NASA CONTRACTOR
REPORT
CO
vO
I NASA CR-673
THE MOLECULAR ASPECTS
OF BIOLOGICAL DEVELOPMENT
Edited by R. A. Deering and Muriel Trask
Prepared by
THE PENNSYLVANIA STATE UNIVERSITY
University Park, Pa.
for
£H )NAL AERONAUTICS AND SPACE ADMINISTRATION • WASHINGTON, D. C. • FEBRUARY 1967
61i "
D34
r
NASA CR-673
THE MOLECULAR ASPECTS OF BIOLOGICAL DEVELOPMENT
Edited by R. A. Deering and Muriel Trask
A Workshop
Held at the Pennsylvania State University, University Park, Pa.
July 19-21, 1965
'"^ Sponsored by /
1-0
a The Biophysics Department of
The Pennsylvania State University
(Grant NsG-324)
and
National Aeronautics and Space Administration
Distribution of this report is provided in the interest of
information exchange. Responsibility for the contents
resides in the author or organization that prepared it.
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
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CONTENTS
Page
Preface v
Participants vi
GROSS, PAUL R. RNA and Protein Synthesis in Developing Sea
Urchin Eggs . o 1
EPEL, DAVID Early Biochemical Events Following Fertilization
of Sea Urchin Eggs 17
KOHNE, DAVID E. Ribosomal Ribonucleic Acid Synthesis in
Rana pipiens Embryos. 35
PAPACONSTANTINOU, JOHN Molecular Aspects of Lens Cell
Differentiation 47
TILL, JAMES E. Proliferation and Differentiation of Stem Cells
of the Blood-forming System of the Mouse 69
MASSARO, EDWARD J. The Structure of Isozyme Systems and
Their Role in Development 77
GREGG, JAMES H. Antigen Synthesis During Reorganization in
the Cullular Slime Molds 93
WRIGHT, BARBARA Control of Enzyme Activities in D.
discoideum During Development 109
KAHN, ARNOLD J. Cell Interactions in Slime Mold (Acrasina)
Development 123
CHALKLEY, ROGER Histones in Relation to Control in Living
Systems 131
CANTING, EDWARD C. Dynamics of the Point of No Return
During Differentiation in Blastocladiella emersonii 149
LOVETT, JAMES S. Nucleic Acid Synthesis During Differentia-
tion of Blastocladiella emersonii 165
TS'O, PAUL O. P. The Molecular Aspect of Nucleic Acid Inter-
actions 183
TS'O, PAUL O. P. The Problems and Promises of Research on
the Molecular Aspects of Development (Workshop Summary) 195
ui
Preface
This is the transcript of an informal work-
shop on "The Molecular Aspects of Develop-
ment" held at the Nittany Lion Inn of The
Pennsylvania State University, University Park,
Pennsylvania, on July 19-21, 1965. It was or-
ganized by The Pennsylvania State University
Biophysics Department under the sponsorship
of the University and the National Aeronautics
and Space Administration. Its purpose was to
bring together scientists actively doing research
in different areas of differentiation and develop-
ment. Researchers from several disciplines
doing work on many different biological systems
were invited to give presentations ofthfeirwork.
These presentations were informal and discus-
sion was invited at all times. In addition to
those invited to give talks, participants were
invited from many related departments at the
University. A complete list of all participants
is given following this preface.
This conference reflects the growing in-
terest in the problems of differentiation and
development as attacked from the molecular
point of view. The combined backgrounds and
methods of many disciplines such as biochem-
istry, biophysics, cell biology, genetics, micro-
biology, physical chemistry, physics, mathe-
matics and others are being brought to bear on
this problem and the potential reward is great.
An interdisciplinary approach to this problem
is necessary and should be emphasized. Free
informal communication between scientists with
differing backgrounds and viewpoints is essen-
tial. We feel that this conference was a success-
ful step in that direction and as such was
valuable to all participants. We hope that this
publication of the presentations and discussions
will be useful to the student, teacher and re-
searcher who is interested in the problem of
development in biological systems.
The conference was taped and transcribed.
Each participant was then given a chance to
rework his contribution, with the directive to
retain the informality and to leave spontaneous
discussion intermixed with presentations. The
slides and blackboard drawings used in most
presentations have been reproduced here as
figures, some of which were redrawn from
photographs of the projected slides or sketches
furnished by the authors. These are sometimes
incomplete and are merely used to illustrate
points in the talks. More complete data can
often be found in the original publications which
are referenced throughout. The attempt to retain
the spontaneous, informal flavor of the workshop
may result in some presentations seeming
incomplete and unpolished. However, since
spontaneity and informality are the values of a
conference of this type, we feel the reader should
be allowed as true a view of it as possible.
We wish to thank those who made this con-
ference and publication possible: in particular,
the National Aeronautics and Space Adminis-
tration (Grant NsG-324), through the efforts of
Dr. George J. Jacobs, Chief, Physical Biology
Biosciences Program; The Pennsylvania State
University Biophysics Department and its
chairman. Dr. Ernest C. Pollard; and The
Pennsylvania State University College of Science
and its Dean, Dr. C. I. Noll. We are indebted
to Dr. Paul Ts'o for his extra effort in pre-
paring the summary presentation which appears
at the end. Finally, we wish to thank all par-
ticipants for their enthusiastic discussion and
their cooperation and hard work in preparing
presentations and manuscripts.
Editors
May 4, 1966
Participants
Edward C. Cantino*
Department of Botany and Plant Pathology
Michigan State University
Roger Chalkley*
Division of Biology
California Institute of Technology
Thomas Coohill
Biophysics Department
The Pennsylvania State University
Rufus Day
Biophysics Department
The Pennsylvania State University
R. A. Deering/?
Biophysics Department
The Pennsylvania State University
David Epel*
Hopkins Marine Station
Pacific Grove, California
Charles Fergus
Botany Department
The Pennsylvania State University
John Freim
Biophysics Department
The Pennsylvania State University
William Ginoza
Biophysics Department
The Pennsylvania State University
James H. Gregg *^
Department of Zoology
University of Florida
Paul R. Gross * i*
Department of Biology
Massachusetts Institute of Technology
Paul Grun
Botany Department
The Pennsylvania State University
Allan Hanks
Biophysics Department
The Pennsylvania State University
Wesley Hymer
Zoology Department
The Pennsylvania State University
Arnold Kahn*
Department of Zoology
Syracuse University
George Kantor
Biophysics Department
The Pennsylvania State University
David Kohne *
Department of Terrestrial Magnestism
Carnegie Institution of Washington
James S. Lovett
Department of Biological Sciences
Purdue University
Charles Lytle
Zoology Department
The Pennsylvania State University
Richard McCarl
Biochemistry Department
The Pennsylvania State University
Edward J. Massaro*
Department of Biology
Yale University
Rainer Maurer
Division of Biology
California Institute of Technology
Mary Osborn
Biophysics Department
The Pennsylvania State University
John Papaconstantinou*
Biology Division
Oak Ridge National Laboratory
Stanley Person
Biophysics Department
The Pennsylvania State University
Ernest C. Pollard*^
Biophysics Department
The Pennsylvania State University
VI
Harald Schraer
Biophysics Department
Tlie Pennsylvania State University
Wallace Snipes
Biophysics Department
The Pennsylvania State University
Greenville K. Strother
Biophysics Department
The Pennsylvania State University
William Taylor
Biophysics Department
The Pennsylvania State University
Daniel Tershak
Microbiology Department
The Pennsylvania State University
* Principal Speakers
# Session Chairmen
James Till *
Department of Medical Biophysics
Ontario Cancer Institute
Paul O. P. Ts'o
Department of Radiological Science
School of Hygiene and Public Health
The Johns Hopkins University
Barbara Wright*
John Collins Warren Laboratory
Huntington Memorial Hospital
Massachusetts General Hospital
James Wright #
Botany Department
The Pennsylvania State University
Leonard Zimmerman
Microbiology Department
The Pennsylvania State University
Vll
RNA AND PROTEIN SYNTHESIS IN DEVELOPING
SEA URCHIN EGGS
Paul R. Gross
Biology Department, Massachusetts
Institute of Technology, Cambridge, Massachusetts
I propose to summarize here what I believe
are some important points emerging from the
recent study of biochemical events, especially
those involved with macromolecule synthesis,
that follow immediately after fertilization of
sea urchin eggs (1). There appears to be a
necessity for the existence of systems control-
ling protein synthesis at the level of translation
of RNA messages (2). Experiments on the
early course of development are now no longer
unique in demonstrating the existence of trans-
lation control. However, in fact, these were
among the first in which the necessity for such
a conclusion appeared. There are a number of
other kinds of developing and differentiating
systems in which evidence of control at this
level is available. Someof these will undoubtedly
be considered as these discussions progress.
The observations that led to the postulation
of translation control follow. Synthesis of pro-
teins, which is an inevitable accompaniment of
early development, may be uncoupled from the
synthesis of new RNA (e.g., 3). This uncoupling
can be absolute and may last for a very long
time. When the observation was first made, it
was surprising because the situat'.on with re-
spect to messenger function of RNA in micro-
bial cells would not necessarily have led to
the prediction of such a level of control, since
in microbes the continuation of protein synthesis
requires concomitant synthesis of RNA mes-
sages whose half life is short, relative to the
length of the cell cycle. In a system allowing
the synthesis of protein to go on in the absence
of new synthesis of messenger RNA, it must be
true that either such synthesis doesn't require
messages, or that the messages are very stable.
The possibility that protein synthesis ac-
companying early development may not require
messenger RNA could be established in a num-
ber of ways. One could, for example, look for
polyribosomes in embryos. One could make
estimates of the fraction of the early synthesis
that occurs on polyribosomes, and if most of
the synthesis does occur there, then it is rea-
sonable to assume that protein synthesis does
require messenger RNA and associated ribo-
somes. Such seems to be the case (4, 5, 6). There
is no primary site that we have been able to
detect for protein synthesis in sea urchin eggs
other than ribosomes associated with a length
of highly nuclease-sensitive RNA. The extent
to which those objects, themselves, are asso-
ciated with other, perhaps larger, structures
is an interesting point that I hope will come up
in the discussion later. At least, the poly-
ribosome, itself, is the unit on which early
proteins are made. Since under uncoupling
conditions new messages are not made, old
ones must supply the information for transla-
tion. That much alone suggests that these mes-
sages must be stable. Although at the time the
observations were made, that was in itself a
moderately radical proposal, the existence of
very stable messages has since been shown in
several cases (e.g., 7). Stable messages seem
now to be not at all exceptional in higher cells,
even in relation to the long inter-mitotic time.
Our starting point was the independence of
new protein synthesis from new RNA synthesis
in embryos; that is, messages directing the
early synthesis must have been present in the
egg before it was fertilized. There is, quite
generally, a long period between the time that
an egg is completed and set aside in a condition
of relative dormancy in the ovary, and the time
it is released from the mother to be fertilized.
Hence, the further suggestion that the templates
for early embryonic protein synthesis are not
only very stable in use, but may be stored for
long periods of time without being used at all.
There are several kinds of developing and
differentiating systems to which the statements
I have just made are now known to apply. This
being so, we conclude that some agency of
control must exist in the cytoplasm to turn
on the reading of stored messages, since it is
demonstrable in all of the systems being studied
that the co-factors necessary for protein syn-
thesis are already available in the unfertilized
egg. Thus, there has emerged from studies of
macromolecule synthesis in development a need
to find out how translation-control systems
work. They clearly exist and they must be
concerned not only with the control of develop-
ment but with the control of decision-making
processes, in general, in differentiated higher
cells.
As far as I know, there is no detailed
scheme that explains as yet how any translation-
control system works. Perhaps we will have
suggestions in the course of this week, as to
where to look for the agencies of control.
In the meantime, there are experiments that
led to the position I have just sketched, which
lead in turn to a closer study of the events of
macromolecule synthesis in early development.
I will discuss three lines of such experimenta-
tion briefly, relying mainly upon slides to sum-
marize the present position in each case. The
three problems with which we will be concerned
are (1) the pattern of synthesis of RNA during
early development, (2) the search for stored
maternal messages whose existence is sug-
gested although not proven by indirect evidence
and (3) a study of the proteins themselves, a
large fraction of which presumably are made on
900
700
--300
500
--200
--I00
Fig. I.
stable messages during the period of cleavage.
The pattern of RNA synthesis is radically
different from what one might have expected
from the behavior of microbial systems. Figure
1 deals with a sucrose gradient and with RNA
labeled for 30 minutes at the blastula stage in
the sea urchin embryo. I have chosen this
pattern to start with because it is characteristic
of the pattern of synthesis of RNA throughout
the course of the period from cleavage to the
late blastula. The sea urchin has ordinary RNA
in bulk, with 28S, 18S and 4S species. (These
are the three major peaks of Fig. 1 from left
to right, respectively, shown by circles.) Radio-
activity incorporated in this case from labeled
uridine is distributed in gradients as shown by
the triangles. The circles are O. D. Such radio-
active material is non-coincident with the stable
pre-existing bulk RNA, except in the 4S region.
The radioactive product is highly heterogeneous
with respect to sedimentation constant. In the
4S region, where coincidence does occur, there
is also, throughout early cleavage, the most
rapidly labeled RNA. There is every reason
to suspect, on the basis of physical behavior
alone, that the non-4S material being labeled
is not ribosomal and is very likely, at least,
to be messenger RNA, or heterogeneous RNA
with possible template function. I should point
out that in these embryos there are no nucleoli
until long after the swimming blastula stage.
Figure 2 is a fortunate tangential cut through
the surface of a quite late blastula, already
ciliated and swimming. It shows nuclear profiles.
Note that there are no nucleoli. As long as there
are none, we see little or no ribosomal RNA
synthesis on gradients, no incorporation of label
that sediments in coincidence with the ribosomal
species and, as we shall see in a moment, base
compositions for the newly-synthesized mate-
rial that differ radically from those of the bulk
ribosomal RNA. When the nucleoli do appear
at the late gastrula stage, it becomes possible
to detect ribosomal RNA synthesis at a steadily
increasing rate.
Figure 3 is another experiment like the one
represented in Fig. 1, but in this case the label-
ing was with radioactive phosphate. Four sets
of fractions were pooled, corresponding roughly
to the centers of gravity of the 28S, IBS, lOS
and 3-1/2S bulk RNA. Base-composition analy-
ses were performed.
Table I will show what the compositions
are. The fractions shown here were indicated
in Fig. 3. This work was done with Arbacia.
DNA of this species has a GC content of slightly
TABLE I
Base Compositions of Sea Urchin RNA Fractions
Sample
A
(Mole
U
G
C
(Mole 7.)
G + C
Source
Fraction I
28.9
24.4
23.6
23.1
46.7
These exp'ts.
Fraction II
28.1
27.4
21.8
22.3
44.6
"
Fraction III
33.8
16.2
18.2
31.8
50.0
"
Fraction IV
14.4
12.9
14.5
58.2
72.7
"
28S rRNA
22.4
18.8
32.8
26.0
58.8
Gross, Malkin
& Hubbard (1965)
18S rRNA
24.4
21.7
30.0
24.0
54.1
"
Bulk RNA
22.3
20.7
29.6
27.4
57.0
Elson, et al.
(1954)
Arbacla DNA
28.4
32.8(T)
19.5
19.3
38.8
Daly, et al.
(1950)
Fractions I-IV from Arbacia embryos exposed to PO4 from fertilization to early blas-
tula. 28S and 18S RNA from.) raiacia eggs labeled with ^ PO4 during oogenesis. Hydrolysis,
separation of nucleotides and determination of base composition according to Salzman,
Shatkin and Sebring (1964), except for compositions of bulk (total) RNA and DNA (sperm),
which are from the literature. Approximate centers of gravity in sedimentation profile
corresponding to fracUons I-IV: 28S, IBS, lOS, 3S.
^ Table I, Gross, Kraemer and Malkin, Biochem. Biophys. Res. Comm. 18, 569, 1965; repro-
duced with permission of Academic Press.
«S«B:>f»73- . Avr~'.-.f^- '
0.25
0.20--
» 0.I5--
0.10
0.05- -
40 60
% FRACTION
Fig. 3.
(Fig. 1, Gross, Kraemer and Malkin, Biochem. Biophys.
Res. Comm. 18, 569, 1964; reproduced with permission of
Academic Press.)
Fig. 2.
under 40%. The bulk RNA, mainly the two
ribosomal species, has a GC content of about
57%. RNA labeled through early cleavage up to
the blastula and all fractions except the lightest
one have the GC content that is markedly lower
than what would be expected for ribosomal
RNA. Remember that this is accumulation of
radioactivity over a period of about seven hours
with the radio-phosphate in the medium being
kept at constant specific activity, so that with
respect to GC content, this is very much a
DNA-like RNA and probably one of considerable
stability.
The base composition of the light fraction
is highly aberrant. It is very rich in cytidylic
acid, and has a roughly equal distribution of
radio-activity among the other bases. This
suggests strongly that the heavy incorporation
of radioactivity coincident with the 4S peak
represents labeling of the terminal CCA se-
quence in transfer RNA. This is the dominant
synthetic process associated with RNA in the
course of earliest development and far out-
weighs the activity associated with internal
synthesis. What the significance of the end-
labeling is, I do not know, and I have not heard
any really useful suggestions about it. It seems
to be a widespread phenomenon in developing
systems and in other systems in which cells do
not grow. I should point out in this connection
that the embryos don't grow in any strict
sense. New cells are forming as a result of
cleavage, but there is no increase in mass.
Indeed, throughout the course of development
to the early larval stages, there is a slow but
Table II (top) shows the base composition
for fractions a, B, J, &, £ as indicated in
Fig. 4. Remember that the DNA has a 40% GC
content and that these are pooled fractions from
heavy to light. Most of them still have a low
GC content except that as one approaches
the light end, GC content rises because there
is still a considerable amount of end-labeling.
There is clearly some ribosomal RNA accumu-
lating during this period. If incorporation is
allowed to take place from the late gastrula
to the prism stage, as shown by Fig. 5 (symbols
as for Fig. 4), which is the beginning of the
differentiation of definitive larval tissues, then
there is a predominant ribosomal RNA synthesis,
quite steady decline in mass, and this is be-
cause some carbon compounds are broken down
to CO 2 and water.
If one allows radioactivity to be incor-
porated into RNA later, for example, with P^^
as the label (Fig. 4), from late blastula to early
gastrula, using a long labeling period (about
seven hours), one gets something that looks as
though there were the beginning of ribosomal
synthesis. (Open circles, OD; closed circles,
counts per minute; triangles, specific activity.)
Notice that the specific activities are minima
where there are optical density maxima, sug-
gesting that coincidence is poor between the
bulk ribosomal RNA (represented by optical
density) and the radioactivity. The base composi-
tion again shows that the heterogeneous RNA
is still present even after a long exposure to
isotope.
O.D.
1.0- -
0.6 --
0.4--
0.2--
-| r
-1 r
-S-i lLi;
--2
SP. ACT.
3 X 10"'
CTS/MIN.
-rSOOO
0 -^0
0 15 20 25
FRACTION NUMBER
30
4000
--3000
I --2000
--I000
5 10 15 20 25
FRACTION NUMBER
30
Fig. 4.
Fig. 5.
TABLE II
Base Composition of RNA Synthesized During Long Exposures of Sea Urch:
Embryos to ^^ P.
Sample
Fraction
a.
A
Composition,
U
Mole Z
G
C
%
G + C
Blastula
29 J
23.1
25.6
21.5
47.3
Gastrula
,
27.7
25.7
29.1
17.5
46.6
7 hrs.
(
32. b
23.8
23.0
20.6
43.6
•>
23.9
23.5
26.2
26.3
52.5
i
18.8
18.0
32.3
30.9
63.2
Gastrula
«
25.7
26.1
23.6
24.6
48.6
Prism
f
23.9
23.2
27.2
25.7
52.9
12 hrs.
r
25.4
22.5
26.5
25.6
52.1
■i
21.5
22.1
28.4
27.9
56.3
*18S rRNA
24.4
21.7
30.0
24.0
54.1
*28S rRNA
22.4
18.8
32.8
26.0
58.8
represented both by the change in base com-
position (Table II, lower part) and by a clear
coincidence of the counts with the absorbancy
pattern. (Notice the constant specific activity
across the ribosomal optical density peak in
Fig, 5.) Thus RNA synthesis begins in this
system under conditions such that little or no
ribosomal RNA is made and the major incor-
poration activity represents labeling of the CCA
terminal in transfer RNA. In time, the rate of
end-labeling falls and the rate of synthesis of
heavy heterogeneous RNA rises steadily from
fertilization onward. At some point, probably
well after the blastula and perhaps as late
as the time of appearance of definitive nucleoli,
the synthesis of ribosomal RNA begins in
quantity. This means that a complicated system
of control operates on the synthesis of RNA, and
specifically, on the utilization of the cistrons
that provide templates for synthesis of the
ribosomal RNA.
This is all heavily descriptive, and I cannot
offer anything in the way of a reasonable ex-
planation for the existence of this pattern, but
it is beginning to be quite a general one. For
example, the situation in the amphibian seems
to be roughly the same, except that there is
some argument about when the synthesis of
new heterogeneous RNA begins. Dr. Kohne will
tell you about this later.
POLLARD: I would like to ask you a couple
of questions. First, as a microbiologist, I'd
like to know what the amount of turnover of
RNA and protein is. In E. coli, for example,
the RNA does turn over to some extent. At least
the uracil label changes.
GROSS: Does it turn over in the ribosomal
RNA?
POLLARD: I don't think it does, but if we
just look at the general cell behavior, there is
a difference between thymine label and uracil
label. To what extent do you see something like
that?
GROSS: There are two different answers,
depending on how you evaluate the available
data. Comb (8) believes that there is some
considerable degradation of ribosomal RNA
in sea urchin embryos from the beginning of
development to the gastrula stage, and that
the products of degradation are possibly used
for resynthesis of messenger RNA. That is the
only information I know that suggests such
a turnover. Other data that we have do not offer
much support for this idea. The egg starts its
life with a large pool of precursors for RNA,
and this pool diminishes slowly but steadily
during development. It doesn't enlarge, as far
as I can tell, at any time. Actual synthesis of
bulk RNA, represented at least by the incor-
poration of radioactivity, seems to be small,
perhaps not exceeding a few per cent of the
original total. Certainly on this basis there
is no need for massive synthesis of bulk RNA.
Finally, by a technique that I'll describe in a
minute, we have been able to label the RNA
in ribosomes of unfertilized eggs. If such
eggs are fertilized and allowed to develop in
the presence of a very large excess of un-
labeled uridine and cytidine in the medium,
there is no detectable loss of counts in the
RNA. This is probably in inadequate "chase"
and certainly not direct evidence for the com-
plete stability of cytoplasmic RNA. There is,
in short, no really adequate answer to your
question at this moment, but one's prejudice
is in the direction of little or no turnover.
POLLARD: How about the case of protein
synthesis?
GROSS: That may really be the more in-
teresting matter. The egg starts its life with a
large pool of amino acids, but a peculiar one
because of its abnormal composition relative
to that of typical proteins. A very large fraction
of the osmolarity of the sea urchin egg is pro-
vided by glycine. Some of the other amino
acids, such as leucine, are in short supply in
the pool. In any case, there is such a pool, but
I would guess that it is probably not adequate
for prolonged synthesis of a variety of proteins,
such as begins at the beginning of development.
Now, I cannot tell you what the real rate of
protein synthesis is following fertilization, be-
cause we don't have proper information about
the pool changes. The total protein of the egg
does fall by about 15% from fertilization until
the larval stage. However, the egg has in it a
very large amount of yolk, most of which is
gone by the end of the larval period. This yolk
is mostly protein, 90% or so. Consequently,
there is a very significant transformation of
protein. There must be a lot of traffic through
the pool, most of it being provided by yolk at
one end and stable new proteins at the other.
KOHNE: May I make a comment on the
first question? In these embryos, it is very
difficult to do quantative studies and it would
be very difficult to determine turnover if it
were occurring.
GROSS: Yes, most of what I said is per-
haps circumlocution. We don't have adequate
pool data, nor suitable "chase" techniques for
obtaining a satisfactory answer to the question.
POLLARD: The protein turnover with re-
spect to yolk wasn't circumlocution, was it?
GROSS: No. It is clear that some protein
disappears and new protein forms. Perhaps in
the course of the discussion, we can come back
to yolk.
GRUN: Is there an obvious simple explana-
tion for this inverse relationship between the
specific activity curves and the OD curves shown
in Fig. 4?
GROSS: Suppose that the counts in each
fraction were invariant, so that the computation
of a specific activity involved a division at each
gradient point of a constant number by a variable.
The variable number, i.e., the optical density,
is alternately high and low. Where it is low,
you get a high value of specific activity, and
where it is high, you get a low value. Therefore,
in such an ideal case, with radioactivity a
straight line of zero slope throughout the gradi-
ent, the computed radioactivity would be a
pattern exactly the inverse of the optical densi-
ties. Now, as the actual radioactivities deviate
from that ideal condition, the oscillations in the
computed specific activity will be damped, and
when the optical density and radioactivity are
completely coincident, the computed specific
activity becomes a straight line. I'm about to
raise this point again in connection with radio-
activity in the unfertilized egg.
HYMER: May I ask about the high specific
activity in the region of the gradient containing
molecules larger than 28S? Is there evidence
for a heavy ribosomal precursor molecule in
your system?
GROSS: No, there isn't. Certainly not at the
beginning of development when there isn't any
evidence of accumulating ribosomal RNA.
KOHNE: A "heavy" ribosomal precursor
has been demonstrated in Xenopus laevis.
MASSARO: I'd like to deviate from the
subject for a second. During the early protein
synthesis, what contribution is the male com-
ponent making? From this type of analysis,
we're developing a type of parthenogenetic
embryo.
GROSS: There is no difference in the pat-
tern of protein synthesis - at least that we can
determine by methods that I'll discuss later -
between a parthenogenetic merogone, which has
neither a sperm nucleus nor an egg nucleus,
and the fertilized egg during the first few
cleavages.
KOHNE: What is very interesting is that
such an active process could go on at the low
levels of sRNA present in eggs.
6
GROSS: It's a maximum of 5% in the sea
urchin.
POLLARD: It is also interesting that this
increase in sRNA seems to be in response to a
need, because breaking down the yolk protein
gives a large supply of amino acids, and this
needs transfer RNA in order to be used, also.
GROSS: Yes, and that suggests, of course,
that putting the CCA on has something to do
with protein synthesis.
POLLARD: Well, a much higher concen-
tration of tRNA will be necessary anyway, and
whether the CCA addition is an essential part
of that process or not I don't know. The con-
centration of transfer RNA has to be raised in
order to actually get the amino acids in the
proper location on the template. Actually, it's
almost certain in a cell which is fairly big like
this that the numbers have to be raised very
considerably unless there is compartmentation,
and protein synthesis occurs only a small
regions. Still, I don't think that answers your
question. You want to know why the sudden
burst in the CCA part of it occurs.
GROSS: Yes, there is no question that there
is a rapid net synthesis of sRNA when the
protein synthesis rates rise for a second time
at gastrulation. There begins a rapid internal
synthesis of sRNA at that point and that is
quite reasonable. However, why the entire
CCA triplet should be knocked off and put
back on, I don't know. At least I know of no
evidence indicating that such an event is neces-
sary in order for the sRNA to transfer and
activate amino acid.
DEERING: Does anyone know whether this
end group is actually present during the earlier
stages or is it just added later?
GROSS: There is some uncertainty about
this, but some evidence that has recently become
available indicates that functional RNA is present
in the unfertilized egg, that is, sRNA with its
CCA triplet intact.
PAPACONSTANTINOU: Did your base
ratio for that RNA give a GC content of 78%?
GROSS: No, but that is just the base com-
position of the new RNA, determined from
hydrolysis of the bulk. It gives information
only about what bases are being incorporated.
It is possible that they are being added to
populations of sRNA molecules that don't have
any CCA on them at all.
PAPACONSTANTINOU: Then how could
you test those for aP32 base ratio of 78%?
GROSS: Think of a piece of RNA in the
presence of medium containing radio-phosphate.
Then add to it pC, pC and pA, all of them
radioactive. These are added to base X on every
molecule. Now, stop the reaction, purify the
material, and hydrolyze it. One residue comes
off with no phosphate because the chain runs
in the wrong direction. The next comes off with
the phosphate and then X comes off with the
radioactive phosphate. Nothing has been labeled
at the next location. Radioactive XP can be any
one of the four bases. Let's assume for the
moment randomly so. That is what the base
composition implies. Measuring the radioac-
tivity of the phosphate, the composition of the
material I have labeled in the way shown here,
will be determined at 75% C and the rest
distributed among A, U and G.
PAPACONSTANTINOU: I still can't see
how you can explain a 70% GC unless you have
an active CC turnover. That's the only way
you can explain it. You have evidence that
there is CCA present in the early embryo
because of the 78% GC. However, suppose you
started out with all the sRNA having no CCA
on it. Would you still get this pattern?
GROSS: Yes.
As to the RNA of the unfertilized egg, there
is strong but indirect evidence that the tem-
plates carrying the information for most or all
of the protein synthesis that occurs during the
period of cleavage are already present in the
unfertilized egg. If that's so, then one is dealing
with RNA templates that are storable under
conditions of non-use and are very stable when
they do begin to be used. In the presence of
actinomycin, in doses sufficient to shut off new
RNA synthesis, the primitive pattern of protein
synthesis persists for a long time. It is a matter
of some interest, therefore, to attempt to dem-
onstrate directly that such a maternal mes-
senger fraction exists, and second, to isolate it.
It would be useful to isolate it because whatever
approach works for the isolation would surely
tell us something about the state of this material
in the cell, and that, in turn, might tell us
something about the control of its translation.
Nothing has been done as yet about isola-
ting this material in bulk. A number of steps
have been taken, however, to demonstrate its
existence more directly. One approach is to
make the RNA of an unfertilized egg radioactive
during oogenesis in order to show that among
the radioactive species there are some that are
not ribosomal or transfer RNA. This has been
done with most success in the amphibian (e.g.,
9), and I'll leave it for Dr. Kohne to discuss.
It has been possible to do the same sort of
thing in sea urchin eggs under more trying
biological circumstances. If radioactive RNA
precursors are injected into a gravid female
sea urchin, no radioactivity is found in the
mature eggs. This indicates that mature eggs
are finished and no longer making any RNA
and none can be forced in when the female is
ready to spawn. An alternative is to make a
female spawn and then let her carry out oogen-
esis, making a new crop of eggs in the presence
of radioactivity. This is not a very practical
procedure, at least with the species available
to us, because it means having animals in
large tanks of sea water, containing high levels
of radiophosphate circulating in the sea water
for weeks. However, there is a simple trick
that can be done. This is to make a female
spawn partially at the height of the normal
reproductive season and then to place her in
a tank that contains radioactive precursors for
about a week. Under those conditions (2), a few
of the oocytes complete their maturation to
replace the ones lost in the partial spawning.
We collect the mature eggs. Some of them are
highly radioactive, as I'll show you, and in
those the distribution of radioactive RNA can
be studied.
Figure 6 is a section of an ovary of a sea
urchin. This is a highly lobulate organ, whose
walls contain an epithelium that gives rise to
the ootids. There are oocytes in this wall in all
stages of development, and an oocyte is identi-
fiable by its large germinal vesicle nucleus.
In some cases, you can see a nucleolus. This
is prominent because the oocytes are growing
and making ribosomes very rapidly. The ulti-
mate product of the differentiation - and I use
I-ig. t).
that word advisedly - of an oocyte into an egg
is an ootid. It is recognizable here by its small
pronucleus. These ootids fill the central parts
of the lumina of the lobes. When the animals
spawn, there is a highly stretched muscle in
the outer layer of the ovary that contracts and
fully mature ootids are extruded while the small
immature eggs remain inside.
Now, if you perform the trick that I have
described - partial spawning and labeling for
a week - you find that it is possible to force
radioactivity into the cells as represented in
the autoradiogram shown in Fig. 7. This shows
a region near the wall. The wall consists of
three layers, an outer and an inner one, and
a muscle layer. The oocyte layer is next to
the wall. You see all of these cells are highly
labeled, both in nuclei and in the cytoplasm,
after a week. At the top of the figure is the
luminad region with the cells getting larger.
Everything in the region of the wall is radio-
active, except for one cell that happens to be
outside the wall and was fixed.
There is an interesting progression, as
shown in Fig. 8. Close to the region shown in
Fig. 7 about three-fourths of the cells are
labeled, indicated by the left part of Fig. 8.
When they are, the number of silver grains
over each one is about the same. Those that
are not labeled have no counts above back-
ground. There seem to be very few interme-
diate conditions of radioactivity between cells
that have been making RNA at some constant
rate during the time of exposure and the
unlabeled ones that have finished before the
radioactivity was supplied. Moving toward the
lumen (left to right in Fig. 8), the number of
labeled cells becomes smaller until finally in
the central lumen, where the oldest eggs are,
there is no label at all. This fact suggests
that we are causing a few eggs to complete
their maturation and labeling them while this
is in progress. Silver grains represent counts
in RNA, because all these sections are DNAse
treated.
We can extract and purify the labeled RNA.
The pattern obtained is shown in Fig. 9. One
thing is at once apparent. During the time that
labeling took place, these eggs were making
all the bulk kinds of RNA. Both ribosomal
species and 4S become radioactive and the radio-
activity (faint solid line) and bulk patterns
(dotted line) are superficially coincident. There-
fore, these eggs, during the late stages of their
maturation, are still making ribosomal RNA
and presumably ribosomal proteins as well.
'■^^.^ W ■.•>^ 'f '■■' '1
(Platell, Gross, Malkln and Hubbard, /. Mol. Biol. 7.?, 463,
1965; reproduced with permission of Academic Press.)
^^^^^^K' M y. '-
•
■ ' /
■' " ■ \,
■ y
Fig. 8.
(Plate III, Gross, Malkin and Hubbard, /. Mol. Biol. 13,
463, 1965; reproduced with permission of Academic
Press.)
They appear to be assembling complete ribo-
somes up to the very end of oogenesis. Now it
is interesting that when one plots specific
activities, determined after careful registra-
tion of counts and optical density for each
fraction, one gets the sort of pattern shown
by the heavy line in Fig. 9. There are several
things that could give rise to deviations from
constancy of specific activity in the manner
shown. If the counts really represent what's
present in bulk, then, of course, there should
be no deviations from constancy. Now one
possibility is that some extra counts are present
throughout the gradient; i.e., that there is not
complete coincidence between the optical density
and the radioactivity. In that case, the pattern
obtained will be of the type with maxima at the
positions of the optical density minima.
There are two other possibilities, both of
them representing technical errors: (a) that
some highly radioactive bacterial RNA is pres-
ent as a contaminant which would sediment
slightly out of coincidence with the sea urchin
RNA because the sea urchin species sediment
at 18 and 28S, whereas the bacterial RNA sedi-
ment at 16 and 23S. On the other hand (b), per-
haps for some unknown technical reason, we've
failed to register the counts and optical densi-
ties accurately. In neither case would the
pattern of deviation from constancy of specific
activity be what is observed. A simple periodic
function ratio shows that the pattern obtained
would be one of constantly varying deviations
across the peak, but no minima under the peak
of optical density. These functional points are,
however, less important than the fact that con-
stancy of specific activity across the ribosomal
density peaks is in fact obtained when the RNA
is labeled late in development at a time when
ribosomal RNA synthesis predominates. This
was demonstrated on an earlier slide. Since
these materials are treated and analyzed in the
same way as those obtained from the labeled
unfertilized eggs, there seems to be no doubt
SP. ACT.
X 10"^
■50
40
--30
--20
10 15 20
FRACTION NUMBER
Fig. 9.
25
10 15 20
FRACTION NUMBER
Fig. 10.
25
(Fig. 1, Gross, Malkin and Hubbard, ]. Mol. Biol. 13, 463.
1965, reproduced with permission of Academic Press.)
that the deviations from constancy of specific
activity do represent the first condition, that is,
the presence of a small amount of RNA of high
specific activity, not coincident with the ribo-
somal species. From the sizes of the specific
activity variations, one can make a crude esti-
mate of the amount of heterogeneous radio-
activity. There is no good theoretical way for
making such an estimate, but it is possible
to make simple models composed of Gaussian
error curves to represent the bulk species and
extra counts distributed in roughly the way one
might expect heterogeneous RNA to be dis-
tributed. You see in Fig. 10 that the order of
maximum deviation of specific activity from
unity is two (circles). Figure 10 shows a real
gradient of specific activity. The reason that
we drew the optical density curves (smooth
curve, solid) continuously is that this is how
they emerge from the Gilford recorder. We do,
however, in each case, select individual frac-
tions, measure their optical densities again,
and then count them so that the specific activity
as plotted results from the division of an ac-
tually measured optical density by an actually
measured count.
With the model shown in Fig. 11, which is
4--
--2.0
J ooo°°:
GO O o
°°po,
--0.5
10 15 20 25
FRACTION
Fig. U.
the closest one that we've been able to construct
to the experimental results, there are 15%
extra counts (large circles) distributed hetero-
geneously among the total in these preparations.
(Specific activity, triangles).
There's only one final objection to this,
and it is another kind of technical error. There
might be absorption, or simply quenching, that
results from the presence of RNA in these
samples, and the amount of quenching could
therefore be directly proportional to the amount
of RNA. This has been checked, and it is not
so. The specific activity deviations are there-
fore real and, on the basis of the model, they
result from the presence of some 10 to 15%
extra radioactivity in these preparations, sedi-
menting out of coincidence with the ribosomal
and transfer RNA's. The suggestion is, there-
fore, that this is the messenger RNA in the
unfertilized egg.
UNKNOWN DISCUSSANT: Let me ask you
a technical question. What label were you using
in these studies?
GROSS: The first one, without data points
(Fig. 9), was labeled with P^-^; the second one
(Fig. 10) was labeled with uridine.
UNKNOWN DISCUSSANT: And did you use
DNA digestion to eliminate any possibility of
DNA labeling?
GROSS: Yes, DNase digestions are done
routinely. There are a number of alternative
possibilities for checking the conclusion that
this represents messenger RNA. One is to
examine the hybridizability of the radioactive
RNA with DNA. We've done this, and it is by
no means an easy thing to do because the
specific activities of these preparations are
10
quite low. However, only a small fraction of
the hybridizable radioactivity in preparations
like this is ribosomal RNA. From competition
experiments, we get a crude estimate of the
fraction of the genome that's involved in the
synthesis of ribosomal RNA and that appears
to be about 0.2%. Most of the counts that do
hybridize appear to be attached to sites on the
DNA for which ribosomal RNA does not com-
pete. I might say finally that unless one is
familiar with this field, one might tend to be
impressed with the result just described. How-
ever, there are, in principle, much better ways
of doing it. The best, by far, seems at the
moment to be an experiment showing that in
the unfertilized egg there is a kind of RNA
capable of supporting protein synthesis in vitro,
an RNA other than degraded ribosomal or
transfer RNA, and if one obtains an in vitro
system that demonstrates this in a reliable
way, then the problem has really been solved
properly. There is a result from Monroy's
laboratory (10) that appeared about a year ago,
showing this to be the case, although the total
incorporated activities were quite low. Never-
theless, their claim, and it seems to be a
justified one, was that there is as much tem-
plate RNA in an unfertilized egg as there is
in an early blastula. That is certainly in accord
with the indirect evidence described earlier.
We come now to the final point, which
concerns proteins. First, there is some reason
to suspect that among the proteins made at the
beginning of development, are some that must
be important for mitosis. Inhibitors of proteins
synthesis, such as puromycin, also inhibit
cleavage (11). They inhibit all of development,
of course, but they do stop an ongoing cleavage
if applied before metaphase. These inhibitors
therefore stop division at a characteristic
cytologic stage - a stage just before the mitotic
spindle is formed and the nuclear membrane
breaks down. All of this suggests that there
are among the early proteins some that have
something to do with mitosis. Autoradiograms
of eggs labeled with amino acids make this
suggestion in another way. We make such
autoradiograms as a control whenever we label
sea urchin eggs. The reason for this is that
when dealing with animal cells, in a medium
like sea water, the problem of bacterial con-
tamination is ever present. One way that one
can be reasonably sure that the radioactivity
being studied is really inside the cells is to
make autoradiograms and, hence, we do so
routinely.
Fig. 12.
(Fig. 7, Gross, Malkln and Hubbard, /. Mol. Biol. 13, 463,
1965; reproduced with permission of Academic Press.)
Examining autoradiograms of cells that
have been labeled with amino acids, during
the first division cycle, we observe the sort
of thing shown in Fig. 12. In cells that were
at metaphase or early anaphase, there was a
heavy concentration and, indeed, an almost ex-
clusive localization of radioactivity in the mitotic
spindle. Shown in the figure is an early anaphase
mitotic apparatus. Now there are two possible
interpretations of this result, and the one you
accept depends on your hypothesis of the or-
ganization of the mitotic apparatus. If you believe
that the mitotic spindle as seen in situ is a
simple structure, most or all of whose protein
is uniquely characteristic of it, then an auto-
radiogram of the type shown proves that most
of the radioactivity goes into one protein, i.e.,
that all protein synthesis at the beginning of
development has to do with the mitotic apparatus.
The alternative arises if you don't believe that
the spindle has in it only spindle proteins, but
that it may have others as well. Then you have
to decide whether the localization may mean
something else. Figure 13, which is an elec-
tronmicrograph, shows why it is our conviction
that the second alternative has to be accepted.
This is a section through an early anaphase
spindle at moderate magnification, and it is
11
Fig. 13.
meant to show the spindle fibres which in the
best preparations occupy, as you can see, a
rather small portion of the total area or volume
of the spindle. There are also chromosomes and
background or matrix material. This matrix is
very densely populated with vesicles, fragments
of membranes and a large number of particles.
The particles are of the same size and of the
same electron density as are the ribosomes
seen elsewhere in the cell, and there is no
reason to believe that they are not ribosomes.
If the characteristic spindle protein is what
makes these fibres, then one would conclude
from a picture like this that most of the protein
in the spindle is not the characteristic micro-
tubular protein. It is ribosomal and soluble
protein.
A second consideration is relevant. If we
were to take a sample volume in an egg without
an organelle, like the mitotic spindle, we would
find that there were a certain number of yolk
particles in that volume. These yolk particles
are solid objects. They don't seem to have the
high degree of crystalline order in sea urchin
yolk that's seen in some other species, but the
particles are nevertheless very dense and have
a high protein content. If the spindle or an
organelle like it is formed, the yolk particles
are extruded and indeed one can see large
particulates such as yolk and mitochondria
extruded from the forming spindle. Hence, the
mitotic apparatus has in it no particles of the
size of yolk and mitochondria. Soluble proteins,
on the other hand, are presumably not extruded
from the forming mitotic apparatus, because
ribosomes are not, and the soluble proteins are
smaller. Thus, if one were to measure the con-
centration of soluble proteins in the mitotic
apparatus, and in the region outside of it, one
would certainly find that the concentration of
soluble proteins is higher within the region
of the mitotic apparatus than it is in the peri-
phery, simply because the peripheral material
has in every volume element a large excluded
subelement occupied by the yolk. On this basis
alone, any report that something is localized
in the spindle should be viewed with caution.
For example, there are reports in the literature
on the cytochemical localization of enzymes
and certain thiol-rich proteins in the mitotic
apparatus, but I would venture to predict on the
basis of the argument just given that at least
some of the observed cytochemical localizations
are localizations by default and not the result
of active processes associated with the as-
sembly of the mitotic apparatus. I wanted to
make this argument clear because it suggests
that the radioactivity seen in the spindle may
have been included in that region in a passive
rather than an active fashion. One possibility
exists for testing this question further, and
that depends on the presence in the spindle
of fibres or microtubules that presumably
represent the definitive working part of the
organelle.
Figure 14 is an optical autoradiogram of an
isolated spindle sectioned at one micron. This
spindle is a member of a population obtained
from eggs that have been pulsed with the amino
acid leucine and "chased" prior to the appear-
ance of the metaphase spindle. You might al-
ready see in the figure a suggestion that the
radioactivity, which is represented by the silver
grains, has a certain tendency to follow the
lines of the fibres. These fibres, which run in
tracts, are visible in sections of this thickness.
Figure 15 is an electron microscope auto-
radiogram made from the same material. At
low magnification, one sees tracts of fibres
running through the center of the spindle and
silver grains distributed over the whole area.
12
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Fig. 14.
(Fig. 1, Mangan, Miki-Noumura and Gross, Science 147,
1575, 1965; copyright 1965 by the Association for the
Advancement of Science.)
K.
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^^
'f^' '^-
Fig. 15.
Now one's impression is certainly that a very
large fraction of these silver grains are either
on or next to the fibres. Does this mean that
the fibres are labeled? I think that it does, for
the following reason: either the fibres are more
radioactive than the region as a whole, or they
are not and the radioactivity is simply randomly
distributed. There are a number of ways to
test such a question, and the next two figures
show the way that we elected to do so. A sheet
of acetate overlay is placed on a print of the
type shown in Fig. 15. A circle, whose diameter
represents the average silver-grain diameter,
is drawn on the overlay over every grain, and
wherever a fibre occurs next to or under such
a grain, the fibre is indicated and that grain
scored as a hit. Figure 16 is the overlay pattern
for the print shown in Fib. 15. Next, the area of
the print is divided into a large number of
coordinates, say 10,000, and then using these
coordinates and the total number of silver
grains in the actual print, a number of points
is selected from a table of random numbers
equal to the number of grains. These points
will, of course, be randomly distributed over
the coordinate grid. Circles representing the
points selected from the random number table
are drawn on a new sheet of overlay, placed
over the print, and a fibre is scored as a hit
when it is adjacent to or under one of the circles.
The result is shown in Fig. 17. Now, it is
always observed that the number of hits obtained
with randomly-placed points is much smaller
Fig. 16.
than it is with the actual prints and grain
patterns. This rather rigorous test suggests,
therefore, that the microtubules are in fact
labeled. There is radioactivity in the interstices,
as one might have expected, but a large fraction
of the radioactivity, a larger fraction than would
be expected on the basis of chance alone,
13
Fig. 17.
appears actually to be on the fibres. Therefore,
one of the first proteins synthesized in the early
development of the sea urchin and presumably
one of those for which the program is stored
in the egg prior to fertilization, is a protein
that has some function in the organization or
operation of the mitotic apparatus.
I believe that my time is up and we will
therefore have to defer a discussion of other
products of early protein synthesis to another
occasion.
POLLARD: Thank you very much. Are
there any questions for Dr. Gross?
CHALKLEY: Do the ribosomes from the
mature egg support protein synthesis under in
vitro conditions?
GROSS: There has been some argument
about whether unfertilized ribosomes are com-
petent to support protein synthesis. An alterna-
tive explanation to the maternal messenger
story might be that there is a lesion in the
ribosomes of unfertilized eggs which is healed
on fertilization. That is indeed a part, at least,
of the point of view of Monroy and his collabor-
ators (12). Nemer (13), on the other hand, has
presented what was, I believe, reasonably good
evidence that ribosomes from unfertilized eggs
work well. In his experiments, they operate
with poly-U and with other synthetic poly-
nucleotides. Monroy explains that the ribosomes
from fertilized eggs respond well to natural
messages, while the ribosomes from unfer-
tilized eggs do not. The point of their recent
paper is that unfertilized ribosomes which
respond very poorly to natural messengers
in vitro can be made to respond normally by
a brief treatment with trypsin. They are sug-
gesting that the unfertilized ribosomes are
blocked, perhaps with a protein, and that one
of the first events of early development is the
removal of that block, possibly by proteolysis.
It should be pointed out, however, that the same
group of investigators have shown that in this
material endogenous mRNA levels are about the
same in unfertilized eggs and blastulae.
DEERING: Do you know what happens to the
RNA situation when you artificially activate an
egg?
GROSS: If you do this successfully, you
turn on both protein and RNA synthesis in the
normal way, since one gets a normal haploid
embryo.
MAURER: What about nuclease activity?
Could it be that the stability of your messenger
is due to a low level of ribonuclease?
GROSS: It could, but it is certainly not so.
These eggs have extremely high levels of
nuclease, so that the problems of handling the
RNA are very complicated, indeed.
MAURER: Can you inhibit by bentonite?
GROSS: Yes. You can inhibit the nuclease
activities sufficiently to make what look like
respectable RNA preparations, but this does
require rather heroic efforts. There is only
one way I know of dealing with the high levels
of nuclease when such activity must be stopped
entirely. We learned of the trick when working
with polyribosomes. This is to add either large
amounts of enucleate HeLa cells, that is to say,
HeLa cell cytoplasm, or large amounts of yeast
RNA. In both cases, what one is doing is provid-
ing the endogenous nucleases with a large excess
of substrate in the hope that the substrate in
which one is interested will remain, to a large
extent, untouched.
CHALKLEY: Wouldn't this raise a very
interesting point, then? First, you have a very
stable RNA in the cell and a lot of nuclease
present and, presumably, not able to attack and
disrupt it; later the problem arises that it can
attack it. One might think of compartmentation
playing a role.
GROSS: Yes, I believe it would be a neces-
sary conclusion. If the nuclease is really there.
14
then either the RNA or the nuclease is seques-
tered.
CHALKLEY: Then the point I'm aiming at
is that the RNA is not in some mysterious way
stabilized.
GROSS: It's not easy to distinguish at this
point between the two proposals.
HYMER: I would like to comment on this
point. Dr. E. L. Kuff and I demonstrated the
presence of an endonuclease within nuclei iso-
lated from murine plasma cell tumors. This
enzyme preferentially attacked rapidly labeled
high molecular weight RNA, and its activity
could be completely inhibited by the addition of
cytoplasmic soluble fraction.
GROSS: Well, in any case, the whole prob-
lem of stability and instability in messages is
both interesting and difficult, and it is by no
means restricted to embryos. On the basis of
a large body of accumulating evidence, one can
now safely conclude that stable and unstable
messages coexist in the cells of higher orga-
nisms.
UNKNOWN DISCUSSANT: You mention that
you are able to hybridize the nucleic acid from
the unfertilized egg. What percentage of hybrid-
ization were you getting and what technique were
you using?
GROSS: Our technique was a modification
of the Nygaard-Hall method, essentially the one
described by McConkey and Hopkins in the
Proceedings of the National Academy of Science
about a year ago (14). The method gives low
values of hybridization. In fact, McConkey and
Hopkins got a value for the size of the ribosomal
fraction that is obviously much too low. Their
method has the one virtue that it reduces so-
called mistaken identity hybrids to the lowest
values that I know without the use of ribo-
nuclease. We use this method, therefore, be-
cause our low specific activities and large
amounts of ribosomal RNA demanded it. With
it, we get something like 1-1/2% hybridization.
That is 1-1/2% of the total counts in a prepara-
tion of the type for which we saw gradients
earlier, hybridized under the conditions of
saturation routinely employed. By using a 5 to
15-fold excess of unlabeled RNA, we can reduce
the counts by only a very small amount - 8 or
10% of the original number. From that reduction,
we got the estimate of the fraction of the genome
occupied by the ribosomal cistrons.
