  drug  that  selectively  blocks  HVA  cal- 
cium channels  in  many  types  of  excitable  cells, 
including  prolactin  secretors.  We  found  that  ni- 
fedipine inhibits  prolactin  secretion  by  preferen- 
tially suppressing  the  LP  lactotropes.  In  fact,  a 
large  proportion  (around  50  percent)  of  LP  lacto- 
tropes behave  functionally  as  SP  lactotropes  in 
the  presence  of  nifedipine.  Our  results,  taken 
together,  indicate  that  calcium  entry  through 
dihydropyridine-sensitive  HVA  calcium  channels 
contributes  to  the  high  basal  rate  of  prolactin  se- 
cretion in  LP  lactotropes. 

Sodium  Channels  in  Lactotropes 

We  have  characterized  an  additional  difference 
in  ion  channel  activity  between  the  two  lacto- 
trope subtypes:  membrane  depolarization  in- 
duces larger  whole-cell  sodium  currents  in  LP 
lactotropes  than  in  SP  lactotropes.  Such  differ- 
ences are  not  related  to  cell-to-cell  variations  in 
the  kinetic  properties  of  the  sodium  currents  and 
persist  after  current  amplitude  is  normalized  by 
cell  capacitance,  which  eliminates  membrane 


489 


Functional  Heterogeneity  in  Prolactin-secreting  Cells 


area  as  a  variable.  Thus  sodium  channels,  like 
HVA  calcium  channels,  are  not  uniformly  ex- 
pressed among  SP  and  LP  lactotropes. 

Sodium  channels  should  be  functionally  im- 
portant for  the  secretory  activity  of  lactotropes,  as 
they  favor  the  triggering  of  action  potentials  and 
thereby  promote  the  opening  of  HVA  calcium 
channels.  Indeed,  we  have  found  that  blocking 
the  sodium  channels  of  cultured  cells  with  exter- 
nal tetrodotoxin  drastically  decreases  the  total 


amount  of  prolactin  secreted.  Furthermore,  popu- 
lation analysis  of  prolactin  plaque  sizes  suggests 
that  tetrodotoxin,  like  nifedipine,  preferentially 
inhibits  prolactin  secretion  from  LP  cells. 

These  findings  raise  the  question  of  what  fac- 
tors regulate  ion  channel  expression  in  pituitary 
cells.  They  also  renew  our  interest  in  the  differ- 
entiation of  lactotrope  subtypes  in  the  develop- 
ing pituitary  gland.  Our  current  research  is  fo- 
cused on  these  topics. 


490 


Ionic  Channels  in  Sea  Urchin  Sperm  Physiology 


Alberto  Darszon,  Ph.D. — International  Research  Scholar 

Dr.  Darszon  is  Professor  of  Biochemistry  at  the  Biotechnology  Institute,  National  Autonomous  University 
of  Mexico,  Cuernavaca,  and  Adjunct  Professor  of  Biochemistry  at  the  Center  for  Research  and  Advanced 
Studies,  National  Polytechnic  Institute,  Mexico  City.  He  received  his  undergraduate  degree  in  chemistry 
from  the  Universidad  Iberoamericana  in  Mexico  City,  and  his  Ph.D.  degree  in  biochemistry  from  the 
Center  for  Research  and  Advanced  Studies.  His  postdoctoral  research  was  done  with  Mauricio  Montal  at 
the  University  of  California,  San  Diego.  His  honors  include  the  National  Science  Award  of  the  Mexican 
Academy  of  Scientific  Research  and  both  Guggenheim  and  World  Health  Organization  fellowships. 


THE  molecular  mechanisms  involved  in  cell 
communication  are  at  the  forefront  of  re- 
search in  biology  today,  since  they  play  a  key  role 
in  determining  the  behavior  of  organisms.  For  the 
past  decade  the  main  goal  in  our  laboratory  has 
been  to  understand  the  vital  egg  signals  that  allow 
sperm  to  fuse  with  the  egg  and  fertilize  it. 

The  sea  urchin  has  proved  to  be  an  excellent 
model  in  which  to  study  reproduction.  From  this 
animal  enormous  quantities  of  sperm  (10^°/ 
male)  can  be  collected  that  respond  to  environ- 
mental stimuli  rapidly,  synchronously,  and  in  a 
compulsory  order.  It  has  been  shown  that  the 
flow  of  ions  through  the  plasma  membrane  of  sea 
urchin  sperm  participates  crucially  in  the  events 
leading  to  fertilization.  Indeed,  these  are  excit- 
able cells  that  quickly  respond  to  components 
from  the  outer  layer  of  the  egg,  the  jelly,  with 
changes  in  their  plasma  membrane  permeability. 

Ionic  fluxes  play  a  fundamental  role  in  the  acti- 
vation of  respiration  and  motility,  in  chemotaxis, 
and  in  triggering  the  sperm  acrosome  reaction 
(AR) .  This  latter  reaction  occurs  within  seconds 
after  sea  urchin  sperm  reach  the  egg  and  interact 
with  its  jelly  coat.  The  AR  involves  important  mor- 
phological changes  that  allow  sperm  to  fuse  with 
the  egg.  Participating  in  AR  induction  is  an  in- 
crease in  the  uptake  of  Ca^"^  and  Na^  and  efflux  of 
K"^  and  H^.  We  would  like  to  learn  how  these 
fluxes  are  related  and  elucidate  the  molecular 
mechanisms  that  orchestrate  them  to  trigger 
the  AR. 

Previous  results  indicated  indirectly  that  some 
ion  fluxes  occur  through  ionic  channels.  These 
are  integral  membrane  proteins  capable  of  form- 
ing hydrophilic  pores  through  the  membrane  that 
allow  the  passive  diffusion  of  ions  at  high  rates 
(10^"^  ions/s).  We  have  used  model  membranes 
(planar  bilayers)  formed  from  sperm  compo- 
nents, and  patch-clamp  techniques  in  whole 
cells,  to  detect  for  the  first  time  the  activity  of 
single  channels  in  the  plasma  membrane  of  sea 
urchin  sperm.  These  techniques,  together  with 
studies  of  membrane  potential,  intracellular  Ca^^ 
([Ca^+]j),  and  intracellular  pH  (pHj)  in  whole 


sperm,  have  established  the  presence  of  K^,  Ca^^, 
and  Cl~  channels  in  this  cell  and  are  allowing  us 
to  explore  their  participation  in  the  AR. 

These  findings  have  led  us  to  propose  a  work- 
ing hypothesis  of  how  egg  jelly-induced  changes 
in  ionic  permeability  might  trigger  the  AR  in  sea 
urchin  sperm.  A  receptor  (to  the  inducing  egg 
factor)  in  the  plasma  membrane  of  the  sperm  cell 
opens  a  Ca^^  channel,  which  deactivates  in  a  few 
seconds.  The  activation  of  this  channel  modu- 
lates the  opening  of  a  second  Ca^^  channel  by 
poorly  understood  mechanisms  that  include  a 
Ca^^-dependent  change  in  pHj.  At  the  same  time 
or  immediately  after  the  first  Ca^"^  channel  opens, 
a  channel  is  activated,  hyperpolarizing  the 
cell  and  activating  a  voltage-dependent  Na^/H^ 
exchange  that  increases  pHj.  This  latter  change  in 
pHj  is  linked  to  the  opening  of  the  second  Ca^"^ 
channel  and  to  a  large  depolarization. 

In  light  of  this  working  hypothesis,  we  thought 
it  should  be  possible  to  hyperpolarize  sperm  arti- 
ficially and  induce  an  increase  in  [Ca^"^]!  after  a 
depolarization  and  AR.  Valinomycin-induced  hy- 
perpolarization  of  sperm  from  the  sea  urchin  Ly- 
techinus  pictus  in  K^-free  sea  water  raised  pHj, 
caused  a  small  increase  in  ^'Ca^"^  uptake,  and  trig- 
gered some  AR.  When  the  cells  were  depolarized 
with  30  mM  KCl  40-60  seconds  after  the  hyper- 
polarization,  the  pHj  decreased  and  there  was  a 
significant  increase  in  ''^Ca^^  uptake,  [Ca^"^];,  and 
AR.  Therefore  the  jelly-induced  hyperpolariza- 
tion  may  lead  to  the  intracellular  alkalinization 
required  to  trigger  the  AR  and  may  modulate,  on 
its  own  or  via  pHj,  Ca^^  channels  involved  in  this 
process. 

The  sea  urchin  sperm  offers  distinct  advantages 
over  more  complex  cell  types  as  a  basic  model  for 
chemotaxis.  As  the  spermatid  matures,  many  in- 
tracellular organelles  and  macromolecules  not 
involved  in  fertilization  are  eliminated.  Sperm 
are  incapable  of  division  and  devoid  of  the  ma- 
chinery for  genome  expression.  However,  they 
retain  systems  for  sensing,  swimming  toward,  and 
fusing  with  the  egg,  as  described  above. 

The  sperm  of  the  sea  urchin  Arbacia  punctu- 
lata  is  attracted  at  nanomolar  concentrations  to  a 


491 


Ionic  Channels  in  Sea  Urchin  Sperm  Physiology 


small  peptide  isolated  from  the  homologous  egg. 
This  peptide,  called  resact,  is  species  specific  and 
induces  in  sperm  a  transient  increase  in  [Ca^^]; 
and  cGMP  and  an  increase  in  pHj.  The  egg  of 
Strongylocentrotus  purpuratus  contains  a  pep- 
tide, named  speract,  that  also  elevates  cGMP  and 
[Ca^^ji  and  induces  an  alkalinization  and  a  K"^- 
dependent  hyperpolarization. 

Sea  urchin  sperm,  however,  are  tiny  cells  (head 
diameter  ~2  ytvci).  This  has  precluded  a  careful 
characterization  of  their  electrophysiological 
properties  that  would  shed  light  on  the  molecu- 
lar mechanisms  determining  their  fascinating 
egg-induced  behavioral  changes.  Recently,  in  col- 
laboration with  Donner  Babcock  and  Martha 
Bosma  from  the  University  of  Washington  in  Seat- 
tle, we  found  that  it  is  possible  to  swell  sea  urchin 
sperm  in  diluted  sea  water.  The  swollen  cells  are 
spherical  (~4  yum  in  diameter),  immotile,  and 


metabolically  active,  and  they  can  regulate  their 
[Ca'^^Ji,  pHj,  and  membrane  potential. 

The  swollen  cells  respond  to  pM  concentra- 
tions of  speract  with  an  increase  in  K"^-selective 
permeability  that  lasts  for  many  seconds.  We 
found  that  this  permeability  change,  as  well  as 
the  changes  in  [Ca^^];  and  pHj  that  are  seen  at 
higher  speract  concentrations,  also  occurs  in 
nonswoUen  sperm.  An  advantage  of  swollen 
sperm  is  that  they  can  be  much  more  easily  patch 
clamped  and  single-channel-activity  recorded. 
We  have  observed  that  pM  speract  activates  a 
small  K"^  channel.  Thus  swollen  sperm  provide 
new  avenues  to  study  ionic  channels  and  their 
regulation  by  egg  factors  and  second  messengers. 

This  work  is  supported  by  grants  from  the  Mex- 
ican Council  for  Science  and  Technology,  the 
World  Health  Organization,  and  the  Miguel  Ale- 
man  Foundation. 


Confocal  immunofluorescent  micrograph  of 
Salmonella  typhimurium  (yellow)  interact- 
ing with  a  cultured  epithelial  cell  and 
causing  rearrangement  of  epithelial  actin 
filaments  (blue)  around  the  invading  bac- 
terium. 

Research  and  photograph  by  B.  Brett 
Finlay. 


492 


Host-Pathogen  Interactions  in  Microbial 
Pathogenesis 


B.  Brett  Finlay,  Ph.D. — International  Research  Scholar 

Dr.  Finlay  is  Assistant  Professor  in  the  Biotechnology  Laboratory  and  the  Departments  of  Biochemistry 
and  Microbiology  at  the  University  of  British  Columbia,  Vancouver.  He  is  a  member  of  the  Canadian 
Bacterial  Diseases  Center  of  Excellence.  After  receiving  his  B.Sc.  and  Ph.D.  degrees  in  biochemistry  from 
the  University  of  Alberta,  Edmonton,  he  conducted  postdoctoral  work  on  microbial  pathogenesis  in  the 
laboratory  of  Stanley  Falkow  at  Stanford  University  as  a  fellow  of  the  Alberta  Heritage  Foundation  for 
Medical  Research. 


IN  all  cases  of  bacterial  disease,  the  bacterium 
or  a  bacterial  product  interacts  with  host  cells 
or  surfaces  in  either  of  two  ways.  It  may  adhere  to 
the  cell  surface  or  may  actually  enter  the  cell  and 
grow  within  (intracellular  pathogen) .  Pathogens 
residing  in  an  intracellular  environment  are  pro- 
tected from  the  host's  immune  systems,  antibi- 
otics, and  other  therapeutic  agents.  They  often 
use  this  safe  niche  to  multiply  before  disseminat- 
ing to  other  sites  and  deeper  tissue. 

Our  laboratory  uses  a  multidisciplinary  ap- 
proach to  define  the  interactions  that  occur  be- 
tween pathogenic  bacteria  and  host  cells.  Essen- 
tial to  continuation  of  these  interactions  is  the 
exchange  of  signals  between  pathogen  and  cell. 
We  are  studying  the  molecular  nature  of  these 
signal  transduction  events  in  an  effort  to  deter- 
mine the  mechanisms  involved.  Knowledge  of 
these  mechanisms  should  point  the  way  to  novel 
therapeutic  strategies. 

Several  bacterial  pathogens  are  used  in  these 
studies,  since  each  organism  has  its  individual 
features.  Additionally,  comparison  of  the  signals 
generated  by  different  pathogens  can  tell  which 
mechanisms  are  common  and  which  are  unique. 

Salmonella  typhimuriumi 

A  Model  for  Intracellular  Parasitism 

For  several  reasons,  5.  typhimurium  provides 
an  excellent  model  for  the  study  of  intracellular 
parasitism.  Salmonella  species  continue  to  cause 
significant  health  problems  in  both  developed 
and  less  developed  countries.  These  organisms 
have  the  capacity  to  enter  into,  survive,  and  repli- 
cate within  host  cells — features  that  contribute 
to  virulence.  Since  5.  typhimurium  is  z  close  velz- 
tive  of  the  nonpathogenic  Escherichia  colt,  we 
have  been  able  to  use  established  molecular  ge- 
netic techniques  to  study  several  aspects  of  Sal- 
monella pathogenesis.  Finally,  5.  typhimurium 
infection  of  the  mouse  closely  mimics  human  ty- 
phoid fever. 

Since  Salmonella  species  (and  many  other 
pathogens)  interact  with  intestinal  epithelial 
cells  following  oral  ingestion,  we  have  been  able 
to  utilize  monolayers  of  polarized  epithelial  cells 


grown  on  permeable  substrates  to  study  the  inter- 
actions. These  systems  have  several  properties  in 
common  with  columnar  epithelial  cells  of  the  in- 
testine. Salmonella  species  have  the  capacity  to 
enter  and  penetrate  through  these  polarized  epi- 
thelial monolayers.  Moreover,  transposon  mu- 
tants that  are  defective  for  such  penetration  have 
been  isolated  from  5.  typhimurium.  Molecular 
characterization  of  the  genetic  loci  that  are 
disrupted  by  these  transposons  will  provide  clues 
about  the  bacterial  products  required  for  cell 
entry  and  penetration. 

Once  inside  a  vacuole  within  an  epithelial  cell, 
5.  typhimurium  finds  itself  within  a  presumably 
quite  different  environment.  Very  little  is  known 
about  the  microenvironment  of  any  intracellular 
pathogen.  To  probe  and  define  aspects  of  the  va- 
cuolar habitat,  we  have  been  using  a  bacterial  re- 
porter gene  (tocZ)  fused  to  several  S.  typhimur- 
ium genes  that  are  variously  regulated.  For 
example,  one  can  measure  expression  of  reporter 
genes  that  are  affected  by  oxygen  levels,  carbon 
source,  pH,  or  iron  or  magnesium  concentra- 
tions. These  studies  are  providing  clues  about  the 
nature  of  this  intracellular  niche.  Additionally, 
we  have  begun  to  search  for  other  S.  typhimur- 
ium loci  that  are  only  induced  when  the  bacte- 
rium is  inside  host  cells,  in  the  hope  of  finding 
other  regulated  genes. 

After  initial  survival  inside  a  host  cell,  5.  typhi- 
murium begins  to  multiply  within  its  intracellu- 
lar vacuole.  Virtually  nothing  is  known  about  its 
requirements  there.  We  have  identified  three  bac- 
terial genes  that  are  needed  for  S.  typhimurium 
to  multiply  within  host  cells  but  not  without. 
Characterization  of  these  genes  and  other  experi- 
ments may  provide  information  about  bacterial 
products  necessary  for  intracellular  growth. 

Signal  Transduction  Between  Host 

and  Pathogen:  Involvement  of  the  Host 

Cytoskeleton  and  Tyrosine  Kinases 

Most  intracellular  pathogens  require  participa- 
tion of  the  host  cell  for  successful  pathogen  inter- 
nalization. Functional  actin  filaments  are  often 
involved  in  bacterial  uptake,  and  5.  typhimur- 


493 


Host-Pathogen  Interactions  in  Microbial  Pathogenesis 


ium  is  one  pathogen  that  requires  host  actin  fila- 
ments for  invasion.  When  S.  typhimurium  enters 
cukured  epitheUal  cells,  there  is  a  large  rear- 
rangement in  intracellular  actin  and  the  function- 
ally related  cytoskeletal  proteins  a-actinin,  tropo- 
myosin, and  talin  that  surround  the  membrane 
engulfing  the  invading  organism.  This  rearrange- 
ment, presumably  needed  for  functional  internal- 
ization, is  triggered  by  bound  extracellular  bacte- 
ria and  occurs  in  the  region  beneath  adherent 
organisms.  Once  the  pathogen  is  inside  a  vacu- 
ole, the  epithelial  cytoskeleton  returns  to  its  nor- 
mal distribution. 

We  have  been  examining  the  signals  trans- 
duced through  the  host  cell  membrane  that  are 
responsible  for  triggering  this  cytoskeletal  rear- 
rangement, and  host  cell  signals  that  are  neces- 
sary for  the  uptake  of  other  pathogenic  bacteria 
into  epithelial  cells.  Host  tyrosine  kinases  appear 
to  participate  in  the  uptake  of  several  pathogens, 
including  Yersinia  species  (causative  agent  of 
gastrointestinal  problems).  Listeria  monocyto- 
genes (a  gram-positive  organism  associated  with 
meningitis  and  serious  infections  of  neonates), 
and  enteropathogenic  Escherichia  coli  (caus- 
ative agent  of  diarrhea).  We  are  using  various 
kinase  inhibitors,  monoclonal  antibodies  that 
recognize  phosphotyrosine  residues,  and  radiola- 
beling  of  host  phosphate  pools  to  begin  to  iden- 
tify the  components  of  the  signal  transduction 
pathways  that  these  bacteria  pirate  for  their 
own  use. 

An  in  Vitro  Blood-Brain  Barrier  to  Study 
Bacterial  Meningitis 

Bacterial  meningitis  (infection  of  the  brain  lin- 


ing) is  a  common  and  serious  disease  in  both 
children  and  adults.  In  an  effort  to  define  more 
clearly  the  molecular  mechanisms  mediating  this 
disease,  we  developed  a  model  in  vitro  blood- 
brain  barrier  (BBB) .  This  model  uses  primary  iso- 
lates of  microcapillary  endothelial  cells  from  bo- 
vine brain.  Reseeded  onto  permeable  substrates, 
these  cells  form  impermeable  monolayers  with 
tight  junctions  and  have  several  features  charac- 
teristic of  the  BBB. 

When  Haemophilus  influenzae  (the  most 
common  cause  of  pediatric  meningitis)  is  added 
to  these  monolayers,  they  are  completely 
disrupted  by  a  cytotoxic  mechanism — similar  to 
the  effects  observed  in  vivo.  We  have  found  that 
this  cytotoxicity  is  mediated  by  bacterial  lipo- 
polysaccharide  (LPS)  in  concert  with  a  soluble 
serum  factor.  Current  research  has  a  twofold  aim: 
first,  to  block  this  cytotoxicity  with  monoclonal 
antibodies  against  the  serum  factor  in  a  primate 
meningitis  model  and  second,  to  define  the  signal 
transduction  pathways  that  are  triggered  in  the 
endothelial  cell  by  the  LPS.  In  addition,  the  role 
of  cytokines  in  this  cytotoxic  event  is  being 
studied. 

Collectively  this  work  provides  several  insights 
as  to  how  pathogenic  bacteria  manifest  disease. 
Studies  with  5.  typhimurium  have  afforded  new 
perceptions  into  the  molecular  biology  of  intra- 
cellular parasitism.  Definition  of  the  signal  trans- 
duction pathways  of  invasive  pathogenic  bacteria 
is  providing  evidence  that  many  pathogens  ex- 
ploit existing  pathways,  often  utilizing  them  to 
pirate  the  host  cytoskeleton  for  their  own  advan- 
tage. Work  with  the  BBB  has  offered  new  opportu- 
nities for  therapeutic  intervention  in  treating 
meningitis. 


494 


Mechanisms  of  Transcriptional  Regulation 


Jack  Greenblatt,  Ph.D. — International  Research  Scholar 

Dr.  Greenblatt  is  Professor  in  the  Banting  and  Best  Department  of  Medical  Research  and  the  Department  of 
Molecular  and  Medical  Genetics  at  the  University  of  Toronto.  He  earned  his  undergraduate  degree  in 
physics  from  McGill  University,  Montreal,  and  his  Ph.D.  degree  in  biophysics  from  Harvard  University, 
where  he  studied  bacterial  gene  regulation  with  Walter  Gilbert.  He  pursued  postdoctoral  studies  with 
Alfred  Tissieres  at  the  University  of  Geneva.  He  has  received  the  Ayerst  Award  of  the  Canadian 
Biochemical  Society. 


THE  ultimate  focus  of  transcriptional  regula- 
tory mechanisms  is  RNA  polymerase,  a  com- 
plex multisubunit  enzyme  whose  activity  is 
guided  by  interactions  with  a  myriad  of  regula- 
tory proteins  and  regulatory  sequences  in  DNA  or 
RNA.  Much  of  our  research  is  devoted  to  the  basic 
enzymology  of  initiation  and  termination  of  tran- 
scription and  to  identifying  and  characterizing 
some  of  the  key  protein-protein  interactions  in- 
volved. In  addition,  we  are  examining  how 
model  regulatory  proteins  interact  with  the  basic 
transcriptional  apparatus.  This  work  is  also  sup- 
ported by  grants  from  the  Medical  Research 
Council  of  Canada  and  the  National  Cancer  Insti- 
tute of  Canada. 

Initiation  of  Transcription  by  Human  RNA 
Polymerase  II 

A  set  of  general  transcription  factors  (TFILA,  B, 
D,  E,  F,  H)  is  necessary  for  RNA  polymerase  II  to 
initiate  the  transcription  of  protein-coding  genes. 
TFIID  is  the  general  factor  that  recognizes  TATA 
sequences  present  in  the  promoters  of  many 
genes.  After  TFIID  binds  to  the  DNA,  TFIIA  and 
TFIIB  recognize  and  bind  to  the  TFIID-DNA  com- 
plex. Subsequently  the  other  general  factors  and 
RNA  polymerase  II  assemble  into  a  multiprotein 
complex  at  the  promoter.  We  use  protein  affinity 
chromatography  as  a  technique  to  identify  direct 
protein-protein  interactions  involved  in  the  as- 
sembly of  this  complex. 

TFIID  has  been  highly  conserved  during  evolu- 
tion. TFIID  molecules  from  most  or  all  eukar- 
yotes,  including  fungi,  insects,  and  plants,  can 
function  in  transcription  reactions  containing 
RNA  polymerase  II  and  other  general  factors  of 
human  origin.  By  using  yeast  TFIID  as  a  ligand  for 
affinity  chromatography,  we  have  identified 
three  human  polypeptides  that  interact  with 
TFIID  and  constitute  human  TFIIA.  Curiously, 
TFIID  columns  do  not  retain  TFIIB,  nor  do  TFIIB 
columns  retain  TFIID,  suggesting  that  a  confor- 
mational change  in  TFIID  induced  by  DNA  bind- 
ing facilitates  its  interaction  with  TFIIB. 

By  using  RNA  polymerase  II  as  a  ligand  for  affinity 
chromatography,  we  identified  human  RAP30  and 


RAP74,  the  small  and  large  subunits  of  TFIIF.  Hu- 
man cDNAs  encoding  RAP30  and  RAP74  have  both 
been  cloned,  the  latter  in  collaboration  with 
Zachary  Burton  (Michigan  State  University) .  Using 
recombinant  RAP30,  we  found  that  RAP30  is  the 
subunit  of  TFIIF  that  binds  RNA  polymerase  II. 
RAP30  prevents  RNA  polymerase  II  from  associat- 
ing with  and  transcribing  nonpromoter  sequences 
in  DNA,  a  property  also  of  bacterial  a  factors.  In- 
deed, we  found  that  RAP30  can  bind  to  Escherichia 
coli  RNA  polymerase  and  be  displaced  by  the  major 
bacterial  a  factor,  known  as  a""^. 

RAP30  also  has  a  central  role  in  promoter  rec- 
ognition by  RNA  polymerase  II.  In  collaboration 
with  Danny  Reinberg  (University  of  Medicine  and 
Dentistry  of  New  Jersey),  we  found  that  RAP30 
can  recruit  RNA  polymerase  II  to  a  preinitiation 
complex  containing  TFIIA,  TFIIB,  and  TFIID.  In 
fact,  recognition  of  a  promoter  containing  a  TATA 
sequence  by  RNA  polymerase  II  can  be  achieved 
with  recombinant  TFIIB,  RAP30,  and  TBP,  the 
TATA  sequence-binding  subunit  of  TFIID,  all 
produced  in  E.  coli.  These  three  general  factors, 
therefore,  constitute  a  minimal  set  of  proteins 
necessary  and  sufficient  for  promoter  binding  by 
RNA  polymerase  II.  However,  this  preinitiation 
complex  containing  RNA  polymerase  II  will  not 
initiate  transcription  unless  supplied  with 
RAP74,  TFIIF,  TFIIH,  and  other  factors.  The  roles, 
subunit  compositions,  and  protein-protein  inter- 
actions of  some  of  these  factors  remain  to  be 
identified. 

Regulation  of  Initiation 
by  RNA  Polymerase  II 

Many  transcriptional  activator  proteins  have 
two  domains.  One  binds  to  regulatory  sequences 
in  DNA,  and  the  other,  known  as  an  activation 
domain,  provides  an  activating  signal  to  the  basic 
transcriptional  apparatus.  How  these  activation 
domains  function  is  a  fascinating  question  in  reg- 
ulatory biology.  It  has  been  the  major  focus  of 
collaborative  studies  with  my  colleague,  C.James 
Ingles  (University  of  Toronto) . 

Many  activation  domains  are  highly  acidic.  A 
particularly  potent  one  is  found  in  the  Herpes 


495 


Mechanisms  of  Transcriptional  Regulation 


simplex  virus  protein  VP  16.  By  using  the  VP  16 
activation  domain  as  a  ligand  for  affinity  chroma- 
tography, v^e  found  that  VP  16  interacts  directly 
with  the  TBP  subunit  of  TFIID .  Mutations  in  VP  1 6 
that  reduce  gene  activation  by  VP  1 6  also  reduce 
its  binding  to  TBP,  and  preliminary  work  has 
identified  a  mutation  in  TBP  that  reduces  its  abil- 
ity to  interact  with  and  respond  to  VPl6.  Interac- 
tion of  VP  16  with  TBP  alters  the  association  of 
TBP  with  the  promoter.  Therefore,  VP  16  may  in- 
fluence the  ability  of  TBP  to  recruit  other  general 
factors  and  RNA  polymerase  II  to  the  promoter. 

A  potent  acidic  activation  domain  is  also  found 
in  p53,  the  product  of  a  human  anti-oncogene  (a 
tumor-suppressing  gene)  that  is  mutated  in  about 
half  of  all  human  cancers.  Like  VP16,  the  p53 
activation  domain  also  binds  TBP.  By  using  affin- 
ity chromatography  to  search  for  other  proteins 
that  interact  with  p53,  we  recently  discovered  a 
new  protein  that  interacts  with  the  p5  3  and  VP  1 6 
activation  domains.  At  least  some  oncogenic  mu- 
tations in  p53  prevent  binding  of  this  protein  to 
the  p53  activation  domain.  The  precise  role  of 
this  novel  protein  in  transcription  is  still  under 
investigation. 

Transcriptional  Antitermination  in  E.  coli 

The  N  protein  of  bacteriophage  X  prevents  ter- 
mination by  RNA  polymerase  during  transcrip- 
tion of  X  operons  expressed  immediately  after  in- 
fection of  E.  coli  cells.  We  have  reconstituted 
antitermination  by  N  in  vitro  in  a  system  contain- 
ing seven  purified  proteins:  E.  coli  RNA  polymer- 
ase, the  E.  coli  transcription  termination  factor 
Rho,  N,  and  four  E.  co/?  cofactors  for  antitermina- 
tion (NusA,  NusB,  SIO,  and  NusG).  By  using  pro- 
tein affinity  chromatography  and  other  methods, 


we  have  identified  many  of  the  protein-protein 
interactions  in  this  system.  Three  factors,  NusA, 
NusG,  and  SIO,  all  bind  to  RNA  polymerase.  NusA 
is  important  for  termination  of  transcription  at 
some  terminators.  Since  N  binds  to  NusA,  NusA  is 
also  an  adapter  that  couples  the  antitermination 
factor  N  to  RNA  polymerase.  In  addition,  NusB 
binds  to  SIO  and  NusG  to  Rho  factor.  In  fact, 
NusG  aids  termination  by  Rho  in  vitro  and  is  es- 
sential for  termination  by  Rho  in  vivo. 

The  stable  association  of  N  with  RNA  polymer- 
ase requires  an  N  utilization  site  (nw?  site)  in  the 
transcribed  DNA  and  all  four  bacterial  cofactors. 
The  nut  site  RNA  in  the  growing  RNA  transcript  is 
recognized  and  bound  by  the  proteins.  N  recog- 
nizes the  boxB  component  of  the  nut  site  RNA, 
while  NusB  and  SIO  recognize  a  better  version  of 
the  box  A  component  of  nut  site  RNA,  which  is 
found  in  the  antiterminator  sequences  of  bacte- 
rial ribosomal  RNA  operons.  Since  mutations  in 
RNA  polymerase  can  prevent  the  formation  of  a 
stable  multiprotein  complex  and  can  prevent 
binding  of  protein  to  the  nut  site  RNA  during 
transcription,  we  infer  that  a  stable  ribonucleo- 
protein  complex  containing  nut  site  RNA  and 
five  antitermination  proteins  assembles  on  the 
surface  of  RNA  polymerase.  This  extensively 
modified  RNA  polymerase  can  then  transcribe 
through  kilobases  of  X  DNA  containing  many  tran- 
scriptional terminators. 

The  human  immunodeficiency  viruses  (HIV-1 
and  HIV-2)  produce  antitermination  factors, 
known  as  Tat,  that  recognize  regulatory  se- 
quences in  viral  RNA,  known  as  TAR.  The  human 
host  factors  involved  in  antitermination  by  Tat  are 
unknown.  Antitermination  of  HIV  transcription 
by  Tat  may  well  resemble  antitermination  of  X 
transcription  by  N  in  many  respects. 


496 


Ionic  Homeostasis  in  White  Blood  Cells 


Sergio  Grinstein,  Ph.D. — International  Research  Scholar 

Dr.  Grinstein  is  Head  of  the  Division  of  Cell  Biology  at  the  Research  Institute  of  the  Hospital  for  Sick 
Children,  Toronto,  and  Professor  of  Biochemistry  at  the  University  of  Toronto.  He  received  his  Ph.D.  degree 
at  the  National  Polytechnic  Institute,  Mexico  City,  where  he  studied  with  David  Erlij.  His  postdoctoral 
training  was  in  two  stages:  initially  at  the  Hospital  for  Sick  Children  under  the  supervision  of  Aser 
Rothstein,  and  later  at  the  Federal  Institute  of  Technology  in  Zurich  with  Giorgio  Semenza.  He  has  received 
the  Ayerst  Award  of  the  Canadian  Biochemical  Society. 


LEUKOCYTES  constitute  the  body's  first  line  of 
defense  against  invading  microorganisms. 
These  white  blood  cells  first  detect  and  engulf 
bacteria  and  other  microbes,  then  secrete  lytic 
enzymes  and  synthesize  reduced  oxygen  metabo- 
lites to  kill  them. 

These  microbicidal  processes  call  for  pro- 
nounced changes  in  the  generation  and  intracel- 
lular distribution  of  acid  equivalents.  First,  the 
phagocytic  vacuole  wherein  the  microorganisms 
are  trapped  becomes  markedly  acidic.  This  evi- 
dently promotes  the  activity  of  the  lytic  enzymes 
released  into  the  phagosome  and  may  also  facili- 
tate its  fusion  with  the  vesicles  containing  bacteri- 
cidal agents.  Then  too,  the  leukocytes'  rate  of 
metabolic  acid  production  increases  greatly  dur- 
ing infection,  threatening  the  stability  of  the  cyto- 
solic  compartment,  which  must  remain  slightly 
alkaline  to  preserve  optimal  cell  function. 

The  purpose  of  our  research  is  to  understand 
the  mechanisms  underlying  phagosomal  acidifi- 
cation, the  pathways  responsible  for  excess  meta- 
bolic acid  during  leukocyte  activation,  and  partic- 
ularly the  processes  involved  in  the  maintenance 
of  the  cytoplasmic  pH  under  both  resting  and  ac- 
tivated conditions. 

Antiports  and  Channels  in  the  Regulation 
of  Cytosolic  pH 

The  pronounced  metabolic  burst  that  leuko- 
cytes undergo  when  confronted  by  microorgan- 
isms or  their  products  can  be  largely  attributed  to 
activation  of  an  otherwise  quiescent  enzyme,  the 
NADPH  oxidase.  The  one-electron  reduction  of 
oxygen  catalyzed  by  this  enzyme  is  accompanied 
by  oxidation  of  NADPH  to  NADP^  and  release  of 
protons.  Regeneration  of  NADPH  through  the 
hexose  monophosphate  shunt  is  a  source  of  fur- 
ther proton  production.  If  uncompensated,  the 
proton  production  by  these  pathways  would  pro- 
duce a  massive  intracellular  acidification,  incom- 
patible with  normal  leukocyte  function  and  possi- 
bly even  their  viability. 

Three  primary  pathways  appear  to  be  involved 
in  proton  (equivalent)  extrusion  in  activated  leu- 
kocytes. The  first  and  perhaps  most  important  is 


an  electroneutral  exchanger  (antiport)  that  trans- 
ports protons  out  of  the  cells  in  exchange  for  ex- 
tracellular sodium.  A  major  isoform  of  this  anti- 
port  has  been  identified  in  fibroblasts  as  a 
1 10-kDa  membrane  glycoprotein.  We  have  found 
that  the  antiport  is  active  not  only  after  stimula- 
tion but  also  in  resting  cells.  Its  activity,  however, 
is  greatly  enhanced  following  the  addition  of  bac- 
terial chemoattractants  or  of  molecules  that 
mimic  events  in  the  intracellular  signaling  cas- 
cade triggered  by  microorganisms. 

