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University  of  California  •  Berkeley 


Regional  Oral  History  Office  University  of  California 

The  Bancroft  Library  Berkeley,  California 


Program  in  the  History  of  the  Biological  Sciences  and  Biotechnology 


Mary  C.  Betlach 

EARLY  CLONING  AND  RECOMBINANT  DNA  TECHNOLOGY  AT  HERBERT  W.  BOYER'S 

UCSF  LABORATORY  IN  THE  1970s 


Interview  Conducted  by 

Sally  Smith  Hughes,  Ph.D. 

in  1994 


Copyright  ©  2002  by  The  Regents  of  the  University  of  California 


Mary  C.  Betlach,  1972. 


Since  1 954  the  Regional  Oral  History  Office  has  been  interviewing  leading  participants  in  or  well-placed 
witnesses  to  major  events  in  the  development  of  northern  California,  the  West,  and  the  nation.  Oral 
history  is  a  method  of  collecting  historical  information  through  tape-recorded  interviews  between  a 
narrator  with  firsthand  knowledge  of  historically  significant  events  and  a  well-informed  interviewer, 
with  the  goal  of  preserving  substantive  additions  to  the  historical  record.  The  tape  recording  is 
transcribed,  lightly  edited  for  continuity  and  clarity,  and  reviewed  by  the  interviewee.  The  corrected 
manuscript  is  indexed,  bound  with  photographs  and  illustrative  materials,  and  placed  in  The  Bancroft 
Library  at  the  University  of  California,  Berkeley,  and  in  other  research  collections  for  scholarly  use. 
Because  it  is  primary  material,  oral  history  is  not  intended  to  present  the  final,  verified,  or  complete 
narrative  of  events.  It  is  a  spoken  account,  offered  by  the  interviewee  in  response  to  questioning,  and 
as  such  it  is  reflective,  partisan,  deeply  involved,  and  irreplaceable. 

************************************ 

All  uses  of  this  manuscript  are  covered  by  a  legal  agreement  between  The 
Regents  of  the  University  of  California  and  Mary  C.  Betlach  dated  October  10,  2001. 
The  manuscript  is  thereby  made  available  for  research  purposes.  All  literary  rights  in 
the  manuscript,  including  the  right  to  publish,  are  reserved  to  The  Bancroft  Library  of 
the  University  of  California,  Berkeley.  No  part  of  the  manuscript  may  be  quoted  for 
publication  without  the  written  permission  of  the  Director  of  The  Bancroft  Library  of 
the  University  of  California,  Berkeley. 

Requests  for  permission  to  quote  for  publication  should  be  addressed  to  the 
Regional  Oral  History  Office,  486  Bancroft  Library,  Mail  Code  6000,  University  of 
California,  Berkeley  94720-6000,  and  should  include  identification  of  the  specific 
passages  to  be  quoted,  anticipated  use  of  the  passages,  and  identification  of  the  user. 
The  legal  agreement  with  Mary  C.  Betlach  requires  that  she  be  notified  of  the  request 
and  allowed  thirty  days  in  which  to  respond. 

It  is  recommended  that  this  oral  history  be  cited  as  follows: 

Mary  C.  Betlach,  "Early  Cloning  and  Recombinant  DNA  Technology  at 
Herbert  W.  Bayer's  UCSF  Laboratory  in  the  1970s"  an  oral  history 
conducted  in  1994  by  Sally  Smith  Hughes,  Regional  Oral  History  Office,  The 
Bancroft  Library,  University  of  California,  Berkeley,  2002. 


Copy  no. 


Cataloguing  information 


Mary  C.  Betlach  (b.  1 945)  Scientist 

Early  Cloning  and Recombinant  DNA  Technology  at  Herbert  W.  Bayer's  UCSF Laboratory  in  the  1970s, 
2002,  v,  79  pp. 

Discussion  of  laboratory  facilities,  personnel,  competition,  and  working  atmosphere  at  Herbert  Boyer's 
laboratory  in  the  early  1970s;  early  work  to  purify  restriction  enzymes  and  plasmids  and  clone  DNA; 
biosafety  concerns;  plasmid  vector  development,  approval  and  certification,  and  conflicts  surrounding 
dissemination  to  colleagues;  perspectives  on  division  between  science  and  industry;  opinions  on 
scientists  who  merit  the  Nobel  Prize;  comments  on  Herbert  Boyer,  Stanley  N.  Cohen,  Robert  Helling, 
Ernest  Jawetz,  Art  Riggs,  William  J.  Rutter,  and  others. 


Interviewed  in  1994  by  Sally  Smith  Hughes  for  the  Program  in  the  History  of  Biosciences  and 
Biotechnology,  Regional  Oral  History  Office,  The  Bancroft  Library,  University  of  California, 
Berkeley. 


TABLE  OF  CONTENTS-Mary  C.  Betlach 

BIOTECHNOLOGY  SERIES  HISTORY  by  Sally  Smith  Hughes  i 

BIOTECHNOLOGY  SERIES  LIST  iii 

INTERVIEW  HISTORY  iv 

BIOGRAPHICAL  INFORMATION  v 

The  Herbert  W.  Boyer  Laboratory  at  UCSF  in  the  Early  1 970s  1 
Old  Facilities  in  the  Microbiology  Department 
Lab  Personnel 

Studying  Enzyme  Restriction  and  Modification  Then  and  Now  3 

Ernest  Jawetz  and  His  Lab  Group  4 
Atmosphere  of  the  Boyer  Lab 

Recombinant  DNA  Technology  8 

Early  Development  8 

Authorship  of  the  First  Paper  10 

Contemplating  Commercial  Applications  1 1 

Cloning  Eukaryotic  DNA  1 1 

Reconstructing  the  Experiments  12 

Reporting  the  Discovery  14 

Biosafety  Concerns  16 

Plasmid  Vectors  20 

Plasmid  Development  in  the  Boyer  Lab  20 

Isolation  of  EcoRl  and  the  Development  of  Betlach  Plasmids  21 

Plasmid  Dissemination  23 

The  Boyer  Lab  Moves  to  the  Biochemistry  Department  25 

Use  of  an  Uncertified  Plasmid,  1 977  26 

Rivalries  28 

UC's  Contract  with  Genentech  on  Somatostatin  29 

Boyer  as  a  Lab  Director  30 

Tension  over  University  Ties  with  Genentech  3 1 

TAPE  GUIDE  34 

APPENDICES  35 

A.  Curriculum  Vitae  36 

B.  Current  Rl  Endonuclease  Purification  Procedure,  December  1972  42 

C.  Betlach-Boyer  Procedures  for  Preparing  Closed-Circular  DNA,  late  '72  or  early  '73  45 

[handwritten  by  Herb  Boyer] 

D.  Photographs  from  Herb  Boyer's  Laboratory  in  the  1970s 

[captions  and  photographs  courtesy  of  Mary  Betlach]  48 


E.  Agarose  Gel  Electrophoresis  of  Linear  Duplex  DNA,  April  1973  56 

F.  Electrophoresis  of  DNA  in  Agarose  Gels,  Boyer  Laboratory  Procedures,  1973  58 

G.  Restriction  Map  of  pBR322  60 

H.  Biolabs  Midnight  Hustler,  newsletter  parody  produced  by  colleagues  of 

Mary  Betlach  at  Harvard  University,  April  1 ,  1 977  61 

I.  Gartland/Fredrickson  Memo  Certifying  pBR322,  July  6,  1977  76 

INDEX  78 


1 
BIOTECHNOLOGY  SERIES  fflSTORY-Sally  Smith  Hughes,  Ph.D. 

Genesis  of  the  Program  in  the  History  of  the  Biological  Sciences  and  Biotechnology 

In  1996  The  Bancroft  Library  launched  the  Program  in  the  History  of  the  Biological  Sciences 
and  Biotechnology.  Bancroft  has  strong  holdings  in  the  history  of  the  physical  sciences—the  papers  of 
E.O.  Lawrence,  Luis  Alvarez,  Edwin  McMillan,  and  other  campus  figures  in  physics  and  chemistry,  as 
well  as  a  number  of  related  oral  histories.  Yet,  although  the  university  is  located  next  to  the  greatest 
concentration  of  biotechnology  companies  in  the  world,  Bancroft  had  no  coordinated  program  to 
document  the  industry  or  its  origins  in  academic  biology. 

When  Charles  Faulhaber  arrived  in  1995  as  Bancroft's  director,  he  agreed  on  the  need  to 
establish  a  Bancroft  program  to  capture  and  preserve  the  collective  memory  and  papers  of  university  and 
corporate  scientists  and  the  pioneers  who  created  the  biotechnology  industry.  Documenting  and 
preserving  the  history  of  a  science  and  industry  which  influences  virtually  every  field  of  the  life  sciences, 
generates  constant  public  interest  and  controversy,  and  raises  serious  questions  of  public  policy  is  vital 
for  a  proper  understanding  of  science  and  business  in  the  late  twentieth  and  early  twenty- first  centuries. 

The  Bancroft  Library  is  the  ideal  location  to  carry  out  this  historical  endeavor.  It  offers  the 
combination  of  experienced  oral  history  and  archival  personnel,  and  technical  resources  to  execute  a 
coordinated  oral  history  and  archival  program.  It  has  an  established  oral  history  series  in  the  biological 
sciences,  an  archival  division  called  the  History  of  Science  and  Technology  Program,  and  the  expertise  to 
develop  comprehensive  records  management  plans  to  safeguard  the  archives  of  individuals  and 
businesses  making  significant  contributions  to  molecular  biology  and  biotechnology.  It  also  has 
longstanding  cooperative  arrangements  with  UC  San  Francisco  and  Stanford  University,  the  other 
research  universities  in  the  San  Francisco  Bay  Area. 

In  April  1996,  Daniel  E.  Koshland,  Jr.  provided  seed  money  for  a  center  at  The  Bancroft  Library 
for  historical  research  on  the  biological  sciences  and  biotechnology.  And  then,  in  early  2001,  the 
Program  in  the  History  of  the  Biological  Sciences  and  Biotechnology  was  given  great  impetus  by 
Genentech's  generous  pledge  of  one  million  dollars  to  support  documentation  of  the  biotechnology 
industry. 

Thanks  to  these  generous  gifts,  Bancroft  has  been  building  an  integrated  collection  of  research 
materials—primarily  oral  history  transcripts,  personal  papers,  and  archival  collections— related  to  the 
history  of  the  biological  sciences  and  biotechnology  in  university  and  industry  settings.  A  board 
composed  of  distinguished  figures  in  academia  and  industry  advise  on  the  direction  of  the  oral  history 
and  archival  components.  The  Program's  initial  concentration  is  on  the  San  Francisco  Bay  Area  and 
northern  California.  But  its  ultimate  aim  is  to  document  the  growth  of  molecular  biology  as  an 
independent  field  of  the  life  sciences,  and  the  subsequent  revolution  which  established  biotechnology  as 
a  key  contribution  of  American  science  and  industry.  The  UCSF  Library,  with  its  strong  holdings  in  the 
biomedical  sciences,  is  a  collaborator  on  the  archival  portion  of  the  Program.  David  Farrell,  Bancroft's 
curator  of  the  History  of  Science  and  Technology,  serves  as  liaison. 

Oral  History  Process 

The  oral  history  methodology  used  in  this  program  is  that  of  the  Regional  Oral  History  Office, 
founded  in  1954  and  producer  of  over  1,800  oral  histories.  The  method  consists  of  research  in  primary 
and  secondary  sources;  systematic  recorded  interviews;  transcription,  light  editing  by  the  interviewer, 
and  review  and  approval  by  the  interviewee;  library  deposition  of  bound  volumes  of  transcripts  with 
table  of  contents,  introduction,  interview  history,  and  index;  cataloging  in  UC  Berkeley  and  national 


11 

online  library  networks;  and  publicity  through  ROHO  news  releases  and  announcements  in  scientific, 
medical,  and  historical  journals  and  newsletters  and  via  the  ROHO  and  UCSF  Library  Web  pages. 

Oral  history  as  a  historical  technique  has  been  faulted  for  its  reliance  on  the  vagaries  of  memory, 
its  distance  from  the  events  discussed,  and  its  subjectivity.  All  three  criticisms  are  valid;  hence  the 
necessity  for  using  oral  history  documents  in  conjunction  with  other  sources  in  order  to  reach  a 
reasonable  historical  interpretation.1  Yet  these  acknowledged  weaknesses  of  oral  history,  particularly  its 
subjectivity,  are  also  its  strength.  Often  individual  perspectives  provide  information  unobtainable 
through  more  traditional  sources.  Oral  history  in  skillful  hands  provides  the  context  in  which  events 
occur—the  social,  political,  economic,  and  institutional  forces  which  shape  the  course  of  events.  It  also 
places  a  personal  face  on  history  which  not  only  enlivens  past  events  but  also  helps  to  explain  how 
individuals  affect  historical  developments. 

Emerging  Themes 

Although  the  oral  history  program  is  still  in  its  initial  phase,  several  themes  are  emerging.  One 
is  "technology  transfer,"  the  complicated  process  by  which  scientific  discovery  moves  from  the 
university  laboratory  to  industry  where  it  contributes  to  the  manufacture  of  commercial  products.  The 
oral  histories  show  that  this  trajectory  is  seldom  a  linear  process,  but  rather  is  influenced  by  institutional 
and  personal  relationships,  financial  and  political  climate,  and  so  on. 

Another  theme  is  the  importance  of  personality  in  the  conduct  of  science  and  industry.  These 
oral  histories  testify  to  the  fact  that  who  you  are,  what  you  have  and  have  not  achieved,  whom  you  know, 
and  how  you  relate  have  repercussions  for  the  success  or  failure  of  an  enterprise,  whether  scientific  or 
commercial.  Oral  history  is  probably  better  than  any  other  methodology  for  documenting  these  personal 
dimensions  of  history.  Its  vivid  descriptions  of  personalities  and  events  not  only  make  history  vital  and 
engaging,  but  also  contribute  to  an  understanding  of  why  circumstances  occurred  in  the  manner  they  did. 

Molecular  biology  and  biotechnology  are  fields  with  high  scientific  and  commercial  stakes.  As 
one  might  expect,  the  oral  histories  reveal  the  complex  interweaving  of  scientific,  business,  social,  and 
personal  factors  shaping  these  fields.  The  expectation  is  that  the  oral  histories  will  serve  as  fertile 
ground  for  research  by  present  and  future  scholars  interested  in  any  number  of  different  aspects  of  this 
rich  and  fascinating  history. 

Location  of  the  Oral  Histories 

Copies  of  the  oral  histories  are  available  at  the  Bancroft,  UCSF,  and  UCLA  libraries.  They  also 
may  be  purchased  at  cost  through  the  Regional  Oral  History  Office.  Some  of  the  oral  histories,  with 
more  to  come,  are  available  on  The  Bancroft  Library's  History  of  the  Biological  Sciences  and 
Biotechnology  Website:  http://bancroft.berkeley.edu/Biotech/. 


Sally  Smith  Hughes,  Ph.D. 
Historian  of  Science 


Regional  Oral  History  Office 
The  Bancroft  Library 
University  of  California,  Berkeley 
August  2002 


1The  three  criticisms  leveled  at  oral  history  also  apply  in  many  cases  to  other  types  of 
documentary  sources. 


IV 

INTERVIEW  HISTORY-Mary  C.  Betlach 


Dr.  Betlach  was  interviewed  in  1994  about  her  position  as  key  technician  in  Herbert  W.  Boyer's 
laboratory  at  UCSF  at  the  time  of  the  creation  and  early  expansion  of  recombinant  DNA  technology. 
Although  the  inspiration  for  the  interview  came  from  an  oral  history  conducted  that  year  with  Dr.  Boyer, 
her  central  role  in  the  development  of  several  procedures  that  made  recombinant  DNA  widely  practicable 
are  historically  as  well  as  technically  important  in  their  own  right. 

Betlach  came  to  Boyer's  lab  in  1972,  eager  to  work  on  restriction  enzyme  modification,  the  lab's 
central  focus.  It  was  to  become  the  reason  for  Boyer's  collaboration  with  Stanley  N.  Cohen  of  Stanford 
in  the  genesis  in  1973-1974  of  a  straightforward  method  for  combining  and  amplifying  DNA.  Betlach 
describes  her  participation  in  the  development  of  recombinant  DNA  technology  and  the  laboratory's  role 
in  disseminating  it  to  molecular  biology  laboratories  worldwide. 

Although  Cohen's  technician  at  the  time,  Annie  Chang,  is  co-author  of  three  papers  on 
recombinant  DNA  published  in  these  years,  Betlach,  who  also  played  a  seminal  technical  role,  is  not  an 
author.  When  the  topic  came  up  in  the  interview,  she  was  characteristically  nonchalant.  Because  as  a 
general  rule,  technicians  are  not  named  as  authors  of  scientific  publications,  Annie  Chang's  position  on 
the  papers  could  be  regarded  as  the  exception  to  the  rule  and  Betlach' s  "omission"  the  more  common 
situation.  Whatever  the  reason,  one  hopes  that  the  oral  history  establishes  for  the  historical  record  that 
Betlach  was  far  from  a  pair  of  hands  in  the  Boyer  laboratory;  she  created  and  modified  procedures 
instrumental  for  the  development  and  expansion  of  recombinant  DNA  technology.    As  she  describes  in 
the  interview,  it  was  Betlach  who  created  some  of  the  earliest  plasmids  critical  to  the  application  of 
recombinant  DNA  and  sent  them  out  to  investigators  around  the  world.  She  also  reflects  on  the 
atmosphere  of  the  laboratory  which,  like  the  man  at  its  head,  was  simultaneously  competitive,  laid-back, 
and  amazingly  productive.  Both  Betlach  and  Chang  went  on  to  earn  doctorates  in  the  biomolecular 
sciences. 

Betlach's  view  of  the  accomplishments  and  culture  of  the  Boyer  lab  at  the  height  of  its 
preeminence  has  obvious  historical  merit.  The  interview  is  also  a  welcome  extension  of  the  Boyer  oral 
history  and  the  oral  histories  in  progress  with  Stanley  Cohen  and  Herbert  Heyneker,  a  postdoctoral 
fellow  in  the  Boyer  laboratory  in  1975-1977.  Together  they  provide  novel  historical  documentation  of 
the  earliest  manifestation  of  a  technology  destined  to  transform  biomedical  science  and  to  become  a 
major  basis  for  the  biotechnology  industry. 


Sally  Smith  Hughes,  Ph.D. 
Historian  of  Science 


Regional  Oral  History  Office 
The  Bancroft  Library 
University  of  California,  Berkeley 
July  2002 


Regional  Oral  History  Office 
Room  486  The  Bancroft  Library 


University  of  California 
Berkeley,  California  94720 


Your  full  name 


BIOGRAPHICAL  INFORMATION 
(Please  write  clearly.  Use  black  ink.) 
CAR.OL<-r/J 


Date  of  birth 


Birthplace 


Father's  full  name 


7~A<>/rj£S<>/O 


Oc  cup  at  i  on     M.  D  . 


) 


Birthplace 


Mother's  full  name 
Occupation 


OU$eu/)/-t 


Birthplace    T3f.  J/f.VJ  //£  ,  WX. 

^~ 


Your   spouse  /partner 
Occupation 


a/y\ 


Birthplace 


Your  children   3o£,AJ 


Where  did  you  grow  up  ? 
Present  community 
Education 


Occupation(s) 


Areas  of  expertise 


Other  interests  or  activities 


c 


Organizations  in  which  you  are  active 


SIGNATURE 


DATE  : 


INTERVIEW  WITH  MARY  BETLACH 


The  Herbert  W.  Bover  Laboratory  at  UCSF  in  the  Early  1970s 

[Date  of  Interview:  March  25,  1994]##' 

Old  Facilities  in  the  Microbiology  Department 

Betlach:  We  were  in  an  old  part  of  the  Medical  Sciences  building  and  the  labs  were  pretty  funky- 
old-fashioned  histology  labs.  We  didnt  have  a  lot  of  space.  The  chairman  of  the 
microbiology  department,  Ernest  Jawetz,  came  to  me  on  more  than  one  occasion  and  asked 
for  my  help  in  cleaning  the  place  up.  Once  he— and  he  made  me  laugh-appealed  to  me  "as 
a  wife  and  a  mother,"  couldn't  I  please  do  something  about  this  place?  And  the  only  reason 
it  was  really  messy  was  because  we  were  crowded.  When  I  came  it  was  disorganized  and 
there  just  wasn't  a  lot  of  space.  And  a  couple  of  the  graduate  students  that  Herb  had,  Joel 
Hedgpeth  in  particular,  were  kind  of  messy. 

So  I  reorganized  the  whole  place  a  little  bit.  As  we  got  more  people,  of  course  it  just  got 
worse,  and  what  could  I  say  or  do?  A  small,  ill-designed  space,  you  can  only  organize  so 
well.  There's  just  not  too  much  more  you  can  do.  We  were  having  to  walk  across  to 
another  building  to  take  our  gel  pictures.  What  is  here  at  Parnassus  Pharmaceuticals  is  a 
paradise  compared  to  what  we  had-three  tiny  rooms. 

Hughes:  Was  that  typical  lab  space  at  UCSF  at  that  time?  Or  do  you  think  that  your  lab  was 
particularly  deprived? 