15
References
1. P. R. Gross, J. Exp. Zool. 157, 21 (1964).
2. S. A. Terman and P, R. Gross. Biochem.
Biophys. Res. Comm. 21, 595 (1965).
3. P. R. Gross, W. Spindel and G. H. Cousineau.
Biochem, Biophys. Res. Comm. 13, 405
(1963).
4. L. I. Malkin, P. R. Gross and P. Romanoff.
Devel. Biol. 10, 378 (1964).
5. A. Monroy and A. Tyler. Arch. Biochem.
Biophys. 103, 431 (1963).
6. D. W. Stafford, W, H. Sofer and R. M.
Iverson. Proc. Natl, Acad. Sci. U.S. 52,
313 (1964).
7. T. Humphreys, S. Penman and E. Bell.
Biochem. Biophys. Res. Comm. 17, 618
(1964).
8. D. G. Comb and R. Brown. Exp. Cell Res.
34, 360 (1964).
9. D. D. Brown and E. Littna. J. Mol. Biol. 8,
669 (1964).
10. R. Maggio, M. L. Vittorelli, A. M. Rlnaldi
and A. Monroy. Biochem. Biophys. Res.
Comm. 15, 436 (1964).
11. T. Hultin. Experientia J 7, 410 (1961).
12. A. Monroy, R. Maggio and A. M. Rinaldi.
Proc. Natl. Acad. Sci. U.S. 54, 107 (1965).
13. M. Nemer. Biochem. Biophys, Res, Comm,
8, 511 (1962).
14. E. H. McConkey and J. W. Hopkins. Proc,
Natl. Acad. Sci. U.S. 51, 1197 (1964).
15. P. R, Gross, L. I. Malkin and M. Hubbard,
J. Mol, Biol, 13. 463 (1965).
16. D. Elson, T. Gustafson and E. Chargaff.
J, Biol, Chem. 209, 285 (1954).
17. M. M. Daly, V. G. Allfrey and A. E. Mirsky.
J. Gen, Physiol, J5,497 (1950).
18. P. R. Gross, K. Kraemer and L. I. Malkin.
Biochem. Biophys. Res. Comm. 18, 569
(1965).
19. J. Mangan, T. Miki-Noumura and P. R.
Gross. Science 147, 1575 (1965).
16
EARLY BIOCHEMICAL EVENTS FOLLOWING
FERTILIZATION OF SEA URCHIN EGGSi
David EpeP
Johnson Research Foundation, Department of Biophysics and
Physical Biochemistry, University of Pennsylvania Medical School,
Philadelphia, Pennsylvania
INTRODUCTION
Fertilization results in a metabolic activa-
tion, similar in certain respects to the activa-
tions occurring upon neurochemical stimulation
of muscle or addition of hormone to target
tissue. It differs from the above, however, in
that fertilization occurs only once during the
lifetime of the organism, initiating a unique
series of reactions leading to rapid cell divi-
sions and embryonic differentiation.
The changes which occur upon fertilization
are dramatic at both the morphological and
molecular levels. Changes in membrane struc-
ture, respiration rate, and rates of DNA, RNA,
and protein synthesis occur, as well as changes
in cation and coenzyme content, and subcellular
location of enzymes. These all occur within
seconds or minutes of insemination, and some-
how are interrelated with each other to yield an
orderly pattern of embryonic development.
Although many post-fertilization changes
have been observed, numerous unresolved prob-
lems still exist. Little is known about how
these changes occur, when they occur, or the
casual connections between them. For example,
it is not known whether synchronous activation
of all enzymes is the case, or whether one or
several changes are triggered which then initiate
the other reactions in a chain or cascade-type
reaction system.
The research to be discussed represents
the beginnings of an intensive study of the
fertilization reactions, aimed at shedding some
light on the above problems. The experimental
approach used is based on the assumption that
the fertilization changes result solely from
enzymic activation. The pertinent evidence for
this is, first, that eggs can be artificially ac-
tivated (artificial parthenogenesis) to develop
without sperm (1). This indicates that the
sperm does not supply some missing enzyme or
substrate to the egg, and hence implies that all
materials necessary for development reside in
the egg. The second piece of evidence is that
eggs can be fertilized in the presence of con-
centrations of puromycin sufficient to inhibit
the bulk of protein synthesis. Under such con-
ditions, they will develop up to the first mitotic
division (90 minutes after insemination in the
eggs of S. purpuratus) before any arrest occurs
(2). This result means that little or no de novo
protein synthesis is required for the earliest
reactions of development, such as pronuclear
fusion or RNA synthesis. These two experiments
indicate that the immediate changes of fertiliza-
tion most probably result from activity of
enzymes already present in the egg.
Enzymes and metabolic pathways activated
by fertilization, as well as physicochemical
changes possibly controlling these activations,
are shown in Table I. This table categorizes
the best described post-fertilization changes in
sea urchin eggs as changes in carbohydrate
and energy metabolism, co-factor and coenzyme
metabolism, synthetic metabolism, and changes
in structure.
Examination of these changes suggests some
possible factors limiting metabolism in the
unfertilized egg. For example, the metabolic
machinery of the egg might be limited by cations
(as evidenced by changes in Ca"*"^ or K+), by
^Supported by Public Health Service grant 5T1 GM2G277
and National Science Foundation grant GB-4206.
^ Present address: Hopkins Marine Station, Pacific
Grove, California.
17
TABLE I
Metabolic and Structural Changes Upon Fertilization of Sea Urchin Eggs:
A. Carbohydrates and Energy Metabolism
1. Respiration rate increase
2. Increased pentose shunt activity
3. Increased content of glycolytic esters
B. Cofactor and Coenzyme Metabolism
1 . TPNH increase
2 . Free Ca'
+2
increase
3 . K increase
-3
i* . PC, uptake increase
C. Synthetic Metabolism
1. Increased rates of protein synthesis
2. Increased rates of RNA synthesis
3. Increased rate of lipid synthesis
D. Structural and Physical Changes
1. Cortical granule breakdown
2. Changes in subcellular localization of
enzymes
3. Fertilization acid excretion
4. Proteolytic activity increase
5. Membrane potential
6. Light-scattering change in cortex
References
41 , 42 (review) ,
14, 6
39, 40
29, 43
9, 12
34
44
45, 46
47-53, 3
54- 5b
57
19, 58 (reviews)
32, 33
59, 22
60
15, 16
13
coenzymes (as evidenced by increased TPNH),
by lack of respiratory substrate (as evidenced
by increased content of glycolytic esters, respi-
ration rate, etc.), by unavailability of substrate
to enzyme (as evidence by both structural
changes in cortex and intracellular location of
enzymes, as well as the transient proteolytic
activity), or possibly by presence of a general
inhibitor (as suggested by acid excretion or
proteolytic activity).
To decide between these alternatives, a
kinetic analysis has been used and will be
described in this paper. Such an analysis,
aimed at describing the temporal sequence of
the fertilization reactions, should yield infor-
mation on possible mechanisms of activation.
Hypotheses derived from the kinetic analysis
can then be tested, hopefully leading to elucida-
tion of any primary reaction or reaction series
of fertilization. These studies should also pro-
vide rigorous testing of hypotheses. As an
example, if the recent hypothesis relating pro-
teolytic activity to the post-fertilization initia-
tion of protein synthesis is correct (3), the
transient activation of proteolytic activity should
occur before the activation of protein synthesis.
To date, we have concentrated on the kine-
tics, mechanism, and metabolic significance of
changes in coenzymes, carbohydrate and res-
piratory metabolism, acid excretion, and struc-
tural changes. The methods we have used
measure in vivo changes in cell suspensions,
using procedures developed at the Johnson
Foundation of the University of Pennsylvania
(4, 5). The basic equipment consists of awater-
jacketted glass cuvette, into which is placed a
concentrated suspension of eggs. From the side
of this cuvette, optical measurement of light-
18
scattering (structural changes) and 366 m^
induced cell fluorescence can be made. This
latter measurement, in all systems so far
described, is specific for detecting changes in
reduced pyridine nucleotide (4). Through the top
of the cuvette can be inserted an oxygen electrode
for measuring respiration rate, and a pH
electrode for measuring excretion of the fer-
tilization acid (see 6 and 12 for experimental
details). Finally, samples can be taken from
the cuvette for analysis of coenzymes, sub-
strates, or enzyme activity. The four para-
meters (light-scattering, fluorescence, respira-
tion, and acid excretion) have been monitored
through low time constant amplifiers, and re-
corded individually on synchronized recorders,
or simultaneously on a multi- channel recorder.
RESULTS
I. Temporal sequence of fertilization changes
A. Pyridine nucleotide changes
TPNH is the coenzyme generally involved
in reductive biosynthesis, as indicated by the
coenzyme specificity of reductive reactions,
as well as by the general correlation between
synthetic activity and both TPNH levels and
TPNH/TPN ratios (7, 8). This compound has
been reported to increase within one hour after
fertilization (9), and hence this change might
be important in initiating and controlling re-
ductive biosynthesis in the egg.
As indicated, 366 m/;f-induced cell fluor-
escence is a sensitive monitor of reduced
pyridine nucleotide in vivo. Measurements of
cell-fluorescence following fertilization, shown
in Fig. 1, indicate an increase in this para-
meter, beginning at 40 seconds after sperm
addition, and ending by 5 minutes with a 1/2
time of 35 seconds. Enzymatic analyses of
reduced pyridine nucleotides in alkaline-
extracted cell homogenates are shown in Fig. 2.
These indicate that the reduced pyridine nu-
cleotide which increases is TPNH, and that this
increase parallels the changes in fluorescence.
Furthermore, the sum of reduced pyridine
nucleotides at various times after fertilization is
linearly related to the cell fluorescence (Fig. 3),
which confirms the relationship between in vivo
fluorescence and reduced pyridine nucleotide.
The increase in TPNH does not result
f'-om reduction of pre-existing TPN, but rather
from phosphorylation of DPN to TPN, and most
probably the subsequent reduction of this TPN
to TPNH. This is shown in Fig. 4 and Table II.
Figure 4 shows that DPN decreases, while TPN
increases in a mirror-image fashion. Similar
behavior is also seen for the TPNH increase
shown in Fig. 2. These changes suggest a
precursor -product relationship, and this sup-
position is further verifiedby the stoichiometric
relationship shown in Table II, which is a
balance sheet of pyridine nucleotide before and
after fertilization. The pertinent point to observe
is that total amount of pyridine nucleotide is the
same before and after fertilization, but that an
interconversion of pyridine nucleotide types has
occurred - total TPN and TPNH increasing,
while total DPN andDPNH decrease. The enzyme
implicated in such an interconversion is DPN
kinase, which catalyzes the reaction:
DPN and ATP ► TPN and ADP(10, 11).
This enzyme, then, is apparently activated by
fertilization. Possible mechanisms of its acti-
vation will be described later.
POLLARD: How does that fit with any
reasonable turnover numbers for the production
Flourescence Of Egg Suspension
— rf-
rj
1
" —
~
■ — ■
Sperm Added
-■^-^
1 1 1
1
1
1000 300
270
240
210
ISO 160
Seconds
120
90
60
30
-30
Fig. 1.
366 mu Induced fluorescence of eggs of S. purpuratus following fertilization. (Fig. 1, Epel,
Biochem. Riophys. Res. Comm. 17, 69, 1964; reproduced with permission of Academic Press.)
19
s
o
T
30
60
I I I 1 I I I r
90 120 150 180 210 240 270 300 330 1050
Seconds After Sperm Addition
Fig. 2.
Analysis of reduced pyridine nucleotide at various times
after fertilization of S. purpuratus. (Fig. 2, Epel, Biochem.
Biophys. Res. Comm. 17, 69, 1964; reproduced witJi per-
mission of Academic Press.)
Totol Reduced Pyridine Nucleotide (10 moles/10 cells)
Fig. 3.
Linearity of cell fluorescence and reduced pyridine nu-
cleotide at various times following fertilization.
E
'o
u
in
O
E
'o
1 <;-
A *
IO-
r TPN
CS-
../
0.6 n
A
04-
02-
0-
' 1 r—
1111
20 40 60 80 100 120 140
Seconds After Sperm Addition
Fig. 4.
Analysis of oxidized pyridine nucleotide following fertili-
zation of 5. purpuraius (Figs. 1, 2 and 4 are from separate
experiments and not strictly comparable).
20
TABLE II
Average Content and Ratios of Pyridine Nucleotides in S. purpuratus^
a 10-1° moles/ 105 cells
TPtm
TPNH
TPN
DPN
DPNH
Unfertilized
6.7 + 1.5
7.0 + 0.1
59.3 + 6.8
3.3 + 2.5
0.96
DPN
17.9
Fertilized
K
atio .fpf,
29.7 + 9.0
10.7 + 3.0
2.8
Rat
DPN
^° DPNH
32.7 + 8.3
5.0 +4.3
6.6
Total
7. DPN & DPNH
7. TPN & TPNH
76.3
827.
187.
78.1
487.
527.
of the TPN? You've got 10^ molecules per cell
formed in about 10 seconds. Isn't that quite
rapid formation? Is there any "miracle" here?
EPEL: Any "miracle"?
POLLARD: I'm referring to the fact that
10^ molecules per cell are made in 10 seconds.
EPEL: The data in the figure is per 100,000
cells.
POLLARD: It's 10^ molecules per cell,
which gives a really very rapid turnover num-
ber of about 10,000 per minute. Why are the
enzymes that good? That would seem to me to
be the exciting thing you've got here. Is it all
right?
EPEL: Actually this is consistent with
maximum activity of the enzyme. For some
reason the enzyme is suddenly activated close
to maximum activity, or at least within a factor
of 2 or 4.
POLLARD: That is a slight miracle. Is it
more than "maximum"?
EPEL: No, it's not more than maximum,
as extrapolated from in vitro experiments under
simulated in vivo conditions.
DEERING: This assumes you know how
much of the enzyme is present.
EPEL: Yes. On the basis of extracting
enzyme from a known amount of cells, and as-
saying kinase activity at ATP and DPN concen-
trations present in vivo. In any case, if it were
grossly aberrant, we would notice it. This is
the most active source of the enzyme that's
ever been found. The maximum activity is only
three times less than the 75-fold purified
enzyme from pigeon liver.
B. Respiratory changes
Simultaneous measurement of respiration
rate and cell fluorescence, shown in Fig. 5,
indicates that the fluorescence change (TPNH
increase) precedes the activation of respiration.
Respiration is measured polarographically, and
an upward deflection indicates a decrease in
oxygen content. Rate is indicated by the slope.
The respiration rate (see Fig. 7) is characterized
by a transiently large burst, followed by a slow
decrease to a rate 4-5 times that of the pre-
fertilization rate. Significance of these kinetics,
as well as possible controlling mechanisms for
respiration, will be described later.
C. Excretion of the fertilization acid
Simultaneous measurements of fluorescence
and extracellular pH indicate that changes in
these two parameters began simultaneously if
measured at similar amplification levels (i.e.,
at amplifications such that the total changes
are of similar magnitude on the chart paper),
21
_J^=J- 1
"■^■;:5::4D
<
A
^i"
15
sec
-_
\&5
— N.,
^
^
i25
Sperm
\
Respiration
i
-«:..
s
K"
■^c^
A
1 ^~ —
. - —
\
I2nv<
DPN
Adde
Fluorescence
-^
"\
(1.89x10" moles/10' cells/sec)
I.I.I
\
d
i
N
X^
t
^~
t
--- —
S
perm
Fig. 5.
Simultaneous measurement of respiration and fluorescence following fertilization of
S. purpuratus. Decrease in O2 content is towards the top of the figure. Respiratory rates
at various times are indicated on the trace, in 10-1' moles O2 consumed/lO^ cells/sec.
Time is from right to left.
as in Fig. 6. If measured at different amplifi-
cations, as in Fig. 8, the timings of the changes
were apparently different, acid excretion pre-
ceding the fluorescence change.
The rate of the acid excretion, in eggs of
all three species of sea urchin examined,
always peaked before the peak respiratory rate
(Fig. 7). This suggests that the reactions re-
sponsible for the acid formation occur very
rapidly, and are essentially over before the
respiratory increase. The source and mechan-
ism of the acid formation will be discussed
later.
D. Light-scattering changes
Light-scattering measurements can be a
sensitive monitor of structural changes. Ac-
cordingly, the kinetics of light-scattering
changes following fertilization were measured
in collaboration with Dr. B. C. Pressman,
using an instrument designed by Dr. Pressman
(5). This instrument can simultaneously record
all the parameters previously described.
The results of one such measurement are
shown in Fig. 8. It is seen that a light- scatter-
ing decrease begins at 45 seconds, and is tem-
porally coincident with the beginning of acid
excretion. Within five seconds the fluorescence
change begins, and this is followed at 60 sec-
onds after sperm addition by the activation of
respiration.
These measurements, then, indicate that
a temporal differentiation of these events does
occur following fertilization. In the remainder
of this paper, I shall discuss first, the reality
and universality of these kinetics, and second,
the possible structural and molecular mecha-
nisms of the observed changes.
II. Possible factors influencing the kinetic de-
termination
Several questions can be raised as to the
degree the observed temporal sequence reflects
the actual sequence. A major biological artifact
could be the kinetics of sperm-egg interaction.
Thus, if the successful contact between egg and
sperm took several seconds or minutes, the
timing and duration of the observed changes
could simply, and uninterestingly, represent
the fertilization time. The experimental condi-
tions which would obviate this argument, how-
ever, are (1) a large redundancy of sperm
were added, and (2) the same kinetics were
obtained in the presence of 10-fold less sperm.
The experimental measurements also pro-
vide an estimate of the time for successful
sperm-egg interaction, which is related to the
duration of that reaction completed in the
shortest interval. From Fig. 8, this is seen
to be the light-scattering change, which has a
% time of only 20 seconds. The % time for
fertilization is probably less than this, how-
ever, since the light-scattering change in a
single cell probably has a finite duration. If
this change is identical to that observed in
single cells, its duration in one cell would be
about 20 seconds (13).
22
i
PH
- —
15
sec
pH=0.067
H=6..
— ^
T
p
57 —
^~-
-~^
--
p
^
Fluorescence
-"-^
v^
x
_pH=6.90 J
"~~
—
=^
^=-
lerm"
ded '
Ad
Fig. 6.
Simultaneous measurements of extracellular pH and cell fluorescence following fertiliza-
tion of S. furpuratus. Note that time is from right to left.
The other question relates to whether the
observed temporal sequence might result from
instrumental artifacts. This is probably the
case in the lag between the light-scattering-pH
change and fluorescence change shown in Fig. 8.
Thus, if the observed light-scattering or acidity
changes were adjusted to give the same ampli-
tude on the chart as the fluorescence change
(as in Fig. 6), the temporal sequence would
be almost identical (within two seconds). It is
probable, therefore, that changes in acid ex-
cretion, light-scattering, and fluorescence all
begin simultaneously, with possibly a slight lag
in the fluorescence change.
The respiratory change, in all cases so
far examined, always begins after the above
changes and does not appear to result from
any instrumental lag. First, when fluorescence
and respiration rate are similarly amplified,
the lag is still apparent. Secondly, when an
nnnoles
'6 20 40 60 80 100 140 180
Seconds After Sperm Addition
-2
Fluorescence Increase!
Light Scattering Decreasty
— I — I — I — 1 — I — I — I — I — [ — I — I 1
0+1 2 3 4 5 6
Minutes After Sperm Addition
Fig. 7.
Derived rates of acid excretion and cell respiration
following fertilization of S. purpuraius. Note that peak acid
excretion occurs before the increase in respiratory rate.
Fig. 8.
Simultaneous measurement of cell fluorescence, extra-
cellular pH, respiration rate and light-scattering in eggs
of S. purpuratus. (Data of Epel and Pressman).
23
uncoupler of respiration is added to fertilized
eggs, there is only a 10-second lag before the
increased respiratory rate is evidenced, as
compared to a 30-second lag between fluores-
cence and respiration when these eggs were
initially fertilized. Finally, the lag is evident
in other species examined (see, e.g.. Fig. 10),
and was also observed by Ohnishi and Sugiyama
(14) in several species of Japanese seaurchins.
These workers, furthermore, were using a bare
platinum electrode with time constants less than
one second, as compared to our membrane-
covered electrodes with time constants of 3-6
seconds.
The present data, then, indicate that the
first discernable event of fertilization - in our
measuring system - is a structural change,
probably related to cortical granule breakdown
(see Sec. IVa). This light-scattering change,
observed in cell suspensions, is probably simi-
lar to that seen by Rothschild and Swann in
single cells under dark field illumination (13).
Although this structural change occurs
early, the first change in the eggs is undoubtedly
related to attachment of the sperm acrosomal
filament, which probably initiates these struc-
tural reactions in a primary, or possibly
secondary, reaction. The structural events might
also be related to changes in electrical prop-
erties of the membrane, as first shown by
Tyler et al (15) and Hiramoto (16). The data of
Hiramoto is shown in Fig. 9, and indicates an
early change in membrane resistance, capaci-
tance, and potential upon successful sperm-egg
contact. This change precedes membrane ele-
vation and might also precede cortical granule
Time \n minucet
Fig. 9.
Data of Hiramoto, showing changes in membrane poten-
tial i , membrane resistance i , and membrane capaci-
tance A T , following fertilization of Peronella. (Fig. 2,
Hiramoto, Exp. Cell Res. 16, 421, 1959; reproduced with
permission of Academic Press.)
' ' ' ' I — I — 1 — I — I — I — I — 1 — I — I — I — I — 1
-60 0 60 120 180 240 300 330 360
Seconds After Sperm Addition
Fig. 10.
Respiration rate and extracellular pH following fertiliza-
tion of Lytechinus variegatus (data of Epel and Iverson).
breakdown, although the temporal relationship
between granule breakdown and membrane ele-
vation is not clearly defined, and might vary in
different species (17).
GROSS: The time is about a minute after
fertilization, isn't that right?
EPEL: Yes.
MASSARO: Is that from the time of adding
the sperm or from the time of contact?
EPEL: 1 believe it's from the time of sperm
addition. However, the important point is that
he shows data that indicate relative time of
membrane elevation.
MASSARO: Well, how long does it take the
sperm to get in? Where is the sperm after 15
seconds?
EPEL: That's a good question. In some or
most organisms an acrosomal filament is ejected
from the head of the sperm. In Hydroides this,
supposedly, takes place within 9 seconds after
you add the sperm.
POLLARD: Isn't that about where the first
indication of change in membrane resistance is
seen? The resistance shows quite a change right
away.
EPEL: The best evidence for a rapid
change is a change in light-scattering of single
cells observed under dark field. This takes place
24
about 10 seconds after sperm-egg contact is
made.
MASSARO: The sperm is on the outside with
the acrosome penetrating?
EPEL: I don't think there is any direct
evidence for that. Certainly the sperm head
does penetrate within a short time. However,
it takes a relatively long time for it to appear
inside the egg.
GROSS: These things are all cortical
changes?
EPEL: Yes. I doubt if the sperm is con-
tributing anything in the initial chemical changes
such as genetic information or enzymes getting
inside the egg are concerned. As you indicated,
the sperm doesn't get in until minutes after.
These are surface reactions.
TS'O: These eggs can only be fertilized
by a single sperm?
EPEL: You can get poly-spermy if you add
a very large redundancy, but normally only one
sperm penetrates.
GROSS: The barrier to poly-spermy takes
about 20 to 45 seconds to develop at normal
temperature. So you'd need a very large multi-
plicity.
EPEL: I think it's more like 10 seconds,
although I wouldn't want to say it's that, defi-
nitely. [(Added in proof): A short note by
Rothschild and Swann (Exp. Cell Res., 2, 137,
1951) indicates that the actual block to poly-
spermy takes at least 25 seconds, and probably
longer. They interpret the failure of the kinetic
calculation to apply to the in vivo situation as
indicating that the limiting factor is the prob-
ability of a "successful" sperm-egg collision.]
There is one, so far unconfirmed, report
which is completely revolutionary. This is a
report by Neyfakh etal.(Biochem. Biophys.Res.
Comm.18, 582, 1965) on fertilization in fish
eggs, which shows that simple contact with
sperm is sufficient to activate synthesis of
cytochrome oxidase. This activation occurs
within one second, and is hence the most rapid
change ever reported.
MAURER: Do we know anything we can do
to the sperm which will eliminate this kind of
surface contact?
GROSS: I don't know of any.
POLLARD: What happens if you ultra-
violate the eggs and sperm in vivo ?
EPEL: They're okay.
POLLARD: They still do it?
EPEL: Yes, you can chemically activate
the egg without any sperm.
MAURER: What pushes the button in the
sperm?
EPEL: Presumably interaction between
sperm and egg result in ejection of the acroso-
mal filament. We have some evidence of in-
creases in respiration when you add a very
dense sperm suspension. In some cases there is
a transient, but definite, increase in respiration
(about double). Sperm with no eggs present
don't give this.
TS'O: Anatomically, does the stimulation
have to be in the head or tail of the sperm?
EPEL: Presumably, only the head can
stimulate.
GROSS: The tail never hits first. There's
apparently a strong chemo-taxis that orients
the sperm in the direction of the egg so that
the head goes first. This is important.
TS'O: Is this because of antibodies?
GROSS: Well, that's what Tyler says.
There's a complicated literature. The assump-
tion is that there is a specific receptor in the
sperm, and that a product of the egg surface
attracts the sperm toward the egg.
EPEL: There is good evidence for lytic
enzymes in the acrosome which may be involved
in getting into the egg. Whether these are in-
volved in the activation isn't clear. In con-
clusion, the in vivo kinetic studies indicate
that the timing of structural changes (light-
scattering), acid excretion, electrical and fluo-
rescence changes (TPNH) cannot at present be
temporally separated from each other, but that
these can all be temporally distinguished from
respiratory activation. This, then, suggests
both parallel and cascade-type reactions upon
fertilization.
III. Universality of the temporal sequence
Because interspecies variations in behavior
of other parameters after fertilization of sea
urchin eggs have been found (18), itis important
to determine whether the above changes occur
in other species of sea urchin, and in the same
sequence, or whether they are unique to the
species so far described.
Figures 10 and 11 provide a partial answer
to this question. The figures depict data, ob-
tained in collaboration with Dr. Ray M. Iverson
of the University of Miami, on the fertilization
changes in the eggs of the sea urchin Lytechinus
variegatus. Figure 10, which depicts respiration
rate and acid excretion, shows the same tem-
poral sequence in these two changes as had
ijeen observed in S . purpuratus . Of interest here
is the rapidity of the acidity changes. In this
species (at 30° C, as compared to 17° C for the
25
120
100-
80-
E
2 60-
'o
40-
20-
DPN
TPN
■ ■ .
I I I I I I I I I
20 40 60 80 100 120 140 160 180
Seconds After Sperm Addition
Fig. U.
Analysis of DPN and TPN following fertilization of L.
variegatus. Arrows indicate initiation of acid excretion and
increased respiration (data of Epel and Iverson).
S, purpuratus), the acid excretion has begun
at 18 seconds after sperm addition. The res-
piratory lag is longer here, O2 consumption not
increasing until 30 seconds after the pH increase.
Figure 11 shows that the DPN decrease
similarly occurs, beginning after acid excretion
and before respiratory activation. Although TPN
does not change (analogous to the sea urchin
Arbacia punctulata), TPNH does increase (data
from separate experiments not shown here).
A similar temporal sequence was also ob-
served in Lytechinus pictus, where measure-
ments were done in the Pressman apparatus
as in Fig. 8. It thus appears from an examina-
tion of two genera and three species, that the
temporal sequence is identical as regards
changes in structure, fertilization acid, fluo-
rescence, and respiration.
IV. Significance and mechanism of observed
changes
A. Light-scattering and acidity changes
The observed decrease in light-scattering
suggested a volume or size increase. Although
the volume of the egg supposedly does not
change, there does occur an elevation of a
"fertilization membrane". This membrane, in
the unfertilized egg, lies closely apposed to a
peripheral ring of granules - the cortical gran-
ules - which rupture upon fertilization, releas-
ing their mucopolysaccharide contents. The
overlying membrane is then presumably pushed
out, or elevated, either by expansion of the
mucopolysaccharide through hydration, through
osmotic forces resulting from these substances,
or molecular unfolding of the precursor mem-
brane (see 19). At any rate, the effective volume
of the egg doubles, which makes this change a
prime suspect as the cause of the light-scatter-
ing change.
This hypothesis can be tested, since the
precursor membrane can be removed with
trypsin. When this was done - to our great
surprise -the identical light- scattering change
was still observed. The scattering change,
therefore, does not result from elevation of the
fertilization membrane. The two most plausible
alternatives are that the scattering change
represents either the breakdown of the cortical
granules (which are trypsin-insensitive), or an
actual change in cytoplasmic structure. The
latter interpretation is suggested by changes in
texture and granularity of the cytoplasm, which
can be seen in stained eggs (20) or in vivo in
extremely transparent eggs (21).
That the change might correspond to break-
down of the cortical granules is suggested by
the similar kinetics of the acid excretion and
light-scattering. Although we had initially
thought the acid resulted from accumulation of
some acidic carbohydrate compound (such as
lactic acid), no compound analyzed was present
in sufficient concentration to account for the
acidity change. This was true for lactate,
pyruvate, glucose-6-phosphate, 6-phosphoglu-
conic acid, isocitrate, and malate. In fact, the
only change so far described which can account
for the acid production is the acidic mucopoly-
saccharide released by the cortical granules
(22). If one assumes that the sulfate moiety of
the mucopolysaccharide exists as sulfuric or
bisulfuric acid in the granules, then the amount
of protons released upon rupture of the granules
would be in the same range as the observed
acid release after fertilization (23). Although
not yet proven, the similar stoichiometry and
kinetics strongly support the conclusion that the
light-scattering and acid increase result from
the same event - the cortical granule break-
down.
Irrespective of interpretation, the kinetic
analysis of the light-scattering changes suggests
that structural changes may be highly critical
in metabolic activation, since they are one of
26
the first observable changes. If they indeed do
represent cortical granule breakdown, the hy-
potheses of Moser (24) and Runnstrom and
Immers (25), relating granule breakdown to
metabolic activation, take on added significance.
B. Respiratory changes
Although intensively studied since Warburg
first observed the dramatic post-fertilization
increase in O2 consumption, the operative
respiratory control mechanism is still unclear.
One possibility, suggested by the work of
Chance (26) and Lardy (27), showing respiratory
control by phosphate acceptor (ADP), is that
fertilization results in increased ATP utiliza-
tion and concomitant ADP formation. The in-
creased ADP level could then result in the
increased respiratory rate. Such a hypothesis
is also suggested by the recent finding that sea
urchin mitochondria exhibit respiratory con-
trol via ADP (28). To check this possibility,
eggs were sampled at rapid intervals after
fertilization, and analyzed enzymatically for
adenine nucleotides. The results of such as-
says, shown in Fig. 12, indicate no significant
changes in these coenzymes. Most importantly,
there are no changes at the time of maximum
respiratory activation. Although this suggests
that ADP-limited respiration (State 4-State 3
transition) is not operative here, it is probable
that ADP produced is immediately rephosphory-
lated, and that perhaps it is the ADP content in
the mitochondrial micro-environment which is
critical.
An alternative possibility accounting for
the low respiration rate in the unfertilized egg
is that respiration is substrate-limited. If so,
the increased respiratory rate following fertili-
zation could result from increased availability
of respiration-linked substrate [i.e., a State
2 -State 3 transition, as defined by Chance and
Williams (26)]. Such a mechanism was first
suggested by the findings of Aketa et al. (29)
that a large increase in the various glycolytic
esters, especially glucose-6-P04, had occurred
by five minutes after fertilization.
To check this possibility simultaneous anal-
yses of respiration and glucose-6-P04 were
carried out. The results of these experiments,
shown in Fig. 13, indicate that such an inter-
pretation might be tenable. It is seen that in
L. variegatus the glucose-6-P04 level does
indeed increase, and begins before the activa-
tion of respiration. This increase is rapid and
large. By six minutes (not shown) it is six
90
80
70
^ 60
o
■g 50
to
I 40
'o 30
20-
I0--
0
ATP
ADP
AMP
0 20 40 60 80 100 120
Seconds After Sperm Addition
Fig. 12.
Adenine nucleotide levels following fertilization of
S. purpuratus.
times the unfertilized level. Changes in glucose-
6-PO4 are nowhere near as marked in S.
purpuratus, however, nor are they so obvi-
ously related to the respiratory activation. These
differences could suggest that different sub-
trates are being utilized in these two species,
or that substrate mobilization is not critical to
the respiratory activation. It could also mean
that the different levels simply reflect differ-
ences in relative enzyme activities and rate of
flux of the glycolytic substrates. For example,
in frog skeletal muscle the glycolytic flux can
increase many fold before any increase in glu-
cose-6-P04 is seen (30), whereas in rat heart
a flux increase is immediately reflected in a
glucose-6-P04 increase (31). Since G-6-P is a
substrate in flux, as opposed to a coenzyme
which can cycle in its various forms, it might
therefore be premature to ascribe too much
importance to the different glucose-6-phosphate
levels. Rather, the comparative results suggest
that fertilization does activate substrate mobil-
ization in both cases.
The enzyme(s) responsible for this mobili-
zation is still not known. Glycogen phosphorylase
is the best candidate, and is indeed present in
both fertilized and unfertilized eggs of S.
purpuratus. Furthermore, preliminary experi-
ments indicate that the activity of this enzyme
is sufficient to account for the peak respiratory
activity of the fertilized egg.
POLLARD: Is all this respiration in the
mitochondria?
27
L.VARIEGATUS
S. PURPURATUS
40 80 120 160 0 40 60 120 160
Seconds After Sperm Addition Seconds After Sperm Addition
Fig. 13.
Comparison of clianges in content of glucose-6-phosptiate,DPN,TPN and rates of respira-
tion in eggs of L.variegatus and S. purpuratus. (From Epel and Iverson, In "Control of
Energy Metabolism," 1965; reproduced with permission of Academic Press.)
EPEL: We don't know yet, but it is another
possibility. We're just looking into this now.
C. TPNH changes
The stoichiometry and kinetics of the pyri-
dine nucleotide changes implicate activation of
DPN kinase by fertilization. That this is the
case is seen in Table III, which shows activity
measurements of DPN kinase in homogenates
prepared from unfertilized and fertilized eggs.
As can be seen, the activity is essentially the
same in both cases. Although this demonstrates
that the enzyme is indeed present in the un-
fertilized egg, and hence activated by fertiliza-
tion, it is disappointing from a heuristic view-
point that these differences could not also be
reflected in the broken cell preparations. This
suggests that the enzyme is either activated
by the homogenization procedures, that the
enzyme is activated by the assay procedure, or
that some substrate, activator, or cofactor
missing in the unfertilized egg is being either
released during homogenization or supplied in
the assay mixture.
The structural changes, as well as several
reports of enzyme translocation following fer-
tilization (32, 33), suggested that the enzyme
might be changing its subcellular site upon fer-
tilization. To check this, the enzyme has been
extracted with numerous different media, and
the activity in particulate and soluble phases
checked. In all cases, the enzyme has always
been found in the supernatant.
Measurement of substrate localization be-
fore and after fertilization were also carried
out, estimating the amounts of DPN and ATP
in the mitochondrial-nuclear fraction and the
post-mitochondrial supernatant. Although not
completely satisfying from the viewpoint of
both leakage of substrates from particles, and
some loss of ATP and DPN during centrifugation,
the results did not indicate any large amount
of binding of ATP or DPN to or in particles.
Briefly, 75% of the DPN and greater than 90%
of the ATP were in the post-mitochondrial
supernatant. As the DPN kinase is also in the
supernatant, it appears that both substrates and
enzyme are present in adequate amounts for the
reaction to proceed. These findings, therefore.
28
TABLE ni
DPN Kinase Activity in Homogenates of S. purpuratus
Unfertilized
Fertilized
3.1 + 0.1
3.2 + 0.04
Eggs homogenized in 0.1 M trlethanolamine buffer. 01 ml
of this extract (1.1 to 1.3 mgms protein) was incubated
for 30 minutes at 30°C in a medium containing 5 //moles
ATP, 5 |/moles DPN, 20 //moles MgCl2 and 180 //moles
trlethanolamine buffer, pH 7.4, in a total volume of 2.0
ml. Assay procedures as In Fig. 14.
suggest that enzyme and substrate are "ap-
parently available" to each other, but do not
interact until after fertilization.
The remaining requirement for DPN kinase
enzyme activity is a divalent cation. Although
we as yet have no data on cation content of
the soluble phase, Mazia (34) has shown that
fertilization results in an increase in free
Ca+2 (as opposed to bound, or non-dialyzable
Ca"'"^). Could the enzyme requireCa'''^, andif so,
could the Ca+2 change account for enzyme acti-
vation?
Studies to test this hypothesis have been
done by assaying enzyme activity in dialyzed
or chromatographically desalted supernatants
at ATP and DPN concentrations paralleling the
in vivo concentrations of substrate. One such
activity curve is shown in Fig. 14. It is seen
that the enzyme exhibits a requirement for a
divalent cation, and is activated more strongly
by Ca^^ at low cation concentrations. Above
3mM, however, it is seen that Mg'''^ activates
20% better than Ca"*"^. Such behavior is rela-
tively unique for a kinase, since most enzymes
of this type are better activated by Mg''"2, and
in some cases are Ca+2 inhibited. For example,
Ca+2 is only 40% as active as Mg+2 in pigeon
liver DPN kinase (11).
What picture emerges from these studies?
The kinetic analysis suggests the following
picture. A light-scattering change occurs, prob-
ably reflecting the breakdown of cortical gran-
ules. Coincident with this is the initiation of
fertilization acid excretion, probably reflecting
the release of sulfated mucopolysaccharides.
Within a second or two of these two changes
DPN kinase is activated. Shortly thereafter (or
simultaneously) carbohydrate flux increases,
possibly through phosphorylase activation, and
when sufficient substrate has reached the res-
piratory chain, respiratory activation occurs.
1 1 1 \ \ I I I r
0 10 20 30 40 50 60 70 80 90 100
CATION CONCENTRATION (10'"* M)
Fig. 14.
Cation dependence of DPN kinase from unfertilized eggs
of 5. purpuratus. 0.1 ml of a 12,500g supernatant, desalted
by passage through a Bio-Gel P-2 column, was incubated
with 0.2 mM DPN, 3.6 mM ATP and the noted concentra-
tion of cation In trlethanolamine buffer, pH 7.4, 0.083 M
for 30 minutes at 30°C. The reaction was quenched by
boiling, and TPN assayed with isocitrlc dehydrogenase.
The above hypothetical scheme is compatible
with the data. The weakest point is the picture
of respiratory activation, since there is not
really good evidence that carbohydrate mobil-
ization via phosphorylase is the responsible
factor.
Although the mechanisms for these changes
are not rigorously defined, the analysis of the
DPN kinase reaction suggests the hypothesis
that the change in free Ca+2 is the primary
activator of this enzyme. Glycogen phosphoryl-
ase can also be Ca+2 activated through the
complex phosphorylase kinase system (35) so
such a hypothesis takes on added interest. A
possible criticism of this interesting theory is
that the kinase is also Mg+2 activated, and the
Mg+2 content in vivo is more than adequate to
activate the enzyme (36). Although no data is
available on the free Mg+2 content, it should be
noted that the amount of RNA in the eggs is
sufficient to completely bind the available Mg+ 2
(37). Obviously much more data has to be ob-
tained in order to prove or negate the Ca+2-
activation hypothesis, and it is presented here
solely to indicate the possible directions of
this research.
In closing, I should like to comment on
how the various post-fertilization reactions
might be involved in initiating the syntheses
characteristic of development. Monroy et al.
(3) have recently reported evidence indicating
29
that activation of protein synthesis might be
controlled through the transient proteolytic
activity at fertilization. Here, the ribosomes are
visualized as being coated by a protein envelope,
thus preventing protein synthesis sterically.
They visualize this envelope as being removed
by the protease, thus resulting in increased
protein synthesis.
The TPNH change could account for the
observed activation of lipid synthesis at fer-
tilization, since this coenzyme is specifically
involved in this synthetic sequence. The TPNH
change, especially the increase in the redox
couple of TPNH/TPN, could also be critical
for protein disulphide interactions believed to
be involved in cell division (38). The total
increase in the triphosphopyridine nucleotides
could also be involved in the channeling of
carbohydrate through the pentose shunt, whose
activity increases following fertilization (39,
40, 9). A change in carbohydrate flux, although
still not rigorously proven, could also be
important in regulating macromolecule synthe-
sis. Besides the important energy yields from
carbohydrate metabolism, a major limiting fac-
tor could be carbon skeletons for synthesis, as,
e.g., ribose for RNA synthesis.
V. Conclusions
The present study of the temporal sequence
and mechanism of the fertilization reactions in
sea urchin eggs has centered on light- scattering
(structural) changes, fertilization acid excre-
tion, and activation of DPN kinase and respira-
tion. The data indicate that changes in light-
scattering and acid excretion begin
simultaneously, followed almost immediately
by activation of DPN kinase. Respiratory ac-
tivity increases last.
Analysis of these changes suggests that the
light- scattering and acid changes reflect the
breakdown of the cortical granules. DPN kinase
activation might be through the free Ca+2 re-
lease known to occur after fertilization since
this enzyme is both Ca+2 and Mg+2 activated.
The mechanism of respiratory activation is still
unclear, but the available data suggest substrate
mobilization, possibly through control of
glycogen phosphorylase.
POLLARD: Is there any possibility of get-
ting at this genetically? Are there any deficient
eggs which require that a large amount of cal-
cium be added to the medium in order to get
fertilization? This sort of thing would be some-
thing you could look at. There might be some-
thing here similar to the findings by Slonimski
on yeast mitochondria, which themselves are
rather specific kinetic things. Is this possible?
Are sea urchins accessible genetically?
EPEL: Yes, generally they are. I think it
would be a very good contribution. There may
be some organisms in which you could do this.
You do require calcium to fertilize invertebrate
eggs in the sea water.
POLLARD: I feel you're trying to describe
a lot of exciting kinetics without quite putting
your finger on the initiating point.
EPEL: That's right.
POLLARD: You think that the best lead so
far might very well be the potentiation of
enzyme by action of calcium or magnesium,
presumably initiated by some membrane com-
ponent that makes this possible. You're refer-
ring, essentially, to a fast physical change,
like chemiosmosis, followed by fairly rapid
concentration of an ion which is favorable to
enzyme X. I would feel that if you're starting
to look at a single enzyme, this is the sort of
thing that you could have missing genetically.
Then you'd have to add a whole lot of other
things to the medium to make it go. Is there
any evidence at all for this sort of thing?
EPEL: Not that I know of.
TS'O: Is this enzyme stimulated by pH
changes? For instance, will a simple change
of pH from 6.9 to 6.5 affect the enzyme activity?
EPEL: No, it appears to have a broad
optimum between pH 7 and 8.
TS'O: Can physical studies be made on
fragments of membrane?
EPEL: There have been some enzyme
studies made on sea urchin egg cell cortexes.
They have a sodium-potassium-activated
ATPase.
PAPACONSTANTINOU: This might impli-
cate a regulation between the hexose mono-
phosphate pathway and the Embden-Meyerhof
pathway of glycolysis. We know that some
substrates from the hexose-monophosphate
pathway will regulate the activity of some
glycolytic enzymes. I wonder whether there
might be some regulation here where sedo-
heptulose-7-phosphate or other metabolites of
this cycle aiffect the activity of this enzyme.
EPEL: Yes, I think this would be very
possible.
30
ACKNOWLEDGEMENT
I thank Professor Britton Chance for in-
valuable advice and support during this work,
as well as my many colleagues at the Johnson
Foundation. I also gratefully acknowledge the
stimulating collaboration of Dr. B. Pressman
and Dr. R. M. Iverson in some facets of the
reported experiments.
31
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33
RIBOSOMAL RIBONUCLEIC ACID SYNTHESIS IN
RANA PIPIENS EMBRYOS
David E. Kohne
Biology Department, Purdue University, Lafayette, Indiana!
One primary reason for the difficulty in
studying the biochemistry of development is
the lack of good genetic information on the
developing systems which are normally used.
It is now possible through the study of ribo-
nucleic acid (RNA) synthesis to investigate the
direct expression of a specific class of genes,
ribosomal RNA (R-RNA) genes, during develop-
ment. By utilizing developing Rana pipiens em-
bryos we have attempted to get an insight into
the gross aspects of the regulatory processes
which control the synthesis of R-RNA during
embryogenesis.
There were two technical problems to be
solved before Rana pipiens could be used for
the experimental animal in this study: 1) The
utilization of standard ribosome isolation pro-
cedures resulted in the ribosomes being irre-
versibly bound to the egg proteins. It was
found that the egg ribosomes could be readily
isolated if the frog eggs were homogenized in
a buffer of high ionic strength and high pH, to
which sodium lauryl sulphate had been added
(1). 2) When used onRana pipiens eggstheusual
methods for the isolation of undegraded high
molecular weight R-RNA resulted in highly
degraded low molecular weight R-RNA as the
isolation product. It was obvious that large
amounts of powerful nucleases existed in these
eggs and a method had to be devised to negate
the «ffect of these enzymes. This procedure
primarily involved maintaining a temperature as
low as possible during the RNA isolation pro-
cedure (1).
Three experimental embryological systems
were used in this work to ask some simple
questions about the regulative phenomena in-
volved in the synthesis of ribosomal RNA during
development. 1) Hybrid embryos were utilized
in order to study the effect of a qualitative
change in the genome of Rana pipiens on R-RNA
synthesis during development. 2) Haploid em-
bryos were employed to ascertain the effect
on R-RNA synthesis during development of a
quantitative change in the frog genome. 3) Em-
bryos reared in a medium lacking in magnesium
were studied to determine the effect of mag-
nesium deprivation on R-RNA synthesis during
development.
In order to have a base line for comparison
of R-RNA synthesis in experimental systems to
that in normal development, it was necessary
to determine the pattern of R-RNA synthesis
in the normally developing iiana/)z7)zen5 embryo.
Figure 1 depicts the pattern of R-RNA synthesis
during normal development in Rana pipiens,
R-RNA synthesis could not be detected during
early development and was first detected at
early gastrula stage (two left peaks in gradients
shown). From early gastrula stage R-RNA
synthesis increases rapidly as development
proceeds. The base ratio of this newly syn-
thesized RNA is high in guanine + cytosine
which is a characteristic of all ribosomal RNA
(Table I).
The first experimental system was picked
in order to investigate the effect of a qualitative
change in the Rarui pipiens genome on the pattern
of synthesis of R-RNA during development.
Hybrid embryos produced by fertilizing Rana
pipiens eggs with Rana catesbeiana sperm were
used for these experiments. These hybrids
developed normally until the onset of gastrula-
tion and at this time development ceased. Al-
though development ceased at the early gastrula
stage, the hybrid embryos continued to live for
several days (2). It was of interest to determine
the pattern of R-RNA synthesis in the hybrid
* Present address: Department of Terrestrial Mag-
netisn;!, Carnegie Institution of Washington, Washington, D.C.
35
10
20
30 0 10
TUBE NUMBER
Fig. 1.
Sedimentation patterns of R-RNA extracted from ribosomes Isolated from 200 ^^ P- labeled:
a) unfertilized eggs, b) blastula embryos, c) gastrula embryos, d) neurula embryos,
e) hatching embryos, f) gill circulation embryos. Sibling embryos were used In this
experiment.
embryos in the hope that it might yield some
clue as to the control of R-RNA synthesis.
Twenty -four hour (early gastrula) and forty-
eight hour (early neurula) 32p_iabeled control
and hybrid embryos were extracted for RNA
and the purified RNA preparation displayed on
a sucrose gradient (Fig. 2). All RNA prepara-
tions were treated with DNase prior to sucrose
density gradient analysis. It is evident from Fig.
2 that the hybrid embryos synthesize much
less R-RNA at 48 hours than do the control
embryos. There is some question as to whether
the hybrid embryos synthesize R-RNA at all.
Stained histological sections of hybrid and con-
trol forth-eight hour embryos showed nucleoli
present in the control embryos but nucleoli
were not observed in the hybrids. The sucrose
density patterns, however, indicated that some
R-RNA was synthesized in the hybrid embryos.
Further work is necessary to resolve this point.
36
TABLE I
Base Compositions of Ribosomal RNA. a. Base composition of 28S and 18S R-RNA sub-
units. The values are expressed as mole per cent of the total RNA. b. The 32 p base
composition of the ■'2p_ia(,giej[ 28S RNA isolated from early neurula embryos. Values
are expressed as the per cent of the total CPM in the ^^P-labeled 28S RNA.
Material
a. Frog Eggs
Adult
Frog Liver
b . Early Neurula
28S
IBS
2'(3')
Uridylic Acid
19.5
22.3
2'(3')
Guanylic Acid
35.5
34.1
2'(3')
Cytidylic Acid
27.6
25.4
2'(3')
Adenylic Acid
17.4
18.2
2'(3')
Uridylic Acid
19.8
25.0
2'(3')
Guanylic Acid
35.1
30.2
2'(3')
Cytidylic Acid
27.4
24.1
2'(3')
Adenylic Acid
17.4
20.8
2'(3')
Uridylic Acid
17.3
2'(3')
Guanylic Acid
34.3
2'(3')
Cytidylic Acid
29.7
2'(3')
Adenylic Acid
18.7
In comparing R-RNA synthesis in R ana
pipiens haploid embryos and the Rana catesbei-
ana x Rana pipiens hybrid embryos it is strik-
ing that the haploid embryos exhibit the normal
pattern of R-RNA synthesis. The addition of a
foreign set of chromosomes to the Rana pipiens
haploid set of chromosomes has poisoned the
hybrid embryo and rendered it incapable of
further development. It is not likely that the
crippling of the hybrid's ability to elaborate
R-RNA was responsible for the developmental
retardation and death of the embryo. Recent
studies have shown that the anucleolate embryos
of Xenopus laevis develop to the swimming tad-
pole stage in the complete absence of R-RNA
synthesis (3).
The relative inability of the hybrids to
elaborate R-RNA prompted us to utilize another
experimental system. A developmental abnor-
mality caused by rearing Rana pipiens embryos
in medium lacking magnesium seemed to offer
an approach to the problem of the control of
R-RNA synthesis during development. Embryos
reared in this manner (magnesium deficient
embryos) develop normally to stage 21-23
(swimming tadpole) after which they undergo
developmental retardation, become edematous
and immobile and die 2-3 days later (4). Brown
initially made several potentially interesting
observations regarding the synthesis of ribo-
somes in these magnesium deficient embryos (4).
The magnesium deficient embryos apparently
contained one-sixth as much R-RNA in the
isolatable ribosome fraction as did control em-
bryos even though the magnesium deficient
embryos contained the same amount of total
RNA per embryo as did control embryos. Since
R-RNA usually comprises 80-90% of the total
cell RNA, it was of interest to investigate the
nature of the RNA from the immobile magnesium
deficient embryos.