Our  current  and  future  research  eff'orts  in  this 
area  deal  with  the  molecular  characterization  of 
the  antiport  in  leukocytes  and  of  the  mechanisms 
underlying  its  activation  during  infection  and  in- 
flammation. We  are  particularly  interested  in  the 
subcellular  localization  of  the  antiports  before 
and  after  stimulation,  in  the  mechanisms 
whereby  chemoattractants  signal  activation,  and 
in  the  segregation  and/or  inactivation  of  anti- 
ports  in  compartments  where  sodium/proton  ex- 
change activity  is  counterindicated  (such  as  the 
phagosome) . 

We  have  recently  detected  a  second  pathway 
that  appears  to  be  important  in  the  extrusion  of 
protons  from  activated  leukocytes,  namely  a  pro- 
ton conductance,  possibly  a  channel.  This  con- 
ductive path  is  essentially  undetectable  in  quies- 
cent cells  but  becomes  clearly  apparent  when  the 
cells  are  stimulated.  Preliminary  data  indicate 
that  this  putative  channel  is  present  in  neutro- 
phils, macrophages,  and  the  human  leukemic 
cell  line  HL60.  The  conductive  proton  pathway 
could  serve  two  important  functions  in  the  stimu- 
lated leukocyte:  it  could  contribute  to  the  extru- 
sion of  net  acid  equivalents  from  the  cell,  and  it 
could  also  serve  as  a  source  for  counterions  to 
neutralize  the  voltage  generated  by  the  NADPH 
oxidase,  proposed  to  be  electrogenic. 

Little  is  known  at  present  about  the  conductive 
pathway.  We  are  interested  in  defining  its  molec- 
ular identity,  physiological  significance,  intra- 
cellular distribution,  developmental  pattern,  and 
the  molecular  basis  of  its  activation.  In  this  re- 
gard, it  is  noteworthy  that  activation  of  the  con- 
ductance closely  mirrors  the  behavior  of  the 


497 


Ionic  Homeostasis  in  White  Blood  Cells 


NADPH  oxidase.  We  are  in  the  process  of  explor- 
ing the  relationship  between  these  events,  with 
particular  interest  in  whether  the  putative  chan- 
nels are  a  component  of  the  oxidase  complex  or 
whether  assembly  of  the  latter  is  required  for  ac- 
tivation of  the  conductance. 

For  this  purpose,  we  have  initiated  studies  us- 
ing-cells  from  patients  with  chronic  granuloma- 
tous disease.  These  cells  are  defective  in  specific 
components  of  the  NADPH  oxidase.  In  future  ex- 
periments we  will  attempt  to  detect  the  conduc- 
tance electrophysiologically  and  to  reconstitute 
its  activity  in  cell-free  systems.  If  a  linkage  be- 
tween the  two  processes  is  established,  purified 
or  recombinant  components  of  the  oxidase  will 
be  used. 

Proton  Pumps  in  Cytoplasmic 
and  Organellar  pH  Regulation 

Recent  experiments  have  also  suggested  that  a 
third  mechanism  of  proton  extrusion  is  opera- 
tional in  stimulated  leukocytes.  Pharmacological 
evidence  indicates  that  this  latter  pathway  may  be 
a  proton-pumping  ATPase  of  the  vacuolar  type.  As 
described  for  the  other  systems,  the  activity  of  the 
pumps  becomes  clearly  apparent  after  stimula- 
tion. At  present,  neither  the  subcellular  location 


of  the  pumps  is  known  nor  the  mechanism  of  ac- 
tivation understood.  We  will  try  to  determine 
whether  activation  results  from  post-translational 
modification  of  pumps  present  in  the  relevant 
membrane  or  whether  translocation  between  in- 
active and  active  compartments  occurs.  Immuno- 
chemical and  molecular  biological  means  will  be 
used  to  localize  the  pumps  and  identify  the 
type(s)  of  isozyme  involved. 

Proton  pumps  are  also  seemingly  responsible 
for  phagosomal  acidification.  We  are  currently  in- 
terested in  the  source  of  the  pumps  that  underlie 
this  process  and  their  mode  of  activation.  We  are 
also  planning  to  study  the  determinants  of  the 
internal  pH  of  the  phagosome  and  other  endo- 
membrane  compartments.  Our  current  evidence 
suggests  that  differential  counterion  permeabil- 
ity, which  was  claimed  to  be  the  main  source  of 
pH  heterogeneity,  is  not  an  important  factor  dic- 
tating intraorganellar  pH.  Differential  pH  sensitiv- 
ity of  the  pumps,  due  to  varying  subunit  compo- 
sition, or  a  regulated  proton  leak  permeability  are 
our  preferred  hypotheses. 

These  studies  are  expected  to  contribute  to  our 
understanding  of  immune  cell  function  and  intra- 
cellular pH  regulation  in  these  and  other  cells. 


498 


Genetic  Basis  of  Multidrug  Resistance 


Philippe  Gros,  Ph.D. — International  Research  Scholar 

Dr.  Gros  is  Associate  Professor  of  Biochemistry  at  McGill  University,  Montreal,  and  a  member  of  the  McGill 
Cancer  Center  and  the  McGill  Center  for  the  Study  of  Host  Resistance.  He  received  his  Ph.D.  degree  from 
McGill  University  and  pursued  postdoctoral  training  in  molecular  biology  and  cancer  research  at 
Massachusetts  General  Hospital  with  Joel  Habener  and  at  the  Massachusetts  Institute  of  Technology  with 
David  Housman. 


TUMOR  cells  in  vivo  and  in  vitro  can  develop 
simultaneous  resistance  to  a  wide  range  of 
structurally  and  functionally  unrelated  cytotoxic 
drugs.  Such  multidrug  resistance  (MDR)  severely 
impedes  the  chemotherapeutic  treatment  of 
many  types  of  tumors.  Structural  or  functional 
characteristics  common  to  drugs  of  the  MDR 
spectrum  are  few.  In  general  these  drugs  are 
small,  hydrophobic  natural  products  that  often 
contain  a  basic  nitrogen  atom  and  penetrate 
the  cell  by  passive  diffusion  across  the  mem- 
brane. MDR  is  associated  with  a  decreased  in- 
tracellular drug  accumulation  and  concomitant 
increased  drug  efflux  from  resistant  cells,  both 
ATP-dependent . 

MDR  is  caused  by  the  overexpression  of  a  high- 
molecular-weight  membrane  phosphoglycopro- 
tein  called  P-glycoprotein  (P-gp) .  P-gp  has  been 
found  capable  of  binding  photoactivatable  ana- 
logues of  ATP  and  cytotoxic  drugs,  suggesting 
that  it  functions  as  an  ATP-driven  efflux  pump 
that  reduces  the  intracellular  accumulation  of 
drugs  in  resistant  cancer  cells.  Recent  studies 
have  shown  that  increased  P-gp  expression  in  neu- 
roblastomas and  soft-tissue  sarcomas  causes  lack 
of  response  to  chemotherapy  and  is  associated 
with  very  poor  prognosis  and  outcome  of  these 
diseases. 

P-gp  is  encoded  by  a  small  family  of  closely 
related  genes,  termed  mdr  or  pgp,  that  share  con- 
siderable sequence  homology  and  common  an- 
cestral origins.  This  gene  family  has  three 
members  in  rodents  (mdrl,  mdr 2,  and  mdr 3) 
and  two  in  humans  {MDRl  and  MDR2)  .We  have 
isolated  and  characterized  full-length  cDNA 
clones  corresponding  to  the  three  mouse  genes 
and  have  deduced  the  amino  acid  sequences  of 
the  three  predicted  polypeptides.  P-gps  share 
considerable  sequence  homology  (80-85  per- 
cent identity)  and  common  structural  features, 
including  1 2  predicted  transmembrane  domains 
and  two  nucleotide  binding  sites. 

Each  P-gp  is  formed  by  two  homologous  halves 
that  show  sequence  conservation  with  a  large 
group  of  bacterial  transport  proteins  participat- 
ing in  the  import  and  export  of  specific  substrates 


in  Escherichia  coli.  This  evolutionary  conserva- 
tion is  in  keeping  with  P-gp's  proposed  drug  ef- 
flux function. 

The  normal  physiological  function  of  P-gps  has 
yet  to  be  elucidated.  Each  P-gp  isoform  is  ex- 
pressed in  a  tissue-specific  fashion,  generally  on 
the  apical  surface  of  secretory  epithelial  cells 
such  as  those  of  the  bile  canalicular,  the  brush 
border  of  the  intestine,  and  the  proximal  tubule 
of  the  kidney.  It  has  also  been  found  in  endothe- 
lial cells  of  the  blood-brain  barrier  and  in  early 
pluripotent  stem  cells  of  the  hematopoietic  sys- 
tem. From  these  findings  it  appears  that  P-gp  ei- 
ther plays  a  normal  detoxifying  role  against  envi- 
ronmental xenobiotics  or  transports  normal 
physiological  substrates  yet  to  be  identified. 

Recently  it  was  shown  that  the  mrfr  gene  family 
is  itself  part  of  a  larger  family  of  sequence-related 
genes  shown  to  play  key  physiological  functions 
in  normal  cells  and  tissues.  These  include  the 
STE6  gene  of  the  yeast  Saccharomyces  cerevi- 
siae,  responsible  for  the  transmembrane  trans- 
port of  the  "a"  mating  pheromone;  the  pfmdrl 
gene  of  the  malarial  parasite  Plasmodium  falci- 
parum, associated  with  chloroquine  efflux  from 
resistant  isolates  of  this  parasite;  and  in  humans, 
the  CFTR  chloride  channel  gene,  mutations  of 
which  cause  cystic  fibrosis,  and  the  RING  family 
genes,  which  code  for  peptide  pumps  implicated 
in  antigen  presentation  by  T  lymphocytes.  There- 
fore it  appears  that  the  mdr  supergene  family 
codes  for  membrane-associated  transport  pro- 
teins that  may  transport  different  types  of  sub- 
strates by  the  same  mechanism. 

We  have  carried  out  functional  analyses  of  indi- 
vidual members  of  the  mouse  mdr  gene  family. 
For  this,  we  have  transfected  and  overexpressed 
cDNAs  that  correspond  to  each  member  of  the 
family.  We  observed  that  mdrl  and  mdr3,  but 
not  mdr2,  could  directly  confer  MDR  to  other- 
wise drug-sensitive  cells.  In  addition,  the  profile 
of  drug  resistance  conferred  by  mdrl  and  mdr3 
appeared  distinct. 

One  of  the  key  unanswered  questions  about  P- 
gp  and  MDR  is  how  a  single  transport  protein  can 
apparently  recognize  and  transport  a  large  group 


499 


Genetic  Basis  of  Multidrug  Resistance 


of  structurally  and  functionally  unrelated  com- 
pounds. The  identification  of  P-gp  segments  and 
residues  implicated  in  drug  recognition  and  trans- 
port is  a  necessary  prerequisite  to  the  rational  de- 
sign of  new  cytotoxic  compounds  capable  of 
blocking  or  bypassing  the  action  of  P-gp  in  drug- 
resistant  tumor  cells.  We  have  exploited  the  high 
degree  of  sequence  similarity  and  striking  func- 
tional differences  detected  among  members  of 
the  mouse  mdr  family  to  identify,  in  chimeric 
and  mutant  proteins,  segments  and  residues  im- 
portant for  drug  recognition.  To  identify  the  do- 
mains of  mdrl  that  are  essential  for  MDR  and  that 
may  be  functionally  distinct  in  the  biologically 
inactive  mdr2,  we  have  constructed  1 1  chimeric 
molecules  in  which  discrete  domains  of  mdr2 
have  been  introduced  into  the  homologous  re- 
gion of  mdrl.  We  have  analyzed  these  chimeras 
for  their  capacity  to  confer  MDR. 

The  two  predicted  ATP-binding  sites  of  mdr2 
were  found  to  be  functional,  as  either  could  com- 
plement the  biological  activity  of  mdrl.  How- 
ever, the  replacement  of  either  the  amino-  or  the 
carboxyl-terminus  transmembrane  (TM)  domain 
region  of  mdrl  by  the  homologous  segment  of 
mdr2  resulted  in  inactive  chimeras.  Replace- 
ment of  as  few  as  two  TM  domains  of  mdrl  from 
either  the  amino-  or  carboxyl-terminal  halves  by 
the  corresponding  segment  of  mdr2  was  suffi- 
cient to  destroy  mdrVs  activity.  These  observa- 
tions suggest  that  the  functional  differences  de- 
tected between  mdrl  and  mrfr2  reside  within  the 
TM  domains  of  the  two  proteins. 

P-gps  encoded  by  mouse  mdrl  and  mdr3 
confer  distinct  drug  resistance  profiles.  While 
both  clones  confer  comparable  levels  of  resis- 
tance to  vinblastine  (VBL),  mdr 3  confers  prefer- 
ential resistance  to  actinomycin  D  (ACT),  and 
mdrl  to  colchicine  (COL). 

To  identify  protein  domains  implicated  in  the 
preferential  drug  resistance  encoded  by  either  pa- 
rental mdr  clone,  homologous  protein  domains 
were  exchanged  in  a  series  of  16  hybrid  cDNA 
clones,  and  the  drug  resistance  profiles  encoded 
by  the  corresponding  chimeric  proteins  were  an- 
alyzed. While  all  chimeric  clones  conferred  simi- 
lar levels  of  VBL  resistance,  the  levels  of  ACT  and 
COL  resistance  conferred  by  the  various  clones 
were  heterogeneous,  being  either  similar  to  the 
parental  mdrl  or  mdr3  clones  or,  in  many  cases, 
intermediate  between  the  two. 

Only  those  chimeric  proteins  carrying  seg- 
ments that  overlapped  both  the  amino  and  car- 


boxyl  sets  of  TM  domains  of  the  respective  parent 
conveyed  the  parent's  full  preferential  drug  re- 
sistance profile.  These  results  suggest  that 
the  resistance  profiles  encoded  by  mdrl  or 
mdr 3,  possibly  representing  sites  of  drug- protein 
interactions,  involve  several  determinants  asso- 
ciated with  TM  domains  from  both  homologous 
halves  of  P-gp.  Recently  we  have  tentatively  iden- 
tified one  of  these  sites.  We  have  observed  that  a 
simple  serine-to-phenylalanine  substitution  at 
position  941  (mdrl)  or  939  (m<^r3),  within  pre- 
dicted TMl  1 ,  had  a  dramatic  effect  on  the  overall 
activity  of  the  two  pumps. 

The  modulating  effect  of  this  mutation  on 
mdrl  and  mdr 3  varied  for  the  drugs  tested.  It  was 
very  strong  for  COL  and  adriamycin  (ADR)  but 
only  moderate  for  VBL.  For  mdrl,  the  serine-to- 
phenylalanine  replacement  produced  a  unique 
mutant  protein  that  retained  the  capacity  to 
confer  VBL  resistance  but  lost  the  ability  to  confer 
ADR  or  COL  resistance.  These  results  suggest  that 
ADR-COL  and  VBL  may  have  distinct  binding  sites 
on  P-gp  and  that  the  serine  residue  within  TMl  1 
plays  a  key  role  in  P-gp's  recognition  and  trans- 
port of  the  former  drugs.  We  are  currently  prob- 
ing drug-P-gp  interactions  at  this  residue,  using 
additional  mutants  together  with  COL  and  ADR 
analogues  modified  at  key  positions  on  their  re- 
spective backbone. 

Other  investigators  have  found  the  same  resi- 
due mutated  in  the  pfmdrl  gene  from  isolates  of 
the  human  malarial  parasite  Plasmodium  falci- 
parum that  are  resistant  to  chloroquine  (CLQ). 
Such  resistance  is  caused  by  an  increased  ATP- 
dependent  CLQ  efflux  and  is  associated  with  mu- 
tant alleles  of  the  mdr  homologue pfmdrl,  map- 
ping near  TMl  or  within  TMl  1 .  Taken  together, 
these  and  our  studies  indicate  that  the  TMl  1  do- 
main of  mdr  and  mdr-like  genes  is  critical  for 
drug  recognition  and  transport.  Besides  their 
high  degree  of  hydrophobicity,  no  significant  ho- 
mology is  detected  between  TMll  domains  of 
mdrl-3  and  pfmdrl  proteins.  Both,  however, 
have  the  potential  of  forming  amphipathic 
helices. 

The  mdr  Ser^'^"^^'  residues  and  the  mutated 
residues  in  pfmdrl  fall  within  the  hydrophilic 
side  of  this  helix,  and  the  mutant  residues  map 
near  what  appears  to  be  the  boundary  of  the  hy- 
drophilic side.  These  amphipathic  helices  may 
be  important  for  the  recognition  of  hydrophobic 
compounds,  such  as  MDR  drugs,  that  readily  par- 
tition within  the  cell's  lipid  bilayer. 


500 


Control  of  Bacterial  Protein  Synthesis 
During  Viral  Infection 


Gabriel  Guameros  Pena,  Ph.D. — International  Research  Scholar 

Dr.  Guameros  is  Professor  of  Genetics  and  Molecular  Biology  at  the  Center  for  Research  and  Advanced 
Studies,  National  Polytechnic  Institute,  Mexico  City.  He  received  his  undergraduate  and  M.Sc.  degrees  in 
microbiology,  chemistry,  and  biochemistry  in  Mexico  City,  and  his  Ph.D.  degree  in  molecular  biology 
from  the  University  of  California,  Berkeley,  where  he  studied  with  Harrison  Echols.  He  joined  the  staff  of 
the  Center  after  doing  postdoctoral  work  in  molecular  biology  at  the  University  of  Geneva,  Switzerland, 
in  Harvey  Eisen's  laboratory.  He  has  been  awarded  fellowships  from  the  Guggenheim  Memorial 
Foundation,  the  Commission  of  the  European  Communities,  and  the  Sistema  Nacional  de 
Investigadores,  Mexico. 


INFECTING  viruses  divert  the  functions  of  the 
host  cell  for  their  own  development.  To 
achieve  this,  the  viral  genome  directs  the  synthe- 
sis of  regulatory  molecules,  which  reorient  cell 
functions  to  conform  to  the  specific  viral  develop- 
ment program.  There  is  abundant  evidence  of 
transcriptional  control  during  phage  X  infection 
of  Escherichia  coli,  where  the  phage  genome  di- 
rects the  synthesis  of  regulatory  proteins  that 
alter  the  cells'  transcriptional  pattern.  Little  is 
known,  however,  about  the  translational  control 
in  X-infected  cells.  Our  laboratory  has  pursued  a 
case  of  translational  control  involving  X  RNA  se- 
quences, named  bar,  and  peptidyl-tRNA  hydro- 
lase (Pth),  a  bacterial  enzyme  essential  for  pro- 
tein synthesis. 

The  Target  in  the  Cell 

Bacterial  mutants  partially  defective  in  the  ac- 
tivity of  Pth  are  unable  to  support  the  growth  of  X 
phage.  We  have  shown  that  the  corresponding 
mutations  are  located  in  the  pth  gene  region.  This 
result  was  further  confirmed  by  sequence  analy- 
sis; the  mutations  are  base  substitutions  within  a 
translational  open  reading  frame  that  corre- 
sponds to  the  pth  gene.  Pth,  the  gene  product, 
was  isolated  and  characterized  chemically  and 
enzymatically. 

Pth  hydrolyzes  peptidyl-tRNAs  to  yield  free 
tRNAs  and  peptides.  It  has  been  proposed  that  the 
enzyme  is  a  scavenger  of  peptidyl-tRNAs  that  have 
dropped  off  the  ribosomes  during  editing  of  mis- 
incorporated  amino  acids  in  polypeptide  chains. 
The  Pth  function  is  essential  for  the  cell,  as  in- 
ferred from  the  fact  that  a  heat-sensitive  mutant  of 
Pth  accumulates  peptidyl-tRNAs  and  stops  pro- 
tein synthesis  upon  shift  to  the  nonpermissive 
temperature.  Moreover,  the  enzyme  is  ubiquitous 
among  organisms  from  bacteria  to  mammals. 

The  possible  role  of  Pth  in  ribosome-bound  hy- 
drolysis of  peptidyl-tRNA  at  the  step  of  polypep- 
tide chain  termination  has  not  been  supported  by 
the  work  of  others.  Preliminary  results,  however, 
indicate  that  Pth  may  be  involved  in  polypeptide 


chain  termination  (see  below),  and  we  aim  to 
investigate  this  possible  participation. 

Nature  of  the  X  Regulator 

We  have  isolated  phage  mutants  that  overcome 
the  bacterial  Pth  defect.  These  mutations  defined 
several  genetic  sites  named  bar.  DNA  sequence 
analysis  of  two  of  these,  barl  and  barll,  revealed 
that  the  mutations  affect  nearly  identical  l6-bp 
segments  having  dyad  symmetry. 

The  inhibition  of  phage  development  by  mu- 
tants defective  in  Pth  requires  transcription 
through  wild-type  X  bar  regions.  Transcripts 
themselves,  not  polypeptides,  seem  to  be  the  ac- 
tive molecules.  Aplasmid  system  in  which  short  X 
bar  sequences  were  cloned  in  front  of  an  active 
promoter  somehow  mimics  the  X  inhibition 
effect. 

Transcription  of  wild-type  bar  in  plasmids  is 
lethal  to  Pth-defective  (but  not  wild-type)  cells. 
This  effect  is  specific,  because  constructs  carry- 
ing mutant  bar  sequences  are  not  lethal.  Tran- 
scription through  a  vector  harboring  a  synthetic 
nucleotide  sequence  as  short  as  20  bp  mimicking 
barl  caused  pth  mutant  lethality;  therefore  we 
think  that  the  1 6-bp  bar  sequence  is  the  core  of 
the  inhibitory  transcripts.  Protein  synthesis  is 
shut  off  soon  after  bar  transcript  induction,  but 
RNA  synthesis  continues  for  several  hours.  Thus 
the  lethal  effect  is  probably  caused  by  a  rapid 
inhibition  of  protein  synthesis. 

How  does  bar  RNA  inhibit  protein  synthesis? 
Preliminary  data  on  nonsense  codon-specific 
suppression,  obtained  in  collaboration  with 
Emanuel  Murgola  (M.  D.  Anderson  Cancer  Cen- 
ter, Houston),  have  led  us  to  propose  a  unifying 
model  that  implicates  foar  RNAand  Pth  in  peptide 
chain  termination.  The  model  postulates,  first, 
that  bar  RNA  can  interfere  with  the  termination 
of  UGA-mediated  translation  by  antiparallel  base- 
pairing  with  ribosomal  1 6S  RNA,  and  second,  that 
mutant  Pth  causes  a  defect  in  polypeptide  termi- 
nation facilitating  bar  RNA- 1 6S  RNA  interaction. 


501 


Control  of  Bacterial  Protein  Synthesis  During  Viral  Infection 


We  are  attempting  to  test  this  model  through  a 
direct  assay  of  bar  RNA  imeraction  with  cell  com- 
ponents and  the  effect  of  Pth  on  polypeptide 
chain  termination.  We  will  also  investigate  possi- 
ble genes  and  gene  products  that  interact  with 
Pth  through  the  isolation  of  second-site  muta- 
tions that  suppress  pth  defect. 

Biochemistry  of  Pth  and  Regulation 
of  pth  Expression 

To  purify  Pth  protein,  we  have  exploited  the 
fact  that  cells  harboring  pth  plasmids  overpro- 
duce the  wild-type  enzyme.  Part  of  our  current 
efforts  are  devoted  to  overproducing  and  purify- 
ing mutant  enzymes  to  compare  their  biochemi- 
cal properties.  Mutant  Pths  may  have  different 
patterns  of  specificity  for  various  amino  acyl- 
tRNAs.  Also  our  laboratory  is  working  on  the  con- 
trol of  pth  expression  by  analyzing  the  transcrip- 
tional and  translational  properties  of  the  gene. 


What  Does  bar  Do  for  X? 

We  have  considered  two  possible  roles  for  bar 
regulation  in  X  biology,  taking  into  account  the 
two  functions  proposed  for  Pth.  First,  bar  RNA 
may  control  the  relative  levels  of  specific  tRNAs 
to  fit  the  profile  of  codon  usage  in  X.  This  func- 
tion, perfectly  tolerated  in  normal  cells,  may  lead 
to  inhibition  of  phage  development  or  to  lethality 
in  cells  defective  for  Pth.  Second,  bar  RNA  may 
act  on  termination  and/or  initiation  of  polypep- 
tide chains  directed  by  phage  transcripts.  Among 
X  genes,  UGA  is  the  most  frequent  termination 
codon  (not  UAA,  as  in  the  host),  and  in  the  X 
genome  it  is  not  uncommon  for  a  gene's  termina- 
tion codon  to  overlap  partially  the  initiation  co- 
don of  the  next  gene,  producing  the  sequence 
AUGA.  The  fact  that  the  bar  core  RNA  contains 
the  sequence  AUGA,  and  the  alleged  interaction 
of  this  sequence  with  ribosomal  1 6S  RNA,  suggest 
a  role  for  these  overlapping  genes  in  polypeptide 
chain-termination  (-initiation)  events. 


502 


Molecular  Genetics  of  Photosynthesis 
and  Carbon  Assimilation  in  Plants 


Luis  R.  Herrera-Estrella,  Ph.D. — International  Research  Scholar 

Dr.  Herrera-Estrella  is  Professor  and  Head  of  the  Department  of  Plant  Genetic  Engineering  at  the  Center 
for  Research  and  Advanced  Studies,  National  Polytechnic  Institute,  Irapuato.  He  received  his 
undergraduate  degree  as  biochemical  engineer  from  the  National  Polytechnic  Institute  in  Mexico  and  his 
Ph.D.  degree  from  the  State  University  of  Ghent,  Belgium,  where  his  thesis  advisors  were  Marc  Van 
Montagu  and  Jeff  Schell.  The  subject  of  his  thesis  was  the  expression  of  foreign  genes  in  plant  cells.  As  a 
postdoctoral  researcher  in  Ghent,  he  studied  the  regulation  of  light- inducible  plant  genes  and  the 
movement  of  proteins  into  the  chloroplast.  His  honors  include  the  Minoru  and  Ethel  Tsutsui  Distinguished 
Graduate  Research  Award  in  Science  from  the  New  York  Academy  of  Sciences  and  the  Javed  Husain  Award 
for  Young  Scientists  from  UNESCO. 


PHOTOSYNTHESIS  and  carbon  assimilation 
are  the  most  important  biochemical  and  mo- 
lecular events  in  the  life  cycle  of  higher  plants 
and,  indeed,  are  key  to  the  provision  of  nutrients 
for  the  entire  food  chain.  Solar  energy  is  first  col- 
lected in  the  chloroplasts  of  photosynthetic  tis- 
sues, mainly  of  leaves,  by  light-harvesting  anten- 
nas composed  of  chlorophyll  and  protein 
molecules.  The  collected  energy  is  then  used  to 
convert  atmospheric  carbon  dioxide  (CO2)  into 
triose  phosphate  molecules.  These  three-carbon 
molecules  proceed  through  a  series  of  reactions, 
called  the  Calvin-Benson  cycle,  that  culminates 
in  the  production  of  sugars  from  v^^hich  all  the 
organic  molecules  required  for  plant  life  are 
synthesized. 

More  specifically,  triose  phosphate  molecules 
are  converted  in  the  cytoplasm  of  photosynthe- 
tic, or  source,  cells  into  sucrose,  which  is  translo- 
cated through  the  phloem  to  feed  the  nonphoto- 
synthetic,  or  consumer,  tissues  (i.e.,  roots, 
flowers,  seeds,  tubers).  Assimilated  carbon  is 
stored  temporarily  or  permanently  in  the  form  of 
starch  in  both  source  and  consumer  tissues.  The 
starch  in  seeds  or  tubers  provides  most  of  the  car- 
bon and  energy  for  the  germination  and  develop- 
ment of  new  plants. 

The  light-dependent  production  of  ATP  and 
NADPH,  the  reductive  assimilation  of  CO2,  and 
sucrose  and  starch  synthesis  are  interlinked  and 
interdependent.  These  processes  must  be  coordi- 
nated in  vivo  at  both  the  biochemical  and  genetic 
level  (i.e.,  at  the  level  of  gene  expression).  The 
balance  between  the  efficiency  of  CO2  fixation, 
sucrose  translocation  and  uptake,  and  assimila- 
tion of  sucrose  in  consumer  tissues  plays  a  funda- 
mental role  in  determining  the  productivity  of 
any  given  plant  species.  This  balance  is  affected 
by  both  genetic  determinants  of  the  individual 
and  its  interaction  with  the  environment. 

Our  laboratory  is  interested  in  studying  the  mo- 
lecular events  that  control  the  biochemical  pro- 
cesses involved  in  carbon  assimilation  in  plant 


cells.  One  aspect  that  we  are  investigating  is  how 
light  regulates  genes  involved  in  photosynthesis. 
Ribulose  1 ,5-bisphosphate  carboxylase  oxygen- 
ase (RuBisCO)  is  a  multimeric  enzyme  composed 
of  eight  identical  small  subunits  (SS)  and  eight 
identical  large  subunits  (LS) .  In  the  so-called  C3 
plants,  RuBisCO  carries  out  the  initial  CO2  fixa- 
tion step,  utilizing  the  five-carbon  sugar  ribulose 
bisphosphate  to  produce  two  three-carbon  deriva- 
tives (triose  phosphate  molecules).  The  gene 
family  encoding  the  SS  is  located  in  the  nuclear 
genome;  the  gene  encoding  the  LS  is  located  in 
the  plastid  genome.  How  genes  located  in  differ- 
ent cellular  compartments  are  coordinately  regu- 
lated (those  for  both  SS  and  LS  are  regulated  by 
light  and  should  produce  the  corresponding  sub- 
units  in  equimolar  amounts) ,  and  how  the  active 
RuBisCO  enzyme  is  assembled  from  its  subunits, 
are  some  of  the  questions  we  wish  to  answer. 

In  collaboration  with  June  Simpson,  we 
showed  previously  that  the  5'-flanking  sequences 
of  genes  encoding  the  small  subunit  of  the  RuBis- 
CO {ss  genes)  and  the  chlorophyll  «/fo-binding 
proteins  (cabSO  gene)  are  responsible  for  the 
light-inducible  transcription  of  these  genes.  We 
have  also  shown  that  enhancer  and  silencer  ele- 
ments are  involved  in  the  tissue-specific  and 
light-inducible  expression  of  the  cabSO  gene. 
The  cis-  and  trans-acting  elements  involved  in  the 
regulation  of  the  cabSO  gene  are  being  analyzed. 
From  these  studies  a  7-base  pair  repeated  ele- 
ment within  the  5'-flanking  region  of  the  cabSO 
gene  has  been  identified  as  the  site  of  interaction 
with  a  putative  regulatory  DNA-binding  protein. 

Many  attempts  to  assemble  RuBisCO  in  vitro 
from  its  isolated  subunits  have  failed,  suggesting 
that  the  plant  cell  in  vivo  provides  other  req- 
uisite components.  It  has  been  suggested  that 
RuBisCO  assembly  requires  the  participation  of 
proteins  that  act  as  molecular  chaperones  to  pro- 
mote the  correct  interaction  between  the  sub- 
units,  ensuring  the  assembly  of  the  catalytically 
active  enzyme.  Two  60-kDa  proteins  termed 


503 


Molecular  Genetics  of  Photosynthesis  and  Carbon  Assimilation  in  Plants 


chaperonins  (Cpn60)  a  and  ^  have  been  sug- 
gested as  participating  in  RuBisCO  assembly.  Since 
the  availability  of  functional  RuBisCO  is  important 
for  carbon  assimilation,  we  have  isolated  two 
cDNAs  and  a  genomic  clone  for  Cpn60  /?. 

To  study  whether  Cpn60  ^  is  indeed  involved 
in  RuBisCO  assembly,  and  whether  it  is  specific 
for'RuBisCO  or  plays  a  more  general  role  in  pro- 
tein assembly,  we  are  currently  generating  trans- 
genic plants  containing  a  chimeric  gene  to  pro- 
duce an  antisense  RNA  for  Cpn60  /3.  The  RNA 
should  interact  with  the  Cpn60  /3  mRNA  and 
arrest  the  production  of  Cpn60  /3  polypeptides, 
thus  producing  transgenic  plants  that  will  allow 
us  to  assess  the  role  of  Cpn60  (8  in  RuBisCO  assem- 
bly. We  are  also  constructing  chimeric  genes  com- 
posed of  the  promoter  of  a  Cpn60  ^  gene  and  the 
coding  sequence  of  the  /^-glucuronidase  bacterial 


reporter  gene,  to  study  the  tissue-specific  and  en- 
vironmental regulation  of  this  gene  in  transgenic 
plants. 

Again,  the  main  assimilates  from  photosyn- 
thesis are  triose  phosphate  molecules  that  are 
converted  into  sucrose  to  be  translocated  to  con- 
sumer tissues.  Several  physiological  and  biochem- 
ical studies  indicate  that  sucrose  6-phosphate  syn- 
thase (SPS)  is  the  limiting  enzyme  for  this 
conversion.  To  study  the  role  of  SPS  in  carbon 
assimilation,  and  the  interrelation  of  the  genes 
encoding  this  enzyme  with  photosynthetic  genes, 
we  are  currently  isolating  SPS  cDNA  clones,  using 
monospecific  SPS  antibodies  and  polymerase 
chain  reaction  technology  in  collaboration  with 
Horacio  Pontis  from  Mar  del  Plata-Argentina. 
(The  work  on  SPS  is  supported  in  part  by  a  grant 
from  the  Rockefeller  Foundation.) 


Opposite:  Light  microscopy  of  transverse  stem 
sections  of  transgenic  tobacco  plants  harbor- 
ing a  chimeric  gene  in  which  the  P-glucuroni- 
dase-coding  sequence  is  under  control  of  the 
chaperonin  60  0  promoter.  The  blue  staining 
indicates  the  cell- type- specific  expression  di- 
rected by  the  promoter  at  the  basal  (A)  and 
apical  (B)  stem  regions  of  transgenic  plants. 

Research  and  photograph  by  Eduardo  Zaba- 
leta  in  the  laboratory  of  Luis  Herrera-Estrella. 


504 


505 


Expression  of  the  mouse  En- 2  gene  in  a  band  of  cells  across 
the  midbrain- hindbrain  junction.  The  10-day  transgenic 
embryo  contains  a  lacZ  reporter  construct  expressed  from 
En-2  DNA  regulatory  sequences.  The  cells  expressing  lacZ 
( dark )  are  visualized  by  histochemical  staining  of  the 
lacZ  gene  product. 

From  Sedivy,  f.,  and  foyner,  A.  1992.  Gene  Targeting. 
New  York:  W.H.  Freeman,  p  161. 