'##  This  symbol  indicates  that  a  tape  or  tape  segment  has  begun  or  ended.  A  guide  to  the 
tapes  follows  the  transcript. 


Betlach:  I  don't  know.  Herb  probably  knows  a  lot  more  about  how  it  was  set  up. 

We  were  in  an  old  part  of  the  building  and  there  were  new  labs  in  the  new  towers, 
Health  Sciences  East  and  West,  which  had  just  been  built.  I  can  remember  they  didn't  even 
have  grass  in  the  courtyard  yet.  But  I  don't  know  the  politics  that  were  involved  in  why 
Herb  got  old  labs  and  not  new  labs.  But  most  of  the  rest  of  the  people  in  the  microbiology 
department  were  in  the  new  towers  and  there  were  teaching  labs  and  the  departmental 
office  in  the  old  building  where  we  were. 

We  didn't  have  a  lot  of  interaction  with  the  other  people  in  the  department.  We  had  a 
dishwashing  facility  in  the  new  building,  a  room.  We  had  some  interaction  with  people  in 
[J.  Michael]  Mike  Bishop's  lab. 

Hughes:  Because  you  were  doing  similar  research? 

Betlach:  Well,  it  wasn't  that  similar.  They  were  doing  tissue  culture  and  working  on  viruses, 

whereas  we  were  working  with  bacteria.  But  they  were  just  nicer  to  us.  [laughter]  What 
can  I  say? 

Hughes:  Do  you  know  why  Dr.  Boyer  was  recruited? 

Betlach:  That  was  before  my  time,  so  I  don't  really  know.  I  came  in  1972. 


Lab  Personnel 

Hughes:  Who  was  there  when  you  came  in  1972? 

Betlach:  Not  very  many  people— Joel  Hedgpeth  was  just  graduating  and  Ned  Mantei  whose  job  I 
filled.  I  came  to  take  his  position.  I  came  in  as  a  technician.  Ned  was  going  back  to 
graduate  school  with  Charles  Weissmann.  We  both  had  previously  worked  for  Hatch 
[Harrison]  Echols  at  the  University  of  Wisconsin.  Let's  see,  Bob  Helling,  Daisy  Roulland- 
Dussoix.  That's  all  that  comes  to  my  mind  right  now. 


Hughes:  What  was  she? 

Betlach:  It  was  so  long  ago  and  there's  been  a  lot  of  postdocs  and  sabbaticals  and  everything  under 
the  bridge  since  then.  I  think  she  was  not  a  postdoc.  I  think  she  was  higher  than  that.  I 
think  she  was  on  sabbatical  or  she  had  some  temporary  appointment.  Herb  should  know 
that.  Bob  Helling  was  definitely  a  sabbatical. 

So  there  weren't  very  many  people  at  first,  but  then  six  months  after  I  was  there,  Pat 
Greene  came  in  as  a  postdoc.  And  then  other  people  started  coming,  like  Paco  Bolivar  and 
Ray  Rodriguez  and  Bob  Tate  and  Herb  Heyneker,  and  we  started  to  really  get  crowded. 

Hughes:  This  was  when  the  research  was  heating  up? 
Betlach:  The  increase  in  people  happened  because,  yes,  we  started  to- 
Studying  Enzyme  Restriction  and  Modification  Then  and  Now 

Betlach:  When  I  first  went  there,  I  was  purifying  restriction  enzymes,  of  which  there  were  not  very 
many  known,  and  there  are  hundreds  now.  Bob  Helling  was  isolating  plasmids  and  running 
them  on  tube  gels.  Pat  [Patricia]  Greene  came  and  started  to  set  up  a  gel  assay  for  our 
restriction  enzyme  purifications,  which  previously  had  been  done  on  sucrose  gradients, 
which  was  laborious. 

Hughes:  What  does  that  change  in  technology  mean? 

Betlach:  That  was  a  big  leap.  Initially,  restriction  and  modifications  systems  were  investigated  in 
vivo.  For  example,  you  could  infect  an  E.  coli  strain  with  unmodified  phage  and  you'd  see 
if  your  phage  titer  decreased  in  comparison  to  control  strains  that  either  contained  the 
restriction  modification  system  or  did  not.  The  presence  of  the  restriction  modification 
system  in  the  strain  you  were  testing  was  indicated  by  a  decrease  in  titer  since  the  phage 
would  be  susceptible  to  digestion  by  the  restriction  enzyme. 


For  purifying  restriction  enzymes  we  used  an  in  vitro  assay.  We  took  radioactive 
lambda  DNA  and  we'd  mix  it  in  a  tube  with  our  enzyme  preps  that  we  were  assaying.  So 
you'd  collect  fractions  across  a  given  column  during  any  purification  step.  There  would  be 
several  different  column  purification  steps  and  you'd  have  these  fractions  and  you'd  take  a 
small  amount  and  you'd  react  it  with  the  radioactive  lambda  DNA  and  then  run  it  on  a 
sucrose  gradient.  So  each  sucrose  gradient  was  one  sample,  one  fraction  we  were  assaying, 
so  it  was  really  labor  intensive.  The  sucrose  gradients  were  run  in  an  ultracentrifuge, 
fractionated,  and  counted  in  a  scintillation  counter.  A  lot  of  work. 

Today  you  take  an  enzyme  sample;  you  react  it  in  the  tube  the  same  way  only  the  DNA 
is  not  radioactive— and  then  you  run  it  in  one  slot  on  a  slab  gel.  You  can  do  forty  at  a  time 
in  half  an  hour,  instead  of  all  day  to  do  six  because  an  ultracentrifuge  would  only  hold  six 
samples. 

Hughes:  Did  you  have  an  ultracentrifuge  by  the  mid-seventies? 

Betlach:  Yes. 

Hughes:  I  know  you  didn't  in  the  beginning. 

Betlach:  Yes,  we  had  one  and  a  cold  room  we  used  for  enzyme  purifications.  I  was  trying  to 
remember  where  our  Sorvall  was.  I  can't  remember  where  it  was. 


Ernest  Jawetz  and  His  Lab  Group 

Betlach:  I  remember  that  sometimes  we  needed  an  extra  Sorvall  centrifuge  and  that  across  the  hall  in 
the  department,  the  chairman  had  a  Sorvall  in  his  lab.  But  he  was  an  old-fashioned  guy, 
Ernie  Jawetz.  His  Sorvall  was  absolutely  spotless  and  never  used  and  never  touched  and  he 
would  not  let  us  use  it.  I  got  to  use  it  sometimes  because  he  thought  that  I  was  neater  and 
cleaner  than  the  rest.  It  was  really  sexist,  [laughter]  The  Sorvall  was  actually  under  a 
plastic  sheet.  Here  we  were  slaving  across  the  hall,  people  lined  up  trying  to  use  this  other 


piece  of  equipment,  hopelessly  overloaded.  That's  the  kind  of  atmosphere;  that  probably 
tells  a  lot. 

Hughes:  Was  there  tension  between  the  two  labs? 

Betlach:  Sure.  Not  only  between  Herb  and  Jawetz,  but  also  with  the  whole  lab. 

Hughes:  You  mean  one  lab  against  the  other? 

Betlach:  Well,  he  didn't  really  have  very  many  people  in  his  lab.  All  I  can  remember  him  doing  was 
walking  around  in  his  white  lab  coat.  I'm  trying  to  remember  the  names  of  the  people 
involved  in  setting  up  the  microbiology  classes,  people  pouring  agar  plates  and  so  on. 

Hughes:  There  was  a  woman  named  Hanna. 

Betlach:  Lavelle  Hanna.  White  hair,  yes.  And  then  there  was  another  one  with  dark  hair  and 

glasses.  I  can't  remember  her  name.  I  liked  her  a  bit  better.  They  were  all  sort  of  remote, 
that  threesome.  Not  very  friendly,  and  like  old-time  microbiologists  from  the  '30s  or 
something.  Just  a  different  generation. 

Hughes:  And  working  on  chlamydia,  weren't  they? 

Betlach:  I  don't  even  know  what  they  were  working  on. 

Hughes:  That  says  something  in  itself  about  the  lack  of  communication. 

Betlach:  Yes,  I  didn't  even  know  what  he  was  working  on.  It  didn't  seem  to  me  like  it  was  working, 
[laughter]  He  was  just  being  the  chairman.  Low  key.  We  were  turned  up  a  couple  of 
notches  from  them.  Some  resentment  there. 

They  did  do  some  remodeling  on  one  of  the  labs  and  made  an  office  for  Herb  when  I 
was  there.  They  did  do  that  for  him. 


Hughes:  So  the  gist  is  that  Dr.  Boyer  didn't  feel  particularly  supported? 

Betlach:  I  think  that's  probably  true. 

Hughes:  A  classical  microbiologist  might  have  recoiled  when  you  mentioned  molecular  biology. 

Betlach:  But  there  really  wasn't  molecular  biology  back  then. 

Hughes:  Why  do  you  say  that? 

Betlach:  I  think  of  molecular  biology  as  being  more  modern,  using  recombinant  DNA  technology 
and  cloning  and  working  on  the  molecular  level.  We  weren't  working  on  the  molecular 
level  at  first.  We  were  doing  restriction  modification  studies  in  vivo,  which  was  more 
similar  to  what  Jawetz  was  doing.  But  then  we  were  starting  to  leap  forward,  doing  in  vitro 
studies,  getting  more  to  the  molecular  level,  see?  And  Jawetz  wasn't  doing  that. 

Hughes:  How  were  you  thinking  of  yourselves? 

Betlach:  What  do  you  mean? 

Hughes:  What  were  you?  Were  you  geneticists? 

Betlach:  Well,  each  person  in  that  group  had  a  different  background.  Bacterial  geneticists  and 

biochemists  are  probably  what  we  thought  of  ourselves  as.  We  were  just  doing  what  we 
thought  was  interesting.  And  you  make  the  leap,  and  you  have  an  idea,  and  you  test  it.  As 
I  said  to  you  on  the  phone,  I  feel  like  the  work  that  I've  been  doing  almost  at  every  time  of 
my  life  has  been  inherently  interesting. 

It  was  really  obvious  when  we  started  to  clone  DNA  from  other  organisms—it  was  the 
first  time  that  that  was  being  done—that  that  was  a  large  advancement.  We  didn't  know  all 
that  was  going  to  come  out  of  it,  but  we  recognized  that  it  was  important.  Herb  really  early 
on  was  talking  about  cloning  insulin.  And  I  used  to  think,  that's  a  little  bit  far-fetched! 


And  all  kinds  of  jokes  about  cloning  frog  DNA.  Are  the  bacteria  going  to  croak?  Are  they 
going  to  be  green?  It  was  fun. 


Atmosphere  of  the  Boyer  Lab 

Hughes:  Talk  about  the  atmosphere  of  the  lab.  Was  there  interchange  at  all  levels?  Everybody 
working  together  and  exchanging  ideas? 

Betlach:  Within  the  lab  it  was  terrific.  It  was  incredible.  I  was  in  that  lab  as  a  technician.  I  didn't 
have  a  Ph.D.  and  I  was  treated  as  an  equal.  There  was  a  free  flow  of  ideas.  It  didn't  matter 
what  level  you  were  at.  There  was  just  a  really  good  combination  of  people  and  there  was  a 
lot  of  free  interchange  of  ideas. 

Any  idea  I  had  was  equal  to  any  idea  Herb  had  or  any  idea  that  any  postdoc  had.  It  was 
a  real  exciting  time. 

Hughes:  Were  you  working  long  hours? 

Betlach:  Yes.  And  weekends. 

Hughes:  Weekends,  really?   That's  just  what  you  did? 

Betlach:  Yes,  right!  The  work  was  interesting.  You'd  come  in  to  follow  something  through  because 
you  had  an  experiment  that  was  in  the  middle  of  being  done.  You  didn't  even  think  twice. 

I  was  married  at  the  time  and  my  husband  was  pretty  mad  about  it.  He  wanted  me  to  be 
home  at  five  or  six  every  night.  I  got  divorced  soon  afterwards.  He  was  sort  of  jealous  that 
my  work  was  so  gratifying  to  me  and  that  the  intellectual  atmosphere  of  the  lab  was  so 
stimulating  to  me.  It  was  an  extremely  interesting  place  to  be. 

Hughes:  Was  it  unusual  to  have  such  free  interchange  regardless  of  who  you  were? 


Betlach:  Not  real  unusual.  I  think  you  could  find  it  in  a  number  of  places.  I  always  sought  out  that 
kind  of  environment,  and  I  had  had  it  in  other  places  before  I  came  to  Herb's  lab.  In 
Jawetz's  lab  it  would  be  unusual. 

Hughes:  When  you  moved  to  the  Department  of  Biochemistry  you  found  the  same  free  flow? 

Betlach:  Sure.  If  it's  a  good  lab,  it  will  be  that  way.  It  could  be  a  good  lab  and  not  be  that  way  but 
then  I  wouldn't  want  to  be  in  it.  When  I  was  at  the  University  of  California,  Santa  Barbara, 
in  Ed  Orias's  lab,  it  was  also  that  way,  but  we  weren't  breaking  ground  in  quite  the  same 
way  as  we  were  in  Herb's  lab.  And  at  Hatch  Echols's  lab  at  Wisconsin  it  was  also  that  way. 

Hughes:  You  felt  that  you  were  at  the  cutting  edge? 

Betlach:  Yes,  but  I  felt  that  at  other  times.  As  I  said,  I  always  feel  like  the  work  I'm  doing  is 

inherently  interesting  and  is  on  the  edge  or  is  interesting  to  me,  and  that's  enough  for  me. 


Recombinant  DNA  Technology 
Early  Development 

Betlach:  The  part  where  it  made  a  difference  was  when  we  could  see  recombinant  DNA  technology 
was  going  to  have  these  really  broad  applications.  And  that  added  another  level  of 
excitement  until  we  started  getting  negative  press.  That  was  kind  of  bad. 

It  was  exciting  to  think  about  what  the  applications  were,  the  type  of  things  we  could  do. 
Everybody  was  sending  us  DNA  which  we  were  cloning  like  mad  once  we  finished  cloning 
the  Xenopus  DNA.  And  so  there  was  just  an  incredible  variety  of  experiments  going  on 
and  people  contacting  us  from  all  over  the  world. 


Hughes:  Follow  that  through  a  bit  more  systematically,  perhaps  beginning  in  1972  when  you 

arrived.  The  first  cloning  paper  was  published  in  1973,1  so  it  appeared  pretty  close  to  your 
arrival. 

Betlach:  Yes.  I  came  and  I  started  to  purify  restriction  enzymes.  Bob  Helling  was  there  and  he  was 
running  plasmids  on  tube  gels.  Herb  had  started  this  collaboration  with  Stan  Cohen.  They 
put  together  the  idea  of  restriction  enzymes,  plasmids,  maybe  we  can  do  something.    Yes, 
that  was  a  pretty  exciting  idea.  Annie  Chang  and  I  both  did  that  experiment.  I  can't 
remember  if  we  cut  it  and  they  ligated  it,  but  I  know  for  sure  that  I  screened  the  clones 
because  I  had  the  original  DNAs  for  a  long  time  and  I  remember  doing  it,  and  we  had  really 
crude  ways  of  doing  it.  We  had  to  work  out  methods  for  purifying  plasmid  DNA  from 
these  clones  in  order  to  characterize  them.  The  technology  has  since  advanced  a 
tremendous  amount. 

Hughes:  So  you  had  to  invent  that  methodology?    It  wasn't  in  the  literature. 

Betlach:  Oh,  yes.  In  fact,  I  still  have  some  of  our  original  procedural  write-ups2,  and  we  kept 

actively  improving  them  all  of  the  time.  When  these  procedures  got  out  into  other  people's 
hands,  they  developed  faster  and  faster.  My  feeling  was  once  I  got  any  given  method  to  the 
point  where  I  could  get  what  I  wanted  out  of  it  at  a  reasonable  pace,  I  didn't  want  to  spend 
any  more  time  working  on  the  methods.  I  wanted  to  move  on  and  actually  do  the  work. 

I  have  a  procedure  handwritten  by  Herb,  "The  Betlach/Boyer  Plasmid  Purification 
Procedure."  It's  two-pages  long  and  involves  many  time-consuming  steps,  such  as  using  a 
flash  evaporator  for  one  sample.  And  now  it's  just  a  lot  more  streamlined  and  you  can  buy 
commercial  kits  to  do  this.  It  was  just  like  the  Dark  Ages  what  we  were  doing.  It  was 
pretty  exciting. 


'  S.N.  Cohen,  A.C.Y.  Chang,  H.W.  Boyer,  and  R.B.  Helling,  "Construction  of  Biologically 
Functional  Bacterial  Plasmids  In  Vitro, "  Proceedings  National  Academy  of  Sciences  1973,  70:  3240- 

44. 

2See  appendix. 


10 


Authorship  of  the  First  Paper 

Hughes:  Why  wasn't  your  name  on  the  paper? 

Betlach:  Well,  Herb  has  said  a  lot  of  times  since  then  that  my  name  should  have  been  on  that  paper, 
and  I  think  it  probably  should  have  been  because  certainly  I  made  at  least  as  equal  a 
contribution  as  Annie  Chang  did.  I  have  to  say  that  I  am  a  different  person  now  than  I  was 
then,  and  I  didn't  think  then  to  speak  up  to  say,  "My  name  should  be  on  this  paper." 
Whereas  now,  I  would.  This  was  unfortunate  for  me,  but  I  was  pretty  young  and  naive. 

Hughes:  It  wouldn't  stand  out  if  Annie  Chang's  name  wasn't  on  the  paper. 

Betlach:  I  know.  I  was  just  trying  to  remember  how  long  I  had  been  there.  Maybe  at  that  time  I  was 
fairly  new  and  Herb  couldn't  predict  how  much  of  a  contribution  I  was  going  to  make  and 
how  dedicated  I  was  going  to  be. 

Hughes:  Well,  that  paper  had  to  have  been  published  before  June  of  1973  when  Dr.  Boyer  talked 
about  cloning  DNA  at  the  Gordon  Conference  [on  Nucleic  Acids].1 

Betlach:  I  started  in  72,  in  the  fall,  so  it  could  very  easily  be  what  I  said. 
Hughes:  That  you  may  not  have  been  in  the  lab  for  more  than  just  a  few  months? 
Betlach:  Yes.  But  I  did  do- 
Hughes:  You  did  the  work. 

Betlach:  Yes,  it  also  may  have  been  what  I  said.  And  as  you  said,  the  omission  wouldn't  be  so 

glaring  if  Annie  Chang  also  wasn't  on  there.  And  also  if  my  contributions  since  then  hadn't 
been  what  they  were. 


'The  first  paper  on  cloning  was  published  in  November  1973. 


11 


Contemplating  Commercial  Applications 

Hughes:  Were  the  potential  commercial  applications  of  what  you  were  doing  immediately  apparent? 

Betlach:  Well,  Herb  kept  saying,  and  I  can't  put  a  date  on  it,  "We  can  probably  clone  insulin."  Or, 
"This  could  have  benefit  for  society."  So  I  have  to  say  he  was  probably  thinking  that  way. 
I  wasn't.  I  was  just  enjoying  the  pure  science. 

Hughes:  But  it  wasn't  clear  for  some  time  that  you  could  actually  express  proteins,  was  it? 

Betlach:  Yes,  probably  not.  But  I'm  telling  you,  Herb  was  saying  insulin  really  early.  You  can  ask 
him  and  I'd  be  curious  if  he  could  remember  exactly  when,  but  I  just  know  he  was  thinking 
about  that  long  before  anybody  else. 

Although  if  you  think,  okay  we  can  clone  human  DNA,  fly  DNA,  whatever  it  is,  in 
bacteria,  it  is  not  a  big  conceptual  leap  to  expression.  You  would  think,  maybe  we  can 
express  it;  we  can  grow  a  lot  of  bacteria  and  they'll  be  little  factories.  Although  we  didn't 
even  know  that  much  about  promoters  or  regulation  of  expression  then! 

Cloning  Eukaryotic  DNA 

Hughes:  Well,  do  you  want  to  talk  about  the  frog  [Xenopus  laevis]  work?  It  did  cause  a  stir. 

Betlach:  Well,  yes.  I  can  remember  it  fairly  clearly.  I  can  remember  what  room  I  was  in  when  I  did 
it,  of  all  the  three  labs.  In  our  tenure  there,  I  moved  around  to  various  rooms  and  I  can 
remember  that  there  weren't  very  many  people  there  yet.  And  for  some  reason  I  can 
remember  what  the  plates  looked  like,  probably  because  it  was  a  unique  experiment  at  that 
time  and  it  was  done  differently  than  other  experiments.  I  remember  handling  the  clones 
and  making  the  DNA.  I  remember  pretty  clearly.  I  guess  that  says  that  it  was  different. 
Because  that  was  a  long  time  ago. 


12 

Hughes:  Was  there  excitement  when  you  saw  that  it  actually  did  work? 
Betlach:  Sure!  Yes.  [laughs] 

Reconstructing  the  Experiments 

Hughes:  Talk  more  about  the  technical  parts  of  it.  How  did  you  single  out  the  clones  that  actually 
had  inserts? 

Betlach:  What  we  had  to  do  was  make  plasmid  DNA  from  the  clones-  This  reminds  me  a  little  bit 
of  those  depositions  that  I've  been  at  when  they're  asking  me  to  reconstruct  experiments, 
[laughter] 

Hughes:  Sorry  about  the  bad  memories! 