Initial studies on the ribosomal content of
magnesium deficient embryos demonstrated that
an almost normal complement of ribosomes (as
compared with control embryos) could be iso-
lated if the ribosome extraction technique de-
signed for Rana pipiens eggs was used. Further
studies in which R-RNA was labeled while the
37
magnesium starved embryos were immobile
indicated that immobile magnesium deficient
embryos (Shumway stages 21-23, swimming
tadpole) made fewer ribosomes than did control
embryos of a comparable age (Fig. 3).
The apparent decrease in R-RNA synthesis
in magnesium starved embryos could be ex-
plained by one or more of the following hypoth-
eses: 1) Ribosomes were made at the normal
rate in the magnesium starved embryos but
ribosomal turnover was accelerated; 2) The
rate of synthesis of R-RNA was slower in
magnesium starved embryos than in controls;
3) There was a failure to assemble all newly
made R-RNA into ribosomes.
The following experiment was performed
to determine the stability of ribosomes in
immobilized magnesium starved embryos. Em-
bryos were grown in 10% Holtfreter's solution
and at Shumway state 20-21 were labeled with
^'*C02 and then incubated for 20 hours in non-
radioactive 10% Holtfreter's solution. At the end
(a) 24 HOUR CONTROL
(DORSAL LIP)
(b) 24 HOUR HYBRID
(DORSAL LIP)
30 0 10
TUBE NUMBER
E
o
CM
d
d
0.9
0.6
0.3
(C) 48 HOUR CONTROL
(EARLY NEURULA)
(d) 48 HOUR HYBRID
(DORSAL LIP)
20,000
15,000
10,000
5,000
f 900
600
-\ 300
0
TUBE NUMBER
Fig. 2.
Sedimentation patterns of RNA extracted from ^^ P-labeled whole control and hybrid em-
bryos: a) 50 twenty-four hour control embryos, b) 50 twenty-four hour hybrid embryos,
c) 55 forty-eight hour control embryos, d) 55 forty-eight hour hybrid embryos. Sibling
embryos were used in this experiment.
38
J 0.75
I 0.6
SO.45
^ 0.3
O
0.15
0
(a) CONTROL EMBRYOS
10
(b)Mg. STARVED EMBRYOS
20 0 10
TUBE NUMBER
loii
Fig. 3.
Sedimentation patterns of RNA extracted from ribosomes isolated from '^C02 -labeled
control and immobilized magnesium starved embryos: a) 25 control embryos, b) 25 mag-
nesium starved embryos. The embryos were incubated for 1 hour in a solution containing
10 //c/ml of Na2'''C03 and then placed in non-radioactive solution.
of the 20-hour "chase" the embryos were sep-
arated into three groups. RNA was extracted
from the ribosomes isolated from one group
(group 1) of embryos. A second group (group 2)
was placed in 10% Holtfreter's solution con-
taining magnesium. The third group (group 3)
was placed in 10% Holtfreter's solution which
lacked magnesium. The second and third groups
of embryos were kept in their respective solu-
tions for three days, at which time ribosomes
were isolated from each group of embryos and
extracted for RNA. The magnesium starved
embryos were immobile by the end of three
days. The RNA obtained from each group of
embryos was analyzed by sucrose density gra-
dient centrifugation. If the ribosomes of the
magnesium deficient embryos were stable, the
amount of radioactivity present in the R-RNA
of immobilized magnesium starved embryos
would be identical to the amount of radioactivity
present in the R-RNA of an equal number of
embryos from each control group (Group 1 and
group 2).
The amount of radioactivity present in the
R-RNA of immobile magnesium deficient em-
bryos was equal to the amount of radioactivity
present in the R-RNA of group 2 embryos and
very nearly equal to the amount of radioactivity
present in R-RNA of group 1 embryos (Figs.
4a, b, c). This demonstrated that the magnesium
starvation syndrome did not affect the stability
of normal ribosomes.
This same experiment also indicated that
the synthesis of ribosomes was slower in mag-
nesium starved embryos as compared to con-
trol embryos. The specific activities, measured
in counts/minute/unit of optical density at 260
mu (CPM/OD), of R-RNA from group 1, 2 and
3 embryos were presumably identical at the
end of the 20-hour chase. Since no more radio-
activity was available for R-RNA synthesis in
group 2 and 3 embryos (the total radioactivity
incorporated into the RNA was nearly the same
for each group), any further synthesis of R-RNA
would result in a dilution of the radioactivity
and a reduction in the specific activity of the
R-RNA. The specific activities reported here
were calculated from the amounts of radio-
activity and optical density present in the peak
tube of the 28S R-RNA component of each of the
three groups. The specific activity of the R-RNA
of group 1 embryos was 16,700 CPM/OD (Fig.
4a). Group 2 R-RNA had a specific activity of
9700 CPM/OD (Fig. 4b), while the R-RNA from
39
(a) GROUP I
20 HOUR CHASE
3.
E
o
u>
CVJ
O
(b) GROUP 2
CONTROL
3 OAY CHASE
10 20
TUBE NUMBER
Fig. 4.
30
6000
4500
3000
1500
0
o
T)
6000
4500 ?
-^3000
1500
0
6000
4500
3000
-1500
Sedimentation patterns of RNA isolated from "CO2-
labeled control and Immobilized magnesium starved
embryos: a) 35 Group 1 control embryos, b) 35 Group
2 embryos, 3 day chase, c) 35 Group 3 magnesium de-
ficient embryos. The embryos were incubated for 1 hour
in a solution containing 10 fic/ml Naj
embryos were used in this experiment.
14
CO,. Sibling
magnesium deficient embryos had a specific
activity of 11,700 CPM/OD (Fig. 4c), Since
the specific activity of the R-RNA of the group
2 embryos was lower than specific activity of
the R-RNA from magnesium deficient embryos
the group 2 embryos were making more ribo-
somes than the magnesium starved embryos.
The question still remained whether the
magnesium starved embryos converted all newly
synthesized R-RNA into ribosomes. When ac-
tinomysin D was used to inhibit RNA synthesis
in HeLa cells, the majority of the newly syn-
thesized R-RNA remained in the nucleus in the
form of 28S and 18S R-RNA subunits and was
not assembled into ribosomes. Some of the
R-RNA was assembled into ribosomes which
were transferred to the cytoplasm (5). An ex-
periment was performed to test the possibility
that a similar situation existed in magnesium
deficient embryos.
Control and immobilized magnesium defi-
cient embryos were labeled with ''^COj for 1
hour and then placed in non- radioactive medium
for a 20-hour "chase". RNA was extracted from
control and magnesium starved embryos and
ribosomes isolated from control and magnesium
starved embryos. The RNA was then analyzed in
a sucrose density gradient. The specific activi-
ties of the 28S R-RNA peaks were determined
for each sample. If the ratio of the specific
activities of whole egg 28S RNA/28S RNA ex-
tracted from isolated ribosomes was appre-
ciably higher for magnesium starved embryos
than the same ratio for control embryos, it
would indicate that the magnesium starved em-
bryos have difficulty in assembling newly made
R-RNA into cytoplasmic ribosomes.
The value of the ratio was 0.98 for mag-
nesium starved embryos and 0.97 for control
embryos. These figures indicated that no more
newly synthesized R-RNA was accumulated in the
nuclei of magnesium starved embryos than was
accumulated in the nuclei of control embryos.
The data presented here indicate that im-
mobilized magnesium deficient embryos contain
almost normal amounts of ribosomes and are
capable of synthesizing ribosomes. These mag-
nesium starved embryos, however, made fewer
ribosomes than did control embryos of the same
chronological age. The experiments on magne-
sium deficient embryos in this report were
based on the assumption that ribosome synthesis
in the magnesium deficient embryos was, some-
how, impaired. It must be remembered, how-
ever, that a characteristic of the magnesium
starvation syndrome is partial developmental
arrest of the magnesium deficient embryos.
Control and magnesium starved embryos of the
same chronological age were not at the same
developmental stage. It is possible that the mag-
nesium deficient condition had no effect at all
on the rate of synthesis of ribosomes and that
the rate of ribosome synthesis observed in the
magnesium deficient embryos was characteris-
tic of all embryos at that developmental stage.
40
Rana pipiens haploid embryos were next in-
vestigated in our search for some clue to the
mechanism of control of R-RNA synthesis during
embryogenesis. These embryos were useful for
studying the effects of a quantitative change in
the Rana pipiens genome on R-RNA synthesis
during development. Haploid embryos were
produced by fertilizing normal Rana pipiens
eggs with ultraviolet irradiated sperm. The
subsequent haploid embryos exhibited all of the
characteristics usually associated with the
"haploid syndrome."
Rana pipiens haploid embryonic develop-
ment is characteristically abnormal and delayed
as compared to control embryos. Development
proceeds normally until late blastula, at which
time the haploid embryos begin to show develop-
mental retardation. Haploids continue to develop
for eight days at which time the majority of the
embryos become edematous and die (6, 7).
Cytological studies demonstrated that the normal
sized cells of the control embryos contained a
diploid set of chromosomes and two nucleoli.
The smaller cells of the haploid embryos
contain one nucleolus and a haploid set of
chromosomes. These haploid cells, as expected
contain one-half as much DNA as diploid cells
(8).
It was possible to study the effect of quan-
titative changes in the gene complement of de-
veloping embryos on R-RNA synthesis by inves-
tigating the synthesis of R-RNA in haploid
embryos. Four- and six-day old ^^p.^^i^gled
control and haploid embryos were analyzed for
RNA, DNA and incorporation of 32p into R-RNA.
Developmental retardation, characteristic of
haploidy, necessitated still another type of
control. Haploid and normal embryos of the
same chronological age were not the same
developmental age since the haploids developed
at a slower rate. The additional control con-
sisted of five-day old normal embryos, which
closely approximated the same developmental
age as the six-day haploid embryos. Both
haploid and control embryos originated from
the same clutch of eggs. Tail tips of these
embryos were also examined cytologically to
determine the number of nucleoli per cell.
Quantitative determinations demonstrated
that considerable RNA and DNA synthesis oc-
curred in both haploid and control embryos
between four and six days of development (Table
II). The RNA increase was almost directly pro-
portional to the DNA increase in both haploid
and control embryos (Table II). Sucrose density
gradient analysis also indicated that R-RNA was
being synthesized in both haploids and controls
TABLE II
The values in this table arise from the experiment
illustrated in Fig. 2. An aliquot was taken from the
whole homogenate of each set of embryos and assayed
for RNA and DNA. All values are given on a per embryo
basis.
Stafie
^S RNA
Jig DNA
Ais RNA
>ig DNA
4-day Haploid
4.1
2.9
1.41
6-day Haploid
7.4
5.6
1.12
4-day Control
5.8
4.2
1.38
5-day Control
6.7
5.6
1.20
6-day Control
14.4
12. &
1.14
(Fig. 5). As expected, control embryos contained
more RNA and DNA than did haploid embryos
of the same chronological age (Table II). Haploid
embryos (6-day) contained nearly the same
amount of DNA and RNA as did control embryos
(5-day) of about the same developmental age
(Table II). Cytological examinations demon-
strated the presence of one normal sized
nucleolus per cell in haploid embryos while
the larger cells of the control embryos contained
two nucleoli.
The RNA/DNA ratios of both haploid and
diploid embryos were approximately the same
at all stages checked (Talbe II). This indicated
that a unit of DNA produced about the same
amount of R-RNA whether it resided in a
haploid cell or a diploid cell. Since the cells
of haploid embryos contained only one-half as
much DNA as the cells of diploid embryos, the
cells of haploid embryos produced only one-half
as much R-RNA as the cells of diploid embryos.
Haploid embryos were developmentally re-
tarded and it was expected that they would con-
tain less RNA and DNA than control embryos
of the same chronological age. It was, however,
surprising that haploid embryos contained ap-
proximately the same amount of RNA as control
embryos of the same developmental age. These
results implied that the amount of RNA syn-
thesized during development was a function of
the stage of development. Brown reached a
similar conclusion in studies on Xenopus haploid
embryos where the haploid embryos also con-
tained the same amount of RNA as control
embryos of a comparable developmental age (9).
Haploid and diploid embryos of the same
developmental age also contained about the same
amount of DNA. Haploid embryos, then, had
41
roughly twice the number of cells as did diploid
embryos at a comparable developmental stage.
It has been shown elsewhere that triploid cells
are 3/2 as large (10) and contain 3/2 as much
DNA as diploid cells. This implies that triploid
and diploid embryos of the same size contain the
same amount of DNA, since triploid embryos
contain two-thirds as many cells as diploid
embryos and each triploid cell has 3/2 as much
DNA as a diploid cell.
Indirect evidence suggests that the amount
of DNA necessary to reach any developmental
stage is a function of the volume of the egg
from which the embryo originated. Frog embryos
originating from small eggs, consisted of a
reduced number of normal sized cells as com-
pared to control embryos originating from
normal sized eggs (12). The smaller embryos
contained fewer cells and, therefore, probably
less DNA than did the larger embryos at the
same developmental stage. A reduction in the
amount of cytoplasm per embryo thus produced
a proportional decrease in the DNA content per
embryo as compared to normal sized controls.
These considerations suggest that during de-
velopment the extent of DNA synthesis is
regulated by the amount of cytoplasm present
in the embryo and that this regulation is re-
flected by the similar DNA/ cytoplasm ratios of
haploid, diploid and triploid embryos.
Since haploid and diploid embryos of the
same developmental stage contain about the same
amount of DNA, it is possible that the stage of
an embryo is dependent on the DNA content of
that embryo. That is, a certain quantity of DNA
(relative to the amount of cytoplasm present)
must be present in an embryo before the
embryo can attain a specific developmental
stage.
We have seen that the DNA content seems
to be controlled by the amount of cytoplasm
present in the egg and that R-RNA synthesis
is apparently stage dependent. With these ob-
servations in mind a hypothesis concerning the
gross regulation of R-RNA synthesis during
development follows. Specifically I would sug-
gest that during development the extent of DNA
synthesis is controlled by the amount of cyto-
plasm present in the embryo and that an inter-
action between the DNA and the cytoplasm
somehow regulates the synthesis of R-RNA.
It is now possible to design experiments to
directly test this hypothesis.
POLLARD: Have you tried any microinjec-
tions? You could just mash up an ordinary
embryo, one that won't arrest in two or three
days, separate out the enzyme part and inject
it into the mutant. This is based on the possi-
bility that a "transcriptase" for making ribos-
omal RNA is missing.
KOHNE: Usually when you inject anything
into these embryos, they arrest all by them-
selves. It's very difficult to put anything into an
egg because you get chromosomal abnormalities.
POLLARD: If these are already arrested,
you've got nothing to lose.
KOHNE: There is something that more or
less approximates what you're asking. I haven't
the vaguest idea what it means but Briggs at
Indiana has an axolotol mutant that he calls
the "00" or something similar. This mutant
even looks different during early development,
but it will develop into a gastrula and then
become arrested. However, if you take normal
egg cytoplasm and inject it into this mutant, it
develops beautifully.
POLLARD: Maybe that "loosens up" the
transcription.
GROSS: With this technique you get a gas-
trula arrest and a failure of the ribosomal
RNA synthesis to turn on? However, you're not
suggesting that it's the failure of ribosomal
RNA synthesis to turn on that is responsible
for the gastrula arrest, are you?
KOHNE: No, I think the evidence from the
anucleolate mutant says that ribosomal RNA
is not needed yet, at least until stage 21.
PAPACONSTANTINOU: Are you familiar
with the experiments that Stanley Cohen did a
few years ago in regard to this arrest? He
looked at the respiratory cycle intermediates
and found an accumulation of malonic acid in
these embryos. I don't know if anybody has
repeated them, but I know they are in the
literature. You may have a lesion in the res-
piratory function and, if this is the case, you
may be able to repeat this with your controls
by adding malonate.
KOHNE : There' s one other comment on this
that I'd like to make with respect to hybrids
of the Rana catesbeiana sperm x Rana pipiens
egg cross, which have a haploid set of Rana
chromosomes but arrest at gastrula. Haploids
which have the Rana pipiens chromosomes de-
velop almost normally until the swimming tad-
poles. Thus, the catesbeiana chromosomes are
doing something that is poisoning the system.
PAPACONSTANTINOU: Does it always
have to be the catesbeiana rasile and the pipiens
female? Can it be the other way around?
KOHNE: Yes, but they arrest, too. There
are a lot of hybrid embryos it would be inter-
esting to work with but the problem is getting
the material. Rana sylxxitica is one where you
42
get a hybrid arrest at gastrula. Nucleoli then
start forming and the embryo lives for several
days but remains at the gastrula stage. This
system would appear to be well suited for this
type of study. We could not, however, obtain
any of the frogs.
PAPACONSTANTINOU: Another question
relates to the volume in these embryos as
they approach gastrula. Is the total volume the
same?
KOHNE: Well, it may not be but if there
is a change it's so imperceptible that you don't
notice it.
PAPACONSTANTINOU: If you looked at
this with an electron microscope, you wouldn't
be able to detect a decrease in the ribosomal
population per cell as it develops from the egg
to the gastrula? You're not synthesizing ribos-
omes, but your cells are dividing. I don't know
how many cells there are in a gastrula state
but I was wondering whether the total volume
of the whole embryo remains the same.
(a) 4- DAY HAPLOID
(b) 4-OAY CONTROL -|I500
(e) 5- DAY CONTROL
1500
900
300
20 30
TUBE NUMBER
Fig. 5.
Sedimentation patterns of RNA extracted from whole •'^P-labeledhaploid and control em-
bryos: a) 20 four-day-old haploid embryos, b) 20 four-day-old control embryos, c) 20
six-day-old haploid embryos, d) 20 six-day-old control embryos, e) 20 five-day-old con-
trol embryos. Sibling embryos were used in this experiment.
43
KOHNE: In histological studies, from what
I've seen, the cells look about the same size.
KAHN: Does developmental arrest of the
catesbeiana-pipiens hybrids occur at the same
stage in reciprocal crosses?
KOHNE: No, it's different. An interesting
thing about hybrids in general is that they
react differently depending on which egg cyto-
plasm is used. Even though the same total
genome is present in the different cytoplasms
the end result may be quite different.
EPEL: Are the magnesium-deficient and
the anucleolate embryos the same?
KOHNE: No, but they are almost pheno-
copies. They act more or less the same way,
except the magnesium-deficients obviously do
synthesize ribosomal RNA.
POLLARD: Do you have any kind of hy-
pothesis? For example, can we say the following?
The idea is that one chromosome or one part
of a chromosome has the mechanism for tran-
scribing the ribosomal RNA. This has to come
off the DNA. The DNA you have is no good, it
won't work. It'll transcribe all right in one, but
it won't in the other, so it's stuck with the
wrong transcription. It keeps pumping this out
and this hooks up with the RNA and it doesn't
work. The stage in which you need this ribosomal
RNA could come quite a bit earlier than when
the cell is desperate for it.
KOHNE: Yes, I agree with you.
POLLARD: The concentrations may be
very critical. You may need just 8 or 10 ribos-
omes to get something started. It seems to me
you've got to have the organism tell you when
you're supplying a deficiency. I would start
with a nice arrested cell with everything in bad
shape and then start firing things into it. I
would use anything I could think of that was
remotely similar to transcriptase, anything
I could get off a DNA, and any kind of histone
material which was somehow associated with
DNA. I'd try to get hold of something that would
unlock this mechanism. What's the matter with
that idea?
KOHNE: Well, I'm overwhelmed.
GROSS: Dr. Pollard, why do you fire in
transcriptase?
POLLARD: Well, it seems to have every-
thing else.
GROSS: It's also got transcriptase.
POLLARD: Yes, but what it has won't work.
GROSS: I think I'd try naked ribosomes.
POLLARD: Would you use ribosomes made
on the DNA in the hybrid?
KOHNE: The haploid is perfectly capable
of making ribosomes; the only thing the haploid
doesn't have that this hybrid has is the other
set of chromosomes.
POLLARD: Maybe you've got the right
transcriptase and the wrong DNA.
KOHNE: Well, each of these things con-
tributes a nucleolus. I think that the cytoplasm
interaction with this cafes ftezana genome is what
stops development. However, what it is really
doing I don't know.
PAPACONSTANTINOU: If the nucleoli don't
appear when they should appear, right before
gastrulation, I don't understand why you think it
has to be cytoplasmic. Do you think something
in the cytoplasm is regulating the appearance
of the nucleolus?
KOHNE: Yes, let me talk about that in a
minute. This is all really pretty much specula-
tion. We don't have many facts to go on.
TS'O: Have any practical chemical tests
been done here?
KOHNE: I don't think anybody's ever done
any on catesbeiana. That's something I've al-
ways wondered about. What differences are
there qualitatively and quantitatively in the
DNA's of these frogs. Amphibians have a lot of
DNA in their cells compared to other things.
GROSS: Let us go back to some of your
other points. Is there a fixed quantitative rela-
tionship between cytoplasm, DNA and RNA?
Maybe there is a cytoplasmic repressor.
KOHNE: Possibly. I'm just saying that cy-
toplasm is somehow involved in control of
nucleic acid synthesis. These cells also divide
extremely rapidly during development.
POLLARD: You're saying that up to tetra-
ploid in your system, the relationship of cyto-
plasmic volume to RNA and DNA seems to be
very constant?
KOHNE: I was quoting other people's work.
In 1925 de Beer quoted experiments related to
cytoplasmic-nuclear ratio. He suggested that
what controls the development of embryos is
the cytoplasmic-nuclear ratio. He stated that
when you get to a certain stage the genome is
turned on and that what triggers it is the ratio
of the nuclear material to the cytoplasmic
material. He pinpointed that stage at gastrula,
the same stage at which we know now that
messenger RNA synthesis is turned on very
rapidly. I have changed the wording a little to
the parlance of molecular biology in saying that
the cytoplasm is controlling the extent of DNA
synthesis and that a DNA-cytoplasm interaction
controls RNA synthesis.
DEERING: Does this 6-day haploid have
twice as many cells as the 5-day control?
KOHNE: That's right.
44
DEERING: Are the cells smaller?
KOHNE: Yes, they are. Again this is a gen-
eralization because I can't say anything about
what happens to the cells of specific parts,
such as those destined to be the liver, brain
or epidermis.
GRUN: There is one thing I was wondering
about. This is the comparison between haploids
and diploids. I wonder, has any attempt been
made to produce inbred lines of frogs? What
I'm concerned with is the question of whether
this is a straight line comparison of the haploid
state as compared with the diploid state or
whether you're saying that the condition is a
genetic effect of exposed recessives that, of
course, would be effective in the haploid state
and cause abnormal development.
KOHNE: Well, as I mentioned, if you start
with a small enough egg, you will get normal
development, so the phenomenon is probably
not caused by the expression of recessives.
KAHN: Have you considered the possible
role of cytoplasmic DNA?
KOHNE: There hasn't been any evidence
as yet that cytoplasmic DNA is in any way active.
Igor Dawid at Carnegie Institution has isolated
a substance that has the characteristics of non-
nuclear DNA, and he thinks it may come from
mitochondria. Whether it's active in the differ-
entiation process I don't know.
KAHN: Isn't it true that DNA synthesis does
not begin until the beginning of gastrulation?
KOHNE: No, it begins immediately. Many
people have thought that the cytoplasmic DNA
might be contributing to the genome, but it's
never been proven. There are several systems
now in which immediate DNA synthesis has
been shown.
GROSS: It doesn't make any difference
quantitatively - the new DNA in the amphibian
doesn't begin to make an impact on the total
DNA per egg for some time.
KOHNE: There is supposed to be about 1000
times more cytoplasmic DNA than nuclear DNA
in Rana eggs and 300 times in sea urchin.
GROSS: At any rate, there is a lot of it
around, and even if each genome is fully repli-
cated from the pool, the impact on the total
DNA will be small until you get about a thousand
cells or so. His ratios are all taken at stages
where, presumably, the cytoplasmic DNA has
been used up.
KAHN: This raises other questions. How
is the cytoplasmic DNA utilized? What is the
function of mitochondrial DNA?
KOHNE: There are two sources of diphen-
ylamine-reacting material (DNA like). One of
them is the mitochondrial DNA, The other one
is an acid soluble fraction and is probably
just nucleotides. There is about 10 times as
much of the latter as there is of the DNA poly-
mer.
GROSS: It may turn out that the thing people
have been overlooking systematically is the
enormous ratio of cytoplasm to nucleus in the
egg. Since the sizes of mitochondria don't
differ greatly between embryonic and somatic
cells, this may mean that in the egg you have
thousands of times as many mitochondria per
nucleus as you have in the somatic cell. And if
all mitochondria do have DNA, then in the egg
the mitochondrial DNAmight make a tremendous
impact on the total, whereas in a somatic cell
it wouldn't.
KOHNE: There is some initial circum-
stantial evidence, in studies on centrifuged
ascidian eggs, that you get two fractions: one
of them with mitochondria and the other without.
The part with mitochondria will develop and the
part without won't. For another type of ascidian
with a light mitochondrial fraction and a heavy
mitochondiral fraction, you get a partitioning
of mitochondria in each fraction and both will
develop: one of them being haploid and the
other being diploid. However, without the mito-
chondria these things don't develop.
45
References
1. D. Kohne. Exptl. Cell Res. 38, 211 (1965).
2. T. J. King and R. Briggs. J. Exptl. Zool.
123. 61 (1963).
3. D. D. Brown and J. B. Gurdon. Proc. Natl.
Acad. Set. U.S. 51, 139 (1964).
4. D. D. Brown and D. Gaston. Devel. Biol. 5,
412 (1962).
5. M. Girard, S. Penman and J. E. Darnell.
Proc. Natl. Acad. Set. U.S. 51, 205 (1964).
6. K. R. Porter. Biol. Bull. 77, 233 (1939).
7. R. Briggs, E. Green and T. J. King. J. Exptl .
Zool. 116, 455 (1951).
8. B. C. Moore. J. Morphol. 101, 227 (1957).
9. D. D. Brown. Annual Report of the Director
of the Department of Embryology, J. D.
Ebert, ed., Reprinted from "Carnegie In-
stitution of Washington Year Book 63,"
p. 503.
10. R. Briggs. J. Exptl. Zool. 106, 237 (1947).
11. R. G. McKinnel and K. Bachmann. Exptl.
Cell Res. 39, 625 (1965).
12. R. Briggs. J. Exptl. Zool. Ill, 255 (1949).
46
MOLECULAR ASPECTS OF LENS CELL DIFFERENTIATION
John Papaconstantinou^
Department of Zoology, The Institute of Cellular Biology,
The University of Connecticut, Storrs, Connecticut
I. Introduction
We spent the first session of this workshop
discussing some of the molecular aspects of
early embryonic differentiation. Through these
discussions it has become obvious that one of
the major problems confronting the investigators
studying the mechanisms of cellular differen-
tiation is how developing cells acquire specific
biochemical characteristics and how these are
linked to morphological development and cellular
function. It is now well documented that as cells
progress through specific stages of differentia-
tion new biochemical traits can be acquired and
some existing traits can be lost. Thus, during
differentiation there occurs a progressive cell-
ular diversification which is characterized mor-
phologically by cellular structure and biochem-
ically by the synthesis of specific structural
proteins and enzymes. The ultimate form of
morphological and biochemical specialization
may be seen in the muscle cell, erythrocyte,
lens cell, etc., which synthesize tissue specific
proteins in the form of myosin, hemoglobin and
crystallins, respectively. This ability of cells
to lose and acquire specific biochemical charac-
teristics during differentiation is attributed to
differential gene action. The mechanisms by
which vertebrate cells can regulate genetic
expression are not known; however, it is these
mechanisms which are believed to be funda-
mental to the regulation of morphogenesis. One
of the approaches to the study of these mecha-
nisms is through studies on the regulation of
synthesis of tissue specific proteins as cells
become more highly differentiated. This after-
noon I would like to start the session by describ-
ing a system in which the regulation of synthesis
of specific proteins is associated with a specific
stage of cellular differentiation, i.e., the dif-
ferentiation of the lens epithelial cell to the
fiber cell. In addition, I would like to describe a
series of changes in the nucleic acids (RNA and
DNA), also associated with fiber cell formation
and possibly associated with the regulation of
protein synthesis. Our studies have been cen-
tered, therefore, on the occurrence of protein
and nucleic acid changes associated with a
specific stage of lens cell differentiation. Before
proceeding to discuss our biochemical data I
would like to go over the morphological changes
which occur in these cells and then associate
these changes with the biochemical events.
n. Morphological Changes in Fiber Cell
Differentiation
A. Structure of the lens
The lens is an avascular tissue composed
of the following distinct cell types: (a) an outer
single layer of epithelial cells; (b) a zone of
elongation, composed of cells which are in the
process of developing into fiber cells; and (c)
the inner fiber cells (Fig. 1). Initiation of the
differentiation of epithelial cells to fiber cells
occurs at the peripheral or equatorial zone of
the lens. It is in this region where the gross
morphological changes associated with fiber
cell differentiation occur, i.e., the transition
from a cuboidal lens epithelial cell to the
elongated fiber cell. After the embryonic lens
has been formed, fiber cells are continuously
laid down throughout the pre-natal and post-
natal life of the animal. The bulk of the lens
is composed of layer upon layer of these fiber
cells, and this continuous formation of fiber
cells accounts for the growth of this tissue.
It can be seen, therefore, that (a) secondary
^ present address: Biology Division, Oak Ridge National
Laboratory, Oak Ridge, Tennessee.
47
epithelial cells
cortex fiber eel Is
region of active
cellular replicotion
zone of
cellular
elongation
Fig. 1.
A diagramatlc presentation of the adult vertebrate lens.
The lens is surrounded by an external non-cellular
capsule. Beneath the capsule are found the lens epithelial
cells. The zone of cellular elongation Is found in the
peripheral area. This is the region of transition where
the epithelial cells begin to elongate into fiber cells. The
fiber cells that are newly laid down represent the cortex
region; the fiber cells laid down during the early growth
period of the lens compose the nucleus region of the
adult lens. (Fig. 1, J. Papaconstantinou, Science, in press;
Copyright 1966 by the American Association for the
Advancement of Science.)
fiber cell formation represents the final stage
of lens cell differentiation and (b) in the adult
lens the fiber cells formed during embryonic
growth compose the central or nucleus region
while the newly formed fiber cells are found
in the peripheral or cortex region.
B. Cytological and cytochemical
observations on the process of
fiber cell formation
The lens epithelial cells are characterized
by their cuboidal shape, their basophilic stain-
ing properties and their ability to replicate (I).
In the zone of elongation (Fig. 2), where the
epithelial cells begin the process of fiber cell
formation the following changes occur in the
intracellular structures: (a) the cell sends out
cytoplasmic processes anteriorly and poste-
riorly beneath the cuboidal epithelial cell layer
to form the fiber cell; (b) the nucleus and nu-
cleoli enlarge (2); (c) the ribosomal population
increases significantly, especially in the cyto-
plasm adjacent to the enlarged nucleus (3, 4).
In the completed fiber cell, (a) the cytoplasm
loses its basophilic properties and takes on
acidophilic properties; (b) the nucleus and
nucleoli reduce in size and the endoplasmic
reticulum, which has a granular appearance in
the epithelial cell, takes on a smoother appear-
ance in the fiber cell; (c) through electron
microscope studies it has been shown that a
significant decrease in the ribosomal population
occurs in the differentiated fiber cell (3, 4).
These differences in staining properties and
changes in intracellular structures indicate
that significant macromolecular changes are
associated with fiber cell differentiation. The
enlargement of the nucleus and nucleoli, for
example, as well as the increase in ribosomal
population are an indication of increased nucleic
acid and protein synthesis during elongation.
Keeping these structural changes in mind, I
would like to describe a series of biochemical
events which are associated with fiber cell
formation, and which may be closely linked with
the cytological observations just described.
III. The Biochemistry of Lens Fiber Cell
Differentiation
A. The association of r-crystallin
synthesis with fiber cell differentiation:
gene activation
I would like to begin this section of my
discussion by describing our observations on
the appearance of a group of lens proteins, the
r-crystallins, during the differentiation of the
lens epithelial cell to a fiber cell (5, 6). This
presents us with an example of the activation
of the synthesis of a specific protein simul-
taneously with the initiation of the morphological
changes associated with the differentiation of a
fiber cell. There are three major groups of
proteins synthesized by lens cells; the a-crystal-
lins, ^-crystallins and y-crystallins. The crys-
tallins were first classified according to their
mobility at alkaline pH; the fastest migrating
group being the a-crystallins, the intermediate
group being the p-crystallins and the slowest
migrating group being the r-crystallins (7, 8).
More recently, through the efforts of my col-
leagues and myself, these structural proteins
have been identified according to their elution
properties on DEAE-cellulose columns (5, 9).
It was essentially through the resolving power
of DEAE-cellulose that the qualitative and quan-
titative differences in the crystallins of the
different lens cells were detected. Typical
patterns showing the stepwise elution of a-, ^-
48
epithelial cell
elongating
cells
cortex fiber
cell region
morphological
characteristics
basophilic
rough endoplasmic
reticulum
cells replicate
biochemical
characteristics
a,P-crystallin synthesis
mhibited by actinomycin
oxidotive metabolism is
efficient
coif; LDH-5>LDH-|
adult: LDH-I > LDH-5
cell volume increases initiation of |f-crystallin
nuclei enlarge synthesis
nucleoli enlarge o,3, j(-crystallin synthesis
increase in ribosomal inhibited by actinomycin
population tronsition from LDH-5 to
cells no longer lDH-| enhanced
replicate
acidophilic
smooth endoplasmic
reticulum
ribosomes break down
m-RNA for crystallins is
stabilized
nuclei decrease in size DNA is metabolically Inactive
nucleoli decrease in actinomycin stimulates
sue crystal! in synthesis
LDH-I > LDH-5
active aerobic glycolysis
Fig. 2.
A dlagramatic presentation of the region of cellular elongation in the vertebrate lens.
The major morphological and biochemical characteristics associated with lens cell dif-
ferentiation are listed and are discussed in detail in the text. (Fig. 2, J. Papaconstantinou,
Science, in press; copyright 1966 by the American Association for the Advancement of
Science.)
and y-crystallins from DEAE columns are
shown in Figs. 3B and 3C. The protein fractions
from the cortex fiber cells (Fig. 3B) and from
the nucleus fiber cells (Fig. 3C) of the adult
lens were precipitated and further character-
ized by free boundary electrophoresis. Their
electrophoretic mobilities are listed in Table I.
The mobility of these fractions was used as a
means of identification of the protein fractions
eluted from the column.
At the time that these studies were initiated
I was impressed by the mechanism of lens
growth, especially, by the existence of many
layers of fiber cells which are systematically
laid down throughout the life of the animal.
Theoretically, therefore, by peeling away the
layers of fiber cells in an adult lens it should
be possible to recover the cells formed at
various ages. Actually, the fiber cells can be
peeled off when the decapsulated lenses are
placed in a buffered solution. The outer cortex
fiber cells, for example, continue to peel off
until the central, nucleus region is reached.
The freed fiber cells can be separated from the
nucleus fiber cells by decanting, and using this
procedure for separating the fiber cells from
the different lens regions, one could look for
any chemical differences between cells that were
laid down throughout the growth periods of the
lens. Since the epithelial cells and elongating
cells from the equatorial zone could be removed
along with the lens capsule, we were now pro-
vided with a method for separating the lens
cells into three groups: (a) the epithelial
cells, (b) the newly formed cortex fiber cells
and (c) the fiber cells of the nucleus region
which had been laid down during the early life
of the animal. These cells were homogenized
in 0.005 M sodium phosphate buffer pH 7.0 and
fractionated on DEAE-cellulose columns. Char-
acteristic elution patterns for each of the regions
were obtained as is shown in Figs. 3A, 3B and
3C. These are not pure fractions as can be seen
from the electrophoresis data (Table I), but this
49
TABLE I
The Electrophoretic Mobility x 10^ (cm^ volts"! sec.'^ ) of the lens a-,p- and y-CrystalUnsfrom Adult
Bovine Lens Cortex and nucleus fibers. The crystallinswere fractionated on DEAE-cellulose columns. The
peaks were precipitated and analyzed by free boundary electrophoresis.
y-crystallins
)S-crystallins
a-, p-
crystallins
a -crystalllns
DEAE fraction
aj + as
b
c
d
e
f
g
h
Cortex
fiber
Fast
component
2.56
3.08
3.48
3.96
4.76
5.23
5.61
6.12
cell
proteins
Slow
component
1.93
2.38
2.41
3.08
3.54
~
~
~
Nucleus
fiber
Fast
component
2.22
~
3.13
3.98
4.30
5.04
5.10
5.22
ceU
proteins
Slow
component
1.72
~
2.93
2.94
3.37
—
~
—
method is quite good for separating the proteins
into the a-, ^- and y-crystallin groups. The
r-crystallins, which are the proteins we are
interested in for this discussion, are eluted
cleanly from the column as peaks a 1,02 and b
in the cortex fiber pattern (Fig. 3B) and as
peak a in the nucleus fiber pattern (Fig. 3C).
POLLARD: What' s the separation process?
Is it on a column?
PAPACONSTANTINOU: This is a DEAE-
cellulose column, using a stepwise elution
system starting with 0.005 M phosphate buffer
pH 7.0 and going to 0.02 M phosphate buffer
pH 5.7. After this, further elution is achieved
by increasing the ionic strength with NaCl.
We have done linear gradients on this more
recently and they are essentially the same.
We've used two linear gradients: the first is a
sodium phosphate gradient ranging from 0.005
M phosphate pH 7.0 to 0.02 M phosphate pH 5.7.
With this, the y- and ^-crystallins are eluted
from the column. Then the phosphate concen-
tration is kept constant at 0.02 M pH 5.7 and a
NaCl gradient is initiated. This results in the
elution of the a-crystallins.
A comparison of these elution diagrams
shows that the epithelial cells (Fig. 3 A) contain
only traces of y-crystallins in comparison to the
amounts found in adult cortex (Fig. 3B) and
adult nucleus (Fig. 3C) fiber cells. Furthermore,
it can also be seen that the y-crystallins of the
adult cortex and adult nucleus fiber cells are
both qualitatively and quantitatively different
with respect to their chromatographic properties
on DEAE-cellulose columns. These observations
indicate, firstly, that the y-crystallins are pro-
teins which are characteristic of the fiber cell
and secondly, that y-crystallins formed in fiber
cells of young animals (cells found in the nucleus
region of the adult lens) are chromatographically ,
and possibly chemically, distinct from
y-crystallins synthesized in fiber cells of older
animals (cells found in the cortex region of the
adult lens).
If the first proposal is correct, i.e., that
y-crystallins are proteins specific to the fiber
cells, then epithelial cells from animals of all
ages should lack these proteins. The elution
pattern of proteins from epithelial cells of 3
month calf lenses (Fig. 4A) indicate that this
is indeed the case. Although traces of y-crystal-
lins are detected by this procedure, the amount
detected is significantly less than that detected
in the fiber cells (Figs. 4B, 4C). In addition, the
traces of y-crystallins that are detected in the
epithelial cells are due to the adherence of the
elongating cells to the lens capsule. It is, we
believe, in these elongating fiber cells, where
the activation of y-crystallin synthesis occurs.
Thus, when we compare the elution patterns of
proteins extracted from epithelial cells, cortex
fiber cells and nucleus fiber cells of adult and
calf lenses, we see that (a) at both ages the
epithelial cells do not contain y-crystallins and
conclude that y-crystallin synthesis is initiated
during fiber cell formation in young and adult
lenses. Similarly, it has been reported that
y-crystallin synthesis is associated with fiber
50
60 120 180 240 300 360
08
(
C
06
-
'
04
-
02
I A
1 1
d
U
h
60 120 180 240 300 360
ml effluent
Fig. 3.
(A) Fractionation of soluble proteins from adult bovine lens
epithelial cells. The cells were homogenized in 0.005 HI
phosphate buffer pH 7.0 and the homogenate was cleared
by centrifuging at 10,000 x g for lOmin. The supernatant
was dialyzed against 0.005 \! phosphate buffer overnight.
74.0 mg of protein were added to 10 g of DEAE-cellu-
lose; 60.29 mg protein were recovered at the end of the
experiment. Buffers were added to the column in the
following sequence: I. 50 ml 0.005 -1/ sodium-phosphate
pH 7; II. 50 ml 0.0075 M sodium-phosphate pH 6.5; III. 50
ml 0.01 M sodium-phosphate pH 6; IV. 75 ml 0.02 M so-
dium-phosphate pH 5.7; V. 50 ml 0.02 M sodium-phos-
phate pH 5.7+ 0.1 M NaCl; VI. 50 ml 0.1 ,V sodium-
phosphate pH 5.7 + 0.1 ,M NaCl; VII. 50 ml 0.1 M
sodium-phosphate pH 5.7 + 0.3 A/ NaCl. The fractions
were collected in 3 ml allquots. (B) Fractionation of
soluble proteins from cortex fibers of the adult bovine
lens. The elution sequence is the same as that shown above.
74.0 mg protein were placed on 10 g of DEAE-cellulose;
65.39 mg protein were recovered at the end of the experi-
ment. (C) Fractionation of soluble proteins from nucleus
fibers of adult bovine lens. The elution sequence is the
same as that shown above. 73.92 mg protein were placed
on 10 g of DEAE-cellulose; 43.77 mg protein were re-
covered at the end of the experiment. (Fig. 1, J. Papacon-
stantlnou, Biochim. Biophys. AcialOT, 81, 1965; reproduced
with permission of Elsevier Publishing Company.)
10-
08-
06-
04-
02
\k±^
i-t—r^
60 120 ISO 210 300 360
60 120 180 240 300 360
60 120 ISO 240 300 360
ml effluent
Fig. 4.
<A) Fractionation of soluble proteins from epithelial cells
of calf lenses. 48.24 mg protein were placed on 10 g of
DEAE-cellulose; 41.63 mg protein were recovered at the
end of the experiment. Buffers were added to the column
in the sequence described in Fig. 3. (B) Fractionation of
soluble proteins from calf cortex fibers. 49.80 mg pro-
tein were placed on 10 g DEAE-cellulose; 51.40 mg pro-
tein were recovered at the end of the experiment. The
elution sequence is the same as that described above.
(C) Fractionation of soluble proteins from calf nucleus
fibers. 50.23 mg protein were placed on 10 g of DEAE-
cellulose; 49.90 mg were recovered at the end of the
experiment. The elution sequence is the same as that
described above. (Fig. 2, J. Papaconstantinou, Biochim.
Biophys. Ada 107, 81, 1965; reproduced with permission
of Elsevier Publishing Company.)
51
cell formation in the regenerating salamander
lens (10).
In my second proposal, I stated that the
variation in DEAE column properties between
adult cortex and adult nucleus r-crystallins
imply distinct differences exist between the
y-crystallins of the nucleus region and cortex
region of the adult lens. Since the adult nucleus
fibers are cells which were formed during the
earlier period of lens growth, these regional
differences in r-crystallins of the adult lens
(Figs. 3B and 3C) may be due to amino acid
differences in the r-crystallins formed by fiber
cell differentiation at differentiation at different
ages. Thus, the y-crystallins from lenses of
younger animals (embryos and young calves)
should have the same chromatographic prop-
erties as y-crystallins from the nucleus fibers
of an adult lens. Evidence for this is presented
by the elution patterns for proteins from calf
cortex (Fig. 4B), calf nucleus (Fig. 4C) and
embryonic lenses (Fig. 5), which show that the
y-crystallins in the fiber cells of these younger
lenses are chromatographically similar to the
40
30
-a
20
-
e
A
10
-
d
i
L av-
-^\
g
h
o
Cl
e
200 400 600 800 1000
ml effluent
Fig. 5.
Fractionation of the soluble proteins from the combined
lenses of U5 day and 130 day embryos, 426.24 mg pro-
tein in a volume of 7.4 ml were added to a DEAE-cellu-
lose column (2 cm x 10 cm). 302.9 mg protein were
recovered at the end of the experiment. The elution se-
quence and volume of buffers used are as follows:
I. 100 ml 0.05 M sodium-phosphate pH 7; II. 200 ml
0.0075 M sodium-phosphate pH 6.5; III. 150 ml 0.01 M
sodium-phosphate pH 6; IV. 200 ml 0.02 M sodium-
phosphate pH 5.7; v. 500 ml 0.02 M sodium-phosphate
pH 5.7 + 0.1 M NaCl; VI. 150 ml 0.1 M sodium-phos-
phate pH 5.7 + 0.1 NaCl; VII. 150 ml 0.1 M sodium-
phosphate pH 5.7 + 0.3 NaCl. The fractions were col-
lected in 10 ml aUquots. (Fig. 3, J. Papaconstantinou,
Biochim. Biophys. Acta 107,81, 1965; reproduced with per-
mission of Elsevier Publishing Company.)
y-crystallins of the adult nucleus fibers
(Fig. 3C). Furthermore, it can be seen that only
in the elution pattern of the calf cortex fibers
(Fig. 4B), where the predominating y-crystallins
are of the "embryonic type", are there indica-
tions of the appearance of the types of y-crys-
tallins observed in the adult cortex fiber cells,
i.e., peaks a 2 and b (Fig. 3B). The patterns for
calf nucleus fiber cell proteins and embryonic
lens proteins show a complete absence of adult
cortex fiber type y-crystallins.
In view of the differences in chromato-
graphic properties of the y-crystallins attempts
were made to obtain further evidence for more
distinct differences between the embryonic and
adult y-crystallins. Further purification of adult
cortex, adult nucleus and embryonic y-crystal-
lins was achieved by DEAE-cellulose fractiona-
tion using tris buffer ranging in pH from 10 to
7 (Fig. 6). In each case the y-crystallins were
resolved into 4 major proteins. Our observations
are similar to those of Bjork (11) who, through
the use of alternative procedures of fractiona-
tion, was able to resolve the y-crystallins into 4
distinct fractions. The purified y-crystallins
from each of these fractions were concentrated
and their relative mobilities were determined
by paper electrophoresis. The electrophoretic
patterns (Figs. 7, 8) show that the y-crystallins
from embryonic and adult nucleus fibers have
the same mobility, whereas the y-crystallins of
the adult cortex have a different mobility.
These data are in agreement with the prelimi-
nary observations on DEAE-columns.
From these observations it can be con-
cluded that (a) y-crystallin synthesis is initiated
during fiber cell formation and is associated
with this specific stage of lens cell differentia-
tion and (b) that the y-crystallins synthesized
during embryonic and early post-natal fiber
cell differentiation are electrophoretically dis-
tinct from those synthesized in the fibers of the
adult lens. Thus, the type of y-crystallin syn-
thesized depends on the age of the animal or
possibly the rate at which fiber cells are laid
down (5, 6).
With respect to the initiation of y-crystallin
synthesis we have an example of gene activation
at the molecular level, and one question which
concerns us now is whether the activation of
y-crystallin synthesis is intimately associated
with the genetic regulation of the morphological
changes in the cell. Furthermore, since fiber
cell formation involves the transition of a
replicative cell to a non-replicative cell, is
y-crystallin in any way associated with this
aspect of lens cell differentiation? We are
52
02
0.1 h
- A
_L
02r B
200 400 600 800
83 84
E
200 400 600 800
200 400 600 800
ml effluent
Fig. 6.
The fractionation of y-crystallins from adult cortex, adult
nucleus and embryonic lenses. The proteins, which had
been stored as an ammonium sulfate precipitate were
spun down and dissolved in 0.01 A/ Tris, pH 10. Ammon-
ium sulfate was eliminated by dialysis against 0.01 M
Tris. The protein solution was placed on a DEAE-
cellulose column and was eluted from the column by the
stepwise addition of Tris-HCl buffers in the following
order: I. 150 ml 0.01 M Tris, pH 10; II. 150 ml 0.02 /W
Tris, pH 9; III. 150 ml 0.04 M Tris, pH 8.6; IV. 150 ml
0.06 M Trls-HCl, pH 8.2; V. 150 ml 0.08 M Trls-HCl,
pH 7.6; VI. 150 ml 0.1 M Tris-HCl, pH 7.2. In each
fractionation the column size was 17 mm diameter x 20
cm height; 5 ml aUquots were collected. (Fig. 4, J.
Papaconstantinou, Biochim. Biophys. Ada 107, 81, 1965; re-
produced by permission of Elsevier Publishing Company.)
presently involved in experiments designed
to determine whether epithelial cells in vivo and
in tissue culture can be induced to synthesize
r-crystallins and at the same time retain both
their epithelial cell structure and their ability
to replicate.
B. The loss of LDH-5 isozyme synthesis
in lens cell differentiation: gene
repression.
I would like to present you with another
example of differential gene action associated
with fiber cell differentiation as well as with
the aging of the epithelial cells, i.e., the specific
repression of one of the lactate dehydrogenase
isozymes. These experiments were carried out
in collaboration with Mr. James A. Stewart (12,
13).
Lactate dehydrogenase isozymes have been
shown to occur in many vertebrate tissues in
5 electrophoretically distinct forms, and to vary
in activity during embryonic and post-embryonic
development (14-19). In addition, it is now well
established that all 5 isozymes are composed
of 4 protein subunits and that only the extreme
cathodal (LDH-5) and anodal (LDH-1) forms of
these enzymes are homogeneous with respect to
their subunits. Furthermore the subunits of
LDH-1 do not have the same amino acid com-
position as the subunits of LDH-5. Thus, by
dissociation and reassociation of the subunits in
a mixture of LDHs-1 and 5, all 5 isozymes can
be formed (20). These experiments show that
LDH's-2, 3 and 4 are composed of combinations
of LDH-1 and 5 subunits. Since there is now
good evidence that the synthesis of LDH-1 and
LDH-5 subunits is genetically regulated (21),
we felt that any alterations in the isozymic
patterns during fiber cell differentiation would
be another indication of the differential regula-
tion of protein synthesis which in the lens could
be localized to a very specific stage of cellular
differentiation, namely, the differentiation of an
epithelial cell to a fiber cell.
Our electrophoretic analyses of the lactate
dehydrogenase isozymes show that the epithelial
cells of the adult lens and calf lens have five
forms of the enzyme. Typical isozymic patterns
of calf and adult epithelial and fiber cells are
diagrammatically presented in Fig. 9. Concen-
trating on the epithelial cell diagrams alone,
a comparison of the patterns from calf and adult
cells shows that a change occurs from pre-
dominantly cathodal forms to predominantly
anodal forms. These data show that there is
a transition of epithelial cell LDH isozyme ac-
tivity during the post-natal aging of these cells.
This is a very interesting change since the
epithelial cells of the lens, during embryonic,
early post-natal and adult life, carry out the
same functions, i.e., to either replicate for the
formation of more epithelial cells or to differen-
53
•••••••"^
■• ♦ ♦*
bcl «c2 «c3 ttc4 »nl «n2 bn3
ADULT CORTEX
ADULT NUCLEUS
Fig. 7.