506 


Gene  Pattern  Expression  in  Early  Embryogenesis 


Alexandra  L.Joyner,  Ph.D. — International  Research  Scholar 

Dr.  Joyner  is  a  Senior  Scientist  at  the  Samuel  Lunenfeld  Research  Institute  of  Mount  Sinai  Hospital, 
Toronto,  and  Associate  Professor  of  Molecular  and  Medical  Genetics  at  the  University  of  Toronto.  She 
received  her  B.Sc.  degree  in  zoology  and  her  Ph.D.  degree  in  medical  biophysics  from  the  University  of 
Toronto.  She  did  postdoctoral  work  in  mammalian  development  with  Gail  Martin  at  the  University  of 
California,  San  Francisco. 


THE  establishment  of  the  basic  body  plan  re- 
quires an  intricate  coordination  of  cell-cell 
interactions  that  appear  to  be  controlled  largely 
by  the  genetic  program  handed  down  from  gener- 
ation to  generation  in  our  DNA.  Many  of  the  genes 
that  run  the  program  of  pattern  formation  have 
been  identified  in  Drosophila  and  shown  by  mu- 
tant analysis  to  regulate  the  development  of  em- 
bryonic regions  rather  than  the  differentiation  of 
cell  types.  In  keeping  with  this,  many  of  these 
genes  are  expressed  early  in  embryogenesis  in 
spatially  defined  patterns. 

The  primary  focus  of  research  in  my  laboratory 
has  been  to  identify  and  study  mouse  homologues 
of  Drosophila  pattern-formation  genes.  This 
work  has  been  based  on  the  premise  that  a  con- 
servation of  gene  structure  through  evolution  re- 
flects a  corresponding  conservation  of  gene  func- 
tion. Work  over  the  last  few  years  in  many 
laboratories,  including  mine,  has  shown  this  to 
be  the  case. 

A  second  research  project  has  involved  devel- 
oping and  applying  a  new  type  of  random  screen, 
called  the  gene  trap,  for  genes  expressed  in  a  spa- 
tially defined  manner  during  early  mouse 
embryogenesis. 

Mouse  Homologues  of  the  Drosophila 
Gene  engrailed 

The  fruit  fly  body  is  divided  into  a  number  of 
repeated  units  referred  to  as  segments,  and  the 
engrailed  (en)  genes  are  known  to  be  required 
for  proper  development  of  the  posterior  half  of 
each  segment.  The  en  gene  has  characteristics  of 
a  "switch"  that  can  direct  cells  down  a  posterior, 
as  opposed  to  anterior,  developmental  pathway. 
Molecular  characterization  of  the  gene's  product 
has  shown  it  to  be  a  transcription  factor  that  binds 
DNA  through  a  motif  called  a  homeodomain.  We 
have  been  studying  En  1  and  En-2,  the  mouse 
homologues  of  the  Drosophila  en  gene,  to  deter- 
mine whether  they  also  act  as  transcription  fac- 
tors controlling  pattern  formation. 

One  striking  difference  between  these  homolo- 
gous mouse  and  fruit  fly  genes  is  that  the  fly  en 
gene  is  expressed  during  embryogenesis  in  14 


stripes  (one  for  each  segment) ,  whereas  the  ver- 
tebrate En  genes  are  first  expressed  in  a  single 
band  across  the  developing  mid-  and  hindbrain 
junction.  En  l  and  En-2  continue  to  have  a  spa- 
tially defined  expression  pattern  in  the  brain  un- 
til the  cells  begin  to  differentiate  into  particular 
cell  types.  En  expression  then  switches  and  be- 
comes cell-type  specific. 

Many  other  mouse  homologues  of  Drosophila 
pattern-formation  genes  have  also  been  analyzed. 
A  recurring  theme  is  expression  in  spatially  de- 
fined patterns  early  in  development,  particularly 
in  the  nervous  system.  This  suggests  that  at  least 
part  of  the  mechanism  for  laying  down  the  basic 
body  plan,  and  especially  for  specifying  different 
regions  of  the  nervous  system  along  the  anterior- 
posterior  axis,  involves  the  coordinate  expres- 
sion of  different  sets  of  these  developmental 
switch  genes. 

One  of  our  objectives  has  been  to  make  trans- 
genic mice  that  lack  the  En  genes.  To  date  we 
have  made  mice  that  are  deleted  for  the  En-2 
homeodomain-coding  DNA  sequences.  These 
mice  do  not  show  a  major  disruption  of  the  whole 
mid-  and  hindbrain  region  but  show  a  distinct 
disruption  in  the  pattern  of  folds  in  the  cerebel- 
lum. We  are  now  studying  the  developmental  pro- 
gression of  the  defect  to  determine  the  cellular 
basis  of  the  mutant  phenotype.  That  the  cerebel- 
lum, uniquely  among  adult  brain  structures,  ex- 
presses En-2  in  the  absence  of  En-1  indicates  a 
functional  redundancy  between  En-1  and  En-2. 
Thus  both  En  genes  must  be  deleted  before  their 
function  in  development  of  the  mid-  and  hind- 
brain can  be  studied. 

A  second  aspect  of  our  En  research  is  to  iden- 
tify other  genes  in  the  same  genetic  pathways  as 
En-1  and  En-2.  One  of  our  approaches  has  been 
to  seek  transcription  factors  that  regulate  En  ex- 
pression. As  a  first  step,  we  have  analyzed  the 
DNA  sequences  around  the  En-1  and  En-2  genes 
and  have  identified  fragments  that  will  direct  ex- 
pression of  the  /acZ  reporter  gene  to  the  mid-  and 
hindbrain  junction.  These  fragments  can  now  be 
further  subdivided  and  used  to  identify  the  tran- 
scription factors  that  bind  these  sequences  and 
regulate  En  expression. 


507 


Gene  Pattern  Expression  in  Early  Embryogenesis 


In  addition,  we  are  using  these  DNA  transcrip- 
tion regulators  to  express  other  pattern-formation 
genes  aberrantly  in  the  mid-  and  hindbrain  re- 
gion. In  this  way  we  can  test,  for  example, 
whether  other  homeodomain-containing  genes, 
expressed  in  more-posterior  regions  than  En,  can 
switch  the  developmental  program  of  mid-  and 
hindbrain  cells  to  more-posterior  regions. 

We  are  also  interested  in  identifying  genes  that 
are  regulated  by  En.  One  approach  we  are  taking 
is  to  analyze  mouse  homologues  of  Drosophila 
genes  that  are  regulated  by  en.  To  this  end,  we 
have  cloned  homologues  of  the  Drosophila  cubi- 
tus interruptus  (Ci)  gene.  The  mammalian  genes 
are  called  Gli,  since  one  member  of  the  family 
was  found  to  be  overexpressed  in  gliomas.  We 
can  now  analyze  the  expression  of  the  Gli  and 
other  potential  £n-regulated  genes  in  mice  lack- 
ing En- 2. 

Screening  for  Genes  Expressed  in  Spatially 
Defined  Patterns 

With  a  view  to  identifying  other  types  of  genes 
involved  in  pattern  formation,  we  devised  a 
screen  that  takes  advantage  of  mammalian  gene 
structure  and  mouse  embryonic  stem  (ES)  cells. 
The  screen  involves  randomly  integrating  a  vec- 
tor we  refer  to  as  a  gene  trap  into  the  ES  genome. 


The  vector  has  a  splice  acceptor  site  upstream  of 
the  reporter  gene  lacZ.  We  reasoned  that  if  the 
vector  integrated  in  the  correct  orientation  into 
the  intron,  then  splicing  of  the  gene  would  be 
directed  to  the  reporter  gene.  We  have  now  con- 
firmed this  by  cloning  and  characterizing  a  num- 
ber of  transcripts  for  /flcZ-endogenous  gene 
fusions. 

Cells  expressing  lacZ  can  be  revealed  with  a 
histochemical  stain.  ES  cells  expressing  the  re- 
porter are  then  reintroduced  into  a  host  embryo 
for  lacZ  expression  pattern  analysis.  We  have 
now  analyzed  the  expression  pattern  of  the 
cloned  endogenous  genes  from  two  lacZ  inser- 
tions and  have  shown  that  the  lacZ  pattern  faith- 
fully mimics  that  of  the  endogenous  gene. 

Finally,  lacZ  insertions  that  show  a  spatially 
defined  expression  pattern  can  be  transmitted 
into  transgenic  mice  and  the  insertions  analyzed 
for  mutant  phenotypes.  Two  out  of  three  such 
mice  showed  mutant  defects.  Thus  we  have  dem- 
onstrated the  feasibility  of  the  gene  trap  approach 
and  are  now  carrying  out  a  larger  screen  for  can- 
didate pattern-formation  genes. 

All  of  this  work  is  also  funded  by  grants  from 
the  Medical  Research  Council  and  the  National 
Cancer  Institute  of  Canada,  the  National  Insti- 
tutes of  Health,  and  Bristol-Myers  Squibb. 


508 


Diagnostic  Use  of  RNA  Replication 
in  Infectious  Diseases 


Paul  M.  Lizardi,  Ph.D. — International  Research  Scholar 

Dr.  Lizardi  is  Professor  of  Biochemistry  at  the  Biotechnology  Institute,  National  Autonomous  University 
of  Mexico,  Cuernavaca.  He  received  his  Ph.D.  degree  from  the  Rockefeller  University  and  conducted 
postdoctoral  research  in  embryology  with  Donald  Brown  at  the  Carnegie  Institution  of  Washington, 
Baltimore.  After  serving  as  Associate  Professor  at  Rockefeller  as  an  Andrew  Mellon  Foundation  fellow. 
Dr.  Lizardi  spent  a  sabbatical  year  at  Massachusetts  General  Hospital,  Boston,  and  held  a  visiting 
professorship  in  genetics  at  Harvard  Medical  School. 


INFECTIOUS  diseases  are  frequently  managed 
by  health  professionals  without  definitive 
identification  of  the  pathogen.  Classical  ap- 
proaches to  diagnosis,  such  as  direct  microscopic 
observation,  cultivation,  or  infection  of  suscepti- 
ble hosts,  are  often  too  slow  or  cumbersome  for 
routine  medical  care.  Modern  laboratory  tests 
based  on  the  use  of  antibodies  provide  effective 
tools  for  diagnosis  of  a  number  of  infectious  dis- 
eases, but  most  antibody  tests  are  designed  to  de- 
tect the  presence  of  a  host  immune  response 
rather  than  the  pathogen  itself.  Thus  there  is  a 
need  for  techniques  permitting  the  rapid  and  reli- 
able detection  of  infectious  agents  so  that  epide- 
miological monitoring  and  patient  management 
can  be  more  effective. 

A  direct  way  to  detect  a  pathogen  is  to  identify 
its  genetic  material,  which  invariably  contains 
unique  sequence  patterns.  However,  the  genetic 
material  is  usually  so  minute  in  a  biological  sam- 
ple that  its  detection  presents  an  extraordinary 
technical  challenge.  Molecular  biologists  have 
been  up  to  the  task,  and  techniques  developed 
recently  permit  the  generation  of  millions  of  cop- 
ies of  DNA  or  RNA  segments  in  the  test  tube  by  a 
process  of  exponential  amplification.  The  best- 
known  amplification  method  is  the  polymerase 
chain  reaction  (PCR) ,  in  which  DNA  strands  are 
sequentially  separated  by  heating  and  then  cop- 
ied with  DNA  polymerase.  The  PCR  limit  of  de- 
tection is  about  50  molecules  of  target,  which  is 
over  100,000  times  as  sensitive  as  a  typical  en- 
zyme-linked immunoassay. 

Amplified  RNA  Binary  Probes 

My  laboratory  is  developing  alternative  ampli- 
fication methods  for  the  detection  of  RNA  or  DNA 
in  biological  samples.  The  work  is  being  carried 
out  in  close  collaboration  with  Fred  Kramer  at  the 
Public  Health  Research  Institute  in  New  York 
City  and  Jack  Szostak  at  Massachusetts  General 
Hospital  in  Boston.  Our  methods  exploit  several 
interesting  properties  of  RNA  molecules: 

•  Relatively  short  molecules  of  RNA  (15-30 
nucleotides)  have  long  been  known  to  form 
stable  helical  structures  when  bound  to  perfectly 


complementary  single  strands  of  DNA  or  RNA. 
The  thermodynamic  stability  of  these  helical 
complexes  distinguishes  them  from  similar  com- 
plexes containing  mismatched  bases.  Hence  one 
can  design  an  RNA  probe  that  will  bind  very 
strongly,  and  uniquely,  to  a  segment  of  the  DNA 
or  RNA  of  an  infectious  agent  in  a  biological 
sample. 

•  RNA  probe  molecules  can  be  joined  to  other 
RNA  molecules  in  a  target-dependent  manner. 
That  is,  joining  will  only  take  place  if  the  mole- 
cules are  aligned  on  a  complementary  target 
strand  that  serves  as  a  guide  for  the  joining  event. 
We  catalyze  the  joining  by  an  enzyme  called  a 
ribozyme  ligase,  isolated  in  Jack  Szostak's  labora- 
tory. The  ligase  is  itself  an  RNA,  derived  from  a 
naturally  occurring  intron  called  group  I,  origi- 
nally discovered  in  Tetrahymena  thermophyla 
by  Thomas  Cech  (HHMI,  University  of  Colorado 
at  Boulder) . 

•  A  specific  class  of  molecules  known  as  repli- 
catable  RNAs  can  be  produced  exponentially 
under  natural  conditions,  so  that  millions  of  cop- 
ies are  generated  in  minutes.  An  enzyme  called 
RNA  replicase  catalyzes  reactions  in  which  RNA 
single  strands,  parent  and  daughter,  are  forced 
apart  during  synthesis,  in  contrast  to  DNA-depen- 
dent  reactions,  in  which  the  two  strands  remain 
annealed.  The  best  characterized  of  these  en- 
zymes is  Q-beta  replicase.  We  have  shown  that 
replicatable  RNAs,  in  the  presence  of  this  en- 
zyme, can  harbor  RNA  probe  inserts  without  loss 
of  replicative  efficiency. 

We  have  devised  schemes  in  which  probe  bind- 
ing, RNA  joining,  and  exponential  replication  of 
the  joined  RNA  are  used  in  an  assay  to  detect  the 
presence  of  a  specific  target  sequence  from  an 
infectious  agent.  Probes  that  are  not  joined  are 
not  replicated.  The  signal  in  these  assays  is  repli- 
cated RNA,  generated  by  joined  probes  and 
readily  quantitated  by  fluorescence  staining.  The 
intensity  of  the  signal  is  proportional  to  the  num- 
ber of  targets  present  in  the  original  sample. 

However,  a  number  of  technical  problems  re- 
main to  be  solved  before  optimal  sensitivity  and 
specificity  can  be  achieved  in  these  assays.  For 
example,  the  ligation  step  involving  the  ribo- 


509 


Diagnostic  Use  of  RNA  Replication  in  Infectious  Diseases 


zyme  ligase  is  still  relatively  inefficient.  (About 
25  percent  of  the  ligation-competent  binary 
probe  molecules  are  joined  in  a  1-hour  incuba- 
tion.) An  actual  assay  would  require  a  ligation 
efficiency  closer  to  85  percent.  Fortunately,  re- 
cent developments  in  RNA  biochemistry  suggest 
a  possible  solution  to  this  problem. 

Directed  Evolution  of  Ribozymes 

We  propose  to  develop  more-efficient  ribo- 
zyme  ligases  for  our  assays  by  directed  evolution 
in  vitro.  Rachel  Green,  in  Jack  Szostak's  labora- 
tory, has  selected  novel  ribozymes  with  altered 
catalytic  efficiency  by  Darwinian  selection  in  a 
test  tube.  A  Darwinian  selection  experiment  be- 
gins with  the  synthesis  of  a  large  number  of  DNA 
molecules,  each  containing  the  sequence  of  a  ri- 
bozyme.  Known  as  a  mutant  pool,  the  population 
of  molecules  contains  point  mutations  generated 
during  chemical  synthesis.  Each  molecule  con- 
tains just  a  few  mutations,  located  at  random  po- 
sitions in  the  sequence,  and  thus  the  population 
contains  over  a  trillion  (10'^)  variants.  The  en- 
zyme T7  RNA  polymerase  is  then  used  to  generate 
RNA  copies  of  the  DNA,  creating  over  a  trillion 
dififerent  mutant  ribozymes.  These  are  briefly  in- 
cubated under  RNA  ligation  conditions,  where 
they  are  given  the  opportunity  to  catalyze  a  chem- 
ical reaction  that  joins  them  to  a  special  piece 
of  RNA. 

Those  ribozymes  that  succeed  in  carrying  out 
the  ligation  reaction  are  chosen  as  survivors  in  a 
subsequent  step,  while  those  that  fail  to  partici- 
pate in  catalysis  are  lost  by  dilution.  Survivors  are 
allowed  to  increase  in  number  by  reverse  tran- 
scription (which  converts  RNA  into  DNA),  fol- 
lowed by  PGR  amplification  of  the  DNA.  This  se- 
ries of  steps  effectively  rewards  all  competent, 
efficient  ribozyme  mutants  by  reconverting  them 
into  DNA  and  copying  them  many  times. 

At  this  point  the  DNA  is  transcribed  again  into 
RNA,  and  a  second  cycle  of  Darwinian  selection 
begins,  exactly  as  above.  After  four  cycles  of  se- 
lection, a  relatively  small  number  of  mutant  ribo- 
zyme sequences  will  be  present  in  the  DNA  popu- 
lation, instead  of  the  original  millions.  These 
molecules  are  the  fittest:  they  were  able  to  cata- 
lyze ligation  reactions  in  all  four  rounds  of  selec- 
tion. The  molecules  are  then  sequenced  in  order 
to  compare  them  to  the  original  parental 
ribozyme. 

Using  the  methods  outlined  above,  Rachel 
Green  isolated  a  number  of  interesting  mutant 
ribozymes  that  display  high  efficiency  in  a  spe- 


cific type  of  ligation  reaction.  The  experiments 
used  to  generate  these  ribozymes  mimic  natural 
variation  and  selection.  While  it  takes  nature 
hundreds,  thousands,  or  even  millions  of  years  to 
evolve  better  enzyme  catalysts,  RNA  enzymes  can 
be  improved  in  the  laboratory  by  a  Darwinian  se- 
lection process  in  a  matter  of  months.  The  term  in 
vitro  genetics  has  been  coined  to  describe  these 
man-made  selection  schemes.  We  are  working  on 
improvements  that  should  enable  us  to  generate 
ribozyme  ligases  better  suited  to  our  assays,  in 
order  to  improve  the  process  of  joining  RNA  bi- 
nary probes. 

Multiplexed  Diagnostic  Assays 

Epidemiological  monitoring  of  infectious  dis- 
eases is  a  complex  undertaking  in  a  developing 
country  like  Mexico.  Diseases  must  be  monitored 
both  in  large,  modern  urban  settings,  like  Mexico 
City,  and  in  sparsely  populated,  rural  areas  like 
the  Ghiapas  countryside.  The  list  of  diseases  caus- 
ing significant  mortality  and  morbidity  includes 
AIDS,  malaria,  amebiasis,  tuberculosis,  hepatitis, 
typhoid  fever,  diverse  intestinal  infections,  and 
more  recently  cholera.  There  is  a  need  for  devel- 
oping accurate,  low-cost  technologies  to  detect 
pathogens  in  the  field,  and  also  a  need  for  ex- 
tremely efficient,  high-tech  tools  for  the  epidemi- 
ological laboratory  and  the  blood  supply  centers. 
A  valuable  epidemiological  tool  would  be  a  diag- 
nostic assay  capable  of  detecting  any  of  a  number 
of  pathogens  in  a  single  clinical  sample.  Such  a 
tool  is  known  as  a  multiplex  assay. 

Under  the  auspices  of  the  Rockefeller  Founda- 
tion and  the  World  Health  Organization,  our  insti- 
tute has  established  a  collaborative  research  pro- 
gram with  Stanford  University  School  of  Medicine 
to  develop  and  implement  state-of-the-art  tech- 
niques for  epidemiological  assessment  in  Mex- 
ico. The  Stanford  group  is  headed  by  Gary 
Schoolnik  from  the  Division  of  Geographic  Medi- 
cine and  HHMI.  Using  a  multiplex  PGR  assay, 
they  have  conducted  a  series  of  experiments  dem- 
onstrating the  simultaneous  detection  of  any  of 
several  bacterial  pathogens  in  diarrheal  stool. 

Assays  based  on  RNA  amplification  could  be 
multiplexed  by  using  a  combination  of  binary 
probes.  Each  binary  probe  pair  would  contain 
RNA  sequences  designed  for  binding  to  an  indi- 
vidual pathogen,  and  as  many  as  10  probe  pairs 
could  be  mixed  together  in  a  single  assay.  Eventu- 
ally multiplexed  assays  could  be  automated,  and 
cost-effectiveness  would  be  very  favorable  be- 
cause several  candidate  infectious  agents  would 
be  assayed  at  once. 


510 


Lineage-Specific  Gene  Expression 
in  Caenorhabditis  elegans 


James  D.  McGhee,  Ph.D. — International  Research  Scholar 

Dr.  McGhee  is  Professor  in  the  Department  of  Medical  Biochemistry  at  the  University  of  Calgary,  Alberta. 
He  received  his  B.Sc.  degree  in  physiology  and  biochemistry  from  the  University  of  Toronto  and  his  Ph.D. 
in  molecular  biology  from  the  University  of  Oregon.  His  postdoctoral  research  was  done  in  the  laboratory 
of  Gary  Felsenfeld  at  the  National  Institutes  of  Health.  He  is  a  Medical  Scientist  of  the  Alberta  Heritage 
Foundation  for  Medical  Research. 


ONE  of  the  most  important  problems  in  devel- 
opmental biology  is  to  understand  the  mech- 
anism of  lineage-specific  gene  expression.  That 
is,  how  does  a  developing  embryo  manage  to  ex- 
press a  particular  gene  in  one  cell  or  cell  lineage 
and  not  in  the  many  other  cells  of  its  body?  Fur- 
thermore, how  does  it  tell  time,  in  order  to  ex- 
press this  gene  only  at  the  correct  point  in 
development? 

We  are  approaching  these  problems  in  the  sim- 
ple nematode,  or  roundworm,  Caenorhabditis 
elegans.  The  reasons  for  choosing  such  a  simple 
animal  are  that  it  comprises  only  about  a  thou- 
sand cells  and,  more  importantly,  that  the  divi- 
sion pattern  and  cell  lineage  of  every  one  of  these 
is  known.  We  are  studying  the  expression  of 
genes  in  the  C.  elegans  intestine,  since  this  is  the 
simplest  lineage  available. 

When  the  developing  embryo  has  only  eight 
cells,  one  of  these  (called  the  E  cell)  is  the  pro- 
genitor of  the  animal's  entire  intestine.  As  a 
marker  for  biochemical  differentiation  of  the  gut, 
we  have  characterized  a  simple  hydrolytic  en- 
zyme, a  nonspecific  carboxylesterase.  As  shown 
in  the  figure,  the  gene  coding  for  this  esterase 
(called  ges-1,  standing  for  gut  esterase)  is  only 
expressed  in  the  developing  intestine.  Presum- 
ably the  natural  function  of  the  enzyme  has  some- 
thing to  do  with  the  worm's  digestion,  but  mu- 
tants that  do  not  express  esterase  appear  to  be 
perfectly  viable. 

The  ges-1  gene  is  transcribed  from  the  genome 
of  the  embryo  at  a  point  in  development  when 
the  embryo  has  only  100-200  cells  and  the  devel- 
oping gut  lineage  consists  of  only  four  cells.  Mi- 
cromanipulation experiments  have  shown  that 
ges-1  is  expressed  normally  in  embryos  in  which 
non-gut  cells  have  been  removed  or  destroyed.  In 
other  words,  we  have  no  evidence  for  any  interac- 
tion between  gut  and  non-gut  cells.  An  intriguing 
feature  of  ges-1  expression  is  that  it  is  completely 
dependent  on  DNA  synthesis  during  the  cell  cycle 
in  which  the  embryo  has  a  total  of  eight  cells,  i.e., 
the  cell  cycle  just  after  the  gut  has  been  clonally 
established. 

The   problem   of  understanding  intestinal- 


specific  expression  of  the  ges-1  gene  is  twofold. 
One  must  define  regions  in  the  DNA  to  which 
transcription  factors  bind,  and  then  identify  and 
characterize  the  transcription  factors  that  bind  to 
these  regions.  To  address  the  first  part  of  the 
problem,  we  have  injected  the  cloned  DNA  from 
the  ges-1  gene  back  into  a  ges-i-nonexpressing 
mutant  and  have  been  able  to  reconstitute  accu- 
rate gut-specific  expression.  We  have  used  this 
transformation  assay  to  identify  sequences  that 
appear  to  be  necessary  for  correct  gut  expression. 

We  have  also  found  regions,  however,  whose 
deletion  causes  the  ges-1  gene  to  be  expressed  not 
in  the  gut  but  in  specific  sets  of  cells  in  the  phar- 
ynx, in  the  body  wall  musculature,  and  in  the 
hypodermis.  These  cells  belong  to  either  the  sis- 
ter or  cousin  lineage  of  the  gut. 

The  simplest  model  to  explain  these  ectopic 
staining  patterns  invokes  lineage-specific  repres- 
sors that  would  normally  keep  the  ges-1  gene  si- 
lent. When  the  repressor-binding  site  is  deleted, 
the  normally  silent  gene  can  be  expressed.  We  are 
testing  this  model  by  attempting  to  identify  short 
discrete  sequences  that  cause  lineage-specific  re- 
pression of  a  gene  that  is  otherwise  ubiquitously 
expressed.  We  are  also  attempting  to  provoke  ec- 
topic expression  of  the  ges-1  gene  by  injecting 
large  amounts  of  putative  repressor-binding  sites 
as  a  competitor. 

There  is  a  curious  and  unexpected  feature  of 
these  ectopic  ges-1  staining  patterns.  With  con- 
structs that  produce  staining  in  pharynx,  mus- 
cles, and  hypodermis,  the  three  staining  patterns 
are  by  and  large  exclusive:  individual  embryos 
show  only  one  of  the  patterns,  and  combinations 
of  patterns  are  rare.  Whatever  the  molecular  ex- 
planation turns  out  to  be,  the  phenomenon  sug- 
gests that  the  embryo  is  making  a  decision  very 
early  in  development  and  stably  propagating  this 
decision  thereafter.  Indeed,  this  decision  must  ac- 
tually be  made  at  or  before  the  two-cell  stage  of 
the  embryo,  since  this  is  the  last  point  when  the 
different  lineages  are  common. 

We  are  also  working  on  the  second  part  of  the 
problem  of  lineage-specific  gene  expression, 
namely  to  identify  the  protein  factors  that  bind  to 


511 


Lineage-Specific  Gene  Expression  in  Caenorhabditis  elegans 


the  ges-1  gene  and  control  its  expression  only  in 
the  gut  and  only  at  the  correct  embryonic  stage. 
Here  one  of  the  few  limitations  of  C.  elegans  as  an 
experimental  organism  becomes  apparent:  it  is 
essentially  impossible  to  obtain  large  amounts  of 
synchronized  embryos  on  which  to  do  biochemi- 
cal experiments.  We  are  taking  two  routes  past 
this'obstacle. 

The  first  route  is  to  turn  part  of  our  efforts  to 
Ascaris  suum,  the  well-known  parasite  of  pig  in- 
testines. Ascaris  adults  are  millions  of  times 
larger  than  adult  C.  elegans,  yet  their  embryos  are 
remarkably  similar.  Furthermore,  Ascaris  fe- 
males produce  hundreds  of  thousands  of  ferti- 
lized eggs  per  day,  which  develop  more-or-less 
synchronously.  We  hope  to  use  this  system  to 
watch  the  arrival  and  departure  of  transcription 
factors  during  early  nematode  development. 

The  second  route  is  based  on  our  finding  that 
C.  elegans  embryos  can  be  blocked  in  mid- 
development  by  exposure  to  the  chemotherapeu- 
tic  agent  fluorodeoxyuridine.  This  has  allowed  us 
to  produce  nuclear  extracts  from  these  blocked 
embryos  and  to  detect  a  number  of  protein  factors 
(possibly  as  many  as  a  dozen)  that  bind  to  the 
5'-flanking  sequence  of  the  ges-1  gene.  We  have 
concentrated  on  two  such  factors. 

The  first  factor  binds  to  a  DNA  sequence  that 
we  have  tentatively  identified  as  a  gut  "activa- 


tor." The  protein  is  not  detectable  in  oocyte  cyto- 
plasm and  thus  must  be  produced  (transcribed  or 
translated)  sometime  during  early  development. 
The  second  factor  binds  to  a  sequence  that  is  com- 
pletely conserved  between  C.  elegans  and  the  re- 
lated nematode  Caenorhabditis  briggsae.  Un- 
like the  first  factor,  this  second  factor  is  present 
in  the  oocyte  cytoplasm  and  must  migrate  at  some 
point  in  early  development  into  the  embryonic 
nuclei.  The  second  factor  would  thus  be  a  candi- 
date for  a  classic  cytoplasmic  "determinant"  pro- 
duced by  the  mother  worm  and  then  segregated 
into  the  gut  lineage  during  early  cell  cycles. 

If  we  can  indeed  describe  the  constellation  of 
transcription  factors  responsible  for  activating 
the  ges-1  gene  in  the  developing  gut,  will  we  re- 
ally be  much  further  ahead  in  understanding  its 
control?  In  some  respects,  all  this  work  will  only 
move  the  problem  one  step  further  back  in  devel- 
opment, to  the  question  of  how  the  transcription 
factors  themselves  got  where  they  did.  However, 
one  of  the  main  advantages  of  ges-1  as  an  experi- 
mental system  is  that  we  only  have  3-5  cell  cy- 
cles through  which  to  regress,  until  we  reach  the 
oocyte.  Thus  it  should  be  possible  to  describe  the 
complete  molecular  logic  that  controls  this  sim- 
ple gene. 

This  work  was  supported  by  the  Medical  Re- 
search Council  of  Canada  and  by  the  Alberta  Heri- 
tage Foundation  for  Medical  Research. 


A  newly  hatched  larva  of  the  nematode  Caeno- 
rhabditis elegans.  The  dark  red  precipitate  re- 
flects the  esterase  activity  of  the  ges- 1  gene,  ex- 
pressed in  all  20  cells  of  the  gut. 

From  Edgar,  L.G.,  and  McGhee,  J.D.  1986. 
DevBiol  114:109-118. 


512 


Cytokine  Regulation  of  Effector  Functions 
in  Immune  Responses 


Tim  R.  Mosmann,  Ph.D. — International  Research  Scholar 

Dr.  Mosmann  is  Professor  and  Chair  of  the  Department  of  Immunology  at  the  University  of  Alberta, 
Edmonton.  He  received  his  Ph.D.  degree  in  microbiology  at  the  University  of  British  Columbia,  Vancouver, 
and  obtained  postdoctoral  training  in  Toronto  and  Glasgow  as  a  fellow  of  the  Medical  Research  Council. 
After  four  years  as  Assistant  Professor  in  Immunology  at  Alberta,  he  spent  eight  years  as  a  senior  scientist 
at  DNAX  Research  Institute  in  Palo  Alto.  He  then  returned  to  the  University  of  Alberta. 


AFTER  infection  the  host's  immune  system 
must  respond  by  specifically  recognizing 
the  invading  organism  and  inducing  the  effector 
mechanisms  that  will  most  effectively  destroy  the 
pathogen.  Determination  of  the  correct  antigen- 
specific  recognition  structures  is  mediated  by  the 
process  of  clonal  selection,  whereby  B  or  T  cells 
are  selectively  activated  if  they  already  express 
receptors  specific  for  the  infecting  agent.  Choice 
of  the  appropriate  effector  mechanism  is  deter- 
mined to  a  large  degree  by  T  cells  and  the  cyto- 
kines they  secrete. 

The  immune  system  can  attack  infectious 
agents  by  a  number  of  means.  Antibody  is  highly 
effective  at  neutralizing  toxins  and  free  virus  and 
at  coating  bacteria  to  enhance  their  recognition 
and  destruction  by  phagocytic  cells.  There  are 
also  various  cell-mediated  cytotoxic  mecha- 
nisms, such  as  killer  T  cells,  macrophages,  and 
granulocytes,  that  are  most  useful  against  intra- 
cellular infectious  agents.  Cytokines  secreted  by 
activated  T  cells  regulate  the  accumulation  and 
activation  of  these  cells  at  sites  of  infection,  and 
some  cytokines  also  have  direct  cytotoxic 
functions. 

Recruitment  and  activation  of  macrophages 
and  granulocytes  occur  strongly  during  a  delayed- 
type  hypersensitivity  (DTH)  reaction,  which  pre- 
sents an  effective  response  against  intracellular 
infections.  In  contrast,  antibody  responses  are 
normally  more  effective  against  extracellular 
pathogens.  It  has  long  been  known  that  antibody 
and  DTH  responses  are  often  "either-or"  re- 
sponses of  the  immune  system,  although  the 
mechanism  of  this  reciprocal  regulation  has  only 
been  partially  resolved. 

Several  years  ago  we  and  others  described  two 
types  of  T  cell  that  secrete  very  different  patterns 
of  cytokines.  THl  cells  produce  interIeukin-2 
(IL-2),  interferon-7  (IFN-7),  and  lymphotoxin 
(LT),  whereas  only  TH2  cells  produce  IL-4,  IL-5, 
IL-6,  IL-10,  and  P6OO.  These  cytokines  have  mul- 
tiple and  profound  effects  on  various  aspects  of 
the  immune  response,  and  so  THl  and  TH2  cells 
have  markedly  different  functions. 

TH2  cells  induce  antibody  production  by  B 


cells.  In  particular,  a  strong  TH2  response  is  asso- 
ciated with  high  production  of  IgE,  the  antibody 
that  causes  allergy.  Although  THl  cells  can  also 
induce  antibody  production  under  some  circum- 
stances, these  cells  are  much  more  effective  at 
inducing  DTH.  Thus  the  choice  of  THl  or  TH2 
responses  influences  the  balance  between  DTH 
and  antibody  responses. 

Although  the  THl  and  TH2  patterns  account 
for  a  major  part  of  the  immune  response  during 
certain  infections,  these  probably  represent  ex- 
tremes, and  there  are  other  T  cells  with  different 
cytokine  secretion  phenotypes.  These  other  pat- 
terns may  be  involved  in  the  many  possible  types 
of  immune  response  that  occur  against  different 
infectious  agents. 

Since  the  type  of  immune  response  induced 
against  a  particular  pathogen  is  often  characteris- 
tic for  that  agent,  the  choice  of  effector  function 
must  be  precisely  regulated.  It  appears  that  cross- 
regulation  by  TH 1  and  TH2  cytokines  plays  a  ma- 
jor role  in  determining  the  effector  functions. 
Some  of  this  cross-regulation  is  now  understood: 
IFN-7  produced  by  THl  cells  can  inhibit  prolifer- 
ation of  TH2  cells,  and  IL-10  produced  by  TH2 
cells  inhibits  the  activation  of  THl  cells. 