Betlach:  No,  not  bad  memories.  It's  just  that  I've  done  a  lot  of  experiments  since  then.  I'm  just 

trying  to  reconstruct  and  I'm  sure  there's  some  gaps,  but  I  can  remember  we  made  plasmid 
DNA.  We  grew  up  a  liter  of  each  clone.  A  liter,  you  know?  Now  we  grow  a  couple  of 
milliliters.  It  was  horrendous. 

Hughes:  Why  so  much? 

Betlach:  Because  there  was  really  no  method  for  doing  it  and  we  were  trying  to  work  out  a 

procedure.  Also,  we  thought  that  the  DNA  could  be  used  as  substrate  for  the  restriction 
enzymes  that  we  were  purifying.  One  of  Herb's  major  interests  at  the  time  was  in 
characterizing  the  EcoRl  restriction  enzyme,  and  these  plasmids  could  be  used  as  a 
substrate  for  the  enzyme.  But  gee  whiz,  one  liter  and  you  have  enough  DNA  forever, 
[laughter] 

Actually  that  became  apparent  to  me  sooner  than  it  did  to  Herb.  Because  I  remember 
grumbling  and  thinking,  we  should  grow  up  less.  He  kept  insisting,  "We  can  use  this  as 


13 


substrate  for  the  enzyme."  So  I  was  already  thinking,  before  him,  that  this  procedure  has 
got  to  be  scaled  down  because  it  was  in  my  hands  and  it  was  really  horrendous. 

Now  what  else  was  I  talking  about?  Making  the  DNA.  You  purify  the  DNA  and  then  it 
would  have  to  be  run  on  the  gel  and  stained.  We  were  doing  these  polyacyrlamide  tube 
gels  and  they'd  have  to  be  stained. 

Hughes:  Why  tube  gels? 

Betlach:  Now,  Bob  Helling  was  the  one  that  set  that  up  in  our  lab.  And  he  was  doing  it  to 

characterize  the  plasmids  found  in  different  bacterial  strains,  and  I  think  he  published  some 
papers  on  that  work.  I'm  just  trying  to  remember  how  that  apparatus  developed.  Maybe 
tube  gels  were  developed  for  some  other  purpose  and  he  adapted  them. 

m 

Betlach:  I  remember  when  Pat  Greene  came,  she  established  an  agarose  method  in  our  lab.  We  were 
trying  to  figure  out  at  first  how  to  take  photographs  of  these  gels.  Now  companies  sell 
special  equipment  for  this.  We  were  going  down  to  the  UCSF  photography  department  and 
trying  different  filters  and  lights  so  we  could  get  good  pictures  and  documentation  of  the 
gels.  We  were  having  to  work  all  of  that  out  every  step  of  the  way.  But  that's  the  way 
science  is.  Now,  it  just  seems  like  the  Dark  Ages  because  these  things  have  evolved  so  far. 
Most  of  the  time  I  don't  think  anything  about  it,  but  other  times  I  just  feel  like  some  old 
fossil.  I  was  one  of  or  the  first  cloner.  And  here  I  am  still  doing  it. 

Hughes:  What  kind  of  reaction  did  you  get  from  the  scientific  community? 
Betlach:  At  what  stage? 

Hughes:  Well,  the  way  I  read  it,  and  I  would  like  your  opinion,  is  that  DNA  cloning  wasn't  making 
too  much  impression  on  the  science  community  until  the  Gordon  Conference. 

Betlach:  When  exactly  was  that?  I  didn't  go  to  the  Gordon  Conference. 


14 


Hughes:  That  was  June  of  1973.  Dr.  Boyer  had  an  agreement  with  Dr.  Cohen  that  Dr.  Boyer  would 
not  say  anything  until  the  paper  was  published,  which  was  in  November  1973.  In  the 
enthusiasm  of  the  moment,  or  whatever— I  don't  know  what  his  motivations  were—he  did 
talk  about  it.  It  fell  flat  until  somebody  at  the  Gordon  Conference  picked  it  up  and  then  the 
implications  became  clear.  A  result  was  the  Singer-Soil  letter. 


Reporting  the  Discovery 

Hughes:  Do  you  remember  talk  about  keeping  the  research  quiet  until  it  was  published? 

Betlach:  No,  I  can't  remember  about  that  specific  instance,  but  I  can  probably  shed  some  light  on 
why  it  might  have  happened  like  that  and  on  Herb's  personality,  which  might  help  you.  I 
don't  like  my  postdocs  to  talk  about  work  unless  it's  actually  been  submitted  for 
publication.  If  it's  submitted,  it's  okay.  It's  better  if  it's  accepted,  but  at  least  if  it's 
submitted,  if  they're  going  to  reject  it,  at  least  we've  got  something  that  we  can  rework  and 
send  back.  But  some  labs  have  policies  that  you're  not  supposed  to  talk  about  work  until 
it's  published.  So  it  sounds  like  there  was  a  difference  in  opinion  between  Herb  and  Stan 
Cohen. 

Herb  is  the  kind  of  guy  that  never  held  anything  back.  He's  not  as  uptight  as  I  am  about 
that.  As  soon  as  something  happens,  he  doesn't  care  if  it's  written  up,  he  wants  to  talk  about 
it.  This  can  be  a  disaster,  but  on  the  other  hand  it  really  helps  the  flow  of  scientific  ideas. 
Also,  Herb  was  not  really  good  about  getting  things  written  up.  [laughs]  Stuff  would  end 
up  being  written  up  in  weird  places,  proceedings  of  meetings  and  stuff  like  that.  I  realized, 
he  would  rather  do  his  science  and  not  the  writing. 

Hughes:  Once  he  wrote  it  up,  he  didn't  necessarily  put  it  in  the  best  journal? 

Betlach:  No.  He  didn't  really  care.  Some  of  our  stuff  was  published  in  the  weirdest,  funkiest  places. 
Some  of  the  stuff  should  have  gone  to  really  premiere  journals,  and  didn't.  Now,  years 
later,  if  I  publish  work  in  meetings  proceedings,  it's  usually  review-like  in  nature  and 


15 


contains  little  really  new  data.  I  don't  think  it's  that  Herb  didn't  feel  confident  in  the  work;  I 
think  it's  just  that  he  didn't  like  to  write  that  much. 

Hughes:  Well,  the  1973  paper  was  published  in  the  Proceedings  of  the  National  Academy  of 
Sciences. 

Betlach:  Well,  that's  good. 

Hughes:  Why  would  they  have  chosen  that  journal? 

Betlach:  That's  a  good  journal  and  it  has  a  really  wide  readership  and  it's  an  appropriate  place.  So 
that  indicates  they  knew  it  was  important— Stan  Cohen,  I'd  say.  I  just  feel  Herb  doesn't 
control  his  contributions  into  the  literature  that  much.  He  likes  to  got  to  meetings  and  talk 
about  it  and  tell  people  about  it.  He  likes  to  do  the  science.  I  bet  that  was  Stan  Cohen's 
decision.  But  don't  tell  Herb  I  said  that,  [laughs] 

Hughes:  Are  there  differences  in  scientific  styles  between  the  two  men? 

Betlach:  Yes.  Stan  Cohen  is  a  little  more  tense  and  controlling.  We  had  Herb's  retirement  party  a 
couple  of  years  ago,  and  Stan  came  to  it  and  it  was  really  nice.  A  lot  of  people  came  to  it. 
It  was  really  fun. 

Hughes:  They  don't  see  each  other  very  much  now? 

Betlach:  No. 

Hughes:  Did  the  collaboration  end  after  the  cloning  work? 

Betlach:  Not  much  happened  after  that.  Maybe  some  minor  things,  but  nothing  that  I  can  remember 
specifically. 


16 


Biosafety  Concerns 

Hughes:  Relating  to  the  recombinant  DNA  debate  is  a  paper  by  Dr.  Cohen  stating  that  transfer  of 

DNA  occurs  under  natural  circumstances.  This  was  supposed  to  allay  the  fear  of  breaching 
species  barriers  and  the  potential  hazard  of  recombinant  DNA  research. 

Betlach:  Yes,  and  that  transfer  does  occur. 

Hughes:  Was  it  a  big  deal  that  he  showed  experimentally  that  it  could  happen? 

Betlach:  I  can't  exactly  place  that  piece  of  work  to  this  time.  But  we  were  not  worried  at  all  at  first, 
and  then  it  became  clear  that  maybe  there  might  be  a  reason  to  worry. 

Hughes:  When  and  why? 

Betlach:  There  was  the  meeting  at  Asilomar  [February  1975].  I  remember,  I  was  doing  a  lot  of 
cloning  of  DNA  from  all  kinds  of  organisms,  [laughs]  It  was  like  the  zoo.  We  just  put 
them  on  the  shelf;  we  just  quit;  we  just  stopped.  I  can't  tell  you  exactly  when. 

Hughes:  You're  not  sure  that  it  was  after  Asilomar? 

Betlach:  I  think,  if  anything,  it  might  have  been  before.  As  soon  as  there  was  any  clue  at  all,  before 
that  meeting.  But  I  just  can't  recall  exactly.  I'd  have  to  look  through  my  old  notebooks, 
which  I  no  longer  have.  Three  years  of  my  notebooks  disappeared  at  some  point. 

Hughes:  Really. 

Betlach:  And  I  am  very  careful  about  my  notebooks.  I  gave  a  lot  of  them  to  Herb  when  I  left  UC. 
In  some  of  the  patent  contests  that  have  been  going  on  between  Lilly  and  Genentech  and 
UC,  I  was  deposed  a  couple  of  times.  The  last  one  I  was  up  for,  they  asked  me,  "Do  you 
know  what  happened  to  your  notebooks  between  the  years  1976  and  1979?"  I  think  those 


17 


were  the  years.  And  they're  just  gone.  There  was  an  earlier  stage  when  I  was  giving 
testimony  when  they  were  there,  and  large  chunks  of  them  were  copied  by  various  people 
involved. 

Hughes:  But  you  got  them  back? 

Betlach:  I  got  them  back  after  parts  of  them  were  copied,  and  then  they  subsequently  disappeared.  I 
never  lose  anything  like  that.  I  have  every  single  notebook  I  ever  had,  except  for  those  that 
were  lost  and  the  ones  I  gave  to  Herb. 

You  asked  if  we  were  aware  of  the  possible  dangers. 
Hughes:  Yes.  Can  you  recreate  the  feeling  of  that  time? 
Betlach:  Were  we  really  worried,  you  mean? 

Hughes:  Yes,  and  how  did  you  react  to  the  dissenting  scientists  and  the  environmental  groups  that 
were  activated  on  this  subject. 

Betlach:  Well,  I  can  only  speak  for  myself.  I  felt  that  there  probably  wasn't  any  danger,  but  I  didn't 
know,  okay?  Especially  human  clones,  you  just  really  didn't  know.  So  we  put  them  on  the 
shelf.  Probably  it  would  have  been  better  to  autoclave  them. 

When  we  got  so  that  we  had  to  certify  vectors  and  things,  we  went  through  some  really 
tedious  testing,  putting  them  in  crippled  bacterial  strains,  to  make  sure  that  there  wouldn't 
be  any  danger.  And  if  you  sat  and  thought  for  a  couple  of  minutes:  these  genes  are  in 
bacteria.  Are  you  going  to  inhale  them?  Or  maybe  you're  going  to  get  them  in  your  gut. 
Your  body's  going  to  have  defenses  against  these  kinds  of  things  and  probably  nothing  is 
going  to  happen. 

We  were  aware  that  there  might  be  mechanisms  that  we  didn't  know  anything  about,  so 
we  were  as  careful  as  we  could  be.  And  I  think  it  was  overkill.  Even  then  I  was  thinking 
what  we  had  to  do  and  all  of  this  furor  about  P3  [physical  containment  lab  level  3].  I  was 


18 


far  more  afraid  of  working  with  hepatitis  virus  or  Rous  sarcoma  virus,  like  Mike  Bishop 
was  working  with  at  the  time.  Tissues  from  diseased  patients,  I  think,  were  much  more 
dangerous. 

Hughes:  So  you  weren't  particularly  afraid  for  yourself? 

Betlach:  No,  I  wasn't  worried.  But  I  was  young  and  did  all  kinds  of  things  in  those  days,  like  not 
using  gloves  when  using  ethidium  bromide  and  using  huge  amounts  of  P32  [radioactive 
phosphorus].  Now  I  wouldn't  do  such  things. 

Hughes:  It  wasn't  an  awareness  in  that  era? 
Betlach:  I  wasn't  the  only  person,  [laughs] 
Hughes:  It  was  standard  behavior,  is  what  I'm  asking;  it  wasn't  that  you  were  a  risk-taking  person? 

Betlach:  No.  Ethidium  bromide,  I  just  didn't  know;  I  thought  it  was  okay.  When  I  learned  otherwise 
I  started  wearing  gloves.  Radioactivity  I  should  have  probably  known  better  and  times 
change  and  people  use  less  radioactivity  in  experiments  now.  But  these  clones— I  didn't 
think  there  was  any  danger.  But  I  didn't  know  for  sure.  I  remember  clearly  having  this 
feeling,  especially  with  human  DNA:  there  may  be  some  mechanisms  I  don't  know. 
Okay,  we'll  just  put  these  away,  and  we've  got  plenty  of  other  things  to  work  on.  And  we 
did.  That's  my  personal  feeling. 

Hughes:  Do  you  know  for  what  period  they  were  put  on  the  shelf? 

Betlach:  Oh,  for  a  long  time.  We  completely  shelved  a  whole  bunch  of  experiments.  We  had  been 
sent  DNA  from  people  to  do  experiments  with.  When  specific  experiments  with  human 
DNA,  for  example,  started  up  again,  they  were  P3,  and  each  experiment  was  usually 
assigned  to  one  postdoc.  It  wouldn't  be  just  me  slinging  hash  on  six  things  at  once.  I  was 
much  more  focused  on  making  vectors  by  that  stage.  By  then  we'd  send  vectors  out  and 
the  recipients  would  be  doing  the  cloning  experiments  themselves  in  their  own  labs. 


19 


And  then  we  had  a  couple  of  projects  where  Herb  thought  that  he  was  going  to  get 
interested  in  immunology.  I  remember  him  saying,  "Immunoglobulins  are  going  to  take  up 
the  next  twenty  years  of  my  life."  We  had  a  couple  of  postdocs  who  were  starting  to  work 
on  that,  and  so  I  just  didn't  work  on  any  of  those  experiments  anymore.  I  started  to  make 
vectors  and  improve  vectors,  which  was  kind  of  fun. 

Hughes:  When  you  were  thinking  about  risk,  what  exactly  were  you  thinking  might  happen? 

Betlach:  I  didn't  know.  I  was  a  bacterial  geneticist.  At  that  time  I  didn't  know  anything  at  all  about 
eukaryotic  systems,  except  in  very  gross  terms.  In  fact,  not  that  much  was  known  at  the 
molecular  level  on  human  systems.  Period.  However,  I  was  afraid  of  Rous  sarcoma  virus 
and  hepatitis  virus.  I  just  thought,  I  don't  know  about  this,  and  there  may  be  mechanisms 
that  we  don't  know  about  and  it's  just  not  worth  it.  I  wasn't  terrified  by  it.  I  recognized  that 
we  didn't  really  know  all  that  was  going  on.  Actually,  if  there  wasn't  a  lot  of  fuss  about 
stopping  it,  I  probably  would  have  just  done  it,  but  it  would  have  made  me  a  little  uneasy. 

Hughes:  Presumably  you  started  again  when  the  NIH  recombinant  DNA  guidelines  were  weakened 
and  such  experiments  were  now  permissible. 

Betlach:  Yes,  but  that's  the  period  when  I  said  that  I  was  focusing  on  something  else  besides  cloning 
the  DNA  everybody  was  sending.  Individual  postdocs  had  projects  where  they  would  work 
with  one  sample  of  DNA.  At  the  early  stage,  before  the  Asilomar  meeting,  we  were  the 
only  place  that  was  doing  it.  So  people  were  sending  us  DNA,  and  we  were  cloning  all 
kinds  of  things.  Later,  we  started  to  focus  on  interesting  new  projects  that  would  use  this 
tool. 


20 
Plasmid  Vectors 

PI  asm  id  Development  in  the  Boyer  Lab 

Hughes:  Well,  talk  about  vector  development. 

Betlach:  Sure.  Now  let  me  shift  gears.  Of  course,  we  wanted  to  develop  vectors  that  would  be 
widely  useful.  I  know  that's  what  Paco  Bolivar  and  Ray  Rodriguez  wanted  to  do.  My 
feeling  was,  I  want  to  make  a  vector  I  can  use  to  do  things  I'm  interested  in.  I  don't  want  to 
make  vectors  for  the  rest  of  the  world. 

Hughes:  They  were  in  the  Boyer  lab  to  make  vectors  rather  than  to  pursue  their  own  research 
interests? 

Betlach:  Many  people  who  joined  the  lab  at  this  time  came  to  learn  the  new  technology.  I  don't 
know  the  specifics  reasons  they  came  for.  When  they  arrived  I  was  already  developing 
new  vectors,  and  then  they  took  ones  I  had  made  and  refined  them  more. 

Hughes:  Go  into  exactly  how  one  does  that.  Do  you  want  to  use  pMB9  as  an  example? 

Betlach:  What  they  wanted  to  do  was  to  make  the  plasmid  easier  to  use,  so  it  would  have  more 

general  utility.  Put  more  antibiotic  resistance  markers  on  it,  so  that  you  could  clone  in  one 
antibiotic-resistant  gene  and  inactivate  it  and  select  for  the  other  marker.  We  could  already 
do  that  with  pMB9,  which  has  the  markers  tetracycline  resistance  and  colicin  immunity. 
However,  colicin  immunity  is  a  tricky  marker  to  use. 

They  wanted  to  use  ampicillin  resistance  and  then  the  idea  was  you  would  clone  in,  for 
example,  the  tet  gene  and  your  transformants  would  be  still  ampicillin  resistant.  But  they'd 
be  tet  sensitive  and  then  you'd  have  a  way  to  tell  which  clones  were  which  without  looking 
at  the  DNA.  So  it's  actually  a  screen.  And  they  wanted  to  make  the  plasmid  smaller,  take  it 
down  to  the  basic  elements  so  you  have  less  DNA  there,  and  make  maps  of  it  so  you  know 
all  the  sites  in  it.  Then  Greg  Sutcliff  sequenced  the  entire  thing,  pBR322. 


21 
Hughes:  What  about  sequencing  techniques  at  that  time? 

Betlach:  Primitive,  primitive!  John  Shine  was  doing  some  and  Joel  Hedgpeth  did  some  too.  It  was 
primitive  and  very  difficult  compared  to  now,  just  like  everything  else  we  were  doing  then. 

But  the  way  we  got  started  on  doing  vectors  was  we  had  originally  pSClOl  which  came 
from  Stan  [Stanley  N.]  Cohen's  lab  that  was  tet  resistant  and  did  not  have  very  many  copies 
per  cell,  so  you  didn't  get  a  lot  of  DNA.  It  was  big  and  difficult  to  use. 

Meanwhile,  the  problem  I  was  interested  in  was,  I  wanted  to  clone  the  EcoRl 
endonuclease  genes  onto  a  multi-copy  plasmid.  Hopefully  then  it  would  be  expressed  at 
higher  levels.  We'd  have  an  over-expressing  strain  so  when  we  made  restriction  enzymes 
we'd  get  a  lot  more  enzyme.  I  tried  to  isolate  and  clone  the  .EcoRl  endonuclease  and 
methylase  genes,  and  it  turns  out,  they  were  already  on  a  multi-copy  plasmid.  Actually, 
that's  an  interesting  story,  too. 

Isolation  of  EcoRl  and  the  Development  of  Betlach  Plasm  ids 

Betlach:  EcoRl  was  originally  isolated  from  a  clinical  isolate  from  the  UC  labs.  You  can  ask  Herb 
about  this.  It  was  isolated  from  someone's  urinary  tract  infection,  [laughs]  Multiple-drug 
resistant  E.  coli  organism.  A  student  of  Herb's  named  Bob  Yoshimori  got  that  strain  and 
worked  on  it  first  a  little,  and  then  left. 

So  I  took  that  strain  and  was  trying  to  clone  these  genes  from  it.  I  characterized  it  and 
we  found,  as  I  said,  that  the  Rl  genes  were  already  on  this  multi-copy  plasmid.  And  then  I 
cloned  the  methylase  gene.  That  was  probably  the  second  cloning  experiment  done 
anywhere.  This  plasmid,  it  turns  out,  was  multi-copy,  and  then  we  also  had  pSClOl,  this 
large  low  copy  number  plasmid  from  Stan  Cohen  which  was  difficult  to  use  but  that  had  tet 
resistance  on  it. 