Electrophoretic analysis of the adult cortex and nucleus y -crystalllns. Electrophoresis
was carried out in 0.5 M Trls-0.021 M EDTA-0.075 W boric acid, pH 8.9, at constant volt-
age (5.8 V/cm) for 17 hrs. The y -crystalllns tested were prepared by DEAE-fracdona-
tion, precipitated in ammonium sulfate and redissolved in 0.05 M Tris. (Fig. 5, J.
Papaconstantinou, Biochim. Biophys. Acta 107, 81, 1965; reproduced with permission of
Elsevier Publishing Company.)
tiate into fiber cells. The only functional varia-
tion between the epithelial cells of young and
adult lenses is their mitotic activity, which is
greater during the early periods of lens growth
(22-25). As the lens reaches its maximum size
in the adult, mitotic activity decreases. It is
during this decrease in mitotic activity that the
major transformation from LDH-5 to LDH-1
occurs. On this basis it might be proposed
that this isozymic transition is associated with
a slowing down of mitotic activity as well as
other metabolic functions. An example which
might be considered similar to the aging of the
lens epithelial cells has been reported by Dawson,
etal., (18). They have found that more LDH-1
and less LDH-5 are found in samples of human
muscle obtained from elderly people and that the
highest concentration of LDH-5 subunits is found
in muscle from healthy adult males.
Now 1 would like to consider our observa-
tions on the LDH isozyme patterns during the
differentiation of epithelial cells to fiber cells.
In addition to the changes in the epithelial cells
alone we found that in both the calf and adult
lens pronounced changes occur during the dif-
ferentiation of the fiber cell. This is the final
stage of lens cell differentiation and results in
the transition from a replicative cell to a non-
replicative cell. During this stage of differen-
tiation LDH-1 persists. In the calf, the fiber
cells contain 5 detectable LDH's in which LDH-1
is predominant. In the adult, the fiber cells
contain essentially just LDH-1 although small
amounts of LDH-2 are detectable. On the basis
that the synthesis of subunits of LDH's-1 and 5
are genetically regulated, the complete loss of
LDH's- 3, 4 and 5 in the adult lens fiber cell and
the intermediate trends toward this loss in the
calf cells might be attributed to the suppression
of LDH- 5 subunit synthesis which would decrease
54
»#• »t2 »t3 M «nl «n2 Wn3
EMBRYO
ADULT NUCLEUS
tfti »t2 b«3 bt4 ttel bc2 be^ be^
EMBRYO ADULT CORTEX
Fig. 8.
Electrophoretic analysis of y -crystallins from adult and embryonic bovine lenses. The
conditions of electrophoresis are the same as described in Fig. 7. (Fig. 6, J. Papacon-
stantinou, Biochim. Biophys. Acta 107, 81, 1965; reproduced with permission of Elsevier
Publishing Company.)
their availability for recombination with LDH- 1
subunits.
We interpret these data to indicate that
during the aging of lens epithelial cells (calf
to adult) there is a regulation of LDH subunit
synthesis such that there is a greater decrease
in the synthesis of LDH-5 subunits than LDH-1
subunits. Furthermore, this tendency for the
persistence of LDH-1 becomes more pronounced
during the differentiation of the epithelial cell
to the fiber cell in both the calf and adult lens.
The extreme case is seen in the adult cortex
fiber cells where LDH-1 is essentially the only
one of the 5 isozymes remaining. Finally, we
would like to correlate this with the replicative
ability of the cell. When these cells, which
retain their ability to replicate, reach a phase
analogous to the stationary phase of a logarith-
mic growth cycle the synthesis of LDH-1
subunits is favored. This is also the case when
the cells reach a stage of differentiation in
which they have lost their replicative capacity
(the fiber cells).
C. LDH isozymes and lens
carbohydrate metabolism
I would like to digress from the main theme
of my talk for one moment to correlate the LDH
isozyme data just presented with the metabolic
activity of the lens epithelial cells and fiber
cells. A possible role of the regulation of
carbohydrate metabolism in skeletal and heart
muscle has been attributed to the persistence of
different LDH isozymes in these tissues. It has
been observed that LDH-1 is the predominant
isozyme in tissues exhibiting high rates of
oxidative metabolism, such as embryonic and
55
Calf
epith cortex
ORGIN
+
1
2
3
4
™
5
^™
Adult
epith cortex
ORGIN
2
3
4
— >■
5
Fig. 9.
A diagramatic presentation of the LDH isozyme patterns
for calf and adult lens epithelial cells and fiber cells.
(Fig. 7, J. Papaconstantinou, Science, in press; copyright
1966 by the American Association for the Advancement
of Science.)
adult heart tissue of mouse and chicken, while
LDH-5 is the predominant form in tissues that
can function under conditions of oxygen debt,
such as adult skeletal muscle. Furthermore, it
has been shown that LDH-1 is more sensitive to
inhibition by high pyruvate concentrations than
LDH-5 (14, 15, 17). On the basis of these ob-
servations it has been postulated that in highly
oxidative tissues such as the heart, the level of
lactic acid is regulated, i.e., kept at a low level,
because of the sensitivity of LDH-1 to pyruvate.
This hypothesis is further borne out by the fact
that skeletal muscle, which is capable of tolerat-
ing a greater variation in oxygen tension than
heart muscle, contains more active LDH-5, the
isozyme which shows less sensitivity to sub-
strate inhibition.
Let us now consider the metabolic proper-
ties of the lens cells. Wanko and Gavin (25, 26)
reported that the epithelial cells have relatively
more mitochondria than the fiber cells and that
the population of epithelial cell mitochondria is
significantly decreased after fiber cells are
formed. Thus, metabolically the epithelial and
fiber cell differs significantly in that the former
cell type exhibits a greater degree of aerobic,
oxidative metabolic pathways. Epithelial cells
have been shown to have higher levels of cyto-
chrome c, greater succinate dehydrogenase
activity, and more active mitochondria (27).
Fiber cells, on the other hand, have been shown
to have a greater degree of aerobic glycolysis
(28). Furthermore, it has been shown that the
most efficient production of ATP from ADP
in calf cortex fibers occurs with fructose-1,
6-diphosphate as substrate (29). Krebs cycle
enzymes are detectable in fiber cells, but their
activity is significantly less than that found in
the epithelial cells. All of these observations
indicate that a major metabolic difference be-
tween epithelial and fiber cells is in their
respiratory and glycolytic activity.
Taking the metabolic properties of lens
cells into account it would appear from the
work on heart and skeletal muscle LDH that
the lens fiber cells should retain LDH-5. Our
data have shown the opposite, i.e., that the
fiber cells retain LDH-1. In addition, even
though LDH-1 is retained, high lactic acid
levels are maintained by these cells.
Several factors such as oxygen tension,
intracellular pools of metabolic intermediates
and cofactors, and predominating pathways of
carbohydrate metabolism have been postulated
to play an important role in the type of LDH
isozymes retained by a specific tissue (30, 31).
Recent work on the LDH isozymes in cultured
chick heart muscle cells has shown that after
6 days in culture LDH-5 is the predominant
form (30). Prior to being placed in culture these
cells have predominantly LDH-1. In fact, chick
heart cells have been shown to retain LDH-1
throughout embryonic and post-embryonic life.
Thus, under tissue culture conditions a new
phase of LDH isozyme distribution, not previ-
ously experienced by these cells, is developed.
This predominance of LDH-5 was significantly
slowed down when placed under conditions of
high oxygen tension or when Krebs cycle inter-
mediates are added to the culture medium.
56
These observations cannot, however, explain
the persistence of LDH-1 in the lens fiber cells,
since the oxygen tension in the lens is lower
than that in the blood and the pathways of
oxidative metabolism are practically negligible.
Even under these conditions the highly glycolyz-
ing fiber cells retain LDH-1 thus showing that
within this tissue some other factor or factors
related to the replicative capacity must also be
considered in explaining the regulation of LDH
subunit synthesis.
I have now come to the end of our observa-
tions on the regulation of synthesis of tissue
specific proteins associated with a specific
stage of cellular differentiation. Our data have
shown that the synthesis of y-crystallins is
specifically associated with the differentiation of
the epithelial cell to the fiber cell. Thus, the
a- and ^-crystallins are structural proteins of
the epithelial cell and the a-, p- and y-crystal-
lins are structural proteins of the fiber cell.
At the beginning of my talk I described some
cytological changes which occur in elongating
epithelial cells such as an enlargement of the
nucleus and nucleoli and an increase in the
ribosomal population. These observations are
indicative of an increase in protein synthesis
and may be associated with the initiation of
y-crystallin synthesis.
The lactate dehydrogenases on the other
hand have shown us the simultaneous "turning
off" of a specific protein which is associated
not only with fiber cell formation, but also
with the aging and replicative activity of the
cell. Thus, the ability of the cell to regulate
LDH subunit synthesis in the absence of mor-
phological changes brings out a significant
difference between the regulation of y-crystallin
synthesis and LDH subunit synthesis. The y-
crystallins are highly tissue specific proteins
whose function may be essentially involved in
the structure of the lens whereas the LDHs are
widespread and are essential for metabolic
activity. In both cases, differential gene action
is required. Whether these regulatory mech-
anisms are similar must await further experi-
mentation.
IV. The Role of Nucleic Acids in Lens Fiber
Cell Differentiation
A. The status of m-RNA in differentiating
lens cells: the stabilization of m-RNA
It has recently been shown that the synthe-
sis of specific proteins such as hemoglobins
(32), feather keratins (33) and lens crystallins
(34-38) occurs on relatively long lived m-RNA
templates. These long lived messengers are
found in highly differentiated cells and are
involved in the synthesis of proteins specific
for these cells. Bacterial m-RNA for example,
which is considered to be short lived has a
half -life of 2 minutes (39), while the half-life of
m-RNA for feather keratin synthesis has been
reported to be longer than 24 hours (33). At
present, the only way to show stable m-RNA is
through the insensitivity of a protein-synthe-
sizing polysomal unit to actinomycin D, and all
the cases described so far are based on the
observation that protein synthesis continues
long after RNA synthesis has been halted by
actinomycin. On the basis of these preliminary
observations, it appears that the stability of
m-RNA is a very important feature of the
differentiated cell in which a large percentage
of the proteins synthesized are tissue specific
proteins. Although many tissue specific proteins
appear in the initial stages of tissue differen-
tiation, the basic question we would like to
consider is whether these proteins are synthe-
sized on "pre-existing" stable templates or
whether there is a progressive transition from
an actinomycin sensitive to an actinomycin
insensitive period of protein synthesis.
In a series of experiments carried out by
Mr. James A. Stewart, Dr. Paul V. Koehn and
myself (36-38), we attempted to determine
whether (a) the lens crystallins of the epithelial
cells and fiber cells are synthesized on long
lived or short lived messenger templates and
(b) if there is some period of lens cell differen-
tiation in which the m-RNA passes from a stage
of actinomycin sensitivity to actinomycin insen-
sitivity, thus associating the stabilization of
m-RNA to a specific stage of cellular differen-
tiation.
In these experiments, intact bovine calf
lenses were incubated in the presence of C^'*-
amino acids with and without actinomycin D
(10 fjg/ml). The epithelial and fiber cells were
separated and the crystallins from each cell
type were fractionated on DEAE-cellulose col-
umns. An elution diagram of the separation of
a-, ^- and y-crystallins of untreated and
actinomycin treated epithelial cells is shown
in Figs. 10 and 11. The incorporation of amino
acids into these proteins is also shown. It can
be seen that incorporation of amino acids into
epithelial cell crystallins could be extensively
inhibited by actinomycin. Both elution diagrams
show similar protein patterns. The specific
57
20
-
a
1
7000
6000
5000
o
■D
3
4000 :;
16
c
o
1 08
a.
E 04
_r
H
P
-
1 1
1
i
o
o
-
VWA
J ^r
Aa
3000 §
i
l'^-4-^.4
I-- ■t--+--i- i-
— I--W
/..-tv
60
120 180 240
300
360
ml effluent
-2000
-1000
120 ISO 240 300 360
ml effluent
Fig. 10.
The fractionation of calf lens epithelial cell proteins on
DEAE-cellulose after incubation in '''C-algal hydroly-
sate (amino acids) for 2 hours at 37°C. The elution sys-
tem is the same as that described for Fig. 3. The solid
lines denote total proteins (mg) per 3 ml fraction. The
dotted lines denote total counts per minute per 3 ml
fraction. (Fig. 8, J. Papaconstantinou, Science, in press;
copyright 1966 by the American Association for the
Advancement of Science.)
activity data (Table H), however, show that
incorporation of amino acids into a-crystallins
was inhibited by 71%, ^-crystallins by 83% and
y -crystallins by 80%.
The same experiments were performed
with the lens fiber cells. Elution patterns of
a-, ;S- and y-crystallins of cortex fiber cells
incubated in the absence and in the presence
of actinomycin D are seen in Figs. 12 and 13,
respectively. The incorporation of amino acids
into these proteins is also shown and, again,
both patterns are essentially identical with re-
spect to the distribution of the crystallins. The
incorporation of amino acids into the crystallins,
however, is significantly greater in the actino-
mycin treated cells. A comparison of the specific
activity of the a-, fi- and y-crystallins from
control and actinomycin treated lenses shows
that there is a significant stimulation of protein
synthesis by the antibiotic which ranges from
66% for the /8 -crystallins to 103% for the
a-crystallins (Table II).
4000
3000
2000 5
1000
Fig. 11.
The fractionation of calf lens epithelial cell proteins on
DEAE-cellulose after incubation in ^''C-algal hydroly-
sate (amino acids) with lO^g/ml actinomycin D. The
experimental conditions are the same as those described
in Fig. 10. (Fig. 9, J. Papaconstantinou, Science, in press;
copyright 1966 by the American Association for the
Advancement of Science.)
A comparison of the specific activity of
actinomycin treated cells shows an 85% inhibi-
tion of r-crystallin synthesis in the elongating
epithelial cells and a 68% stimulation of this
same group of proteins in the fiber cells. Thus,
at the time of y-crystallin appearance the syn-
thesis of this protein, as well as of the a- and
)8-crystallins, is still sensitive to inhibition by
actinomycin D, whereas in the completed fiber
cell the synthesis of these same proteins is
stimulated. The mechanism by which actinomy-
cin D stimulates protein synthesis is unknown.
The mechanisms which have been proposed for
this effect are as follows: first, the stimulation
might be attributed to the availability of more
ATP for protein synthesis as a result of the
inhibition of RNA synthesis by actinomycin (40).
Thus, in the fiber cell the ATP normally used
for RNA synthesis could be channeled into the
synthesis of the proteins being formed on stable
RNA templates.
POLLARD: Quite apart from that, however,
if you're just going to use one protein, aren't
you only using the t-RNA more efficiently on
that one protein when you shut off the others?
If all you're doing is just using one protein and
if you've got a long-lived message, isn't there
every reason why it would go up?
PAPACONSTANTINOU: Yes, that's right;
if that's the explanation for it.
58
TABLE II
The Effect of Actinomycin D on Lens Protein Synthesis in Calf Lens Epithelial and Fiber Cells.
Epithelial
Cells
Cortex Fiber Cells
cpm/mg Protein in
Control
Act. D % Inhibition
Control Act. D
% Stimulation
a-crystallins 1980
572 71
a -crystallins 340 690
103
^-crystallins 963
166 83
^-crystallins 99 165
66
y-crystallins 1590
314 80
y -crystallins 235 396
68
POLLARD: How can you avoid having that
happen if the t-RNA is there? It seems to me
you've got to have some stimulation if there is
a long-lived message present.
GROSS: We reported, in 1962, stimulation
by actinomycin in the sea urchin and we sug-
gested that it was the sparing of ATP.
PAPACONSTANTINOU: Tomkins and his
coworkers have recently presented their ob-
servation on the stimulatory effect of actino-
mycin on liver enzyme activity. They have
shown that the induction and early periods of
enzyme synthesis are inhibited by actinomycin
whereas after a given period of time enzyme
activity is stimulated by actinomycin. They
believe that at the time these enzymes are
stimulated by actinomycin their m-RNA is
stable and a repressor is inhibiting further
synthesis of this m-RNA. The stimulation occurs
only when actinomycin inhibits further synthe-
sis of the repressor. Pollock has also shown
a stimulation of penicillinase by actinomycin in
B. subtilis. He also showed that ^-galactosidase
is not stimulated. One of his explanations for
this is a difference in binding of actinomycin
to the bacterial genome, thus explaining the
differential sensitivity of these two enzymes
to actinomycin. He proposes that this differen-
tial binding of actinomycin may be a function of
the GC content of the individual genes.
GROSS: That's a fine idea if you can show
that all the RNA synthesis is lost. Pollock used
very low levels of the drug and he did not
present fully convincing evidence that he had
shut down all synthesis of template RNA.
PAPACONSTANTINOU : Well, it didn' t mat-
ter whether he shut it down or not. He showed
he got a stimulation of the penicillinase. If he'd
60
-
50
-
<u
"
e-4n
—
E
i
^
:!
.^^n
—
il -
{
O
_
II
Q.
n 20
—
—
o
h-
1 \ \
,1 ~
10
-
\\\
fk
\ ' r'-\--' 1 S-- r 'I- 1 "" i' -■V~i--4--i-
-1600
200 I
800 a
-400
60 120 180 240 300 350
ml
Fig. 12.
The fractionation of calf lens cortex fiber cell proteins.
The experimental conditions are the same as those
described for Fig. 10. (Fig. 10, J. Papaconstantlnou,
Science, in press; copyright 1966 by the American
Association for the Advancement of Science.)
gotten no effect I could accept that argument;
but he got an effect.
GROSS: He might have had the penicillinase
messenger still coming out, but the outflow of
others seriously depressed; so that you're
synthesizing the proteins from templates that
remain at a selectively higher rate.
PAPACONSTANTINOU : Right, What you' re
essentially saying is that the penicillinase
region may not have as high a GC content as
some of the other regions.
59
180 240
ml
Fig. 13.
The fractionation of calf lens cortex fiber cell proteins
Incubated with l^C-algal hydrolysate and lO^g/mlac-
tinomycin D. The experimental conditions are the same
as described for Fig. 10. (Fig. 11, J. Papaconstantlnou,
Science, in press, copyright 1966 by the American Asso-
ciation for the Advancement of Science.)
GROSS: Well, whether its GC or not, they're
not as sensitive.
PAPACONSTANTINOU: Right. I'm just
presenting all these ideas which have come out
in the literature. They're not my ideas, and I'm
just trying to fit some of our data into any one
or all of these as we go along. However, I think
the evidence does seem to indicate that we can
pinpoint the stage at which the messenger RNA
becomes stabilized, and that is at the time when
the epithelial cells are starting to elongate.
The y-crystallin is still sensitive to actino-
mycin at that stage, but when they've finally
elongated, it's no longer sensitive. This holds
true, also, for the a- and ^-crystallins. The
period of stabilization seems to fall in con-
currently with the breakdown of ribosomes and
the decrease in the size of the nucleus and
nucleoli.
Secondly, this stimulation might be attrib-
uted to inhibition of the synthesis of a repressor
protein by actinomycin (41). It has been shown
that the actinomycin D stimulation of tryptophan
pyrrolase and tyrosine transaminase occurs
after these enzymes have been induced by
hydrocortisone, when their m-RNA is relatively
stable. By inhibiting the m-RNA responsible
for the synthesis of repressor protein the level
of this repressor is decreased and a stimula-
tion of these enzymes results. Finally, the
lens epithelial cells are essential for the active
transport of nutrients into the lens fiber cells.
It is, therefore, possible that actinomycin alters
these properties such that there is an increase
in the transport of amino acids into the fiber
cell layer, resulting in a stimulation of protein
synthesis on stable RNA templates.
B. Ribosomal breakdown in lens fiber cells
In this final phase of my talk I would like
to describe a phenomenon, again associated with
fiber cell differentiation which may explain the
reduced rate of protein synthesis observed in
the final cell. I shall start by reiterating that
Eguchi and Karasaki (3, 4) showed by electron
microscope studies that during fiber cell forma-
tion, in the elongating epithelial cell there is an
increase in ribosomes, whereas in the completed
fiber cell the ribosomal population is decreased.
We feel that we have been able to show essen-
tially the same thing in our chemical analyses of
the ribosomal RNA in the fiber cell. In these
studies we have used methylated albumin col-
umns (42) to fractionate nucleic acids from lens
epithelial cells and fiber cells and to detect any
qualitative or quantitative differences that may
occur in the nucleic acids of these cells. The
fractionation procedure involved the use of a
linear NaCl gradient in 0.05 M Na-phosphate
buffer pH 6.8. An elution patternof the epithelial
cell nucleic acids is shown in Fig. 14. The t-RNA
(peak A) is eluted between 0.4 M - 0.6 M NaCl;
DNA (peak B) is eluted between 0.6 M - 0.8 M
NaCl; and ribosomal RNA (peak C) is eluted
between 0.8 M - 1.0 M NaCl. This sequence of
elutions compares well with a similar elution
system used to fractionate t-RNA, DNA and
ribosomal RNA from E. coli (43). A pattern
for the RNA extracted from fiber cells is super-
imposed over the epithelial cell pattern to
facilitate comparisons between them. It can be
seen that there are striking differences between
the two patterns: (a) the pattern for fiber cell
RNA shows a significantly larger amount of
material eluted in peak A (t-RNA) with respect
to the amount of material in the ribosomal RNA
(peak C); (b) in addition, there is a sharp
decrease in the DNA (peak B) of the fiber cell
pattern. The quantitative differences between
t-RNA and R-RNA of epithelial and fiber cells
are better seen in the patterns of Fig. 15. Phenol
extracted nucleic acids from epithelial and
fiber cells were DNase treated to remove DNA,
prior to fractionation on methylated albumin
columns. The DNA (peak B) is completely lost,
60
o
14
o
lU
I — 1
(/)
z
Q
-a
O
06
I — 1
o
o
B-
o
0 2
0 60 120 180 240 300 360
ml
120 180 240 300 360
Fig. 14.
The fractionation of phenol extracted nucleic acids from
calf lens epithelial cells ( ) and calf cortex fiber
cells ( ) on methylated albumin columns (MAK).
A total of 35.4 O.D. 2^0 units from epithelial cells were
placed on the column; 45.0 O.D. 250 units from the fiber
cells were placed on the column. The nucleic acids were
eluted with a linear salt gradient ranging from 0.2 M to
1.4 M NaCl in 0.05 M sodium-phosphate pH 6.8. (Fig. 12,
J. Papaconstantlnou, Science, in press; copyright 1966 by
the American Association for the Advancement of
Science. 1
and now the epithelial and fiber cell patterns
are almost alike except for the quantitative
differences between peaks A and C (Fig. 15).
Our next step was to determine whether
the RNA of peak A is t-RNA or a mixture of
t-RNA and a ribosomal RNA breakdown product.
(This mixture will be referred to below as
total soluble RNA). In his studies of the RNA
fractions from E. coli Midgely showed that
t-RNA and ribosomal RNA could be separated
on DEAE columns using 0.05 M tris pH 7.4 with
an increasing NaCl gradient (44). We carried
out a similar fractionation to determine whether
peak A is a mixture of t-RNA and ribosomal
RNA. We used this procedure to determine
whether the total soluble RNA from lens
epithelial and fiber cells could be resolved
into two fractions. In one experiment, phenol-
extracted RNA was first placed on a sucrose
gradient to eliminate the ribosomal RNA. The
material remaining at the top of the gradient
was dialyzed against tris buffer and was then
fractionated on a DEAE column. The elution
diagrams in Figs. 16A and 16B show a fiber
Fig. 15.
The fractionation of DNase-treated phenol-extracted
nucleic acids from calf lens epithelial cells ( ) and
calf cortex fiber cells ( ). The conditions of
fractionation are exactly as described in Fig. 14. (Fig. 13,
J. Papaconstantinou, Science, in press; copyright by the
American Association for the Advancement of Science.)
cell and epithelial cell pattern respectively.
Firstly, it can be seen from the OD26O readings
that there is a small amount of RNA eluted by
0.5 M NaCl, which corresponds to the region
where bacterial t-RNA is eluted. Another larger
fraction is eluted by 0.7-0.8 M NaCl, which
corresponds to the region where bacterial
ribosomal RNA is eluted. Secondly, it can be
seen that there is a significant increase in the
ribosomal RNA fraction in the fiber cell pattern.
In another experiment the total soluble RNA
from calf cortex fiber cells was separated from
ribosomal RNA by fractionation on a MAK
column. The soluble RNA fractions (peak A)
were pooled, dialyzed against tris buffer and
fractionated on DEAE-cellulose. This fractiona-
tion is shown in Fig. 16C. It can be seen that
this elution pattern is identical to that obtained
for the total soluble RNA from the sucrose
gradient (Fig. 16A). These preliminary data
indicate that the increase in total soluble RNA
(peak A) of the fiber cell (Figs. 14, 15) is due
to the accumulation of a nondialyzable ribosomal
breakdown product which has chromatographic
(MAK) properties similar to t-RNA. Further
evidence that this may be a ribosomal break-
down product was obtained by a base ratio
61
30 90 150 210
ml
Fig. 16.
The fracnonatlon of total soluble RNA from epithelial
cells and fiber cells on DEAE-cellulose. (A) Total
soluble RNA from calf cortex fiber cells. The rlbosomal
RNA and soluble RNA were first separated by sucrose
gradient. The soluble RNA fractions from the top of
the gradient were combined and dialyzed against 0.01 M
Tris-HCl pH 7.3 + 0.01 M MgCl2 +5|/g/ml PVS before
being placed on the column. (B) Total soluble RNA from
calf epithelial cells. The experimental procedures were
the same as in A. (C) Total soluble RNA from calf
cortex fiber cells. The rlbosomal RNA and soluble RNA
were first separated by fractionation on a MAK column.
The soluble RNA (peak A) fractions were combined and
dialyzed as described in Fig. 16A before being placed on
the column. (Fig. 14, J. Papaconstantinou, Science, in
press; copyright by the American Association for the
Advancement of Science.)
analysis of the rlbosomal RNA eluted from the
DEAE column. This fraction has a GC content
of 64%, which is typical of rlbosomal RNA. As
a further characterization of these fractions
we are presently studying the extent to which
they can be charged with C^"* -amino acids.
C. Inactivation of DNA: the loss of nuclear
activity in lens fiber cell
The MAK column patterns in Fig. 14
indicate that there is a significant decrease in
the DNA in the cortex fiber cells. These ob-
servations are in agreement with cytological
reports that the nucleus of the cortex fiber
cell decreases in size and is ultimately lost in
180 240 300 360
Fig. 17.
The fractionation of phenol-extracted nucleic acids from
calf lens epithelial cells incubated in ^H-thymidine.
CD. 260 ' ; counts per minute/ml . (Fig. 15,
J. Papaconstantinou, Science, in press; copyright by the
American Association for the Advancement of Science.
the older fiber cells.
As I stated previously, the lens fiber cells
do not have the ability to replicate. Although
small amounts of DNA could be detected in the
nucleic acids extracted from the fiber cells it
is not known whether this DNA is metabolically
active. In view of this, we performed experi-
ments to determine whether ^H-thymidine is
incorporated into the DNA of the fiber cells.
After incubating calf lenses in ^H-thymidine,
the nucleic acids from epithelial cells and fiber
cells were phenol extracted and fractionated
on MAK columns (Fig. 17). The DNA (peak B)
fractions were counted and it was found that
the ^H-thymidine was incorporated into the
DNA of the epithelial cells. On the other hand,
the corresponding experiment with the fiber
cell DNA fraction showed that there is no
incorporation of ^H-thymidine into the fiber
cell DNA. We concluded from these experiments
that although the DNA of the epithelial cells is
metabolically active, this activity is lost in the
fiber cell. This observation, we feel, has an
important and obvious bearing on our observa-
tions that the m-RNA in the fiber cell is stable.
V. Summary and Conclusions
I would like to return to the beginning of
my lecture, where I listed the cytological events
marking the differentiation of the lens epithelial
cell to the fiber cell and now attempt to corre-
late these events with our biochemical observa-
tions. The morphological and biochemical alter-
ations characteristic of all stages of lens cell
62
differentiation are shown in Fig. 2. Firstly, tlie
epithelial cells have basophilic staining prop-
erties whereas the fiber cell has acidophilic
staining properties. This change in staining
properties may have been brought about by the
synthesis of r-crystallins. These proteins are
slightly basic having isoelectric points ranging
from pH 7.5 - 9.0. In view of their basic prop-
erties the r-crystallins may bind to the nucleic
acids of the nucleus and cytoplasm thus effect-
ing the observed alteration in staining
properties. It should also be pointed out that
the isoelectric points of the a-crystallins is
5.2 and of the ^-crystallins ranges from 6.0 to
7.0. Both these groups of proteins as well as
the rich population of ribosomes would con-
tribute to the basophilic staining properties of
this cell.
In addition to the change in staining prop-
erties there is also a conversion from a rough
endoplasmic reticulum in the epithelial cell to
a smooth endoplasmic reticulum in the fiber
cell. The breakdown and subsequent decrease
in the ribosomes may be directly related to
the appearance of the endoplasmic reticulum.
At present I cannot present any information
on the mechanism of this ribosomal breakdown.
The fact, however, that there is also an overall
decrease in the rate of protein synthesis in the
fiber cells may be a consequence of the ribosomal
breakdown.
The increase in the size of the nucleus and
nucleoli and the increase in the ribosomal
population at the time of cellular elongation
might indicate the initiation of an overall syn-
thesis of materials required for the morpho-
logical changes of the cell. Although we know
nothing of the function of r-crystallins, it has
been shown that the synthesis of this major
group of proteins is initiated during cellular
elongation. In addition to the increase in protein
synthesis there must also be an increase in the
synthesis of nucleic acids, both ribosomal and
m-RNA. The synthesis of these two classes of
RNA may account, therefore, for the morpho-
logical changes in the nuclei and nucleoli.
In the fiber cell it has been observed that
there is a gradual decrease in the size of the
nucleus and as the cell gets older the nucleus
disappears. Through the use of ■^H-thymidine
we have shown, as would be expected in a
replicative cell, that the epithelial cells contain
metabolically active DNA whereas the fiber
cells no longer have the capacity to incorporate
precursors into its DNA. Furthermore, the data
from our MAK columns have shown that there
is a significant loss of DNA in the fiber cell.
Both of these observations are in complete
agreement with the cytological observations
on the fate of the nucleus in lens fiber cell
differentiation.
The loss of nuclear activity brings up the
question of the synthesis of m-RNA for the
continuation of protein synthesis. Upon inacti-
vation of the nucleus, the synthesis of m-RNA
stops and the cell would require some mecha-
nism for the conservation of existing m-RNA
for the continuation of protein synthesis. The
stabilization of m-RNA in these fiber cells
has been shown; the mechanism of stabiliza-
tion, which has not been worked out, should
prove to be an important one for understanding
the molecular aspects of terminal cellular
differentiation.
I have presented just a limited spectrum
of the macromolecular interactions which occur
during the terminal stages of lens cell differ-
entiation. There are many cell types which
undergo similar morphological alterations dur-
ing their terminal stages of differentiation.
These cells are also involved in the synthesis
of highly specific proteins such as hemoglobin,
myosin and keratin and there are indications
that the same molecular alterations described
for the lens may also occur in these cells.
Thus, a specific series of macromolecular
interactions such as those described above
may be a basis for the biochemical definition
of the terminal stages of cellular differentia-
tion. The differentiation of the reticulocyte, for
example, involves inactivation of the nucleus
and stabilization of m-RNA. It remains to be
seen whether there also occurs a ribosomal
breakdown and the accumulation of a breakdown
product such as I have described here. Further-
more, the elucidation of the mechanisms of
reactions involving nuclear inactivation, the
stabilization of m-RNA and the breakdown of
the ribosomes may be the basis of the mecha-
nisms of terminal cellular differentiation. This
is important because most cells exhibiting the
property of synthesizing highly tissue specific
proteins, enter terminal stages of differentia-
tion and exhibit molecular properties similar
to those described above.
The lens cell has reached its highest form
of cellular differentiation when it has formed
the fiber cell, and as it approaches this stage
it develops very specific metabolic activities.
With respect to the mechanism of lens fiber
cell formation, therefore, one would ask how
much of this metabolic activity is dependent
upon the morphological changes and whether
63
these biochemical interactions are intimately
associated with the genetic regulation of mor-
phogenesis. To be more specific, we would like
to know whether y-crystallin synthesis is in-
timately linked to fiber cell formation and
whether the r-crystallins are required to bring
about the formation of a fiber cell. The poten-
tial for synthesizing y-crystallins is inherent
in the genome of the cell. This part of the
genome is non-functional in the epithelial cell.
Can these genes be activated without bringing
about a simultaneous (a) cellular elongation;
(b) loss of cellular replication (c) stabilization
of m-RNA and (d) breakdown of the ribosomes?
The degree of coupling or uncoupling of tissue
specific protein synthesis to morphogenesis is
an important part of the mechanism of cellular
differentiation. We feel that we have now reached
the stage where we can begin to answer these
questions.
MASSARO: I have a couple of questions if
you wouldn't mind going back to LDH. What
other data do you have besides the pyruvate
inhibition curves to show that the LDH-1 of your
lens system is different from the LDH-1 of the
skeletal muscle, heart, brain, glands system?
PAPACONSTANTINOU : Well, we don' t have
any other evidence.
MASSARO: Recently we've found that in
fish the LDH system of the eye apparently
differs from the skeletal muscle, heart, glands
and CNS system. I would be kind of shaky about
making a very strong statement concerning the
results Cahn got in tissue culture and the results
one obtains with aging muscles because we just
don't know much about what goes on during the
aging process. Also, there are dedifferentia-
tion and other problems in tissue culture.
GROSS: Did you say that the behavior of
your LDH-1 with respect to pyruvate was
different from muscle LDH?
PAPACONSTANTINOU: No, it's the same.
We have no evidence that they' re different at all.
MASSARO: Doesn't your LDH-1 differ from
the LDH-1 of the muscle tissue?
PAPACONSTANTINOU: No, it's the same;
it's sensitive to pyruvate. The difference is that
the fiber cells retain LDH-1 although they have
a high rate of aerobic glycolysis, and they should
have LDH- 5.
MASSARO: Are all of the properties of
this LDH-1 similar to the LDH-1 of the muscle?
PAPACONSTANTINOU: They are similar
to those of the heart muscle.
MASSARO: The only difference is that in
this particular environment you have an LDH-1
which is sensitive to a clearly known pyruvate
concentration?
PAPACONSTANTINOU: That's right. What
we're trying to point out here is that the fiber
cells are highly glycolytic and according to
the theory that's been proposed for heart and
skeletal muscle, the fiber cells should retain
LDH-5; instead they retain LDH-1.
MASSARO: Then, the only difference that
you see here is in the pyruvate inhibition
curves?
GROSS: The proposal about LDH and the
oxidative level is, as I understand it, not a
theory, but an explanation of why you have
more of one enzyme in one tissue than another.
You are suggesting that differential gene action
results from the influence of the environment
of the cell. In the classical cases, the influence
was pinpointed as oxygen tension or as the state
of carbohydrate metabolism. Now, as I under-
stand it, Papaconstantinou's evidence showed
that, in his system, this explanation is invalid.
My conclusion from that would be that the
initial explanation is not universally applicable.
MASSARO: This conclusion is on the basis
of the pyruvate inhibition?
GROSS: Yes.
PAPACONSTANTINOU: It's based on the
fact that LDH-1 persists in a highly glycolyzing
system that can go into oxygen debt.
MASSARO: You see, I think we're running
into some semantic problems here by labeling
this LDH-1.
PAPACONSTANTINOU: WeU, I can't call
it heart-type LDH because it's not in the heart.
MASSARO: We're crossing over systems
and using the LDH terminology of the heart
and skeletal system in the eye system. I don't
think this is valid because I don't think they're
comparable. I think, perhaps, from this data
that the eye LDH system may be quite different
from that of skeletal muscle and heart muscle.
GROSS: What you're saying, then, is that
there are, in fact, different cistrons involved.
MASSARO: That's right.
GROSS: The initial assumption in this story
was that there are two genes involved and they' re
the same in every genome. Now, you're sug-
gesting, in fact, that there are other genes
involved.
MASSARO: We know this for a fact in the
case of the so-called "X-bands of gonadal LDH"
PAPACONSTANTINOU: I would not expect
to find differences in amino acid composition
between lens LDH and heart LDH from the same
animal. Genetically the two LDH's should be
64
the same. I think, however, that the point I am
trying to make with respect to the regulatory
mechanism has nothing to do with the genes;
it's at an entirely different level. The only
genetic involvement in this work is the regula-
tion of subunit synthesis.
MASSARO: On the basis of the pyruvate
inhibition curve alone, I don't think you can
emphasize that these LDH's are one and the
same.
GROSS: It seems to me if you have two
different genes, then the original explanation is
wrong on the basis of Papaconstantinou's re-
sults. If you don't have two genes, then this
is another complicated case of differential gene
action and not very much more can be said
about it.
HYMER: Have you studied the process of
ribosomal breakdown during differentiation to
the fiber cell by electron microscopy?
PAPACONSTANTINOU: With electron mi-
croscopy there is a decrease in the ribosomal
population. In other words, if you count the
ribosomes in a unit area, you'd find a decrease
in the actual number of ribosomes.
HYMER: Are these membrane bound?
PAPACONSTANTINOU: No, they're not
membrane bound, especially in the fiber cells.
KOHNE: The one criticism that you could
make of this is that RNA breakdown is some-
thing other than ribosomal breakdown since all
you've got is phenolyzed RNA.
PAPACONSTANTINOU: Well, there is no
ribonuclease in the lens system, that we can
detect. I apologize to everybody who has ribo-
nuclease troubles, but we just don't have them.
We used 5 gammas/ml of polyvinylsulfate in
our phenol extractions but it doesn't make any
difference whether we use it or not.
POLLARD: How does ribosomal RNA break
down?
PAPACONSTANTINOU: We don't knowyet.
It's been a very interesting phenomenon. If
ribonuclease is present we ought to get break-
down products smaller than what we observe,
unless it's a special ribonuclease, which I'd
like very much to find out.
GROSS: You remember Nemer's paper on
RNA synthesis during the early development of
the sea urchin, in which he showed gradients
with RNA's that looked like 28s, 18s, 13s, 10-13s
and 4s? The 28s RNA in animal cells of this
sort does, in fact, break down into things that
sediment roughly at 10- 13s.
PAPACONSTANTINOU: Because of the high
GC content of the breakdown product I am in-
clined to believe that it comes from the 50s
ribosomal particle. This is the part of the ribo-
some that 28s RNA is derived from. However,
this is just speculation right now. We are trying
to characterize the 50s and 30s ribosomal RNA
to determine if the breakdown product originates
specifically from either one or both.
EPEL: Can you say a little more about the
temporal relationship between the ribosomal
breakdown and the lens protein synthesis?
PAPACONSTANTINOU: Although we don't
really have kinetics or good turnover data,
indications are that lens protein synthesis
decreases about 10-fold in going from an
epithelial cell to a fiber cell. Paul, did you
mention the fact that there was a protein that
was involved in the stabilization of messenger
RNA in the early embryo?
GROSS: It's an idea that's been suggested.
Monroy and his colleagues found that trypsin
treatment of unfertilized ribosomes allowed
them to work.
PAPACONSTANTINOU : If we prepare ribo-
somes from epithelial cells and fiber cells and
do an RNA-protein ratio, the RNA-protein ratio
in the epithelial cells is about 0.5 to 0.8 (they're
good ribosomes). When you do it in the fiber
cell, it goes down to 0.1 and sometimes less.
There' s an indication that there' s protein being
stuck onto the ribosomes of the fiber cells
which may explain that smooth endoplasmic
reticulum and possibly the stabilization of
messenger.
GROSS: Couldn't there also be isolation
artifacts since you're making lots of the crys-
tallins and what not?
PAPACONSTANTINOU: Well, no. We're
extracting under the same conditions. Why
shouldn't we get a lot of protein on these ribo-
somes then?
GROSS: Because it's a different kind of
protein.
PAPACONSTANTINOU: The only differ-
ence we have is the formation of the gamma
crystallin. The gamma crystallin is a basic
protein relative to the others. The gamma
crystallins could be sticking onto those ribo-
somes. They may be a stabilizing factor. This
is a highly specific protein that is formed in
fiber cell formation. This is a real speculation,
though. It's a highly specific protein that's
associated mainly" with fiber cells and it comes
up just before the stabilization of the messenger.
I don't know why they would want so much
gamma crystallin for the stabilization.
65
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67
PROLIFERATION AND DIFFERENTIATION OF STEM CELLS
OF THE BLOOD-FORMING SYSTEM OF THE MOUSE
James E. Till
Department of Medical Biophysics, University of Toronto and
The Ontario Cancer Institute, Toronto 5, Ontario, Canada
First, I'll describe the system with which
we've been working, and, then, in the remain-
ing time, I'll tell you something of what we've
been doing with it. The work I'll describe was
done in collaboration with Dr. Ernest A.
McCulloch and Dr. Louis Siminovitch.
The method we use to detect and count
stem cells has been described in detail else-
where (1, 2). The method is based on the
transplantation of cells into heavily irradiated
recipient animals. The irradiation converts the
recipients into culture vessels in which the
transplanted cells can grow by destroying the
proliferative capacity of the animals' own cells.
Inbred strains are used to avoid transplantation
difficulties. One can regard this as a form of
cell culture in vivo. The irradiated recipient
is well designed for this purpose, since it has
a built-in temperature control, a built-in pH
control and a built-in medium supply.
Cells are taken from a normal donor,
suspended, counted and injected intravenously
into the irradiated recipients. Cells from any
blood-forming tissue may be used; we usually
use marrow. If you wait 10 days and look at
the spleens of these animals, you find colonies
in their spleens. These are colonies of cells
that have grown up from those cells of the trans-
plant which lodged in the spleens of the irradi-
ated animals. When the colonies are fixed in
Bouin's solution, they turn yellow, and you can
count them very easily. The number you get is
proportional to the number of cells you put in.
We find about 10 colonies per spleen per 10 ^
cells injected. Why is there this rather low
efficiency? We think it is because most of the
cells that go into the mouse are fully differen-
tiated, or almost fully differentiated; that is,
they are cells that don't have much prolifera-
tive capacity left. Certainly, they do not have
enough to make a colony of this size which con-
tains something like a million cells.
POLLARD: That's not straight dilution, is
it?
TILL: No, we've measured that. About a
fifth go to the spleen, so it's not that only a
small number get there (3), The spleen is a
pretty efficient filter of cells put into the blood
stream.
The other point I want to make is that the
relationship between the number of colonies
formed and the number of cells injected is
linear and extrapolates back through the origin.
This suggests that the colonies are formed by
single entities which lodge in the spleen (1, 2).
POLLARD: Have you done the Poisson test
on this?
TILL: Yes. The distribution of the number
of colonies per spleen, for colonies formed by
transplanted cells, appears to be a Poisson
distribution.
If you look at the colonies using histological
methods, you find that they contain differentiated
cells. Thus, these colonies are not like the
colonies that are formed by bacteria, for ex-
ample, where the cell composition is fairly
uniform. The cell composition of these colonies
is heterogeneous; they contain differentiated
cells and often more than one kind of dif-
ferentiated cell. These differentiated cells are
blood cells or their precursors, that is, erythro-
blasts, granulocytes and megakaryocytes. So
this raises the question, are these colonies
formed by the differentiation of more than one
initiating cell or does this mixture arise from
a single precursor? It's a rather important
question, so we have tried to find out about this.
Dr. Andrew Becker did these experiments
(4). What he did was to obtain spleen colonies
from irradiated marrow cells. The irradiation
69
inactivates some of the colony-forming ability
of these cells, so he had to start with more
cells (about 100 times as many) in order to end
up with discrete colonies. Then individual col-
onies were picked out of the unfixed spleen. The
individual colonies were separately dispersed
and chromosome preparations were made and
examined.
At the dose that was used (650 rads) about
10% of the colonies contained chromosome aber-
rations. The point of interest was that when-
ever a chromosomal aberration was found in a
dividing cell in a colony, the same aberration
was present in more than 95% of the other
dividing cells in that colony. The interpretation
was that the irradiation of a single precursor
had generated the chromosomal aberration which
was then passed on to all its descendants, and
that, in fact, the colony was a clone formed from
this original damaged precursor. With ionizing
radiation, aberrations are formed in a random
fashion, so each aberration that you get is dif-
ferent. He found a total of 8 marked colonies.
Each one had a different kind of aberration, but
all appeared to be clones because all the divid-
ing cells of each colony carried the marker
characteristic of that colony.
This doesn't really settle the question of
whether the different kinds of differentiated
cells have come from a common precursor be-
cause here we have just looked at dividing cells.
On the other hand, most of the differentiated
cells no longer are dividing. We haven't con-
clusively proven that the different kinds of dif-
ferentiated cells we find in a single colony have
arisen from a single precursor but this is fairly
good indirect evidence.
We've taken as a working hypothesis that
the spleen colonies are clones, and that these
clones are formed by some sort of precursor
cell which can differentiate in multiple ways.
What one would like to know is, what governs
that choice? What determines whether the cell
will give rise to erythrocytic precursors or
granulocytic precursors or megakaryocytes?
This is what we're primarily interested in.
Perhaps I should just refresh your mem-
ories for a moment about the organization of a
renewal system like this. It's been postulated
for a long time that there exist stem cells which
have two functions: one is to maintain their own
numbers; the other is to begin the pathway of
differentiation so one gets a series of divisions
resulting in cells with different functional
characteristics, thus ending up with a wholly
differentiated, fully specialized cell such as the
red blood cell. This latter cell can't divide; it
hasn't even got a nucleus. Its immediate pre-
cursors can divide a few times, but capacity for
proliferation is limited and they have begun to
differentiate and form hemoglobin. The ones
nearest the stem cell can divide a lot and contain
no hemoglobin. The fully differentiated cells are
continually lost from the system, and they must
be replaced somehow. Since they can't divide
themselves, they must be replaced by the divi-
sion of the precursor cells. The ultimate cell,
the cell that has no precursors after the em-
bryonic stages, is the stem cell. Although this
type of cell was postulated to exist, it proved
difficult to obtain clearcut ways of recognizing
it experimentally. The spleen-colony technique
appears to be one way of doing it. This method
makes use of the major function of the stem
cells, which is to proliferate, and demands that
they be able to multiply through a number of
cell generations sufficient to give rise to a
colony of cells that you can see with the naked
eye. It's an arbitrary criterion, so we probably
don't detect all the stem cells by this criterion;
but we do see some. It gives us a class of stem
cells that we can look at.
If this is true, we would like to know what
regulates the proliferation and differentiation
patterns of the stem cells. The stem cell, if it
is really multipotent, has several choices open
to it. One can postulate four of these. First, it
may choose to proliferate so as to maintain its
own numbers. We could call this, for the lack of
a better term, "self-renewal," although there is
evidence that the daughter stem cells may not
be exactly like their parents (5). If the system
is to keep going, the stem cells need to be able
to perform some sort of self-renewal, because,
by definition, there is no precursor for them in
the adult animal. Second, the stem cell may
differentiate to give rise to cells of the red cell
series. Third, it may give rise to cells of the
granulocyte series. Fourth, it may give rise to
megakaryocytes. Thus, four different pathways
of proliferation or differentiation are available
to the stem cell. One would like to find out what
governs whether or not a particular choice is
made.
How can one go about trying to solve this
problem? Professor Pollard suggested this
morning that one way of approaching a problem
of this type is by the use of genetic methods, and
this is what we have tried to do. If you can find
a single gene mutation that affects some step
in the process, then you can assume that the
molecular basis for that particular effect is
70
probably relatively simple. If it were not, it
wouldn't be under the control of a single gene.
In the mouse, there are several mutations which
are known to produce anemia, i.e., to affect in
some way one of the pathways of differentiation
in which we are interested. Some of these have
already been studied in considerable detail by
Dr. Elizabeth Russell and Dr. Seldon Bernstein
at The Jackson Laboratory, Bar Harbor, Maine
(6). The question is, how do these genes affect
the pathways of differentiation of the stem cell?
Let's imagine what kinds of genetic changes
one could see, speaking of gross changes rather
than individual steps. Each one of the four sug-
gested pathways open to the stem cell, pre-
sumably, could be regulated by a separate gene.
There might be a gene that regulates self-
renewal. For example, there maybe some mole-
cule that triggers off the self-renewal division,
since there is evidence that it isn't happening
all the time (7). Most of the stem cells are not
dividing and one may postulate that some of
them are triggered off by a gene product. You
can imagine four classes of these genes cor-
responding to these four possible pathways that
this stem cell can go into. You can imagine,
also, two general ways that regulation could
occur. It could be a property intrinsic in the stem
cell itself which regulates it or the regulator
could come from outside. Let me just take an
example; let's say that regulation might depend
on the permeability of the membrane of the cell.
That would be something I would call intrinsic
to the cell itself. On the other hand, the regulator
could be a hormone that comes in from outside
and tells the cell to do something. That I would
call external because the mutation would then be
a mutation that stops the supply of that hormone,
whereas the mutation in the first case would be
one that alters the membrane. So there are two
classes, intrinsic and extrinsic, each of which
can be applied to each of the four suggested
pathways.
We have looked at three mutations in some
detail so far; and we think that each falls into a
different class. One of these is extrinsic in its
action and the other two are intrinsic. I'll
describe the evidence for this next.
The mutations we've looked at have been
W, SI and/. Let's consider IV and SI first.
The animals that we studied were of genotype
W/W and Si/Sid, obtained from Drs. Russell
and Bernstein. Animals of these two genotypes
have very similar phenotypes. Both have a
severe macrocytic anemia, the coat color is
affected, the animals are sterile and they are
very radiation-sensitive. Superficially, the
phenotypes appear almost identical. However,
we know they're different because they map at
different genetic sites. If the cells that form
colonies in the spleen are a kind of stem cell
and the basis for these anemias is a defect in
the stem cell, then we should find deficient
colony formation when we test the cells from
these animals for their ability to form colonies
in irradiated normal hosts.
For W/W^ we found deficient colony for-
mation (8), and the subsequent work that's been
done indicates that this may be a defect in the
ability of the stem cell - the colony-forming
cell - to renew itself. Therefore, this is a
mutation whose effects are intrinsic to the
stem cell. It's apparently a defect at the cel-
lular level in the ability of the cell to produce
more cells like itself. Because the phenotype
was similar, we expected that the stem cells
in animals of genotype Sl/Sl d would be similar
in their properties to those of animals of geno-
type W/W. In fact, they turned out to be very
different. When we tested cells from Sl/Sl ^
mice for their ability to form colonies in our
standard test system (irradiated normal mice),
we found that they formed colonies perfectly
well (9). Every attempt that we made to find
a defect in the composition of these colonies
failed; they are apparently perfectly normal.