Effector  T  cells  such  as  TH  1  and  TH2  are  proba- 
bly derived  from  precursor  cells  that  secrete  only 
IL-2.  After  initial  stimulation  with  antigen,  these 
cells  presumably  differentiate  into  THl,  TH2, 
and  other  cells.  This  process  is  influenced  by  cy- 
tokines— e.g.,  IL-4  encourages  the  production  of 
more  cells  that  secrete  IL-4.  However,  the  full 
regulation  of  this  process  is  largely  unknown,  and 
this  is  currently  an  active  area  of  investigation  in 
our  laboratory  and  others.  Since  the  differentia- 
tion process  is  quite  rapid  (e.g.,  a  few  days),  we 
are  developing  single-cell  cytokine  detection 
methods  so  that  we  can  follow  differentiation  of 
single  clones  of  cells. 

Two  methods  are  currently  possible:  the  mRNA 
for  different  cytokines  can  be  measured  in  single 
cells  after  amplification  by  polymerase  chain  re- 
action, and  cytokine  protein  secreted  by  single 
cells  can  be  analyzed  by  a  colorimetric  spot  assay 
using  anticytokine  monoclonal  antibodies.  Both 


513 


Cytokine  Regulation  of  Effector  Functions  in  Immune  Responses 


methods  are  feasible  for  detecting  single  cyto- 
kines produced  by  one  cell,  and  we  are  currently 
adapting  them  to  simultaneous  measurement  of 
multiple  cytokines.  Since  there  is  considerable 
evidence  of  several  different  cytokine  secretion 
phenotypes,  5-10  cytokines  must  be  analyzed 
from  each  cell  to  obtain  a  true  picture  of  T  cell 
diversity.  Once  developed,  this  methodology 
will  be  applied  to  the  differentiation  of  T  cells 
during  normal  immune  responses,  allowing  the 
signals  that  influence  differentiation  to  be 
determined. 

The  characteristic  cytokines  of  THl  and  TH2 
are  major  determinants  of  the  function  of  these 
cells.  For  example,  IL-4,  IL-5,  and  IL-10,  pro- 
duced by  TH2  cells,  contribute  to  the  production 
of  IgE,  mast  cells,  and  eosinophils — all  asso- 
ciated with  the  development  of  allergy.  IFN-7  is  a 
major  contributor  to  DTH  reactions,  inhibits 
many  of  the  functions  of  IL-4,  and  inhibits  TH2 
cells  directly.  We  have  been  interested  for  some 
time  in  additional  cytokines  of  the  two  T  cell 
types. 

Recently  we  have  been  working  on  P6OO,  origi- 
nally discovered  as  a  clone  in  a  cDNA  library  of 
activated  TH2  cells.  P600  is  particularly  interest- 
ing, since  it  is  expressed  only  after  activation  and 
is  not  produced  by  THl  cells.  The  sequence  of 
the  open  reading  frame  in  the  P600  cDNA  clone 
encodes  a  small  protein  with  a  leader  sequence 


characteristic  of  secreted  or  membrane  proteins. 
Since  all  of  these  properties  suggest  that  P600 
may  be  an  additional  TH2-specific  cytokine,  we 
decided  to  test  for  possible  functions  of  the  P6OO 
protein. 

We  transfected  the  P6OO  cDNA  clone  into  mon- 
key cells  and  observed  secretion  of  a  new  protein 
of  the  expected  size.  After  screening  in  a  wide 
variety  of  biological  assays,  we  found  that  P600 
induces  the  production  of  large  numbers  of  ad- 
herent cells  from  bone  marrow.  The  functions  of 
these  cells  are  currently  under  investigation  to 
determine  how  the  effects  of  P6OO  fit  into  the 
overall  pattern  of  TH2  functions  during  immune 
responses. 

The  different  T  cell  cytokine  secretion  pheno- 
types were  originally  discovered  in  the  mouse, 
and  initially  there  was  some  doubt  that  similar 
phenotypes  exist  in  humans.  However,  recent 
data  on  human  T  cell  clones  and  immune  re- 
sponses have  made  it  clear  that  the  THl  and  TH2 
patterns  are  also  important  in  himian  diseases.  Ap- 
parently several  parasitic  diseases  strongly  in- 
duce TH 1  -  or  TH 2 -biased  responses,  and  only  one 
of  these  responses  is  usually  able  to  clear  the  in- 
fectious agent.  Recent  data  indicate  that  these 
two  cytokine  patterns  are  also  important  in  lep- 
rosy. Thus  a  knowledge  of  the  regulation  of  these 
patterns  will  have  considerable  potential  for  the 
design  of  better  immunomodulatory  treatments 
and  vaccines. 


514 


Cellular  and  Molecular  Basis  of  Variability 
in  Entamoeba  histolytica 


Esther  Orozco,  Ph.D. — International  Research  Scholar 

Dr.  Orozco  is  Professor  of  Genetics  and  Molecular  Biology  in  the  Department  of  Experimental  Pathology  at 
the  Center  for  Research  and  Advanced  Studies,  National  Polytechnic  Institute,  Mexico  City.  She  received 
her  bachelor's  degree  in  chemistry  and  biology  from  the  University  of  Chihuahua,  Mexico,  and  her  Ph.D. 
degree  from  the  National  Polytechnic  Institute.  She  has  been  a  visiting  professor  in  several  institutes 
around  the  world,  including  Harvard  School  of  Public  Health  in  Boston  and  the  Weizmann  Institute  of 
Science  in  Israel.  Dr.  Orozco  counts  among  her  honors  a  Guggenheim  Foundation  Award  and  the 
Dr.  J.  Rosenkranz  1991  Award  given  by  Syntex. 


MORE  than  500  million  people  throughout 
the  world  are  infected  by  Entamoeba  his- 
tolytica, the  protozoan  parasite  responsible  for 
human  amebiasis.  Ninety  percent  of  those  in- 
fected do  not  present  clinical  symptoms,  while 
the  rest  develop  colitis,  dysentery,  or  hepatic 
abscesses. 

In  1925  Brumpt  proposed  the  possible  exis- 
tence of  two  E.  histolytica  species,  one  patho- 
genic (P)  and  the  other  nonpathogenic  (NP),  and 
he  and  others  have  continued  to  advance  this  pos- 
sibility. There  are  data  both  supporting  and  op- 
posing this  concept.  For  instance,  migration  pat- 
terns of  several  enzymes  in  a  high  number  of 
amebic  isolates  has  led  to  the  establishment  of 
more  than  20  E.  histolytica  zymodemes.  Interest- 
ingly, the  zymodemes  of  P  and  NP  trophozoites 
differ. 

According  to  the  opinion  of  several  groups,  zy- 
modemes and  virulence  are  stable  phenotypes. 
However,  experimental  evidence  against  the  sta- 
bility of  zymodemes  and  virulence  has  also  been 
documented.  Resolution  of  this  controversy  is  of 
fundamental  importance  clinically.  If  E.  histoly- 
tica comprises  two  species,  cyst  passers  spread- 
ing the  NP  form  are  not  a  public  health  menace, 
and  asymptomatic  carriers  need  not  be  treated. 
However,  if  the  harmless  E.  histolytica  can  turn 
into  a  virulent  one,  a  different  decision  may  be 
indicated.  In  any  case,  the  molecular  basis  of  con- 
version, if  it  occurs,  would  be  a  highly  interesting 
phenomenon  to  study. 

We  have  focused  this  controversy  by  1 )  investi- 
gating cloned  E.  histolytica  populations  after 
their  isolation  from  asymptomatic  carriers  through- 
out the  axenization  process,  2)  comparing  E.  his- 
tolytica clones  with  diverse  origins,  and  3) 
detecting  and  cloning  DNA  sequences  present 
only  in  E.  histolytica  clones  that  express 
pathogenicity. 

Study  of  NP  E.  histolytica  Throughout 
Axenization 

Cloned  trophozoites  (MAV-1),  with  an  initial 
NP  zymodeme  I  and  isolated  from  an  asymptom- 


atic carrier,  were  cultured  under  polyxenic, 
monoxenic,  and  axenic  conditions.  Zymodeme  I 
of  polyxenic  MAV-1  trophozoites,  cultured  in 
Robinson's  medium,  presented  an  NP  zymodeme 
XII  when  trophozoites  were  cultured  in  Jones' 
medium.  Monoxenic  trophozoites  grown  in  the 
presence  of  Fusobacterium  symbiosum  re- 
tained NP  zymodeme  I  (with  slow-running  hexo- 
kinases  [HKs]),  an  a-phosphoglucomutase  (PGM) 
band,  and  lack  of  pathogenicity.  Surprisingly, 
when  MAV-1  trophozoites  were  grown  under 
axenic  conditions,  the  original  NP  zymodeme 
switched  to  a  P  zymodeme,  with  fast-running  HKs 
and  a  /3  PGM.  Virulence,  defined  as  the  tropho- 
zoites' ability  to  infect  experimental  animals  and 
damage  target  cells,  was  also  expressed. 

The  analysis  of  E.  histolytica  zymodemes  indi- 
cates that  P  as  well  as  NP  trophozoites  have  a  set 
of  genes  for  a  given  enzyme.  Excluding  the  fast- 
running  HKs  and  the  /3  PGM  that  have  not  been 
reported  for  NP  isolates,  all  other  isoenzymes  are 
expressed  in  P  and  NP  trophozoites,  including 
slow-running  HKs.  A  certain  type  of  bacteria  in 
the  medium  could  be  participating  in  the  regula- 
tion of  isoenzyme  expression.  We  will  continue 
our  studies  on  the  factors  involved  in  the  expres- 
sion of  isoenzymes  in  P  and  NP  trophozoites,  in 
order  to  look  for  conclusive  evidence  on  zymo- 
deme and  virulence  switching — evidence  that 
should  dissipate  skepticism  about  conversion. 

Differences  in  Genetically  Related  Clones 

Functional  and  biochemical  dissimilarities 
mark  clones  of  diverse  origin.  P  trophozoites 
show  a  high  rate  of  phagocytosis,  destroy  cell  cul- 
ture monolayers,  and  produce  hepatic  abscesses 
in  experimental  animals.  On  the  contrary,  NP  tro- 
phozoites generally  do  not  show  these  virulence- 
related  properties.  Fortuitously,  they  express  low 
virulence. 

Monoclonal  and  polyclonal  antibodies  against 
certain  antigens  can  distinguish  between  P  and 
NP  trophozoites.  The  presence  of  the  major  ame- 
bic cysteine  protease  involved  in  the  cytopathic 
effect  of  trophozoites  is  also  specific  to  P  tropho- 


515 


Cellular  and  Molecular  Basis  of  Variability  in  Entamoeba  histolytica 


zoites.  However,  the  so-called  P  trophozoites 
form  a  heterogeneous  group  of  E.  histolytica  iso- 
lates, which  show  virulence  in  a  broad  spectrum. 
This  indicates  that  virulence-involved  genes  are 
differentially  regulated. 

On  the  other  hand,  amebic  strains  are  consti- 
tuted by  trophozoites  displaying  different  pheno- 
types.  Clones  from  a  given  strain  express  viru- 
lence in  the  same  broad  spectrum  as  amebic 
strains.  Some  clones  are  avirulent  (are  they  NP?); 
others  are  highly  virulent,  and  specific  monoclo- 
nal and  polyclonal  antibodies  are  able  to  discrimi- 
nate between  them. 

The  simplest  interpretation  for  this  is  that 
strains  are  a  mixture  of  genetically  unrelated  tro- 
phozoites living  together  in  the  human  intestine. 
However,  clones  derived  from  cloned  popula- 
tions differ  phenotypically  and  genotypically. 
The  differences  include  virulence,  antigens,  and 
genetic  divergence.  Clones  isolated  from  clone  A 
(strain  HM1:IMSS)  show  the  P  zymodeme  but 
differ  in  virulence  and  in  the  expression  of  the 
1 12-kDa  adhesin,  involved  in  both  phagocytosis 
and  virulence  of  E.  histolytica. 


Detection  and  Cloning  of  DNA  Sequences 
Present  Only  in  Pathogenic  Trophozoites 

Several  E.  histolytica  genes  show  high  similar- 
ity among  P  strains  but  differ  from  their  homolo- 
gous genes  in  NP  strains.  Molecular  studies  have 
given  the  strongest  evidence  to  suppon  the  exis- 
tence of  two  E.  histolytica  species.  To  date,  there 
is  no  explanation  for  the  genomic  divergence 
shown  by  P  and  NP  trophozoites.  However,  reti- 
cence to  accept  that  there  are  two  species  based 
on  their  polymorphism  is  generated  by  the  re- 


markable genomic  plasticity  of  this  parasite,  even 
among  clones  derived  from  cloned  populations. 

Genomic  divergence  is  showed  by  strains  and 
clones  cultured  under  different  conditions.  Ge- 
netically related  clones  obtained  from  a  cloned 
population  show  polymorphism  in  several  genes 
of  the  same  clone  cultured  in  different  laborato- 
ries (clone  A,  strain  HM1:IMSS).  They  also  differ 
in  their  molecular  karyotype  and  in  the  chromo- 
somal location  of  some  genes,  indicating  genome 
rearrangements.  Finally,  we  have  identified, 
cloned,  and  sequenced  a  1.6-kilobase  DNA  frag- 
ment from  clone  A  (virulent)  that  is  altered 
in  nonvirulent  (NP?)  clones  derived  also  from 
clone  A. 

We  are  currently  studying  genetic  mechanisms 
underlying  this  intriguing  phenomenon.  One  hy- 
pothesis, highly  speculative,  is  that  there  are 
genes  in  E.  histolytica  that,  under  pressure,  are 
selectively  rearranged,  modified,  or  amplified 
before  being  transcribed.  If  certain  genes  were 
amplified  in  P  trophozoites  and  not  in  NP,  single- 
copy  or  slightly  modified  genes  could  not  be  de- 
tected in  comparative  experiments.  If  this  were 
true,  it  would  explain,  at  least  in  part,  the  poly- 
morphism found  in  P  and  NP  strains. 

The  presence  of  "cassettes"  carrying  different 
genes  that  could  be  amplified  and  expressed  se- 
lectively is  another  possibility.  We  are  trying  to 
develop  a  genetic  transformation  system  in  E.  his- 
tolytica to  introduce  foreign  genes  as  landmarks 
to  follow  up  the  genome  modifications  that  will 
explain  the  erratic  behavior  of  this  parasite, 
or  at  least  the  differences  between  P  and  NP 
trophozoites. 

This  work  is  supported  by  a  grant  from  the  Na- 
tional Council  for  Science  and  Technology, 
Mexico. 


516 


Phosphorylation  and  Protein-Protein  Interactions 
in  Signal  Transduction 


Tony  Pawson,  Ph.D. — International  Research  Scholar 

Dr.  Pawson  is  Senior  Scientist  at  the  Samuel  Lunenfeld  Research  Institute  of  Mount  Sinai  Hospital, 
Toronto,  where  he  holds  the  Apotex  Chair  of  Molecular  Oncology;  Professor  of  Molecular  and  Medical 
Genetics  at  the  University  of  Toronto,  and  a  Terry  Fox  Cancer  Research  Scientist  of  the  National  Cancer 
Institute  of  Canada.  He  received  his  Ph.D.  degree  from  the  University  of  London  while  working  at  the 
Imperial  Cancer  Research  Fund.  Prior  to  moving  to  Toronto,  Dr.  Pawson  did  postdoctoral  work  at  the 
University  of  California,  Berkeley,  and  was  a  faculty  member  of  the  University  of  British  Columbia. 


MANY  of  the  polypeptide  hormones  that  con- 
trol development  in  the  embryo  and  cell 
growth,  differentiation,  and  metabolism  in  the 
adult  bind  to  the  extracellular  region  of  receptors 
that  span  the  plasma  membrane.  These  receptors, 
in  their  cytoplasmic  region,  contain  a  protein  ki- 
nase domain  that  phosphorylates  tyrosine  resi- 
dues. Normally  these  catalytic  receptors  are  only 
active  when  engaged  by  the  appropriate  growth 
factor.  However,  structural  alterations,  resulting 
from  cancer-causing  mutations  in  the  relevant 
genes,  can  render  them  active  even  in  the  absence 
of  a  growth  factor.  Such  aberrantly  active  recep- 
tors can  trigger  unrestricted  cancerous  cell 

Activated  growth  factor  receptors  can  elicit 
many  changes  in  stimulated  cells,  including 
changes  in  gene  expression,  cellular  architec- 
ture, cell-cell  communications,  and  cellular  me- 
tabolism. An  important  question  involves  the 
identities  of  the  intracellular  targets  of  receptor 
tyrosine  kinases — targets  that  control  signal 
transduction  pathways  within  the  cell. 


SH2 -containing  Proteins  Bind  Activated 
Growth  Factor  Receptors 

The  first  thing  growth  factor  receptors  do  upon 
activation,  apparently,  is  phosphorylate  them- 
selves on  tyrosine.  Receptor  autophosphoryla- 
tion  serves  as  a  switch  to  elicit  the  tight  binding 
of  intracellular  signaling  proteins  to  the  receptor. 
These  receptor-binding  proteins  include  Ras 
GTPase-activating  protein  (GAP),  phospholipase 
C-7,  phosphatidylinositol  (PI)  3'-kinase,  and  Src- 
family  cytoplasmic  tyrosine  kinases.  They  have 
the  attributes  expected  of  signaling  proteins,  in 
the  sense  that  they  regulate  specific  intracellular 
signal  transduction  pathways  and  in  some  cases 
are  known  to  be  phosphorylated,  and  activated, 
by  receptor  tyrosine  kinases.  How  are  these  func- 
tionally diverse  signaling  proteins  all  able  to  rec- 
ognize autophosphorylated  receptors?  They  each 
contain  one  or  two  copies  of  a  protein  domain, 
the  Src  homology  2  (SH2)  domain,  which  recog- 


nizes specific  autophosphorylated  sites.  Thus  ty- 
rosine phosphorylation  of  the  receptor  itself 
creates  a  tight  binding  site  for  SH2  domains  and 
promotes  a  strong  interaction  between  the  acti- 
vated receptor  and  its  target  SH2-containing 
proteins. 

There  is  evidently  some  specificity  to  these 
SH2-receptor  interactions.  Different  SH2  do- 
mains bind  tenaciously  to  distinct  phosphory- 
lated sites  on  a  receptor,  suggesting  that  the 
amino  acid  sequence  surrounding  the  phospho- 
tyrosine  residue  is  important  in  determining  tar- 
get specificity. 


A  Network  of  SH2 -mediated  Interaction 

An  increasing  number  of  SH2-containing  pro- 
teins that  can  bind  to  activated  growth  factor  re- 
ceptors have  been  identified.  These  are  all  likely 
to  regulate  some  aspect  of  intracellular  signaling, 
though  in  several  cases  the  relevant  biochemical 
pathways  have  not  been  defined. 

The  association  of  SH2  domains  with  auto- 
phosphorylated growTih  factor  receptors  is  one  in- 
dication of  their  general  ability  to  bind 
tyrosine-phosphorylated  sites.  As  an  example, 
considerable  data  suggest  that  the  SH2  domain  of 
the  c-Src  cytoplasmic  tyrosine  kinase  interacts 
with  an  inhibitory  tyrosine  phosphorylated  site  in 
its  own  carboxyl-terminal  tail,  thereby  repressing 
c-Src  tyrosine  kinase  activity.  However,  once 
c-Src  is  enzymatically  (and  oncogenically)  acti- 
vated by  removal  of  the  carboxyl-terminal  phos- 
phorylation site,  the  SH2  domain  has  a  positive 
transforming  effect,  probably  by  virtue  of  its  abil- 
ity to  bind  substrates  of  the  kinase  domain  and 
participate  in  the  formation  of  signaling  com- 
plexes. Thus  the  c-Src  SH2  domain  has  multiple 
functions.  GAP  is  another  example  of  a  signaling 
protein  whose  SH2  domains  can  interact  with 
several  different  phosphotyrosine-containing 
proteins. 

We  are  pursuing  the  structure  and  function  of 
SH2  domains,  using  a  variety  of  biochemical,  cel- 
lular, and  genetic  approaches. 


517 


Phosphorylation  and  Protein-Protein  Interactions  in  Signal  Transduction 


Receptor  Tyrosine  Kinases 
in  Embryonic  Development 

We  have  recently  cloned  cDNAs  for  two  novel 
receptor  tyrosine  kinases,  elk  and  nuk,  which  are 
primarily  expressed  in  the  mammalian  nervous 
system.  During  formation  of  the  nervous  system 
in  the  early  mouse  embryo,  nuk  assumes  a  very 
specific  and  interesting  pattern  of  expression.  We 
are  using  genetic  manipulation  in  the  mouse  to 
determine  whether  nuk  is  involved  in  control- 
ling neural  differentiation. 

Dual-Specificity  Protein  Kinases 

Protein  kinases  are  typically  divided  into  those 
that  can  phosphorylate  serine/threonine  residues 
and  those  that  phosphorylate  tyrosine.  We  have 
recently  identified  two  mammalian  protein  ki- 


nases that  can  phosphorylate  both  serine/threo- 
nine  and  tyrosine  when  artificially  expressed  in 
bacteria.  We  are  investigating  the  significance  of 
this  observation  for  mammalian  cells. 

One  of  these  kinases,  Clk,  is  related  to 
members  of  the  cdc2  protein  kinase  family.  The 
second.  Nek,  is  similar  to  the  product  of  the 
nimA  gene,  required  for  initiation  of  mitosis  in 
the  fungus  Aspergillus  nidulans.  The  identifica- 
tion of  nek  as  a  mouse  relative  of  nimA  suggests 
that  this  gene  may  have  been  conserved  in  evolu- 
tion, as  have  other  members  of  the  cell  cycle  ma- 
chinery. These  observations  prompt  the  specula- 
tion that  control  of  the  cell  cycle  involves  protein 
kinases  with  dual  specificity. 

Additional  funding  for  this  work  was  provided 
by  the  National  Cancer  Institute  of  Canada  and 
the  Medical  Research  Council  of  Canada. 


518 


Chemical  and  Functional  Characterization 
of  Scorpion  Toxins 


Lourival  Domingos  Possani,  Ph.D. — International  Research  Scholar 

Dr.  Possani  is  Professor  and  Chairman  of  the  Department  of  Biochemistry  at  the  Biotechnology  Institute, 
National  Autonomous  University  of  Mexico,  Cuernavaca.  He  received  his  B.S.  degree  in  natural  history 
from  the  Universidade  Federal  do  Rio  Grande  do  Sul,  Porto  Alegre,  Brazil,  and  his  Ph.D.  degree  in 
molecular  biophysics  from  the  University  of  Paris,  France.  He  completed  the  academic  requirements  for 
his  Ph.D.  degree  in  biochemistry  at  the  Rockefeller  University,  working  with  Edward  Reich  on  the  isolation 
of  the  acetylcholine  receptor. 


SCORPIONISM  is  a  serious  public  health  prob- 
lem in  certain  areas  of  the  world.  In  Mexico 
alone,  more  than  200,000  people  are  stung  by 
scorpions  each  year.  Thanks  to  serotherapy  with 
horse  antisera,  the  number  of  deaths  is  kept  to 
approximately  700-800  annually.  Not  all  scor- 
pions are  dangerous  to  humans.  Among  the  136 
species  known  to  live  in  Mexico,  only  6  present  a 
real  risk.  They  belong  to  the  family  Buthidae, 
genus  Centruroides.  The  species  dangerous  to 
humans  are  Centruroides  elegans,  C.  infamatus 
infamatus,  C.  limpidus  limpidus,  C.  limpidus 
tecomanus,  C.  noxius,  C.  sculpturatus  (which 
occurs  also  in  Arizona),  and  C.  suffusus  suffusus. 

Half  of  the  casualties  are  due  to  one  species, 
represented  by  two  subspecies:  C.  limpidus  lim- 
pidus from  the  states  of  Guerrero  and  Morelos, 
and  C.  limpidus  tecomanus  from  the  state  of  Co- 
lima.  All  dangerous  species  abide  in  the  Pacific 
coast  region.  No  dangerous  scorpions  are  found 
in  the  Gulf  of  Mexico  region,  the  Yucatan  Penin- 
sula, or  Mexico  City. 

Envenomation  by  the  sting  of  these  arachnids  is 
due  to  low-molecular-weight  peptides  called 
toxins.  More  than  1 00  such  toxins  have  been  puri- 
fied and  characterized.  There  are  three  well- 
known  classes  of  scorpion  toxins:  short-chain 
peptides  (38-39  amino  acid  residues  long), 
which  are  K"^  channel  blockers;  medium-chain 
peptides  (61-66  amino  acid  residues),  specific 
for  Na^  channels;  and  long-chain  peptides  (about 
70  amino  acid  residues),  toxic  to  insects  and 
crustaceans.  Recently  a  fourth  class  seems  to  have 
emerged  with  the  discovery  of  low-molecular- 
weight  proteins  (about  1 20  amino  acid  residues) 
that  affect  the  Ca^^  release  channel  from  sarco- 
plasmic reticulum. 

The  study  of  scorpion  toxins  is  scientifically, 
medically,  and  biotechnologically  interesting. 
They  represent  several  families  of  structurally  re- 
lated polypeptides  with  exquisite  preference  for 
ion  channel  molecules,  which  makes  them  a 
model  for  future  new  drugs  to  control  cell  excit- 
ability or  for  new  types  of  specific  insecticides. 
And  they  represent  excellent  tools  for  studying 
cellular  responses,   permitting  discrimination 


among  receptor  molecules  in  the  membranes  and 
investigation  of  various  types  of  ion  channels. 

The  contribution  of  our  research  group,  which 
comprises  a  multidisciplinary  interaction  with 
investigators  from  several  countries,  began  with 
the  isolation  and  chemical  characterization  of 
toxin  gamma,  a  potent  Na^  channel  blocker,  from 
the  Brazilian  scorpion  Tityus  serrulatus.  Our 
work  was  extended  at  the  beginning  of  the  1 980s 
by  the  discovery  of  the  first  peptide  capable  of 
blocking  the  delayed  rectifier  channel  from 
the  squid  axon.  It  is  a  short-chain  peptide  (39 
amino  acid  residues)  and  was  subsequently 
shown  to  affect  other  types  of  channels.  This 
peptide  was  named  noxiustoxin,  after  the  scor- 
pion C.  noxius. 

Our  studies  continued  with  the  purification 
and  characterization  of  more  than  25  different 
peptides  from  scorpion  venoms,  specific  block- 
ers of  Na^  channels  from  a  variety  of  different 
tissues.  At  least  eight  additional  small-chain  pep- 
tides similar  to  noxiustoxin  were  also  isolated 
and  characterized.  To  study  the  function  of  these 
toxins,  we  used  neurotransmitter-release  and 
-binding  studies  with  brain  synaptosomes  and 
mainly  electrophysiological  studies  with  a  vari- 
ety of  excitable  cells.  Our  more  recent  progress 
in  this  field  is  focused  on  the  three  lines  of  re- 
search mentioned  below. 

Chemical  Synthesis  of  Peptides 
and  Monoclonal  Antibodies 

We  have  recently  synthesized,  by  the  solid- 
phase  method  of  Merrifield,  more  than  1 00  dif- 
ferent peptides  corresponding  to  segments  of  the 
amino  acid  sequence  of  the  scorpion  toxins. 
These  peptides  and  the  preparation  of  a  dozen 
distinct  monoclonal  antibodies  allowed  us  to 
probe  for  specific  structural  regions  of  the  toxins. 
For  example,  the  nonapeptide  at  the  amino-ter- 
minal  region  of  noxiustoxin  was  shown  to  recog- 
nize and  affect  channels  in  a  manner  similar  to 
the  native  peptide.  These  results  are  promising  in 
the  context  of  possible  development  of  new 
drugs  aimed  at  controlling  cellular  excitability 
through  channels. 


519 


Chemical  and  Functional  Characterization  of  Scorpion  Toxins 


Using  monoclonal  antibodies,  we  found  four 
main  distinct  antigenic  determinants  in  the  Na"'" 
channel  target  toxins  of  eight  different  species  of 
scorpion  from  the  American  continent.  One  of 
the  antibodies  was  shown  to  produce  an  impor- 
tant protection  to  experimental  animals  enveno- 
mated  with  highly  purified  toxin.  This  result  is 
also  important  for  developing  possible  synthetic 
peptides  for  vaccination. 

Cloning  of  Scorpion  Toxin  Genes 

The  most  recent  advance  of  our  laboratory,  in 
collaboration  with  Francisco  Bolivar  and  Baltazar 
Becerril,  was  the  successful  isolation  of  several 
clones  that  code  for  peptides  corresponding  to 
the  amino  acid  sequence  of  scorpion  toxins.  A 
cDNA  library  was  prepared  from  the  telsons  of  the 
scorpions  C.  noxius  and  C.  limpidus  limpidus. 
In  the  future  we  hope  to  use  directed  mutagene- 


sis to  obtain  specific  modified  toxins  in  order  to 
study  the  structure-function  relationship  of  these 
ligands  with  specific  ion  channels. 

Search  for  New  Toxins 

New,  unknown  peptides  toxic  to  insects  and 
crustaceans  were  purified  and  are  being  se- 
quenced. These  are  from  several  species  of  scor- 
pion, including  some  not  dangerous  to  humans. 
Also,  in  collaboration  with  Hector  Valdivia  and 
Roberto  Coronado  from  Madison  University,  we 
are  conducting  a  search  for  specific  toxins  that 
target  the  Ca^"'"  release  channel  from  the  sarco- 
plasmic reticulum. 

This  research  is  supported  in  part  by  grants 
from  the  Consejo  Nacional  de  Ciencia  y  Techno- 
logia,  Mexico,  and  Direccion  General  del  Per- 
sonal Academico,  from  the  National  Autonomous 
University  of  Mexico. 


Venom  extraction  by  electrical  stimulation  of  a  scorpion,  species  Hadrurus  concolorous. 
Photograph  by  Manuel  Varela  and  Lourival  Possani. 


520 


Protein  Crystallography  in  the  Study 
of  Infectious  Diseases 


Randy  J.  Read,  Ph.D. — International  Research  Scholar 

Dr.  Read  is  Assistant  Professor  of  Medical  Microbiology  and  Infectious  Diseases  and  of  Biochemistry  at  the 
University  of  Alberta,  Edmonton.  He  obtained  his  undergraduate  and  doctoral  training  in  the  Department 
of  Biochemistry  at  the  University  of  Alberta,  then  pursued  postdoctoral  training  in  protein  crystallography 
with  Wim  Hoi  at  the  University  of  Groningen  in  the  Netherlands,  before  returning  to  Edmonton. 


INFECTIOUS  organisms — viruses,  bacteria,  and 
parasites — must  overcome  many  obstacles  to 
cause  disease.  They  must  gain  entry  to  their  fa- 
vored niche  in  the  host,  obtain  nutrients,  evade 
attacks  of  the  immune  system,  and  spread  to  new 
hosts.  These  are  complex  problems  with  com- 
plex and  varied  solutions.  But  the  pathogenic 
mechanisms  used  by  microorganisms  to  infect 
and  cause  disease  are  gradually  coming  to  light, 
in  some  cases  at  the  level  of  the  key  molecules. 
The  interest  in  pathogenesis  is  more  than  aca- 
demic, since  the  understanding  of  these  pro- 
cesses can  be  exploited  to  prevent  or  treat  disease. 

Our  interest  is  in  advancing  the  understanding 
of  infectious  disease  at  the  molecular  level,  using 
the  technique  of  x-ray  crystallography  to  study 
the  three-dimensional  structure  of  important 
proteins.  There  are  two  major  aspects  to  our 
work.  We  are  studying  the  crystal  structures  of  a 
number  of  proteins  that  are  involved  in  pathogen- 
esis, some  of  which  are  described  below,  and  we 
are  developing  methods  to  exploit  this  kind  of 
structural  information  in  the  design  of  new  drugs. 


Bacterial  Toxins 

Many  pathogenic  bacteria  produce  toxins  that 
cause  cell  and  tissue  damage  and  can  be  responsi- 
ble for  the  most  severe  effects  of  the  illness.  Bacte- 
rial toxins  often  belong  to  the  A-B  class,  having  a 
two-part  structure  in  which  the  B  (binding)  sub- 
unit  binds  to  the  surface  of  a  target  cell  and  the  A 
(active)  subunit  enters  the  cell,  carrying  out  the 
toxic  action.  We  are  involved  in  studying  the 
crystal  structures  of  pertussis  toxin  (in  collabora- 
tion with  Glen  Armstrong  at  the  University  of  Al- 
berta, and  with  Connaught  Laboratories  in  To- 
ronto) and  verotoxin  (in  collaboration  with 
James  Brunton,  University  of  Toronto). 

Pertussis  toxin  (PT)  is  produced  by  Bordetella 
pertussis,  the  bacterium  that  causes  whooping 
cough.  There  is  a  major  interest  in  the  role  of  this 
toxin  in  improved  vaccines.  Currently,  killed 
whole-cell  vaccines  are  used  for  whooping 
cough.  They  are  effective,  but  have  an  undesir- 
able level  of  toxicity.  The  side  effects  should  be 
reduced  or  eliminated  in  a  defined  vaccine  pro- 


duced from  genetically  engineered  proteins.  It  has 
been  shown  that  PT  is  a  necessary  component  of 
effective  whooping  cough  vaccines,  but  it  must  be 
rendered  nontoxic  for  safe  use.  A  three-dimensional 
structure  of  PT  would  help  show  how  to  remove  its 
toxicity  while  preserving  the  surface  features  of  the 
molecule  recognized  by  the  immune  system.  In  ad- 
dition, we  would  gain  a  better  understanding  of  how 
the  B  component  of  the  toxin  recognizes  the  surface 
of  target  cells,  how  the  A  subunit  enters  the  cell,  and 
how  it  carries  out  its  toxic  action.  Exploiting  the 
intense  radiation  produced  by  synchrotrons,  we 
have  collected  x-ray  data  from  crystals  of  this  toxin 
and  from  several  potential  heavy-atom  derivatives, 
but  the  determination  of  the  phases  needed  for  the 
visualization  of  the  structure  is  still  in  progress. 

The  Shiga  toxin  family  is  a  group  of  closely  re- 
lated toxins  produced  by  Shigella  dysenteriae 
type  1  (Shiga  toxin)  and  by  certain  strains  of  Esch- 
erichia coli  (verotoxins,  or  VTs).  Shiga  toxin  is 
associated  with  bacterial  dysentery,  a  serious 
problem  in  many  developing  countries.  The 
strains  of  E.  coli  that  produce  VTs  cause  a  disease 
often  referred  to  as  "hamburger  disease,"  be- 
cause it  can  be  acquired  from  contaminated  ham- 
burger. VTs  can  provoke  the  hemolytic  uremic 
syndrome  and  are  thereby  the  major  cause  of 
acute  kidney  failure  in  children. 

We  have  crystallized  and  solved  the  structure 
of  the  B  subunit  of  VT-1 ,  a  member  of  the  Shiga 
toxin  family.  The  B  subunit  forms  a  pentamer  that 
recognizes  target  cell  surfaces  by  binding  to  the 
carbohydrate  component  of  a  cell-surface  glyco- 
lipid,  globotriaosylceramide  (Gbj).  Since  this  in- 
teraction largely  determines  which  cells  the 
toxin  will  attack,  we  are  interested  in  under- 
standing its  molecular  details.  Comparison  of  the 
amino  acid  sequences  of  all  the  toxins  in  this  fam- 
ily has  enabled  us  to  predict  that  a  surface  cleft 
between  B-subunit  monomers  will  prove  respon- 
sible for  binding.  Crystallographic  binding  stud- 
ies should  allow  us  to  test  this  prediction. 