The  first  plasmid  that  I  isolated  from  the  clinical  isolate  I  named  pMB  1 .  It  was  big  too, 
but  it  was  multi-copy,  so  we  tried  to  get  it  down  a  little  bit  in  size.  We  didn't  have  very 


22 


many  restriction  enzymes  at  that  time,  so  I  took  pSClOl  and  pMB  1  and  did  an  EcoR.1  star 
digest  which  is  a  decreased  specificity  for  the  EcdRl  site.  This  probably  all  means  nothing 
to  you.  It  was  like  a  witch's  brew,  okay?  We  didn't  have  a  lot  of  tools  to  work  with  and  I 
just  sort  of  mixed  things  together,  selected  for  tet  resistance,  and  hoped  for  the  best.  Out 
came  a  bunch  of  clones,  one  of  which  I  named  pMB9,  that  contained  the  tet  gene  from 
pSClOl,  but  the  origin  of  replication  from  the  multi-copy  plasmid,  pMBl. 

Hughes:  Do  you  do  the  procedure  very  deliberately  so  that  you  know  that  you're  going  to  get  the  tet 
gene? 

Betlach:  Not  exactly.  I  selected  for  tet  resistance,  but  a  fairly  random  mixture  of  fragments  was 
used.  There  were  not  very  many  restriction  enzymes  and  no  restriction  maps  yet.  There 
was  Hind3  and  there  was  EcoRl.  If  you  react  EcoR.1  under  certain  conditions,  you  get  a 
decreased  specificity,  so  it  hits  in  more  places.  But  it's  almost  random,  so  I  digested  a 
mixture  of  pSClOl  and  pMBl  with  this  fairly  random-cutting  enzyme.  I  put  a  selection  on 
for  tetracycline  resistance.  I  selected  for  that  so  it  would  pull  a  little  fragment  containing 
tet  resistance  out  of  the  mixture.  I  always  felt  like  it  was  a  witch's  brew,  and  I  guess  I  was 
the  witch,  [laughter] 

I  remember  I  got  this  set  of  clones  and  I  ran  them  out  on  a  gel  and  there  was  only  one 
that  looked  any  good.  I  thought,  oh,  I  can't  wait  to  check  that  one  out,  and  I  went  on 
vacation  for  a  month  because  it  was  in  the  middle  of  the  summer  and  I  had  already  made 
these  vacation  plans  and  I  couldn't  find  out  the  result  until  I  got  back.  I  came  back  and  then 
I  characterized  it  and  the  one  promising  clone  (which  I  named  pMB9)  was  good  for  a  lot  of 
things.  I  could  do  a  lot  of  things  with  it  and  so  that's  why  I  stopped  developing  it.  It  was 
small,  it  was  multi-copy,  it  had  tetracycline  resistance  on  it.  It  was  useful.  And  we  gave 
pMB9  out  to  a  lot  of  people. 


23 
Plasmid  Dissemination 

Hughes:  What  is  the  protocol  in  science  for  exchange  of  materials? 

Betlach:  Well,  usually  once  you  publish  information  on  a  strain  or  plasmid  etc.,  you're  morally 

obliged  to  give  it  out.  Before  publication,  you're  really  not.  In  fact,  some  journals  stipulate 
that  you  have  to  give  out  anything  that's  published  in  their  journal.  Not  everybody  does, 
but  we  freely  gave  out  all  sorts  of  materials.  We  gave  out  pMB9  all  over  the  world  before 
anything  was  published  on  it.  It  didn't  bother  me.  I  didn't  care  because  I  wanted  it  to  use  it 
for  experiments  and  it  was  fine.  And  if  it's  useful  for  other  people's  purposes  that  aren't  in 
competition  with  you,  then  why  should  it  bother  you?  So  we  gave  it  out  all  over  the  place. 

Hughes:  Were  there  any  restrictions? 
Betlach:  None  that  I  can  remember. 

Hughes:  Did  you  stop  distribution  for  a  while  when  there  began  to  be  the  concern  about  recombinant 
DNA? 

Betlach:  Well,  we  had  pMB9,  pBR3 13,  and  pBR322,  and  they  were  being  tested.  I  think  we  did 

stop  sending  them  out  until  we  got  the  plasmids  certified.  Or  I  may  have  given  them  out  to 
some  people,  but  told  them  that  we  were  waiting  for  certification  and  they  couldn't  use 
them  before  we  got  it.  I  can't  remember  exactly,  but  I  think  it  makes  sense  that  we  just 
stopped  sending  it  out. 

Hughes:  That  leads  into  the  pBR322  episode.  My  main  source  is  Stephen  Hall's  book,  Invisible 
Frontiers. 

Betlach:  That  one  is  pretty  good.  Accurate. 


24 

Hughes:  Yes.  The  Race  to  Synthesize  a  Human  Gene  is  the  subheading.  He  maintains  that  Dr. 
Boyer  in  January  of  1977  was  very  clear  on  the  distinction  between  an  "approval"  and 
"certification."1  Remember  that  two-step  process? 

Betlach:  I  remember  it.  It's  very  clear  in  my  mind. 

Hughes:  It  apparently  was  not  clear  to  other  people  in  the  department. 

Betlach:  That  may  have  been  the  case. 

Hughes:  Namely,  to  Howard  Goodman  and  William  Rutter. 

Betlach:  Well,  I  don't  know  if  they  were  clear  on  it  or  not,  but  I  definitely  knew  that  it  was  a  two- 
step  process.  People  were  calling  me  up  all  the  time.  I  remember  Herb  was  down  in 
Miami  or  someplace  at  a  meeting,  and  the  plasmid  was  at  NIH  and  they  were  deciding  it 
had  been  approved  and  not  certified,  or  certified  and  not  approved,  now  I'm  temporarily 
confused. 

Hughes:  "Approved"  comes  first.  Certification  was  by  [Donald]  Fredrickson,  the  NIH  director. 

Betlach:  What's  the  name  of  the  other  guy,  Gartland? 

Hughes:  William  Gartland.  He  was  head  of  the  NIH  Recombinant  DNA  Advisory  Committee. 

m 


1  Stephen  S.  Hall,  Invisible  Frontiers:  The  Race  to  Synthesize  a  Human  Gene,  (Redmond, 
WA:  Tempus  Books,  1987),  pp.  116-17. 


25 
The  Bover  Lab  Moves  to  the  Biochemistry  Department 

Hughes:  What  difference  did  you  find  between  the  Department  of  Microbiology  and  the  Department 
of  Biochemistry  when  you  moved  at  the  end  of  1976? 

Betlach:  When  we  moved  up  to  the  fifteenth  floor  in  the  Health  Sciences  Towers? 
Hughes:  Right. 

Betlach:  Well,  we  had  a  giant  new  lab,  and  that  was  great,  but  we  were  up  on  the  fifteenth  floor  and 
we  were  fairly  isolated  at  first.  For  me  it  wasn't  really  that  much  different  because  we 
interacted  just  as  much  with  people  in  the  Biochemistry  Department  [ninth  floor]  when  we 
were  in  the  Microbiology  Department.  There  was  actually  quite  a  bit  of  cross-talk  between 
Microbiology  and  Biochemistry.  The  departments  overlap  a  lot  now.  They  have  the  PIBS 
program—Program  in  Biological  Sciences;  the  graduate  programs  are  shared  in  a  lot  of 
ways.  But  it  was  nice  to  be  away  from  that  oppressive  Microbiology  Department  feeling. 
But  otherwise  I'd  say  the  interaction  with  Biochemistry  stayed  the  same.  And  we  had  these 
gorgeous  labs. 

Hughes:  You  didn't  have  more  collegial  support? 

Betlach:  We  already  had  a  lot  of  collegial  support  from  the  Biochem  department.  So,  I  think  it  was 
the  same.  [William  J.]  Rutter  did  an  incredible  job  of  building  up  that  department.  And 
there's  been  a  lot  of  excitement  and  development  into  other  scientific  areas  since  then.  The 
UCSF  Biochemistry  Department  is  ranked  first  or  second  in  the  nation. 


26 
Use  of  an  Uncertified  Plasmid.  1977 

Hughes:  Well,  we  were  talking  about  the  pBR322  episode.  You  said  you  were  very  clear  on  the 
distinction  between  "approval"  and  "certification." 

Betlach:  Yes,  I  knew  that  we  needed  both.  People  would  call  me  up.  A  lot  of  times  I'd  be  busy  and 
they'd  ask  me  if  pBR322  was  okay  to  use,  and  I'd  say,  "It's  approved,  it's  approved!"  Not 
necessarily  would  they  hear  that  you  need  both  "approved  and  certified."  So  you  could  see 
people  might  take  my  answer  the  wrong  way. 

Hughes:  All  of  this  was  verbal  at  this  stage? 

Betlach:  Yes,  phone  calls. 

Hughes:  What  about  the  insulin  clones? 

Betlach:  All  I  know  is  hearsay.  I  know  that  people  were  calling  me  up  and  I  told  them  what  I  told 
them.  Everybody  had  the  plasmids  because  we  were  so  freely  giving  everything  out. 
You're  asking  me  whether  or  not  the  plasmid  was  used  ahead  of  NIH  certification? 

Hughes:  Yes. 

Betlach:  I  can  give  you  an  opinion.  People  could  have  misunderstood  what  I  told  them  on  the  phone 
and  could  have  used  pBR322  before  it  was  certified.  One  could  imagine  a  lot  of  scenarios. 
And  then,  like  you,  everyone  is  saying  this,  that,  and  the  next  thing.  All  I  can  tell  you  is  my 
personal  feeling  and  I  don't  really  have  anything  to  substantiate  it:  Probably  pBR322  was 
used  and  probably  used  by  mistake  because  there  was  a  misunderstanding. 

Hughes:  And  then  destroyed?  Once  it  was  revealed  that  the  plasmid  had  been  used  before 
certification,  Axel  Ullrich  supposedly  destroyed  the  clones. 

Betlach:  Yes,  but  the  problem  with  that  was  the  experiment  had  to  be  redone. 


27 


Hughes:  Some  people  didn't  believe  that  the  experiment  could  be  repeated  and  submitted  to  Science 
within  three  weeks.  They  questioned  whether  the  original  clones  had  been  destroyed. 

Betlach:  The  experiments  were  pretty  quick  to  do  and  Axel  would  have  all  the  materials  to  do  it. 
He'd  have  the  RNA.  This  is  a  guy  that  worked  night  and  day. 

It  was  really  sort  of  an  incestuous  situation  at  the  time.  All  of  the  postdocs  in  the 
department  were  socializing.  So  you  were  living  with  these  people  all  the  time,  and  there 'd 
be  big  parties,  and  we  partied  pretty  heavily.  All  of  this  was  going  on.  I  knew  Axel  pretty 
well.  He  was  dating  my  roommate  at  the  time.  My  marriage  had  broken  up  and  I  was 
sharing  an  apartment  with  another  divorced  woman. 

I  remember  when  this  other  woman  and  Axel  and  I  watched  this  Nova  show  on  TV, 
recreating  all  of  this.  We  just  sat  there,  silent,  [laughter]  Itwasreally- 

Hughes:  Tense? 

Betlach:  Not  tense.  Just  quiet,  thoughtful. 

Hughes:  Was  the  program  accurate? 

Betlach:  I  can't  remember  how  accurate  it  was.  I  just  know  we  each  knew  our  own  truths,  if  you 
know  what  I  mean. 

Hughes:  You  didn't  ask,  "Axel,  did  you  destroy  the  clones?" 

Betlach:  No,  I  did  not  because  by  that  time  I  think  Bill  Rutter  had  been  sending  around  little  notices 
to  everyone  saying,  "What  do  you  know  about  this?"  Because  he  was  trying  to  figure  out 
what  went  on,  maybe  before  going  to  the  Senate  hearing.  I  didn't  reply  because  I  felt  I 
really  didn't  know  anything,  except  hearsay  and  the  fact  that  when  people  called  me  I  told 
them  X,  Y,  and  Z.  So  I  wasn't  going  to  contribute  to  the  general  paranoia  and  weirdness 
that  was  going  around,  so  I  never  talked  to  Bill  Rutter  about  it. 


28 

Hughes:  Well,  I  saw  Dr.  Ullrich  at  the  Rutter  symposium,  and  I  asked  him  if  he  would  talk  with  me.1 
Betlach:  His  future  wife  Suzanne  also  socialized  with  us. 
Hughes:  What  lab  was  she  in? 

Betlach:  She  was  a  graduate  student.2  Whose  lab  was  she  in?  Reg[is]  Kelly's?  And  now  Axel's  at 
the  Max  Planck  Institute  in  Munich.  I  was  there  at  a  meeting  last  spring  and  I  stopped  by 
his  lab,  but  he  was  in  Spain.  I  just  left  him  a  little  note.  I  haven't  seen  him  in  a  long  time. 
And  the  little  note  I  left  said,  "Here's  a  blast  from  your  past."  [laughter] 

Rivalries 

Hughes:  There  was  quite  a  bit  of  intra-departmental  rivalry  at  that  time,  for  instance,  between 
Goodman's  group  and  Rutter's  group.  There  was  also  Harvard. 

Betlach:  Oh,  yes,  Harvard.  I  remember  we  felt  the  competition  with  Wally  [Walter]  Gilbert's  group 
at  Harvard.  They  put  out  these  little  newsletters  "The  Midnight  Hustler,3"  talking  like  we 
were  sports  teams  or  something.  Those  competitions  were  all  very  good-natured. 

Hughes:  Even  within  the  department? 

Betlach:  Well,  Herb  and  Howard  had  a  falling  out.  We  never  had  any  problem  with  Bill  Rutter,  and  I  have 
a  very  high  opinion  of  him.  Any  problems  there  were  between  him  and  Howard,  I  don't  know 
much  about.  But  I  know  Herb  had  a  falling  out  with  Howard.  We  had  a  lot  of  interaction  with 
everybody  in  Howard's  lab,  up  to  a  certain  point  in  time.  In  fact  it  was  almost  like  the  two  labs 
were  one  lab.  We  had  joint  seminars  and  we  shared  supplies.  There  was  a  lot  of  camaraderie. 


1  An  interview  with  Dr.  Ullrich  was  conducted  on  April  5,  1994  and  will  be  available  after  a 
second  interview  is  recorded. 

Subsequently,  she  got  a  faculty  position  at  Stanford.  [MB] 

3C 


3See  Appendix  H. 


29 
UC's  Contract  with  Genentech  on  Somatostatin 

Hughes:  Did  you  have  any  part  in  the  work  on  somatostatin? 

Betlach:  I  had  a  more  peripheral  role  in  somatostatin.  At  this  time  Herb  had  been  talking  to  Bob 
[Robert  A.]  Swanson.  Genentech  had  been  formed  and  they  wanted  to  start  doing  some 
work.  Somatostatin  was  one  thing  they  wanted  to  start  working  on  and  a  contract  was  set 
up  somehow—all  kosher  with  the  university.  There  was  a  little  bit  of  money  that  was  made 
available  to  the  lab  to  do  this. 

Hughes:  From  Genentech? 

Betlach:  Yes.  And  so,  I  was  put  on  it  at  first,  along  with  Art  Riggs.  He  came  up  from  southern 
California.  Art  Riggs  didn't  know  much  molecular  biology  at  the  time,  and  so  I  had  to 
teach  him.  We  were  doing  the  somatostatin  research  together.  He  sort  of  drove  me  nuts. 

Hughes:  Why? 

Betlach:  Well,  he's  one  of  these  real  methodical  guys.  He's  too  slow.  I  was  very  impatient  with 

him,  and  I'm  not  very  good  at  teaching,  either.  So  we  started  to  work  on  it  for  a  while  and 
then  I  got  off  the  project.  Actually,  I  remember  it  fairly  clearly  because  I  was  deposed  for 
this  pretty  recently  and  they  went  through  all  the  experiments.  "Do  you  remember  you  did 
this  experiment,  etc.?"  but  that's  about  all  there  is  to  it  that  would  probably  be  of  interest  to 
you.  My  role  was  more  peripheral. 

Hughes:  Congress  at  the  time  was  debating  whether  legislation  should  be  passed  to  regulate 
recombinant  DNA  research.  Which,  of  course,  the  scientists— 

Betlach:  Did  not  want. 

Hughes:  The  cloning  of  somatostatin  was  announced  in  a  Senate  subcommittee  hearing.  The  point 
was  to  show  the  commercial  and  medical  possibilities  of  this  technology  and  allay  some  of 
the  scare  talk.   Do  you  remember--? 


30 


Betlach:  No,  I  don't  remember  much  about  that.  All  I  know  is  Rutter  was  collecting  data  points 

from  people  before  he  went  to  the  Senate  hearing.  I  was  talking  to  Herb  every  day,  and  he 
didn't  discuss  with  me  any  strategy  about  what  he  was  going  to  say  or  what  they  were  going 
to  do. 

Hughes:  Did  you  get  the  feeling  that  they  were  preparing  for  the  hearing? 
Betlach:  Just  Rutter. 
Hughes:  But  not  Dr.  Boyer? 

Betlach:  I  didn't  have  any  feeling  that  he  was.  But  I  don't  know.  Herb  has  this  laid-back  style,  so  he 
may  be  preparing  for  something  and  you  don't  have  a  clue,  [laughter]  That's  the  way  he  is. 

Bover  as  a  Lab  Director 

Hughes:  How  true  is  that  laid-back  style? 

Betlach:  It's  true,  but  he's  wonderful  to  work  with  because  he's  really  intuitive.  When  he  was 

interested  in  science  it  was  wonderful  to  have  him  around  because  he's  really  intuitive  and 
he  allows  people  working  under  him  to  have  an  incredible  amount  of  independence  and 
encourages  it.  And  he  encourages  you  to  be  intuitive,  too!  So  there's  these  wonderful  ideas 
bouncing  back  and  forth.  But  he's  very  laid  back.  He's  not  going  to  come  and  tell  you  how 
to  do  things.  I've  heard  it  said  about  him  that  he  gives  you  enough  rope,  just  don't  hang 
yourself  with  it.  I  functioned  really  well  in  that  environment  because  I  never  could  stand 
somebody  breathing  down  my  neck.  Stan  Cohen  seemed  a  little  bit  more  like  that. 
However,  with  Herb  it  was  take  the  ball  and  run. 

Hughes:  How  did  his  laid-back  style  work  with  graduate  students? 

Betlach:  It  depended  upon  the  graduate  student.  Actually,  he  didn't  have  that  many  graduate 

students.  Postdocs  did  better-they  are  more  independent.  Postdocs  thrive  in  that  kind  of 


31 


environment,  but  for  graduate  students  it's  a  little  tough.  Usually  they  floundered,  unless 
they  were  taken  under  a  postdoc's  wing. 

Hughes:  Dr.  Boyer  told  me  he  didn't  like  teaching  medical  students. 

Betlach:  Yes,  he  didn't  like  giving  formal  lectures  to  medical  students,  but  he  was  really  good  at 
teaching  postdocs  in  this  indirect  way  because  he  encourages  independence. 


Tension  over  University  Ties  with  Genentech 

Hughes:  One  more  question  as  I  know  I'm  taking  a  lot  of  your  time:  Talk  about  the  controversy  that 
grew  up  in  the  early  days  of  Genentech  when  it  was  functioning  in  the  biochemistry 
department. 

Betlach:  Bad  feelings.  There  were  a  lot  of  people  in  the  department  looking  down  their  noses  at 
Herb,  saying  that  science  and  industry  should  be  separate.  He  was  blacklisted  by  a  lot  of 
people  in  the  department,  to  be  perfectly  frank.  Finally,  I  think,  they've  come  around  and 
they're  all  involved  in  new  companies  now.  But  it  was  pretty  bad  and  pretty  crummy 
treatment  of  him.  I  thought  Genentech  was  a  pipe  dream,  to  tell  you  the  truth.  I  thought  it 
wouldn't  go  anywhere.  But  it  seemed  to  me,  if  Herb  followed  all  of  the  rules  that  he  had  a 
right  to  try  something  like  that.  If  he  was  fulfilling  his  obligations  at  the  university,  nobody 
had  the  right  to  any  say  about  it.  That's  what  I  thought. 

Hughes:  And  he  was  fulfilling  these  obligations? 

Betlach:  And  he  was.  It  was  really  irritating  to  me  that  these  people  had  this  attitude.  I  continued  to 
be  irritated  about  it  for  the  next  twenty  years  after  that.  Some  of  them  stayed  that  way  for  a 
long  time. 

Hughes:  How  was  it  expressed? 
Betlach:  A  lot  of  it  indirectly. 


32 

Hughes:  Was  Dr.  Boyer  cut  out  of  scientific  interchange? 

Betlach:  No,  people  that  were  interested  in  the  same  things  that  we  were  would  still  interact  with  us. 
No,  it  was  more  people  that  were  working  in  other  areas  of  research.  All  I  can  remember  is 
disapproving  looks,  and  I  don't  know  specifically  what  things  were  done  or  said  to  Herb.  I 
do  know  some,  I'm  sure,  but  I  can't  come  up  with  any  specific  instances  right  now  that  can 
properly  convey  the  feeling  that  there  was. 

Hughes:  How  did  he  react? 

Betlach:  Well,  it  wasn't  fair.  I  remember  I  was  hurt.  I'm  not  exactly  sure  if  he  didn't  give  a  damn  or 
if  it  bothered  him.  Some  of  the  people  he  really  didn't  think  that  much  of,  anyway.    So  if 
they  were  going  to  have  that  opinion,  it  was  their  problem.  But  then  maybe  there  were 
certain  individuals  who  felt  that  way,  and  maybe  it  bothered  him,  so  probably  it  was 
dependent  upon  the  individual. 

Hughes:  Does  he  talk  about  it? 