So then we did some experiments the other
way around. We used these animals as recipi-
ents for normal cells. We had found previously
that W/W animals are good hosts for the
growth of normal cells (8). You can put normal
stem cells into them and they grow well. In
fact, you don't even have to irradiate the hosts
first. You can put normal cells into unirradi-
ated mice of this genotype and they'll still form
spleen colonies. In fact, they'll cure the anemia
of the animal permanently (10). Thus, animals
of genotype W/W" exhibit a defect at the cellu-
lar level, but the host is capable of supporting
the growth of normal blood-forming cells. How-
ever, if you put normal cells into Sl/Sl ^ mice,
they don't form colonies. Even if you irradiate
these animals with large doses, they won't
support detectable growth of normal cells (9).
In this case, the cells seem to be normal, but
the mouse does not support their growth nor-
mally. Thus, this mutation affects a process
which is extrinsic to the stem cells.
One would expect, if all this is right, that
the cells from Sl/Sl '^ would grow in W/W "
hosts. In other words, one should be able to
take cells from an anemic animal of genotype
71
Sl/Sl '^, put them into an anemic animal of
genotype W/W and cure the anemia. This
experiment has been done by Russell and
Bernstein, with whom we collaborated in these
experiments, and it worked perfectly well. The
animals are cured of their anemia at least as
well with cells from Sl/Sl '^ animals as with
genetically normal cells (9). This is comple-
mentation of the cellular level.
KAHN: Where did the stem cells come from
in the SI type? If you have no stem cell renewal,
how do you get the spleen to pick up cells?
TILL: We don't know the answer to that.
Maybe this is not as serious a defect in the
embryo and enough multiplication of the stem
cells can occur. Also, we can't rule out that
there's slow multiplication. Perhaps over a
long period of time these cells can build up in
numbers. We don't know yet which is right.
Our experiments on growth in these hosts have
been carried out over a fairly limited period
of time - about two weeks. I think the fact that
these animals are alive and that they do have
normal numbers of stem cells in them means
that there must be slow growth of stem cells
until the equilibrium level is reached. However,
the evidence does suggest that the W gene acts
intrinsically and the SI gene extrinsically to
the stem cells.
In the Sl/Sl '^ animals one would like to
know, what is the external regulation? Is it by
means of a molecule? Can the molecule be iso-
lated? We did standard experiments to test for
such a molecule. An Sl/Sl '^ animal was joined
in parabiosis with a normal animal of the same
inbred strain; that is, they had a shared cir-
culation. We demonstrated the existence of the
shared circulation by putting chromium-labeled
erythrocytes into one animal and showing that
they appeared in the other. Then both were
irradiated and inoculated with the same number
of normal bone marrow cells. If the failure of
cells to grow in the Sl/Sl'^ irradiated host is
due to an inhibitor which is circulating in the
peripheral blood, then we should get growth in
neither member of the parabiotic pair. If, on the
other hand, it's the lack of a stimulatory factor
that accounts for the failure of cells to grow in
Sl/Sl'^ hosts, in the parabiotic situation this
factor should be supplied by the normal member
of the pair and there should be growth in both.
In fact, what we got was exactly the same
situation as if they hadn't been joined to-
gether (9). In other words, the cells grew in
the normal animal and they didn't grow in the
mouse of genotype Sl/Sl ^ . Either there is not
a factor that circulates in the peripheral blood
or it's so unstable that before it can get from
the normal to the anemic mouse it's gone. We
don't know which it is, but this just makes the
whole system difficult to study, because it
means one is looking at relatively short range
factors. We haven't yet had any good ideas on
how to investigate a short range factor of this
type.
The third type of mutant that we have studied,
f/f has quite a different kind of anemia. It's
a transitory anemia which just shows up in the
embryo and the new born and apparently cures
by the time the animal is two weeks of age (11).
The first experiment we did on mice of geno-
type f/f was as follows : we thought a transitory
anemia might be more interesting because we
might see shifts in the properties of the cell
depending on the age of the animal we took them
from, so we took animals of this genotype of
various ages and tested their marrow cells for
ability to form colonies, as compared with cells
from normal animals of the same inbred strain.
We found that marrow cells from the controls and
from the mutant animals produced spleen col-
onies with the same efficiency. In other words,
we got the same number of colonies from the
same number of marrow cells transplanted.
However, in the case of cells from /// donors,
independent of their age, the colonies were dif-
ferent in composition. They were smaller, and
when we tested them in various ways, they
showed fewer erythrocyte precursors. Experi-
ments done in collaboration with Dr. Margaret
W. Thompson and Dr. John Fowler (12, 13), have
indicated that this deficiency is specific, in that
both the number of granulocytes and the number
of new stem cells, produced by rapidly pro-
liferating transplants derived from /// donors,
appears to be normal. Apparently, the defect in
cells from mice of genotype f/f causes them to
be late in arriving at the stage where they begin
hemoglobin synthesis. If the cells are stimulated
to proliferate at their maximum rate, then this
defect appears in cells from f/f donors, inde-
pendent of their age. Thus, the "curing" of the
anemia in adult mice is not due to repair of
the defect, but is apparently, the result of its
being masked by the decreased demand for pro-
duction of new erythrocytes in the adult animal.
In any event, the defecting// mice appears
to be intrinsic to the stem cells, and seems to
affect their ability to differentiate toward the
production of red cells. What the exact nature
of the defect is, we don't know. Of course, there
is a hormone which specifically stimulates cells
of the blood-forming system to proliferate and
72
to produce red cells. That is the hormone ery-
thropoietin (14). It's possible that this mutation
is affecting the cells that have committed them-
selves to erythropoietic differentiation but have
not actually begun to synthesize hemoglobin, and
that what it does is to decrease the ability of
these cells to respond to erythropoietin. This is
one possible hypothesis - that they do not respond
normally to erythropoietin, so they are late in
initiating hemoglobin synthesis.
Now, if this view is correct, the defect in
mice of genotype f/f is manifested at a stage
prior to the initiation of hemoglobin synthesis.
Apparently, here is a case of a cell being al-
ready committed to erythropoietic differentia-
tion, since the defect appears to be specific for
that pathway; and yet the defect is manifested
at a stage prior to the synthesis of hemoglobin.
It seems that there must be a whole set of con-
trols of early differentiation which act well be-
fore the cell actually begins to differentiate to
synthesize hemoglobin. Thus, these controls
may have nothing to do with regulation of the
stable messengers or ribosomes involved in
hemoglobin synthesis, since they're acting at
stages much earlier than that. One would like
to know whether or not this type of early dif-
ferentiation involves a different means of regu-
lation.
GROSS: Why do you think that kind of
regulatory process would involve things other
than ribosomes and messenger and what's your
suggestion for the other things?
TILL: Simple prejudice. I have no evidence
whatsoever.
GROSS: I've heard other people make that
statement. It would be very nice if one knew
about these things. However if you want to make
hypotheses that one can test about what makes
a cell decide to do something, you have to use
things that you know about.
TILL: Let me take a possible example.
There is a system in the mouse which regulates
whether or not a transplant will grow, if you
take it from one inbred strain to another. It's
the histocompatibility system. There are sev-
eral genes that have been mapped that are con-
cerned with this. The principal one is the H-2
locus. This is a complex locus but apparently
one of its functions is to control the synthesis
of an antigen on the surface of the cell. This
antigen is what the new host reacts to if you
transplant the cells into an unrelated host. Since
such transplantations of cells do not occur in
nature, one may ask: What is the normal func-
tion of the surface antigen, if any? One sugges-
tion which has been made is that the real func-
tion of the H-2 locus is a regulatory function in
the normal animal (15). If so, the antigen we
detect when we transplant cells from one animal
to another is part of a normal regulatory sys-
tem. This antigen is known to be on the cell
surface and it's possible that cell surfaces are
what are involved in regulation of this level.
GROSS: I'm still afraid that it is just a
matter of principle which is under discussion.
It's entirely conceivable that cell surfaces do
differ dramatically from one differentiation path
to another, and that if they do differ, they differ
because their macromolecular content is dif-
ferent. If they have different proteins, then the
initial event still has to arise in the genome.
TILL: I think there is a misunderstanding
here. I'm certainly not suggesting that all the
recent work on the mechanisms of regulation
of protein synthesis is incorrect, nor that there
is some new system of protein synthesis that
has nothing to do with messengers or ribo-
somes. It is possible that the initial stages in
the regiilation of the proliferation and dif-
ferentiation of erythropoietic cells may have
nothing directly to do with the production of
messenger for the synthesis of hemoglobin.
The synthesis of hemoglobin may represent
a very late stage in the differentiation process.
If so, then perhaps studies of the control of
the formation of messenger for the synthesis
of hemoglobin by cells may tell one relatively
little about the very early steps of differentia-
tion. The initiation of hemglobin synthesis may
be just one byproduct of other events which
took place long before, in the history of the
progenitors of those cells.
POLLARD: Have you done any fractiona-
tion at all of the cells at this stage where ery-
thropoietin might take over?
TILL: No. A number of people are doing
this, though.
POLLARD: If it's the surface that you're
talking about, then this is a place where a lot
of material is located.
TILL: Oh yes, I think one could get at this
experimentally.
EPEL: Can you transfer these cells to
other parts of the body? Do they go any other
place and proliferate?
TILL: Yes, the cells that we put into ir-
radiated recipients seem to proliferate in any
place where hemopoietic cells should pro-
liferate.
PAPACONSTANTINOU: Do these others
show the same composition that you get in the
spleen?
73
TILL: Yes. They're just easier to see in
the spleen. Fetal liver, which is a blood forming
tissue, also will make spleen colonies which
look very much like the colonies you get from
adult marrow.
This brings up another point about fetal and
adult hemoglobin. I was quite interested in your
suggestion that you get a different number of
your LDH set predominating, depending on
whether the cells are proliferating or not, be-
cause this same thing has been suggested for
adult and fetal hemoglobin (16). It's not the
adult versus the fetus so much that counts; it's
how fast the cells are multiplying. This is
hard to test but it has been suggested.
PAPACONSTANTINOU: We might be able
to use our system for such a test if we could
place fetal cells in culture and get them to dif-
ferentiate.
TILL: I'm hoping we'll be able to test this
for fetal versus adult hemoglobin.
GRUN: There's a possibility that you could
get at this question of inhibition versus the pos-
sibilities of supplying the missing ingredients by
crossing these two mice. Then, if there were an
inhibitory substance produced by one mouse, you
might expect the hybrid, also, to have this in-
hibitory process produced by one of the alleles.
TILL: The trouble with the hybrid (con-
taining two mutant SI and two mutant W alleles)
is that it's got both things wrong with it. It's
got a defect in the cell aiid a defect in the host.
So it's not the same as doing a transplant.
J. WRIGHT: Have you used hybrids?
TILL: Hybrids have been made at The
Jackson Laboratory, and I believe that they
die right away.
GRUN: I suppose you could try to go at it
by crossing the mutant with the normal.
TILL: The cross is a different thing. The
transplant lets you vary the genetic composition
of the cell independently of the genetic composi-
tion of the host, which you cannot do by any kind
of a cross that you can devise.
KOHNE: Can the mosaics of these mice
live?
TILL: That Idon'tknow about. We've essen-
tially made a mosaic but I don't know of a
naturally occurring one. Igather it is technically
feasible in a developing system at a very early
stage, after fertilization, to make a composite
embryo and get a mosaicthat way (17). However,
I don't think it's been done with these mutants.
KOHNE: How long do the chimera live?
TILL: Dr. Beatrice Mintz has raised them,
but she works primarily with a t mutation. I
don't think she's tried this with these mutants.
EPEL: Are the kinetics of the iron uptake
in the ///cells the same as the controls once
it starts?
TILL: The uptake per synthesizing cell ap-
pears to be the same in rapidly proliferating
cells from /// animals as in cells from controls.
The proportion of cells which are undergoing
synthesis is less for the cells iromf/f mice
than in the controls (13).
GRUN: Maybe I'm missing a point in this
thing. The question that you're asking is, does
this Sl/Sl '* mouse form something which is in-
hibitory in the developing animal or not? Now, if
you formed an Slsl the SI allele in the het-
erozygote would presumably still be forming
inhibitory substance if there is an inhibitory
substance there and the SJ is a dominant con-
dition.
TILL: It isn't. One sees a very nearly nor-
mal blood picture. Now, this suggests to me
that SI is failing to supply some nutritive re-
quirement to the stem cell rather than that SI is
forming an inhibitory substance.
GRUN: In that case, in the parabiotic it
should have been filled.
TILL: It might still be unstable. We were
very disappointed in the parabiosis experiment.
74
References
1. J. E. Till and E. A. McCulloch. Rad. Res
14, 213 (1961).
2. E. A. McCulloch and J. E. Till. Rad. Res.
16, 822 (1962).
3. L. Siminovitch, E. A. McCulloch and J. E.
TILL. J. Cell. Comp. Physiol. 62, 327
(1963).
4. A. J. Becker, E. A. McCulloch and J. E.
Till. Nature 197, 452 (1963).
5. L. Siminovitch, J. E. Till and E. A. McCul-
loch. J. Cell. Comp. Physiol. 64, 23 (1964).
6. E. S. Russell. In "Methodology in Mam-
malian Genetics," W. J. Burdette, ed.
(Holden-Day, San Francisco, 1963), p. 217.
7. A. J. Becker, E. A. McCulloch, L. Simino-
vitch and J. E. Till. Blood 26, 296 (1965).
8. E. A. McCulloch, L. Siminovitch and J. E.
Till. Science 144, 844 (1964).
9. E. A. McCulloch, L. Siminovitch, J. E. Till,
E. S. Russell andS. E. Bernstein. Blood 26,
399 (1965).
10. E. S. Russell, S. E. Bernstein, F. A. Lawson
and L. J. Smith. J. Natl. Cancer Inst. 23,
557 (1959).
11. H. Griineberg. "The Genetics of the Mouse"
(Martinus Nijhoff, The Hague, 1952), p. 239.
12. M. W. Thompson, E. A. McCulloch, L.
Siminovitch and J. E. Till. Brit. J.
Haematol. 12, 152 (1966).
13. J. H. Fowler. Personal communication.
14. L. O. Jacobsen and M. Doyle, eds. "Ery-
thropoiesis" (Grune and Stratton, New York,
1962).
15. G. Moller and E. Miiller. Nature 208, 260
(1965).
16. C. Baglioni. In "Molecular Genetics," J. H.
Taylor, ed. (Academic Press, New York,
1963), p. 405.
17. B. Mintz. Amer. Zool. 2, 432 (1962).
75
THE STRUCTURE OF ISOZYME SYSTEMS AND THEIR
ROLE IN DEVELOPMENT
Edward J. Massaro
Department of Biology, Yale University, New Haven, Connecticut
In recent years, the study of isozymes has
expanded to such a degree that an attempt to
cover the field at a conference of this nature
would probably be only of minimal value. There-
fore, what I intend to do today is to use the
lactate dehydrogenase system of isozymes as a
model and present to you a fairly detailed view
of some of the work that has been and is being
pursued in this area.
The individuality of cells, that is their
phenotype, is expressed in large measure by
the activitiesof their constituent enzymes. These
enzymes are the products of a complex series
of metabolic events that are under genetic con-
trol. In the broadest terms, enzyme biosynthesis
involves DNA transcription into RNA and RNA
translation into the linear amino acid sequence
or primary structure of a polypeptide chain.
Each polypeptide chain then assumes the charac-
teristic three dimensional conformation of its
secondary and tertiary structure. In numerous
instances these intricately folded polypeptide
chains are enzymatically inactive until they be-
come aggregated into more complex units (the
quarternary structure of the enzyme).
The essence of the relationship between
DNA and enzyme structure has been summed-up
in the so-called one gene-one enzyme hypothesis.
A logical consequence of this hypothesis is that
the cells of a homozygous organism should
synthesize identical replicas of all of their con-
stituent protein molecules. But it has become
abundantly evident in recent years that numer-
ous proteins, including many enzymes, exist in
several physically distinctforms within the cells
of a single organism. The multiple molecular
forms of enxymes have been termed "iso-
zymes" (1).
The isozymes of LDH exhibit both species-
and tissue-specific patterns (2). Furthermore,
during the course of embryonic development,
these patterns undergo profound, albeit gradual,
changes (3). From such observations it seems
reasonable to propose that the remarkably
characteristic isozyme pattern of each tissue
reflects a physiological uniqueness of the indi-
vidual isozymes which is superimposed upon
their essential similarities. The existence of
isozymes then poses important questions con-
cerning their biosynthesis and their enzymatic
and physiological activities.
LDH is ubiquitously distributed in isozy-
matic forms among vertebrates and also occurs
as such in numerous other organisms. It is an
oxido- reductase catalyzing the inter conversion
of lactate to pyruvate. This reaction is mediated
through the cofactor nicotinamide adenine di-
nucleotide (NAD). During periods of relative
anaerobiosis the enzyme functions to provide a
reservoir for the storage of hydrogen by form-
ing lactate which occupies a metabolic dead end.
This aids in maintaining the supply of NAD needed
at an earlier step in the glycolytic pathway. When
adequate supplies of NAD are again available,
lactate is oxidized to pyruvate. These reactions
are summarized in Fig. 1. It should be noted
that all the enzymes indicated on this chart, ex-
cept the three denoted by an asterisk, have been
shown to exist in multiple molecular forms. Re-
cent evidence indicates that triosephosphateiso-
merase also probably exists in isozymic forms.
Although early investigations had un-
doubtedly demonstrated the existence of multiple
molecular forms of LDH, their biological sig-
nificance was not at first recognized (4, 5). In
1957 Vesell and Beam (6) described the existence
of three LDH isozymes in human serum and
changes in their proportions during various
disease states. About the same time, Wieland
and Pfleiderer (7) independently discovered the
existence of multiple molecular forms of LDH
and demonstrated tissue specific patterns of the
77
KOSPHORYLASE
GLUCOSE METABOLISM
giuco3«
KJNASE
HEXOKJ
G6 PDH
NADP* ^^NADPH, *-^^''
.b 6 - phosphogluconott
6PG0H
NAD PH. *
froctosp-6- phosphofc
ffucfose -1,6 -diphosphote
NADPH
flbulose-5- phosphate
{Jihydfo< yocetone *
phosphofe
t ALOOLAS
Pentose Shuni
glyceroldehyde - 3 -
phosphate
Fig. 1.
The glycolytic pathway. Under anaerobic conditions pyruvate is converted to lactate with
the concomitant oxidation of NADH 2 toNAD. (FromMarkert, in The Harvey Lectures, Series
59, 187, 1965; reproduced with permission of Academic Press.)
isozymes. In 1959 a very sensitive and con-
venient method for analyzing LDH in tissue
homogenates was developed by Markert and
Miller (1). They coupled starch gel electro-
phoresis with a histochemical staining pro-
cedure for visualizing dehydrogenases. This
facilitated the clear demonstration of tissue,
ontogenetic, and species specificity of LDH
isozyme patterns as well as the isozymes of a
variety of other enzymes. With these data the
biological significance of isozymes received
general recognition As more data were accumu-
lated it became apparent that most mammalian
tissues contain five principle LDH isozymes and
that the electrophoretic mobility of isozymes
exhibits a high degree of specificity, as illus-
trated in Fig. 2.
Although there are readily discernible dif-
ferences in electrophoretic mobility among the
homologous LDH isozymes of widely different
vertebrate species, it is observed that the over-
all isozyme patterns of homologous tissues are
remarkably similar. This is clearly illustrated
by comparing the LDH isozyme patterns of a
given tissue, such as heart muscle, from several
species as in Fig. 3. In general, vertebrate
heart muscle is richest in the more rapidly
migrating isozymes, LDH-1 and LDH- 2, while
vertebrate skeletal muscle is richest in LDH- 5
and LDH-4. At the present time, the major
apparent exceptions to this generalization are
found among the fishes. For example, as shown
in Fig. 4, the heart muscle of the whiting ex-
hibits a remarkably bizarre pattern of LDH iso-
zymes which is difficult to interpret. A few
other exceptions should be noted. Skeletal muscle
78
(+)
• ••—
Fig. 3,
Zymogram demonstrating the similarities In LDH iso-
zyme pattern of heart tissues from 4 different species:
the mouse, cow, rabbit and chicken. Heart muscle LDH
from most vertebrate classes consists mainly of the more
anodally migrating Isozymes, LDH-1 and -2.
Fig. 2.
Zymogram of the Isozyme patterns of mixed tissue homo-
genates from 10 mammalian species. Note the differences
In electrophoretic mobility of homologous Isozymes
among the different species. LDH-1 is the fastest or
most anodally migrating (most negatively charged) band.
The slower moving bands, possessing a progressively
decreasing negative charge, are designated LDH- 2,
LDH-3, LDH-4, LDH-5, respectively. In our electro-
phoretic system, LDH-5 is essentially neutral and its
apparent cathodal movement is the resultant of electro-
endosmosis within the electrophoretic medium (starch
gel). (From Markert, in The Harvey Lectures, Series 59,
187, 1965; reproduced with permission of Academic
Press.)
which is capable of indefinitely sustained
("heart- like") activity, such as the breast
muscles of certain birds (8), and, as we have
recently observed, the flight muscles of bats (9),
contains a predominance of rapidly migrating
LDH isozymes. The similarities in LDH isozyme
patterns among homologous tissues are em-
phasized by the differences in the isozyme pat-
terns among heterologous tissues. This is
PIKE
WMf
TING
/
4
i
i
mm «
\
ORIGIN -* — *Jii 'w
C-)
i
MIWHW ■
1
Fig. 4.
Zymogram of the LDH patterns of representative tissues
of the pike (left) and the whiting (right). Both fish are
members of the order Ostelchthys. Note the complex pat-
tern of Isozymes found in heart tissues of the whiting.
79
illustrated by comparing Fig. 5, the isozyme
patterns of various tissues of the Rhesus
monkey, with Fig. 3, the isozyme patterns of
the heart tissues of several other vertebrates.
The predominance of LDH-1 and LDH-2 in
heart muscle and LDH-5 and LDH-4 in skeletal
muscle suggests that the different isozymes have
different physiological roles. In general, tissues
possessing a highly aerobic metabolism (e.g.
heart, brain, kidney cortex) contain mainly the
most negatively charged isozymes, LDH-1 and
LDH-2, while those tissues possessing a high
anaerobic metabolism contain mainly LDH-5
and LDH-4. These observations seem to indi-
cate that the net charge on an isozyme mole-
cule may be important in determining its intra-
cellular localization and may thus be a reflec-
tion of its metabolic role.
The stable isozyme pattern of adult tissues
must have arisen at some time during develop-
ment through a sequence of orderly changes.
This has been clearly demonstrated by the
direct analysis of tissues of the mouse at dif-
ferent stages of development (3) and is illus-
trated in Fig. 6 and 7. LDH-5 is the predomi-
nant isozyme in embryonic mouse tissues (Fig.
7). As development progresses the isozyme
pattern migrates, in effect, toward the anodal
end of the electrophoretic spectrum so that in
most tissues an increasing proportion of enzyme
activity becomes localized at the LDH-1 end
of the spectrum. Only in those adult tissues,
such as liver and skeletal muscle (Fig. 6), in
which LDH-5 is the predominant isozyme, is
the redistribution of enzyme activity during
development relatively insignificant.
From these studies there was no indication
that the isozyme patterns of the different tis-
sues shift synchronously, and it is quite obvious
that all do not shift to the same extent. How-
ever, the direction of the shift, when it occurs
at all, is the same for all tissues. In some tis-
sues, such as mouse heart muscle, the change
in isozyme pattern was sufficiently rapid to
exclude a corresponding change in cell popula-
tion. Therefore it would seem that isozyme
patterns must change within individual cells.
It is interesting to note that LDH- 1 is the pre-
dominant isozyme in embryonic birds and that
during development, in contrast to the situation
in mammals, the isozyme patterns shift toward
the LDH-5 end of the spectrum so that in adults
the LDH patterns in homologous tissues of
birds and mammals are reasonably similar.
From the standpoint of the one gene-one
enzyme hypothesis, the discovery of multiple
molecular forms of enzymes presented per-
plexing problems concerning the genetic con-
trol of protein biosynthesis. To reconcile the
phenomenon of isozymes with this hypothesis,
it was proposed that a single gene controlled the
synthesis of a single protein which could be
folded into five alternative configurations each
possessing a different net charge. In order to
test this hypothesis an attempt was made in our
laboratory to reversibly unfold and refold the
LDH molecule. Beef heart LDH was treated
with urea or guanidine-HCl to disrupt the hydro-
gen bonds maintaining the characteristic tertiary
structure of the molecule. LDH is readily de-
natured and inactivated by these reagents but all
attempts to reactivate the molecule by removal
of the denaturing reagents were unsuccessful.
A study of the nature of the products of de-
naturation was then undertaken. Denaturation of
a preparation containing all five isozymes re-
sulted in the appearance of only two protein
bands following electrophoresis in acrylamide
gel. During the denaturation procedure, three
bands had disappeared. This unanticipated re-
sult combined with sedimentation data opened
the door to our present understanding of the
structure of LDH. From previous ultracentri-
fugal and other studies the molecular weight of
native LDH had been calculated to be about
135,000 and the 5 major isozymes were all
shown to possess identical molecular weights.
However, when the guanidine denatured prepa-
ration was analyzed in the ultracentrifuge, the
molecular weight was shown to be about 35,000
or approximately one-fourth that of the native
protein. This data is summarized in Table I.
The conclusion drawn from these results was
that LDH exists in the native state as a tetramer
composed of four equal sized (approx. M. W. =
35,000) subunits (10). As shown from acryla-
mide electrophoretic data these subunits exist
as two electrophoretically distinct species
designated A and B (11). It is obvious that ran-
dom assortment of the two kinds of subunits
into all possible combinations of four yields
five isozymes of the composition shown in Fig. 8.
Several tests can be performed to verify
the subunit hypothesis of LDH isozyme struc-
ture. This hypothesis assumes that LDH-1 con-
sists entirely of B subunits, while LDH-5 con-
sists of only A subunits. It follows that LDH- 1
and LDH-5 must be distinct protein species.
This could easily be verified by a complete
amino acid analysis of each of these isozymes.
Accordingly, both LDH-1 and LDH-5 were pre-
pared in pure form by electrophoresis of crys-
80
(+)
LDH - I
2
ORIGIN
(-)
It
V 1^
v.^-.^^.
>'^J^^^
LDH ISOZYME
PATTERNS
OF MOUSE TISSUE
(+)
:9
M
S
P
1
3 'dgttiHHfefc'
••
4MHB
»
9 ^^^^^^^^^1
1
m
(-)
,^^'^
Fig. 6.
Zymogram of the LDH Isozyme patterns of selected tis-
sues of the adult mouse. Note that each of the tissues
possesses a distinct proportion of the Isozymes. Equal
allquots of total enzyme activity from each tissue were
applied to the origin.
Fig. 5.
Zymogram of the LDH Isozyme patterns of adult Rhesus
monkey tissues. Each tissue Is distinguishable by the
relative proportions of the isozymes that it contains.
Adult heart muscle is rich In LDH-1 and -2 while the
proportion of these fast moving loszymes is strikingly
reduced or essentially absent in adult skeletal muscle
In which LDH-3, -4 and -5 predominate. Note, however,
that all five of the major isozymes are present in most
tissues albeit in different proportions. (From Markert,
in The Harvey Lectures, Series 59, 187, 1965; reproduced
with permission of Academic Press.)
talline preparations which contained several of
the isozymes. The pure isozymes were subse-
quently hydrolyzed and their constituent amino
acids were analyzed by the method of Moore,
Spackman, and Stein (12). The results of such
analyses, shown in Table II, establish unequiv-
ocally that from the standpoint of amino acid
composition the two kinds of subunits are dif-
ferent proteins. In addition, in full accord with
the subunit hypothesis, LDH-3 consisting of 2
A and 2 B subunits, was shown to have an
amino acid composition intermediate between
that of LDH-1 and LDH-5 (13, 14). The amino
acid analyses also revealed that beef heart
LDH-1 contains 128 arginine and lysine resi-
dues calculated on the basis of a molecular
weight of 135,000. Consequently, denaturation
followed by trypsin digestion would be expected
DIAPHRAGM
(+)
•SSSto 2
mm '
a ■ s a:
-1 +3 +21 ADULT '"'
HEART
(+)
wm^'.^ ^^B
Wi^'
viBfl
f^^^^Kk^Mmjjk
-9 -5 -1
1 5
(-)
+ 12 +21 ADULT
Fig. 7.
Zymogram demonstrating the shift in LDH isozyme pat-
terns during development of representative tissues of the
mouse. The negative numbers along the abscissa indicate
days before birth and the positive numbers indicate days
after birth. The numbers along the ordinate designate the
isozymes. With time, there is an increase in LDH activity
at the anodal end of the electrophoretic spectrum and a
concomitant decrease at the cathodal end.
81
to produce about 128 peptides. The actual num-
ber of peptides found by this technique was about
thirty or one-fourth the expected number. This
result certainly reinforces the proposal that
LDH- 1 is a tetramer composed of four identical
TABLE I
Molecular Weights of Lactic Dehydrogenases Determined
in the Multichannel Short Column Equilibrium Cell, Using
Schlleren Optics
LDH-1
i
aOb*
LDH- 2
I
A^b3
LDH- 3
J.
A2b2
Fig. 8.
LDH-i*
i
LDH- 5
1
a'*bo
Proposed subunit composition of the five major isozymes
of LDH. LDH-1 consists entirely of B subunitsandLDH-5
consists entirely of A subunits. The intervening iso-
zymes, LDH-2, -3, and -4, are the various combinations of
the A and B subunits.
Phosphate Buffer
Guanidine-HCl*
pH 7.2
Beef Heart LDH-1
134, 000
34, 000
Beef Heart LDH-V
140,000
35, 000
Pig Heart LDH-1
132,000
34, 000
* a V of 0. 740 has been assumed in all calculations.
TABLE II
Amino acid composition of LDH isozymes from
beef muscle.
Amino Acids
Lysine
Hist idine
Arginine
Aspartic Acid
Threonine
Serine
Glutamic Acid
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Number of amino acid residues per molecule of enzyme
LDH-1 LDH- 5
94
25
34
123
56
92
124
42
91
72
135
32
86
130
26
19
95
34
52
104
62
61
135
63
100
122
82
20
73
118
35
26
Based upon a molecular weight of 135,000 (assuming 12 residues of cysteine
and 30 residues of tryptophan in each isozyme).
subunits. Beef LDH-5 was subjected to the same
type of analysis and also yielded about thirty
peptides. A comparison of the peptide maps of
LDH-1 and LDH-5 of beef revealed that some
of the peptides were common to both of these
ioszymic forms, but most were clearly dif-
ferent. It may be concluded from these observa-
tions that the A and B polypeptides are related,
but long stretches of the primary structure must
be quite different.
Perhaps the best test of a subunit hypo-
thesis of isozyme structure is the dissociation
of the active polymers into their constituent
monomers and reassociation of the monomers
into new active configurations. It was discov-
ered in our laboratory that this can be readily
achieved by freezing and thawing equal quan-
tities of LDH-1 and -5 in neutral phosphate buf-
fer which is one molar in NaCl (15). After
thawing, an aliquot of the preparation is analyzed
by electrophoresis in starch gel and subsequent
staining of the gel slab for LDH activity. Prepa-
rations treated in this manner show all five
isozymes in the proportions of 1:4:6:4:1, the
expected binomial distribution of isozymes as-
suming the A and B subnits associated in a
random manner.
In contrast to the irreversible denaturation
obtained by treatment of LDH with urea or
guanidine, the salt-freezing technique is quite
mild. It seems possible that the subunits main-
tain their tertiary configurations essentially
intact during this mild dissociative procedure.
Although the salt-freezing technique is by far
the most efficient method for recombining iso-
zymes, saturated salt solutions in the absence
of freezing as well as repeated freezing and
thawing in buffer alone will gradually produce
recombination. The recombination of LDH is not
influenced by NAD and NADH, lactate, or
pyruvate, and is independent, within wide limits,
of the concentration of LDH, The optimum salt
82
concentration for attaining equilibrium recom-
bination ranges from 0.1 to 4.0 M. Concentra-
tions lower than 0. 1 A7 are much less effective.
It is of interest to note that only a few salts
promote recombination and that both cations
and anions play an important role in the process.
Among the effective cations are sodium, potas-
sium, lithium, magnesium, and zinc. Chloride,
bromide, iodide, nitrate and phosphate are
effective anions. Certain other ions, for ex-
ample, borate, sulfate, and tris, inhibit recom-
bination.
From elementary genetic considerations,
since the A and B subunits of LDH are dif-
ferent proteins, they must be under the control
of different genes. Recent genetic evidence bears
this out. An LDH mutant has been discovered in
the deer mouse Peromyscus maniculatus (16).
In these animals, the mutation occurred at the B
locus, and, as theory predicts, the heterozy-
gote produced fifteen isozymes. During the
screening of several diverse human populations
mutants were found at either the ^ or B loci
(17). To our knowledge, no double heterozygotes
have yet been reported.
A third gene controlling the synthesis of a
third type of LDH subunit, designated the C sub-
unit, was discovered by Zinkham and co-
workers (18). C polypeptides appear to be
formed mainly (perhaps exclusively) in the
sperm. Isozymes containing C polypeptides are
responsible for the so-called "X-bands" of LDH
activity found on zymograms of testis homo-
genates. In some mammals only one X-band is
observed and it is assumed to be a tetramer of
C subunits. Several X-bands have been detected
in testis homogenates of other mammals. How-
ever, in these cases it has been shown that the
additional bands are the result of the polymeri-
zation of C subunits with either ^ or B sub-
units (19). More recently, Zinkham and co-
workers have shown that, in pigeons, the C gene
exists in two widely distributed allelic forms
designated C and C' (20). From testicular
homogenates resolved by the technique of starch
gel electrophoresis, they have been able to
classify each pigeon into one of three phenotypic
classes designated CC, CC, and C'C.
Although it is theoretically possible to form
fifteen isozymes from three different subunits
no such number has been observed in sperm
homogenates. The following interpretations of
this may be brought forth. It is possible that
the freedom of combination necessary for the
formation of the fifteen isozymes does not exist
or that the gene controlling the C polypeptide
biosynthesis may be turned on only when the
A and B genes are turned off. It is also pos-
sible that certain hybrid molecules cannot be
formed for purely physical reasons or that
certain hybrid combinations are inactive. How-
ever, a mixture of a, B, and C subunits will
readily recombine in vitro to yield the expected
fifteen different tetramers.
LDH zymograms of many different animals,
especially the rabbit, show that several of the
basic five isozymes exist as two or more distinct
bands of enzyme activity (Fig. 9). An entirely
satisfactory interpretation of the phenomenon
of subbanding is not yet available although
(+)
LDH-
<
a
ORIGIN
(-)
Fig. 9.
Zymogram of LDH patterns of various tissues of the
rabbit. Note the multiple banding, termed subbanding, of
most of the isozymes and the variation in subbanding
which exists among homologous isozymes of different
tissues. The subbanding Is relatively constant for any
particular species but varies considerably among dif-
ferent species. {From Markert, in The Harvey Lectures,
Series 59, 187, 1965; reproduced with permission of
Academic Press.)
83
several hypotheses have been considered. Among
these is the proposal by Fritz and Jacobson
that the subbanding in mouse tissues is the
result of the differential binding of NAD by the
subunits (21). The possibility that a small mole-
cule such as NAD, by becoming attached to the
subunits, can change the net charge, and hence
the mobility of an isozyme, is certainly not un-
reasonable. However, this hypothesis was not
supported in identical experiments with rabbit
LDH. Another interpretation proposes that sub-
bands represent permutations of the tetrameric
combinations. This is supported by the observa-
tion that the mixing and recombination of rabbit
LDH-1 and LDH-5, which in themselves show no
subbanding, yields subbanding at the LDH- 3 posi-
tion. Kaplan and Costello have advanced the
hypothesis that the subbanding in mouse LDH re-
sults from the existence of two different A sub-
units each of which is under the control of a
separate gene (22). This interpretation is
strongly supported by numerous observations of
the existence of the subbanding in the pattern
predicted for two different A subunits. The
presence of such patterns in inbred strains of
mice rules out heterozygosity as an alternative
and suggests the existence of a fourth gene con-
trolling the synthesis of LDH polypeptides.
Clearly no single one of these interpretations
fits all of the data. Indeed, there may be no
all- encompassing explanation.
The existence of the X-bands and subbands
tends to emphasize the fact that starch- gel
electrophoresis resolves mammalian LDH into
five major zones of activity. However, the iso-
zyme pattern can differ considerably among
mammals (Fig. 2) and among different vertebrate
classes, as shown in Fig. 10. It is of interest to
note that the net charge on the B subunit (the
more negatively charged subunit) of mamalian
LDH is apparently greater than that on the
homologous subunit of most other vertebrate
classes as reflected in the greater mobility of
mammalian LDH-1. However, the A and B sub-
units of the vertebrate classes, excepting some
fish, must be remarkably complementary in that
most can be combined to form functional hybrid
molecules (tetramers) of LDH by means of the
salt-freezing technique as illustrated in Fig. 11.
PAPACONSTANTESfOU: Aren't there more
than five bands in the sixth column from the
left? Yet you started out with pure LDH.
MASSARO: Yes, there are more than five
bands. I started out with pure beef LDH- 1 which
was hybridized with rattlesnake muscle LDH and
electrophoresed. Muscle of this species of
rattlesnake contains several LDH isozymes,
seven, in fact.
PAPACONSTANTINOU: Then are the com-
plementary LDH-l's combining?
MASSARO: Yes, however when complemen-
tary LDH-l's are hybridized, if they have very
close mobilities, the hybrid isozymes do not
separate into distinct bands in our electro-
phoretic system.
Let us divert for a minute to a fish story.
We have studied, to date, approximately thirty
species of fish and have found that they can be
placed conveniently into three categories
according to the number of isozymes of LDH
that they possess and the hybridization charac-
teristics of these isozymes. Those fish pos-
sessing a single band of LDH activity, as re-
vealed by starch gel electrophoresis, are placed
in one category. This group consists of the
fluke and related flatfish. In another category
are place those fish possessing either two or
three bands of LDH activity. There are some
twenty-plus species in this group, evenly dis-
tributed between the two and three banded varie-
ties. The third category consists of those fish
possessing more than three bands of LDH
activity. So far we have placed only three species
in this group, the herring (Alosa aestivalis) , the
shad (Alosa sapidissma), and the whiting (Mer-
luccius bilinearis).
Under our conditions, any two of the LDH
isozymes of the herring will readily hybridize
with one another to form the expected auto-
genous hybrid molecules. This is also true for
the isozymes of the whiting, and the shad (i.e.,
those fish possessing three or more bands of
LDH activity). The fluke, having only one band
of LDH activity, obviously does not show auto-
genous hydridization. All four of these species
will also form hybrid molecules with one another
and with mammalian LDH. Significantly, those
species possessing two or three bands of LDH
activity will not form autogenous hybrid mole-
cules although they will hybridize with mam-
malian LDH and LDH from the two other groups
of fish. The factors underlying the lack of auto-
genous hybridization within this group are under
investigation in our laboratory.
Another interesting aspect of this study was
the discovery of a very rapidly migrating band
of LDH activity in the eye of many species of
fish. This band has a mobility greater than that
of mammalian LDH-1 and, like the C tetramers
of sperm LDH, may represent another type of
LDH isozyme.
Further evidence of the remarkable com-
84
ORIGIN
#
i
C-)
i
% % '^^^ % \ \ ^-1
Fig. 10.
Zymogram of the LDH pattern of a representative of
each of the classes of vertebrates. The representative
species are human (mammal), Adelie penguin (bird),
rattlesnake (reptile), Amphiuma (amphibian), fluke (bony
fish), sand shark (cartUagenous fish), and the lamprey.
Note the considerable variation in the number and mo-
bility of LDH isozymes present in each of these orga-
nisms. (From Markert, in The Harvey Lectures, Series 59,
187, 1965; reproduced with permission of the Academic
Press.)
plementarity of the subunits of vertebrate LDH
is presented in Fig. 12, which shows the iso-
zyme patterns obtained when horse LDH is
hybridized with lamprey, fish, or salamander
LDH. These patterns are relatively simple.
More complex patterns are obtained in hybridi-
zations involving other organisms. This is
illustrated in Figs. 13 and 14 in which the
results of the hybridization of chicken LDH
with horse, snake, cow, or rabbit are shown.
TILL: Why are you doing all this?
MASSARO: For kicks: Seriously, one of
our major interests is finding out how the LDH
tetramer is put together. We feel that a study
of various aspects of the phenomenon of inter-
specific hybridization is a valid approach to the
problem.
McCARL: Do you always get the same
patterns of hybridization?
MASSARO: Yes, they are very constantbe-
tween any two given species.
It would be expected that the catalytic prop-
erties of the hybrid molecules differ from those
of the parental types. This is analogous to the
situation encountered with the heteropolymeric
isozymes formed by recombination of LDH-1
and -5 from the same species. From our data,
it appears that, in closely related animals,
H C H C H
(+)
BECr LDH-I flP^^'J^^
C H C H C
# • •
0«l
t
*1 m
0RI6IN
(-)
\ / \ / \
\* *
0 - HYBRID liOZYMC
M - HYBRIDIZED
C - CONTROL
1*1
/ \ — / \ /
X^., %X %>.
<9
Fig. 11.
Zymogram illustrating the hybrid Isozymes of LDH
formed between beef LDH- 1 and LDH from several classes
of vertebrates (lamprey, fish, rattlesnake, penguin LDH-
5, and pig LDH-5). The hybrids are indicated by small
circles. (From Markert, in Ideas in Modern Bi logy, 1965;
reproduced with permission of the National Academy of
Sciences).
85
(+)
LDH
I
LDH
(-)
0= HYBRrO
JSOZYMES
s \ % %^ % % \ \ \
\ \ \ V \
'H^.
\
Fig. 12.
Interspecific hybridization of horse LDH. The Isozymes
of native LDH are designated by the numbers 1, 2, 3, 4,
5, while the hybrid isozymes are designated 0. Fish re-
fers to the herring, -l/osa aestivalis, and salamander to the
newt, Diemictytus viridescens. (From Markert, in The
Harvey Lectures, Series 59, 187, 1965; reproduced with
permission of Academic Press.)
Fig. 13.
Interspecific hybridization of horse LDH. Chicken refers
to White Leghorn chicken heart LDH and snake to the
LDH of pooled tissues of the diamondback rattlesnake
(Crotalus adamanteus). The numbers along the ordinate
designate the isozymes of horse LDH.
interspecific hybrid molecules have catalytic
properties analogous to those of the intraspecif ic
hybrid molecules. However, as the evolutionary
distance between species increases, the
enzymatic activity of the hybridized preparation
decreases. This loss in activity may be due to
the formation of completely or partially inactive
polymers.
Mammalian LDH-1 (the tetramer composed
of B subunits) will combine with LDH- 5 (the
tetramer composed of A subunits) from any of
the other six vertebrate classes to form at
least three hybrid molecules. In an identical
hybridization in which there are two kinds of
B subunits with respect to charge, theoretically,
fifteen isozymes can be formed. If , in addition,
two differently charged A subunits are involved,
then thirty-five different isozymes should be
formed. However, the resolution of thirty-five
isozymes may exceed the capabilities of the
starch-gel electrophoretic system. In any event,
as many as twenty-five distinct bands have been
counted on the zymograms of certain hybridiza-
tions such as between chicken and horse.
The occurrence of isozymes of LDH in
nearly all vertebrates which have been ex-
amined strongly suggests that, for certain en-
zymes, multiplicity of form is evolutionarily
advantageous and does not represent simple
heterogeneity of no biological value. The impli-
cation is that the individual isozymes subserve
a specialized role in the economy of the orga-
nism. This is supported by the fact that, although
all isozymes of LDH catalyze a characteristic
86
chemical reaction, they possess markedly dif-
ferent physical and chemical properties. In the
light of this evidence, it seems reasonable to
conclude that isozymes are groups of molecules
of common origin that have become differentiated
to meet highly specific requirements within the
cell. The specialization of the individual iso-
zymes indicates that they may be located indif-
ferent places within the cell, or concentrated
in different kinds of cells and tissues. Evidence
in this regard has been brought forth by several
investigators (23, 24, 25).
An insight into the physiological role of
individual isozymes has been provided through
determinations of their optimal substrate con-
centrations. The earliest investigation, by Plage-
mann et al., revealed that the optimal pyruvate
concentration for human LDH- 1 is considerably
lower than that for human LDH- 5 (26). This has
now also been established for LDH- 1 and LDH- 5
from other vertebrates (27). For example, in a
series of experiments carried out in our labora-
tory (summarized in Fig. 15), it was observed
that the optimal pyruvate concentration of horse
LDH-1 is distinctly lower than that of horse
LDH- 5. In the case of the fluke whose tissues
reveal only a single band of LDH activity as
analyzed by starch gel electrophoresis, both
heart and skeletal muscle LDH appear to have
identical substrate optima. This and other data
(28) indicate that fluke heart LDH is identical
to fluke skeletal muscle LDH. Since the pyru-
vate optimum of fluke LDH is similar to that of
horse LDH- 5, it seems reasonable to assume
that other properties of fluke LDH would be
similar to vertebrate LDH- 5 and that fluke LDH
is, in effect, an LDH- 5.
These observations are significant in that
LDH- 5 is found mainly in tissues, such as
skeletal muscle, which are subject to periods
of relative anaerobiosis and consequently are
subjected to relatively high concentrations of
pyruvate and lactate due to an increased func-
tioning of the glycolytic pathway and decreased
functioning of the tricarboxylic acid cycle. On
the other hand, LDH-1 is found mainly in well
oxygenated tissues with a high aerobic metabo-
lism such as heart and brain in which high con-
centrations of pyruvate and lactate are not
encountered.
An interpretation of this data involves the
effect of high concentrations of lactate on muscle
tissue. As is well known, during violent€xercise,
lactate can accumulate in skeletal muscle until
the muscle is paralyzed. Obviously, this cannot
be allowed to occur in heart muscle. The inhibi-
tion of heart muscle LDH at relatively low con-
centrations of pyruvate, then, acts as a check
valve which functions to shunt pyruvate into the
( + )
Xcv %. ^xv X^ %. 'V «> .
Fig. 14.
Interspecific hybridization of chicken LDH. The hybridi-
zations were performed with LDH obtained from pooled
tissues of each of the organisms. Note the complexity of
these hybrid patterns as compared to those illustrated in
Fig. 12.(FromMarkert, in Ideas in Modem Biology, 1965;
reproduced with permission of the National Academy of
Sciences.)
87
>
<
X
<
10-" 10"' 10"^
[pyruvate]
Fig, 15.
Pyruvate Inhibition curve for horse LDH-1 and -5 and
fluke LDH-1 and-5. Experiments were performed at 23° C
In 0.1 M sodium phosphate buffer, pH (apparent) 7.0. The
optimal pyruvate concentration for horse LDH- 5 Is higher
than that for horse LDH-1, Fluke heart and skeletal
muscle LDH, which are electrophoretlcally indistinguish-
able, have identical pyruvate optima. These optima are
similar to that of horse LDH- 5,
Krebs cycle so that lactate cannot accumulate
in heart muscle.
As previously mentioned, this correlation
extends to embryonic life. The tissues of mam-
malian embryos which have a relatively poor
oxygen supply contain large amounts of LDH- 5
whereas the well- oxygenated tissues of avian
embryos contain mainly LDH-1.
A fundamental aspect of theinterconversion
of pyruvate and lactate as catalyzed by LDH is
the oxidation- reduction of nicotinamide adenine
dinucleotide. And this may be the most important
function of LDH, The maintenance of the proper
ratio of oxidized to reduced NAD is of con-
siderable importance in that NAD is involved
in numerous metabolic ractions.
In conclusion then, since the intracellular
environment must surely vary from place to
place from time to time within the cell, the
existence of a spectrum of functionally distinct
types of a particular enzyme would allow for a
more efficient and precise control of a metabolic
step. Since the discovery of the isozymes of LDH
more than 100 other enzymes have been shown,
at least tentatively, to exist in isozymic form.
DEERING: Do the isozymes of LDH always
exist with four subcomponents?
MASSARO: Yes, So far as we know.
DEERING: Is this also true of some of the
enzyme systems other than LDH? Are there
always four or do you, perhaps, get combina-
tions of three subunits there? Is there any
reason to expect that they can't exist as dimers
or trimers in some systems?
MASSARO: Isozyme systems other than the
LDH system may be constructed on a dimer or
trimer basis. The isozymes of MDH, malate
dehydrogenase, for example, are dimers.
DEERING: You mentioned when you went
through it the first time that the whiting pattern
was very complex. Can you explain it in terms
of ^, B, and C subunits?
MASSARO: This is quite possible. However,
we have not yet attempted the analysis. The
banding pattern in this fish may be related to the
subbanding in rabbit LDH. The multiple banding
may have something to do with permutations of
the tetramic structure of the individual iso-
zymes. Such permutations could conceivably
change the electrophoretic mobility of the ios-
zymes resulting in the very complex pattern
that we find.
CANTINO: I have a question about the fish
story in general. Do you work exclusively with
frozen fish or freshly caught fish or mixtures
of the two?
MASSARO: We use both fresh and frozen
fish and never mix them unless we are certain
that freezing has had no effect on the LDH iso-
zyme patterns.
CANTINO: You stressed the importance of
freezing and thawing upon recombination.
MASSARO: For tne most part, in intact
tissues, and let me stress intact tissues, not
homogenates, LDH is quite stable. Intact tis-
sues can usually be frozen and thawed without
altering their LDH patterns. In a very few
cases, however, we have seen entirely dif-
ferent patterns between frozen fish and fresh
fish and, I am sure, this can also occur with
tissues from other animal species. Some tis-
sues we have studied could not possibly have
been obtained fresh. For example, we have ob-
88
tained whale tissues from Alaska and penguin
and seal tissues from Antarctica. From our
experience, however, we feel confident that we
are looking at essentially unaltered LDH pat-
terns in frozen tissue.
CANTINO: Does it ever happen that you
get a change in pattern?
MASSARO: If you mean, "can the pattern
be changed experimentally?," there is some
evidence in the affirmative. Kaplan's group
appears to have done this in tissue culture by
varying oxygen tensions.
PAPACONSTANTINOU: The point with oxy-
gen tensions was that they didn't get any changes
unless they used abnormally high oxygen con-
centration. You never would find that in living
tissue. Isn't that about right?
MASSARO: That is true.
LOVETT: May lask a rather naive question,
perhaps? Has anyone looked carefully at some
of these purified isozymes from the point of
view of small molecules that might be function-
ing in a regulatory sense, at different stages
differently? For example, in an embryo as
compared with an adult?
MASSARO: Not that I know of.
LOVETT: There might be something like
intermediates of other pathways, or some other
coordinating system. I' m thinking about function.
For example, how rapidly can it turn over?
MASSARO: To my knowledge, nothing has
been done on this particular aspect of the
problem.