The  most  surprising  result  is  an  unexpected 
structural  similarity  to  the  B  subunit  of  members 
of  the  cholera  toxin  family.  This  is  unexpected 
because  the  associated  A  subunits  of  the  two 
toxin  families  are  completely  unrelated,  the  B 


521 


Protein  Crystallography  in  the  Study  of  Infectious  Diseases 


subunits  are  very  different  in  size,  and  the  degree 
of  amino  acid  sequence  similarity  is,  if  anything, 
less  than  what  one  would  expect  for  two  random 
sequences.  Nonetheless,  the  structural  similarity 
implies  some  distant  evolutionary  relationship 
between  these  families. 

Pseudomonas  aeruginosa  Pilin 

Pseudomonas  aeruginosa  is  an  opportunistic 
pathogen  that  infects  burn  victims  and  immuno- 
compromised patients.  It  is  also  one  of  the  major 
pathogens  infecting  the  lungs  of  cystic  fibrosis 
patients.  Filaments  on  its  surface,  termed  pili,  at- 
tach to  epithelial  cell  surfaces,  promoting  colon- 
ization. Pili  are  formed  from  a  helical  array  of 
identical  pilin  subunits.  We  have  crystallized  pi- 
lin from  this  organism  (in  collaboration  with  Wil- 
liam Paranchych,  University  of  Alberta)  and  are 
attempting  to  improve  the  quality  of  the  crystals 
to  allow  us  to  determine  its  structure.  The  three- 
dimensional  structure  would  help  us  understand 
the  details  of  cell-surface  binding,  which  could 
be  used  to  devise  strategies  to  interfere  with 
colonization. 

Computer-aided  Drug  Design 

Most  of  the  drugs  in  use  today  were  discovered 
by  trial  and  error,  and  many  of  these  have  un- 
known mechanisms  of  action.  Those  with  known 
mechanisms  usually  act  by  binding  specifically  to 
a  drug  receptor,  often  a  protein  molecule.  Since 
crystallography  allows  us  to  examine  the  struc- 
tures of  receptors  and  the  details  of  their  interac- 
tions with  drugs,  it  should  help  in  improving  ex- 
isting drugs  or  even  in  inventing  new  ones. 
Progress  has  been  made  through  crystallography 
in  the  former  objective,  but  designing  new  drugs 
from  scratch  is  still  extremely  difficult.  We  are 
trying  to  address  this  problem  with  the  aid  of 
computers. 


To  design  a  new  drug,  one  must  first  choose  an 
appropriate  receptor  or  target.  In  the  design  of  an 
antibacterial  drug,  the  key  factor  is  selective  tox- 
icity-, the  drug  should  poison  the  pathogen  but 
not  the  patient.  One  might  choose,  as  a  drug  tar- 
get, an  essential  enzyme  in  a  biochemical  path- 
way unique  to  the  bacterium,  because  that  would 
minimize  the  chances  of  side  reactions  with  host 
enzymes.  In  our  work  on  drug  design,  we  are  as- 
suming that  a  good  potential  drug  target  has  been 
chosen  and  its  crystal  structure  determined.  The 
problem,  then,  is  how  to  exploit  the  structural 
information.  There  are  probably  billions  of  com- 
pounds that  might  be  used  as  drugs;  determining 
which  of  these  might  bind  to,  and  interfere  with, 
the  target  protein  is  far  from  trivial. 

We  have  chosen  a  "divide  and  conquer"  ap- 
proach to  drug  design  to  reduce  the  magnitude  of 
this  problem.  The  vast  set  of  possible  compounds 
is  made  up  of  various  combinations  of  a  much 
smaller  set  of  molecular  fragments.  We  propose 
to  design  drugs  by  using  a  computer  to  dock 
members  of  a  library  of  fragments  to  the  region  of 
the  desired  binding  site,  then  to  combine  docked 
fragments  to  form  chemically  sensible  mole- 
cules. We  have  tested  the  feasibility  of  fragment 
docking,  and  the  results  are  sufficiently  promis- 
ing that  we  will  go  on  to  test  the  feasibility  of  the 
next  step.  Fragment  docking  requires  the  calcula- 
tion of  binding  energies,  which  as  yet  can  only  be 
approximate  and  requires  a  great  deal  of  com- 
puter time.  But  we  believe  that  this  method  can 
provide  a  useful  tool  for  drug  design  in  the  not- 
too-distant  future,  in  view  of  the  acceleration  in 
the  speed  of  computers  and  the  improvements 
that  are  continually  being  made  in  the  under- 
standing of  molecular  interactions. 

Our  work  is  also  supported  by  the  Medical  Re- 
search Council  of  Canada  and  the  Alberta  Heri- 
tage Foundation  for  Medical  Research. 

A  schematic  illustration  of  the  crystal  structure 
of  the  B  (binding)  subunit  of  verotoxin-1, 
viewed  from  the  top  of  the  pentamer.  Arrows 
indicate  strands  of  j3-sheet,  and  cylinders  indi- 
cate ahelices. 

From  Stein,  P.E.,  Boodhoo,  A.,  Tyrrell,  GJ., 
Brunton,  f.L.,  and  Read,  R.J.  1992.  Nature 
355:748-750.  Copyright  ©  1992  Macmillan 
Magazines  Limited. 


522 


Representations  and  Transformations  of  Tactile 
Signals  in  Somatic  and  Frontal  Motor  Cortices 


Ranulfo  Romo,  M.D.,  Ph.D. — International  Research  Scholar 

Dr.  Romo  is  Professor  of  Neurosctence  at  the  Institute  of  Cellular  Physiology,  National  Autonomous 
University  of  Mexico,  Mexico  City.  He  received  his  M.D.  degree  from  the  National  University  of  Mexico  and 
a  Ph.D.  degree  in  neuroscience  from  the  University  of  Paris,  France.  His  postdoctoral  work  was  done  with 
Jacques  Glowinski  at  the  College  of  France  in  Paris,  Wolfram  Schultz  at  the  University  of  Pribourg, 
Switzerland,  and  Vernon  B.  Mountcastle  at  the  Johns  Hopkins  University  in  Baltimore.  Dr.  Romo  has 
received  the  Demuth  Prize  of  the  Swiss  Medical  Research  Foundation  and  a  Guggenheim  Memorial 
Foundation  fellowship. 


PRIMATES  have  access  to  events  occurring  in 
the  external  world  through  specialized  sen- 
sory systems.  The  events  are  first  transduced  by 
the  sensory  receptors  and  encoded  and  transmit- 
ted to  the  central  nervous  system  by  the  primary 
afferent  fibers.  These  messages  are  orderly  distrib- 
uted and  processed  in  brain  centers,  where  the 
external  events  are  represented  and,  under  some 
conditions,  lead  to  sensation,  perception,  mem- 
ory, and  purposeful  motor  acts.  These  phenom- 
ena can  only  be  studied  in  highly  evolved  brains. 
Our  laboratory  is  investigating  the  representation 
of  sensory  signals  in  the  brain  and  the  mecha- 
nisms by  which  the  motor  centers  process  them 
in  order  to  guide  behavior. 

The  somatic  sensory  system  of  subhuman  pri- 
mates appears  to  be  an  appropriate  model  for  ap- 
proaching the  question  of  how  tactile  signals  are 
represented  in  the  cerebral  cortex,  since  the 
hands  and  relevant  brain  structures  are  much  like 
those  of  the  human.  Similar  sensory  performance 
in  somatesthetic  tasks  has  been  observed  in  both. 
Moreover,  the  exploratory  hand  movements  of 
both  primates  have  similar  characteristics,  re- 
flecting the  fact  that  their  somatic  and  motor  sys- 
tems are  similarly  linked.  These  parallels  set  the 
base  for  studying  the  dynamic  neural  operations 
of  the  sensory-motor  interface — in  other  words, 
the  way  sensory  representations  guide  motor 
behavior. 

We  have  selected  an  experimental  paradigm  in 
which  monkeys  discriminate  among  sensory  stim- 
uli delivered  to  the  skin  of  their  hands  as  we  re- 
cord the  associated  cortical  activity.  This  allows 
us  1)  to  define  the  relevant  stimuli  among  which 
monkeys  can  discriminate,  2)  to  follow  the  trans- 
formation of  the  initial  cortical  display  in  the  dis- 
tributed cortical  system  separating  it  from  those 
cortical  areas  that  drive  the  difi'erential  motor  re- 
sponses required  for  successful  execution  of  the 
task,  and  3)  to  study  the  details  of  intracolumnar 
operations  in  the  sensory-association  areas  of  the 
parietal  lobe.  I  will  refer  to  the  first  point. 

Experiments  are  in  progress  to  define  how  the 


direction  and  speed  of  a  probe  moving  across  gla- 
brous skin  of  behaving  monkeys  is  represented  in 
the  activity  of  somatosensory  cortex  neurons 
(areas  3b  and  1).  For  this,  we  have  designed  and 
constructed  a  Cartesian  robot  that  allows  me- 
chanical stimuli  to  be  presented  to  the  skin  of  an 
awake  primate's  hand  at  specified  traverse  dis- 
tance, speeds,  and  directions.  We  have  quantita- 
tively studied  many  neurons  in  areas  3b  and  1  of 
two  alert  monkeys  performing  a  behavioral  task 
unrelated  to  the  tactile  stimuli.  The  receptive 
fields  were  scanned  with  a  probe  moving  at  dif- 
ferent speeds  in  eight  directions  at  preselected 
levels  of  force  exerted  by  the  probe  in  the  skin. 

The  first  objective  in  the  analysis  of  the  re- 
sponding neurons  was  to  reconstruct  the  initial 
representations  of  the  peripheral  events  in  areas 
3b  and  1  and  to  identify  the  possible  transforma- 
tions occurring  in  these  stages  of  the  somatic  pro- 
cessing system.  The  preliminary  analysis  indi- 
cated that  it  was  possible  to  quantify  the 
representation  of  the  physical  aspects  of  the  stim- 
uli in  the  discharges  evoked:  velocity  and  posi- 
tion (kinematics)  and  force  (dynamics) .  The  ac- 
tivity of  a  large  percentage  of  the  recorded  cells 
in  areas  3b  and  1  varied  with  the  speed  of  the 
stimulus  and  displayed  directional  preference. 
This  finding  suggests  that  certain  sets  of  neurons 
of  the  primary  somatic  areas  respond  and  make  a 
neural  replica  of  the  mechanical  stimuli  and  that 
there  are  already  some  transformations  (prefer- 
ence for  direction)  at  this  level.  Moreover,  the 
effects  of  these  two  variables  (speed  and  direc- 
tion) were  modulated  by  the  force  exerted  by  the 
stimulus  on  the  skin. 

What  is  interesting  in  our  data  is  the  fact  that 
directionality  can  already  be  detected  at  the  very 
beginning  of  the  cortical  somatic  processing  sys- 
tem. This  finding  may  explain  the  presence  of 
some  nonisomorphic  images  of  complex  tactile 
signals  in  cortical  areas  3b  and  1 . 

We  are  presently  studying  the  representation  of 
these  signals  in  areas  2,  5,  and  7b,  applying  the 
same  experimental  protocol.  We  are  also  imple- 


525 


Representations  and  Transformations  of  Tactile  Signals  in  Somatic 
and  Frontal  Motor  Cortices 


meriting  a  method  for  recording  the  simultaneous  3b  and  1  as  animals  detect  and  categorize  be- 
activity  of  up  to  eight  neurons  in  order  to  study  tween  different  directions  and  velocities  of  the 
the  input-output  operations  of  columns  of  areas    stimuli  moving  across  the  skin. 


524 


Anterior-Posterior  Patterning 
in  the  Early  Mammalian  Embryo 


Janet  Rossant,  Ph.D. — International  Research  Scholar 

Dr.  Rossant  is  Professor  of  Molecular  and  Medical  Genetics  at  the  University  of  Toronto  and  Senior 
Scientist  at  the  Samuel  Lunenfeld  Research  Institute  of  Mount  Sinai  Hospital,  Toronto.  She  received  her 
undergraduate  training  in  zoology  at  Oxford  University  and  her  Ph.D.  degree  from  Cambridge  University. 
Her  postdoctoral  training  was  in  Richard  Gardner's  laboratory  at  Oxford.  Before  joining  Mount  Sinai 
Hospital,  she  was  Associate  Professor  of  Biological  Sciences  at  Brock  University,  St.  Catherines. 


SHORTLY  after  implantation  in  the  mother's 
uterus,  the  mammaHan  embryo  is  transformed 
from  an  undifferentiated  group  of  cells  with  no 
axis  of  symmetry  into  a  trilayered  structure  with 
anterior-posterior  (A-P)  and  dorsal -ventral  (D-V) 
polarity  and  the  beginnings  of  segmentation.  One 
of  the  main  challenges  in  mammalian  develop- 
ment is  to  understand  the  cellular  processes  that 
underlie  these  events  and  how  they  are  geneti- 
cally controlled. 

Studies  in  vertebrates  other  than  the  mouse 
have  implicated  inductive  interactions  between 
tissue  layers  as  important  in  both  determining 
new  tissue  types  and  establishing  regional  do- 
mains along  the  A-P  axis.  We  have  devised  an  ex- 
plant-recombination  system  that  allows  us  to 
address  the  importance  of  such  inductive  interac- 
tions in  the  mouse.  Using  the  mouse  homeobox- 
containing  engrailed-like  (En)  genes  as  markers 
for  a  specific  anterior  domain  of  the  nervous  sys- 
tem, we  have  shown  that  expression  of  these 
genes  in  ectoderm  depends  on  interaction  with 
underlying  mesoderm. 

Isolated  anterior  ectoderm  from  premesoderm 
stages  of  development  will  not  initiate  En  gene 
expression  in  vitro  but  will  express  En  genes 
after  aggregation  with  later  anterior  mesoderm. 
Anterior  mesoderm  will  also  induce  En  expres- 
sion in  posterior  ectoderm,  which  never  nor- 
mally expresses  En  proteins.  Posterior  mesoderm 
is  incapable  of  En  induction  when  combined 
with  either  early  ectoderm  or  later  posterior 
ectoderm. 

These  experiments  and  others  in  progress  indi- 
cate that  there  is  regionalization  in  the  capacity 
of  mesoderm  to  induce  specific  neural  struc- 
tures. Furthermore,  preliminary  evidence  sug- 
gests that  the  basic  patterning  of  the  forebrain, 
midbrain,  and  anterior  hindbrain  structures  is 
laid  down  by  mesoderm  induction  at  the  early 
neural  plate  stage  and  that  the  later  interac- 
tions that  lead  to  the  localized  expression  of 
other  anterior  genes  may  be  confined  to  the 
neuroectoderm . 

We  are  continuing  these  kinds  of  studies  on 
both  mesoderm  and  neural  induction  in  the 


mouse,  making  use  of  the  increasing  number  of 
early  marker  genes  for  the  processes  of  A-P  pat- 
terning. We  expect  these  studies  to  define  in 
more  detail  the  temporal  and  spatial  parameters 
of  inductive  interactions.  The  data  will  help  in 
the  search  for  the  underlying  molecular  basis  of 
patterning  in  the  embryo. 

One  factor  thought  to  play  a  role  in  A-P  pattern- 
ing is  retinoic  acid  (RA).  There  is  considerable 
circumstantial  evidence  to  implicate  RA  in  help- 
ing to  establish  the  boundaries  of  specific  A-P  do- 
mains in  the  developing  embryo.  We  have  pro- 
vided more  such  evidence  by  showing  that 
transgenic  mice,  carrying  an  RA-responsive  ele- 
ment upstream  of  a  neutral  promoter-/<2cZ  con- 
struct, express  the  bacterial  lacZ  gene  in  a  spe- 
cific posterior  domain  of  the  embryo. 

The  boundary  of  this  expression  domain  coin- 
cides, at  the  early  neural  plate  stage,  with  the 
anterior  boundary  of  the  Hox  gene,  Hox-2.9.  As 
development  proceeds,  the  boundary  of  expres- 
sion of  the  transgene  recedes  in  concert  with  the 
establishment  of  more-posterior  //ox  gene  bound- 
aries in  the  hindbrain.  Hox  genes  are  thought  to 
be  involved  in  A-P  patterning,  especially  in  defin- 
ing the  identity  of  the  segmental  hindbrain  rhom- 
bomeres.  The  coincidence  of  Hox  gene  bound- 
aries and  the  receding  RA-responsive  transgene 
boundary  suggests  a  possible  involvement  of  RA 
in  establishing  Hox  gene  expression  domains. 

We  have  shown  that  anterior  members  of  the 
Hox- 2  gene  cluster  respond  within  four  hours  to 
exogenous  RA  in  vivo.  This  response  involves  an 
anterior  shift  in  their  expression  boundaries,  and 
the  altered  expression  domain  persists  in  later 
embryos  and  can  be  correlated  with  the  specific 
teratogenic  effects  of  RA  in  the  hindbrain.  Further 
experiments  are  planned  to  confirm  that  endoge- 
nous RA  is  important  for  Hox  gene  patterning. 

Our  knowledge  of  the  genetic  control  of  pat- 
terning in  the  gastrulating  mouse  embryo  is  still 
very  limited.  If  one  could  screen  rapidly  through 
large  numbers  of  genes  for  their  pattern  of  ex- 
pression at  gastrulation,  one  could  hope  to  iden- 
tify overlapping  expression  patterns  that  indicate 
fundamental  developmental  domains.  In  coUabo- 


525 


Anterior-Posterior  Patterning  in  the  Early  Mammalian  Embryo 


ration  with  Alexandra  Joyner  (HHMI,  Interna- 
tional Research  Scholar),  we  are  using  gene-trap 
vectors  inserted  into  embryonic  stem  (ES)  cells  as 
one  strategy  for  identifying  expression  domains. 

In  these  vectors  the  bacterial  lacZ  gene  lacks  a 
promoter  but  has  a  splice  acceptor  sequence  up- 
stream, such  that  /«cZ  can  be  activated  as  a  fusion 
transcript  when  the  vector  inserts  in  the  intron  of 
an  active  host  gene.  Many  /acZ-expressing  ES 
clones  with  different  integrations  are  being  as- 
sessed in  chimeras  for  their  pattern  of  expression 
at  gastrulation.  We  have  screened  more  than  200 
such  lines  to  date  and  find  about  20  percent  with 
spatially  restricted  expression  patterns.  The 
screen  will  continue,  and  the  most  interesting 
lines  will  be  cloned  from  the  fusion  transcript 


and  taken  through  the  germline  to  analyze  the 
mutant  phenotype  induced  by  the  vector 
integration. 

Other  more  rapid  screens  for  expression  pat- 
terns using  whole-mount  in  situ  hybridization 
with  cDNA  clones  are  in  the  pilot  stages.  The  com- 
bination of  large-scale  searches  for  new  patterns 
of  gene  expression  with  embryonic  manipulation 
experiments  using  such  genes  as  markers  should 
give  new  insights  into  the  basis  of  patterning  in 
the  mouse  embryo.  This  broad  approach  does 
not,  of  course,  eliminate  the  need  to  study  further 
the  role  of  specific  genes  already  identified  as 
potential  regulators  of  development,  and  we  are 
also  undertaking  the  targeted  mutagenesis  of  a 
number  of  such  genes. 


Mouse  embryos  at  day  8.5,  showing  expression  domains  of 
RAREhsplacZ  (top),  Hox-2.9  (middle),  andKxox-20  (bottom). 

Research  and  photograph  by  Ron  Conlon  in  the  laboratory  of 
fanet  Rossant. 


526 


Response  of  the  Cerebral  Cortex  to  Spatial 
Information 


Jean-Pierre  Roy,  M.D. — International  Research  Scholar 

Dr.  Roy  is  Assistant  Professor  of  Neurology  and  Neurosurgery  at  the  Montreal  Neurological  Institute, 
McGill  University,  and  Neurologist  at  Montreal  Neurological  Hospital,  Montreal,  Quebec.  After  receiving 
his  M.D.  degree  from  Laval  University  in  Quebec  City,  he  spent  two  years  in  Mircea  Steriade's  laboratory 
at  Laval  investigating  the  origin  of  rhythmic  oscillations  in  the  thalamic  relay  cells.  He  then  went  on  to  a 
neurology  residency  at  the  Montreal  Neurological  Hospital.  This  was  followed  by  fellowship  studies  at 
NIH  with  Robert  Wurtz  on  neuronal  sensitivity  to  visual  stimuli  in  the  cerebral  cortex. 


THE  main  goal  of  my  work  is  to  understand  the 
transformation  of  simple  information,  such  as 
the  speed  and  direction  of  motion  registered  by 
individual  neurons,  into  information  about  the 
motion  of  the  subject  and  the  spatial  properties  of 
his  environment.  My  approach  is  to  examine  the 
response  of  single  cells  in  one  area  of  a  rhesus 
monkey's  parietal  cortex — the  medial  superior 
temporal  area  (MST) — to  different  visually  pre- 
sented stimuli.  The  neurons  in  the  MST  are  inter- 
esting in  that  about  half  of  them  respond  to  mo- 
tion in  one  direction,  for  example  left,  when  the 
motion  is  in  front  of  where  the  subject  is  looking, 
and  respond  to  motion  in  the  opposite  direction, 
right  in  this  case,  when  the  motion  is  behind 
where  the  subject  is  looking.  This  corresponds, 
we  think,  to  the  apparent  motion  of  the  environ- 
ment experienced  during  self-motion. 

When  a  subject  moves  in  the  environment 
while  looking  at  an  object  to  the  side,  the  objects 
in  the  foreground  will  move  in  the  direction  op- 
posite to  that  of  self-motion  while  those  in  the 
background  will  move  in  the  same  direction  as 
the  self-motion.  The  correlation  between  direc- 
tion of  motion  and  disparity  in  these  neurons  sug- 
gests that  they  detect  these  environmental  mo- 
tions. But  more  than  that,  it  suggests  that  these 
cells  are  involved  not  so  much  in  indicating  the 
movement  of  the  environment  as  such,  but  rather 
the  movement  of  the  subject  himself.  Indeed, 
these  cells  will  respond  if  foreground  moves  left 
or  if  background  moves  right,  or  both.  Each  of 
these  three  conditions  will  be  observed  when  the 
subject  moves  to  the  right.  Hence  we  propose 
that  those  neurons  signal  motion  of  the  subject 
himself. 

Within  one  direction  and  one  disparity  re- 
sponse, we  have  evidence  that  speed  is  another 
property  the  cells  record.  Indeed,  there  appears 
to  be  a  preference  for  lower  speeds  when  the  dis- 
parity of  the  motion  is  of  a  small  absolute  value, 
i.e.,  when  it  corresponds  to  motion  close  to  the 


point  of  fixation,  and  a  preference  for  higher 
speeds  when  the  disparity  is  of  a  large  absolute 
value,  i.e.,  when  it  corresponds  to  motion  far 
from  the  point  of  fixation.  This  last  property  is 
very  interesting  in  that  it  suggests  that  those  cells 
could  be  signaling  not  only  the  direction  of  the 
self-motion  but  possibly  also  the  structure  of  the 
environment. 

This  requires  elaboration.  I  am  proposing  that 
the  preliminary  evidence  of  a  differential  re- 
sponse of  cells  to  different  speeds  could  repre- 
sent a  role  for  them  in  transforming  the  speed 
information  into  depth  (or  more  precisely,  rela- 
tive depth)  information. 

During  the  condition  described  above,  objects 
in  the  environment  that  are  very  near  or  very  far 
will  move  fast,  while  those  that  are  close  to  the 
plane  where  the  subject  fixates  will  move  slowly. 
Speed,  then,  contains  information  about  the 
depth  of  objects  in  the  environment.  In  order  to 
test  this  hypothesis,  individual  planes  moving  in- 
dependently, as  were  used  before,  are  inade- 
quate. What  is  needed  are  multiple  planes  mov- 
ing simultaneously  at  different  speeds.  To 
explore  whether  MST  neurons  do  respond  to 
these  stimuli  representing  speed  and  disparity 
gradients,  we  have  developed  stimuli  with  ap- 
propriate characteristics. 

With  those  stimuli,  we  should  be  able  to  exam- 
ine the  response  of  neurons  to  different  simple 
properties  of  the  stimulus.  Our  prediction  is  that 
the  cells  should  respond  optimally  to  a  stimulus 
that  has  the  correct  direction  of  motion  of  the 
foreground  versus  the  background,  but  also  a 
speed  gradient  and  possibly  a  disparity  gradient. 
This  would  suggest  that  those  cells  are  indeed 
involved  in  transforming  the  information  they  re- 
ceive about  speed  into  information  about  depth. 
It  would  not  be  the  ultimate  answer,  which  could 
only  come  from  lesion  studies  we  are  planning 
for  the  future. 

This  work  is  also  supported  by  a  grant  from  the 
Medical  Research  Council  of  Canada. 


527 


Molecular  Studies  on  Neuronal  Calcium  Channels 


Terry  p.  Snutch,  Ph.D. — International  Research  Scholar 

Dr.  Snutch  is  Assistant  Professor  of  Zoology  and  Neuroscience  at  the  Biotechnology  Laboratory,  University 
of  British  Columbia,  Vancouver.  He  obtained  a  B.Sc.  degree  in  biochemistry  from  Simon  Fraser  University, 
Vancouver,  and  remained  there  to  complete  his  Ph.D.  degree  for  studies  on  the  molecular  genetics  of  the 
heat- shock  response  o/ Caenorhabditis  elegans  with  David  Baillie.  After  further  research  training  in  the 
laboratory  of  Norman  Davidson  at  the  California  Institute  of  Technology,  Dr.  Snutch  joined  the  newly 
formed  Biotechnology  Laboratory.  He  recently  received  a  fellowship  in  neuroscience  from  the  Alfred  P. 
Sloan  Research  Foundation  and  the  Killam  Research  Prize  from  the  University  of  British  Columbia. 


THE  entry  of  calcium  ions  (Ca^^)  into  cells 
mediates  a  wide  variety  of  cellular  and  physi- 
ological responses,  including  muscle  contraction 
and  hormone  secretion.  In  the  nervous  system,  an 
increase  in  intracellular  Cd}^  concentration  di- 
rectly affects  the  electrical  properties  of  neurons. 
Ca^"^  entry  has  also  been  shown  to  have  a  role  in 
regulating  gene  expression,  modulating  Ca^^- 
dependent  enzymes,  and  mediating  nerve  grov^h 
and  regeneration.  Furthermore,  an  increase  in 
C?}*  concentration  at  the  presynaptic  nerve  ter- 
minal triggers  the  release  of  neurotransmitter,  in- 
hibiting or  exciting  postsynaptic  neurons. 

The  rapid  entry  of  Ca^"*^  into  neurons  is  me- 
diated by  membrane  proteins  called  Ca^"*^  chan- 
nels. These  diverse  molecules  respond  to  voltage 
changes  across  the  cell  membrane  by  opening 
Ca'^"'^-selective  transmembrane  pores.  Besides  the 
wide  variety  of  normal  physiological  effects  that 
Ca^"^  channels  mediate,  they  are  implicated  in  a 
number  of  disorders,  such  as  angina,  hyperten- 
sion, migraine,  and  certain  arrhythmias.  The  clin- 
ical treatment  of  these  disorders  is  aided  by  Ca^"^ 
channel-blocking  drugs.  Our  present  studies  uti- 
lize molecular  cloning  techniques  to  address  ba- 
sic questions  concerning  the  structure,  function, 
and  expression  of  Ca^"^  channels  in  the  mamma- 
lian nervous  system. 

Molecular  Diversity  of  Neuronal 
Ca^*  Channels 

Four  types  of  Ca^"^  channel,  called  T,  L,  N,  and 
P,  have  been  identified,  with  differing  electro- 
physiological and  pharmacological  properties. 
Among  their  subunits  is  the  aj-subunit,  which 
both  responds  to  voltage  changes  and  forms  the 
ion-conducting  pore.  Although  the  differences 
among  various  Ca^^  channels  may  be  due  to  a 
number  of  factors,  we  hypothesize  that  distinct 
a  1 -subunits  account  for  most  Ca^^  channel 
diversity. 

Utilizing  molecular  cloning  techniques,  we 
have  isolated  cDNAs  encoding  four  distinct 
classes  of  Ca^^  channel  from  rat  brain:  classes 
A,  B,  C,  and  D.  Their  amino  acid  sequences  show 


that  they  are  large  proteins  (2,100-2,300  amino 
acids)  and  that  they  share  a  number  of  conserved 
features  with  cloned  sodium  and  potassium 
channels. 

The  class  C  channel  found  in  brain  is  nearly 
identical  to  a  Ca^^  channel  previously  found  in 
heart  and  lung,  while  classes  A,  B,  and  D  repre- 
sent novel  forms.  Hybridization  of  the  clones  to 
rat  genomic  DNA  indicates  that  each  of  the  classes 
of  brain  Ca^^  channel  is  encoded  by  a  distinct 
gene. 

One  interesting  result  of  these  studies  is  that 
within  each  class  of  aj-subunit  cloned,  several 
varieties  have  been  identified,  and  the  actual 
number  of  distinct  a, -subunits  is  much  larger 
than  previously  thought.  Using  molecular  ge- 
netic techniques,  we  have  demonstrated  that 
these  varieties,  at  least  in  the  class  C  and  D  in- 
stances, are  generated  by  alternative  splicing.  For 
class  C  Ca^"^  channels,  two  distinct  isoforms  are 
expressed  at  different  levels  in  various  regions  of 
the  rat  brain  and  thus  may  make  unique  contribu- 
tions to  distinct  populations  of  neurons. 

Differential  Localization  of  Ca^^  Channel 
Subtypes 

In  an  attempt  to  provide  information  concern- 
ing the  physiological  roles  of  the  neuronal  Ca^"*" 
channel  subtypes,  we  are  determining  their  cel- 
lular and  subcellular  localizations.  Studies  local- 
izing Ca^"^  channel  gene  expression  by  examina- 
tion of  RNA  levels  show  that  the  various  classes  of 
Ca^^  channel  are  differentially  expressed  in  the 
brain.  For  example,  in  the  cerebellum  the  class  A 
channel  is  mostly  localized  to  Purkinje  cells, 
while  class  C  channels  are  more  widespread.  In 
addition  to  their  different  spatial  distributions  in 
the  brain,  studies  using  antibodies  generated 
against  the  cloned  Ca^*  channel  aj -subunits  show 
that  the  various  Ca^^  channel  proteins  have  dis- 
tinct subcellular  localizations  on  individual  neu- 
rons. For  example,  some  Ca^*  channel  subtypes 
appear  to  be  localized  to  dendritic  regions,  while 
others  are  located  on  both  cell  bodies  and  den- 
drites. We  believe  that  the  distinct  cellular  and 


529 


Molecular  Studies  on  Neuronal  Calcium  Channels 


subcellular  distributions  of  the  Ca  channel  sub- 
types reflect  their  unique  contributions  to  neuro- 
nal physiology.  A  long-term  goal  of  this  research 
is  to  define  how  the  individual  subtypes  uniquely 
contribute  to  neuronal  functioning. 

Functional  Expression  of  Neuronal 
Ca^^-Channels 

We  are  taking  two  approaches  to  determining 
the  functional  properties  of  the  cloned  Ca^"^ 
channel  aj-subunits.  First,  full-length  clones  for 
the  four  main  classes  of  rat  brain  aj-subunit  are 
being  introduced  into  mammalian  cell  lines  that 
normally  do  not  express  any  Ca^"^  channels.  The 
electrical  and  pharmacological  properties  of  the 
Cd}^  channels  will  then  be  determined  with  the 
patch-clamp  technique.  Second,  we  are  using  the 
DNA  sequences  derived  from  the  cloned  Ca^^ 


channels  to  generate  small  synthetic  pieces  of 
DNA  (oligonucleotides)  that  will  hybridize  to 
mRNAs  encoding  these  molecules. 

Microinjection  of  oligonucleotides  into  cells 
that  already  express  defined  Ca^^  channels  re- 
sults in  the  oligonucleotide  hybridizing  to  Ca^"*" 
channel  mRNA  and  inhibiting  expression.  By  in- 
hibiting the  expression  of  a  single  type  of  Ca^"*" 
channel  in  cells  that  normally  express  several 
types,  we  hope  both  to  identify  the  specific  type 
of  Ca^"^  channel  blocked  by  the  oligonucleotide 
and  also  to  obtain  some  insight  into  the  physiolog- 
ical roles  that  individual  Ca^"^  channel  types  con- 
tribute to  neurons.  Using  this  technique  we  have 
found  that  the  rat  brain  class  C  aj-subunit  en- 
codes a  dihydropyridine-sensitive  (L-type)  Ca^"^ 
channel.  Similar  studies  with  the  other  classes  of 
brain  Ca^"^  channel  are  under  way. 


Detection  of  single- copy  sequences  in  fluores- 
cence micrographs  of  metaphase  chromo- 
somes, interphase  nuclei,  and  free  chromatin, 
a )  Metaphase  chromosomes  from  a  human  dip- 
loid lymphocyte  culture,  hybridized  with  four 
cosmid  probes  (cM58-3-6,  CF14,  cf21,  and 
cWlO-20)  together,  spanning  341  kb.  b)  Hy- 
bridization of  the  same  probes  with  interphase 
nucleus.  Arrows  indicate  two  sets  of  hybridiza- 
tion signals,  c-f)  Results  of  hybridization  with 
different  combinations  of  cosmids  in  the  7q31 
region,  c)  cNH24  and  cf21;  d)  cM58-3-6  and 
cf21;  e)  CM58-3.6,  CF14  and  cf21  (note:  two 
sets  of  hybridization  signals);  f)  cM58-3-6, 
CF14,  cf21,  and  cW  10-20. 

Research  and  photograph  by  Henry  Heng  in 
the  laboratory  of  Lap-Chee  Tsui. 


530 


Cystic  Fibrosis,  Gene  Expression  in  the  Mammalian 
Lens,  and  Mapping  of  Chromosome  7 


Lap-Chee  Tsui,  Ph.D. — International  Research  Scholar 

Dr.  Tsui  is  Senior  Scientist  and  Sellers  Chair  of  Cystic  Fibrosis  Research  in  the  Department  of  Genetics  at 
the  Research  Institute  of  the  Hospital  for  Sick  Children,  Toronto,  and  Professor  of  Molecular  and  Medical 
Genetics  at  the  University  of  Toronto.  He  was  born  in  Shanghai,  raised  and  educated  in  Hong  Kong,  and 
there  awarded  degrees  from  the  Chinese  University.  His  Ph.D.  degree  is  from  the  University  of  Pittsburgh, 
where  his  thesis  was  on  the  structure  and  assembly  of  bacteriophage  X  (with  Roger  Hendrix).  After 
training  briefly  in  the  Biology  Division  of  Oak  Ridge  National  Laboratory,  he  joined  the  laboratories  of 
Manuel  Buchwald  and  Jack  Riordan  at  the  Hospital  for  Sick  Children  to  work  on  cystic  fibrosis.  Dr.  Tsui's 
honors  include  the  titles  of  Scientist  of  the  Medical  Research  Council  of  Canada,  Fellow  of  the  Royal  Society 
of  Canada,  and  Fellow  of  the  Royal  Society  of  London. 