Betlach:  No,  he  doesn't.  I've  known  him  for  a  long  time  and  I  feel  like  I  know  him  pretty  well.  He's 
laid  back,  but  he  keeps  a  lot  of  things  inside.   I  don't  think  very  many  people  know  him 
very  well,  and  I  feel  like  I'm  probably  one  of  the  people  who  knows  him  the  best,  and  I 
don't  know  him  very  well. 

Every  year  when  Nobel  Prize  time  comes  around,  I  feel  upset  for  him.  I  think  now  he's 
gotten  over  a  lot  of  this  because  he's  doing  so  many  different  things  in  his  life  now.  About 
three  months  ago  I  finally  sat  down  and  told  him  how  I  felt,  that  I  felt  like  it  wasn't  right, 
that  I  felt  like  he  should  have  the  Nobel  Prize.  A  lot  of  these  things  had  been  unsaid  and  I 
just  said  them. 

Hughes:  How  did  he  react? 

Betlach:  I  think  he  appreciated  the  support,  but  he  wasn't  forthcoming.  I  could  see  how  he  felt  from 
his  body  language  and  knowing  him,  rather  than  by  what  he  was  saying.  It's  easy  to  scratch 


33 


the  surface  with  him  but  hard  to  go  deep.  So  I  can  see  how  it  would  be  easy  to  interview 
him  but  difficult  to  get  some  things  out  of  him.  Kary  Mullis  got  the  prize  for  PCR 
[polymerase  chain  reaction].  Herb  Boyer  should  have  the  Nobel  Prize.  I  think  Paul  Berg 
deserved  the  Nobel  Prize;  I  don't  know  if  it  should  have  been  for  recombinant  DNA.  I 
think  for  recombinant  DNA  and  cloning,  Herb  deserves  the  Nobel  Prize. 

Hughes:  Is  there  anything  more  you  want  to  say? 

Betlach:  I  think  I've  said  a  lot.  I  don't  know  what  else  to  say,  except  I'm  glad  I  was  there.  It  was 
fun. 


Transcribed  by  Michael  J.  McArdle 

Final  Typed  by  Julie  Allen  and  Kathy  Zvanovec-Higbee 


34 
TAPE  GUIDE--Mary  C.  Betlach 


Date  of  Interview:  March  24,  1994 
Tape  1,  Side  A 

Tape  1,  Side  B  13 

Tape  2,  Side  A  25 

Tape  2,  Side  B  unrecorded 


35 


APPENDICES 

A.  Curriculum  Vitae  36 

B.  Current  Rl  Endonuclease  Purification  Procedure,  December  1972  42 

C.  Betlach-Boyer  Procedures  for  Preparing  Closed-Circular  DNA,  late  '72  or  early  '73  45 

[handwritten  by  Herb  Boyer] 

D.  Photographs  from  Herb  Beyer's  Laboratory  in  the  1970s 

[captions  and  photographs  courtesy  of  Mary  Betlach]  48 

E.  Agarose  Gel  Electrophoresis  of  Linear  Duplex  DNA,  April  1 973  56 

F.  Electrophoresis  of  DNA  in  Agarose  Gels,  Boyer  Laboratory  Procedures,  1973  58 

G.  Restriction  Map  of  pBR322  60 

H.  Biolabs  Midnight  Hustler,  newsletter  parody  produced  by  colleagues  of 

Mary  Betlach  at  Harvard  University,  April  1,  1977  61 

I.  Gartland/Fredrickson  Memo  Certifying  pBR322,  July  6,  1977  76 


36  APPENDIX  A 


Mary  C.  Betlach,  Ph.D. 
341  Oyster  Point  Boulevard 
South  San  Francisco,  CA  94080 
Phone:  (650)  266-3612 


POSITIONS/EMPLOYMENT 

2001-  Director,  Grant  Research  Collaborations,  Sunesis  Pharmaceuticals,  SSF,CA 

1993-  Adj.  Associate  Professor,  Pharmaceutical  Chemistry,  UCSF 

1999-2001  Director,  Scientific  Operations,  Kosan  Biosciences,  Hayward,  CA 

1995-1999  Director,  Molecular  Biology,  Kosan  Biosciences,  Hayward,  CA 

1993-1994  Sr.  Scientist,  Cell  Genetics,  Parnassus  Pharmaceuticals,  Alameda,  CA 

1983-1993  Research  Specialist,  Biochemistry  &  Biophysics,  UCSF 

1972-1983  Staff  Research  Associate,  Microbiology,  Biochemistry  &  Biophysics,  UCSF 


EDUCATION 

1963-1967       Undergraduate  (Biochemistry),  University  of  Wisconsin,  Madison,  WI 
1992  Ph.D.  (Biochemistry /Molecular  Biology),  UCSF 


HONORS/AWARDS 

1963-1966       University  of  Wisconsin  Scholarship 

1967  University  of  Wisconsin  Leadership  Scholarship 


EDITORIAL  AND  GRANT  REVIEW  PANEL  SERVICE 

1981-  Ad  Hoc  Research  Grant  Reviewer  for  National  Science  Foundation. 

1983-  Ad  Hoc  Editorial  Reviewer  for  /.  Mol.  Bio/.,  Nucleic  Acids  Res.,  J. 

Bacterial,  and  others 

1986,1987       FASEB  LSRO  (ONR)  Grant  Review  Panelist:  Archaebacteria 
1987  FASEB  LSRO  (ONR)  Grant  Review  Panelist:  Molecular  Marine  Biology 

1990  Ad  Hoc  Research  Grant  Reviewer  for  Canadian  Research  Council 

1992  Ad  Hoc  Research  Grant  Reviewer  for  Department  of  Energy 


PATENTS 

1977    Expression  of  Heterologous  Polypeptides  in  Halobacteria,  U.S.  Patent  #  5,641,650. 
2000    Sorangium  Polyketide  Synthase,  U.S.  Patent  #6,090,600. 

2000  Recombinant  Narbonolide  Polyketide  Synthase,  U.S.  Patent  #6,117,659. 

2001  Recombinant  Oleandolide  Polyketide  Synthase,  U.S.  Patent  #6,251,636. 
2001     Production  of  Polyketides  in  Plants,  U.S.  Patent  #6,262,340. 


37 


2001    Sorangium  Polyketide  Synthases  &  Encoding  DNA,  U.S.  Patent  #  6,280,999. 
2001    Six  pending  patent  applications  on  various  aspects  of  combinatorial  biosynthesis 
and  polyketide  production. 


PUBLICATIONS 

1.  Hartmann,  H.  A.,  Lin,  ].,  and  Shively,  M.  C.  RNA  of  nerve  cell  bodies  and  axons 
after  p,p-Iminodipropionitrile.  Ada  Neuropathologica,  11:  275-281, 1968. 

2.  Orias,  E.  Gartner,  T.  K. ,  Lannan,  J.  E.,  and  Betlach,  M.  C.    Close  linkage  between 
ochre  and  missense  suppressors  in  Escherichia  coli.  J.  Bacterial.  109: 1125-1133, 1972. 

3.  Schneider,  E.  L.,  Epstein,  C.  J.,  Epstein,  W.  L.,  Betlach,  M.  C.,  and  Abbo- 
Halbasch,  G.  Detection  of  mycoplasma  contamination  in  cultured  human 
fibroblasts.  Exper.  Cell  Res,  79:  343-349, 1973. 

4.  Greene,  P.  J.,  Betlach,  M.  C.,  Goodman,  H.  M.,  and  Boyer,  H.  W.  The  Eco  Rl 
restriction  endonuclease.  In  DNA  Replication  (Methods  in  Molecular 
Biology),  R.  B.  Wickner  (ed.),  Marcel  Dekker,  Inc.,  New  York,  7,  p.  87-105, 1974. 

5.  Roulland-Dussoix,  D.,  Yoshimori,  R.,  Greene,  P.,  Betlach,  M.,  Goodman,  H. 
M.,  and  Boyer,  H.  W.  R  factor-controlled  restriction  and  modification  of 
deoxyribonucleic  acid.  In  Microbiology  1974.  D.  Schlesinger  (ed.).  ASM, 
Washington,  D.  C.,  p.  187-198, 1975. 

6.  Boyer,  H.  W.,  Greene,  P.  J.,  Meagher,  R.  B.  Betlach,  M.  C.,  Russel,  D.,  and 
Goodman,  H.  M.  The  methylation  of  DNA  as  the  biochemical  basis  of  host 
controlled  modification  of  DNA  in  bacteria.  FEES  Symposium  (1974). 
Budapest,  Hungary,  v.  34:  p.  23-37, 1975. 

7.  Betlach,  M.,  Hershfield,  V.,  Chow,  L.,  Brown,  W.,  Goodman,  H.,  and  Boyer  H. 
A  restriction  endonuclease  analysis  of  the  bacterial  plasmid  controlling  the 

Eco  Rl  restriction  and  modification  of  DNA.  (FASEB  Symposium,  April  1974),  Fed. 
Proc.  35:  2037-2043, 1976. 

8.  So,  M.,  Boyer,  H.  Betlach,  M.,  and  Falkow,  S.  Molecular  cloning  of  an 
Escherichia  coli  plasmid  determinant  that  encodes  for  the  production  of  heat- 
stable  enterotoxin.  /.  Bacterial.  128: 463-472, 1976. 

9.  Rodriguez,  R.  L.,  Bolivar,  F.,  Goodman,  H.  M.,  Boyer,  H.  W.  and  Betlach,  M. 
Construction  and  characterization  of  cloning  vehicles.  In  Molecular  Mechanisms 
in  the  Control  of  Gene  Expression,  ICN-UCLA  Symposia  on  Molecular  and 
Cellular  Biology,  Vol.  V),  D.  P.  Nierlich,  W.  J.  Rutter,and  C.  F.  Fox  (eds.), 
Academic  Press,  New  York,  p.  471-477, 1976. 

10.  Meagher,  R.  B.,  Tait,  R.  C.,  Betlach,  M.,  and  Boyer,  H.  W.  Protein  expression 
in  E.  coli  minicells  by  recombinant  plasmids.  Cell  10:  521-536, 1977. 

11.  Boyer,  H.  W.,  Betlach,  M.,  Bolivar,  R.,  Rodriguez,  R.  L.,  Heyneker,  H.  L.,  Shine,  J., 
and  Goodman,  H.  M.  The  construction  of  molecular  cloning  vehicles.  In 
Recombinant  Molecules:  Impact  on  Science  and  Society,  R.  F.  Beers  and  E.  G.  Bassett 
(eds.),  Raven  Press,  New  York,  p.9-20, 1977. 


38 


12.  Rodriguez,  R.  L.,  Tait,  R.,  Shine,  J.,  Bolivar,  F.,  Heyneker,  H.  L.,  Betlach,  M, 
and  Boyer,  H.  W.  Characterization  of  Tetracycline  and  ampicillin  resistant 
plasmid  cloning  vehicles.  In  Molecular  Cloning  of  Recombinant  DNA,  vol.  13: 
Academic  Press,  New  York,  p.  73-84, 1977. 

13.  Bolivar,  F.,  Rodriguez,  R.  L.,  Betlach,  M.  C,  and  Boyer,  H.  W.  Construction 
and  characterization  of  new  cloning  vehicles:  I.  Ampicillin  resistant 
derivatives  of  the  plasmid  pMB9.  Gene  2:  75-93, 1977. 

14.  Bolivar,  F.,  Rodriguez,  R.  L.,  Greene,  P.  J.,  Betlach,  M.  C.,  Heyneker,  H.  L.,  Boyer, 
H.  W.,  Crosa,  J.  H.,  and  Falkow,  S.  Construction  and  characterization  of  new 
cloning  vehicles:  II.  A  multipurpose  cloning  system.  Gene  2:  95-113, 1977. 

15.  Bolivar,  F.,  Betlach,  M.,  Heyneker,  H.  L.,  Shine,  J.,  Rodriguez,  R.  L.,  and  Boyer, 
H.  W.  Origin  of  replication  pBR345  plasmid  DNA.  Proc.  Natl.  Acad.  Sci.  USA 
74:  5265-5269, 1977. 

16.  Tait,  R.  C.,  Heyneker,  H.  L.,  Rodriguez,  R.  L.,  Bolivar,  F.,  Covarrubias,  A., 
Betlach,  M.,  and  Boyer,  H.  W.  Tetracycline  resistance  conferred  by  pSClOl, 
pMB9  and  their  derivatives.  In  Microbiology  1978,  ASM,  Washington,  D.  C., 
p.  174-176, 1978. 

17.  Greene,  P.  J.,  Heyneker,  H.  L.,  Bolivar,  F.,  Rodriguez,  R.  L.,  Betlach,  M.  C., 
Covarrubias,  A.,  Backman,  K.,  Russel,  D.,  Tait,  R.,  and  Boyer,  H.W.  A  general 
method  for  the  purification  of  restriction  enzymes.  Nucl.  Acids  Res.  5:  2373- 
2380, 1978. 

18.  Backman,  K.,  Betlach,  M.,  Boyer,  H.,  and  Yanofsky,  S.  Genetic  and  physical 
studies  on  the  replication  of  ColEl-type  plasmids.  Cold  Spring  Harbor  Symp. 
Quant.  Biol  43:  p.69-76, 1979. 

19.  Betlach,  M.,  Pfeifer,  F.,  Friedman,  J.,  and  Boyer,  H.  W.  Bacterio-opsin  mutants 
of  Halobacterium  halobium.  Proc.  Natl.  Acad.  Sci.  USA  80: 1416-1420, 1983. 

20.  Pfeifer,  F.,  Betlach,  M.,  Martienssen,  R.,  Friedman,  J.,  and  Boyer,  H.  W. 
Transposable  elements  of  Halobacterium  halobium.  Mol.  Gen.  Genet.  191: 182-188, 
1983. 

21.  Pfeifer,  F.,  Friedman,  J.,  Boyer,  H.  W.,  and  Betlach,  M.  Characterization  of 
insertions  affecting  the  expression  of  the  bacterio-opsin  gene  in  Halobacterium 
halobium.  Nucl.  Adds  Res.,  12:  2489-2497, 1984. 

22.  Betlach,  M.,  Friedman,  J.,  Boyer,  H.  and  Pfeifer,  F.  Characterization  of  a 
halobacterial  gene  affecting  bacterio-opsin  gene  expression.  Nucl.  Acids  Res. 
12:  7949-7959, 1984. 

23.  Pfeifer,  F.,  and  Betlach,  M.  Genome  organization  in  Halobacterium  halobium:  a  70 
kb  island  of  more  (AT)  rich  DNA  in  the  chromosome.  Mol.  Gen.  Genet.  198:  449- 
455, 1985. 

24.  Pfeifer,  F.,  Boyer,  H.  and  Betlach,  M.  Restoration  of  bacterio-opsin  gene 
expression  in  a  revertant  of  Halobacterium  halobium.  J.  Bacterial.,  164:  414-420, 
1985. 

25.  Betlach,  M.,  Leong,  D.,  and  Boyer,  H.    Bacterio-opsin  gene  expression  in 
Halobacterium  halobium.  System.  Appl.  Microbiol.  7:  83-89, 1986. 


39 


26.  Betlach,  M.,  Leong,  D.,  and  Boyer,  H.  Bacterio-opsin  gene  expression  in 
Halobacterium  halobium.  In:  Archaebacteria  1985 ,  O.  Kandler  and  W.  Zillig  (eds.). 
Fisher  Verlag,  Stuttgart.  1986. 

27.  Betlach,  M.,  Leong,  D.,  Pfeifer,  F.,  and  Boyer,  H.  Bacterio-opsin  gene  expression 
in  Halobacterium  halobium.  In:  Microbiology  1986,  S.  Silver  (ed.),  ASM, 
Washington,  D.  C,  p.  363-369, 1987. 

28.  Betlach,  M.,  Leong,  D.,  and  Boyer,  H.    E.  coli  expression  vectors  for  bacterio- 
opsin.  In:  Retinal  Proteins  1987.  (Proceedings  from  the  Second  International 
Conference  on  Retinal  Proteins.)  VNU  Science  Press.  The  Netherlands,  p.  317- 
331, 1987. 

29.  Harley,  C.,  Lawrie,  J.,  Betlach,  M.,  Crea,  R.,  Boyer,  H.,  and  Hedgpeth,  J. 
Transcription  initiation  at  the  tet  promoter  and  effect  of  mutations.  Nucl.  Acids. 
Res.  16:  7269-7285, 1988. 

30.  Leong,  D.,  Pfeifer,  F.,  Boyer,  H.,  and  Betlach,  M.  Characterization  of  a  second 
gene  involved  in  bacterio-opsin  gene  expression  in  a  halophilic 
archaebacterium.  /.  Bacterial.  170:  4903-4909, 1988. 

31.  Leong,  D.,  Boyer,  H.,  and  Betlach,  M.  Transcription  of  genes  involved  in 
bacterio-opsin  gene  expression  in  mutants  of  a  halophilic  archaebacterium.  /. 
Bacterial  17: 4910-4915, 1988. 

32.  Betlach,  M.  C.,  Shand,  R.  F.,  and  Leong,  D.  M.  Regulation  of  the  bacterio-opsin 
gene  of  a  halophilic  archaebacterium.  Can  J.  Micro.    35: 134-140, 1989. 

33.  Betlach,  M.  C.,  and  Shand,  R.  F.  Molecular  Biology  and  regulation  of  bacterio- 
opsin  gene  expression  in  Halobacterium  halobium.  In:  General  and  Applied 
Aspects  of  Halophilic  Microorganisms  (FEMS-NATO),  Plenum,  p.  259-264, 
1991. 

34.  Shand,  R.  F.,  Miercke,  L.  J.  W. ,  Mitra,  A.  K.,  Fong,  S.,  Stroud,  R.  M.  and 
Betlach,  M.  C.    Wild-type  and  mutant  bacterio-opsins  D85N,  D96N  and  R82Q: 
High  level  expression  in  Escherichia  coli.  Biochemistry.  30:  3082-3088, 1991. 

35.  Miercke,  L.  J.  W.,  Betlach,  M.  C.,  Shand,  R.  F.,  Fong,  S.  K.  and  Stroud,  R.  M. 
Wild-type  and  mutant  bacteriorhodopsins  D85N,  D96N  and  R82Q:  purification 
to  homogeneity,  pH  dependence  of  pumping  and  electron  diffraction. 
Biochemistry.  30:  3088-3098, 1991. 

36.  Lin,  S.W.,  Fodor,  S.P.,  Miercke,  L.J.,  Shand,  R.  F.,  Betlach,  M.C.,  Stroud,  R.M. 
and  Mathies,  R.  A.  Resonance  Raman  Spectra  of  bacteriorhodopsin  mutants 
with  substitutions  at  Asp-85,  Asp-96  and  Arg-82.  Photochemistry  and 
Photobiology.  53:  341-346, 1991. 

37.  Shand,  R.  F.  and  Betlach,  M.  C.  Expression  of  the  bop  gene  cluster  of 
Halobacterium  halobium  is  induced  by  low  oxygen  tension  and  light.  /. 
Bacteriol.  173:  4692-4699, 1991. 

38.  Thorgeirsson,  T.,  Milder,  S.,  Miercke,  L.,  Betlach,  M.,  Shand,  R.,  Stroud,  R.  and 
Kliger,  D.    Effects  of  Asp96  — »  Asn,  Asp85  — »  Asn,  Arg82  -»  Gin  single-site 
substitutions  on  the  photocycle  of  bacteriorhodopsin.  Biochemistry.  30:  9133- 
9142, 1991. 


40 


39.  Gropp,  R.,  Gropp,  F.  and  Betlach,  M.  Association  of  the  halobacterial  7S  RNA 
to  the  polysome  correlates  with  expression  of  the  membrane  protein  bacterio- 
opsin.  Proc.  Natl.  Acad.  Sci.  USA  89:  1204-1208, 1992. 

40.  Turner,  G.,  Miercke,  L.,  Thorgeirsson,  T.,  Kliger,  D.,  Betlach,  M.  and  Stroud,  R. 
Bacteriorhodopsin  D85N:  Three  spectroscopic  species  in  equilibrium. 
Biochemistry.  32:  1332-1337,1993. 

41.  Mitra,  A.,  Miercke,  L.,  Turner,  G.,  Shand,  R.,  Betlach,  M.,  and  R.M.  Stroud. 
Crystallization  of  Escherichia  co/z-expressed  bacteriorhodopsin  and  its  D96N 
variant:  High  resolution  structural  studies  in  projection.  Biophys.  J.  65:  1-12, 
1993. 

42.  Gropp,  F.,  Gropp,  R.  and  Betlach,  M.  C.  A  fourth  gene  in  the  bop  gene  cluster 
is  co-regulated  with  the  bop  gene.  System.  Appl.  Microbiol.  16:  716-724, 1994. 

43.  Betlach,  M.  C.  and  F.  Gropp.  The  bop  gene  cluster  of  Halobacterium 
halobium.System.  Appl.  Microbiol.  16:  712-715, 1994. 

44.  Gropp,  F.  and  Betlach,  M.  C.  The  bat  gene  of  Halobacterium  halobium 
encodes  a  trans-acting  oxygen  inducibility  factor.  Proc.  Natl.  Acad.  Sci.  USA. 
91:  5475-5479,1994. 