GRUN: I had the impression that the idea
was that there were two genes and that the
tetramers you got were random combinations
of polymers of these two genes. If A were
active, you'd expect to find only, or mostly,
LDH-5. If B were active, you'd expect the re-
verse of this. Some of the patterns that were
on your figures made it look as though there
wasn't a maximum at one end tapering off to the
other end, as though there was something
missing.
MASSARO: Well, you have to keep one thing
in mind when you work with these zymograms.
Each of these isozymes, LDH-1, LDH-5, and
-2, -3, and -4, has different kinetic properties.
When they are placed together into a reaction
mixture which has a certain level of substrate
and allowed to react, those having a higher turn-
over rate are going to show up as bigger,
heavier blobs than those which have a very
slow turnover rate.
PAPACONSTANTINOU: How much varia-
tion in turnover rate is there?
MASSARO: There is about a two-fold dif-
ference between beef LDH-1 and -5.
GRUN: Are -2,-3 and -4 intermediate?
MASSARO: Present evidence suggests that
the properties of these heteropolymers reflect
the proportions of the parental monomers which
they contain.
DEERING: Do you always find that you get
the proper ratios of isozymes if you know the
relative amounts of A and B? Take a situation
in which the amounts of A and B are not equal;
you wouldn't expect a 1:4:6:4:1 ratio in that
case. If you get the actual amounts of A and
B you can always predict the amounts of 1, 2,
3, 4, and 5, or is there the possibility of some
active mechanism which skews this in one direc-
tion or another?
MASSARO: Theoretically you can predict
the distribution of isozymes, all things being
equal.
TS'O: At least, Kaplan thinks so.
DEERING: It's merely a function of the con-
centration of the two?
MASSARO: It looks that way. However,
in vitro the distribution can be skewed by un-
known factors.
PAPACONSTANTINOU: Under all situa-
tions the recombination seems to follow the
binomial theorem. The reason you get the dif-
ferent combinations may be because one is
turning over a thousand times more rapidly than
the other. At least, my impression from Markert
was that they never had any conditions under
which they didn't follow recombination explain-
able by the binominal theorem.
MASSARO: I don't really think that this is
worth pursuing to any great length.
GROSS: You look at a zymogram and you
see a gene product; your conclusions about
the amounts of these gene products depend on
the rate of the reaction in the gel. Has anyone
ever measured how much LDH-1, LDH-2, etc.,
are present in a homogenate? The question
that's implied by this is, does the difference
that you see in an isozymic pattern really re-
flect the difference in quantity?
MASSARO: If we know the turnover rates
of the isozymes under our conditions, it does.
This is the big problem. Of course, one way to
find out is to resolve the isozyme mixture by
electrophoresis, cut out the individual isozymes
and measure the quantities and turnover number
of each. If you get good recoveries for each iso-
zyme you have the answer.
Now, we have done this. Unfortvinately, this
kind of analysis is usually unsatisfactory if
89
etc., a group
evolutionary
evolutionary
My student,
starch gel is employed because recoveries from
starch gel are ridiculously low. Recently we have
been working with an acrylamide gel system
which is quite satisfactory. From the limited
data which we have obtained I would say that
there seems to be a reasonably good relation-
ship between what you see and the quantities
present in the original mixture.
GROSS: Are the genes contiguous?
MASSARO: We don't know.
EPEL: Are the shad, herring,
of fishes that are in the same
family?
MASSARO: Yes.
J, WRIGHT: In terms of the
scale, I think there's no pattern.
Novak, did a survey of LDH in various tissues
in various species and among those, gar and
bowfin are supposedly the most primitive. We
get 5 bands for the gar and only two bands for
the bowfin in almost all tissues looked at. In
contrast, the perch and bass would be further
up on the scale, and these have low numbers
of bands and it varies considerably.
GROSS: Are these stages samples of these
species?
J. WRIGHT: Yes, and there are individual
differences within some of these species.
ZIMMERMAN: I just wonder how you can
explain the two bands in some species. Is this
explained in terms of an A and a. B? Don't you
need a minimum of 5 bands?
MASSARO: The structure of the isozymes
of those species possessing two or three bands
of LDH activity has not been worked out. It is
conceivable, but improbable, that the LDH
molecule of these species is a dimer; if so,
one would not expect to find 5 bands of activity.
Also, it does not necessarily follow that tetra-
meric molecules will produce five bands of
activity since certain combinations of mono-
mers may not be allowed.
TS'O: Did you study the mammalian case?
Do you know whether these subunits have to
function co-operatively or can each individual
subunit function separately?
MASSARO: We don't know, but we are in the
process of attacking this problem.
EPEL: Relating to what forms exist in vivo,
perhaps, in breaking up the cells you're selec-
tively causing some compartmental exchange?
MASSARO: That is possible.
EPEL: If you take tissue which specifically
has LDH-5 and one which has LDH-1, mix the
two homogenates together and then do a zymo-
gram, do you just get 1 and 5 or do you get
intermediates? Do you have to salt-f reeze to get
hybridization?
MASSARO: In our experience you have to
either salt them heavily and freeze them or
salt them tremendously with a very high con-
centration of salt and let them sit around for a
long time before you'll get any hybridization.
J. WRIGHT: What is the relationship of
these movements on starch and acrylamide?
MASSARO: At comparable pH's and gel
densities the movements are reasonably similar
with the exception of LDH-5 which runs toward
the cathode in starch gel under our conditions
and toward the anode in acrylamide.
J. WRIGHT: How about the cathode area of
insertion, now? Do you get LDH-5 moving back-
ward in the area of insertion?
MASSARO: In starch, yes. Although, under
our conditions, LDH-5 is negatively charged,
a strong electroendosmotic effect propels it
cathodally. In acrylamide you do not have an
electroendosmotic effect so it moves toward
the positive pole.
FERGUS: In regard to hybridizing, have
any attempts been made to use some non-LDH
protein?
MASSARO: Yes, we tried it with MDH and
IDH, but got no results.
90
References
1. C. L. Markert and F. MfUler. Proc. Nat.
Acad. Set. U.S. 45, 753 (1959).
2. C. L. Markert. In "The Harvey Lectures,"
Series 59. (Academic Press, New York,
1965), p. 187.
3. C. L. Markert and H. Ursprung. Develop.
Biol. 5, 363 (1962).
4. A. Meister. J. Biol. Chem. 184,111 (1950).
5. J. B. Neilands. Science 115, 143 (1962),
6. E. S. Vesell and A. G. Beam. Proc. Soc .
Exptl. Biol. Med. 94, 96 (1957).
7. T. Wieland and G. Pfleiderer. Biochem. Z.
329, 112 (1957),
8. A. C. Wilson, R. D. Cahn and N. O. Kaplan.
Nature 197, 331 (1963).
9. E. J. Massaro and C. L. Markert. Unpub-
lished (1965).
10. E. Appella and C. L. Markert. Biochem.
Biophys. Res. Comm. 6, 171 (1961).
11. C. L. Markert. In "Hereditary, Develop-
mental, and Immunologic Aspects of Kidney
Disease," J. Metcoff, ed. (Northwestern
University Press, Evanston, Illinois, 1962),
p. 54.
12. S. Moore, D. H. Spackman and W. H. Stein,
Anal. Chem. 30, 1185 (1958).
13. C. L. Markert. In "Cytodifferentiation and
Macromolecular Synthesis," 21st Symp.
Soc. Study Develop. Growth, M. Locke, ed.
(Academic Press, New York, 1963), p. 65.
14. T. P. Fondy, A. Pesce, L Freedberg, F.
Stolzenbach and N, O, Kaplan, Biochem. 3,
522 (1964).
15. C. L. Markert. Science 140, 1329 (1963).
16. C. R. Shaw and E, Barto. proc. Nat. Acad.
Sci. U.S. 50, 211 (1963),
17. E. S. Vesell. In "Progress in Medical
Genetics," A. G. Steinberg and A. G. Beam,
eds. (Grune and Stratton, New York, 1965),
p. 128.
18. A, Blanco and W. H. Zinkham. Science 139,
601 (1963),
19. W. H. Zinkham, A. Blanco and L. Kupchyk.
Science 142, 1303 (1963).
20. A. Blanco, W. H. Zinkham and L. Kupchyk.
J. Exp. Zool. 156, 137 (1964),
21. P. J. Fritz and K. B. Jacobson. Science 140,
64 (1963).
22. L. A. Costello and N. O. Kaplan. Biochim.
Biophys. Acta 73, 658 (1963).
23. J. M. Allan. Ann. N.Y. Acad. Sci. 94, 937
(1961),
24. J. L. Conklin. J. Exptl. Zool. 155, 151
(1964),
25. M, Van Wijhe, M. C, Blanchaer and S. St,
George-Stubbs. J. Histochem. Cytochem.
12, 608 (1964),
26. P. G. W. Plagemann, K. F. Gregory and
F. Wroblewski. J. Biol. Chem. 235, 2288
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91
ANTIGEN SYNTHESIS DURING
REORGANIZATION IN THE CELLULAR SLIME MOLDS
James H. Gregg
Department of Zoology,
University of Florida, Gainesville, Florida
Perhaps most of you are familiar with the
details of the development of the slime molds.
However, I'd like to emphasize certain steps in
their development before continuing with the
remainder of the talk. Figure 1 is a diagram
of the development of two species of slime mold,
Dictyostelium mucoroides and Dictyostelium
discoideum. Aggregation of a homogeneous group
of D. discoideum vegetative amoebae occurs,
which, through morphogenetic movements, forms
itself into a migrating pseudoplasmodium or
slug. Further morphogenetic movement results
in the formation of a mature sorocarp consisting
of a small mass of cells supported by a slender
stalk. Development is similar in D. mucoroides
with the exception that D. mucoroides forms a
stalk as it migrates. Eventually, a fruiting body
is formed, again consisting of a mass of cells
supported by a slender stalk.
If we examine a fruiting body of D. discoi-
deum closely, we find that it has developed pro-
portionally; that is, regardless of the size of the
cell mass, about 70% of the cells differentiate
into spores and the remaining 30% differentiate
into stalk cells. The basis for this proportional-
ity arises by the time of the migration stage.
At this time two types of cells have differentiated:
the so-called prespores and prestalks. Now, in
D. mucoroides as stalk formation occurs con-
tinually during migration new prestalk cells
are formed from the prespore mass. Thus, at
any point during migration there is a constant
proportionality between the prespore cells and
the prestalk cells, which results in the forma-
tion of a proportional sorocarp.
The question arises, what is the mechanism
involved in establishing this proportionality?
Obviously it's a problem with the differentiation
of two types of cells initially. More specifically,
it's a problem in which two types of cells must
D. mucoroides
6
0
^
J}=^ A
k.
Vegetotive A«gr«gotlon Migrotion Preculmlnofion Culminotien
O^
Moturt
Sportt
Fig. 1.
The developmental stages of D. discoideum and D. mucoroides.
93
differentiate in particular numbers.
One of the ways in which cell differentiation
may be studied in these two slime molds is by
immunological methods; and in this seminar
today I want to talk about the use of fluorescent
antibody in studying differentiation. This par-
ticular method was first employed by Takeuchi
(1) in studies on Dictyostelium. This seminar is
based upon a study which has recently been
accepted for publication (2).
The first step in doing an immunological
study involves the production of antisera (Table
I). Antibody was produced to three species or
strains of slime molds: D. discoideum, D, mu-
coroides (strain TYP) and a mutant of D. muco-
roides isolated and reported by Filosa (3),
These antisera were made to vegetative amoe-
bae, migrating pseudoplasmodia and mature
sorocarps, in each instance; that is, all three
stages were used in producing the antisera of
any one species. Now, the antiserum was con-
jugated with fluorescein iso-thiocyanate by more
or less conventional means, the salient points
of which involved the precipitation of gamma
globulin by cold methanol, weighing of a small
sample of the globulin solution with a micro-
balance in order to determine the total amount
of globulins in the sample, and mixing the
globulins with 0.0188 mg of fluorescein per mg
of globulin (an amount we found to be optimum).
Following conjugation at 5^C for 15 to 18 hours,
the samples were centrifuged and then run
through a Sephadex column to remove nonin-
corporated fluorescein from the labeled globulin.
Such serum was used in staining various stages
of the slime molds. Unless otherwise indicated
homologous antiserum was used in the staining
procedure.
Figure 2 shows D. mucoroides amoebae
removed from an aggregating stream. We find
that such cells, or such groups of cells, re-
moved from the stream will stain with various
intensities. Note the two extremes here: very
dark cells which stained with little intensity and
other cells which stained with a considerable
intensity. I believe these correspond to the so-
called 'Tjright" and "dark" cells which Takeuchi
(1) reported. I'll discuss the possible signifi-
cance of these cells later on.
The early aggregates were sectioned at
about 5 microns. Although bright and dark cells
appear in the aggregating stream, once the cells
aggregate to form a cell mass in the early
aggregate the stain is more or less homogeneous
TABLE I
Preparation of Conjugated Antisera '
1. Ganuna globulins precipitated from 1.0 volume serum by cold methanol.
Reagents and fractionation procedure described by Dubert _et ^. (8) .
2. Globulins redissolved in 0.85 volumes of 1.0% NaCl .
3. 100 ;ul aliquots of globulin solution dried and weighed, on Cahn ultra-
micro balance. Correction calculated for weight of NaCl in aliquot.
4. Globulin solution dilutea with 0.15 volumes of 1.0 M carbonate-
bicarbonate buffer at pH 9.0.
5. Globulin solution placed in 250 ml Erlenmeyer flask. Ice crystals
produced in globulin solution by immersing flask in dry ice-methyl
cellosolve bath (9) .
6. In presence of ice crystals 0.0188 mg fluorescein iso-thiocyanate
added per mg globulin and mixed with magnetic stirrer at 5°C for
15-18 hours (10) .
7. Centrifuged 20 minutes at 3000 X G in refrigerated centrifuge to
remove particulate niatter resulting from conjugation.
8. Purification of f luorescein-conjugated globulins utilizing a G-25
fine Sephadex column (Pharmacia Fine Chemicals, Inc.) (11).
' From Gregg, 1965 (2), reproduced with permission of Developmental Biology, published
by Academic Press.
94
(Fig, 3). You cannot detect that prestalk cells
have differentiated at this stage. In the late
aggregate prestalk cells begin to differentiate
(Fig. 4). These prestalk cells are characterized
by the fact that they tend to lose their cyto-
plasmic antigens. Consequently, they do not
stain with high intensity. At the same time you
see spots of intense staining in the prespore
cells which mark the synthesis of prespore
antigen. Consequently, all the cells in this area
form prespore cells and the anterior cells which
stain the least become prestalk cells.
Figure 5 shows a migrating pseudoplas-
modium of D. mucoroides. This has been stained,
however, with the normal conjugated antiserum.
Little or no staining was found with normal con-
jugated serum. The preparation itself tends to
transmit light in such a way that it appears to
be bright, but the fluorescent staining is rela-
tively low.
LOVETT: Is that region in the center
the stalk?
GREGG: Yes. Figure 6 is another D. muco-
roides slug stained with the antiserum. You can
see that stalk formation is occurring; the stalk
runs down through the center of the slime mold.
The prestalk cells in the anterior area are fully
differentiated now, resulting in the formation of
a proportional slug. The prespore antigen in-
creases in the prespore cells throughout the
entire area. This results in a sharp delineation
between the prespore cells and the prestalk
cells. Thus, by this time these two types of cells
have developed with the prestalk cells always
in the anterior or leading end of the slug. The
question arises, how does this polarity develop?
Takeuchi has suggested that the bright-staining
and dark-staining cells that he found - and that
I have seen - in the aggregating streams sort
out during aggregation. Simultaneously, the
dark-staining cells lose even more of their
staining and eventually end up in the anterior
tip, thus composing the prestalk area. Conse-
quently, the brightly- staining cells form the
prespore area.
MASSARO: What is the magnification here?
GREGG: That's about 120X.
DEERING: What's that film along the edge
of the slug in some figures? Is it something that
peeled off?
GREGG: It's a slime track or slime sheath
that's produced along the edges of the slime
mold.
Now, it's possible that bright-staining and
dark-staining cells sort out to form these two
areas. However, the slug has developed propor-
tionally, and in order to account for this we
would have to assume that the prestalk and the
prespore cells differentiated during the aggre-
gation stage and aggregated in numbers suitable
to form this proportionality in a cell mass of a
certain size. It's a little difficult to conceive of
this occurring. It would seem more obvious that
proportionality results after the cells come
together. However, since these cells can revers-
ibly differentiate, it's possible that they differ-
entiate in either direction, depending upon the
necessity, in order for the proportionality to be
established. Before continuing, however, in this
discussion let's look at the situation in another
system, D. discoideum.
TS'O: Excuse me for asking a question on
the biology of this organism. Can you take a
single cell and generate a mass like this or do
you have to always start with lots of cells?
GREGG: Yes, it is possible. Either a single
mature spore cell or a single amoeba will pro-
duce innumerable colonies.
In D. discoideum we have also been bright-
and dark-staining cells in the aggregating
stream. However, in the early aggregate we
again see no evidence that the prestalk cells
have differentiated (Fig. 7). This upper margin
is what we call an edge effect, which you get in
certain fresh preparations. This artifact does
not represent the differentiation of prestalk
cells.
Figure 8 shows a late aggregate of D. dis-
coideum. This is the orientation of a late aggre-
gate on an agar plate. They stand up just prior
to flopping over and migrating about on the agar.
Even at this relatively late stage one usually
cannot see a differentiation of prestalk cells.
On occasion there is a small tip end of prestalk
cells which have differentiated, but otherwise
the cell mass appears to be uniformly stained.
It's obvious that the form and the polarity of the
cell mass is independent of the differentiation
of the prestalk cells. Thus, prestalk cells need
not differentiate in order to produce this par-
ticular shape. Consequently, this suggests that
subtle differences exist in the cell mass, prior
to prestalk and prespore cell differentiation.
Now, I suggest that one of these subtle differ-
ences is that of acrasin production which is at
its greatest intensity in the anterior tip. Bonner
(4) has shown this in D. discoideum. Perhaps
such differences as this result in the differen-
tiation of the cell according to the point at which
it happens to be located.
Immediately after the late aggregate it is
obvious that prestalk differentiation has oc-
95
curred (Fig. 9). Exactly what the mechanism
is that caused the differentiation, of course, is
a problem.
CHALKLEY: Is this a sharp or slow tran-
sition?
GREGG: If you look at enough of these, you
can see small areas in the late aggregate that
have begun to differentiate. Presumably, be-
tween the time they are standing up like this
and the time they flop over they differentiate
most of their prestalk cells. Now, it's impossi-
ble to say in this preparation how long this
particular slug has been migrating.
KAHN: It might be worth pointing out that
this process of tipping over takes no more than
a few minutes.
GREGG: Yes. So differentiation may begin
just prior to flopping and is completed in a
relatively short time.
B. WRIGHT: Do you think this difference
in staining intensity could be a difference in
permeability to the stain?
GREGG: I don't think so because these are
histological sections, of course, and I wouldn't
think that cell permeability is involved here.
B. WRIGHT: Perhaps the spore cells have
a more resistant coating.
GREGG: Since these are no longer whole
cells, having been sectioned, I don't think a
permeability factor could be involved.
LOVETT: You showed the two kinds of
amoebae in the aggregating stream of D. muco-
roides. Are they the same in this respect?
GREGG: Yes, they' re present both in D. dis-
coideum and Z). mucoroides.
LOVETT: Can't you see them at all when
it's still erect?
GREGG: You cannot distinguish two types
of cells once the aggregate has formed. You can
detect them only when you look at the separate
amoebae taken from a late interphase or an
aggregate.
LOVETT: If they're just lost in the mass,
I wonder if they could creep up.
GREGG: Yes, and that is just the reason
you cannot exclude sorting out. However, it's
just amazing that by the late aggregate in
D. discoideum you see very little evidence of
prestalk cell differentiation. I would think that
if sorting out was going to occur, it would occur
as part of this process in elongating the cell
mass. It's surprising, if it is one of the proc-
esses of slug formation, that you do not see
more prestalk cell differentiation at this time.
KAHN: I want to ask another question about
the staining. Are the classes of light and dark
Plate I. Figures 2 through 13
Fig. 2. D. mucoroides aggregating myxamoebae exhibit-
ing different degrees of staining with homologous fluores-
cent antiserum (HFAS).
Fig. 3. D. mucoroides early aggregate exhibiting uniform
staining throughout the cell mass (HFAS).
Fig. 4. D. mucoroides late aggregate exhibiting the ini-
tial differentiation of the anterior prestalk cells as indi-
cated by the decreased cytoplasmic staining. Prespore
antigen synthesis has started In certain cells of the
posterior prespore area. Staining observed on all cell
surfaces (HFAS).
Fig. 5. D. mucoroides slug exposed to homologous fluo-
rescent normal serum. Staining Is completely negative.
Fig. 6. D. mucoroides slug exhibiting Intense staining
with homologous fluorescent antiserum (HFAS) in the pre-
spore area but lacking cytoplasmic staining In the pre-
stalk and stalk cells. All surfaces show staining.
Fig. 7. D. discoideum early aggregate exhibiting uniform
staining with homologous fluorescent antiserum (HFAS).
Fig. 8. D. discoideum late aggregate exhibiting uniform
staining throughout the cell mass (HFAS).
Fig. 9. D. discoideum slug exhibiting Intense staining in
the prespore area but lacking cytoplasmic staining In the
prestalk area. All cell surfaces show staining (HFAS).
Fig. 10. D. discoideum preculm exhibiting an Intense
staining in the prespore area but negligible cytoplasmic
staining in the prestalk, stalk and basal disc cells. All cell
surfaces are stained (HFAS).
Fig. 11. D. discoideum culminating exhibiting a de-
creased Intensity of cytoplasmic staining In the prespore
area as compared to the preculm. Prestalk and stalk cells
stained on cell surfaces (HFAS).
Fig. 12. Group of D. discoideum spores exhibiting a de-
creased degree of cytoplasmic staining as compared to
preculm. Cell surfaces retain their staining capacity
(HFAS).
Fig. 13. MV slug exhibiting the lack of well defined pre-
stalk and prespore regions as noted in D. mucoroides slugs
(D. mucoroides FAS).
(Figs. 2, 3, 7-12 from Gregg, Devel. Biol. 12, 377, 1965;
reproduced with permission of Academic Press.)
97
cells absolute or do you have a graded series?
GREGG: In my opinion it is a graded
series. The ones I pointed out were the ex-
tremes. Although photographs may be mislead-
ing I noticed in Takeuchi's black and white
photos that there appear to be more darker-
staining cells than bright cells. This is strange
if these cells are going to sort out to form a
slug with certain proportions.
KAHN: If you were to establish criteria for
classifying light and dark cells and were to
score the cells found in the aggregate do you
think you would find the 30 (prestalk):70 (pre-
spore) ratio?
GREGG: No, I don't think so. As a matter
of fact, you might find various transitions and
not necessarily this final proportion of very
bright and very dark cells. You may find all
intermediates in about equal proportions. Ta-
keuchi has also referred to the various grades
of staining caused by the granules in the cells.
KAHN: One final question. What was your
antigen?
GREGG: All three stages injected into a
rabbit. In theory we had antibodies to vegetative
amoebae, slugs and mature stalks and spores.
They were homogenized before being injected.
B. WRIGHT: How do you prepare these
sections initially?
GREGG: They are fixed in Carney's and
run through an alcohol series.
B. WRIGHT: Yes, but how do you kill them?
What's the initial step? Do you freeze them?
GREGG: No, the fixation kills them. Car-
ney's is essentially acetic acid alcohol and
chloroform. These are paraffin sections.
B. WRIGHT: I see. Could this treatment
itself differentially leach the two cell types?
GREGG: That's a possibility. However,
Takeuchi used methanol and we have obtained
identical results with these two methods.
GROSS: You're presumably looking at pro-
teins with the fluorescence. Carnoy's fixer is
3:1 acetic acid and chloroform. It's a very
effective protein fixer. It's unlikely that it would
wash out antigens.
GREGG: This is more or less a conven-
tional histological technique when using fluo-
rescent antisera.
PAPACONSTANTINOU: Well, there's one
thing that bothers me. Although it may be a
little trivial here, I'd like to find it out. Dr.
Deering asked you what was the staining mate-
rial along the outside and you said it was slime.
Is there any protein in that?
GREGG: The slime sheath could be poly-
saccharide.
PAPACONSTANTINOU: Well, would that
stain?
GREGG: Perhaps the polysaccharides are
antigenic.
PAPACONSTANTINOU: Oh, I see; you've
got polysaccharide as well as protein antigens.
GREGG: Oh yes, that's very likely.
PAPACONSTANTINOU: You don't know
whether that difference in staining is due to
polysaccharide or to something else?
GREGG: No. That cannot be determined
yet. I'm not sure exactly what the composition
of the slime sheath is, but I would say it's
probably polysaccharide.
ZIMMERMAN: Could you tell us once more
how long it takes this thing to flop over?
GREGG: It's a matter of a few minutes.
I'm quoting Dr. Kahn on this. At any rate, by
the time the migrating pseudoplasmodium has
formed, prestalk cells have developed such that
this will result in the development of propor-
tional fruiting bodies. Again you can see that
one of the characteristics of prestalk cells, in
both D. discoideum and D. mucoroides , is that
they tend to lose their cytoplasmic stain. Con-
sequently, the cytoplasmic antigen must be lost
at the anterior end. Obviously development of
the slime mold depends upon differentiation of
these two types of cells, prespore and prestalk
cells. How do we account for the loss of cyto-
plasmic antigens in these prestalk cells?
DEERING: I have one more point I'd like
clarified. Is this a mixture of the antibodies to
all three stages injected at the same time?
GREGG: Yes.
Figure 10 shows a preculmination stage.
There is no drastic change in prespore staining
in the preculmination stage. The stalk has begun
to develop; the lack of cjrtoplasmic antigens is
seen to continue in the prestalk cells.
DEERING: Is that the disc at the bottom?
GREGG: Yes, the basal disc has begun to
form here, and it also loses its cytoplasmic
antigens, and consequently loses its staining
capacity.
KOHNE: Are the cells rapidly dividing as
it's falling over?
GREGG: No, there is very little, if any,
cell division once the aggregate is formed.
For those of you who are not familiar with
the way the slime mold develops, the prestalk
cells in this area move up and flow down into a
funnel-shaped area formed by the stalk. The
cells pile up on top of one another in the same
process by which a chimney is formed, and this
results in the raising of the spore mass.
98
EPEL: One point I don't quite understand
is, if these are antibodies against all tliree
stages, what is the mechanism of differential
staining?
GREGG: You mean why is there no stain in
the prestalk or stalk cells? Apparently the
antigens are lost and when you build up anti-
bodies, there are apparently no antigens present
in here which are specific to build antibodies
which would in turn stain these cells.
EPEL: In other words, these antibodies are
differential against the various proteins.
GREGG: Yes. U you build up antibodies to
all three stages, and for some reason these
cells lose their antigens, or lose the antigens
that they had at an earlier stage, the antibodies
would not stain these cells because the antigens
are gone.
KAHN: If they are antibodies against all
cell types, why shouldn't the prestalk cells show
the same staining response?
GREGG: They stain on the cell surfaces
but not in the cytoplasm. Ifthereareno antigens
inside a cell, there is no reason to believe they
would be antigenic.
KAHN: What do you see at a higher mag-
nification?
GREGG: The cell surfaces are stained, but
otherwise no essential differences.
GROSS: Are there vacuoles in the prestalk
cells?
GREGG: They become vacuolated when
they differentiate into mature stalk cells.
GROSS: How much of the area of a section
of such a cell would be vacuoles?
GREGG: What proportion?
GROSS : Let me phrase it this way. Are the
prospore cells and prestalk cells about the same
size?
GREGG: Prestalk cells are inclined to be
a little larger in the slug.
GROSS: Now, is there a difference in the
amount of vacuolated space in these two types
of cells?
GREGG: The mature stalk cell is more
vacuolated. It's a characteristic of stalk cells.
GROSS: Then it might simply reflect, not
a difference in the number of antigens in the
dry weight of the cells, but simply that there' s
a lot of empty space.
GREGG: Do you mean that the antigens are
crowded out?
GROSS: Yes.
LOVETT: Is that actually true during the
migrating stage when you get the same staining?
GREGG: No, the vacuolation does not occur
until stalk cell differentiation.
TS'O: Have you tried mixing different
antibodies together? If you have, do you see any
different results?
GREGG: Takeuchi (1) used antisera made
only to spores, and he gets exactly the same
results as I found with antisera prepared from
all three stages.
TILL: What would happen if you used anti-
body only to stalk?
GREGG: I think you could. In the first
place, the stalk cells stain on their surfaces so
there are some antigens there.
LOVETT: The only trouble with that is
you'd have to be able to separate just the stalk.
There is no stage at where there is only stalk.
TILL: Well, you could take prestalk cell
area that doesn't stain very well.
GREGG: You can separate the stalks from
the spores after fruiting body formation.
Figure 11 shows a culminating slime mold,
D. discoideum. We notice that the intensity of
the staining begins to diminish in the prespore
area. This probably reflects the fact that the
cells are about to form mature spores. I can't
account for it otherwise. This is the prestalk
area which, of course, remains relatively un-
stained.
Figure 12 shows a section of a mature
spore mass of D. discoideum. The trouble with
this sort of a preparation (cut at 5 microns) is
that it's difficult to determine how many of the
cells have been cut. The spores that are cut
stain in their cytoplasm to a certain extent.
Takeuchi believes they were stained in the cyto-
plasm, whereas certain cells appear to be
stained only on the outside. He believed these
particular cells were not sectioned. In general,
however, the cytoplasmic staining seems to be
reduced in the spores in my preparations. If
you cut through a mass of spores, a consider-
able number of them must be cut. So my feeling
is that the cytoplasmic antigen is relatively
decreased and that, consequently, the staining
is reduced in the spore cells.
SCHRAER: How large are the spores?
GREGG: They're about 5 microns. They're
smaller than the vegetative amoebae or the cells
in the later stages.
SCHRAER: Can you separate the spores
and place them on a glass slide without sec-
tioning them?
GREGG: Yes.
Now, as I mentioned a moment ago, ob-
viously development of the slime molds depends
on differentiation of these two types of cells
99
which are characterized by the loss of prestalk
antigens in the prestalk cells and the synthesis
of additional antigens in the prespores. To what
this may be attributed is a little bit of a puzzle.
However, we know from previous work that
antigens are synthesized during the transition
from the amoebae to the pseudoplasmodium.
Also, antigens are lost during this transition.
Some of these antigens which are lost may be
the antigens from the prestalk cells, resulting
in the loss of standing capacity in the prestalk
area, whereas the additional antigens which are
synthesized during the transition may be repre-
sented by the prespore antigens which are syn-
thesized in the prespore cells. Additional anti-
gens are lost and also synthesized in the
transition from the slug to the mature spores.
Now, Sonneborn et al. (5) have shown that
mucopolysaccharide begins to increase, begin-
ning at about the late aggregation stage in
D. discoideum, and reaches a peak during cul-
mination. Perhaps the synthesis of prespore
antigen is represented by a rise in mucopoly-
saccharide. However, this is speculation. Bonner
et al. (6) have shown that such polysaccharides
are confined to the prespore cells with very
little polysaccharide staining in the prestalk
cells. So, it's possible that some of the antigens
we're dealing with are polysaccharides and that
accounts for the particular staining we find with
fluorescent antibody in the slug.
Now, there are some mutant forms of slime
molds which appear to be inhibited in their abil-
ity to form prespore cells and prestalk cells.
Filosa (3) isolated the mutant MV from D. mu-
coroides-ll, which is characterized by the fact
that it forms a relatively small slug. D. muco-
roides-11 migrates long distances before form-
ing the spore mass, whereas MV may migrate
without forming a stalk or may migrate for a
short distance and form a short stalk bearing a
small spore mass. Thus MV appears to be in-
hibited in their migrating ability, and this may
be tied up with the fact that they are inhibited
in their ability to form prestalk cells and pre-
spore cells. Figure 13 is a cross section of an
MV slug, which in this instance has begun to
produce a small stalk. Now, when we stain MV
slugs with either MV serum or with wild type
antiserum, we observe essentially the same
staining pattern. Generally, the staining pattern
is not as intensive as in the wild type and, on
many occasions, the staining is spotty and gives
a patchy appearance. There is no sharp delinea-
tion between the prestalk cells and the prespore
cells. It appears that the normal complement of
prestalk cells has not differentiated, at least by
this stage. 1 might add that the antiserum pro-
duced by the MV will stain the wild type per-
fectly normally. So it appears that there are
similar antigens in MV which can result in
antiserum which will stain the wild type nor-
mally; but there apparently are not a sufficient
number of antigens in the MV to enable it to
stain intensively.
There is a mutant form of D. discoideum
known as Fr-17 which is similar to MV in that
it fails to develop normally (5). Usually it forms
amorphous mounds of cells but under certain
circumstances it forms an aberrant looking
fruiting body, a stalk bearing a small spore
mass. They have found this mutant, Fr-17,
produces mucopolysaccharide in normal quan-
tities but at a much earlier stage than the
normal wild type. In other words, the production
of mucopolysaccharide appears to be accelerated
in the Fr-17. Incidentally, the mucopolysaccha-
ride is antigenic, also (5). They've tested it with
spore antiserum. So there's a possibility, since
this polysaccharide(s) is antigenic, that we are
dealing with polysaccharide as well as protein.
Now, Takeuchi (1) has reported that MV
cells removed from the interphase stage, that
is just prior to aggregation, still retain, what
he terms, their ring-like staining. This means
that they have more or less of a diffuse staining
at interphase, whereas the wild type cell will
have developed small granules which stain
prominently. There appears to be a delay inMV
at the interphase stage in the synthesis of these
small granules. Whether this has anything to do
with the polysaccharide synthesis we cannot say
at the moment, but it appears that the differen-
tiation of these cells into prestalk and prespores
may be related in some way to the fact that they
have delayed formation of these small granules
which is normal to the wild type.
Most of the material that I've presented so
far was a necessary prelude to the main point
which I hope to make. I had to study normal
development first in order to interpret the
transection experiments which I shall discuss
now.
Raper (7) performed an experiment with
D. discoideum in which he transected prestalk
cells and a portion of the prespore area and
isolated the two fragments. If he allowed suffi-
cient time to go by, each of these portions (the
prespore area and the prestalk area) regulated
to form a normal fruiting body. This means that
each of these portions of the slime mold has the
capacity to regulate. Consequently, each type of
100
zone removed,
Post.
isolate can produce the missing cell type and
produce a fruiting body of normal proportions.
Bonner et al. (6) following Raper's experi-
ment in which they transacted the anterior tip
and posterior prespore area in D. discoideum
(Fig. 14). They discarded the center section in
which the two types of cells were adjacent. I
might add that the anterior tip is devoid of
nonstarch polysaccharide staining (PAS) but
the posterior tip is heavily stained. They allowed
the isolated fragments to reorganize for one
hour before fixing and staining again. They noted
that in the isolated posterior prespore area a
margin of cells had begun to lose staining which
apparently marked the differentiation of prestalk
cells. Later on the prestalk cells are more
clearly established, and it appears that pro-
portional development has been reestablished.
In the isolated anterior tip the staining has begun
in the lower region marking the beginning of the
formation of prespore cells. By 6 hours pre-
spore cell differentiation was well advanced.
Thus, it appears that morphological reorgani-
zation or regulation of the slime mold occurs
simultaneously with regulation of the biochemi-
cal entities.
We performed similar experiments with
D, mucoroides and D. discoideum with the idea
of staining the fragments with fluorescent anti-
body to determine the antigen patterns appearing
during reorganization (Fig. l^).laD. discoideum
transections we obtained about 2/3 of the an-
terior prestalk area and allowed it to reorganize
for three hours before fixing it, running it
through the sectioning process and staining it
with antiserum. In D. mucoroides we isolated
the anterior 2/3 of the prestalk area, trying
to avoid the region we assumed to be close to
the junction of the prestalk-prespore area. You
cannot, of course, see the junction of the two
types of cells in the living slime mold, unless
they have been stained with some sort of vital
dye beforehand. In other transections we cut as
close as possible to the assumed position of the
junction. Each of these fragments was allowed
to reorganize for two hours before fixation.
A third type of transection was made which
isolated the entire prestalk area and approxi-
mately an equal amount of prespore cells. This
type of preparation was allowed to reorganize
for two hours before fixation whereas the pos-
terior prespore areas reorganized for 2 to 5
hours.
Figure 16 shows an isolated D. mucoroides
anterior tip which was allowed to reorganize
for about two hours. Now, prespore cells have
.-«>r.-.'...'.;^'''"'^-'VVl--^>-.VJ^-'>''-'jt"
hour
6 hours
Fig. 14.
A diagram illustrating the experiment In which a par-
tially differentiated migrating cell mass is bisected and
each portion is examined by the PAS technique after one
and 6 hours, respectively. Note that the anterior end of
each fragment reversed its PAS staining properties; in
one case from the light prestalk condition to the dark
prespore condition and vice versa in the other. (Fig. 4,
Bonner, Chiquoine and Kolderle, J. Exp. tool. 130, 147,
1955; reproduced with permission of the Wlstar Institute
of Anatomy and Biology.)
□ PRESPORES
O PRESTALKS
□ STALKS
0 discoideum
D. mucoroides
Fig. 15.
Diagram describing the transection of D. discoideum and
D. mucoroides slugs and their developmental stages at-
tained before fixation and staining with fluorescent anti-
serum. (Fig. 1, Gregg, Devel, Biol. 12, 377, 1965;
reproduced with permission of Developmental Biology,
published by Academic Press).
not differentiated in this particular preparation-
It appears, although we did not make a detailed
study of this, that the number of prespore cells
that differentiate seems to depend upon the re-
gion in which the transection was made. The
closer we get to the junction of the two types of
101
Plate II. Figures 16 through 21
Fig. 16. Transected D. mucoroides prestalk massfoUow-
Ing a period of reorganization exhibiting an increased de-
gree of staining in the prestalk and stalk cells as compared
to a normal D. mucoroides slug (HFAS).
Fig. 17. An Isolated D. mucoroides cell mass composed
of approximately equal proportions of prestalk and pre-
spore cells. Following reorganization the isolate was stained
with HFAS. The prestalk cells did not stain cytoplasmically,
apparently due to the presence of the prespore cells.
Fig. 18. Transected D. discoideum prestalk mass follow-
ing a period of reorganization exhibiting an increased de-
gree of cytoplasmic staining in the prestalk and stalk cells
as compared to the same areas in a normal D. discoideum
preculm (HFAS).
Fig. 19. Transected D. mucoroides prespore mass, fol-
lowing a period of reorganization, exhibiting intense stain-
ing in the prespore area and cell surfaces but lacking
stain in the newly formed prestalk cells (HFAS).
Fig. 20. Transected D. mucoroides prestalk mass, fol-
lowing a period of reorganization, exposed to D. mucoroides
vegetative myxamoebae absorbed HFAS. Fluorescent stain-
ing is completely negative.
Fig. 21. The identical histological described section in
Fig. 20 but stained with HFAS. Staining exhibited by pre-
stalk, stalk cells and cell surfaces.
(Fig. 18 from Gregg, Devel. Biol. 12, Zll, 1965; repro-
duced with permission of Academic Press.)
cells the more we were apt to obtain prespore
cell differentiation. Now, if we allowed such
fragments as these to complete culmination and
form fruiting bodies, we observed small num-
bers of spore cells, sometimes undifferentiated
cells, and, of course, stalk cells. Thus, the
isolates produce prespore cells but the abun-
dance depends to a certain extent upon the
proximity of the transition to the prestalk-
prespore junction.
The most striking thing about the reorgan-
ized anterior tip was the tremendous increase
in the amount of antigen that reappeared. Gen-
erally, mature stalk cells do not contain such a
tremendous amount of antigen as this. The pre-
stalk cells in many of the preparations were
completely uniformly stained. So apparently an
antigen reappears during the reorganization
process. The fact that it appears much more
intensely in the stalk cells may simply result
from a difference in the geometry of the cells
relative to the prestalk cells. The antigen is
probably resynthesized in the prestalk cells
which, of course, form the stalk cells during
the reorganization process.
Figure 17 shows a fragment that was iso-
lated, composed of about the same number of
prespore cells and prestalk cells. Now, we find
that these prestalk cells in the presence of the
prespore cells do not synthesize the antigen. It
102
appears that the presence of the prespore cells
in some way inhibits the synthesis of this anti-
gen that appears in the isolated prestalk cells.
Figure 18 shows an isolated D. discoideum
prestalk area which was allowed to reorganize
for a couple of hours. Again you find a reappear-
ance of the antigen in the prestalk cells and in
the stalk cells. In D. discoideum prespore cells
seem to differentiate more readily. I believe
that these are newly differentiated prespore
cells and not prespore cells which were acci-
dentally removed at the time of the transection.
So, I believe that proportional reorganization
was initiated in this preparation.
TS'O: Excuse me, one thing is not very
clear to me. Is the rearrangement or reorgani-
zation involved with synthesizing new types of
cells or transformation of old types to new
types?
GREGG: In an isolated prestalk area, in
order for them to regain their proportionality,
they must differentiate new prespore cells from
existing prestalk cells.
TS'O: There is new synthesis going on,
too, isn't there? Don't you get new cells?
GREGG: No, there's no increase in cell
number.
TS'O: Then the transected one would be
smaller in size?
GREGG: Oh yes, it would be smaller. The
size depends upon the total number of cells
isolated.
Figure 19 shows a reorganizing D. muco-
roides posterior prespore area. Now if we fixed
and stained this immediately following transac-
tion, the entire area would be stained uniformly.
After two hours of reorganization the cells at
the anterior or uppermost part are beginning to
lose their stain or cytoplasmic antigen. Evi-
dently, they are forming prestalk cells in order
that they can form a stalk and consequently a
fruiting body. If we allow reorganization to go
on for about five hours, the normal slug shape
is regained and the normal proportion of cells
is restored.
In order for a slime mold prestalk isolate
to regain its proportionality it must differentiate
a certain number of prespore cells. The antigen
that reappears must be a necessary entity in the
formation of new prespore cells. The prepara-
tion in Fig, 20 was stained with antiserum which
was absorbed with vegetative amoebae from
cultures of about 17 hours of age. We were
interested in determining whether nor not the
new antigen which reappeared was the same
antigen which was present in the cells of an
earlier age. If it was present in younger cells
it probably was necessary in the initial differen-
tiation of prespore cells and prespore antigens.
However, the absorbed serum produced no stain-
ing whatsoever in this preparation. Now, had
this preparation contained prespore cells, the
prespore cells would be stained to a certain
degree. If the antiserum is absorbed with vege-
tative amoebae, the prespore staining is not
removed. It removes all the staining in the pre-
stalk cells, however.
Figure 21 shows exactly the same section
with the exception that it has been stained with
the non-absorbed serum simply to show that the
antigen had been synthesized in this particular
preparation. Now, it was of interest to us that
this antigen appeared throughout the entire pre-
stalk area. U it is necessary for the slime mold
to produce prespore cells to regain their pro-
portionality and if this antigen is necessary in
the reorganization, it's strange that it was not
confined only to a certain number of prestalk
cells which would be likely to form prespore
cells in the lower area. Thus, it appears that
the isolated prestalk cells cannot immediately
integrate their size with the necessity to dif-
ferentiate a particular number of prespore cells.
This is based on the assumption that this antigen
is an antigen necessary in the differentiation of
prespore cells.
How does this proportionality arise? Well,
I suggest that proportionality arises from the
differentiation of prespore cells; and as a result
of the differentiation of prespore cells, there is
an interaction between the two types of cells
which results in an equilibrium. Consequently,
in some way, the differentiation of the missing
cell types is limited such that the cells are not
over-produced. Now, the same idea may be
applied to the isolated prespore areas. Their
"task", of course, is to produce new prestalk
cells. As prestalk cells are produced, again I
suggest that there is an interaction between the
two cell types which results in a control of cell
differentiation and eventually results in pro-
portionality being established.
FERGUS: Would you care to comment on
why those prespore amoebae could not increase
in number in your transection techniques?
GREGG: Well, I could not state categori-
cally that cell division does not occur. However,
attempts have been made to find whether or not
the cells increase in number during the normal
development, not particularly in transection.
There has been no finding which definitely estab-
lished that there was a tremendous increase in
103
cells, if any at all. You see occasional mitotic
figures but apparently there is no significant
increase in cell number.
FERGUS: Most of this has occurred prior
to the formation of the slug?
GREGG: Yes. As a matter of fact, division
apparently does not occur after aggregation.
Furthermore they utilize only endogenous food-
stuff during morphogenesis.
FERGUS: You were working here with no
external source of food?
GREGG: Yes. They feed upon bacteria dur-
ing vegetative amoebae stage; and once they
aggregate, they can carry out this whole devel-
opmental process in the complete absence of
foodstuffs.
PERSON: Is this a buffered medium?
GREGG: This is on agar; they're buffered
at about 6.2. Complete morphogenesis occurs
on this medium.
GROSS: This certainly ought to dispel one
of the pet ideas of a number of embryologists
that is still quoted very widely: namely, that
differentiation and dedifferentiation are proc-
esses that are intimately linked with cell divi-
sion. Dedifferentiation itself is not demonstrated.
GREGG: This appears to be a form of
dedifferentiation.
DEERING: You have type a (prespore)
changing to type b (prestalk) or type b changing
to type a, either way?
GREGG: Yes, and this occurs in the ab-
sence of an increase in the mass of cells.
KAHN: I think that this is a very interest-
ing point. I must confess that I've always felt
that cell differentiation (morphogenesis) in these
organisms was independent of cell division, but
I'm beginning to think, in terms of this experi-
ment, that this point should be tested. After all,
these amoebae do have a fair amount of endo-
genous reserve. For example, if spores are
placed in a suitable environment they will ger-
minate and may complete the life cycle a second
time in the absence of exogenous nutrient.
GROSS: Are you implying that there is cell
replication?
KAHN: I'm implying that it's possible.
GROSS: However, in order to have some-
thing that approximates the old hypothesis that
the decision is made at mitosis, you'd have to,
at least, double the number of cells. I should
think that could easily be seen.
GREGG: I don't believe that cell division
is necessary, because you can cause fruiting
body formation from small quantities of cells.
As a matter of fact, fruiting bodies have been
obtained from as low as 7 cells,
GROSS: Is that an adult fruiting body?
GREGG: Yes. Obviously cell division isn't
necessary here although it's true no one has
examined a larger isolated anterior tip.
KAHN: I think there's apoint worth stress-
ing about regulation (developmental) in cellvilar
slime mold development. For example, Bonner
has shown that normal development can occur
in aggregates containing fewer than 100 cells as
well as in aggregates containing many thousands
of cells. In the slime mold, Acytostelium, even
a single amoeba may show developmental regu-
lation. In this case the cell gives rise to a struc-
ture composed of a single spore perched on an
acellular stalk.
GROSS: At any rate, I think it's helpful to
the state of the problem so as to have these
things discussed more widely than they are.
Most people don't know about this particular
point. It's such a clear case of a switch in the
choice that the cell makes about what it's going
to do, a switch that can be produced externally
without any massive cell replication.
GREGG: It's one of the most striking things
about cellular slime molds.
GROSS: If it's true that these cells are
really not replicating, then all of this may hap-
pen during interphase. This immediately rules
out any proposal about sequential nature of
transcription in microorganismal cells like
this. If these cells decide to go back and become
another cell type, they're really making dif-
ferent antigens which means different genes
are being transcribed. On the basis of the biol-
ogy of this system it would very unlikely that
they would go back and transcribe the whole
genome in order.
LOVETT: Could they go back and start in
the middle?
GROSS: It seems to me it doesn't argue
against the sequential transcription so much as
it does that it's obligatory that it starts at one
end and can't do anything until it reaches the
other end, and then starts over again.
TS'O: That model you have in mind, Paul,
must be a linear one and not a circular one.
GROSS: Yes.
GREGG: It's hard to say whether they start
at the beginning or in the middle. If you examine
a cell mass, you see what appears to be a sort
of a background fluorescence, and then when
you get prespore differentiation in the late ag-
gregate, you see spots of prespore antigen. The
antigen that reappears in the isolated one appears
to be this background antigen which is present
104
in the vegetative amoebae and easily observed
in the early aggregate. So, the background
antigen appears first; and this is followed up
by the synthesis of the prespore antigen. I
doubt if the cells would synthesize prespore
antigen in the absence of this background anti-
gen. I think the isolated prestalk cells try to
establish conditions as they were in the early
stages of normal development as a prelude to
differentiating into prespore cells.
EPEL: Relating to Paul's point, maybe
this is a difference between microorganisms
and metazoans; if this is a microorganism.
GREGG: It is claimed taxonomically by
both the botanists and zoologists.
GROSS: Are you referring to the capacity
for dedifferentiation?
EPEL: Yes. Is this like a bacterial spore
or protozoan, which forms a spore under cer-
tain environmental conditions?
DEERING: Can you take these things after
you've cut them once, have them change, and
cut them again and have them change back again?
GREGG: If you don't wait too long, I should
think you could.
DEEFUNG: In other words, you can change
5 to a and then back to b? I wonder how long you
could keep this up?
GREGG: Probably until you get down to a
very few cells.
KAHN: A good deal of our discussion has
centered around cell metaplasia, the ability of
a cell to exist in different states. Recently,
Dr. Lindsay Olive (Columbia University) de-
scribed an amoeboid microorganism that is
capable of assuming amoeboid, flagellate, cyst
or spore form. It would be very interesting to
know whether this organism can make these
transformations in the absence of cell division.
MASSARO: Isn't it possible, let us say, that
certain of these cells in a particular area are
like reserve cells, not being particularly com-
mitted at any one time to any one tissue; and
these cells perform the reorganization?
LOVETT: I don't think it's necessary to
assume that the cells in layer X are identical
to the cells in layer Y; but cells in layer X may
be dedifferentiated, undifferentiated, or less
differentiated cells which are in reserve to be
committed to the reorganization or formation
of the structure.
GREGG: You mean this is the case just in
the event someone comes along and cuts one in
half?
LOVETT: Certainly. I respect the potential
of these cells.
GREGG: With regard to your remarks I
can only say this: in D, mucoroides we've
thought about this to a certain extent. Prestalk
cells have to be continually replaced by the
prespore cells as the slug crawls along because
the prestalk cells are continually forming stalk.
So in order to keep the proportions of these
cells constant it has to keep replenishing the
prestalk cells. Now, there appears to be a
gradient of differentiation between these two
regions. In other words, the further anterior
you cut, the more apt you are to get fewer spore
cells and more undifferentiated cells following
a reorganization period. The closer you cut to
the prestalk-prespore junction the greater num-
ber of cells you get which have just crossed
the border into the prestalk area. Consequently,
it's much more likely that they can dedifferen-
tiate to form prespore cells. I don't know
whether this answers your question about re-
serve cells or not.