THE  research  interests  of  my  laboratory  consist 
of  three  general  topics  in  the  molecular  biol- 
ogy of  mammalian  gene  regulation  and  function. 

Molecular  Genetics  of  Cystic  Fibrosis 

Through  classical  genetic  linkage  analysis  and 
various  molecular  cloning  strategies,  the  gene  re- 
sponsible for  cystic  fibrosis  (CF) ,  the  commonest 
severe  autosomal  recessive  disorder  among  Cau- 
casians, has  been  localized  and  identified.  We 
have  shown  that  the  major  CF  mutation,  account- 
ing for  approximately  70  percent  of  all  mutant 
alleles,  is  a  deletion  of  the  phenylalanine  residue 
at  amino  acid  position  508  of  the  predicted  poly- 
peptide, which  is  named  cystic  fibrosis  trans- 
membrane conductance  regulator  (CFTR) . 

To  investigate  the  basic  defect  in  CF,  we  are 
continuing  our  search  for  the  other  CFTR  muta- 
tions. Since  the  CFTR  gene  contains  27  exons  and 
spans  230  kb  of  DNA,  detecting  a  microscopic 
mutation  is  not  straightforward.  Furthermore,  the 
lack  of  a  convenient  functional  assay  for  CFTR 
makes  it  difficult  to  distinguish  a  truly  disease- 
causing  mutation  from  a  benign  amino  acid  sub- 
stitution. To  coordinate  the  detection  eff'ort,  our 
laboratory  has  taken  a  central  role  in  the  forma- 
tion of  an  international  consortium  of  90  groups 
of  researchers  from  26  countries.  Through  active 
exchange  of  gene  sequences  and  mutation  data 
before  submission  for  publication,  the  consor- 
tium has  already  identified  more  than  150  appar- 
ent CF  mutations  and  30  sequence  variations.  The 
collective  data  from  two  years  of  operation  also 
show  that  most  of  the  remaining  30  percent  of 
mutant  alleles  are  individually  rare  and  highly 
heterogeneous  among  different  populations. 

Despite  our  intensive  DNA  sequencing  effort, 
covering  each  of  the  exons  plus  their  flanking 
regions,  we  have  not  been  able  to  identify  the 
mutations  for  6  of  the  94  CF  chromosomes  exam- 
ined. These  alleles  probably  harbor  different  mu- 
tations located  in  regions  that  affect  transcription 
or  RNA  processing.  Nevertheless,  we  have  devel- 


oped a  highly  informative  marker  for  use  in  ge- 
netic counseling  of  CF  families.  The  observed  he- 
terozygosity for  this  marker,  a  dinucleotide 
repeat  polymorphism  in  one  of  the  introns  of  the 
CFTR  gene,  is  over  95  percent.  In  a  case  of  prena- 
tal diagnosis,  we  applied  the  marker  successfully 
after  all  other  available  tests  failed. 

The  common  CF  symptoms  include  chronic 
obstructive  lung  disease,  pancreatic  enzyme  in- 
sufficiency, and  elevated  sweat  electrolytes. 
Other  organs  and  tissues,  such  as  the  hepatobili- 
ary tree,  intestines,  and  vas  deferens,  may  also  be 
involved.  The  varied  degree  of  severity  of  the 
symptoms  among  CF  patients  suggests  that  the 
phenotypes  are  at  least  partly  conferred  by  the 
genotypes  at  CFTR.  Furthermore,  information 
about  genotypes  and  phenotypes  should  also  pro- 
vide important  clues  to  the  function  of  CFTR  and 
the  basic  defect  in  the  various  affected  organs  and 
tissues.  Based  on  this  assumption  and  the  use  of 
the  extensive  clinical  data  collected  at  the  CF 
clinic  in  our  hospital,  we  have  demonstrated  a 
good  correlation  between  pancreatic  involve- 
ment and  genotypes  at  the  CFTR  locus. 

To  facilitate  direct  biochemical  and  physiologi- 
cal analysis,  we  have  constructed  a  full-length 
cDNA  for  CFTR  in  several  expression  vectors. 
Site-directed  mutagenesis  has  been  used  to  re- 
move sequences  that  are  toxic  to  the  host  bacte- 
rium as  well  as  to  generate  mutant  constructs  for 
functional  evaluation.  Using  DNA  transfection 
into  heterologous  cell  types,  we  showed  that  this 
cDNA  could  confer  a  cAMP-regulated  chloride 
channel  activity  de  novo,  suggesting  that  CFTR  is 
a  chloride  channel  itself.  Preliminary  data  from 
the  analysis  of  constructs  reproducing  some  of 
the  naturally  occurring  mutations  appear  to  be  in 
good  agreement  with  those  predicted  from  the 
severity  of  pancreatic  involvement. 

In  order  to  understand  the  regulation  of  the 
CFTR  gene,  we  have  performed  a  series  of  dele- 
tion and  transfection  studies  to  determine  the  se- 
quence elements  responsible  for  basal  promoter 


531 


Cystic  Fibrosis,  Gene  Expression  in  the  Mammalian  Lens,  and  Mapping 
of  Chromosome  7 


activity  and  tissue  specificity.  Our  current  data 
suggest  that  the  basal  promoter  element  is  within 
250  bp  of  the  major  transcription  initiation  site. 
There  also  seems  to  be  a  negative  regulatory  ele- 
ment immediately  upstream  of  this  sequence, 
and  proper  expression  of  CFTR  in  vivo  may  re- 
quire additional  cis-regulatory  element(s)  yet  to 
be  identified.  A  thorough  understanding  of  the 
regulation  of  CFTR  transcription  may  provide  ad- 
ditional means  for  treatment  of  CF,  particularly  in 
some  mild  cases  where  an  increase  of  CFTR  syn- 
thesis may  compensate  for  the  partial  defect. 

Experiments  are  also  in  progress  to  exploit  the 
yeast  STE6  gene  as  a  genetic  system  to  gain  some 
insight  into  the  structure  and  function  of  the  first 
ATP-binding  domain  (NBFl)  in  CFTR.  A  system- 
atic survey  of  second  site  mutations  (which  may 
rescue  the  primary  mutation)  should  provide  im- 
portant information  about  the  structure  of  NBFl 
and  the  possibility  of  its  application  in  drug 
design. 

Lastly,  in  order  to  generate  an  animal  model  for 
the  study  of  CF,  we  have  been  trying  to  inactivate 
the  mouse  Cftr  gene  via  homologous  recombina- 
tion in  embryonic  stem  cells.  Our  attempt  to  in- 
terrupt exon  1 0  has  so  far  been  unsuccessful,  and 
our  current  targets  are  exons  1  and  13.  The  avail- 
ability of  a  mutant  mouse  strain  should  greatly 
facilitate  studies  to  clarify  the  pathophysiology  of 
CF  and  improve  means  of  treatment. 

Regulation  of  Gene  Expression 
in  Mammalian  Lens  Development 

Transparency  of  the  vertebrate  eye  lens  is  at 
least  partly  conferred  by  the  short-range  ordering 
of  water-soluble  crystallin  molecules  in  the  lens 
fiber  cells.  In  order  to  understand  the  role  these 
proteins  play  in  maintaining  lens  transparency, 
one  of  our  interests  has  been  to  identify  the  muta- 
tion responsible  for  a  dominant  lens  defect  in  a 
mouse  strain  called  Elo  {Eye  lens  obsolescence) . 
We  have  performed  genetic  linkage  analysis  to 


show  that  the  Elo  mutation  is  closely  linked  to  the 
7-crystallin  gene  cluster  and  have  excluded  the 
first  five  of  the  six  genes  in  the  cluster  as  being 
the  location  of  the  mutation.  Through  subse- 
quent sequence  analysis,  we  have  identified  a 
frameshift  mutation  in  the  gene,  the  last 
member  of  the  six-membered  cluster.  Although 
the  significance  of  the  latter  observation  is  pres- 
ently unknown,  it  strongly  argues  that  7-crystal- 
lin plays  a  major  rather  than  a  generally  assumed 
passive  role  in  lens  development. 

Physical  Characterization  of  Human 
Chromosome  7 

In  order  to  generate  a  more  complete  set  of 
reagents  for  the  study  of  genes  on  human  chro- 
mosome 7,  a  chromosome-specific  yeast  artificial 
chromosome  (YAC)  library  has  been  constructed. 
Using  a  human-hamster  somatic  cell  hybrid  with 
a  single  human  chromosome  7,  we  have  isolated 
more  than  1,000  YAC  clones  containing  human 
DNA  inserts  averaging  475  kb.  The  clones  are  be- 
ing mapped  to  specific  chromosome  regions  by 
hybridization  with  previously  localized  DNA  seg- 
ments and  with  a  somatic  cell  hybrid  mapping 
panel.  Over  100  clones  have  thus  far  been  identi- 
fied. In  addition  to  the  generation  of  a  long-range 
physical  map  of  chromosome  7,  we  are  develop- 
ing efficient  techniques  for  detecting  gene  se- 
quences based  on  YAC  cloning. 

We  have  also  developed  a  novel  in  situ  hybrid- 
ization procedure  to  facilitate  our  physical  map- 
ping effort.  Using  chromatin  fibers  released  from 
interphase  nuclei  with  specific  reagents  (mostly 
known  as  topoisomerase  II  inhibitors)  and  an  al- 
kaline buffer,  we  have  been  able  to  perform  fluo- 
rescent in  situ  hybridization  to  order  DNA 
segments  less  than  20  kb  apart.  Immediate  appli- 
cation of  this  technique  (named  free  chromatin 
mapping)  should  allow  us  to  estimate  physical 
distance  between  any  given  DNA  segments  and  to 
study  complex  sequence  arrangement,  such  as 
that  in  the  centromeric  regions. 


532 


Index 


abd-A  gene,  Drosophila  development,  549 

ABL  oncogene,  lymphocyte  growth  regulation,  452-454 

Acetylcholine,  neuronal  function,  Iv 

Acetylcholine  receptor  (AChR) 

ion  channel  function,  463-464 

neuronal  function,  Iv,  lix 
achaete  scute  gene  complex  (AS-C),  12 
Acquired  immune  deficiency  syndrome  (AIDS) 

CD4  and  CDS  molecules,  182 

gene  expression,  cellular  transcription,  285-286 

immune  response  and,  lii-liii 

mycobacterial  disease,  200-202 

retroviral  replication,  53-54 

T  cell  development  biology,  265-266 
Acrosome  reaction,  sperm  physiology,  491-492 
a-Actinin,  neuromuscular  disease,  233-234 
Action  potentials 

definition  of,  Iv 

generation  in  olfaction  and,  332 

research  on,  1-2 
Activin,  transforming  growth  factor  and,  275-276 
Adducin 

egg  structure-function  studies,  386 

spectrin  skeleton  assembly,  34 
Adenomatous  polyposis  coli  (APC),  441-442 
Adenosine,  neuromodulation,  caffeine  consumption, 

401-402 
Adenosine  deaminase 

deficiency 

blood  cell  formation,  446 
gene  therapy,  69-  70 

structure/function  studies,  329-330 
Adenovirus 

gene  expression  control  model,  369-370 

transcriptional  regulation,  oncogenesis,  293-294 
Adrenal  cortex,  steroid  hormone  gene  expression,  317-318 
Adrenergic  receptors 

molecular  biology,  25/-252 

structure  and  function,  lix,  229-230 
tVj-Adrenergic  receptor,  structure  and  function,  229-230 
/3-Adrenergic  receptor  kinase  (/3ARK),  252 
(32-Adrenergic  receptor,  cellular  biology,  230 
Adult  respiratory  distress  syndrome  (ARDS),  in  inflammation 

and  metastasis,  40 
Affective  disorders,  neurological  dysfunction  and,  Ix 
Agammaglobulinemia,  immune  system  development  and,  84 
agouti  genes,  mouse  development  and,  19-20 
Agrin 

molecular  neuroimmunology,  319-320 

synaptic  transmission,  355-356 
AIDS.  5ee  Acquired  immune  deficiency  syndrome 
Albinism,  tyrosinase  and,  307-308 
Aldose  reductase,  structure/function  studies,  329-330 
Aldosterone,  cardiovascular  disease  and,  244 
Allelic  exclusion 

B  cell  development,  299-300 

immune  response  and,  /// 
Allosteric  enzymes,  structural  studies,  388 
al  protein,  DNA  interactions,  309-311 
Alternative  splicing,  protein  diversity  and,  288 
Amblyopia,  neurophysiology  of  visual  systems,  284 
Amino  acids 

protein  structure,  xxx 

transport,  retroviral  infections,  97-98 
Aminopeptidase  A  (APA),  84 
Animal  cells 

example  of,  xxx-xxxi 

gene  regulation,  293-294 
Ankyrin,  structure,  34 
Ankyrins,  plasma  membranes,  33-34 
Antennapedia  locus,  morphogenesis,  223-224 
Anterior-posterior  patterning,  embryogenesis,  525-526 


Antibodies 

complement  system,  15-16 

gene  sequences  and,  xlviii,  li 
lymphocyte  difl'erentiation,  110 

molecular  mimicry  by,  110 
Antibody-antigen  interactions,  structure/function  studies, 

329-330 
Antigens 

cytotoxic  T  cells,  37-38 

determinant,  immune  response  and,  li 

gene  sequences  and,  xlix,  li 

immune  response  and,  xlvi,  li-liii 

processing  mechanisms,  93-94 
Antigen-specific  receptors 

generation  of,  351-352 

genetic  mechanisms,  9-10 
Antiports,  leukocyte  homeostasis,  497-498 
Aplysia.  cell  biological  studies  of  memory,  219-220 
Apolipoprotein  E,  chromosome  structural  studies,  4 
Apoptosis 

inflammatory  cytokines,  73-  74 

visual  systems,  neurogenesis  and,  398 
Arachidonic  acid,  phospholipids,  165-166 
Archaebacteria,  protein  folding,  189-190 
Atherosclerosis,  platelet-derived  growth  factor  (PDGF), 
447-448 

Atrial  natriuretic  peptides  (ANPs),  151-152 
aWTw  7  attachment,  transposition  mechanism,  91-92 
Autoantibody  probes.  See  also  Small  nuclear 
ribonucleoproteins  (snRNPs) 

gene  expression,  391-392 
Autoimmune  disease 

biology  of  T  cell  development,  265-266 

CD4  T  cell  activation,  209-210 

complement  system,  15-16 

immune  response,  11,  136-138 

mechanisms,  171-172 

T  cell  function  in,  273-274 

tissue-type  plasminogen  activator  (t-PA),  157-158 
Autosomal  dominant  polycystic  kidney  disease  (ADPKD), 

333-334 
Avian  leukemia  virus  (ALV),  95-96 
Axenization,  Entamoeba  histolytica,  515-516 
Axon  guidance 

neural  development,  207-208 

structure  and  function,  liv 

visual  systems,  neuronal  cell  recognition,  396-398 
B  cells 

complement  receptors,  185-186 

development,  9-10 

gene  regulation,  379-380 

immune  response,  xlvi 

immune  system  development  and,  83-84 

immunoglobulin  heavy-chain  gene  control.  215-216 

immunological  self-tolerance  and  autoimmunity,  /  71- 1 72 

molecular  regulation,  299-300,  415-416 

signal  transduction  pathways,  159-160 
B7  molecule,  T  cell  activation,  209-210 
Bacterial  toxins,  protein  crystallography,  521-522 
fcrt/- genetic  sites,  phage  X,  501-502 
Bare  lymphocyte  .syndrome  (BLS),  325-326 
Base  pairing,  DNA  structure  and,  xxxix 
bed  gene,  transcriptional  activation,  403 
BCG  (bacille  Calmette-Guerin)  vaccine 

leprosy  and  tuberculosis  immunity  and  pathogenesis, 
4  7-48 

mycobacterial  disease,  200-202 
BCR/ABL  protein,  lymphocyte  growth  regulation,  452-454 
Becker  muscular  dystrophy 

gene  identification  and  correction,  69 


533 


Index 


Becker  muscular  dystrophy  {continued  ) 

molecular  genetics,  233-234 

neuronal  function,  Ix 
Beckwith-Wiedemann  syndrome  (BWS),  131 
Betaglycan,  transforming  growth  factor  and,  275-276 
i3-turn,  genetic  analysis  of,  140 
6«co/rf  gene,  transcription  control,  112-114 
Binary  probes,  RNA  replication,  509-510 
Biological  clock,  biochemical  actions  of,  465-466 
Bioph^ics,  eukaryotic  gene  regulation,  59-60 
Bithorax  complex 

Drosophila  melanogaster,  349-350 

limb  development  genetics  in  Drosophila,  80 
Blazing  a  Genetic  Trail,  xix 

Blood-brain  activators,  host-pathogen  interactions,  493-494 
Blood  cells 

formation  mechanisms,  445-446 

molecular  genetics  of,  305-306 
Blood  clotting 

cell  adhesion  and,  195-196 

genetic  regulation,  344-346 

molecular  genetics,  161-162 

prevention  mechanisms,  123-124 
Blood  groups,  glycosyltransferase  molecular  genetics  and, 
267-268 

Blood  vessels,  paracrine  control,  459-460 
Bone  marrow 

blood  cell  formation,  445 

transplants,  coagulation  genetics,  162 
Borrelia  burgdorferi,  immune  tolerance,  136-138 
Breast  cancer,  gene  mapping  techniques  and,  82 
bride  of  sevenless  (boss)  gene,  retinal  cell-cell  interactions, 
471-472 


C4b-binding  protein  (C4bBP),  clotting  prevention,  123- 124 
Cadherin,  neuron  development,  335-336 
Caenorhabditis  elegans 

developmental  genetics,  187-188 

lineage-specific  gene  expression,  511-512 

molecular  genetics,  399-400 

neuronal  function,  lix 

research  with,  xliv 
Caffeine,  neuromodulation,  401-402 
Calcitonin  gene-related  peptide  (CGRP) 

neuroendocrine  system,  339-340 

synaptic  transmission,  192 
Calcium 

hormone  mediation,  129-130 

nerve  cell  electrical  activity,  1-2 

signal  transduction,  visual  systems,  193-194 
Calcium  channels 

dystrophin-glycoprotein  complex,  61-62 

molecular  engineering,  426-428 

molecular  studies,  529-530 

neurotransmitter  storage  and  release  mechanisms,  203 
phototransduction  mechanism,  retinal  rods  and  cones, 

461-462 
prolactin  secretion,  489-490 
scorpion  toxin  characterization,  519-520 
structure-function  studies,  409 
Calmodulin,  protein  structure  and  folding,  139-140 
Calmodulin  kinase  II  (CaMKII)  gene,  mammalian  memory, 
424 

cAMP  (cyclic  AMP) 

chemical  communication,  sex  pheromones,  257-258 
cystic  fibrosis  transmembrane  conductance  regulator 

(CFTR),  439-440 
DNA-binding  proteins,  gene  expression,  175-176 
molecular  engineering,  426-428 
olfactory  system,  ion  channel  regulation,  373-374 
second  messenger  system,  lix 


cAMP  recognition  element  (CRE) 

cell  biological  studies  of  memory,  219-220 
T  cell  receptor  regulation  and,  255-256 

cAMP-responsive  enhancer-binding  proteins  (CREBs),  176 

Campylobacter  jejuni,  molecular  biology,  487-488 

Cancer 

cell  adhesion  and,  195-196 
genetic  modification,  247-248 
growth  factors,  289-290 

lymphocyte  development,  molecular  genetics,  231-232 

mammalian  development  and,  314,  315-316 

multidrug  resistance,  499-500 
Carbohydrate  compounds,  inflammation  and,  40 
Carbohydrate  ligands,  selectins  and,  40 
Carbon  assimilation,  photosynthesis  genetics,  503-505 
Carboxyl-terminal  domain  (CTD),  RNA  polymerase  II 

structure  and  function,  85-86 
Carboxypeptidase  Y  (CPY),  lysosomal  hydrolase  sorting,  121 
Cardiovascular  disease,  genetic  studies,  243-244 
CCAAT  displacement  protein  (CDP),  molecular  genetics  of 

blood  cells  and,  306 
CcN  motif,  cell  fate  control,  14 
CD2  molecule,  T  cell  recognition,  101-102 
CD4  molecule 

activation,  209-210 

cellular  immune  response,  181-182 

development  and  infection,  263-264 

human  immunodeficiency  virus  (HIV),  178 

immune  response  and.  Hi 

in  T  cells,  51-52 
CDS  molecule 

cellular  immune  response,  181-182 

development  and  infection,  263-264 

immune  response  and.  Hi 
CD  18  molecule,  genetic  disease  and,  26 
CD28  pathway,  lymphocyte  development,  415-416 
CD45  antigen,  protein-tyrosine  phosphatases,  413-414 
cdc2  gene,  cell  cycle  control  and,  271-272 
cdk2  protein,  cell  cycle  control  and,  272 
cDNA  clones 

calcium  channel  molecular  studies,  529-530 

cerebellar  gene  expression  and  cell  cycle,  180 

Down  syndrome  and,  237-238 
Cell  adhesion 

cell  structure  and,  xxxviii 

glycosyltransferase  molecular  genetics  and,  267-268 

molecular  basis  for,  195-196 

motor  neuron  differentiation,  211-212 

neuron  development,  335-336 
Cell  adhesion  molecules  (CAM),  26 
Cell  asymmetry,  molecular  genetics,  399-400 
Cell  biology  and  regulation,  research  programs  in, 

xxix-xxxviii 
Cell-cell  interaction 

Drosophila  retina,  471-472 

early  embryogenesis,  oncogenes,  297-298 

neural  development,  207-208 

spectrin  skeleton  a.ssembly,  34 

transforming  growth  factor  and,  275-276 
Cell  cycle 

cell  division  and,  xxxv-xxxvi 

colony-stimulating  factor  1  receptor  (CSF-IR),  371-372 

DNA  replication  and,  21-22 

gene  expression  and,  179-180,  271-272 

research  trends  in,  xxx 
Cell  death.  See  also  Apoptosis 

Caenorhabditis  elegans  development,  187- 188 

visual  systems,  neurogenesis  and,  396-398 
Cell  density,  differentiation  and,  166-168 
Cell  fate 

differentiation  and,  166-168 

Drosophila  retina,  471-472 


554 


Index 


Cell  growth 

cell  structure  and,  xxxv-xxxvi 

liver  regeneration,  411-412 
Cell  injury,  inflammatory  cytokines,  73-74 
Cell  lineage,  Caenorhabditis  elegans  development, 
187-188 

Cell  migration,  Caenorhabditis  elegans  development,  188 
Cell  motility,  defined,  xxxv 
Cell  regulation 

chemistry,  375-576 

phospholipids,  165-166 
Cell  signaling,  Caenorhabditis  elegans  development, 

187-188 
Cell-mediated  immunity 

CD4-bearing  T  lymphocytes,  51-52 

research  on,  xlvi 
Cellular  metabolism,  peptide  activation,  175-176 
Central  nervous  system  (CNS),  growth  cone  guidance  and, 

169-170 
Centromere,  position  efi'ects,  184 

Cerebellum  development,  gene  expression  and  cell  cycle, 

179-180 
Cerebral  cortex 

sensory  representations,  visual  system,  367-368 

spatial  information,  527 
Cervical  cancer,  papillomavirus  molecular  biology,  241-242 
c-Fos  protein,  transcription  control,  470 
CGG  repeat,  fragile  X  syndrome,  431-432 
cGMP  (cyclic  GMP) 

phototransduction  mechanism,  retinal  rods  and  cones, 
461-462 

second  messengers  and  cell  regulation,  151-152 
Chagas  disease,  kinetoplast  genome,  378 
Chaperone  genes,  heat-shock  proteins  (HSPs),  261-262 
Charcot-Marie-Tooth  disease,  gene  mapping,  141 
Chemical  communication,  sex  pheromones,  257-258 
Chemotaxis,  sperm  physiology,  491-492 
Chloramphenicol  acetyltransferase  (CAT),  35-36 
Chloride  channels 

cystic  fibrosis  transmembrane  conductance  regulator 
(CFTR),  439-440 

purification  and  reconstitution,  281-282 
Chloroplasts,  protein  translocons  and,  45-46 
Cholecystokinin,  molecular  biology  of,  143-144 
Cholera  toxins,  protein  crystallography,  521-522 
Cholesterol,  metabolism,  4 
Choroideremia,  molecular  genetics,  295-296 
Chromaffin  cells,  cell  fate  control,  11-12 
Chromatin 

cell  cycle  control,  21-22 

three-dimensional  structure,  eukaryotic  chromosomes, 
362 

Chromosome  7,  mapping  in  cystic  fibrosis  (CF),  531-532 
Chromosome  puffs,  metamorphosis,  417-418 
Chromosomes 

end  structure,  71-72 

structure  and  function,  position  efl'ects,  183-184 
three-dimensional  studies,  3-4 

eukaryotic  chromosomes,  360-362 
unstable,  position  effects,  183-184 
Chylomicronemia  syndrome,  genetic  studies  of 

cardiovascular  disease,  243-244 
Cilia,  ion  channel  activation,  87-88 
Circular  dichroism,  extracellular  matrix,  49 
c-jun  gene,  epidermal  growth  factor  (EGF),  104 
Clathrin,  Yersinia  pseudotuberculosis,  197-198 
C-less  permease,  energy-transducing  membrane  proteins, 
213-214 

Clonal  deletion,  immune  response  and,  // 

Clonal  selection  hypothesis,  immune  response  and,  // 

Cloning 

DNA,  yeast  artificial  chromosomes  (YACs),  303-304 


genetically  related.  Entamoeba  histolytica,  515-516 

scorpion  toxin  receptor  genes,  520 
c-myc  gene 

epidermal  growth  factor  (EGF),  104 

transcription  control,  470 
Collagen,  hereditary  kidney  disease,  333-334 
Colon  cancer,  APC  gene  and,  441-442 
Colony-stimulating  factor  (CSF),  myeloid  cell  growth 

control,  371-372 
Colony-stimulating  factor  (CSF)  1  receptor  (CSF-IR), 
371-372 

Colorblindness,  X-linked  disorders,  163-164 
Common  variable  immunodeficiency  (CVID),  84 
Complement  component  C3,  gene  regulation,  185-186 
Complement  receptor  2  (CR2),  185-186 
Complement  system 

immune  response  and,  lii-liii 

research  on,  15-16 
Complementation-determining  region  (CDR),  101 
Computational  modeling 

sensory  representations,  367-368 

visual  systems  neurophysiology,  283-284 
Computer-aided  drug  design,  protein  crystallography, 
521-522 

Constant  (invariant)  domain,  protein  chains,  xlvi 
a-Constant  Spring  (aCS),  a-globin  gene  expression,  260 
Contractile  protein  genes,  transcriptional  regulation, 
287-288 

Co-receptors.  See  also  CD4  and  CDS  molecules 

immune  response  and.  Hi 
Coronavirus,  replication  and  pathogenesis,  239-240 
couch  potato  {cpo)  gene,  peripheral  nervous  system 

development,  29-30 
Cro  repressor  protein,  macromolecular  interaction,  278 
c-src  proto-oncogene,  383-384 

Cyclic  nucleotide-gated  channel  (CNG),  second-messenger 

ion  channel  regulation,  373-374 
Cyclins 

cell  cycle  control,  21-22 

G2  cyclins,  271-272 
Cyclosporin  A,  T  cell  activation  and  differentiation,  90 
Cysteinyl  residues,  energy-transducing  membrane  proteins, 

213-214 
Cystic  fibrosis  (CF) 

gene  expression,  chromosome  7  mapping,  531-532 

linkage  analysis  and,  81 

molecular  studies  of,  25-26 

somatic  gene  therapy,  449-451 
Cystic  fibrosis  transmembrane  conductance  regulator  (CFTR) 

function  and  regulation,  439-440 

mapping  in  cystic  fibrosis  (CF),  531-532 

multidrug  resistance,  499-500 

somatic  gene  therapy,  449-451 
Cytochrome  b/c,  complex,  photosynthesis  and  respiration,  107 
Cytochrome  P-450  enzymes,  macromolecular  structure,  108 
Cytokines 

CD4-bearing  T  lymphocytes,  51-52 

effector  functions  in  immune  responses,  513-514 

inflammation  and,  73-74 
Cytoplasm,  structure,  xxxv 
Cytoskeleton 

cell  adhesion  and,  196 

host-pathogen  interactions,  493-494 

structure,  xxxv 
Cytotoxic  T  cells 

immune  response  and,  xlvi 

recognition  of,  37-38 

D-type  cyclins,  371-372 

DCoH  dimerization  cofactor,  genetic  regulatory 

mechanisms,  8V-90 
Decay-accelerating  factor,  complement  system,  16 


535 


Index 


"Decoding  code,"  mRNA,  155-156 
Deformed  gene,  Drosophila  morphogenesis,  24 
Delayed-early  (DE)  genes 

growth  factors,  289-290 

viral  gene  regulation,  279-280 
Deletion  mutagenesis,  cell  fate  control,  13-14 
Delta  genes,  cell  fate  control,  13-14 
Dendrites,  structure  and  function,  liv 

Density-sensing  factor  (DSF),  differentiation  and,  166-168 
Desensitization,  epidermal  growth  factor  (EGF),  328 
Developmental  genetics,  mutagenesis  projects,  383-384 
Diabetes  mellitus 

aldose  reductase  functions,  330 

immune  tolerance,  136-138 

IRE-A  DNA-binding  protein,  8 

molecular  genetics,  27-28,  143-144 

stiff-man  syndrome  and,  106 

tissue-type  plasminogen  activator  (t-PA),  157-158 

type  II,  polygenic  inheritance,  144 
Diacylglycerol  (DAG) 

calcium-mediated  hormones,  130 

sex  pheromones,  257-258 
Diarrhea,  guanylyl  cyclase  receptors,  152 
Dictyostelium  discoideum,  differentiation  and,  166-168 
Differentiation 

developmental  control  of  gene  expression  and,  /  73 

genetic  regulatory  mechanisms,  89-90 

keratin  expression,  skin  development,  146-148 

kidney  cells,  transcription  factors,  407-408 

in  lymphocytes,  109-110 

motor  neurons,  211-212 

T  cell  recognition  and,  101-102 

visual  system  development,  341-342 
DiGeorge  syndrome,  gene  targeting  and,  63-64 
Dihydropyridine  receptor 

calcium  channels,  409 

voltage-gated  calcium  channels,  61-62 
Disease  genes.  See  Genetic  disease 
Distal-less  gene,  limb  development  genetics,  79-80 
DNA 

breakage,  bacterial  transposition  reaction,  91-92 
fingerprinting,  gene  mapping,  xliv 
genome  sequencing,  77 

probes,  adenomatous  polyposis  coli  (APC),  441-442 

repair,  antibody  genes,  9-10 

replication  mechanisms,  301-302 

strand  transfer,  bacterial  transposition  reaction,  91-92 

structure  of,  xxxix-xl 

synthesis,  cell  cycle  control  and,  271-272 
DNA  photolyase,  DNA  repair  and,  107 
DNA  polymerase  III 

(3-subunit  structural  studies,  235-236 

replication  mechanisms,  301-302 
DNA-binding  proteins.  See  also  Protein-DNA  interaction 

Drosophila  melanogaster,  developmental  genetics, 
349-350 

immunoglobulin  heavy-chain  gene  control,  215-216 
structural  studies,  309-311,  393-395 
transcription  regulation,  421-422 
dorsal  gene,  signal  transduction  in  B  cells,  159-160 
Down  syndrome,  molecular  analysis,  237-238 
Drosophila  melanogaster 

axis  formation  and  germline  determination,  253-254 
behavior  and  neuromuscular  development,  465-466 
cell  adhesion  and,  196 
cell  fate  control 

embryonic  development,  13-14 

regulatory  molecules,  12 
developmental  genetics,  349-350 
egg  structure-function  studies,  385-386 
embryogenesis 

oncogenes,  297-298 

transcription  control,  112-114 


eukaryotic  chromosomes,  three-dimensional  structure, 

360-362 

growth  cone  guidance  and  neuronal  development, 
169-170 

heat-shock  proteins  (HSPs),  261-262 
ion  channels,  molecular  mechanisms,  5-6 
limb  development  genetics,  79-80 

morphogen  gradients  and  pattern  development,  403-404 
morphogenesis,  genetic  control,  23-24,  223-224 
neural  development,  207-208 
neuronal  function,  lix 

voltage-sensitive  channels,  205-206 
pattern-formation  genes,  65-67,  507-508 
peripheral  nervous  system  development,  29-30 
position  effects,  183-184 
research  with,  xliv 

retinal  cell-cell  interactions,  471-472 
sensory  transduction,  473 
signal  transduction 

intercellular  communication,  323 

visual  systems,  193-194 
visual  systems 

embryogenesis,  341-342 

pattern  formation  and  neuronal  cell  recognition, 

396-398 

Drugs,  receptors,  molecular  biology,  251-252 
Duchenne  muscular  dystrophy  (DMD) 
dystrophin,  62 

gene  identification  and  correction,  69-70 

molecular  genetics,  233-234 

neuronal  function,  Ix 
Dysentery.  See  also  Diarrhea 

Entamoeba  histolytica  variability,  515-516 

protein  cr^'stallography,  521-522 
Dystroglycan,  Duchenne  muscular  dystrophy  (DMD),  62 
Dystrophin 

Duchenne  muscular  dystrophy  (DMD),  62 

identification  and  correction,  69-70 

neuronal  function,  Ix 

research  on,  xliii 
Dystrophin-associated  glycoproteins  (DAGs),  62 

calcium  channels,  61-62 
Dystrophin-related  protein  (DRP),  neuromuscular  disease, 
233-234 


ElA  protein,  gene  expression,  369-370 

E2  transcription  factor,  cellular  regulation,  375-376 

E74  gene,  metamorphosis,  417-418 

Early  B  cell  factor  (EBF),  gene  expression  and,  174 

EBERs,  autoantibody  probes,  392 

Ecdysone,  metamorphosis,  417-418 

Egg  cells,  structure-function  studies,  385-386 

Egg-laying  hormone  (ELH),  neuropeptide  processing  and 

packaging,  355-356 
Egr  gene  family,  kidney  cell  growth  and  differentiation, 

407-408 

Electron  microscopy  (EM),  chromosome  structure,  3-4 
Electrostatic  interactions,  ligand  molecular  recognition,  330 
Elliptocytosis,  hemoglobin  synthesis,  218 
Embryogenesis 

anterior-posterior  patterning,  525-526 

oncogenesis  in,  297-298 
Embryonic  induction  mechanisms,  vertebrates,  269-270 
Embryonic  stem  (ES)  cells 

anterior-posterior  patterning,  525-526 

developmental  genetics  and,  20,  383-384 

Drosophila  embryogenesis,  508-509 

mammalian  memory,  423-424 

molecular  studies  of  cystic  fibrosis,  25-26 
Emery-Dreifuss  muscular  dystrophy,  X  chromosome, 
431-432 