45.  Shand,  R.  F.  and  Betlach,  M.  C.  bop  gene  cluster  expression  in 
bacteriorhodopsin  overproducing  mutants  of  Halobacterium  halobium. 
J.  Bacterial.  176: 1655-1660, 1994. 

46.  Gropp,  R.  and  Betlach,  M.  C.  Effects  of  upstream  deletions  on  light  and  oxygen 
regulated  bacterio-opsin  gene  expression  in  Halobacterium  halobium.  Molecular 
Microbiology.  16:  357-364, 1995. 

47.  Betlach,  Mary,  C.  and  Shand,  R.  F.  Growth  of  halophilic  archaebacteria  under 
conditions  of  low  oxygen  tension  and  high  light  intensity.  In:  Archaea,  a 
laboratory  manual.  F.  Robb  (ed.)  Cold  Spring  Harbor  Laboratory,  Cold  Spring 
Harbor,  N.  Y.  p.  17-21, 1995. 

48.  McDaniel,  R.,  Kao,  C.,  Fu,  H.,  Hevezi,  P.,  Gustafsson,  C.,  Betlach,  M.,  Ashley,  G., 
Cane,  D.  and  Khosla,  C.  (1997)  Gain-of-function  mutagenesis  of  a  modular 
polyketide  synthase.  /.  American  Chemical  Society.  119:  4309-4310, 1997. 

49.  Liu,  L.,  Thamchaipenet,  A.,  Fu,  H.,  Betlach,  M.  and  Ashley,  G.  Biosynthesis  of  2- 
nor-6-deoxyerythronolide  B  by  rationally  designed  domain  substitution.  /. 
American  Cltemical  Society.  119: 10553-10554, 1997. 

50.  Betlach,  M.  Combinatorial  Biosynthesis  of  Polyketides.  In:  Natural  Products  II. 
New  Technologies  to  Increase  Efficiency  and  Speed.  D.  Sapienza,  (ed.)  IBC,  Inc., 
Southborough,  MA.  P.  279-309, 1998. 

51.  Kealey,  J.,  Liu,  L.,  Santi,  D.,  Betlach,  M.  and  Barr,  P.  Production  of  a  polyketide 
natural  product  in  nonpolyketide-producing  prokaryotic  and  eukaryotic  hosts. 
Proc.  Natl.  Acad.  Sci.  USA.  95:  505-509, 1998. 

52.  Betlach,  M.,  Kealey,  J.,  Betlach,  M.,  Ashley,  G.  and  McDaniel,  R.  Characterization 
of  the  macrolide  P-450  hydroxylase  from  Streptomyces  venezuelae  which  converts 
narbomycin  to  picromycin.  Biochemistry,  37: 14937-14942, 1998. 


41 


53.  Ziermann,  R.  and  Betlach,  M.  C.  Recombinant  polyketide  synthesis  in 
Streptomyces:  Engineering  of  improved  host  strains.  BioTechniques,  26: 106-110, 
1999. 

54.  McDaniel,  R.  Thamchaipenet,  A.,  Gustafsson,  C.,  Fu,  H.,  Betlach,  M.,  Betlach,  M. 
and  Ashley,  G.  Multiple  genetic  modifications  of  the  erythromycin  polyketide 
synthase  to  produce  a  library  of  novel  "unnatural"  natural  products.  Proc.  Natl. 
Acad.  Sri.  USA.  96: 1846-1851, 1999. 

55.  Turner,  G.  J.,  Reusch,  R.,  Winter- Vann,  A.  M.,  and  Betlach,  M.  C.  Heterologous 
gene  expression  in  a  membrane-protein-specific  system.  Protein  Expression  & 
Purification  17:  312-323,  1999. 

56.  Turner,  G.  J.,  Miercke,  L.  J.,  Mitra,  A.  K.,  Stroud,  R.  M.,  Betlach,  M.  C.  and  Winter 
Vann,  A.  Expression,  purification,  and  structural  characterization  of  the 
bacteriorhodopsin-aspartyl  transcarbamylase  fusion  protein.  Protein  Expression 
&  Purification  17:  324-338, 1999. 

57.  Ziermann,  R.  and  Betlach,  M.  C.  A  two-vector  system  for  the  production  of 
recombinant  polyketides  in  Streptomyces.  Journal  of  Industrial  Microbiology  & 
Biotechnology.  24:  46-50,  2000. 

58.  Shah,  S.,  Xue,  Q.,  Tang,  L.,  Carnery,  J.,  Betlach,  M.  and  McDaniel,  R.  Cloning, 
characterization  and  heterologous  expression  of  a  polyketide  synthase  and  P-450 
oxidase  involved  in  the  biosynthesis  of  the  antibiotic  oleandomycin.  Journal  of 
Antibiotics,  53:  502-508,  2000. 


42  'APPENDIX  B  ,x. 

December  1972 


CURRENT  RI  ENDONUCLEASE  PURIFICATION  PROCEDURES 


For  300  gm  wet-packed  cells  (RY  13)  .   All  steps  at  4°C. 

1.  Suspend  in  1  L  EB  and  sonify. 

2.  Centrifuge  for  70  rnin,  35,000  RPM,  Beckman  35  Rotor. 

3.  Add  300  ml  fresh  5%  streptomycin  sulfate  (in  H20)  ,  centrifuge  low 

s^e.ed,  30  .min. 

'i  .   Md  an  equal  volume  of  cold  saturated  (NH^)^  SO   (in  H20)  and  stir 

for  30  lain.   Centrifuge  at  low  speed. 

;  i  •  • 

5.  ^Suspend  in  300  ml  EB  and  dialyze  for  about  3  hr  against  4  L  of  EB  . 
' 


.(••    Aptiy  -'•"mediately  to  PC  column  (2  X  60)  equilibrated  with  0.2  M 

EB.  'Run  "012  to  0.6  M  NaCl  EB  gradient  total  volume  of  4  L.  •?.-45~ 


Pool,  fractions  with  RI  endonuclease  activity  (which  elates  around 

0.4  M  NaCl  EB.  At  this  stage  the  RI  endonuclease  will  aggregate 
(presumably  because  of  its  hydrophobicity  )  below  NaCl  concentra 
tions  of  0.2M. 

8.  Dialyze  the  pooled  RI  '  endonuclease  fraction  against  0.2  M  NaCl  EB  . 

9.  Apply  to  hydroxyapaiftite  column  (2x20  cm)  equilibrated  with  0.2M 

NaCl  EB.   Run  K-P04  (pH  7.0)  gradient  of  10-400  mM  in  0.2M 
NaCl.   Endonuclease  activity  elutes  around  0.25  M  K-PO/  . 

^T0^  t«l   lWir- 


Dialyze  against  0.2  M  NaCl  EB  +  0.15%  triton  X,  then  against  EB  + 
0.15%  triton  X. 

Apply  to  DE-52  column  (0.9  x  10)  equilibrated  with  EB  +  triton  X  and 
elute  with  a  NaCl  gradient  of  0  -  0.3  M,  total  volume  of  1  L. 
The  RI  endonuclease  elutes  about  0.15  M  NaCl. 

Dialyze  against  EB-triton  X  and  concentrate  on  a  small  DE-52  column. 
Elute  with  EB-triton  X-0.3  M  NaCl.   Dialyze  against  EB-0.3  M 
NaCl.   Store  at  4°C. 


43 
'  December    1972 

'  .  •   •  •  . 

CURRENT  RII  ENDONUCLEASE  PURIFICATION  PROCEDURES 

For  300  gm  wet-packed  cells  (RY  22).   All  steps  at  4°C. 

1.  Suspend  in  1  L  EB  (10  mM  K-PO,,  pH  7.0,  ImM  EDTA,7  mM  mer captoethanol ) 

and  sonify. 

2.  Centrifuge  for  70  min,  35,000  RPM,  Beckman  35  Rotor. 

3.  Add  300  ml  fresh  5%  stre.ptomyci-n   -salfat-e  (in  H^^))  ,  centrifuge  low 

speed,  30  min. 

4.  Add  an  equal  volume  of  cold  saturated  (NH^)2  SO/  (in  H20)  ,  stir  30 

min.   Centrifuge  low  speed. 

5.  Suspend  in  300  ml  EB  and  dialyze  against  EB  (usually  8  L  overnight). 

6.  Apply  to  Whatman  DE-52 (DEAE-cellulose)  column  CSx60  cm)  in  EB. 

Gradient  of  0-0.5  M  NaCl  in  EB ,  total  gradient  volume  of  4  L, 

7.  Pooled  fractions  with  endonuclease  activity  has  0.1-0.14  M  NaCl. 

8.  Dialyze  DE-52  pool  against  EB. 

9.  Apply  to  phosphocellulose  (1  x  20  cm).   Wash  with  0.2  M  NaCl  EB  and 
run  gradient  of  0.2-0.5M  NaCl  in  EB;  total  volume  of  gradient  is  1  L. 

10.  Pooled  endonuclease  activity  has  NaCl  concentration  of  about  0.26-0.30  M, 

11.  Dialyze  against  EB  and  apply  to  (2  x  20  cm)  hydroxyapat ite  column 

(Clarkson  Chemical  Co.,  Wilkes-Barre ,  Pa.).   Run  a  1  L  gradient 
of  10-400  mM  K-PO^,  pH  7.0. 

12.  Pool  fractions  with  highest  activity  (the  endonuclease  .elutes  about 
0.20-0.25  M  K-P04)  and  dialyze  against  EB . 

13.  Concentrate  by  adsorbing  to  small  DE-52  column  (in  Pasteur  pipette) 

equilibrated  with  EB  plus  10-20%  glycerol.   Elute  with  0.25  M 
NaCl  EB  +  10-20%  glycerol.   Store  as  eluted  at  4 ° C . 

Note:  'NaCl  inhibits  the  endonuclease  activity  at  concentrations  above 
0.1M  NaCl. 

Final  prep  should  have  about  25  mg  protein,  depending  on  column  cuts,  and 
enough  endonuclease.  to  cleave  about  5  mg  X  DNA. 


44 
ASSAYS  FOR  RI  AND  RII  ENDONUCLEASKS 


1.  Centrif ugation  assay.   A  reaction  volume  of  100  yl  containing  100 

mMolar  TRIS,  pH  7 . 5  ;  5m  Molar  MgCl  ;  about  1  yg  of  unmodified 
HJ  X  DNA  and  0.1  to  1.0  yg  of  P32  modified  DNA;  with  1-50  yl 
of  enzyme  fraction. 

S?» 

Incubate  15  min   at  37°C  and  add  10  yl  10%  SDS.   Centrifuge  2.25  hr 
at  55,000  RPM.in  SW  56  rotor,  linear  5-20%  sucrose  gradients 
in  0.1  mKHP04,  pH  7.0,  0.1  M  NaCl.   Fractionate  (15-17  fraction) 
and  determine  amount  of  H3  sedimenting  slower  than  P32. 

2.  Use  electron  m,lcros.co.p.e  -to  f-oll-ow  'Conversion  o-f  circular  molecule  of  SV40 

DNA  to  linear  molecule.   One  double  strand  cleavage  per  molecule 
for  RI  endonuclease . 


APPENDIX   C 


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46 


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47 


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Appendix  D 


Three  shots  of  Herb  Boyer  in  his  Microbiology  Dept  lab.  One  shot  showing  him  in  his  office  sitting  at  his  desk. 
A  second  shot  showing  him  in  front  of  the  fume  hood,  which  has  his  running  clothes  hanging  in  it.  A  third  shot 
of  him  flanked  on  the  left  by  Steve  Yanofsky  (graduate  student)  and  on  the  right  by  Keith  Backman  (postdoc). 

The  top  right  photo  (dated  1977)  shows  Pat  Greene  (left)  who  was  pregnant  at  the  time  and  wearing  a  T-shirt 
labeled  "Recombinant  DNA"  with  an  arrow  pointing  to  her  abdomen  and  me  (right)  in  the  Grateful  Dead  T- 
shirt.  We  had  just  competed  in  a  Biochemistry  Dept.  running  race  in  which  the  Boyer  lab  had  just  won  the 
"beer  relay  race"  event  in  which  five  of  us  had  competed  against  other  labs  in  a  relay  race  characterized  by 
drinking  beer  while  you  ran.  The  bottom  right  photo  is  of  me  in  my  flat  at  a  lab  party  I  had  organized,  which 
was  a  going  away  party  for  Keith  Backman. 


49 


The  three  photos  on  the  left  of  the  page  are  from  Keith  Backman's  going  away  party.  In  the  photo 
at  top  we  had  given  Keith  a  going  away  present  which  was  in  a  small  box  labeled  "recombinant 
DNA  kit."  Inside  was  a  condom  with  a  hole  in  it  which  Keith  is  displaying  on  his  little  finger. 
Others  in  the  photo  are  Herb,  Eli  Elahi  (Iranian  postdoc),  and  Dave  Russel  (research  associate)  and 
his  girlfriend.  The  middle  photo  shows  (half  of)  Tom  Kornberg  (Biochemistry  Dept.  professor)  and 
Jon  Lawrie  (postdoc).  The  bottom  photo  shows  Keith  Backman,  John  Rosenberg  (professor  and  x- 
ray  crystallographer  collaborator  on  EcoRl  project),  a  postdoc  from  Tom  Kornberg's  lab,  and  Pat 
Greene  (lower  right).  The  photo  on  the  right  is  of  Esther  Lederberg  and  Werner  Goebel  (professor 
from  Univ.  of  Wurzberg  in  Germany  who  did  a  sabbatical  in  the  Boyer  lab  in  the  late  70s). 


The  top  two  photos  are  from  Keith  Backnian's  going  away  party  and  show  Herb 
drinking  a  beer  next  to  Keith;  and  Herb  standing  next  to  Pat  Greene.  The  bottom 
photo  was  taken  at  a  lab  party  at  an  Italian  restaurant  in  North  Beach  and  shows 
Wes  Brown  and  Herb. 


51 


The  top  photo  is  of  Francisco  (Paco)  Bolivar  who  had  been  a  postdoc  in  the  Boyer  lab  and  was  back  visiting.  (At  his 
going  away  party  we  gave  him  a  hula  hoop  with  the  sequence  of  pBR322  DNA  on  it.)  He  was  staying  at  my  flat  and 
had  bought  some  lab  equipment  (pipetman,  etc.)  when  visiting  and  was  boxing  it  all  up  for  his  return  to  Mexico. 
Middle  photo  is  of  a  lab  party  at  Pat  Greene's  house.  In  the  photo  (left  to  right)  are  Linda  Robinson  (postdoc),  Herb, 
me,  Paco  Bolivar,  my  son  John,  and  a  visiting  scientist  from  Iceland  (I  can't  remember  his  name).  The  bottom  photo  is 
of  a  lab  Thanksgiving  day  party  (-1977)  at  my  flat.  On  the  left  side  of  the  table  are  Will  Spiegelman  (Pat  Greene's 
husband)  and  Keith  Backman.  On  the  right  side  of  the  table  are  Dan  Vapnik  (visiting  scientist  in  the  Boyer  lab  at  the 
time;  later  of  Amgen  fame),  Jon  Lawrie  (postdoc),  and  Kirby  Alton  (worked  for  Dan  V.). 


52 


Top  two  photos  taken  at  lab  party  at  Pat  Greene's  house.  Top  photo  back  row  shows  wife  of  Icelandic 
postdoc,  Herb,  Franny  DeNoto  (Howard  Goodman's  research  associate),  Keith  Backman,  daughter  of  Ice 
landic  postdoc,  and  Icelandic  postdoc.  Front  row  shows  Linda  Robinson  (postdoc),  Paco  Bolivar,  and  my 
son  John.  Center  photo  shows  Herb  and  Pat  Greene.  Bottom  photo  shows  Herb  in  his  new  biochemistry 
lab  on  the  1 5th  floor. 


53 


Photos  from  new  Boyer  lab  on  the  15th  floor.  Top  photo  shows  Herb,  Pat  Greene,  and  Keith 
Backman.  Center  photo  shows  unknown  graduate  student,  David  Russel,  Axel  Ulrich  (Goodman 
lab  postdoc),  Herb  Boyer,  Icelandic  postdoc  and  Keith  Backman. 

Bottom  photo  shows  Axel  Ulrich,  Franny  DeNoto,  and  Herb  clustered  around  my  lab  desk  in  the 
lab,  drinking  and  eating. 


Top  photo  shows  Boyer  lab  meeting  on  the  15th  floor.  Left  to  right:  Jon  Lawrie,  Kirby 
Alton,  Jordan  Konisky  (visiting  scientist),  Pat  Greene,  me  (with  beer  can  in  front  efface), 
Keith  Backman  (in  front  of  me),  Jan  Maat  (postdoc  from  Netherlands),  Eli  Elahi,  and  I  don't 
know  who  the  guy  is  behind  the  pole.  Middle  photo  is  of  Kirby  Alton  and  Dan  Vapnik 
working  in  the  15th  floor  lab.  Bottom  photo  is  of  Madhu  Gupta  (Pat  Greene's  research  asso 
ciate),  Pat  Greene,  and  me  in  front  of  my  desk  in  the  lab. 


55 


These  were  taken  for  the  Smithsonian  article  and  show  (clockwise  starting  upper  left)  Herb  looking  at  a  bacterial  plate;  Ray  Rodriguez 
collecting  bands  from  a  CsCl  gradient  for  a  plasmid  prep;  me  looking  at  a  bacterial  plate;  me  tracing  plasmid  DNA  spreads  in  the  Electron 
Microscopy  room;  and  Herb  looking  at  bacterial  plates. 


APPENDIX  IS 
56 


.'  R.  Helling 

April  1973 

AGAROSU  GEL  ELIiCTROPHORESIS  OF  LINEAR  DUPLEX  QB  DNA 

Double-stranded  linear  DNA  of  molecular  weight  1  x  105 
and  higher  is  routinely  separated  on  0.7%  agarose  gels.   There 
is  a  linear  relationship  bctween.log  molecular  weight  and  mobility 
up  to  5  -  6  x  106,  and  separations  and  molecular  weight  estimates 
of  larger  DNA  can  be  made.   Molecular  weights  are  estimated  from 
internal  DNA  standards  such  as  endonuclease  RI  fragments  of  <j>80 
and  X.   The  error  in  molecular  weight  estimate  is  less  than  5% 
in  the  linear  range.   Where  the  DNA  of  interest  is  below  1  -  2  x  10 
MW,  higher  concentration  agarose  should  be  used.   Relative  mobili 
ties  are  unaffected  by  temperature  over  the  range  3°C  -  24°  C 
(although  absolute  mobility  increases  with  temperature).   Thus 
separation  appears  to  be  based  on  molecular  size  and  not  on  base 
composition. 

TEA  -  NaCl  Buffer 

40  mm  Tris  acetate,  pH  8.05 
20  mm  Na  acetate 

2  mm  Na2-EDTA 
18  mm  NaCl 

Gels 

15  x  0.6  cm  gels  are  formed  in  disposable  5  ml  pyrex  pipets, 
cut  so  as  to  leave  a  constriction  at  the  bottom  Cto  retain  the  gel)  . 
"0--   Agarose  in  buffer  is  melted  in  the  autoclave.   A  small 
amount  is  pipetted  around  the  outside  of  the  tip  of  the  tube  (held 
in  a  rack)  to  seal  it,  and  after  hardening,  additional  agarose  is 


57  2. 


added  to  form  a  15.5  cm  column.   The  gels  are  allowed  to  harden 
for  at  least  40  minutes.   Subsequently,  the  upper  end  of  each  is 
extruded  (using  a  water-filled  bulb  to  avoid  air  bubbles)  and 
sliced  evenly  to  form  a  15  cm  gel. 
Loading  Sample  and  Running 

The  sample  is  heated  5  minutes  at  65°  C  to  separate  loosely 
associated  DNA  molecules  (e.g.,  "sticky  ends"),  and  quenched  on 
ice.   Bromphenol  blue  and  sucrose  are  added  (to  20%  sucrose)  to 
give  a  final  sample  volume  of  20  yl  (optimally)  to  100  yl. 

The  sample  is  run  into  the  gel  5  minutes  at  100  v,  and  then 
at  22.5  v  (1.5  v/cm  gel).   After  about  20  hours  at  room  tempera 
ture  ^the  dye  marker  should  be  at  the  gel  tip.   The  dye  mobility 
is  equivalent  to  that  of  DNA  of  1  -  3  x  105  MW. 
Staining 

The  gels  are  extruded  into  5  yg/ml  ethidium  bromide.   After 
a  half  hour  the  stained  bands  may  be  visualized  over  long  wave 
length  ultraviolet  light  ("black  light").   (Protective  glasses 
should  be  worn.)   A  band  containing  50  -  100  ng  is  prominent; 
as  little  as  10  ng  can  be  seen. 

The  gels  are  immediately  photographed  beside  a  ruler,  using 
a  yellow  (Kodak  No.  9  Wratten  gelatin  filter)  or  orange  filter, 
and  measurements  are  taken  from  the  prints  (Polaroid  55  P/N  or 
52). 


APPENDIX  F 
58  -77^ 

ELECTROPHORESIS   OF    DNA    IN   AGAROSE    GELS 
BUFFER:  Tris-Borate 


10. 8   g  Tris  base 

.93  g  Na£  EDTA 
5.5   g  Boric  acid 
Bring  to   1.0   liter 

Agarose.   SeaKem  (MCi  Biomedical:  Division  of  Marine  Colloids,  Inc. 