DEERING: Can we really eliminate the
possibility that there is a third type of cell that
can go either way and that this is what always
leads to appearance of new types?
GROSS: I think you can.
GREGG: I suppose it would be possible.
DEERING: In other words, you can't really
eliminate that possibility. I think it' s important.
GROSS: But the requirement is that if you
had such a population of cells, they would have
to be uniformly distributed throughout the slime.
MASSARO: Why is it necessary to have a
uniform distribution?
GROSS: Because you get regulation wher-
ever you cut. If they were restricted to one end,
then you wouldn't get regulation at the other
end.
LOVETT: However, don't you get varying
degrees of reorganization depending on where
you cut?
GREGG: Yes, fruiting body formation in-
variably occurs although the proportions of the
two types of cells composing it depends upon
where you cut and the amount of time the pre-
stalk mass requires to reorganize.
LOVETT: All pieces of the slug cut at the
proper stage will eventually reorganize and
regulate the proper proportion between prestalk
and prespore cells?
GREGG: Presumably if they're allowed to
migrate long enough they reestablish their pro-
portions. I think one of the reasons that you did
not see prespore cells immediately in the an-
terior tips that I showed you was due to the fact
that the anterior tips rush right into fruiting;
105
and I think they rush into fruiting so fast that
they do not have time to form prespore cells
proportionally in all instances.
TILL: Will the spores that you get from
these transections give a normal organism?
GREGG: Oh, I'm sure they would.
MASSARO: To go back for a minute. What
did you mean by uniform distribution? Do you
mean X number of cells surrounded by Y number
of undifferentiated ones?
GROSS: Yes. If there's another type of cell
that is neither prestalk nor prespore, they have
to be somewhere in the slug. Now, when you cut,
you can cut any part of it, and in principle, you
can get back the whole thing.
MASSARO: You could have the third type
of cell anywhere.
GROSS : That' s the point; they are anywhere.
They're a population of finite size. Now, as you
reduce the sizes of pieces you cut, the fraction
of the uncommitted cells relative to those cells
that have already differentiated is going to change
depending on where you cut. If you cut in the
anterior end, you're going to have a large num-
ber of prestalk cells and a very small number
of prespore cells; and you still have a small
number of undifferentiated cells. There's no
replication, so you've got a large number of
prestalk cells that can't go anywhere and no
prespore cells; now, the small number of un-
committed cells must, in that instance, all
differentiate to form prespore cells. Suppose
you don't have enough. It seems to me that as
the piece gets smaller, like 7 cells, you're not
going to have enough of those relatively uncom-
mitted cells.
MASSARO: Well, maybe these cells have
only a certain degree of noncommittedness.
Maybe we're looking at the noncommitted cells
too harshly and saying we have a cell here which
is definitely noncommitted. Maybe certain pre-
stalk cells are less committed than other pre-
stalk cells.
GROSS: This is an argument that extends
far beyond the slime molds. It's one that has
plagued embryologists for many years.
KAHN: Jim, did you look at Polysphon-
dylium at all?
GREGG: No, I didn't.
KAHN: Well, this might be worthy of men-
tion along these lines. If you do the same sorts
of things that Gregg has done with fluorescent
technique with various histochemical techniques,
you do get a differential staining between the
presumptive stalk and the presumptive spore
areas. This is true in Dictyostelium discoideum,
also.
GREGG: The presumptive stalk region is a
very small area.
KAHN: I was going to get to that. The in-
teresting thing about Polys phondy Hum is that
you don't see these differences until very late;
so, in effect, the whole mass is uncommitted
until the very last moment.
GREGG: You can differentiate between the
types of cells in a number of ways: PAS stain-
ing, vital stains, antibodies.
PERSON: Is there a vital stain that can
differentiate between the two types of cells so
that you could keep an individual cell alive and
look at it?
GREGG: Yes. Bonner's used stains such as
Nile blue sulfate, neutral red and Bismarck
brown.
GRUN: Do they all produce a darker stain-
ing in the nonstalk area and a lighter staining
in the stalk area?
GREGG: I believe the staining is more in-
tense in the anterior end with most of these
stains.
ACKNOWLEDGEMENTS
The meticulous histological preparations
which were made by Mrs. Doris Gennaro during
the course of this investigation are gratefully
acknowledged by the author.
This investigation was supported in part by
a Public HealthService Career Programs Award
5-K3-HD-15, 780 from the National Institute of
Child Health and Human Development, Research
Grants E-1452 and GM-10138 from the National
Institutes of Health.
106
References
1. I. Takeuchi. Develop. Biol. 8, 1 (1963).
2. J. H. Gregg. Develop. Biol. 12, 377 (1965).
3. M. F. Filosa. Amer. Naturalist 96, 79
(1962).
4. J. T. Bonner. J. Exptl. Zool. 110, 259
(1949).
5. D. R. Sonneborn, G. J. White and M. Suss-
man. Develop. Biol. 7, 79 (1963).
6. J. T. Bonner, A. D. Chiquoine and M. Q.
Kolderie. J. Exptl. Zool. 130, 133 (1955).
7. K. B. Raper. J. Elisha Mitchell Sci. Soc.
56, 241 (1940).
8. J. M. Dubert, P. Slizewcz, P. Rebeyrotte
and M. Macheboeuf. Ann. Inst. Pasteur 84,
370 (1953).
9. J. D. Marshall, W. C. Eveland and C. W,
Smith. Proc. Soc. Exptl. Biol. Med. 98,
898 (1958).
10. C. W. Griffin, T. R. Carski and G. S. War-
ner. J. Bacteriol. 82, 534 (1961).
11. H. Peters. Stain Technol. 38, 260 (1963).
107
CONTROL OF ENZYME ACTIVITIES IN D. DISCOIDEUM
DURING DEVELOPMENT
Barbara Wright
John Collins Warren Laboratory, Massachusetts General Hospital
Boston, Massachusetts
I believe the usual concept of morphogenesis
includes a visible change in the form or struc-
ture of an organism. This implies a gradual
accumulation or redistribution of structural
material, such as connective tissue, bone or
cell wall polysaccharides, for example. This,
in turn, implies alterations in the activity of
enzymes responsible for the synthesis of these
materials. A number of possible mechanisms
for changing the activity of an enzyme appear
in Fig. 1. This figure summarizes various ways
in which the product characteristic of a par-
ticular differentiated cell might be made to
accumulate during development. The rate of
product accumulation could be enhanced by an
increased level of the enzyme, substrate, acti-
vator or RNA template used in the synthesis of
the enzyme. The accumulation of any of these
types of molecules, of course, implies nothing
with respect to the mechanism. The three possi-
bilities are a) an increased rate of synthesis,
b) a decreased rate of destruction or c) the ac-
tivation of a preformed inactive form of the
molecule. Thus, for each of the mechanisms
listed in the figure the problem is simply pushed
back to another level of analysis.
Although our present state of knowledge
allows the discussion of these three possibilities
only with respect to enzyme levels as indicated
in the figure, levels of the other types of mole-
cules would be altered, also, by similar mecha-
nisms. Finally, it must be kept in mind that an
observed increase in level of any of these fac-
tors would be critical to the formation of a
product of differentiation only if it were already
limiting the process in the cell. Such informa-
tion is exceedingly difficult to obtain. Changing
levels of an enzyme or a substrate may only be
correlated with, and an indirect result of, the
morphogenetic process observed and may be
due to causes quite unrelated to our naive and
prejudiced interpretation. The fact that DNA
and RNA play an important part, at some point,
in controlling the details of cellular differentia-
tion need not be documented. The question con-
cerns the time at which their action is necessary
relative to the unfolding of a particular develop-
mental process.
DNA
I
RNA
-^ PRODUCT OF DIFFERENTIATION
Coenzyme, Activator
Reaction may be stimulated by:
1. Level of enzyme
a) Increased synthesis (RNA and/or DNA activity)
b) Decreased degradation (stabilization)
c) Unmasking or activation (of preformed protein)
2. Level of substrate
3. Level of coenzyme, activator or inhibitor
Fig. 1.
From Wright, Barbara E.: Control of Carbohydrate
Synthesis in the Slime Mold. In Developmental and Meta-
bolic Control Mechanisms and Neoplasia (A Collection of
Papers Presented at the Nineteenth Annual Symposium
on Fundamental Cancer Research, 1965), p. 297. Balti-
more, The Williams and Wllkins Company, 1965.
109
Activity at the enzyme and substrate level
must necessarily be correlated in time with the
accumulation of the product characteristic of the
differentiated cell. This need not be true, of
course, for the nucleic acid template responsible
for the presence of these enzymes. In fact,
recent studies of Brown in the amphibian. Gross
in the sea urchin and Sussman in the slime mold
indicate that certain stages of differentiation do
not, in fact, depend upon the concurrent forma-
tion of messenger RNA. This situation brings
renewed interest to other types of control, such
as the activation of preformed mRNA, enzyme
accumulation through lack of degradation, en-
zyme relocation within the cell, the availability
of a substrate or an enzyme activator, etc.
Regardless of the relative importance of nucleic
acid control during a particular process of dif-
ferentiation, the cellular environment of the
enzymes involved is critical, of course, in de-
termining the nature and extent of their activity.
In other words, since the action of an enzyme is
entirely dependent upon levels of specific sub-
strates, activators, inhibitors and the like,
knowledge of these variables in the intact cell
is essential in attempts to evaluate the signifi-
cance either of a constant or a changing enzyme
level to a reaction important to development.
Let me illustrate this point by mentioning
just two examples in the slime mold. The mor-
phogenesis of this microorganism depends, in
part, upon the breakdown of endogenous protein
and its eventual conversion to carbohydrate. As
protein degradation intensifies during develop-
ment, the intracellular concentration of gluta-
mate increases an order of magnitude. Oxidation
of this amino acid and its entry into the Kreb's
cycle is a necessary step in its utilization for
carbohydrate synthesis. The enzyme responsible
for this oxidation, glutamic dehydrogenase, is
very stable in extracts prepared throughout
development. Although the concentration of this
enzyme does not change, its activity when meas-
ured in vivo using radioactive glutamate in-
creases 7-fold during development. The dehy-
drogenase was purified and its affinity for
glutamate was determined; knowing the effect
of substrate concentration on the rate of this
reaction, it was shown that the accumulation of
glutamate in vivo could fully account for the
enhanced rate of the reaction in differentiating
cells. Thus, data at the enzyme level was insuf-
ficient in interpreting the in vivo activity of this
enzyme during development (1).
The slime mold offers another example of
an enzyme which does increase in concentration
during development (some 6-fold), yet this
change is not reflected in its activity in vivo.
Dr. Gezelius has studied an alkaline phosphatase,
highly specific for 5' -AMP, which reaches a
maximum concentration at the end of differen-
tiation. However, inhibition of the enzyme by
increasing levels of inorganic phosphate in vivo
results in maximum activity of the enzyme not
at the end but in the middle of differentiation (2).
Thus, observed alterations in the concentration
of an enzyme may not bear a direct relationship
to its actual activity in the differentiating cell.
This is probably the rule rather than the excep-
tion.
Enzymes are usually measured under con-
ditions of pH, inonic strength, substrate con-
centration, co-enzyme, activator or inhibitor
concentrations, which do not reflect the condi-
tions in the differentiating cell. Much more
data are needed in which enzyme activities are
measured both in vivo and in vitro and in which
levels of relevant substrates, co-enzymes and
activators are determined in vivo at various
stages of development. All these data, taken
together, may then give a consistent picture of
the activity of an enzyme in differentiating
cells.
To facilitate the following discussion, I
will very briefly summarize the life cycle of
D. discoideum (Fig. 2). Upon starvation, the
cellular slime mold passes from the vegetative
stage, during which it exists as a homogeneous
population of myxamoebae lacking a cell wall,
through an aggregation process to become a
differentiated multicellular organism. Succes-
sive stages which I will refer to are known as
aggregation, pseudoplasmodium, preculmina-
tion, culmination and sorocarp or fruiting body.
In the terminal stages of development the cells
are ensheathed in a cell wall composed of a
cellulose-glycogen polysaccharide complex, the
synthesis of which will be the subject of a good
portion of my presentation. All of the experi-
ments I will talk about were done with cells
which were starving on 2% agar throughout the
differentiation cycle.
Figure 3 summarizes the general area of
metabolism with which we will be concerned.
Endogenous material, such as protein, is de-
graded and gluconeogenesis begins. Hexose
phosphates are formed andglucose-1-phosphate
together with UTP unite to form uridine di-
phosphoglucose (UDPG), an essential precursor
to cell wall material. Phosphoglucomutase,
interconverting G-l-P and G-6-P, is very
active throughout development, as is pyro-
110
tMOltt N
ft '
1
6v?
A ^
MUt.Ti#>^lC*TiO«l
V
\
i
-9^
\
ttlOCHTOX
\
rStUOOKlSHOOUH A
cuunmATiON
1/ jH
'^^
^'tl-
^^^ m
Fig, 2.
phosphatase. This would tend to aid the accumu-
lation of UDPG by removing pyrophosphate.
Neither of these enzymes changes strikingly
during development, but UDPG synthetase does
increase about threefold at culmination.
A number of precursors of cell wall ma-
terial increase and then decrease prior to
sorocarp construction. Figure 4 shows data
obtained by Mr. Beers in our laboratory on
glucose-6-phosphate accumulation in a number
of stage studies. As you can see, there is a good
deal of variation from one stage to another, but
in general, glucose-6-phosphate reaches a peak
at culmination.
Figure 5 is a schematic summary of the
accumulation pattern of a number of poly-
saccharide precursors and of some end products
of differentiation. Gluscose, glucose-6-phos-
phate, glucose- 1 -phosphate and UDPG increase
and decrease in the cells during development
as cellulose, mucopolysaccharides, trehalose
and an alpha-1, 4 polymer, which I will discuss,
accumulate. Since cell wall construction occurs
only at the terminal stages of development, it
represents an excellent index of differentiation.
We wanted to know exactly what conditions in
the starving, differentiating cells set the stage
for cell wall accumulation. This work was done
in collaboration with Carole Ward and Donna
Dahlberg.
CELL WALL
UDPG + PP-
4^
GIP + UTP
-T^P
^GLUCOSE
KREBS CYCLE
CULM SORO
Fig. 4,
From Wright, Barbara E.: Control of Carbohydrate
Synthesis in the Slime Mold. In Developmental and Meta-
bolic Control Mechanisms and Neoplasia (A Collection of
Papers Presented at the Nineteenth Annual Symposium
on Fundamental Cancer Research, 1965), p. 301. Balti-
more, The Williams and Wllkins Company, 1965.
In order to study cell wall synthesis in
vitro, husk preparations of culminating or ter-
minal stage cells were made by passing the
cells through a French pressure cell and then
washing extensively in tris-EDTA buffer. These
cells were then incubated with radioactive UDPG,
111
100- -
z
o
<
= 75-1-
<
<
Z
X 50--
<
2
2 25-t7
X
o
0.
a.
4
/ GIP
AM AGG PS CULM SORO
STAGE
EXPERIMENT
I
TABLE I
Stability of Enzymatic Product '
Boiled
10 min.
57. NaOH
9U0
Boiled
20 hrs.
1% NaCH
947
Boiled
20 hrs.
n NaOH +
2 hrs.,
2.5
N HjSO^
55
Boiled
10 mln.
water, NO
NaOH
1,584
Boiled
10 mln.
30^ NaOH
1.494
From Wright, Barbara E.: Control of Carbohydrate
Synthesis in the Slime Mold. In Developmental and Meta-
bolic Control Mechanisms and Neoplasia (A Collection of
Papers Presented at the Nineteenth Annual Symposium
on Fundamental Cancer Research, 1965), p. 303. Balti-
more, The Williams and Wilkins Company, 1965.
Fig. 5.
TABLE II
Substrate Specificity
the reaction stopped by boiling, carrier cellu-
lose added and the material washed repeatedly,
boiled in alkali, wash some more and finally
counted in a scintillation counter.
Table I indicates the alkaline and acid .«!ta-
bility of the radioactive, alkali-insoluble prod-
uct. Gezelius and Ranby isolated comparable
material from D. discoideum, and the most rig-
orous treatment in their purification was twenty
hours at 100°C in 1% alkali. They studied this
material very carefully by x-ray diffraction
and other types of analyses and concluded that
it was an amorphous form of cellulose (3). They
found only glucose on acid hydrolysis. In con-
firmation of this, we found only radioactive
glucose on acid hydrolysis of our radioactive
cell wall material. A substrate specificity study
revealed that UDPG was by far the preferred
substrate (Table II). GDPG, which has been
recently shown by Hassid's group to be a pre-
cursor to cellulose synthesis in plants (4), was
only about 1/10 as active.
We were able to carry the purification of
cell wall material one step further than Gezelius
and Ranby, and separate it into two fractions,
4 and B, by solution in a cuprammonium hy-
droxide solution known as SchweizSr's reagent
(Table III). We will be talking now just about
fraction A and soluble fraction B, not insoluble
fraction B. After solubilization in the cupric
ammonium hydroxide solution, fractional pre-
cipitated out on neutralization and the addition
UDPG
1,0
G6P
1.0
GLUCOSE
1.0
UDP-GAL
1.0
UDPG
0.2
GDPG
0.2
ADPG
0.2
EXPERIMENT SUBSTRATE juMOLES jiMOLES GLUCOSE INCORP. (x lO-*)
39.0
0
0
0
5.6
0.5
0.2
of water. This material is not water soluble.
Fraction B precipitated out from the supernatant
following the addition of ethanol. Chemical,
enzymatic and chromatographic analyses of the
radioactive and nonradioactive fraction A and
fraction B have identified the latter as an alpha-
D-l,4-linked polymer and fraction^ as cellu-
lose. Some of the enzymatic analyses are sum-
marized in Table IV. Oyster glycogen was used
as a control. The expected limit dextrin was
made from nonradioactive, insoluble fraction B
by phosphorylase treatment. Complete degrada-
tion was achieved by further attack of amylo-1,
6 glucosidase. Analysis of radioactive material
revealed that most of the radioactivity is in-
corporated into fraction B and that fraction A is
contaminated with the alpha-D-1, 4- linked
polymer. Thus our studies have led to the con-
clusion that the alkali-insoluble cell wall ma-
112
TABLE ni
Fractionation of Radioactive Cell Wall Material with Schweizer's Reagent
Total cpm
Exp. Original Undissolved Frac . A Frac . B Frac. B % Total
2,140
residue
400
l
soluble)
1,267
( insolubl
IL
recovery
I
132
85
I
138,000
200
2,770
70,000
10,000
60
II
128,900
-
19,790
68,620
-
68
IV
130,345
8
,310
8,074
77,850
3,770
82
TABLE IV
Hydrolysis of Fractions A and B
.012
Sample
Oyster glycogen
Oyster glycogen
Non- radioactive Frac.
Non-radioactive Frac.
Radioactive Frac. B
Radioactive Frac. A
Enzyme Treatment
<-l,4-phos- amylo- 1 , 6-gluco
phorvlase sldase
present
present
present
present
present
present
absent
present
absent
present
absent
absent
7, Hydrolysis
38
100
41
90-100
100
60-80
terial is composed not of cellulose only, but
rather of a 50-50 mixture of cellulose and
glycogen polysaccharides in intimate associa-
tion. During synthesis of this material in vitro
most of the radioactive glucose in UDPG-^'*Cis
incorporated into the glycogen polymer (5).
Let us now turn to some properties of the
enzyme system catalyzing the synthesis of cell
wall material from UDPG (Fig. 6). We deter-
mined the activity of the enzyme as a function
of UDPG concentration. The UDPG concentra-
tion does not change significantly during poly-
saccharide synthesis in vitro in the presence
of a well-washed particulate preparation. There-
fore it seems justified to consider 1.3 x 10"^ to
be the approximate K^, for UDPG in the synthesis
of cell wall material. During differentiation in
the slime mold the intracellular concentration of
UDPG is well below 10-3 M except in culminating
cells which are rapidly accumulating cell wall
polysaccharides. Assuming that the UDPG values
approximate the concentration available to the
enzyme in vivo, it would appear that UDPG is
one limiting factor to the initiation of cell wall
synthesis in the differentiating cell. Conversely,
I 10 100
UDPG ( p MOLES /ml x 10)
Fig. 6.
From Wright, Barbara E.: Control of Carbohydrate
Synthesis in the SUme Mold. In Developmental and Meta-
bolic Control Mechanisms and Neoplasia (A Collection of
Papers Presented at the Nineteenth Annual Symposium
on Fundamental Cancer Research, 1965), p. 311. Balti-
more, The Williams and Wilkins Company, 1965.
the depletion of UDPG, which occurs very rapidly
during sorocarp construction, would of course
be a determining factor in the termination of
polysaccharide synthesis.
Both glucose-6-phosphate and magnesium
stimulate cell wall polysaccharide synthesis
in vitro (Table V). G-6-P is known to lower the
Km for UDPG in a number of other systems in
which glycogen synthesis has been studied. If
one adds magnesium extracellularly in the 2%
agar in which the slime mold is differentiating,
it increases the rate of overall differentiation
113
TABLE V
Stimulation by G6P and Mg
+ 2
Additions
None
G6P (2 X 10" 3m)
MgCl2 (1 X 10- 3m)
G6P + MgCl
c .p.m.
, in
alkali- insoluble
material
Day 1
Day
_2
261 485
973 2,158
309 603
1,508 2,317
TABLE VI
Stimulation of Polymer Synthesis by Trehalose '
Additions c .p.m.
...
518
G6P (10" 3 M)
730
Trehalose (lO'^ m)
471
G6P + Trehalose
910
* From Wright, Barbara, E.: Control of Carbohydrate
Synthesis in the Slime Mold. In Developmental and Meta-
bolic Control Mechanisms and Neoplasia (A Collection of
Papers Presented at the Nineteenth Annual Symposium
on Fundamental Cancer Research, 1965), p. 312. Balti-
more, The Williams and WiUcins Company, 1965.
ably be a limiting factor at culmination for cell
wall synthesis.
Table VII shows a complex relationship
between UDPG concentration and glucose -6-
phosphate concentration in their effect on cell
wall synthesis. It can be seen that G-6-P only
stimulates cell wall synthesis at a low level of
UDPG, but not at a high level. Therefore, their
effects are interdependent. Although it isn't
shown in this table, the concentration of G-6-P
which maximally stimulates is about 10-3 M and
in the intact cell it never reaches a level higher
than 10 ""^M. Glucose-6-phosphate would, there-
fore, presumably be limiting in the cell for cell
wall synthesis. For unknown reasons intracellu-
lar UDPG levels vary significantly from one
stage study to another. Although the maximum
concentration is always at culmination, the level
throughout differentiation in a particular stage
study may be unusually high or unusually low.
Thus, G-6-P could serve as a buffering agent,
exerting strong stimulation at low UDPG levels
and less stimulation in cells which are not as
limited with respect to their UDPG levels.
It is apparent from these and many other
studies that the existence in vivo of many limit-
ing factors for the synthesis of materials im-
portant to differentiation may be the rule rather
than the exception. It is known that even in fully
differentiated cells enzymes are usually operat-
ing far below their potential activity due to sub-
strate limitation. Cells undergoing differentia-
tion are frequently dependent entirely upon
endogenous metabolism and have very limited
resources from which to obtain the necessary
energy and building blocks for the many syn-
thetic processes required in morphogenesis. If,
in fact, it is true that multiple limiting factors
considerably. It is possible, therefore, that mag-
nesium is limiting cell wall synthesis during
development. In the experiment shown in Table V
the enzyme was prepared on day 1 and assayed
immediately in the presenceof UDPG- i**C; when
the enzyme was aged for a day and assayed on
day 2, it had become activated, but the require-
ments remained comparable. I'll discuss activa-
tion later.
Table VI shows that trehalose stimulates
cell wall synthesis, but only in the presence
of G-6-P. We don't understand the mechanism
for trehalose stimulation, but I want to make the
point that trehalose, according to Filosa, accu-
mulates very late in development during soro-
carp construction, so that trehalose would prob-
TABLE VU
Interdependence of G-6-P and UDPG
Final
mo
• larity
juHo
lies incorporated
G-6-P
UDPG
(x 103)
None
10-^
0.29
10-3
10-^
1.10
None
10-2
44.0
10-3
10-2
40.5
114
are always associated with development, one
might well inquire into the possible advantage
this situation could bring to the differentiating
cell. I would like to suggest that the least pre-
carious approach for a differentiating cell
actually may reside in its dependence upon a
complex interplay of many limiting factors. In
this way, unusual deficiencies or abundances in
the cell or the cell's environment need not
necessarily upset the process of differentiation.
Let me elaborate on this concept briefly,
using some recent work with hexokinase( Fig. 7).
In the figure on the left the reciprocal of the
glucose concentration is expressed on the
abscissa and the inverse of the velocity of the
reaction on the ordinate. Velocity is seen to
increase with increasing levels of ATP from 0.1
to 1.2 millimolar. In other words, the reaction
is stimulated by ATP in the presence of limiting
levels of glucose. Similarly in the right part of
this figure increasing levels of glucose stimulate
the reaction in the presence of limiting levels
of ATP. When both substrates are limiting, in-
creasing the concentration of either increases
the rate of the reaction (6). Thus, if this situation
prevailed in the cell, increasing levels of either
ATP or glucose could increase the level of
glucose-6-phosphate. If, on the other hand, either
substrate were in excess, G-6-P accumulation
would depend upon the concentration of the other
substrate. In this sense the system would be less
flexible than if both substrates limited. Such
flexibility may be very important to the stability
and reproducibility of differentiation.
Let us now turn from complications at the
substrate level to even greater complications at
the enzyme level. We have said nothing as yet
concerning the enzyme activity during the earlier
stages of development. Figure 8 shows one of
our earlier experiments in which we compared
enzyme activity at various stages of development
with the amount of alkali-insoluble material
present. The stages of development are amoeba
(A), aggregation (agg), preculmination (PC), a
combination of culmination and fruit (CF) and
fruit (F). Aliquots of cells were harvested at
various stages of development and the percent
dry weight of the cell wall material determined.
This is indicated on the left ordinate. At each
stage, also, a particulate enzyme of cell wall or
cell membrane preparations was prepared in
tris buffer and 10""* M EDTA and was incubated
with radioactive UDPG. The alkali-insoluble
radioactive product was isolated, counted and
related to the dry weight of the sample. The
specific enzyme activity was thus determined.
ATP
GLUCOSE
0.04 mM
['glucose]
atp + glucose
Fig. 7.
(Fig. 3, From Silverstein and Boyer, /. Biol. Chem. 239,
3645, 1964; reproduced with permission of the American
Society of Biological Chemists, Inc.)
<
3
O
<
_i
<
I5--
I0--
5--
-tt-
.f..A...t,-5r-
--I000 t
KlF + Agg)
X
liJ
>-
cc
a
--500 S
Q.
U
>-
0
HOURS
STAGE h
gg
PC
H 1
CF F
Fig. 8.
From Wright, Barbara, E.: Control of Carbohydrate
Synthesis in the Slime Mold. In Developmental and Meta-
bolic Control Mechanisms and Neoplasia (A Collection of
Papers Presented at the Nineteenth Annual Symposium
on Fundamental Cancer Research, 1965), p. 304. Bald-
more, The WiUlams and Wllkins Company, 1965.
and is expressed on the right ordinate. Mixed
preparations of an enzyme that was active with
an inactive preparation gave relatively little
inhibition. The data in Fig. 8 exhibit a striking
correlation in the activity of an enzyme and the
accumulation of the product of the activity of
this enzyme. The correlation suggests a causal
115
relationship, but we shall see that this is not
justified. We found that if one goes from lO-^M
EDTA to 0.1 MEDTA, it is possible to partially
stabilize enzyme at the earlier stages of develop-
ment and detect activity.
Table VIII is a stage study in which we
harvested, killed the cells and isolated enzyme
at two different stages, late aggregation and
culmination, in the presence of 0.01 M EDTA
and 0.1 M EDTA. In other words, we had four
enzyme preparations: two at aggregation, two
at culmination. The enzyme preparations were
made and assayed as quickly as possible; then,
they were stored in an ice bath at 5° and were
assayed again at two hours and at 24 hours. As
you can see, at both concentrations of EDTA
the enzyme activity at aggregation decreased
with time, but was spared to a greater extent
at O.l M EDTA. At culmination, however, the
enzyme activity was not only spared but in-
creased. This enzyme is strikingly activated
by high concentrations of EDTA. Clearly in-
activation of the enzyme prepared at aggrega-
tion was more rapid between hour 0 and hour 2
than from hour 2 to hour 24. In other words,
the inactivation curve drops rapidly at first and
then more gradually. Seeing this, one wonders
what happens between the time that the en-
zyme was first prepared and the time that the
0 hour value was obtained. In other words, in
that period of preparing the enzyme inactiva-
tion may have been even more rapid. At any
rate, enzyme prepared at one stage is more in-
activatable than the enzyme prepared at a later
stage.
We call this phenomenon differential in-
activation. It is an in vitro artifact, fairly com-
mon in the slime mold. We are very impressed
with the extreme difficulty of detecting it, since
it took us almost a year in this case. In each
stage study the period in time and in stage at
which the enzyme activity can first be detected
and shown to be unstable varies and is very
short-lived. Until this enzyme is stabilized, we
cannot determine specific enzyme activity as
a function of developmental stage. Our experi-
ence with differential enzyme inactivation makes
us very suspect of the absence of any enzyme
activity and prone to place faith in changes in
enzyme activity only when (1) the enzyme is
detected and (2) is relatively stable or capable
of being stabilized. It is possible that the cell
wall enzyme under study is always present in
the cell membrane but is undetectable in vitro
due to the absence of stabilizing primer, for
example. We have some preliminary data on this
point, but before presenting it, I would like to
summarize the facts briefly (Table IX).
We have recently found 4 enzyme activities
involved in cell wall or glycogen synthesis or
both (see Table IX). They may be the same
enzyme or some of them may be different, at
TABLE VIII
Effect of EDTA Concentxatlon on Initial Enzyme Activity and Stability^
Stage
EDTA molarity
0 hr.
C.P.M.
2 hr.
24 hr.
Late aggregation
0.01
10
5
5
ti ft
0.10
117
59
35
Culmination^
0.01
28
75
112
ti ft
0.10
169
210
216
0.35 mg dry weight
"0.50 mg dry weight
* From Wright, Barbara E.: Control of Carbohydrate Synthesis in the Slime Mold. In
Developmental and Metabolic Control Mechanisms and Neoplasia (A CoUectlon of Papers Pre-
sented at the Nineteenth Annual Symposium on Fundamental Cancer Research, 1965),
p. 308. Baltimore, The Williams and Wilkins Company, 1965.
116
least with respect to their location within the
cell. Now, the enzyme we've been talking about
until now is the one found in the cell husk
fraction of the sorocarp and the acceptor for
the radioactive UDPG is in the cell wall; the
enzyme is bound to the acceptor. The product
is cell wall. That's the alkali-insoluble com-
plex of cellulose and glycogen.
Also, we have been studying for some time
an enzyme in the 100,000 x g pellet. This is
the typical glycogen synthetase using UDPG and
it depends upon glycogen as primer. Now, if
this enzyme preparation is coaxed, it will use
alkali-insoluble cell wall material as acceptor.
Furthermore, cell wall primer is a competitive
inhibitor of glycogen synthesis. Thus, the same
enzyme catalyzes both reactions. In Table X
this enzyme is described. Here we see it is
possible to use an enzyme in the cytoplasm of
the amoeba to synthesize alkali -insoluble cell
wall polysaccharides. This enzyme is in the
100,000 X g pellet and, as you can see, it's
completely dependent upon G-6-P and primer,
the primer being alkali- and cellulase-treated
cell wall material. Finally, we have detected
an enzyme in the amoeba cell membrane. This
enzyme will use glycogen as an acceptor but
is unable to use alkali-insoluble primer. It
responds to EDTA in a manner similar to the
cell wall enzyme (Table XI). Perhaps prolonged
incubation of this amoeba cell membrane frac-
tion with partially soluble acceptors, such as
cellodextrins, will reveal a capacity to synthe-
size an insoluble product. It's our hope to
determine if these 4 enzymes (Table IX) are
all different or, perhaps, all the same except
for their localization in the cell and the primer
to which they are bound.
In summary, we've seen that no single
event could possibly trigger cell wall synthesis
since a complex array of primer, substrates,
activators and enzymes are not only limiting
but must interact to bring about the accumula-
tion of cell wall material. The relative contribu-
tion of these factors and of RNA and genetic
control as well as the time at which each acts
relative to the differentiation process are ques-
tions for the future. The probable interaction
and interdependence of all of these mechanisms
presents a challenging problem, to say the
least.
PAPACONSTANTINOU: Are the glycogen
enzymes the ones responsible for the linkage
of glycogen and cellulose later?
B. WRIGHT: Right.
Enzyme Source
TABLE IX
Acceptor of UDPG-^^C
Sorocarp cell wall Cell wall (bound)
Amoeba pellet Cell wall (added)
Amoeba pellet Glycogen (bound and added)
Amoeba cell membrane Glycogen (added)
TABLE X
100,000 X g Pellet Enzyme Donating to AlkaU-Treated
CeU WaU Primer
Condit ion cpm
0.2 mg primer 1,395
0.1 mg primer 589
No primer 8
No G-6-P 11
TABLE XI
Amoebae Membrane Preparation Catalyzing Incorporation
of UDPG-i^C into Glycogen.
EDTA
Absent
Present
Total cpm
Day 1 Day 2
14
555
0
223
PAPACONSTANTINOU: So, if it' s the same
enzyme, you're going to have to postulate some
mechanism for the change in function?
B. WRIGHT: By the same enzyme I mean
we may only be hooking on glucose in alpha- 1,
4 linkages to the cell wall material. We're
looking at an artificial system; in the cell the
ratio is about 1:1 of cellulose to glycogen in
cell wall material, but in vitro we get 80% of it
in the glycogen fraction. So that when I' m talking
about this 100,000 x g pellet enzyme, it may
just be adding to the glycogen moiety of the cell
wall material. However, that is an alkali-
insoluble material because it is intimately
associated with the cellulose. Now, there is a
big problem about the origin of the insoluble
117
primer. We axe going to look for enzymes which
could accumulate cellodextrins during develop-
ment.
PAPACONSTANTINOU: Does this cell wall
preparation include both spore and stalk?
B. WRIGHT: Yes, we've looked at both and,
from staining reactions with iodine and various
other things, we feel that the glycogen moiety is
present in both equally. Thus, we think that the
material that Gezelius and Ranby studied was,
in fact, the material we are studying. This
could explain their description of amorphous
cellulose, if it really was 50% amorphous
glycogen.
PAPACONSTANTINOU: How can you postu-
late the linkage of the glycogen and the cellu-
lose? How do you picture it?
B. WRIGHT: Well, we tried to separate
them physically with urea and high salt concen-
trations, etc., with very little success. Maybe
you could get a very tight physical binding be-
tween the cellulose and the glycogen.
PAPACONSTANTINOU: What I'm wonder-
ing is, is it possible that there's an enzyme that
is actually attaching alpha- 1, 4 linkages to some
part of the cellulose in a straight line of beta-1,
4's?
B. WRIGHT: Yes.
PAPACONSTANTINOU: You have a free
hydroxyl in the 6 position of the hexoses in
cellulose and you may be getting an alpha-1,
6 to start off the glycogen which will then be a
series of alpha-1, 4 linkages.
B. WRIGHT: We have preliminary evidence
for contaminating maltose in the cellulose frac-
tion and cellobiose in the glycogen fraction.
PAPACONSTANTINOU: Oh, fine.
B. WRIGHT: However, this is all very ten-
tative because you don't know how clean the
preparations are. There is a soluble fraction
and an insoluble fraction, but in each there
could be small amounts of the other that were
not actually attached. The amount of the radio-
active cellobiose is so small that we don't like
to make any definite statements until we get
more of it. Maybe we can trick the in vitro sys-
tem into making more of the cellulose fraction
and really analyze it.
GROSS: How much galactose is in the cell
wall?
B. WRIGHT: I don't know. I guess Maurice
Sussman has data on that. Now, his material is
soluble, of course; ours is an insoluble poly-
saccharide. We've looked for galactose in our
preparations and found none. This cell wall
material has been accounted for by weight, and
it is pretty well characterized as a 50-50 mix-
ture of cellulose and glycogen.
GROSS: Well, where is the product of that
UDP-galactose transferred?
B. WRIGHT: That's on the surface, isn't
it?
HANKS: I believe it's associated with the
cell wall.
B. WRIGHT: Yes.
CANTING: Do you know anything about the
average chain length of the glycogen?
B. WRIGHT: We are now doing that en-
zymatically with a combination of phospho-
rylase and amylo-1, 6-glucosidase, determining
glucose and glucose- 1 -phosphate. Wedon'tknow
yet.
CANTING: I wondered whether it might be
changing at the spore stage as compared to the
other stages.
B. WRIGHT: We want to look into that and
compare the cell wall glycogen, after it's been
separated from cellulose, to the pellet glycogen.
Perhaps the cell could be insolubilizing the
pellet glycogen, so to speak, as a primer. It'll
be interesting if the amoeba membrane enzyme
is similar to the cell wall enzyme. It reacts to
EDTA the same, and it may be that we can't
detect it in its potential role in cell wall syn-
thesis because of the lack of alkali-insoluble
material. The enzyme may be there earlier,
but not bound to insoluble material.
TS'O: I'd like to raise some, perhaps, naive
questions which have been bothering me. In dif-
ferentiation, probably the most interesting event
is the decision-making process. You have dis-
cussed the enzyme-inhibitor levels and rate-
limiting processes. I wonder, how do these
relate to the real decision-making process?
B. WRIGHT: I think it is wrong to think in
terms of an important decision-making phe-
nomenon; I think this never exists. This is a
very complex interaction of many things, and
it's misleading to look for the one cause.
GROSS: However, that might be precisely
why decision- making is absolutely important.
There may be two alternative steps, two stable
states, each self-stabilizing as it matures, but
one small thing may be the deciding factor.
B. WRIGHT: However, here we have shown
there are numerous small things that are de-
ciding factors, since they are all limiting.
GROSS: Yes, but in vivo presumably only
one of them is active.
B. WRIGHT: No. This is a very complex
steady state situation which is as stable as it
is and as reproducible as it is precisely be-
118
cause there isn't one thing that's going to be
important. If there's a little bit lacking of one
thing, another will make up for it. That's why
I used this model of the ATP-glucose-hexo-
kinase system.
TS'O: That's another question I have. What
you're saying is that you have a pretty good
idea about how the glucose and the ATP to-
gether maintain a stabilizing effect for a steady
state. You would think in terms of differentia-
tion, however, unless the state is allowed to
change its course, presumably the dynamics
of the cell would not allow you to jump from
one stable state to another.
B. WRIGHT: Cell wall construction is a
big jump. There is no alkali-insoluble ma-
terial, and suddenly you've got alkali-insoluble
material. Let's just start with cellodextrins.
You've got cellobiose in the amoeba. More
complex cellodextrins are slowly building up so
now you get 6 or 7 glucoses in a chain. It's
getting almost insoluble. At the same time
G-6-P and UDPG levels are rising. Glycogen
is being broken down more rapidly because
inorganic phosphate is accumulating, and you
get a big build-up of precursors. Trehalose is
starting to accumulate, also. Magnesium is be-
coming available by the breakdown of something
else. All these things occur together at about
the same time, buffering each other an inter-
acting with each other. When the UDPG level
is very low, G-6-P comes to the rescue. There's
clear data for that. All these things occur to-
gether at about culmination and suddenly we've
got the insoluble chains of beta-linked material;
and now, the glycogen primer is at a state
where it can be used for cell wall synthesis and
the enzyme is being transferred, or perhaps is
in the cell membrane already. This is pure
speculation, but all these things together now
give us what we consider to be quite a jump.
It's really not a "jump" at all.
GROSS: Yes, you're goingto see a dramatic
change at some point from a system in which
the product is soluble to a system in which it
is insoluble.
B. WRIGHT: Right, and this can be a very
gradual build-up of ten different things in order
to create what we call a very abrupt change.
CHALKLEY: Wouldn't this, then, suggest
that this is a modification of the differentiation
process rather than a complete new change from
one differentiated cell to another differentiated
cell.
TS'O: Your point seems to be the following:
in your system there is a mainstream flowing
through slowly and it is the accumulation of the
stream that gives the momentum for this
"abrupt" change. However, I think in many other
systems - not being a biologist I couldn't give
you specific examples - probably one could have
a diversion of the stream, i.e., it can go one way
or the other. It is the diversion of the stream,
a new choice and not just a continuation, which
I would consider a differentiation.
B. WRIGHT: You have to be more specific
or we can't discuss this.
ZIMMERMAN: How would the antigen sys-
tem that we have just discussed relate to this?
B. WRIGHT: There are differences in en-
zyme levels. Alkaline phosphatase, as I said,
increases seven-fold.
EPEL: I think what Dr. Ts'o would like to
know is, is there some point when you start
initiating this? Is there some earliest point at
which you synthesize a real enzyme?
PAPACONSTANTINOU: Well, aren't you
going from a glucose-6-phosphate independent
enzyme to a glucose-6-phosphate dependent
enzyme?
B. WRIGHT: The low UDPG level requires
the G-6-P.
PAPACONSTANTINOU: Your culmination
stage is very much analagous to the glycogen
phosphorylase story in muscle in which one has
the regulation starting with the cyclase. It looks
like what you've got here is a situation where
you may have to go one step further and look
for some kind of cyclic -3', 5' AMP which is
hormonally regulated.
B. WRIGHT: Oh yes. An nucleotide levels
change also during differentiation. Now, if we
could bring in the phosphorylase story which
is very much involved, the reactions we've
discussed may very well depend on glycogen
breakdown. We could make it even more com-
plicated. However, I think if we want to discuss
the point we shouldn't complicate it further by
bringing in more reactions.
PAPACONSTANTINOU: However, the point
is that you've got, also, reaction dependence
here.
B. WRIGHT: Right. There's an intense
competition and interaction among all the reac-
tions which are going on.
PAPACONSTANTINOU: My only point is
this (I'll try and make it as simple as possible):
it appeared to me that you were going from a
system in which the enzymes showed more of
a substrate independence to a differentiated
state in which the enzymes showed more of a
substrate dependence.
119
B. WRIGHT: No, I showed that G-6-P stim-
ulated at low UDPG not high UDPG. This
doesn't make the enzyme different. All I said
was that the combination of increasing G-6-P
and UDPG would enter into their effect on cell
wall synthesis; and if the UDPG level happened
to be unusually low, the G-6-P would stimulate
the cell wall synthesis more. This is not in-
volved in the reaction although it's a modifier.
It stimulates cell wall synthesis more when
UDGP is low, no matter what stage the enzyme
is taken from.
PAPACONSTANTINOU: No matter what
stage you take this enzyme from you always
get the same reaction?
B. WRIGHT: Right.
GRUN: Am I wrong in thinking that this
organism at the time that it is aggregating is
a syncytium?
B. WRIGHT: That's wrong. There are in-
dividual cells.
FERGUS: There's still one nucleus per
cell.
TS'O: I think that the kind of differentia-
tion process which I have in mind is different
from what you have described. For instance, I
could pose a decision-making process like that
in the determination of sex. Once the decision
is made, the organism will carry this decision
to its grave. That decision is made in the early
cell and you cannot change it.
B. WRIGHT: I don't think you can consider
such complex examples if we're going to talk
about it. This is why I introduced the talk by
saying morphogenesis is a change in structure;
therefore, we can look for a simple example
that we can talk about. A lot of biologists like
to talk and think in such complex terms about
morphogenesis, it's difficult to analyze it.
GROSS: Are there any specific differences
where you can find rate-limiting reactions or
something that does one thing that does give
control such as induction in E. colt?
B. WRIGHT: UDPG is limiting, soisG-6-P,
and several other things here are also limiting.
If you studied one of them alone, it might look as
though you'd found the answer.
GROSS: The general trend of what you're
saying is that epigenetic considerations may be
more central to differentiation than genetic ones.
B. WRIGHT: No, if you don't have the gene,
you don't have the enzyme. All I'm saying is that
if you want to know what the immediate control
of this process is, it may or may not be genetic:
you may have gotten the synthesis of the relevant
enzyme a long time ago; you may already have
the enzyme, or at least the message for it. In
glutamic acid dehydrogenase you have the en-
zyme throughout the differentiation, but it be-
comes 7 times more active when it gets more
endogenous substrate. This is another thing I
think ought to be stressed to clarify the situa-
tion. What we are talking about when we say:
this is essential; this is important. This is one
minor cause here, and we're just beginning to
clearly see that you may have a lot of causes
at one time. You know before a particular dif-
ferentiation process, for example, you've got
templates. They are one kind of cause. Now,
where does the immediate control lie? Maybe
it's on the activation of the message. Maybe
that's not it at all. Maybe the enzyme is syn-
thesized all the time and it simply accumulates
because the substrate is stabilizing. There
could be other explanations, and are probably
many of them.
TILL: Am I right that you're arguing that
what you've studied is all an inevitable con-
sequence of the starvation?
B. WRIGHT: We know that definitely; if it
gets fed, it doesn't differentiate.
TILL: Then the decision is whether or not
it gets hungry.
CHALKLE Y: It' s the concept of differentia-
tion that we're mixing up. Differentiation at the
epigenetic level defined in terms of morpho-
logical changes. One of you is talking about that
and one of you is talking about genetic control
in an already differentiated system.
B. WRIGHT: They're both essential and we
should just define which one we're talking about.
I think it's important to stress that there is no
one important thing here at all.
FERGUS: I don't think that fruiting neces-
sitates starving because you can obtain fruits
right on the same plate with a large supply of
bacteria still present.
B. WRIGHT: Yes, but they're not eating it.
FERGUS: Well, if they aren't eating, there
must be some other factors, then, that prevent
their ingestion, rather than that they're being
starved. They're not being starved; they're
already full of bacteria.
B. WRIGHT: When they're aggr eating,
they are essentially starving. They'll do the
same thing whether you have them on nutrient
agar or 2% agar. An important factor in their
starvation may be that the permeability is ter-
rible in these amoebae. The permeability for
some compounds is l/20th as good in the amoeba
as it is at culmination; by that time you can't
interest them at all in eating.
120
FERGUS: I'm sorry; you left me. There
are bacteria present, and these cells can ingest
bacterial cells.
B. WRIGHT: Yes, there are bacteria that
these cells like.
FERGUS: All right, they're still there be-
cause you can get sorocarps with plentiful num-
bers of bacterial cells present.
B. WRIGHT: Nol
KAHN: No, I agree; you can'tl
FERGUS: You certainly can; I've been able
to do it.
B. WRIGHT: They're not the kind of bac-
teria that these cells like.
DEERING: It's a question of whether
they're taking the bacteria up or not.
FERGUS: Well, then there is some factor
that is controlling the failure of the amoebae
to ingest.
DEERING: If you plate the myxamoebae out
on a lawn of bacteria, you can get colonies
of aggregation and culmination with bacteria
between the colonies. If you put only a few
amoebae down on a plate, they will divide and
then go to a final stage, but there will still be
bacteria that will be physically inaccessible to
them. You get clear regions in the bacterial
lawn that have been eaten out by the amoebae.
GREGG: You can get aggregation among
bacteria; there's no question about that. Prob-
ably the differentiation mechanism overrides
the feeding one; you get aggregation in the
presence of bacteria, and they stop feeding at
that time.
TS'O: Back to the original controversy.
Usually, I would think one of the chief purposes
of people working together in development is
trying to find the most important factor which
determines why a certain event will occur in a
certain way. On the other hand, some may think
that all factors involved are equally important.
It seems to me, therefore, there is a funda-
mental difference in philosophy and that's what
we are arguing about and what this workshop is
about.
B. WRIGHT: It certainly is. It's a very
fundamental difference because people go looking
for the cause of morphogenesis when there are
many.
TS'O: It's naive, but the systems we're
working with clearly ask that question.
B. WRIGHT: We have not picked small
enough problems to be able to find out wnether
it's naive or not. I mean, if you look at some
gross change, if you look at a sea urchin egg,
you know nothing about what's going on during
metabolism. Here in the slime mold, it's so
simpleminded that the main thing it's doing is
converting protein to carbohydrate, and you can
study a simple reaction in this process and
this has some meaning. If you attack a complex
system, you will not know what questions to ask,
or get around to knowing the answer to the ques-
tions, because you don't know enough about the
thing you' re studying.
GROSS: But suppose, for the sake of argu-
ment, that somebody were interested in hemo-
globin synthesis. It's a very complicated sys-
tem. Suppose you're lucky enough to show that
at a certain time in the development of a chick,
for example, product x is to come off the shell.
This product becomes soluble and is a specific
inducer for the messengers that are involved in
the heme part of hemoglobin. Hemoglobin begins
to be synthesized and that, in turn, is responsible
for the aggregation or the differentiation of the
blood islands.
B. WRIGHT: All right, you can make an
isolated observation like that and in this compli-
cated system that's as far as you'll go with it.
TS'O: The question in my mind is whether
or not this organism has made an internal deci-
sion at this point to start differentiating or just
that it starts to differentiate when it has used up
its food. Look at all the synthesis of the cell
wall material. A tremendous amount of chemi-
cal energy is being used there.
B. WRIGHT: There are many processes
begun when it starts starving at 0 hours and at
15 hours it makes cell wall; if you look at what's
going on inside there, you see the proteins de-
creasing, the amino acid pool is diminishing, the
glucose is increasing, and the cell wall is being
made.
KAHN: Pseudoplasmodia (slugs) can under
the appropriate conditions migrate for several
days. It is not until the slugs cease migrating
that final cytodifferentiation begins. Clearly,
the "cue" which triggers differentiation cannot
be "starvation" alone.
121
References
1. B.Wright. In "Biochemistry and Physiology
of Protozoa," S. Hutner, ed. (Academic
Press, Inc., New York, 1964), ///, p. 341.
K. Gezelius and B. Wright.
biol. 38, 309 (1965).
</. Gen. Micro-
3. K. Gezelius and B. G. Ranby. Exp. Cell Res.
12, 265 (1957).
4. A. D. Elbein, G. A. Barber and W. Z. Hassid.
J. Am. Chem. Soc. 86, 309 (1964).
5. C. Ward and B. E. Wright. Biochemistry 4,
2021 (1965).
6. H. J. Fromm, E. Silverstein and P. D. Boyer.
J. Biol. Chem. 239, 3645 (1964).
7. B. Wright. In "Developmental and Metabolic
Control Mechanisms and Neoplasia," 19th
Annual Symposium on Fundamental Cancer
Research (Williams and Wilkins Co., Balti-
more, 1966).
122
CELL INTERACTIONS IN SLIME MOLD
(ACRASINA) DEVELOPMENT
A.J. Kahn
Department of Zoology, Syracuse University
Syracuse, New York
Ontogenetically meaningful exchanges occur
between cells and tissues during the develop-
ment of all multicellular organisms. In the
discussion to follow, evidence will be presented
that cellular slime molds are not exceptional
in this regard.