Endocrine  cells,  synaptic  vesicle  traffic  in,  105-106 


536 


Index 


Endoplasmic  reticulum  (ER) 

cytotoxic  T  cells,  37-38 

protein  translocons  and,  45-46 
Endothelial  cells 

blood  clotting  regulation,  344-346 

blood  vessel  function,  459-460 

in  inflammation  and  metastasis,  39-40 
Endothelial  leukocyte  adhesion  molecule- 1  (ELAM-1), 
39-40 

Endothelins,  blood  vessel  function,  459-460 
engrailed  {en)  genes 

DNA-protein  interactions,  309-311 

Drosophila  morphogenesis,  23-24 

embryogenesis,  507-508 

anterior- posterior  patterning,  525-526 
Enhancer  genes 

peripheral  nervous  system  development,  29-30 

viral  gene  regulation,  279-280 
Enhancer  of  split  code,  cell  fate  control,  13-14 
Entamoeba  histolytica,  variability  in,  515-516 
Enteropathogenic  bacteria 

molecular  biology,  487-488 

pathogenicity  studies,  357-359 
Enzyme  specificity,  chromosome  structure,  3-4 
Epidermal  growth  factor  (EGF) 

action  mechanisms  in,  327-328 

cell  fate  control,  13-14 

signal  transduction  of  receptor,  103-104 
Epidermolysis  bullosa  simplex  (EBS),  148 
Epigenetic  changes,  tumor-suppressor  genes,  132 
Epinephrine,  receptors  for,  251-252 
Epithelial  cells 

cystic  fibrosis  transmembrane  conductance  regulator 
(CFTR),  439-440 

papillomavirus  molecular  biology,  241-242 
Epitope,  immune  response,  xlvi 
Epstein-Barr  virus  (EBV) 

autoantibody  probes,  392 

complement  receptors,  185-186 

familial  hypertrophic  cardiomyopathy  (FHC),  363-364 

T  cell  function,  273-  274 
erbB  oncogene,  epidermal  growth  factor  (EGF),  104 
Escherichia  coli 

chromosome  structural  studies,  4 

enteropathic  (EPEC),  357-359 

genome  sequencing,  77 

protein-DNA  interaction,  394-395 

transposition  mechanism,  91-92 
E-selectin,  glycosyl transferase  molecular  genetics  and,  268 
ets  genes,  hematopoiesis  genetics,  486 
Eukaryotic  chromosomes,  three-dimensional  structure, 
360-362 

Excitation-contraction  (E-C)  coupling,  409 
Exocytosis,  synaptic  vesicle  mechanisms,  106 
Exons,  structure,  xxx 
Extracellular  matrix 

cell  structure  and,  xxxvi-xxxvii 

molecular  biology,  49 

neuron  development,  335-336 

Factor  VIII 

coagulation  genetics,  161-162 

X-linked  disorders,  163-164 
Familial  hypercholesterolemia  (FH) 

genetic  studies  of  cardiovascular  disease,  243-244 

somatic  gene  therapy,  449-451 
Familial  hypertrophic  cardiomyopathy  (FHC),  363-364 
Fasciclin,  growth  cone  guidance  and,  170 
FBPase,  intracellular  protein  transport,  353-354 
Fc  receptors,  cell  surface  recognition,  41-42 
Fibrinogen,  tyrosine  phosphorylation  in  platelets,  55-56 
Fibroblast  growth  factor  (EGF) 

blood  vessel  formation,  448 


cell  fate  control,  11-12 

embryonic  mouse  development,  gene  targeting,  63-64 

macromolecular  interaction,  278 

structural  studies,  387-388 

tyrosine  phosphorylation,  56 
Fibronectins,  cell  adhesion  and,  195-196 
Finding  the  Critical  Shapes,  xix 
FK-506  immunosuppressants,  90 

Flavin-adenine  dinucleotide  (FAD),  DNA  repair  and,  107 
Floor  plate-specific  genes,  211-212 
Fluorescence,  molecular  engineering,  426-428 
FMR  l  gene,  fragile  X  syndrome,  431-432 
Follicle-stimulating  hormone  (FSH),  gene  mapping,  142 
Foreskin  epithelial  cells,  papillomavirus  molecular  biology, 
241-242 

Fas  genes,  kidney  cell  growth  and  differentiation,  407-408 
Fragile  X  syndrome,  431-432 

gene  identification  and  correction,  69-70 
Free-energy  perturbation,  structural  biology,  58 
Free  R  value,  NMR  accuracy,  57-58 
Friend  leukemia  virus,  hematopoiesis,  485-486 
From  Egg  to  Adult,  xix 

Fructose  transport,  molecular  genetics  of  diabetes  and,  28 
Fucosyltransferase,  molecular  genetics,  267-268 
Fused  gene,  mammalian  development,  419-420 

G  protein 

adrenergic  receptors,  structure  and  function,  229-230 
basic  fibroblast  growth  factor  (bFGF) 

structural  studies,  387-388 
calcium-mediated  hormones,  129-130 
cellular  regulation,  375-376 

molecular  genetics,  nematode  development,  399-400 
nerve  cell  electrical  activity,  1-2 
neuronal  function,  lix 
olfaction  and,  331-332 
signal  transduction 

transmembrane  transduction,  347 

visual  systems,  193-194 
visual  pigments,  291-292 
G,  cyclins,  colony-stimulating  factor  1  receptor  (CSF-IR), 
371-372 

GABA  neurotransmitter,  synapse-like  microvesicles  (SLMVs), 
106 

GA-binding  protein  (GABP),  viral  gene  regulation,  279-280 

GAD,  stiff-man  syndrome  and,  106 

GAL4  protein,  macromolecular  assembly,  177-178 

gap  genes,  Drosophila  embryogenesis,  transcription  control, 

112-114 
Garrod,  Archibald,  xxxix 
GCN4  molecule 

structural  predictions,  58 

yeast  transcriptional  regulator,  macromolecular  assembly, 
177-178 

Gene  cloning,  research  trends  in,  xliii 
Gene  expression 

adenovirus  as  control  model,  369-370 

animal  cells,  293-294 

autoantibody  probes,  391-392 

B  cell  development,  379-380 

calcium  channel  molecular  studies,  529-530 

cell  cycle  and,  179-180 

cell  differentiation  and  activation,  12,  255-256 

cell  lineage  in  Caenorhabditis  elegans,  511-512 

chromosome  7  mapping,  531-532 

developmental  control,  173-174 

embryogenesis,  507-508 

eukaryotic,  59-60 

hemoglobin  .synthesis,  217-218 

hormonal  regulation,  75-76 

keratin,  146-148 

liver  regeneration,  411-412 

lymphocyte  development,  381-382 


537 


Index 


Gene  expression  {continued^ 

mammalian  development,  419-420 

mechanisms  of,  in  animal  cells,  421-422 

neural  development,  208 

pattern  regulation  in  Drosophila  and,  65-67 

protein-RNA/DNA  interaction,  393-395 

research  on,  xliii 

retroviral 

cellular  transcription,  285-286 
rggulation  of,  in  humans,  95-96 

steroid  hormones,  biosynthesis,  317-318 

viruses  and,  279-280 

visual  pigments,  291-292 
Gene  isolation,  Down  syndrome,  237-238 
Gene  mapping 

genetic  basis  of  hearing  loss  and,  1 19-120 

genetic  disease  and,  141-142 

protein  structure  and  folding,  139-140 

retroviral  replication  and,  54 
Gene  regulation.  See  Gene  expression 
Gene  segments,  receptor  structure  and,  xlvi-xlvii,  li 
Gene  structure,  xxx,  xxxii 

antibody  genes,  9-10 
Gene  targeting 

antibody  genes,  9-10 

embryonic  mouse  development,  63-64 
Gene  therapy.  See  also  Somatic  gene  therapy 

albinism  and,  307-308 

human  disease  and,  249-250 

inherited  disease,  69-70 

T  cell  receptor  functions,  435-436 
Gene  transfer,  blood  cell  formation,  446 
Gene  trapping,  developmental  genetics  and,  20 
Genetic  disease 

cancer  as,  247-248 

gene  identification  and  correction,  69-70 

gene  mapping  techniques,  81-82 

metabolic  disorders,  gene  therapy,  457-458 

molecular  studies,  25-26 

research  trends  in,  xlii-xliii 

skin,  keratin  gene  expression,  146-148 

X-linked  disorders,  163-164 
Genetic  engineering,  structural  biology  and,  xli 
Genetics,  research  programs  in,  xxxix-xlv 
Genomes 

DNA  structure  and,  xxxix,  Ixi 

fragments,  Down  syndrome,  237-238 

mapping  of,  xliv 

rearrangement,  antibody  gene  structure,  9-10 
sequencing  projects,  77 
structure,  xxx 
ges- 1  gene,  cell  lineage  in  Caenorhabditis  elegans, 
511-512 

Glanzmann's  thrombocytopenia,  55-56 
GLI  protein,  DNA  interactions,  310 
Glial  cells,  function,  Iv 

Globin  genes,  structural  determinants,  259-260 

a-Globin  gene  expression,  structural  determinants,  259-260 

Glomerular  basement  membrane,  hereditary  kidney  disease, 

333-334 
Glucagon,  gene  expression,  175 

Glucocorticoid-remediable  aldosteronism  (GRA),  244 
Glucocorticoids,  steroid  hormone  gene  expression,  317-318 
Glucose  transport,  molecular  genetics  of  diabetes  and,  28 
Glyceraldehyde-3-phosphate  dehydrogenase  (GAPDH), 

transcriptional  regulation,  7-8 
Glycogen  phosphorylase,  structural  studies,  388 
Glycolysis/gluconeogenesis,  IRE-A  DNA-binding  protein,  8 
Glycoproteins 

T  cell  recognition,  443 

viral  structure  and  replication,  intracellular  transport, 

245-246 

Glycosyltransferases,  molecular  genetics,  267-268 


Golgi  apparatus,  structure,  xxv 

Gonadotropin-releasing  hormone  (GnRH),  gene  mapping, 
142 

Goodpasture  syndrome,  molecular  basis  for,  333-334 
GP  Ilb-IIIa  receptor,  tyrosine  phosphorylation  in  platelets, 
55-56 

Graft-versus-host  disease  (GVHD),  coagulation  genetics,  162 

Grants  for  Science  Education,  xix 

gRNA,  RNA  editing,  377-378 

groEL  protein,  protein  folding,  189-190 

Growth  cone  guidance,  neuronal  recognition  and,  169-170 

Growth  factors.  See  also  specific  growth  factors 

cell  proliferation  and,  447-448 

genomic  response,  289-290 

insulin  mechanisms  and,  43-44 

signal  transduction,  517-518 

transcription  control,  469-470 
Growth-regulation  proteins,  blood  cell  formation,  446 
GTP,  transmembrane  signal  transduction,  347 
GTP-binding  protein 

neurotransmitter  storage  and  release  mechanisms,  203 

olfaction  and,  331-332 
Guanylyl  cyclase,  second  messengers  and  cell  regulation, 
151-152 

Gyrate  atrophy  (GA),  urea-tricarboxylic  acid  pathways, 
429-430 


HI  transcription  factor  (H1TF2),  179-180 
H-2  molecules,  major  histocompatibility  complex  and, 
153-154 

1119  gene,  mammalian  development,  419-420 
Haemophilus  influenzae,  host-pathogen  interactions, 

493-494 

hairy  gene,  pattern  regulation  in  Drosophila  and,  66 
Hearing  loss,  genetic  basis  for,  1 19-120 
Heart  function,  neuronal  excitability,  206 
Heat-shock  proteins  (HSPs) 

protein  60  (hsp60),  189-190 

stress  tolerance  and,  261-262 

T  cell  epitopes,  358 
heavy-chain  (a-chain)  segments,  li 
hedgehog  gene,  Drosophila  morphogenesis,  23-24 
Helix-helix  association  and  stability,  57-58 
Helix-loop-helix 

cell  fate  control,  14 

DNA  interactions,  311 

MyoD  gene  activation,  433-434 
Helper  T  cells.  See  also  T  cells 

immune  response  and,  xlvi 
Hemagglutinin,  influenza  virus,  157-158 
Hematopoiesis 

gene  expression,  381-382 

normal  and  leukemic  genetics,  485-486 
Hematopoietic  stem  cells 

blood  cell  formation,  445 

genetic  manipulation,  31-32 

lymphocyte  life  cycle,  437-438 

molecular  genetics  of,  305-306 
Hemoglobin 

genetic  disease  and,  xlii-xliii 

molecular  genetics  of  blood  cells  and,  305-306 

synthesis,  genetic  control,  217-218 
Hemophilia 

coagulation  genetics,  161-162 

X-linked  disorders,  163-164 
Heparin,  basic  fibroblast  growth  factor  (bFGF)  and,  387-388 
Hepatitis  B  virus  (HBV) 

replication  and  pathogenesis,  149-150 

RNA  replication  and  pathogenesis,  240 

surface  antigens,  315-316 
Hepatitis  delta  antigen  (HDAg),  RNA  replication  and 
pathogenesis,  240 


538 


Index 


Hepatitis  delta  virus  (HDV),  RNA  replication  and 

pathogenesis,  240 
Hepatocellular  carcinoma,  149-150 

Hepatocellular  transplantation,  gene  therapy  and,  249-250 
Hepatocyte  nuclear  factor  3,  eukaryotic  gene  regulation, 
59-60 

Heterochromatin,  position  effects,  183-184 
Heterogeneous  nuclear  ribonucleoproteins  (hnRNPs), 
117-118 

High-mobility  group  (HMG)  box,  7-8 
Hippocampus,  neuronal  excitability,  206 
Histidine,  visual  pigments,  291-292 
Histocompatibility  antigens  (H  antigens) 

genetics,  structure  and  function,  1 33- 1 34 

molecular  structure,  153-154 

T  cell  receptors,  41 
HLA-B27  glycoprotein,  443 
HNF-1|8  transcription  factor,  89-90 
Holoenzyme,  DNA  replication  and,  301-302 
Homeobox  genes 

Drosophila  melanogaster,  349-350 

embryonic  induction  mechanisms,  269-270 

motor  neuron  differentiation,  211-212 
Homeodomain  proteins 

Drosophila  morphogenesis,  23-24 

DNA  interactions,  crystal  structures,  309-31 1 
DNA-binding  specificity,  112-114 

genetic  regulatory  mechanisms,  89-90 

structural  biology  and,  Ixiii 
Homeotic  genes 

Drosophila  morphogenesis,  23-24 
genetic  control.  223-224 

limb  development  genetics  in  Drosophila,  79-80 
Homologous  chromosomes,  three-dimensional  structure, 
360-362 

Homologous  recombination,  research  trends,  xliv 
Hormone  response  elements  (HREs),  126 
Hormones 

calcium-mediated,  molecular  mechanisms,  129-130 
gene  expression  regulation,  75-76 
molecular  biology,  251-252 
Host-pathogen  interactions,  microbial  pathogenesis, 
493-494 

"Housekeeping"  genes,  complement  system,  16 
Hox  genes,  embryogenesis 

anterior- posterior  patterning,  525-526 

induction  mechanisms,  269-270 

mouse  development,  gene  targeting,  63-64 
hox- 1 .5  muvmt,  63-64 
hox- 1 .6  mutant,  63-64 
Human  foamy  virus  (HFV),  95-96 
Human  growth  hormone  (hGH),  256 
Human  immunodeficiency  virus  (HIV) 

AIDS  and.  Hi 

CD4  molecule  and,  178 

gene  expression,  cellular  transcription,  285-286 
protein-DNA  interaction,  394 
retroviral  replication,  53-54 

HIV- 1  and  HIV-2,  95-96 
surface  glycoproteins,  263-264 
T  cell  epitopes,  358 
trans-activation,  325-326 
Human  leukocyte  antigens  (HLA),  processing  mechanisms, 
93-94 

Human  myeloid  leukemias,  lymphocyte  growth  regulation, 
452-454 

Human  T  cell  leukemia  virus  (HTLV-I  and  HTLV-II) 

retroviral  gene  expression,  95-96 

surface  glycoproteins,  development  and  infection, 
263-264 
Humoral  immune  response 

CD4-bearing  T  lymphocytes,  5/- 52 

research  on,  xlvi 


hunchback  {hb)  gene,  Drosophila  embryogenesis 

segmentation,  113-114 

transcription  control,  113-114,  403 
Huntington  disease,  gene  mapping,  81-82 
Hyperlipidemia,  genetic  studies  of  cardiovascular  disease, 

243-244 
Hypertension 

blood  vessel  function,  endothelins  and,  459-460 

genetic  studies,  243-244 

steroid  hormone  gene  expression,  317-318 
Hypoxanthine  guanine  phosphoribosyltransferase  (HPRT), 
69 

IgA  deficiency  (IgA-D),  84 

Imaginal  wing  discs,  pattern  regulation  in  Drosophila  and, 
66-67 

Immediate-early  genes  (lEGs) 
growth  factors,  289-290 

kidney  cell  growth  and  differentiation,  407-408 
viral  gene  regulation,  279-280 
Immune  response 

CD4  and  CDS  molecules,  181-182 
cytokine  regulation  of  effector  functions,  513-514 
genetic  mechanisms  in  antibodies,  9-10,  171-172 
genetic  research  and,  136-138 

leprosy  and  tuberculosis  immunity  and  pathogenesis, 
47-48 

research  on,  xlvi-xlvii 
Immune  system 

cell  development,  83-84 

cell  surface  recognition,  41 

complement  system,  15-16 

research  programs  on,  xlvi 
Immunodeficiency 

gene  regulation  and,  325-326 

immune  system  development  and,  84 
Immunofluorescence  techniques 

gene  mapping,  81 

post-transcriptional  regulation,  118 
Immunoglobulin 

antigen  receptor  molecule  studies,  351-352 

B  cell  development,  gene  expression,  379-380 

developmental  control  of  gene  expression  and,  173-174 

heavy-chain  gene  control,  215-216 

K  light-chain  gene,  159-160 

lymphocyte  development,  415-416 
activation  and,  109-110 
neoplasia  and,  231-232 
Immunology,  research  programs  in,  xlvi-liv 
Immunoprecipitation,  synaptic  transmission,  356 
Immunosuppression,  lymphocyte  life  cycle,  437-438 
Imprinting,  cancer  as  genetic  disease  and,  248 
In  situ  hybridization,  eukaryotic  chromosomes,  360-362 
In  vivo  gene  expression,  cellular  and  retroviral  genes,  286 
Inactivation  process,  ion  channels,  5 
Inborn  errors  of  metabolism,  gyrate  atrophy,  429-430 
Inducible  cell  adhesion  molecule- 1 10  (INCAM-1 10),  39-40 
Infectious  disease 

protein  crystallography,  521-522 

protein  synthesis,  501-502 

RNA  replication,  509-510 
Inflammation,  vascular  endothelium  and,  39-40 
Influenza  virus 

glycoprotein  mechanisms,  443 

structure  and  replication,  245-246 
Inositol  1,4,5-trisphosphatc  (IP3),  257-258 
Insertional  mutation 

albinism  and,  307-308 

developmental  genetics  and,  20 
Insulin.  See  also  Insulin-responsive  element  A  (IRE-A) 

mechanisms  of  action,  43-44 

molecular  genetics  of  diabetes  and.  27-28 

production  mechanisms  for,  389-390 


539 


Index 


Insulin  {continued) 

receptor  studies,  390 
Insulin-like  growth  factors,  evolution  of,  390 
Insulin-responsive  DNA-binding  protein  (IRP-A),  7-8 
Insulin-responsive  element  A  (IRE-A),  7-8 
Insulinotropic  peptides,  gene  expression,  /  76 
int-1  gene,  embryonic  mouse  development,  63-64 
int-2  gene,  embryonic  mouse  development,  63-64 
INT-1  oncogene,  limb  development  genetics,  79-80 
Integrase,  structure,  53-54 
Integration,  retroviral  replication,  53-54 
Integrin 

cell  adhesion  and,  195-196 

neuron  development,  335-336 

Yersinia  pseudotuberculosis,  197-198 
Intercellular  communication,  signal  transduction,  J23 
Interferon-7 

cytokine  regulation  of  effector  functions,  513-514 

immunodeficiency  and,  325-326 
InterIeukin-1  (IL-1 ) 

B  cell  development,  379-380 

immune  response  and,  xlvi-xlvii 

inflammatory  cytokines,  73-74 
Interleukin-6  (IL-6),  73-74 
Interleukin-7  (IL-7),  84 
lnterleukin-10  (IL-10),  513-514 
Interleukins 

as  host  defense  in  cancer,  248 

lymphocyte  activation  and,  109-110 
Intermediate  filaments,  cytoskeleton  structure,  xxxv 
Intermediate  voltage  electron  microscopy  (IVEM) 

tomography,  3-4 
International  Research  Scholars  Program,  xix,  481 
Intracellular  adhesion  molecule-1  (ICAM-I),  26 
Intracellular  microorganisms 

cytotoxic  T  cells,  38 

molecular  genetics,  197-198 
Intracellular  parasitism,  microbial  pathogenesis,  493-494 
Intracellular  signals,  molecular  engineering,  428 
Intracellular  transpon 

proteins,  353-354 

viral  structure  and  replication,  245-246 
Introns,  structure,  .v.r.v 
Invariant  chains,  antigen  processing,  94 
Invasin,  Yersinia  pseudotuberculosis,  197-198 
Ion  channels 

function,  Iv,  Iviii 

leukocyte  homeostasis,  497-498 

mechanical  activation,  87-88 

molecular  mechanisms,  5-6,  463-464 

nerve  cell  electrical  activity,  1-2 

proteins,  functional  mechanisms,  281-282 

scorpion  toxin  receptor  genes,  519-520 

second  messenger  control,  373-374 

sperm  physiology,  491-492 

synaptic  transmission,  191-192 

viral  structure  and  replication,  245-246 
Islet  amyloid  polypeptide  (LAPP)  (amylin) 

biosynthesis,  390 

molecular  genetics  of  diabetes  and,  27-28 
Islets  of  Langerhans,  insulin  production,  389-390 

Junk  DNA,  xxx 

K  locus,  signal  transduction  in  B  cells,  159-160 
Keratin,  gene  expression,  146-148 
Keratinocyte-stimulating  factor  (KRF-1),  242 
Kidney 

cell  growth  and  differentiation,  407-408 

hereditary  disease  in,  333-334 
Kinase,  See  Protein  kinases 
Kinetics,  T  cell  recognition,  101-102 
Kinetoplast  DNA,  rr)'/)««osowe  genomes,  377-378 


"knocked-out"  genes,  265-266 
kreisler  genes,  19-20 

l(  1  Jcorkscrew  gene,  signal  transduction,  323 
l(  1  )pole  hole  gene,  signal  transduction,  323 
labial  gene,  morphogenesis,  224 

Lactate  dehydrogenase  (LDH),  inflammatory  cytokines, 
73-74 

Lactotropes,  prolactin  secretion,  489-490 

lacZ  gene,  anterior-posterior  patterning,  526 

Lamina  neurons,  visual  systems,  398 

Lamins,  eukaryotic  chromosomes,  362 

Large  dense-core  vesicles  (LDCVs),  105-106 

Laron  syndrome,  gene  mapping,  142 

Lateral  geniculate  nucleus  (LGN),  283-284 

a-Latrotoxin,  neuronal  secretory  pathways,  405-406 

Lectins,  carbohydrate  ligands  and,  40 

Legionella  pneumophila,  molecular  genetics,  197-198 

Leishmaniasis 

immune  evasion  and,  115-116 

kinetoplast  genome  targeting,  3  78 
Leprosy 

genetic  control  of,  202 

immunity  and  pathogenesis,  47-48 
Lesch-Nyhan  syndrome,  69 
Leucine  zipper  proteins 

growth  factors,  289-290 

MyoD  gene  activation,  433-434 

protein  folding,  225-226 

structural  biology,  58 
Leukemia 

immune  system  and,  liv 

lymphocyte  development,  231-232 
Leukemia  inhibitory  factor  (LIF),  31-32 

Leukocyte  adhesion  deficiency  (LAD),  genetic  disease  and,  26 
Leukocytes 

genetic  disease  and,  26 

ionic  homeostasis,  497-498 
Light-chain  segments,  gene  sequences  and,  li 
Linkage  analysis,  gene  mapping  and,  81-82 
Lipoprotein  lipase  (LPL),  243-244 
Listeria  monocytogenes,  cytotoxic  T  cells,  38 
Liver,  molecular  biology,  411-412 
Liver  regeneration  factor  (LRF-1),  412 
Long  terminal  repeat  (LTR) 

developmental  genetics  and,  20 

human  immunodeficiency  virus  (HIV),  325-326 
Long-term  potentiation,  mammalian  memory,  423-424,  425 
Low-density  lipoprotein  (LDL),  449-451 
Lowe's  syndrome,  molecular  genetics,  295-296 
LRP  transmembrane  phosphatase,  413-414 
Luciferase  reporter  mycobacteriophages  (LRMs),  200-202 
Luteinizing  hormone  (LH),  gene  mapping,  142 
Lyme  disease,  immune  tolerance,  136-138 
Lymphocyte  function  antigen  1  (LFA-1),  102 
Lymphocyte  receptors,  immune  response,  xlvi 
Lymphocytes 

activation  mechanisms,  109-110 

development 

antibody  genes,  9-10 
gene  expression,  381-382 

life  cycle,  437-438 

molecular  regulation,  415-416 

normal  and  abnormal  growth  regulation,  452-454 

signal  transduction,  321-322 
Lymphoid  enhancer-binding  factor  1  (LEF-1),  174 
Lymphoid-specific  regulation,  173-174 
Lymphoma 

immune  system  and,  liv 

molecular  genetics,  231-232 
Lysosomal  hydrolase,  sorting,  121-122 
Lysosome  structure,  xxxi> 
a-Lytic  protease,  chromosome  structure,  3-4 


540 


Index 


M  channel,  nerve  cell  electrical  activity,  1-2 

M2  protein,  viral  structure  and  replication,  245-246 

M3  molecule,  histocompatibility  antigen  research,  133-134 

Macromolecules 

recognition,  protein  folding,  225-226 
structural  basis  of  interaction,  277-278 
structural  studies,  Ixi,  177-178 
three-dimensional  structures,  107-108 
Macrophages,  immune  system,  xlvi 
Major  histocompatibility  complex  (MHC) 
antigen  processing,  93-94 
B  cell  development,  299-300 
CD4  and  CDS  molecules,  182 
class  I  molecules 

antigen  processing,  93-94 
histocompatibility  antigen  research,  133-134 
class  II  molecules 

antigen  processing,  93-94 
immune  tolerance,  136-138 
macromolecular  assembly,  /  78 
cytotoxic  T  cells,  37-38 
immune  response  and,  li-lii 
mammalian  memory,  423-424 
molecular  genetics  of,  153-154 
surface  glycoproteins,  263-264 
T  cells 

activation,  209-210 
development  biology,  265-266 
receptor,  435-436 
recognition,  101-102 
Mammalian  development 
disease  and,  313-314,  315-316 
gene  regulation,  419-420 
genetics,  19-20 

X  and  Y  chromosomes,  312-314 
MAP  kinases,  tyrosine  phosphorylation,  56 
MARCKS  (myristoylated  alanine-rich  C-kinase  substrates), 
43-44 

Marfan  syndrome,  gene  mapping,  142 
Master  regulatory  genes,  207-208 
Maternal  genes,  transcription  control,  112-114 
Maternally  transmitted  antigen  (Mta),  133-134 
Maturation-promoting  factor  (MPF),  271-272 
MCAT  protein,  retroviral  infections,  97-98 
mdr  gene  family,  multidrug  resistance,  499-500 
Mechanical  adjustment  hypothesis,  87-88 
Mechanotransduction,  sensory  cells,  473 
Medial  superior  temporal  area  (MST),  527 
Medical  Research  Organization  (MRO),  xix 
Membrane  proteins 

cofactor  protein,  complement  system,  16 

energy  transduction,  213-214 
Membrane-bound  growth  factors,  276 
Memory 

cell  biological  studies,  219-220 

T  cells  and,  423-424 
in  vitro  generation,  102 
Mendelian  genetics,  genetic  structure  and,  xxxix 
Mental  retardation,  X-linked  disease,  295-296 
Messenger  RNA  (mRNA) 

decoding  code,  155-156 

gene  expression  and,  117-118 

a-globin  gene  expression,  259-260 

splicing  studies,  337-338 

transcription  and,  xxx,  xxxiii 
Metamorphosis,  molecular  regulation,  417-418 
Metastasis 

tissue-type  plasminogen  activator  (t-PA),  157-158 
vascular  endothelium  and,  39-40 
Methyl-malonyl  CoA  mutase  (MCM),  gene  therapy  with, 
249-250 

Microfilaments,  cytoskeleton  structure,  xxxv 
/Sj-Microglobulin,  polymorphism,  134 


Microtubules 

cytoskeleton  structure,  xxxv 

linkage  analysis  and,  81 
Missense  mutations,  familial  hypertrophic  cardiomyopathy 

(FHC),  363-364 
Mitochondria 

histocompatibility  antigen  research,  134 

matrix,  protein  folding,  189-190 

structure,  xxxv 

Try panosome  genome,  378 
Mitosis,  cell  cycle  control,  21-22 
Molecular  biology,  research  trends  in,  xxx 
Molecular  engineering,  cell  structure  and  neurobiology, 
426-428 

Molecular  fractionation,  synaptic  transmission,  355-356 
Molecular  recognition 

eukaryotic  gene  regulation,  59-60 

structural  biology  and,  Ixiii 
Moloney  murine  leukemia  virus  (MoMuLV) 

developmental  genetics  and,  20 

hematopoietic  stem  cell  regulation,  31-32 

retroviral  infections,  97-98 
Monoclonal  antibodies,  scorpion  toxin  receptor  genes, 
519-520 

Monocytes,  HIV  gene  expression,  285-286 

Morphogen  gradients,  Drosophila  body  patterns,  403-404 

Morphogenesis,  genetic  control,  223-224 

Motor  neurons,  differentiation,  211-212 

Mouse  development 

antibody  genes,  9-10 

gene  targeting  and,  63-64 

genetics,  19-20 
Multiplex  sequencing,  genomes,  77 
Muscle  development  and  function 

molecular  analysis,  287-288 

MyoD  gene  family,  433-434 
Muscular  dysgenesis  (rndg)  gene,  409 

Muscular  dystrophies.  See  also  specific  muscular  dystrophies 

gene  mapping,  141 

molecular  genetics,  233-234 

neuronal  function,  Ix 
Mutations 

detection,  gene  mapping,  141-142 

gene  cloning  and,  xliii 

ion  channels,  molecular  mechanisms,  5 
Myasthenia  gravis,  neuronal  function,  Iv 
Mycobacteria 

genetic  control  of  disease,  200-202 

leprosy  and  tuberculosis  immunity  and  pathogenesis, 
4  7-48 

Myeloid  cells,  growth  control,  371-372 

Myoblasts,  as  recombinant  protein  delivery  system,  256 

Myocyte-specific  enhancer-binding  factor  2  (MEF2), 

287-288 
MyoD  gene  family 

muscle  cell  lineage,  433-434 

transcriptional  regulation,  287-288 
Myosin  heavy  chain  (MHC) 

familial  hypertrophic  cardiomyopathy  (FHC),  363-364 

transcriptional  regulation,  287-288 

nana  gene,  Drosophila  development  and,  253-254 
Nectins,  cell  adhesion  and,  195-196 
Nematode  development 

genetic  control,  187-188 

molecular  genetics,  399-400 
Nerve  cell  structure,  liv 
Nerve  grov^^h  factor  (NGF) 

cell  fate  control,  11-12 

transcription  control,  469-470 

tyrosine  phosphorylation,  56 
Nerve  impulses,  structure  and  function,  Iv 

541 


Index 


Nervous  system 

biological  clock  system,  465-466 

cell  fate  in,  13-14 
Neural  crest  cells,  cell  fate  control,  11-12 
Neural  networks,  sensory  representations,  visual  system, 
367-368 

Neuroendocrine  system,  molecular  mechanisms,  339-340 
Neurogenesis 

cell  pattern  control,  211-212 

pattern  regulation  in  Drosophila  and  proneural  genes, 

66-67 

visual  systems,  341-342,  396-398 
Neuroimmunology,  molecular  studies,  319-320 
Neuromodulation,  caffeine  consumption  and,  401-402 
neuromusculin  (nrm)  gene,  29-30 
Neuron  doctrine,  liv 
Neuronal  development 

calcium  channel  molecular  studies,  529-530 

cell  fate  control,  11-12 

extracellular  matrix,  335-336 
Neuronal  excitability,  voltage-sensitive  potassium  channels, 

205-206 
Neuronal  recognition 

growth  cone  guidance  and,  169-170 

visual  systems,  396-398 
Neuronal  survival,  neuron  development,  335-336 
Neurons 

differentiation,  olfaction  and,  332 

molecular  engineering,  426-428 

secretory  pathways,  405-406 

structure  and  function,  liv 

synaptic  vesicle  traffic  in,  105-106 
Neuropeptides,  synaptic  transmission,  355-356 
Neurophysiology,  visual  systems,  283-284 
Neuroscience,  research  programs  in,  liv-lx 
Neurotransmitters 

function,  Iv 

neuronal  secretory  pathways,  405-406 

receptors,  synaptic  transmission,  192 

storage  and  release  mechanisms,  203 

synaptic  vesicle  proteins,  105-106 

transcription  control,  469-470 
Neurotrophic  factors,  cell  fate  control,  11-12 
NF-kB  protein,  signal  transduction  in  B  cells,  159-160 
Nitric  oxide,  retroviral  infections,  97-98 
Non-self  lymphocytes,  immune  response,  xlvi 
nos  gene,  translation  repression  of  bb,  403-404 
Notch  gene 

biological  clock  system,  465-466 

cell  fate  control,  13-14 

viral  gene  regulation,  280 
Nuclear  envelope,  eukaryotic  chromosomes,  360-362 
Nuclear  factor 

of  activated  T  cells  (NFAT),  90 

gene  expression  regulation,  75-76 
Nuclear  lamina,  protein  translocons  and,  45-46 
Nuclear  magnetic  resonance  (NMR) 

accuracy  of  crystal  and  solution,  57 

chromosome  structure,  3-4 

extracellular  matrix  structure,  49 

protein  structure  and  folding,  139-140 

structural  biology  and,  Ixiii 
Nuclear  pore  complexes  (NPC) 

protein  translocons  and,  45-46 

structure  and  function,  99-100 
Nuclear  structures,  xxx 

post-transcriptional  regulation,  117-118 
Nucleoporins,  nuclear  pore  complex  (NPC),  99-100 
NUPl  gene,  nuclear  pore  complex  (NPC),  99-100 

Obesity 

IRE-A  DNA-binding  protein,  8 
molecular  biology  and,  143-144 


Oct-2  protein,  B  cell  development,  379-380 
Olfaction 

molecular  approaches  to,  331-332 

molecular  biology,  17-18 

second-messenger  ion  channel  regulation,  373-374 
Oligonucleotide,  calcium  channel  molecular  studies, 