P.O.  Box  748 
Rockland,  Maine  04841  ) 

The  appropriate  concentration  of  agarose  is  dictated  by  several  fac 
tors.   We  usually  use  0.7%  to  1.2%  agarose  made  up  in  the  Tris  Borate 
buffer.   The  agarose  is  melted  by  autoclaving  for  5  min,  refluxing  or 
simply  heated  in  a  boiling  water  bath. 

Agarose  gels  can  be  made  in  standard  tubes  or  slab  apparatuses. 
Tubes  can  be  made  from  5  ml  disposable  pyrex  pipets  or  glass  tubing. 
Cut  the  delivery  end  of  the  glass  pipet  to  leave  a  tapered  end  which 
prevents  the  gel  from  slipping  out  of  the  tube.   Stands  are  avail 
able  for  placing  the  tubes  in  an  upright  position  with  the  tip  of 
the  tube  in  a  small  depression  in  the  stand.   Add  molten  agarose  to 
the  depression  and  allow  to  harden  before  filling  the  tube  with  agarose 
After  solidification  of  the  gel,  the  top  must  be  sliced  evenly  to  pro 
vide  a  horizontal  flat  surface  for  the  sample.   This  can  be  done  by 
filling  a  small  rubber  bulb  with  HgO  or  buffer,  and  use  the  bulb  to 
force  the  gel  part  way  out  of  the  tube  which  can  then  be  cut  with  a 
razor  blade,  etc.,  and  retracted  into  the  tube.   Standard  electro- 
phoresis  chambers  for  tube  gels  can  be  used. 

Slab  gels  can  be  made  with  the  Studier  or  EC  apparatus.   Standard 
power  supplies  with  the  proper  connections  are  sufficient.   The  sample 
is  diluted  with  a  Brom-phenol-blue-glycerol-SDS  solution  C-°7%,  33%, 
71  SDS;  1  part  glycerol  solution  to  5  parts  sample).   Sample  volumes 
are  determined  by  the  area  of  the  sample   slot.   Usually  the  sample 
volumes  for  the  tube  gels  are  not  greater  than  50  yl.   For  large 
preparative  slab  gels  one  can  apply  ml  quantities. 

Electrophoresis  conditions  are  variable.   For  best  resolution  of  DNA 
fragments  on  tube  gels  we  use  a  constant  voltage  of  1.5  V/cm  of  gel, 
which  is  usually  around  22-23  V.   Running  times  for  fragments  of  0.5  x 
106  -  20  x  106  amu  are  about  24  hr,  room  temperature.   Preparative 
slab  gels  are  usually  run  at  75V  for  18-20  hr.   Analytical  gels  for 
fast  separation  of  fragments  with  large  mobility  differences  is 
1  -  1.5  hr. 

Gels  are  stained  by  soaking  in  ethidium  bromide  O  1  yg/ml  in  buffer 
or  H  0  for  about  5-10  min.   The  fluorescing  DNA  bands  can  be  visual 
ized  on  a  long-wave  or  short  wave  transilluminator  (.San  Gabriel  UV 
Products).  [Note:  We  routinely  use  plastic  or  rubber  gloves  to  handle 
the  stained  gels  since  Dr.  B.  Ames  informed  us  that  ethidium  bromide 
has  the  molecular  features  of  many  carcinogens.]   Photographs  and 


-  2-  - 


negatives  can  be  made  with  a  standard  Polaroid  Camera  set:up  for 
making  lantern  slides.   Use  NP  55  positive  and  negative  film  which 
are  exposed  In  a  1545  Land  Film  Holder.   A  double  filter  gives  the 
best  Sntrast.   A  J344  filter  (San  Gabriel  UV  Products)  is  P^ced 
closest  to  the  UV  source, and  the  second  filter  is  a  #9  Wratten  yel 
low  filter;  with  the  diaphragm  wide  open,  exposures  of  ^^t  10-lb 
sec  should  be  sufficient.   Negatives  are  PJePareYL.  *e^urine 
Polaroid.   These  can  be  used  for  reproductions  and  for  measuring 
relative  mobilities  of  gel  fragments.   The  latter  is  done  by  en 
larging  the  image  and  making  a  tracing  of  the  projected  image. 

Gels  can  be  stored  in  the  cold  Ci£  wrapped  in  saran  wrap) ^or.24- 4 8 

hr  without  too  much  diffusion  of  DNA  fragments  in  1 

range. 


10  x  10 6  amu 


REFERENCES 

1.  Sharp,  P.,  Sugden,  W.  and  Sambrook,  J.  (1973) ,  Biochemistry 

1_2,  3055-3063. 

2.  Cohen,  S. ,  Chang,  A.,  Boyer,  H.W.  and  Helling,  R.  (1973), 

Proc.  Natl.'Acad.  Sci,  USA  70,  3240-3244. 


••• 


60 


APPENDIX  G 


61 
MIDNIGHT  HUSTLING,  INC. 

HARVARD     UNIVERSITY 
THE  BIOLOGICAL  LABORATORIES 

16    DIVINITY   AVENUE 
CAMBRIDGE,    MASSACHUSETTS    O2I3B 


APPENDIX  H 


June  1,  1977 


Dear  Colleagues: 

It  has  come  to  my  attention  that  you  are  in  arrears 
for  your  BLMH  subscription.  Please  remit 
$4,000,000.00  or  one  insulin  producing  clone 
immediately. 


Sincerely, 

' 


Circulation  Manager. 


-  JrKT^A- 


Rfil 

•33?* 


STL 


-;-.!.::  «.T.  V'wi.I.'JC.'l  i 
MA  you 


CITY    OF   r:  A;/,  r:: R » r:« c  £•: 


May  I o,   1977 


Mr.  Phil  JpM  land  lor 
Pro.r.irk-nt,  .Nafciqnal'  Academy 


.  . 

?.''.0i  O'iirstituKicn  Avenue,  "N.W 

' 


As  M.-'.yor.of1  thcrGity^pf  Canbrid^o,   I  would  like  to  respectfully  malai 

;-.   ir..-;;,:v!!i  i.  of  you.        *-t  "•-•.••    "-". '..*.•      .,    ;.      •'...-  .-.-.  ;.•'.    ,  •;   •  . 

In  tc.-hiy's  edition  of  the  Boston  Herald •  Arueri can,  a  Hearst  Publication^    ."''. 
thcro  «ro  tv.:o  reports  which  concern  me  greatly.  .  In  Dover >  M*\,   a 
"strange,- oxTiiigCreycd  creature"  v/as  sighted  arttl.  in  Itollis,       v 
Ne-w  Ibi^s'hirc,  n  )!=nn  and.  his  two  sons  \-:cre  coi>rron;:c-i  by  a  "hairy, 
n5.ne  foot  crc-Ature   "  (see.  attached  news. photo  from  the  Herald-American}  .     . 

I  v/c"jl-j  respectfully  ask  that  your  prcr-tig-iaw.*  institution  investigate: 
tl:o:-e  ;7in;Ujios.     I  v/ould  liope  as  well  that  you  might  check  to  sec 
vhe! her  or  not   tliesc  "strange  creatures/'  (should  they  in  fuct  exist) 
are:  5n  auy  way  connected  to^recnmbinant  K\A  erpf-riiticnts"  LaTc-Jjig  place  in 
the  !<{,•!/  rnr-Lr.nd  area. 

Thai)]".."!1.-!^  yon  in  advance  for  your  cooperai.ioat.in  this  matter,  T  rcm?in 

*  *  •  . 

Very  truly  yours, 


A1 1'rcc 
Mayor 


AW:  mi-. 
Enclosure 


Boston  Herald-American  photo 


63 


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CLASSIFRiED       ADS 


BIOLABS  MIDNIGHT  HUSTLER 


Friday,   April  1,  1977 


3n    the  road    to 

Stockholm 

(ITPI)  Cambridge,  MA,  April  1,  1977 — 
The  American  Association  of  Pseudo- 
intellectuals  today  lauded  the 
achievement  of  Dr.  William  A.  Hasel- 
tine  of  the  Sidney  Farber  Cancer 
Center,  Boston,  Massachusetts. 
After  a  search  of  many  years  Dr. 
Haseltime  has  found  a  way  to  get 
his  name  into  the  pages  of  the 
New  York  Times,  and  in  so  doing 
to  cure  cancer  in  chickens.   The 
scope  of  this  achievement  has 
stunned  the  scientific  community 
here.   "I  never  thought  he'd  get 
farther  than  Science  for  the 
People , "  an  unidentified  source 
at  the  Center  for  the  Study  of 
Transient  Fame  said  yesterday. 

A  source  at  the  Sidney 
Farber  Center  described  the 
ingenious  cure  at  a  news  confer 
ence  held  yesterday  at  Harvard's 
Phillips  Brooks  House.   It 
essentially  involves  feeding  the 
affected  chickens  massive  doses 
of  a  chemical  called  "Magic  Spot" 
isolated  by  Dr.  Haseltine.   The 
chemical  is  obtained  from  the  . 
bodies  of  graduate  students  that 
have  died  from  the  the  strain  of 
collaboration  with  Dr.  Haseltine, 
and  is  administered  to  the  chickens 
daily  for  a  period  of  two  months. 
"If  the  chicken  can  survive  that, 
it  can  survive  anything"  the  source 
was  quoted  as  saying  when  asked 
to  explain  the  mechanism  of  the 
cure. 

The  response  to  the  cure  has 
been  overwhelming.  The  Institute 
of  American  Geeks  has  awarded  Dr. 
Haseltine  its  Frank  Perdue  Memorial 
Poultry at ism  Award.  Dr.  Walter 
Gilbert  stated  yesterday  that  the 
cure  was  "intuitively  obvious"; 
Nobel  Laureate  Dr.  George  Wald 
when  contacted  at  the  International 
Symposium  for  Obfuscation  in 
Science  stated  "I  never  understood 
a  word  he  said."  Response  from 
the  rest  of  the  scientific  world 
has  been  equally  gratifying  to 
""  Dr.  Haseltine. 


A  CHICKEN  IN  EVERY  TEST  TUBE 

FOWL  PLAY  BY  MOLECULAR  BIOLOGIST  AND 
NCI  CHICKEN  VIRUS  SUPPLIER  IN  MASSIVE 
GET  RICH  QUICK  SCHEME 

Chicago  Mercantile  Exchange. . .March  31, 
1977... Iced  broilers  led  the  plunge  to 
day  in  the  commodity  pits,  down  the  li- 
• ..  mit  to  6<t  a  Ib.  The  decline,  a  sur 
prise  to  many  traders,  was  related  by 
Mike  Chirigos,  analyst  with  Jack  Gruber 
Associates  of  Bethesda,  to  the  recent 
FDA  decision  allowing  St.  Petersburg 
processor  William  Beard  access  to  the 
iced  broiler  market.  Dr.  Beard's 
chickens  had  been  termed  "bloodless" 
by  the  rest  of  the  industry,  but  the 
FDA  found  Beard's  suggestion  to  dump 
8000  chicken  carcasses  a  week  on  the 
market  "entirely  legal."  In  other 
trading,  platinum  futures  closed 
slightly  higher,  due  to  increased 
demand  by  DNA  sequencers. 


BIOLABS  MIDNIGHT  HUSTLER  65  Friday,   April  1,  1977 


***) 


Salt  City,  Utah.  March  25,  1977 
After  the  first  full  day  of  neg 
otiations  in  the  new  round  of  SALT 
talks,  Harvard  delegate  W.  Gilbert 
said  the  discussions  had  been  useful. 
Because  heads  of  lafcs  are  attending 
the  current  SALT  meeting,  it  was 
expected  that  only  issues  of  para 
mount  importance  would  be  brought 
up,  and  this  proved  to  be  the  case 
today.   "The  first  order  of  business," 
Gilbert  reported,  "was  sodium  chloride, 
and  there  was  general  agreement  that 
0.0666  molar  concentrations  are  not 
required  by  restriction  enzymes."  This 
was  the  salt  breakthrough  everyone  had 
been  waiting  for,  and  which  had  eluded 
previous  SALT  negotiators.   When  quest 
ioned  by  a  Hustler  columnist  as  to  why 
it  had  taken  so  long  to  reach  an  under 
standing  on  salinity  of  buffers,  Gilbert's 
only  reply  was,  "Your  column  needs  more 
salt." 

With  regard  to  on-site  DNA  inspection, 
a  salient  proposal  was  set  forth  by  K.G.B. 
Skryabin,  one  of  the  Moscow  representatives 
to  SALT.   "It  is  a  well  known  fact,"  he 
pointed  out,  "that  the  hydrazine-powered 
deoxynuclear  cruise  missle  requires  salt 
to  suppress  T-cells  in  its  nose  cone." 
Succint  as  it  was,  this  statement  of  the 
Soviet  position  admittedly  confused 
Gilbert  and  the  Harvard  observers,  who 
adjourned  to  study  the  proposal  with 
the  view  that  it  turned  on  detection  of 
cytosine  residues  in  table  salt  by 
their  smell.  An  announcement  of  the 
Harvard  response  is  expected  later 
today  after  consultation  with  experts 
back  at  the  salt  mines  in  Cambridge. 


HELPFUL  HIA/TS  FOR  LABORATORY  GARPENERS 

Now  that  Spring  i*  on  <Lt*  way  and  you. 
can  open  youA  tab  tucndowts,  you.  c.an't 
excuse  the.  pitiful  appe.oAanc.e.  o&  youA 
Lab  ptant&  by  blaming  the.  teAAible. 
6ume*  -at  the.  tinom&  .  -.  .  {AfiteA  aJUL,  yoa'Ae. 
&tUUL  aLive.  asie.n't  you?)  HeAe.  axe. 
&ome.  heJtp^uJL  hint*  fan.  making  youA  lab 
plant*  look  huaLtky  and  gie.e.n: 

1)  WateA  them  only  when  the.  leave* 
be.Q<Ln  to  dfioop. 

2)  Make,  yoan  wateAing  solution  IM 
KCt;  thi*  &eAve*  to  make.  the.  osmotic. 
pA.e*4uAe.  <in  the.  boU,  g/ieateA  than  that 
hi  the,  JLeaveA  oi  the.  plant  theAe.by  uiith- 
dsuwlng  wateA  and  aaa&tng  the.  leave*  to 
dfioop.  you.  OJKL  the.n  ke.pt  buty  by 
conAtantty  watering  you*,  plant*. 

3)  Spiay  the.  leave*  with  a  mlt>teA  at 
l.ea*t  once  a  weefe.  A  I/TOO  solution 

o£  Csi&oAol  Gie.e.n  and  vuateA.  ai.ve*  a 
v<ibiant,  Qtie.en  appearance,  to  the. 


4}  At  odd  <inteA.val&  du/ung  the. 
day  and  night  ^Lick  youA.  lab  light* 
on  and  o  66  napidJty.  Thi*  Mill  heJtp 
youA.  ^towojujiQ  plantd  b£o44om  mone. 
pfio^m*eJiy  by  g<iv£ng  tkejn  moie.  than 
one.  photopeAiod  a  day. 


Lab  regulation 

ONLY  I  AM  AUTHORIZED  TO  AUTHORIZE 
AUTHORIZATIONS.   ANYONE  WHO 
AUTHORIZES  AN  AUTHORIZATION  MUST 
HAVE  IT  AUTHORIZED  BY  ME. 


PAT 


BIOLABS  MIDNIGHT  HUSTLER 


66 


Friday,      April   1,    1977 


*  NOSTALGIA.... 

The  way  it  was 


Cartridge,  Ma... March  31,  1967... The  Cant- 
bridge  social  scene  was  highlighted  by  the 
party  last  weekend  at  Prof.  Jim  Watson's 
Apian  Way  apartment,  given  for  Mario  Cap- 
pechi,  the  junior  fellow  who  finally  de 
cided  to  write  up  his  thesis.   SOK  par 
ticularly  inters ting  couples  were  noted, 
not  the  least  of  the*  involving  Ji»  hia- 
self  (currently  one  of  Canb  ridge's  nost 
eligible  bachelors)  a^d  Lorraine  Larrison, 
a  post-doc  in  Denhardt's  group.  Lorraine 
carae  over  early  for  dinner,  before  the 
party  got  going,  but  our  reporter  was 
there  to  take  in  the  scene.  Dare  Dres 
sier  arrived  with  Ursula  Johnson,  and 
Chris  Weiss  with  a  non-scientist. 
(Young  Janice  Pero- cane  unescorted. )  Once 
the  Music  and  dancing  got  underway,  it 
was  just  like  any  typical  Cambridge  par 
ty.  A  group  of  party  crashers,  led  by 
Mike  Sinensky,  a  first-year  graduate 
student  in  Biocheaistry,  tried  to  enter 
around  nidnight,  but  Jim  rose  to  the 
occasion  and  threw  them  out.   Sinensky, 
laboring  under  the  false  impression 
that  this  was  an  official  party  of  the 
department,  identified  himself  as  M-ty^ 
Sinensky,  first-year  graduate  student. 
Jii>  vas  unBoved,  and  undoubtedly  re— 
•sabered  the  name. 


BLMH  PUZZLE 


Unscraable  the  following  words.     Then 
use  the  circled  letters  to  spell  out 
a  two-word  phrase. 

:0C  E  N  6  I@C  0 
L0D  R  I  M  A  Y  C  A 

r  00B  A  R  0  L  A  R 

H  A  N©M  A  T 


m 


OR  WHAT  GOES  OH  WKILE  WAITING  FOP. 

THE  MACHINE  TO  WARM  OP 


Cambridge.,  fa...Hy4tesUcatx.ej)0'i£i>  o{  Hhat{- 
majt,   hati-a&Ugatoti"  >iejcombinont  PWA  inon 
contour  to  {itteJi  out  o{  tine.  Biotoaicat 
LaboiatoJii&A  despite.  a.  tight  contain  o{  &e.- 
cn&cy  iapo&ed  by  the.  dinejcton  o{  the.  tab*, 
"Iionpant&"  Vottitt.     ?>uo{eA*o>L  Kith  Hubband 
catted  today  {ox.  a.  {utt  <Lnvi&t<iaa£ion, 
claiming  that  {emote.  &ta{{  mejnbin&  have,  been 
a&&au£tejL  white.  UAJJIQ  copying  truchineA  and 
in  photographic  danknoom&.     ?oULitt,  how- 
eveA,  x&poit&  that  aJUL  ca&e*  o{  a&tautt 
can  be.  traced  to  noHmjJULy  LiJbJjcLinowt>  mate. 
{acutty  mejfbesiA.     Vi^itLng  tejctwieA  V*.. 
Je.{{eAy  HWLvi  teamed  the.  ti&wtion  "&e.- 
ductive..  "     Reached  {on  coimext  white,  at 
tending  the.  Wo*td  Heatth  Onganizatitin 
ttMinan  on  cannibatL&m  in  Papua.,  Wew 
Guinea,  Dn.  WatteA  Gitbeat  catted  Hubbasid 
"iAAZ&pon&ihte.."     "It'&  a  known  {act," 
GitbeAt  wa&  quoted  a&  toying,   "that  the. 
de-Acsupticn  o{  thej>e.  40  catted  mon&teM 
{it&  many  o{  my  anaduate.  &tudent&  and 
po&tdoctaxat 


Quotation  of  the  week 

"My  iQ  is  -35.' 


paper  of  the  month: 

A  new  method  fer  promoting  DNA 

it^jl  ndfzte  •J^-~r*/1'r^'  •  '  inr/pipr  liJai  ) 


ALLAN  M-  MAXAM  AND  WALTER  GILBERT 


i  MM 


CotMbuted 


b,  WelterCObert,  December  8.  1979 


BIOLABS  MIDNIGHT  HUSTLER  67  Friday,   April  1,  1977 


SPEAKS  OUT    FOR 
NEW    YOUTH   DIET 


March  28,  1977..   The  National  En 
quirer  reported  today  that  Dr.  Ruth 
Hubbard  of  Harvard  University  is  the 
first  U.S.  scientist  to  speak  out  in 
•support  of  Dr.  Frank's  controversial 
youth  diet.  Dr.  Frank's  diet  is 
based  on  the  use  of  RNA,  a  molecule 
chemically  related  to  DMA.  The  diet 
is  considered  controversial  because 
tests  leaked  from  Canada  indicated. 
that  RNA  induces  tumor  formation  in 
rats.  Dr.  Hubbard,  in  an  informal 
interview,  gave  her  personal  testi 
monial.  "This  diet  is  the  fountain 
of  youth.  I've  been  on  it  for  15 
years—I'm  living  proof!"  She  also 
stressed  that  rumors  of  recombinant 
RNA  experiments  were  totally  false. 
"No  one  at  Fort  Detrick  ever  died 
while  on  this  diet,Vshe  stated. 


.  i     DR.  HUBBARD  'S 

.•  ^^^^^  Hia.  U.  •    FAT.  OFF. 

GVeaeta&le/ 

}  •:  •;;•  '  BRAND 

:\\  GERMICIDE 


.8  ' 


This  product  has  no  connection  whstto- 

fvtr  tvilh  American  National  Red  Crott.  • 

AN  ANTISEPTIC 
NON-POISONOUS  GERMICIDE 

CONTENTS  3  FLUID  OUNCES 

.'      '        ACTIVE    INGREDIENTS 

85%  Alcohol.  10%  Mixture  of  Thymol, 
Oil  Origanum,  Oil  Caleput,  OH  Laven 
der,  Oil  Rosemary  and  Oil  Pine,  5% 
Inert. 

ATOMIZER    COMPLETE 

J.  Hubbard  Co.,  Inc. 
Nashua,  N.  H. 

ESTABLISHED      1869 


The  aibove  questionable  object  was 
discovered  in  the  abandoned 
laboratory  of  Dr.  T.  Maniatis. 
Conclusive  identification  has 
provesrv  to  be  more  difficult  than 
expected,  but  the  experts  have 
offered  some  persuasive  hypotheses : 

W.  Gilbert:  Half-eaten  nude  mouse. 

A.  Ef stratiadis :  It's  Jonathan 

King. 

M.  Ptashne:  Where's  the  key 

to  the  Xerox  room? 

A.  Maxam:  Looks  like  a  G,  but  it 
could  be  an  A. 

C.  Williams:  Why  that's  an 

Assonorium  pharoanis 

M.  Pasek:  Two-fold  rotational 
symmetry . 

A.  Worcel:  Nu  body. 


BIOLABS  MIDNIGHT  HUSTLER 


Friday,   April  1,  1977 


•^MWi'lJfW 


.  fiSt$5^«?5!!31 

^m^f-s^0!^^itf 


BIOLABS  MIDNIGHT  HUSTLER 


69 


Friday,   April  1,  1977 


Dear  Debbie: 

I'm  in  love  with  a  geneticist  from  Geneva, 
although  sometimes  I  think  he's  lacking. 
Talk  about  the  cowboy  kissing  the  horse, 
he  sleeps  with  his  strain  collection  un 
der  his  pillow  and  he  won't  even  kiss  me 
because  he  says  we  can't  use  sterile 
technique.   Is  this  a  hopeless  romance? 

f 


say  it's  hopeless,  but 


it 

n  need 

try 


Dear  I  : 
I  wouldn' t 

sounds  as  though  your  romance  is 
of  an  active  promoter.   You  might 
telling  him  that  some  of  the  most  in 
teresting  bugs  around  result  from  using 
non-sterile  technique,  and  if  that  doesn't 
work,  you  could  threaten  to  put  saltpeter 
in  his  broth.  As  for  his  sleeping  with 
his  strain  collection  under  his  pillow, 
don't  complain  too  much,  my  dear.  There's 
one  woman  who  wrote  me  that  her  husband 
sleeps  with  a  lighted  cigar  in  his  mouth 
and  that  she  counts  smoke  rings  in  order 
to  go  to  sleep.   But  if  worse  comes  to 
worst,  better  move  upstream  to  a  faster 
operator. 


Dear  Debbie: 

My  old  lab  buddy  has  left  me,  and  a  new  one 

has  moved  in.   My  problem  is  that  the  new 

buddy  is  a  neat  freak  (i.e.,  anal.) 

What  can  I  do  about  it? 

Sloppy  but  Happy 

Dear  Sloppy  but  Happy: 
My  anal-ysis  of  the  situation  is  this: 
the  core  of  the  whole  problem  will  be 
for  you  to  learn  to  be  happy  with  your 
own  mess  and  not  depend  on  others  to 
create  disarray  for  you.  Since  you 
didn't  outline  the  sequence  of  events 
which  caused  your  old  lab  buddy  to 
leave,  they  may  have  been  rather  trau 
matic,  and  you  may  still  feel  denatur 
ed.  Give  yourself  some  renaturation 
time;  your  neat  freak  lab  partner  may 
test  out  okay.   In  the  meantime,  con 
tinue  to  be  happy  while  Piling  it 
higher-  and  Deeper. 


Dear  Debbie: 

I'm  an  investigator — a  private  in 
vestigator  these  days.  Recently  I 
have  been  trying  to  tail  a  plasmid, 
but  every  time  I  get  close,  it  under 
goes  some  strange  transformation  and 
I  loose  it.   I've  tailed  it  in  Cacody- 
late  and  in  Cobalt,  I've  even  tried 
a  p~32  homing  device.  Nothing  seems 
to  work!  Well,  if  you  think  this  sounds 
bad,  you  should  try  tailing  a  lac 
fragment  with  the  "dT's."  I  know 
there's  some  kind  of  promotion  opera 
tion  upstream  but  it's  a  puzzle  -  the 
pieces  of  which  haven't  yet  annealed. 
Any  advice? 

Turning  Blue  on  an  XG  Plate 

Dear  Turning  Blue  on  an  XG  Plate: 
Get  out  fast! 

Dear  Debbie: 

Why  do  we  have  glass  pipes  in  our 

hallway? 

Unsigned 

Dear  Unsigned: 

The  better  to  see  it  with,  my  dear. 


And  now  I'm  going  to  take  a  poll 
that  I'm  sure  will  interest  every 
one.  The  question  is,  "If  you  had 
It  to  do  over  again,  would  you  go 
to  graduate  school?"  Answer  only 
on  3  x  5"  notecards  please.  Simply 
state  "Yes"  or  "No,"  and  indicate 
your  year  in  graduate  school.   If 
your  answer  is  "No,"  please  state 
what  you  wish  you  had  done  instead. 
The  cards  can  be  deposited  in  the 
"Dear  Debbie"  box  outside  Room  386, 
and  the  deadline  is  the  day  before 
the  next  edition  of  the  Biolabs 
Midnight  Hustler. 


70 


X 

LU 


HP 
is 


CD 


BIOLABS  MIDNIGHT  HUSTLER 


71 


Friday,   April  1,  1977 


THE     CASE    OF    THE 
POSTHUMOUS     POST-D 


Another  BLHH  crime-stopper  story  in  the  continuing  saga  of  WG-manl 

by  Agatha  Crick-y 


The  man  walked  purposefully  down  the 
dimly  lit  hall.  Dressed  in  a  crumpled  green 
suit,  he  had  a  firm,  proprietary  tread.  He 
had  a  job  to  do.  He  wasn't  sure  what  the 
job  was  yet,  but  he  knew  that  by  the  follow 
ing  morning,  a  murderer  would  be  caught,  a 
burglary  stopped,  a  spy  discovered:  the  -job 
would  be  done.   Suddenly,  he  ducked  into  the 
darkroom  and  shut  the  door.  Five  minutes 
(with  agitation) ,  six  minutes,  seven  minutes 
— and  he  emerged,  dressed  in  sandals,  jeans 
shorts,  a  bright  orange  shirt,  a  long,  flow 
ing  purple  cape  and  glasses  removed.  The 
Green  Hornet?  Wonder  Woman?  No,  it's — 
WG'-man I   By  day,  a  mild-mannered  biochemist 
and  molecular  biologist,  selflessly  serving 
humanity  seeking  the  cure  for  cancer  (or 
the  Nobel  Prize,  whichever  comes  first). 
By  night,  the  GREATEST  CRIME-SOLVER  OF  THEM 

ALL:: 

Last  week,  we  left  our  hero  at  the 
Moon  Villa,  celebrating  with  a  pastry  lunch 
the  capture  of  the  notorious  Reznikoff  Rip 
per  and  his  gang.  But  today  was  another 
day.   WG-man  strode  into  his  office,  swept 
papers  and  journals  off  his  desk,  climbed  up 
and  struggled  to  open  the  window.  Just  as 
it  opened,  he  heard  a  strange  and  ominous 
feound.   "That  sounds  strange  and  ominous1,1  he 
noted  sagely  and  clambered  off  his  desk,  un 
answered  letters  and  fluorescent  chalks  fly 
ing  as  he  fell.   Picking  himself  up,  he 
noticed  when  he  squinted  that  a  pair  of 
shoes  were  sticking  out  of  the  doorway  to 
room  386.   Intrigued,  he  went  toward  them, 
musing,  "Hm,  it  seems  my  post-doc  has  left 
his  shoes  in  the  lab."  As  he  approached  the 
doorway,  his  body  tensed.   The  post-doc's 
shoes  were  there,  alright,  but  with  the 
post-doc  still  wearing  them:  The  post-doc 
was  flat  on  his  back,  possibly  dead,  but  WG- 
man  couldn't  tell— for  there  was  a  300-lb. 
crystal  of  represser-operator  complex  on 
the  chest  of  the  junior  scientist".   "Heavens 
to  Betsy:1'  exclaimed  WG-man,  though  he 


quickly  corrected  himself,  remembering  that 
she  had  left.   "By  Benno,."  he  gasped  and 
this  time  grabbed  the  microscope  to  see 
the  size  and  shape  of  the  wonderous  crystal. 
"Holy  coli:"  he  shouted,  "That's  a  300-lb. 
crystal  of  repressor-operator  complex:11 
Then  he  remembered  the  post-doc  crushed 
beneath  it.   Before  the  next  and  immortal 
words— "what1 s  up?" — could  even  be  uttered, 
WG-man  found  his  cape  was  being  thrown  over 
his  head  and  tied  securely  around  his 
waist.    HE  WAS  CAUGHT:: 

Who  had  been  so  clever  ag. 
to  trap  WG-man  at  his  weakest—when  his 
back  was  turned?  What  nefarious  mind  had 
created  the  much -wanted  crystal  to  bewitch 
his  faculties  and  lower  his  defenses?  Who 
didn't  know  that  he  was  blind  without  his 
glasses  anyway?  And — what  did  this  latest 
mishap  have  to  do  with  the  brutal  murder 
of  his  colleague  and  young  friend?  What 
next  in  this  sinister  plot  against  our 
hero,  WG-man? 

Be  sure  to  read  the  next  issue  of 
the  Bio  Labs  Midnight  Hustler: 


WANTED 


Tea  Biochemist 


Apply  Monday  through 
Friday  4  p.m. 


Room  375 
(tea  room) 


DiUUMDO     Hi  JJIM1  OH  I 


BlOLAUb    MiUNlbHI    MUb  I  LhK     73      rriuay,       aprio.    ±, 


CLASSIFRIED       ADS 


/  / 


NEW::  From  BRINKPERSON  INSTRUMENTS,  inc. 

The  LATEST  in  nonsexist  automatic  pipetting — 
the  PIPETTE   INDIVIDUAL 

Endorsed  by  Emily  Friedan's  mother  and  sold  to  you  by  a  salesperson 
formerly  an  editor  of  Ms.  Why  let  other  labs  be  groovier  than  you? 
Order  today  and  receive  free.  your. own  autographed  copy  of  Fear  of  Flying 
and  a  flammable  brassiere  (suitable  for  burning) . 


Harvard  Biological  Laboratories 
16  Divinity  Ave 
Loony  Bin 
March  28,  1977 


Classifieds  Editor 
Biolabs  Midnight  Hustler 
388  Biolabs 
Cambridge ,  Mass 
USA 

Dear  Sir  or  Madam  (whichever  you  prefer,  no  skin  of^my  nose)  , 

C  would  be  very  grateful  if  you  would  publish  the  following 
classified  ad.  Needless  to  say,  I  would  appreciate  your  discresion(sp?) 

in  this  matter. 


WANTED:  WM,  5G,  companionable , seeks  lab  partner.   Interests  include 
gerbil  propagation,  biochemistry.  Object,  co-existence.   Reply  MH  box 

PE40 . 


Regards , 


74 


©  BIO  LABS 


IGHT  HUSTLER 


U  Know  who 


DEFENDING  THE  LAD  REQUIRES  BUTTONING  ONE'S  LIP  AT  MEETINGS 


Courtesy  Biolabs 
Tribune 


B10LABS  MIDNIGHT  HUSTLER   75  Friday,   April  1,1977 


Well,  well  recombo-readers, 
guess  who's  name  is  appearing 
in  that  imf amous  gene  maker 
magazine,  Gene  Gene  Gene-Jelly 
Bean  again  this  week?   That's 
right,  Serb  Hoyer  and  Rill  Butter 
once  again.   This  time  the  lucky 
number  is  three  —  are  you  ready 
—  Full  Length  Reverse  Transcript 
of  Bovine  miRNA,  Selection  and 
Amplification  of  Bovine  Secretory 
Control  Genes,  and  Bacterial 
Expression  of  Bovus  Lactus  Locus  I 
Hoyer  said  that  the  full  signi 
ficance  of  this  work  would  be 
realized  only  after  Micro- 
Milkers  had  been  developed — 
"They're  cute,  but  they're 
small,"  he  said.   Another 
researcher  questioned  about 
this  retorted,  "That's  Udder 
Bullshit."   We  won't  mention  any 
names  but  his  initials  are  G.W. — 
not  necessarily  in  that  order. 


clone  precipitation  test.   He's 
now  trying  to  work  out  a  method 
for  resuspending  the  precipitatedd 
anti-Body.   His  colaborator, 
Kourilsky,  was  unable  to  comment 
from  under  the  cover  of  his  agar 
plate.   I  hope  he's  good  at 
anaerobic  growth. 

Ta-ta  for  now  Chimeric  fans. 


Well,  we  don't  know  much  about 
that,  G.W.,  but  it  does  seem  to 
this  journalist  that  Hoyer  is 
trying  to  clone  the  10 2n'  element 
in  his  hat  —  the  kind  made  of 
Au  and  indiginous  to  Stockholm. 


Meanwhile,  over  in  France  • — 
P.  Tiollais  announced  the 
completion  of  the  whole  human 


76 


APFEEDDt"*! 


Director,  NIH 
Through:  ES/NIH  -/,'• 

Deputy  Director ™£6f  Science,  NIH 


PUBLIC:  HEALTH  SERVICE 

N \T10NAI    INSTITUTF  OP  HEALTH 


DATE:   July  6,  1977 


FROM    .- 


Director 

Of  fee  of  Recombinant  DNA  Activities,  NIGMS 


SUBJECT: 


El'  ,   lost- 

Vec-or  Systems  Based  on  X1776  and  Plasmids  pBR313  and  pBR322 


'     ^P  ?5  ^  Anivers!tv  of  California,  San  Francisco. 

fi  thLU±e'sity  °f  Washln9ton  submitted  data  on 

n      ra'  °3  P^P0??3  EK2  host-vector  systems  based  on  E.  coli 
K-12  strain  X1776  and  plasmids  pMB9,  pBR313  and  PBR322  (Attachment  lT~ 

toleSlP  Pr^  CeV^  by  a  su^°™littee  of  the  Secombinant  oS 
Molecule  Program  Advisory  Committee  on  January  14,  and  by  the  full 

°n       15 


, 
15'    ^  Conn,ittee  recomended        t 


you 


The  systems  X1776  (PBR313)  and  X1776  (pBR322)   tentatively  were 
fST^I  f°r  C%rtifi?ation  P«^ing  the  receipt  of  SheTdata 
ofThese't  H?,^/alkS!  conc!rni1^  mobilization  and  transmission 
of  these  two  plasmids.     The  Working  Group  for  EK2  Host-Plasmid 
Systems,  which  met  on  March  21,  prepared  a  report  (Attachment  II) 
oBW22CwCuld  S      ^J6rtain,  additional  test  data  for  PSi  and 

fof  sutTsHon^off  ?  ln  °rder  t0  Satisfy  the  revised  requirements 
submission  of  data  on  proposed  EK2  systems  (Attachment  III). 

31 


roun  Pr?ram  Mvisory  Committee  reviewed  the 

Group's  report  on  June  23.     The  Committee  voted  9  to  0  to 

o  £YS?£  fd  thS  "Instructi0^  to  investigators  Concerning 
Data  to  be  Submitted  on  Host-Plasmid  Systems  Proposed  for  EK2 
Certification."     Dr.  Curtiss  abstained  from  the  vote. 


po  H    data.s^itted  by  ^.  Falkow  (Attachment  V) 

pointed  out  that  all  the  required  tests  are  met  by  these  systems 

eCOimiend  that  *1776  (PB^3)  Sd 
EK2  host-vector  systems.     Dr.  Curtiss 

of 


77 


Recommendation:     1  recommend  that  you  certify 
and  xl?76  (p3R322)  as  EK2  host-vector  systems 


Attachments 


(PBR313) 


~Willia¥"j.  Gfertland,  "Jr.T"Ph'.Dl 


Concurrence: 


Dated 


Donald  S.  Fredrickson,  M.D. 


'-1-IS.2J- 


78 


INDEX--Mary  C.  Betlach 

Asilomar  Conference  on  Recombinant  DNA 
Molecules,  16,  19 

Bishop,  J.  Michael,  2,  17-18 

Bolivar,  Paco,  3,  20 

Boyer,  Herbert  W.,  10,  16,  19,  24,  29-30 

and  Cohen,  13-14 

and  Goodman,  28 

and  Genentech,  29,  31-32 

and  Nobel  Prize,  32-33 

and  students,  30-3 1 

prospect  for  cloning  insulin,  6,  1 1 

personality,  14,  30 

scientific  publication,  14 
Boyer  laboratory 

atmosphere,  7-8 

Boyer-Cohen  recombinant  DNA 
experiments/publication,  6-7,  9,  10, 
11-13,15 

early  DNA  cloning  experiments,  8-9,  19, 
21 

move  to  biochemistry  department,  25 

organization  &  space  in  microbiology 

department,  1-2,  5 

tension  with  Jawetz  lab,  4-6 

centrifuge/ultracentrifuge,  4 
Chang,  Annie,  9,  10 
Cohen,  Stanley  N.,  21 

personality,  15,  30 

recombinant  DNA  research/publication, 
9,13-14,15 

DNA  sequencing,  21 

Echols,  Harrison,  2,  8 

enzyme  restriction  &  modification  research, 
3-4,  6,  9,  12,  20-22 
ethidium  bromide,  18 

Fredrickson,  Donald,  24 

Gartland,  William,  24 
Genentech,  29,  31-31 
Gilbert,  Walter,  28 
Goodman,  Howard,  24,  28 


Gordon  Conference  on  Nucleic  Acids,  1973, 

10,  13 
Greene,  Patricia,  3,  13 

Hanna,  Lavelle,  5 
Hedgpeth,  Joel,  1,2,21 
Helling,  Robert,  2,  3,  9,  13 
Heyneker,  Herbert,  3 

insulin  clone  controversy,  UCSF,  23-24, 
26-27 

Jawetz,  Ernest,  1,4-6 

Kelly,  Regis,  28 

litigation,  Eli  Lilly/Genentech/UC,  16-17 

Mantei,  Ned,  2 

"Midnight  Hustler,"  28 

molecular  biology  as  new  discipline,  6 

Mullis,  Kary,  33 

NTH  Guidelines  for  Recombinant  DNA 
Research,  19 

Orias,  Ed,  8 

Parnassus  Pharmaceuticals,  1 
physical  containment  laboratory,  17 
plasmids,  12 

development,  20-22 

dissemination,  23,  26 

isolation,  3,  9 

pBR322,  NTH  approval/certification,  23- 

24,  26-27 

resistance  markers,  20-22 
specific  plasmids,  20-23 

reagent  exchange  protocol,  23 
recombinant  DNA/cloning  technology 
biosafety,  16-18,  19,23,29 
Boyer-Cohen  experiments/publication,  6- 

7,9,10,11-13,  15 
commercial  applications,  11,  29 
controversy  over  commercialization,  31- 
32 


79 


recombinant  DNA/cloning  technology  (continued) 
early  DNA  cloning  experiments,  8-9,  19, 

21 

Riggs,  Arthur,  29 
Rodriguez,  Ray,  3,  20 
Roulland-Dussoix,  Daisy,  2-3 
Rutter,  William  J.,  24,  25,  27,  28,  30 

Shine,  John,  23 

Senate  subcommittee  hearing  on  recombinant 
DNA,  1977,  29-30 
somatostatin  research,  29-30 
Sutcliff,  Greg,  20 
Swanson,  Robert  A.,  29 

Tate,  Bob,  3 

Ullrich,  Axel,  26-28 
Ullrich,  Suzanne,  28 

University  of  California,  San  of  Francisco 
biochemistry  department,  8,  24,  28 
contract  with  Genentech,  29 
controversy  over  commercialization,  31- 

32 
Health  Sciences  Towers  East  &  West,  2, 

25 

microbiology  department,  1-2,  25 
photography  department,  13 
Program  in  Biological  Sciences,  25 

vectors,  17,  18 
Weissmann,  Charles,  2 
Yoshimori,  Robert,  21 


Sally  Smith  Hughes 


Graduated  from  the  University  of  California,  Berkeley,  in  1963  with  an  A.B. 
degree  in  zoology,  and  from  the  University  of  California,  San  Francisco,  in  1966 
with  an  M.A.  degree  in  anatomy.  She  received  a  Ph.D.  degree  in  the  history  of 
science  and  medicine  from  the  Royal  Postgraduate  Medical  School,  University 
of  London,  in  1972. 

Postgraduate  research  histologist,  the  Cardiovascular  Research  Institute, 
University  of  California,  San  Francisco,  1966-1969;  science  historian  for  the 
History  of  Science  and  Technology  Program,  The  Bancroft  Library,  1978-1980. 

Presently  research  historian  and  principal  editor  on  medical  and  scientific  topics 
for  the  Regional  Oral  History  Office,  University  of  California,  Berkeley.  Author 
of  The  Virus:  A  History  of  the  Concept,  Sally  Smith  Hughes  is  currently 
interviewing  and  writing  in  the  fields  of  AIDS  and  molecular 
biology/biotechnology.