529-530 
Oligosaccharide 

epitopes,  protein  structure  and  function,  329-330 

glycosyltransferase  molecular  genetics  and,  268 
Onchocerciasis,  immune  evasion  and,  116 
Oncogenes.  See  also  specific  genes,  e.g.,  src  gene 

adenovirus  transcriptional  regulation,  293-294 

cell  structure  and,  xxxviii 

colony-stimulating  factor  1  receptor  (CSF-IR),  371-372 
early  embryogenesis  and,  297-298 
gene  regulation  mechanisms  and,  421-422 
lymphocyte  development,  molecular  genetics,  231-232 
mammalian  development  studies  and,  315-316 
Optical/electron  microscopy,  eukaryotic  chromosomes, 

360-362 
Organelles,  structure,  xxxv 
Organogenesis,  transcription  control,  114 
Ornithine  transcarbamylase  (OTC)  deficiency,  70 
Ornithine-5-aminotransferase  (OAT),  429-430 
Outer  membrane  proteins  (OMP),  487-488 
Outer  surface  protein  (OspA  &  B),  138 
Oxidative  stress,  redox  proteins  and,  236 
Ox}'R  protein,  oxidative  stress  mechanism,  236 
Oxytricba  nova.  DNA  molecular  structure,  72 


p53  gene 

hematopoiesis,  486 

transcription  regulation  mechanisms,  496 
paired  ^ene,  Drosopbila  embryogenesis,  114 
Pair-rule  segmentation  genes,  65-67 
Pancreatic  /3-cell,  diabetes  and,  27-28 
Papillomaviruses,  human  (HPV),  molecular  biology  of, 
241-242 

Paramyxovirus,  structure  and  replication,  245-246 

Parasites,  immune  evasion  by,  115-116 

patcbed  gene,  Drosopbila  morphogenesis,  23-24 

Pathway  recognition,  growth  cone  guidance  and,  169-170 

Pattern  formation 

Drosopbila  body  patterns,  morphogen  gradients,  403-404 
limb  development  genetics  in  Drosopbila,  79-80 

fax  genes,  embryonic  induction  mechanisms,  269-270 

PC  12  cells,  tyrosine  phosphorylation,  56 

Peptides 

neurotoxins,  ion  channels,  281-282 

structure,  protein  folding,  225-226 
Peptidyl-tRNA  hydrolase  (Pth),  protein  synthesis,  501-502 
per  gene 

biological  clock  system,  465-466 

mRNA  processing  and  behavior,  337-338 
Perception,  neurophysiology,  283-284 
Peripheral  nervous  system 

Drosopbila  development,  29-30 

growth  cone  guidance  and,  169-170 

olfaction  and,  17-18 
Peyer's  patches,  gene  regulation  of,  437-438 
P-glycoprotein  (P-gp),  multidrug  resistance,  499-500 
pH  regulation,  leukocyte  homeostasis,  498 
Phage  X,  protein  synthesis,  501-502 
Phagocytes,  Legionella  pnuemopbila  growth  in,  198 
Phase  determination,  structural  biology  and,  Ixi 
Phenotype,  neuronal  function,  lix 

Phenylalanine  hydroxylase  (PAH),  somatic  gene  therapy, 
457-458 

Phenylketonuria  (PKU),  somatic  gene  therapy,  457-458 
Phosphatidic  acid  (PA),  calcium-mediated  hormones,  130 
Phosphatidylcholine  (PC),  calcium-mediated  hormones,  130 


542 


Index 


Phosphatidylinositol  (PI) 
metabolism,  327-3-28 

phospholipids  and  cell  regulation,  165-166 
Phosphatidylinositol  3-kinase  (PI3-kinase),  122 
Phosphatidylinositol  4,5-bisphosphate  (PIP2),  129-130 
Phosphatidylinositol  monophosphate  phosphatase,  327-328 
Phospholipase  A^,  cellular  regulation,  375-376 
Phospholipase  C,  calcium-mediated  hormones,  129-130 
Phospholipids,  cell  regulation,  165-166 
Phosphorylation.  See  also  Tyrosine  phosphorylation 

epidermal  growth  factor  (EGF),  103-104 

RNA  polymerase  II  structure  and  function,  85-86 

signal  transduction,  517-518 
Phosphotyrosine  protein  phosphatase,  synaptic  transmission, 
192 

Photosynthesis,  molecular  genetics,  503-505 
Phototransduction,  visual  systems,  193-194 
piebald  gene,  mammalian  development,  419-420 
Pilin,  protein  crystallography,  521-522 
Pit-1  gene,  neuroendocrine  system,  339-340 
Plasma  cells,  immune  response  and,  xlvi 
Plasma  membranes 

ankyrins  in,  33-34 

protein  translocons  and,  45-46 
Plasminogen  activator  inhibitor- 1  (PAI-1),  161-162 
Platelet-derived  growth  factor  (PDGF) 

Drosophila  melanogaster  signal  transduction,  323 

genomic  response,  289-290 
Platelets,  tyrosine  phosphorylation,  55-56 
Pole  plasm,  Drosophila  development  and,  253-254 
Poliovirus,  genetics,  227-228 
Polyadenylation,  molecular  mechanisms,  293-294 
Polymerase  chain  reaction  (PGR) 

gene  mapping,  xliv 

genetic  basis  of  hearing  loss  and,  119-120 

hematopoietic  stem  cell  regulation,  32 

major  histocompatibility  complex,  153-154 

viral  genetics,  227-228 
Polypeptide  growth  factors,  action  mechanisms  in,  327-328 
Polypeptide  hormones,  gene  regulation,  175-176 
Ponder-Richards  rotamers,  chromosome  structure,  4 
Population  dynamics,  PKU  gene  distribution,  457 
Positional  cloning 

gene  mapping  and,  81-82 

obesity  and  diabetes,  143-144 
Potassium  channels 

cystic  fibrosis  transmembrane  conductance  regulator 
(CFTR),  440 

functional  mechanisms,  281-282 

insulin  production,  390 

molecular  mechanisms,  5-6 

nerve  cell  electrical  activity,  1-2 

neuronal  excitability,  205-206 

scorpion  toxin  receptor  genes,  519-520 
POU  domain,  neuroendocrine  system,  339-340 
Prader-Willi  syndrome,  141 
Pre-B  cell,  gene  expression  and,  175 
Pre-messenger  RNA,  splicing  mechanisms,  337-338 
Prenatal  diagnosis,  genetic  disease,  457-458 
Prepattern  genes,  neural  development,  207-208 
proboscipedia  gene,  morphogenesis,  223-224 
Prohormone-converting  enzymes,  389-390 
Prokaryotic  repressors,  protein-DNA  interactions,  309-3 1 1 
Prolactin  secretion,  functional  heterogeneity,  489-490 
Promoters 

peripheral  nervous  system  development,  29-30 
trapping,  developmental  genetics,  383-384 
Proncural  genes,  pattern  regulation  in  Drosophila  and, 
66-67 

Protein  kinase  A  (PKA),  gene  expression  and  cell  cycle, 
179-180 

Protein  kinase  C,  sensory  transduction,  473 


Protein  kinases 

carboxyl-terminal  domain  (GTD),  85-86 
cell  cycle  control,  21-22 
cell  division  and,  xxxvi 

epidermal  growth  factor  (EGF)  desensitization,  328 

insulin  mechanisms  and,  43-44 

protein  sorting  and,  1 22 

signal  transduction,  dual-specificity,  518 

synaptic  transmission,  192 

tyrosine  phosphorylation  in  platelets,  55-56 
Protein-DNA  interaction.  See  also  DNA-binding  proteins 

cAMP-dependent,  gene  expression,  175-176 

Drosophila  embryogenesis,  114 

macromolecules,  278 

RNA  molecular  structure,  72 
Protein-protein  interactions,  signal  transduction,  517-518 
Proteins 

design,  protein  folding,  225-226 
folding 

chromosome  structure,  4 

genetic  research  and,  139-140 

histocompatibility  antigen  research,  133-134 

in  vivo  research,  189-190 

macromolecular  interaction,  276-278 
intracellular  transport,  353-354 
ion  channels,  functional  mechanisms,  281-282 
macromolecular  interaction,  276-278 
phosphorylation 

cell  cycle  control  and,  27 1-272 

synaptic  transmission,  191-192 
regulatory  proteins,  387-388 
sorting,  molecular  genetics,  121-122 
structure/function  studies,  xxx,  xxxiv,  Ixi,  329-330 
synthesis,  viral  infection,  501-502 
as  transcriptional  factor,  xxxv 
translocons,  nuclear  organelles  and,  45-46 
Protein-tyrosine  kinase  (PTK) 

lymphocyte  signaling,  molecular  basis  for,  321-322 
T  cell  receptor  functions,  435-436 
Protein-tyrosine  phosphatases,  lymphocyte  activation, 
413-414 

Proton  pumps,  leukocyte  homeostasis,  498 

Proto-oncogenes,  embryonic  mouse  development,  63-64 

P-selectin  gene,  genetic  disease  and,  26 

Pseudoknots,  mRNA  decoding,  1 56 

Pseudomonas  aeruginosa,  protein  crystallography, 

521-522 
pumilio  gene,  253-254 

Q-beta  replicase,  RNA  replication,  509-510 

R  values,  NMR  accuracy,  57 

R7  cells,  retinal  cell-cell  interactions,  471-472 

RAG  genes 

antibody  genes,  9-10 

lymphocyte  differentiation,  110 
RAG-1  protein,  351-352 
RAG-2  protein,  351-352 
ras  protein 

tyrosine  phosphorylation,  56 

visual  system  development,  342 
reck  protein,  protein-DNA  interaction,  394-395 
Receptor-ligand  interaction 

chromosome  structural  studies,  4 

macromolecules,  278 
Receptors.  See  also  specific  receptors,  e.g.,  T  cell  receptor 

adrenergic  receptors,  biosynthesis,  229-230 

antigen-specific,  351-352 

cell  structure  and,  xxxvi 

gene  segments  and,  xlvi,  li 

macromolecular  assembly,  178 

olfaction  and,  17-18 

periplasmic,  active  transport  and  chemotaxis,  330 


Index 


Receptors  {continued) 

retroviral  infections,  97-98 

transforming  growth  factor  (TGF),  103-104 
Recombination 

antigen  receptor  molecule  studies,  351-352 

DNA  technology,  xxxix,  xliii 

Down  syndrome  and,  237-238 

genetic  disease  research,  25-26 

protein-DNA  interaction,  394 

RNA  viruses,  239-240 

transposition  mechanism,  91-92 
Recoverin 

signal  transduction,  visual  systems,  193-194 

urea/tricarboxylic  acid  cycles,  430 
Redox  proteins,  transcriptional  response,  236 
Regenerating  liver  inhibitory  factor  {RL/IF-1),  411-412 
Regulation,  molecular  mechanisms,  339-340 
Regulatory  molecules 

cell  fate  control,  12 

structural  studies,  387-388 
Replication 

cell  cycle  control,  21-22 

in  DNA,  301-302 

protein-DNA  interaction,  393-395 

viral  pathogenesis  and,  149-150 
Resolvase,  protein-DNA  interaction,  394-395 
Restriction  fragment  length  polymorphism  (RFLP) 

cerebellum  development,  180 

gene  mapping,  xliv 

neurological  disorders,  Ix 

obesity  and  diabetes,  143-144 

tuberculosis  infections  and,  200-202 

Wilms'  tumor  genes,  131 
Retinal  degeneration,  molecular  genetics,  295-296 
Retinal  rods  and  cones,  phototransduction  mechanism, 
461-462 

Retinitis  pigmentosa,  gene  expression  in  visual  pigments, 
292 

Retinoblastoma,  adenovirus  transcriptional  regulation, 

293-294 

Retinoic  acid  (RA),  embryogenesis,  525-526 

Retinoic  acid  receptors  (RARs),  response  pathway,  125-127 

Retinoid  X  receptors  (RXRs),  response  pathway,  125-127 

Retrovirus 

correction  of  methyl-malonyl  CoA  mutase  (MCM), 
249-250 

developmental  genetics  and,  20 

gene  expression,  95-96 

cellular  transcription,  285-286 

hematopoietic  stem  cell  regulation,  31-32 

infection  mechanisms,  97-98 

replication  and,  53-54 
Rev  protein,  retroviral  gene  expression,  95-96 
Rev  response  elements  (RRE),  96 
"Reverse  genetics."  See  also  Positional  cloning 

genetic  basis  of  hearing  loss  and,  119-120 
Rheumatoid  arthritis  (RA) 

immune  tolerance,  136-138 

T  cell  function  in,  273-274 
Rhodopsin,  transmembrane  signal  transduction,  347 
Rhombomeres,  developmental  genetics  and,  19-20 
Ribosomal  jumping,  mRNA  decoding,  155-156 
Ribosomal  pausing,  tRNA  molecules,  455 
Ribosome  frameshifting,  translational  regulation,  455 
Ribozymes,  RNA  replication,  509-510 
Ring  complexes,  protein  folding,  190 

RMA-S  mutant  cells,  histocompatibility  antigen  research,  134 
RNA 

catalysis,  71-72 

chromosome  end  structure,  71-72 
editing,  Trypanosome  genomes,  377-378 
recombination,  viral  genetics,  227-228 
replication  in  infectious  disease,  509-510 


synthesis,  RNA  viruses,  239-240 
viruses 

genetics,  227-228 

replication  and  pathogenesis,  239-240 
RNA  polymerase,  structural  biology  and,  Ixi 
RNA  polymerase  II 

post-transcriptional  regulation,  118 

structure  and  function,  85-86 

transcription  regulation  mechanisms,  495-496 
RNA-binding  domain  (RBD),  post-transcriptional  regulation, 

117-118 
RNA-protein  interaction 

post-transcriptional  regulation,  117-118 

structural  biology  and,  Ixi,  393-395 
Rotavirus  diarrhea,  control  epidemiology,  483-484 
Rough  endoplasmic  reticulum  (RER),  xxxv 
RPS4X,  314 
RPS4Y,  314 
RuBisCO 

NMR  accuracy,  57 

photosynthesis  genetics,  503-505 


Saccharomyces  cerevisiae 
DNA  cloning,  303-304 
heat-shock  proteins  (HSPs),  261-262 
lysosomal  hydrolase  sorting,  121 
nuclear  pore  complex  (NPC),  99-100 
research  with,  xliv 

RNA  polymerase  II  structure  and  function,  85-86 

secretory  process,  353-354 
Salmonella  typhi.  See  Typhoid  fever 
Sarcomere,  muscle  development  and  function,  287-288 
Scleroderma,  autoantibody  probes,  392 
Second  messenger  systems 

cell  regulation,  151-152 

cell  structure  and,  xxxvi 

ion  channel  regulation,  373-374 

neuronal  function,  lix 

caffeine  consumption,  401-402 

synaptic  transmission,  192 
Secretory  process 

intracellular  transport,  353-354 

neuronal  pathways,  405-406 

vesicle  transport,  354 
Segment  polarity  genes 

cell-cell  interactions,  298 

Drosophila  morphogenesis,  23-24 
Segmentation 

developmental  genetics  and,  19-20 

Drosophila  embryogenesis,  113-114 

Drosophila  morphogenesis,  23-24 
Selectins 

cell  adhesion  and,  196 

in  inflammation  and  metastasis,  39-40 
Self  antigens,  immune  response  and,  li 
Self  lymphocytes,  immune  response,  xlvi 
Self-reactive  T  cells,  136-138 
Self-tolerance 

biology  of  T  cell  development,  265-266 

mechanisms,  171-172 
Selfish  (junk)  DNA,  xxx 
Sensory  perception,  olfaction  and,  17-18 
Sensory-motor  interface,  representation-transformation, 

523-524 
Sequencing 

gene  cloning  and,  xliii 

recent  technology  trends,  77 
Serotonin,  molecular  engineering,  426-428 
seven-in-absentia  (sina)  gene,  342 
sevenless  gene,  visual  system  development,  341-342 
Severe  combined  immunodeficiency  (SCID) 

antibody  gene  structure,  9-10 


544 


Index 


blood  cell  formation,  446 
lymphocyte  growth  regulation,  452-454 
Sex  combs  reduced  gene,  223-224 

Sex  determination,  X  and  Y  chromosomes  and,  312-314 
Sex  pheromones,  chemical  communication,  257-258 
Sexual  dimorphism,  IRE-A  DNA-binding  protein,  8 
SH2  proteins,  signal  transduction,  517-518 
Shaker  gene 

ion  channels,  molecular  mechanisms,  5-6 

neuronal  excitability,  205-206 
Shiga  toxins,  protein  crystallography,  521-522 
Shock,  tumor  necrosis  factor  (TNF)  and,  35-36 
Sialyl-Lewis  X  determinant,  carbohydrate  ligands  and,  40 
Sickle  cell  anemia,  prenatal  diagnosis,  217-218 
Signal  recognition  factor  (SRF),  protein  translocons  and, 
45-46 

Signal  recognition  particle  (SRP) 

antigen  processing,  93-94 

protein  translocons  and,  45-46 

translational  regulation,  455 
Signal  transduction 

Bcell  pathways,  159-160 

CD4  and  CDS  molecules,  181-182 

colony-stimulating  factor  (CSF)  1  receptor  (CSF-IR),  370 

Drosophila  melanogaster,  323 

epidermal  growth  factor  (EGF)  receptor,  103-104 

IRE-A  DNA-binding  protein,  8 

lymphocytes,  molecular  basis  for,  321-322 

microbial  pathogenesis,  493-494 

olfaction  and,  331-332 

ion  channel  regulation,  373-374 
phosphorylation  and  protein-protein  interactions, 
517-518 

transmembrane,  G  protein-coupled  receptors,  347 

visual  system,  193-194 
development,  341-342 
Signaling  systems,  neural  development,  208 
Simian  virus  40  (SV40) 

macromolecular  assembly,  178 

T  antigen,  immune  tolerance,  138 
Simple  sequence  repeats  (SSRs) 

diabetes  and,  144 

genetic  basis  of  hearing  loss  and,  1 19- 120 
Site-directed  mutagenesis,  214 
situs  inversus,  gene  therapy  and,  308 
Skin,  keratin  expression,  146-148 
Small  eye  (Sey)  gene,  270 
Small  nuclear  ribonucleoproteins  (snRNPs) 

gene  expression,  391-392 

post-transcriptional  regulation,  117-118 

RNA  processing  and  behavior,  337-338 
Smell.  See  Olfaction 
Sodium  channel 

lactotropes,  489-490 

molecular  mechanisms,  5-6 

nerve  cell  electrical  activity,  1-2 

scorpion  toxin  receptor  genes,  519-520 
Somatic  gene  therapy 

blood  cell  formation,  446 

genetic  disease  and,  449-451 

metabolic  disorders,  457-458 
Somatic-motor  cortices,  representation-transformation, 

523-524 
Somatostatin 

gene  expression,  175 

molecular  genetics  of  diabetes  and,  27-28 
Spectrin,  membrane  skeleton  proteins,  33-34 
S-peptide  mutants,  structural  biology,  58 
Sperm  physiology,  ion  channels,  491-492 
Spermatozoa,  second  messengers  and  cell  regulation, 
151-152 

Spherocytosis,  hemoglobin  synthesis,  218 
Spinal  muscular  atrophy  (SMA),  233-234 


Spleen  focus-forming  virus  (SFFV),  486 
Split  genes,  RNA  editing  and,  378 
src  gene  family,  235-236 

protein-tyrosine  phosphatases,  413-414 
SRY  genes 

insulin-responsive  element  A  (IRE-A),  8 

X  and  Y  chromosomes,  312-314 
Steel  gene 

blood  cell  formation,  445-446 

lymphocyte  growth  regulation,  454 

normal  and  leukemic  hematopoiesis,  485-486 

rotavirus  diarrhea,  484 
Stem-loop  structures,  mRNA  decoding,  156 
Steroid  hormones 

gene  expression  in,  317-318 

receptor  genetics,  125-127 
Stiff-man  syndrome,  diabetes  and,  106 
Stress  tolerance,  heat-shock  proteins  (HSPs),  261-262 
Structural  biology,  research  programs  in,  Ixi-lxiii 
Structure-function  studies 

calcium  channels,  409 

egg  cells,  385-386 

energy-transducing  membrane  proteins,  213-214 

neuronal  excitability,  205-206 

T  cell  receptor,  435-436 

visual  pigments,  291-292 
Substance  P,  molecular  neuroimmunology,  319-320 
Substrate  specificity,  chromosome  structure,  3-4 
Sucrose  6-phosphate  synthase  (SPS) ,  504 
Superantigen 

T  cell  function  in  health  and  disease  and,  273-274 

T  cell  repertoire,  221-222 
Superfragment  cloning,  tumor-suppressor  genes,  131 
Surface  membranes,  cell  structure  and,  xxxvi 
SV40.  See  Simian  virus  40 

"Switch  kinases,"  tyrosine  phosphorylation,  56 
Sympathetic  neurons,  cell  fate  control,  11-12 
Synapse-like  microvesicles  (SLMVs),  105-106 
Synapses,  function,  Iv-lvii 
Synaptic  plasticity,  219-220 
Synaptic  potential,  Iv 
Synaptic  transmission 

development  and  function,  355-356 

molecular  mechanisms,  191-192 
Synaptic  vesicles  (SVs) 

neuromodulation,  caffeine  consumption,  401-402 

neuronal  secretory  pathways,  405-406 

traffic  in  neurons  and  endocrine  cells,  105-106 
Synchrotron  x-ray  sources,  Ixi 

Synthetase-tRNA  complex,  protein-RNA  interaction, 

393-395 

Synthetic  peptides,  scorpion  toxin  receptor  genes,  519-520 
Systemic  lupus  erythematosus  (SLE),  391-392 


T  cell  receptor  (TCR) 

antigen  receptors,  101-102 

lymphocyte  activation,  413-414 

molecule  studies,  351-352 
immune  system  development  and,  138 

/3  diversity,  83-84 
mammalian  memory,  423-424 
structure-function  studies,  435-436 
superantigens  and,  221-222 

suppression,  leprosy  and  tuberculosis  immunity  and 
pathogenesis,  47-48 
T  cell  receptor  a  enhancer 

gene  expression  and,  175,  255-256 
IRE-A  DNA-binding  protein,  8 

lymphocyte  development  and  neoplasia,  231-232 
molecular  structure,  41 
V/3  portions,  221-222 
T  cell-specific  DNA-binding  protein,  175 


545 


Index 


T  cells.  See  also  Cytotoxic  T  cells;  Helper  T  cells 
activation  and  differentiation,  89-90,  209-210 

cell  surface  molecule  regulation,  436 
afi,  mammalian  memory,  423-424 
biology  of  development,  265-266 
CD4-bearing,  51-52 
development,  9-10 

epitopes.  Salmonella  typhi  attenuated  strains  as,  358 
76,  mammalian  memory,  423-424 
gene.sequences  and,  li 
HIV  gene  expression,  285-286 
immune  system  development  and,  xlvi,  83-84 
memory  and.  423-424 
molecular  regulation,  415-416 
recognition  and  differentiation,  101-102 
role  of,  in  health  and  sickness,  273-214 
subsets,  cytokine  regulation  of  effector  functions, 
513-514 

surface  glycoproteins,  development  and  infection, 
263-264 

V/3  superantigen,  function  in  health  and  disease,  273-274 
Target  recognition 

growth  cone  guidance  and,  170 

neural  development,  207-208 
Tat  protein,  retroviral  gene  expression,  95-96 
TATA  box,  lymphocyte  development,  382 
TATA-binding  protein  (TBP),  transcription  regulation,  422 
TdT  (terminal  deoxynucleotidyltransferase)  gene,  381-382 
Telomeres 

hereditary  kidney  disease,  333-334 

RNA  molecules,  7/- 72 
Testis-determining  factor,  IRE-A  DNA-binding  protein,  7-8 
Tetrahymena  thermophila  enzyme,  71 
TflllA,  macromolecular  assembly,  177-178 
Thalassemia 

o:-globin  gene  expression,  259-260 

prenatal  diagnosis,  hemoglobin  synthesis,  217-218 
(3-Thalassemia  (Cooley's  anemia),  305-306 
Three-dimensional  structure,  eukaryotic  chromosomes, 
360-362 

"3-4-5"  rule,  hormone  receptor  genetics,  126 
Thrombin,  blood  clotting  regulation,  344-346 
Thrombolysis,  tissue-type  plasminogen  activator  (t-PA), 
157-158 

Thrombomodulin,  blood  clotting  regulation,  344-346 
Thrombosis,  prevention  of  clotting,  123-124 
Thymidine  kinase  (TK),  viral  gene  regulation,  279-280 
Thyroid  hormone,  receptor  genetics,  125-127 
Thyroid  hormone  receptors  (TRs),  75-76 
Thyroid  hormone-responsive  elements  (TREs),  75-76 
Thyroid-stimulating  hormone  (TSH) 

gene  expression  regulation,  75-76 

post-transcriptional  action,  76 
Time-resolved  imaging,  structural  biology  and,  Ixiii 
Tissue  factor,  blood  clotting  regulation,  344-346 
Tissue  specificity,  epidermal  growth  factor  (EGF),  104 
Tissue-type  plasminogen  activator  (t-PA) 

coagulation  genetics,  161-162 

human  disease  and,  157 
Tn7  transposon,  91-92 

toll  gene,  signal  transduction  in  B  cells,  159-160 
Topoisomerase  II,  eukaryotic  chromosomes,  361 
torso  gene,  embryogenesis  transcription  control,  112-114 
Trans-activating  proteins,  gene  regulation  mechanisms  and, 
421-422 

Transacylase,  phospholipids  and  cell  regulation,  165-166 
Transcription 

cell  structure  and,  xxx 

cellular 

gene  expression,  285-286 

cellular  regulation,  375-376 

developmentally  regulated  genes,  422 

Drosophila  embryogenesis,  112-114 


genetic  disease  and,  xlii-xliv 

insulin  mechanisms  and,  43-44 

metamorphosis,  417-418 

molecular  mechanisms,  339-340 

muscle  development  and  function,  287-288 

MyoD  gene  activation,  433-434 

promoter-specific  regulation,  421-422 

regulation  mechanisms,  495-496 

RNA  polymerase  11  structure  and  function,  85-86 

transmembrane  signals,  469-470 
Transcription  factor  IID  (TFIID),  59-60 
Transcription  factors.  See  also  Proteins 

gene  expression,  adenovirus  as  control  model,  369-370 

genomic  response  and,  289-290 

kidney  cell  growth  and  differentiation,  407-408 

molecular  genetics  of  blood  cells  and,  305-306 

polypeptide  hormones,  /  75-  /  76 

viral  gene  regulation,  279-280 
Transcriptional  regulatory  complexes,  177-178 
Transducin 

signal  transduction,  visual  systems,  193-194 

transmembrane  signal  transduction,  347 
Transfer  RNA  (tRNAs) 

ribosomal  pausing,  455 

structural  biology  and,  Ixi 
Transforming  genes,  papillomavirus  molecular  biology,  242 
Transforming  growth  factor-a  (TGF-a:) 

cell  regulation,  275-276 

signal  transduction  of  receptor,  103-104 
Transforming  growth  factor-/?  (TGF-/3) 

cell  regulation,  275-276 

limb  development  genetics  in  Drosophila,  79-80 
Transgenic  mice 

albinism  and,  307-308 
B  cell  development,  299-300 
developmental  genetics,  383-384 
extracellular  matrix  structure,  49 
genetic  basis  of  cancer  and,  247-248 
a-globin  gene  expression,  260 

immunological  self-tolerance  and  autoimmunity,  1 71- 1 72 
mammalian  development  studies  and,  313-314,  315-316 
phagocytic  cells,  molecular  genetics  of  blood  cells  and, 
306 

research  with,  xliv 

shock  pathogenesis,  tumor  necrosis  factor  (TNF)  and, 
35-36 

Transgenic  plants,  photosynthesis  genetics,  503-505 
Transient  gene  expression,  gene  therapy  and,  250 
Trans-inactivation,  position  effects,  183-184 
Translation 

cell  structure  and,  xxx 

insulin  mechanisms  and,  43-44 

mRNA  protein  synthesis,  455 
Translocation 

intracellular  protein  transpon,  353-354 

lymphocyte  development,  molecular  genetics,  231-232 
Transmembrane  signaling,  cellular  regulation,  375-376 
Transmitters,  cell  structure  and.  xxxvi-xxxvii 
Transplantation 

antigens,  immunodeficiency  and,  325-326 

coagulation  genetics,  162 

complement  system,  16 
Transport  functions,  egg  structure-function  studies,  386 
Transporters,  cell  structure  and,  xxxvi 
Transposition,  mechanism  of,  91-92 
Trembler  gene,  Charcot-Marie-Tooth  disease  and,  141 
Tricarboxylic  acid  cycle,  metabolic  pathways,  429-430 
Trophozoites,  Entamoeba  histolytica  detection  and 
cloning,  516 

cv-Tropomyosin  (TM)  gene,  protein  diversity  and,  288 
Trypanosoma  brucei  rhodesiense,  140 
Trypanosomes,  mitochondrial  genome,  377-378 
Trypanosomiasis,  immune  evasion  and,  115-116 


546 


Index 


Tuberculosis 

epidemiological  analysis,  200-202 

immunity  and  pathogenesis,  47-48 
Tumor  necrosis  factor  (TNF) 

inflammatory  cytokines,  73-  74 

regulatory  proteins,  387-388 

shock  pathogenesis,  35-36 
Tumor  necrosis  factor-a  (TNF-a),  structural  studies, 
387-388 

Tumor  necrosis  factor-/3  (TNF-/J),  structural  studies, 
387-388 

Tumors,  cholecystokinin  regulation  and,  144 
Tumor-suppressor  genes 

current  research  on,  131-132 

p53  protein,  tissue-type  plasminogen  activator  (t-PA), 
157-158 

Turner  syndrome,  X  and  Y  chromosomes,  314 
Type  1  collagen,  extracellular  matrix,  49 
Typhoid  fever 

microbial  pathogenesis,  493-494 

molecular  biology,  487-488 

vaccines  with  attenuated  typhoid  strains,  358 
Tyrosinase,  albinism  and,  307-308 

Tyrosine  hydroxylase  (TH),  transcription  control,  469-470 
Tyrosine  kinases 

cell  cycle  control,  21-22 

host-pathogen  interactions,  493-494 

signal  transduction,  518 
Tyrosine  phosphorylation,  cell  regulation,  55-56 

Ultrabithorax  gene ,  Drosophila  morphogenesis,  24 
Upstream  stimulatory  factor  (USF),  59-60 
Urea  cycle,  metabolic  pathways,  429-430 

Vaccines 

immune  response  and,  liv 

leprosy  and  tuberculosis  immunity  and  pathogenesis, 
47-48 

rotavirus  diarrhea,  483-484 

Salmonella  typhi  attenuated  strains  as,  358 

T  cell  repertoire,  superantigens,  221-222 
Variability,  Entamoeba  histolytica,  515-516 
Variable  domains,  protein  chains,  xlvi 
Variable  surface  glycoprotein  (VSG),  115-116 
Vascular  disease,  prevention  of  clotting,  123-124 
Vascular  smooth  muscle  cells,  blood  vessel  function, 
459-460 

Vasospasm,  blood  vessel  function,  459-460 

VDJ  recombination,  antigen  receptor  molecule  studies, 

9-10,  351-352 
Vertebrates 

embryonic  induction  mechanisms,  269-270 

nervous  system,  cell  fate  control,  11-12 
Vesicle  transport,  secretory  pathways,  354 
vestigial  gene .  pattern  regulation  and,  66-67 
Viral  packaging,  RNA  viral  genetics,  227-228 
Viral  protein  16  (VPl6),  279-280 
Viral  replication  and  pathogenesis,  molecular  basis  of, 
149-150 

Viral  snRNPs,  autoantibody  probes,  392 

Viruses.  See  also  specific  viruses,  e.g..  Papillomaviruses 

genetics,  RNA,  227-228 

macromolecular  assembly,  178 

mammalian  gene  regulation  and,  279-280 
Visual  systems 

chromosome  7  mapping,  531-532 

Drosophila 

development  in,  341-342 

pattern  formation  and  neuronal  cell  recognition, 
396-398 
neural  foundations,  283-284 

phototransduction  of  retinal  rods  and  cones,  461-462 


sensory  representations,  367-368 

signal  transduction,  193-194,  473 

spatial  information,  527 

visual  pigment  molecular  biology,  291-292 
Vitamin  D  receptor  (VDR),  hormone  receptor  genetics,  126 
Voltage-clamp  technique,  nerve  cell  electrical  activity,  1-2 
Voltage-dependent  sodium  channels,  33-34 
Voltage-gated  calcium  channels,  61-62 
Voltage-gated  ion  channels,  5-6 
von  Recklinghausen  neurofibromatosis  (NFl),  81 
von  Willebrand  disease 

blood  clotting  regulation,  344-346 

molecular  genetics,  161-162 
von  Willebrand  factor 

blood  clotting  regulation,  344-346 

molecular  genetics,  161-162 
VPl  protein,  macromolecular  assembly,  178 
VP4  protein,  rotavirus  diarrhea,  483-484 
VP7  protein,  rotavirus  diarrhea,  483-484 
Vpsl5  protein  (Vpsl5p),  122 
Vps34  protein  (Vps34p),  122 

White  blood  cells,  molecular  genetics  of,  305-306 

white-spotting  (W)  gene,  normal  and  leukemic 
hematopoiesis,  485-486 

Wilms'  tumor  (WT),  131-132 

wingless  gene 
Drosophila  morphogenesis,  23-24 
limb  development  genetics  in  Drosophila,  79-80 
oncogenesis,  297-298 

Wnt-\  gene  family,  early  embryogenesis,  297-298 

Wound  healing,  cell  adhesion  and,  195-196 

X  chromosome 

disorders,  gene  identification  and  correction,  69-  70 

mammalian  development,  312-314 

molecular  genetics,  295-296 

structure,  xxx,  xxxiii 

X-linked  disorders,  163-164,  431-432 
X-linked  disorders,  163-164,  431-432 
Xq28  chromosome  region,  X-linked  disorders,  163-164 
X-ray  crystallography 

cell-surface  recognition,  41 

structural  biology  and,  Ixi,  57-58 

Y  chromosomes,  mammalian  development,  312-314 
Yeast  artificial  chromosomes  (YACs) 

chromosome  7  mapping,  531-532 

cloning  techniques,  82 

DNA,  large-scale  analysis,  303-304 

gene  expression  and  cell  cycle,  cerebellum  development, 
180 

mitochondria,  protein  translocons  and,  45-46 

molecular  genetics,  neuromuscular  disease,  233-234 

obesity  and  diabetes,  144 

research  with,  xliv 

RNA  viral  genetics,  227-228 

tumor-suppressor  genes,  131 

urea/tricarboxylic  acid  cycles,  429-430 
Yersinia  pseudotuberculosis.  197-198 
YY-1  transcription  factor,  gene  expression,  369-370 

Zellweger  syndrome,  urea/tricarboxylic  acid  cycles,  430 

ZFX  genes,  sex  determination,  312-314 

ZFY genes,  sex  determination,  312-314 

zif 268  gene,  DNA  interactions,  309-31 1 

Zinc  finger  proteins 

DNA  interactions,  crystal  structures,  309-31 1 
kidney  cell  growth  and  difl'erentiation,  408 
structural  biology  and,  Ixiii 

Zygotic  genes,  Drosophila  embryogenesis,  112-114 


547 


I 


I