The now rather familiar life cycle of cellu-
lar slime molds is shown in Fig. 1. While this
drawing was prepared to illustrate Dictyo-
stelium purpureum, in essential features, it is
representative of all members of the family
Dictyosteliaceae. Our attention today will be
focused upon the early stages of the cycle,
beginning with spores (Fig. lA) and terminating
with the formation of the pseudoplasmodium
(aggregate) (Fig. ID).
A nutrient agar plate inoculated with bac-
teria and Dictyostelium (or Polysphondylium)
spores will generally show all stages of the
life cycle after two or three days of incubation
at 25°C. To obtain greater developmental uni-
formity, amoebae may be pregrown in liquid
culture, harvested at the end (or during) the
growth phase, washed and dispensed upon a
non-nutrient substrate. D. purpureum amoebae,
under these conditions, begin to aggregate
after a few hours and complete development in
less than twenty-four hours. Furthermore,
since most of the cells aggregate at about the
same time, substantial synchrony is achieved.
In many respects, a spore is like a zygote —
each spore possessing the ability to germinate,
grow and develop into a complete multicellular
unit. Thus, if spores are isolated and cultivated,
genetically pure clones can be derived.
Table I presents a summary of the various
types of cellular interaction that have been
detected during early slime mold development.
Some of these phenomena are much better known
than others but all, I believe, are worthy of
inclusion in this survey.
The first item in Table I indicates that
spores may interact to limit spore germina-
tion. Russell and Bonner (1) showed that a sig-
nificantly higher percentage of germination
occurs in sparse (dilute) groups of spores than
in dense groups. There are two possible ex-
planations for this observation. When a spore
germinates, it may release into the environ-
Flg. 1.
Life cycle of cellular slime molds. A) Spores; B) germi-
nated amoebae; C) feeding and cell division; D) aggrega-
tion; E) culmination (differentiation of fruiting body);
F) sorocarp (fruUng body) consisting of stalk and spore
mass (sorus).
123
TABLE I
The Types of Cellular Interaction Which Take Place During the Early Development of
Slime Molds.
CELLULAR:
SPORE
BACTERIUM
BACTERIUM
VEGETATIVE AMOEBA
AGGREGATIVE AMOEBA
=^ SPORE (INHIBITION)
-> SPORE (STIMULATION)
-^ VEGETATIVE AMOEBA (ATTRACTION)
VEGETATIVE AMOEBA ,( REPULSION)
AGGREGATIVE AMOEBA (ATTRACTION)
Implemented by
A) Chemotaxis; relay amplification
B) Contact following; adhesion
MULTICELLULAR:
CENTER
-^
CENTER (INHIBITION)
ment a spore germination inhibitor. Or, spores
may compete for some essential factor during
germination. Thus, the first spores to become
active would remove this factor from the en-
vironment and limit the germination of the re-
maining spores. No evidence is available to dis-
tinguish between these two possibilities.
The next item in Table I suggests that bac-
teria may stimulate spore germination. The
evidence for this phenomenon is limited to
some observations that I made several years
ago. I found that six to ten times more spores
would germinate in the presence of bacteria
than in their absence. How bacteria influence
germination is, unfortunately, not known.
As is indicated by the next item in the table,
bacteria may also influence the movement of
amoebae. Samuel (2) demonstrated that amoebae
migrate toward bacteria probably in response
to a chemical released by the bacteria. The
possible relationship of bacterial-amoebal
chemotaxis to aggregation is of interest. It is
well established that aggregation in cellular
slime molds is largely the result of chemo-
taxis. Therefore, during the evolution of these
organisms, chemoreceptors must have evolved
for the receipt and translation of chemical sig-
nals. The first receptors were probably used
to detect and capture bacteria. If this is so, then
perhaps the receptor(s) that operates in aggre-
gation might be nothing more than a modified
version of that used to detect bacteria and, as
such, still is somewhat sensitive to bacterial
attractant. This last assumption could account
for the observed absence of aggregation in the
presence of bacteria. Since the attractant re-
leased by the bacteria would compete for or
occupy receptor sites, no clear aggregation sig-
nal could be received until the bacteria were
removed.
The next item in Table I indicates that
vegetative amoebae repulse one another. Samuel
(2) found that if amoebae are dispensed in small,
dense groups on an agar surface, they will
migrate from the group along rather direct
paths. This migratory activity is probably the
result of a "repellent" that accumulates when
vegetative amoebae are present at high density.
Aggregation is the most complex series of
interactions that takes place in early slime
mold development. It is characterized by the
formation of migrating streams of cells (Fig.
ID). Stream formation is the result of two
mechanisms; chemotaxis (and related "relay
amplification") and "contact following." Relay
amplification describes Shaffer' s model of slime
124
mold aggregation. In this model, the attractant
(acrasin) produced by one cell causes adjacent
cells to migrate toward the source of acrasin
and to, in turn, produce acrasin. The acrasin
produced by the affected cells stimulates other
cells to do likewise resulting in the "relay"
and "amplification" of the aggregation message.
Contact following is a term used by Shaffer (3)
to indicate that cells in a stream adhere and
follow one another. Like circus elephants, the
cells in a row follow the lead cell. How informa-
tion regarding speed and direction of movement
is relayed from cell to cell is not known.
Our attention, to this point, has been focused
on those interactions which take place between
cells. The final item in Table I refers to an inter-
action at the multicellular level. This interaction
is manifest in the disposition of centers (cen-
ters of aggregation) with respect to one another.
More precisely, certain evidence indicates that
the presence of one center may dictate whether
a second center can form within the immediate
area.
Before discussing this phenomenon, are
there any questions?
GREGG: Arnold, would you care to com-
ment on the fact that you can get aggregations
within a mass of bacteria on occasion?
KAHN: I haven't seen this occur myself,
but I can think of a possible explanation. If the
bacterial attractant is short-lived (acrasin is
short-lived under normal conditions), then a
point may be reached where it would no longer
compete with acrasin and aggregation could
proceed.
GRUN: If you take an amoeba from a
colony which is aggregating and if you put it
into the middle of a colony which is vegetative,
does it pass the message to the others?
KAHN: No. However, Sussman has shown
the aggregative phase amoebae can stimulate
aggregation in developmentally younger cells.
If there are no further questions, I should
like now to return to the last item in the table.
My interest in this problem arose as the result
of several investigations carried out by Bonner
and co-workers (4-6). Their studies indicate
that the orientation of fruting bodies and the
number of aggregates formed per unit area of
substrate may be under the control of a factor
present in the gaseous phaseof the environment.
They termed thisfactor the "spacing substance."
I began my study in the hope of answering
two questions. First, does the spacing of aggre-
gates occur in Poly sphondy Hum pallidum? Sec-
ond., if such spacing does occur, is it the result
^
4< ^ ^
sV Vv W vJ<
A
clustered
random
Fig. 2,
C
spaced
Three types of possible spatial distribution of aggrega-
tion centers. A) Clustered, centers appearing In groups;
B) random, centers distributed as expected on the basis
of chance; C) spaced, centers placed at equal distances
from one another. The density of centers is the same in
all three examples.
of a spacing substance present in the gaseous
phase of the environment? Previous work with
Polys phondy Hum indicated that this species was
responsive to those factors (charcoal, mineral
oil) used by Bonner to reduce or eliminate the
spacing substance.
Spacing may be defined as the distribution
of centers of aggregation on a substrate. A
"spaced" distribution is one in which the cen-
ters tend to form at equal distances from one
another (Fig. 2C). ^ A "clustered" distribution,
on the other hand, is one in which the centers
tend to appear in groups (Fig. 2A). The method
of Clark and Evans was used to determine the
distribution of centers. This method consists of
calculating the nearest neighbor distance ex-
pected if the distribution is at random and com-
paring this value with one derived by actual
measurement. If the distribution of centers is
random, the ratio of observed to expected is
unity. If the distribution is spaced, values
greater than one are derived; if clustered, the
values are less than one. Figure 3 is a graphic
illustration of the relation between nearest
neighbor distance and the density of aggregation
centers. Note that deviations to the right of the
curve indicate a spaced distribution while devia-
tions to the left indicate clustering.
In these experiments, the cells were pre-
grown in liquid culture, washed free of residual
bacteria by differential centrifugation, sus-
pended in a saline solution, and dispensed in
1 Figures 2-6 are sketches of data which will appear in
Developmental Biology, 1966.
125
spaced
10
random
25 50
Nearest neighbor distance
Fig. 3.
The relation between the density of aggregates and nearest
neighbor distance. The curve depicts the relationship ex-
pected if the spatial distribution of centers is at random.
Deviations to the right of the curve indicate a "spaced"
distribution; deviations to the left, a "clustered "distribu-
tion.
drops on buffered non-nutrient agar. Counts
of the number of aggregates were made in all
cases after 24-26 hours of incubation. In some
cases, counts were also made at hourly inter-
vals to determine the rate of center formation.
Nearest neighbor distances were obtained with
an ocular micrometer.
When counts and measurements were made
on a number of aggregating populations, it was
found that all three types of distribution oc-
curred. Random distributions were the most
frequent, followed by spaced and then clustered.
Since spaced distributions occur in the presence
of charcoal (an agent that should remove the
spacing substance), it suggests, but does not
prove, that such spaced distributions are not the
result of a gaseous spacing substance. In-
terestingly, spaced distributions were most
often observed in low center density situations
while clustered distribution were associated
with high density.
The correlation between center density and
distribution led to a consideration of those
c
o
o
E
c
o
D
o
10 100
Log density of centers
Fig. 4.
The relationship between the rate of center formation
(the number of centers appearingper unit of time) and the
final density of aggregation centers on the substrate
(surface). Note that the faster the rate of center forma-
tion, the higher the final density.
factors or phenomena that determine center
density. One, apparently fundamental, relation-
ship is illustrated in Fig. 4. Note that the
faster the rate of center formation, the higher
the density of centers.
The next step, then, was to ascertain those
factors which play a role in determining the
rate of center formation. The influence of a
number of such factors are shown in the graphs
in Fig. 5.
Figure 5A illustrates the rate of center
formation as a function of stage in the growth
cycle. Note that stationary phase cells begin to
aggregate the moment they are placed on the
substrate, while logarithmic phase cells do so
only after a lag of two hours. Furthermore, once
aggregation begins, log phase cells proceed
at a much slower rate than do stationary phase
cells.
Amoebae which are incubated in the light
and in the presence of charcoal or mineral
oil, aggregate much faster than comparable
amoebae incubated in the dark and in the ab-
sence of these two factors (Fig. 5B). Charcoal
and mineral oil are believed to remove a center
suppressing factor present in the environment
while light is believed to mitigate the effect of
this factor (7).
126
Q.
O
c
o
ST. PH.
LOG. PH.
LITE, MIN.OIL
LITE, CHARCOAL
LITE
DARK
B.
6(
25 X 10^
5 X 10" CELLS /ML
6
1.25 X 10
. X 10^ C.
12 3 4 5 6
Time (hours)
Fig. 5.
The influence of various environmental and biological
factors on the rate of center formation. The data are
plotted as the number of centers per drop (group or
colony of cells) against time. Graph A Indicates that
cells taken from the stationary phase of growth aggregate
sooner and at a faster rate than do logarithmic phase
cells. Graph B illustrates that the rate of center forma-
tion is faster in the light and in the presence of charcoal
and mineral oil than in the dark and in the absence of
these two agents. Graph C shows that the rate of center
formation is faster at high cell density than at low cell
density.
Figure 5C shows the relationship between
the rate of center formation and cell density.
The higher the density the faster the rate. This
result would be expected if increasing the density
of eells also increased the number of cells
ontogenetically ready to aggregate.
CELL
DENSITY
ADSORBANTS
LIGHT
GROWTH
PHAiSE
RATE OP CENTER PORMATION
f
DENSITY AND DTSTRTBTITTGN
CiV f^KNTF.RS
CELL POOL SIZE
Fig. 6.
The Inter-relatlonshlps between various environmental
and biological factors, the rate of center formation and
the distribution and density of centers. Note that the rate
of center formation and cell pool size are the primary
factors In determining center density and distribution.
Figure 6 summarizes the inter-relation-
ships between the various factors that influence
the rate of center formation, and the distribution
and density of centers. One final variable, not
previously mentioned, is "cell pool size", the
number of cells available for aggregation. If
the pool of cells is large, then after the initial
wave of aggregation, the cells remaining could
aggregate to form additional centers. This would
result in an increase in center density and would
favor the establishment of random or clustered
distributions since these "secondary" centers
could form at any distance from the first. Con-
versely, if the pool is small, few if any cells
would remain after the first wave of aggrega-
tion and no secondary centers could form. This
situation would minimize center density and
favor a spaced distribution.
Two models satisfactorily account for the
relationship between the rate of center forma-
tion, and center density and distribution. In one
127
model, it is proposed that an inhibitor produced
by a center inhibits the formation of other cen-
ters in the immediate area. In the other model,
no inhibitor is postulated and the distribution of
centers is accounted for by the removal of
cells, since without cells no centers can form.
In either model, the distribution and density
of centers depends upon the area that initially
formed centers control (either by withdrawing
cells from the surrounding substrate or through
the spread of inhibitor). Thus, if the time inter-
val between the appearance of centers is long
(a slow rate of center formation), a substantial
area would be occupied and later appearing
centers would be displaced at some distance
from those centers that form first. This situa-
tion would favor a spaced distribution of centers
and low center density (Fig. 7A). On the other
hand, if the time interval is short, initially
formed centers would have little opportunity to
establish territories before other centers would
appear. Since later appearing centers could
form at almost any distance from the first, this
situation would favor the establishment of ran-
dom, if not clustered, distributions (Fig. 7B).
While we cannot, with the data at hand, distinguish
between these models, the cell withdrawal hypo-
A Slow rote of center formation-
TERRITORY
B. Fast rote of center formation-
TERRITORY
Fig. 7.
The consequences of the rate of center formation on center
density and distribution. The faster the rate, the smaller
the area (territory) controlled by first formed centers.
thesis is favored since it does not require the
postulation of an additional, unknown factor.
We may conclude, then, that the non-random
distribution of centers (spaced or clustered)
occurs in Polysphondylium pallidum; that cen-
ter distribution is probably not the result of a
"spacing substance" present in the gaseous phase
of the environment; that what is involved in
establishing center density and distribution is the
rate of center formation and the number of cells
available for aggregation, i
GRUN: It might be possible to find out
whether there is an inhibitory substance or sup-
pressor simply by taking strips of agar these are
growing in from between the centers and putting
them on a petridishbetween strips of agar which
have not had centers growing near them, "undif-
ferentiated" agar, and then see if amoebae placed
on this surface will stay off the experimental
strips.
KAHN: Shaffer has done an experiment
similar to the one you suggest. Aggregates were
allowed to form on opposite sides of a thin agar
membrane. Under these conditions, it was pos-
sible to note that aggregates tend to organize
in the space between aggregates located on the
opposite side of the membrane. This suggests
that some sort of diffusable inhibitor (spacing
substance) may be produced that determines the
spatial distribution of aggregates.
GRUN: It would be diffusing upward in this
case?
KAHN: Yes.
GREGG: Did you say that the centers form
in between the original centers?
KAHN: Yes.
GREGG: How does this correspond to Sus-
sman's thin membrane experiment?
KAHN: I don't know. The observations are
certainly contradictory.
EPEL: Do these centers all have varying
numbers of cells in them or does that vary
under these conditions, too?
KAHN: In a rapidly aggregating population
of cells, one tends to get numerous aggregates
of "moderate" and approximately equal size.
In a slowly aggregating population, fewer, but
larger, aggregates are formed.
GRUN: You didn't talk about the mineral oil.
KAHN: No one really knows how mineral
oil influences aggregation. Perhaps it is behaving
as an absorbant (adsorbant?). Personally, I feel
1 The data presented above will appear in full in Devel-
opmental Biology, 1966.
128
more confident about the effect of charcoal.
TS'O: I'd like to ask a question about the
data on aggregation. Is there a possibility that
some of the influencing substances are physical
in nature?
KAHN: There's a very good possibility.
POLLARD: Has anyone tried to prevent this
phenomenon in an electric field?
KAHN: No, but I think it would be a very
good idea to check for possible bioelectric
phenomena in aggregation. In a single trial, we
were able to detect a potential difference be-
tween the front and back end of the slug.
POLLARD: However, if this thing is alter-
nating very rapidly, perhaps you might not be
able to interfere with it.
KAHN: The apparent rapidity of cell move-
ment in aggregation (note: as seen in a film
shown during this talk) is an illusion created
by showing time lapse photographs at normal
projection speeds. Actually cell movement is
quite slow.
UNKNOWN DISCUSSANT: One last question
while we're on this subject of potential. Has
anyone tried the effect of chelating agents on
this phenomenon?
KAHN: DeHaan did this with EDTA.
UNKNOWN DISCUSSANT: Wouldn't this in-
terfere with the adhesion?
KAHN: It does. Apparently the aggregates
formed without streams. That's why I think this
ought to be looked at in detail.
GREGG: Gerisch also did this and he found
an EDTA sensitive stage and an EDTA insensi-
tive stage. After aggregation occurs, they're
EDTA insensitive so they stick together.
UNKNOWN DISCUSSANT: Is there any
morphological polarity in these cells?
KAHN: During aggregation, there is at least
transient morphological polarity.
GREGG: Does your curve imply that founder
cells may occur as a result of aging of the cell?
KAHN: "Developmental" age is probably
one of the factors that plays a role in the estab-
lishment of a founder cell. In this case, the
transition period between the end of feeding and
the onset of aggregation is probably the most
significant.
EPEL: Is there any possibility they're
going anaerobic under mineral oil?
KAHN: Mineral oil does permit the dif-
fusion of gases and you must bear in mind that
the layer used in these experiments was not
very thick.
GREGG: Well, won' t they aggregate anaero-
bically anyway?
B. WRIGHT: Yes, but what is called anaer-
obic sometimes is not strictly anaerobic.
References
1. G. Russell andJ. T.Bonner. Bull. Torr. Bot.
Club 87, 187 (1960).
2. E.V/. Samuel. Develop. Biol. 3, 317(1961).
3. B. M, Shaffer. In "Advances in Morpho-
genesis," M. Abercrombie and J. Brachet,
eds. (Academic Press, New York, 1962),
2, 109.
4. J. T. Bonner and M.R.Dodd. Biol. Bull. 122,
13 (1962).
5. J. T. Bonner and M. R. Dodd. Develop.
Biol. 5, 344 (1962).
6. J. T. Bonner and M, E. Hoffman. J. Embryol.
Exptl. Morph. 11, 571 (1963).
7. A. J. Kahn. Biol. Bull. 127, 85 (1964).
129
HISTONES IN RELATION TO CONTROL IN
LIVING SYSTEMS
Roger Chalkley
Division ot Biology, California Institute of Technology,
Pasadena, California
As this is a workshop, what I plan to do is
provide a broad outline of some of the things
which are being studied in Professor James
Bonner's laboratory at the California Institute
of Technology. We are concerned with the
molecular aspects of control mechanisms in
differentiated tissues. The strategy of attack
is first to isolate the chromosomal material
in a pure form.
In Fig. 1 is shown a general scheme for
the isolation of chromatin. This scheme is appli-
cable to mammalian tissues and slight modifi-
cations are necessary for plant tissue, but the
general principle is the same. The tissue is
disrupted in a Waring blendor in increasing
volumes of the grinding medium and at increas-
ing speeds. The grinding medium consists of:
0.25 M sucrose, 0.003 M calcium chloride and
0.005 A/tris, pH 7.3. Grinding at increasing
volumes and increasing speeds removes peri-
nuclear contamination and gives rise to what
we think are reasonably pure nuclei. These
nuclei can be used for amino acid incorpora-
tion studies in vitro. The nuclei are washed
once with grinding medium and then with saline
EDTA. This inhibits the action of degrading en-
zymes and also removes the calcium that is
stabilizing the nuclear membranes. This makes
the next step, lysis in 0.01 M tris, more con-
venient. The lysed material is centrifuged
through a rough sucrose gradient at 22,000
rpm for two hours. This gives rise to a gel-like
pellet which, after dialysis against low con-
centrations of tris at pH 7.3, is known as "puri-
fied chromatin". Chromatin so prepared has a
high Svedberg constant and for the purpose of a
number of experiments it has proved advan-
tageous to shear the material and remove larger
aggregates by low speed centrifugation. The
nucleoprotein remaining in solution (90%) is
commonly referred to as nucleohistone.
The chemical compositions of some of the
chromatins that have been isolated are shown
in Table I. The histone:DNA ratio is roughly
1:1. In addition there is a very small amount
of RNA which is difficult to remove. This RNA
is partially resistant to RNase (1, 2). In the
case of pea cotyledon there is a more than
normal quota of RNA, but one has to recognize
that it is a rapidly developing system. It has
also been impossible to remove all of the non-
histone protein and this may have an important
contribution to make toward the chromosomal
apparatus. The histones themselves are acid-
soluble and this frequently provides a method
for their isolation. The molecular weight of the
acid- extracted material appears to be less than
10^. The molecular weight of lysine-rich his-
tones is usually estimated to be about 10,000
CHRQMATIM ISOLATION!
Washed Tissue
Grindinq procedures in Wanna Blendor
I
Washed in qrindinq medium (2 X)
Wa5hed in 0.15 M saline-EDTA
I
Lysed into Tris pH/.s
Purified chromatin isolated after
centrifuqation through a sucrose qradient
Fig. 1.
131
6000
CALF THYMUS HISTONES (COLUMN STANDARDIZATION]
REFR INDEX— ^ 1
-
nb /
-
-
' 1 ^^H-ARGININE
-
m
1 Va
02
C'*-LEUCINE^
RO la lb
\ w
-
01
\ \
-
13700
CPM
300 1 3600
13550
200 I 3500
II 21 31 41 SI a 71 81 91 101 III 121 131 l<X 151 ISI 171 181 191
Fig. 2.
Elutlon of hlstones from the cation exchange resin GC-50,
using a gradient of guanidinium chloride (8-40%, meas-
ured as refractive index). The quenching of C ( a ) or
H3 (A ) during the increasing salt concentration in the
eluted fractions is shown.
and the arginine-rich histones are somewhere
in the order of 25,000 (3). Elevated ionic
strengths dissociate histones from the chromo-
somal apparatus (4). They contain neither try-
ptophan nor cysteine (5). Within a given species
the electrophoretic pattern obtained on an acryl-
amide gel is comparable from organ to organ,
but between species there are sometimes small
but characteristic changes in patterns. Histones
can be identified further by elution from a cation
exchange resin, and this has proven to be a very
useful tool.
A typical pattern is shown in Fig. 2. This
shows acid-extracted histones from calf thymus.
They were applied to the resin in 8% guani-
dinium chloride and eluted with the gradient
shown. One invariably finds a run-off peak
(R.O.), the nature of which is a matter for con-
siderable speculation right now. Histones la and
lb are very lysine-rich, while lib is moderately
lysine-rich and HI and IV are arginine-rich.
One of the earlier studies done in the group
was to see if the isolated chromosomal material
could do some of the things that one would expect
of the in vivo material. One of these was to see
if it could act as a template for DNA-dependent
RNA synthesis and to compare the template
activity of the chromatin with that of DNA which
had not been isolated from an identical prepara-
tion of chromatin (Fig. 3). Here one sees the
4000
2000
a,
i.
Q
<
a:
g 6C00
a:
o
o
a
<
NTP(x 10
4000 -
2000
B
1
1
--^
--DNA(8/xg)
0 002
-
Y
1
(RNA)
0001
y*
xY
0
/
1°
^DNA(l/j.q)
1
^
' — """^
K„ = 2 8« lO'*
10 20
--(nIpi'^'o' -
1
05
NTP(xlO M)EACH
Fig. 3.
DNA-dependent RNA synthesis - a comparison of the tem-
plate activities of liver chromatin and rat liver DNA.
(a) The effect of increasing nucleoside triphosphate
(NTP) concentration upon the template activity of DNA
and chromatin (present in equal amounts); (b) the effect
of Increasing NTP concentration upon the template activity
of different concentrations of DNA. (Fig. 7, Marushlga
and Bonner, j. Mol. Biol. 15, 160, 1966; reproduced with
permission of Academic Press.)
incorporation of AMP into RNA using as tem-
plate either chromatin or DNA isolated from an
equivalent batch of chromatin. It appears that
the chromatin is unable to make RNA at the same
rate as an equal amount of DNA. These particu-
lar experiments were performed by Dr.
Marushiga. In Table n you see some more simi-
larities with in vivo experiments. The synthesis
132
TABLE I
Chemical Composition of Chromatin (mass ratios)
Calf "niynius Calf Endometrium Rat Liver Pea Embryo Ascites TXimor
LNA
1.0
1.0
1.0
1.0
1.0
RNA
0,022
0.11
0.0U3
0.55
0.12
HI 8 tone
l.lU
0.91
1.0
1.07
1.20
Non- hi stone
protein
0.33
0.66
0,67
0.57
0.98
of RNA is seen to be sensitive to actinomycin D.
It is reduced in the presence of DNase but as the
chromatin is fairly resistant to DNase the effect
is more striking following pre-incubation of the
chromatin with DNase.
Now, we know there are histones associated
with this template, and it was intriguing to see if
an appreciable quantity of histone could be re-
moved without dissociating too much of the non-
histone proteins that were present. So Dr.
Marushiga examined salt extraction of rat liver
chromatin and I show the results of some of
these experiments in Fig. 4. He extracted with
sodium perchlorate. First of all, as the concen-
tration of sodium perchlorate increases, his-
tones are released. Then in the region at about
0.4 M he began to dissociate a sizeable amount
of non-histone protein. If one looks at salt con-
centrations where there is not a great deal of
non-histone protein removed, but a considerable
amount of histone is removed, it is possible to
see in all three of these cases that RNA syn-
thesis in the in vitro experiments has been con-
siderably elevated. There appears to be a posi-
tive correlation between histone removed and
increase in RNA synthesis.
Does the RNA made in these in vitro sys-
tems have any biological significance? Can it
direct protein synthesis? In Table HI you see
the results of some work done by Dr. Bonner
and Dr. Huang. They isolated chromatin from
two sources: the pea cotyledon and from pea
apical buds. They used the chromatin to gene-
rate RNA and then coupled it withafull protein-
synthesizing system. Then, after incubation, they
used immunological precipitation techniques to
detect the formation of globulins. In the case of
cotyledons which make globulin in vivo, the
chromatin is able to direct (via RNA synthesis)
TABLE II
Inhibition of the RNA Synthesis Directed by Rat Liver
Chromatin
No.
experiment
System
AMP incorporated
jifiM/O. 25 ml
incubation mixture
I
Complete
" +
" +
Actinomycin D (5
DNAase {5 tig)
chromatin
eg)
710
128
416
30
U-
Complete
" +
DNAase (5 ^g)
chromatin
1640
225
60
m
Complete
" +
RNAase (2. 5 iig)
chromatin
860
288
105
^Chromatin was preincubated with DNAase at 37" C for 10 nnin.
(Table 3, Marushiga and Bonner, /. Mol. Biol. 15, 160,
1966; reproduced with permission of Academic Press.)
a reasonable percentage of globulin syn-
thesis. However, the apical bud chromatin, when
treated in identical circumstances, appears to
be able to synthesize only a very small amount.
They ran some parallel experiments with T4
phage (which so far as we know do not make pea
seed globulin) and the cross reaction they ob-
tained would suggest a low background effect
comparable to that obtained from apical bud
chromatin. Apical buds in vivo do not make pea
seed globulin. Thus there is a correlation be-
tween the protein made by a specific tissue and
the ability of chromatin derived from that tissue
to make messenger for that protein.
PAPACONSTANTINOU: Why is there that
variability in the amount of protein that is being
synthesized in the first column? You have about
133
0.25 0.50 1.0 2.0 0.25 0.50 1.0 2.0 0.25 0.50 1.0
SODIUM PiRCHLORATE COtiC.CM)
Fig. 4.
Relation between protein dissociated from rat liver chromatin and template activity (AMP
Incorporated). (Fig. 4, Marushlga and Bonner, ] . Mol. Biol. 15, 160, 1966; reproduced with
permission of Academic Press.)
TABLE III
Synthesis of Pea-Seed Globulin by Messenger RNA
Dependent Ribosomal System in Response to Messenger
RNA Generated by Two Different Kinds of Pea-Plant
Chromatin
Template for RNA
synthesist
C"-leucine incorporated
into protein
Total soluble
protein Globulin
(cpm) (cpm)
Globulin/total
protein
(°0
Apical bud chromatin
15650
16
010
Apical bud chromatin
41200
54
013
Cotyledon chromatin
8650
623
7-2
Cotyledon chromatin
6500
462
69
t The reaction mixture contains all materials required for both RNA and protein
synthesis. Incubation for 30 minutes at 37^ All particulate material was then
centrifuged off at 105000 ■ g and pea-seed globulin content of soluble protein
synthesized determined by immunochemical assay.
five-fold difference when comparing the coty-
ledon chromatin and the apical bud chromatin.
Do the preparations vary that much?
CHALKLEY: Well, 1 suspect that in these
cotyledons the overall in vivo synthesis of RNA
may be considerably below the overall RNA
synthesis in apical buds and this may be mirrored
in the capacity of the chromatin to make RNA.
The results also reflect different scales of ex-
periments.
PAPACONSTANTINOU: Did you treat with
perchlorate?
CHALKLEY: No, that's exactly as it was
isolated. They haven't been treated at all.
In order to identify the protein as precisely
as possible they applied the following ap-
proaches. The in vitro synthesized protein was
purified using the procedure applicable to pea
seed globulin. If this material is synthesized
from C^* -labeled amino acids and then diluted
with cold globulin isolated from pea cotyledon,
it has been shown to have exactly the same
Svedberg constant as native globulin when studied
in the ultracentrifuge. In addition, radioactive
globulin was digested with trypsin and the re-
sulting peptide fragments separated by two-
directional paper chromatography, and com-
pared with the tryptic digestion pattern from
native pea seed globulin. Radioactivity was found
in positions corresponding to every peptide spot
(from native globulin) and no trace of radio-
activity was located in other regions of the
chromatogram. So it appears that it is really
genuine globulin.
POLLARD: Have you done anything with
antibodies? Does it precipitate?
134
CHALKLEY: Yes.
Dr. Maurer and myself have been concerned
with the problems of hlstone metabolism. This
is an interesting field in which the results tend
to depend upon the methods employed for the
isolation of the different histone fractions. We
have been fortunate to have the advantage of
using the relatively standardized IRC- 50 micro-
separation techniques which have been developed
at Cal Tech. In order to keep the system as
simple as possible, we decided to investigate
systems in which no DNA was being made. In
the presence of DNA replication, as the DNA:
histone ratio appears to be efficiently main-
tained, all histones must be synthesized. One
promising system that we had available to us,
in connection with some hormone studies which
we were doing, was the endometrium tissue iso-
lated from immature calves. We can get a great
deal of this tissue which, in the absence of
estradiol treatment, is not involved in DNA
replication. The tissue was incubated in the
presence of CI-* -leucine and histones were iso-
lated directly from the nucleus by acid extrac-
tion. The results of the subsequent fractionation
of the histones are shown in Fig. 5. Again the
optical density pattern is similar to that de-
scribed previously. However, the most striking
thing is that histones la, lb, and lib, so far as
we can see, do not incorporate radioactive
label to any significant degree. Peaks III and
IV and the material in the run-off peak (R) do
incorporate label. In order to demonstrate that
this was standard protein synthesis, we ran con-
trol experiments with puromycin present in the
incubation medium and the incorporation was
decreased by a large amount in both instances
(Fig. 6).
We were interested in seeing if this labeled
histone was really attached to the chromatin
which we isolated following procedures de-
scribed earlier. We combined this with the
study of the incorporation of amino acids into
peptides in isolated nuclei. Figure 7 shows the
results of a nuclei incubation followed by chro-
matin isolation from the nuclei. Histones were
obtained by acid extraction of the chromatin.
Again, there is incorporation of the label into
III and IV and into the run-off peak. I should
add that if one measures the specific activity
in the peaks from the whole tissue incorpora-
tion, the specific activity approaches that of the
whole cytoplasmic protein.
We wondered if this was a general effect or
whether it was just a rather unusual result
found in the one tissue. In Fig. 8 you see what
O 04
t
? ''
-^ : ^ ] \ ] r-P :
HISTONES FROM CALF ENDOMETRIUM MUCLEI I
R
if
(tissue rncuDaiion ) J
4
O
o
; 1
12
8C
'1'
r\
1 1
1
o
o
9
6
no '
M
1 \
/ ♦ ^
,1 , \
I
, ' ifil
, lb ' ', 'A'
.
.1
L
1
1
V-
1400
1200
1000
800
80 I OU 120
fra:tion mumber
Fig. 5.
The biosynthesis of histones of incubated endometrium tissue In the absence of DNA repli-
cation. (Fig. 2, Chalkley and Maurer, Proc. Natl. Acad. Sci. U.S. 54, 498, 1965; repro-
duced with permission of the National Academy of Sciences.)
135
O 0 4
T
-^ ' 1 1 \ 1 r-9 1 1
HISTONES FROM CALF ENDOMETRIUM NUCLEI
c
20 o
(tissue incubotion, puromycin inhibition)
CL
_
1
--o GuCI
..^-,-'—'--^
-
.-o-
nb
' 1
; I
' 1 *
A
' » III*IZ
f 1
lb ,' \ f I
10 A / \ I \
'1
■uS
^
0 20 40 60 80 100 120 140 160 180 200
FRACTION NUMBER
Fig. 6.
Puromycin inhibition of hlstone biosynthesis in the absence of DNA replication. (Fig. 3,
Chalkley and Maurer, Proc. Natl. Acad. Sci. U.S. 54, 498, 1965; reproduced with per-
mission of the National Academy of Sciences.)
- HISTONES FROM CALF ENDOMETRIUM CHROMATIN
S (nuclei incubotion)
*ni*ix
FRACTION NUMBER
Fig. 7.
Biosynthesis of hlstones in incubated. Isolated nuclei. (Fig. 1, Chalkley and Maurer,
Proc. Nail. Acad. Sci. U.S. 54, 498, 1965; reproduced with permission of the National
Academy of Sciences.)
136
0 15-
0.05 -
c
-20 S
1
HISTONES
FROM RAT LIVER CHROMATIN
r
1
GuCI CONC, 1
(animal injection)
1
1
1
1
1
-
D
-12 1
1
a
-o---°
- -D-
-□-'
J
-a
.
1 1
«
1 w V
<t
/\
• >
fc
i\^
1 1
1 1
'/"H
M
•■•-►
sa^^
1
■ \
20 40 60 80 100 120 140 160 180 200
FRACTION NUMBER
Fig. 8.
Biosynthesis of rat liver hlstones. (Fig. 5, Chalkley and Maurer, Proc. Natl. Acad. Sci.
U.S. 54, 498, 1965; reproduced with permission of the National Academy of Sciences.)
happens if we inject C^^ -leucine into a rat and
isolate histone from liver chromatin. The first
thing 1 would like to point out here is that the
column elution pattern of histones has now
changed a little. This isn't surprising as slight
variations are found from species to species;
though the similarities between histones are
often more impressive than the differences.
However, again there is labeling in in and IV
and the run-off peak and no real elevation above
background for the remaining histones. We then
wished to examine the patterns of synthesis in
the plant kingdom. We selected two systems: pea
cotyledons and cultured tobacco cells. In order
to avoid the problems of concomitant DNA syn-
thesis we employed pea cotyledons from which
the growing embryonic axis was cut off im-
mediately prior to the experiment. The pea
cotyledons were incubated in a sterile medium
in the presence of antibiotics and C^"* -leucine.
The pattern of labeling found in this type of
experiment is shown in Fig. 9. Again there is a
slightly different optical pattern indicating
slightly different histones. However, we see also
the same general pattern found before; that is,
a small amount of label in the run-off peak and
in the III and IV peaks.
We had one more system which we could
conveniently investigate. Here we had tobacco
cells growing in exponential growth in a chemi-
cally defined medium. DNA synthesis continued
apace. They were allowed to incorporate C^*-
leucine to study the incorporation into all his-
tone fractions. The pattern of histone biosyn-
thesis is shown in Fig. 10. The next step was
to take these cells and treat them with 5-FDU.
We knew from the work of Birnstiel and Flamm
(7) that in this system within two hours after a
treatment with lO'^ M 5-FDU, we would totally
inhibit DNA synthesis, without a serious reduc-
tion in RNA synthesis. We could now study a
system where we had artificially inhibited DNA
synthesis. We must bear in mind that the only
thing we've done to alter the system is to im-
pose a metabolic block to the formation of
thymidine. Figure 11 shows the result of this
treatment upon histone biosynthesis. There has
been a change to the pattern observed in cells
in which DNA synthesis was not normally being
synthesized. Thus, it appears that by applying a
137
o
i
1 1 1 1 1 TT
HISTONES FROM PEfl COTYLEDON CHROMATI
(tissue incubation ) ,
FRACTION NUMBER
Fig. 9.
Biosynthesis of pea cotyledon hlstones. (Fig. 6, Chalkley and Maurer, Proc. Natl. Acad.
Sci. U.S. 54, 498, 1965; reproduced with permission of rhe National Academy of Sciences.)
T 0.5
"I
12 ;
HISTOIMES FROIl^ TOBACCO CELL CHROIi«ATlN
(cell incubotion. 5-FDU absenti
*^ **^
Fig. 10.
Biosynthesis of histones in cultured tobacco ceUs growing exponentially. (Fig. 8, Chalkley
and Maurer, Proc. Natl. Acad. Sci. U.S. 54, 498, 1965; reproduced with permission of
the National Academy of Sciences.)
138
HISTONES FROM TOBACCO CELL CHROMATIN i
(cell incubation, 5- FDU inhibition) '
0 20 40
80 100 120 140
FRACTION NUMBER
Fig. 11.
Biosynthesis of hlstones In cultured tobacco cells after inhibition of DNA synthesis with
5-FDU. (Fig, 7, Chalkley and Maurer, Proc. Natl. Acad. Sci. U.S. 54, 498, 1965; repro-
duced with permission of the National Academy of Sciences.)
very simple block to DNA replication, we are
inhibiting the formation of certain types of his-
tones.
A somewhat allied topic concerns the repli-
cation of DNA in the presence of histones. Al-
though it has been suggested that histones might
repress the function of RNA polymerase it is
evident that many cells are quite capable of
maintaining the function of DNA polymerase in
the presence of this ubiquitous protein. Thus
an in vitro study of the effect of DNA poly-
merase upon nucleohistone seemed an exciting
realm for study. This has been pursued by
Dr. S. Schwimmer. Of particular interest was
the fate of the histone associated with the tem-
plate. How would it distribute itself among the
progenv molecules?
The plan of his experiments was some-
what similar to that adopted for the in vitro
analysis of RNA synthesis. He isolated nucleo-
histone from calf thymus. This was incubated
in the standard fashion for DNA synthesis.
The products were examined on a free boundary
electrophoresis apparatus (8) previously stand-
ardized relative to the electrophoretic mobili-
ties of DNA and nucleohistone. The results are
shown in Fig, 12. This shows that some of the
radioactive precursor has been incorporated
into material with the mobility of deproteinized
DNA. In addition some radioactivity is seen
in the region with the mobility expected for
nucleohistone. An important question was to
find out if the newly synthesized DNA had any
histone associated with it. This was answered
by exploiting the well known resistance of
nucleohistone to DNase during a time period
in which DNA alone is extensively degraded.
Experiments showed that the newly synthesized
material (measured in terms of cpm) is
readily solubilized by DNase. Thus a rather
curious result arises from these in vitro ex-
periments, namely that the daughter strands of
DNA are not associated with histone. If the
parent molecule was in fact in such an associa-
tion it is hard to explain this circumstance.
So far there has been no resolution of this
apparent inconsistency.
Evidence exists that the steroid hormones
exert their primary effects at the genetic level
and thus these hormones seem a useful tool
with which to examine the molecular mechan-
isms of control in higher organisms. Some
139
200
1.0 2.0
MOBILITY
Fig. 12.
Free boundary electrophoresis of the product of nucleo-
hlstone-primed DNA synthesis. The faster component has
the mobility of DNA (peak at 2.2), the slower has that of
nucleohistone (peak at 1.6).
studies to this end have been initiated at Cal
Tech. In particular I wish to discuss a number
of experiments related to the release of dor-
mancy in the potato tuber, to the increase in
enzyme activity of the liver induced by hydro-
cortisone, and to the effects of estradiol in
preparing the endometrial layer of the uterus
for implantation following fertilization. The
first experiments were performed by Dorothy
Tuan (9). The system she has studied is the
dormant bud in the potato tuber, the dormancy
of which is relieved by ethylene chlorohydrin.
Dormant buds of potato tubers are treated with
ethylene chlorohydrin for three days and im-
mediately there is an increase in DNA and RNA
synthesis (Fig. 13). The RNA synthesis is
actinomycin-D sensitive. If RNA synthesis is
inhibited at this time in the development, DNA
synthesis is also stopped. Therefore, the
strategy of her next experiments with the sys-
tem was to isolate chromatin from the buds at
an early period where it is making very little
RNA, and to compare its in vitro template activ-
ity with that of chromatin isolated from the
buds at a later period in the development where
RNA synthesis was much increased in vivo.
Table IV shows the result of this type of in-
vestigation. Potato tuber chromatin was found
to have an exceedingly low template activity.
However, this is not necessarily significant since
isolation of the chromatin from the tuber pre-
sents considerable technological difficulties due
to the almost infinite amount of starch present.
In the case of chromatin isolated from the
dormant bud, we see that chromatin can direct
the synthesis of RNA, but at a very low level.
At the end of three days' treatment with ethylene
chlorohydrin, the template activity of the bud
chromatin is seen to increase. A significant
increase in the amount of RNA synthesized is
observed. Thus the template activity of isolated
chromatin has mirrored its change in the pat-
tern of RNA synthesis in development.
A similar approach has been applied to the
study of the effect of hydrocortisone upon RNA
synthesis in the liver of the rat. These studies
have recently been reported by M. Dahmus and
J. Bonner (10). The experimental design was to
compare the template activity of liver chromatin
adrenalectomized rats before and after hor-
mone administration.
A characteristic result of this type of ex-
periment is shown in Fig. 14. The template ac-
tivity (rate of RNA synthesis) is plotted as a
function of the increase in concentration of the
DNA or purified chromatin in the incubation mix-
ture. In the in vivo experiments the increase in
RNA is some 300% (11). However, in this sort
of study it is not nearly so dramatic. This may
be due to a difficulty of getting some of the RNA
in this system to leave the template. Due to the
relatively small increase in chromatin template
activity, statistical studies were applied (10) to
this system and the difference shown to be sig-
nificant at the 95% level. The possibility that an
increase in RNA synthesis following hormone
administration might be due to less RNase or
ATPase in the in vitro system was checked and
found not to be the case. The size of the DNA in
the induced and noninduced chromatin was the
same, as deduced from analytical ultracentri-
fuge studies. The deproteinized DNA from both
types of chromatin were identical in their ability
to direct DNA-dependent RNA synthesis.
In our experiments with estradiol we have
adopted a different approach. Estradiol, applied
in vitro to the endometrial cells of immature
calves, stimulates RNA and protein synthesis
followed by DNA synthesis and mitosis about
44 hours after the initial hormone applica-
tion (12). The fact that it is possible to demon-
strate such mitosis by microphotography (12)
shows that the tissue incubated in vitro appears
to be responding in the same way that the
endometrial tissue is in the calf. One thing which
interested us and which is pertinent to the
problem of histones and the relationship of
140
uu
1
1 1 1
1
80
-
p
60
-
/ -
40
-
dna/
y
/RNA
20
fi^rr^j:--'
1 1 1
<
0 Z 4 6 8 10
DAYS AFTER TREATMENT
RNA and DNA content of buds of potato
tubers at varying times after 3-day pretreatment with
ethylene chlorohydrin.
Fig. 13.
(Fig. lA, Tuan and Bonner, Plant Physiol. 39, 768, 1964;
reproduced with the permission of the American Society
of Plant Physiologists.)
E
CM
d
<
IT
O
a.
a:
o
CJ
z
a.
<
4.
1200
-
1 I
I
900
-
/
^l -
600
-
-
300
/
1 1
1 a
0 10 20 30
|ig DNA per 0.25 ml
Fig. 14.
Template activity of rat Uver chromatin Isolated 4 hours
after treatment with hydrocortisone(-o-)or saline (-a-).
histories to control was as follows. It has been
reported recently in the literature that cells
can be treated with hormones in such a way as
to give a histone-hormone linkage, and it was
implied that hormones might be pulling the his-
tones off DNA. We had an excellent system with
which to examine this hypothesis since we were
able to study large amounts of target organ
tissue.
We were anxious to see if endometrial tissue
incubated in vitro followed some of the rules
that one would expect from the in vivo endome-
trial material. Figure 15 gives an account of the
uptake of hormones into the endometrial cell.
There appears to be some degree of additional
concentration of estradiol and progesterone.
Progesterone is also a hormone which has the
endometrium as a target tissue during preg-
nancy, and so it is not surprising that it is also
concentrated into the tissue. Incorporation of
hydrocortisone which, of course, has liver as its
target organ was low. In Fig. 16 you see the
uptake into the cytoplasmic fraction. It follows
the overall pattern of the previous figure. How-
ever, now when we looked at the lysed nuclei
[which I will refer to as crude chromatin (bottom,
Fig. 16)] we began to see a very dramatic dif-
ference. Again, I stress that as yet lam discus-
sing hormone uptake and not binding. There are
large amounts present of the hormones for which
TABLE IV
Effectiveness of Chromatin of Dormant and of
Non-dormant Potato Buds in the Support of DNA-
dependent RNA Synthesis by Exogenous RNA Polymerase
For composition of reaction mixture see Materials and
Methods.
50 Mg of DNA
supplied to system as :
RNA synthesized
/iyumole AMP incorp
per 10 min
Potato DNA (deproteinized)
Chromatin of potato tuber
Chromatin of dormant buds
Chromatin of buds from tubers at
end of 3-day treatment with
ethylene chlorohydrin
Chromatin of buds from tubers
10 days after 3-day treatment
with ethylene chlorohydrin
3370*
0
122
1412
1538
* Incorporation due to polymerase alone (150 /u/imole)
subtracted.
(Table I. Tuan and Bonner. Plant Physiol. 39, 768, 1964;
reproduced with permission of the American Society of
Plant Physiologists.)
this is the target tissue, and small amounts of
the other steroids. I should add that the specific
activity of testosteroneandestradiol were within
2% of each other. Hydrocortisone was somewhat
141
<
q:
o
Q.
cc
o
o
o
36
24
12
uptake of hormones into
calf endometrium cells
h'-estradiol
h'-progesterone
h'-testosterone
h'- HYDROCORTISONE
Fig. 15.
40
30-
O 20
<
o
Q.
cr
o
(J
z
LU
o
q:
LU
Q.
10 -
-
UPTAKE OF HORMONE INTO
CYTOPLASMIC FRACTION
H'-FSTRflnini
h'-PR06ESTER0NE
h'-testosterone J
h'-hydrocortisone
2 -
UPTAKE OF HORMONE INTO
CRUDE CHROMATIN FRACTION
H^-ESTRADIOL
H -progesterone
h^-testosterone
1 , h'-hydrocortisone
Fig. 16.
lower so it makes the interpretation of that re-
sult rather more difficult.
In the final figure of this trio we see the
specific activity of hormone actually bound (fig.
17). I define "bound" as that hormone which
can be centrifuged through a sucrose gradient
along with the chromatin into the pellet and
which isn't removed by subsequent exhaustive
dialysis. Again, specific activity of the incorpo-
ration of the target tissue specific hormones is
high and that of testosterone and hydrocortisone
relatively low. More recent experiments dem-
onstrated an even more dramatic effect with an
equivalent technique.
GRUN: What tissue was this in?
CHALKLEY: This is the calf endometrium,
the epithelial layer of the uterus, which had
been scraped off and incubated.
SCHRAER: Do you analyze it for the hor-
mone or just for the label?
CHALKLEY: Just for the label.*
SCHRAER: Do you know if it has hormones
in those parts of the cells?
CHALKLEY: We put in labeled hormone and
later on we follow the behavior of the counts.
Now, it may be that this is being degraded and
then the degraded material is being bound. So
far we haven't found that out.
In Table V we see the effects of very dif-
ferent treatment on the chromat in containing
the hormone. Organic solvents appear to rea-
sonably efficiently extract a hormone. Sulfuric
acid (2N) which we know will extract histones
virtually quantitatively, also solubilized a por-
tion of the counts. Guanidinium chloride, which
we also know dissociates histones and de-
natures proteins, released over 50%. Sodium
chloride (2 M) released only a small fraction of
these counts. However, the real clue to the pos-
sibility of binding to histones was given, first
of all, from histones isolated from this and put
through the IRC -50 column. Not a single count
above background was found and in fact essen-
tially all of the hormone, after treatment with
acid, was fully dialyzable. I should add, also that
recently we've found that the hormone appears
to be thermolabile. In 30 minutes at 37° about
50 to 60% of the hormone can be thermolabil-
ized (13).
As a further check on what the binding
really involved we suspended the chromatin in
2.09 M cesium chloride, which is of sufficiently
high ionic strength to dissociate the histones and
other chromosomal proteins, and then centri-
fuged the solution at high speed. You can actually
band the histone component (14) (Fig. 18). The
bulk of the non-histone protein bands at a lower
density than the histone. This material aggre-
gated as a very, very thin skin in the tube, A
considerable number of the counts were localized
in this skin which we homogenized and counted.
EPEL: Before you go into this new subject,
could you clarify the conclusion from the
estradiol experiment?
•Subsequent studies have demonstrated that 99% of the
bound h3 was present as unchanged estradiol- 17>9 (13).
142
<
o
Q.
q:
o
o
a:
1,2
0.9
0 6
03
PERCENT TOTAL HORMONE BOUND TO
PURIFIED CHROMATIN
h'-estradiol
H -PROGESTERONE
-
h'- TESTOSTERONE
M -HYUKUUUMIIbUNt
4000
3000
O
o>
-E 2000-
S
a.
o
1000
H -ESTRADIOL
BINDING OF HORMONE
TO PURIFIED CHROMATIN
H^- PROGESTERONE
H^'-TESTOSTERONE
H -HYDROCORTISONE
Fig. 17.
CHALKLEY: Well, the conclusion is that it
appears to be bound to something which is
lighter than histones. It's not bound, apparently,
to any great extent to histones, as far as we
can see. Possibly it's not bound to histone at
all. It is bound to something which precipitates
at this concentration of CsCl, and our efforts
have been to try and isolate it further.
EPEL: Have you made any estimates of
how many molecules of estradiol there are per
nucleus?
CHALKLEY: No, we haven't yet.*
Well, now I want to think a little about the
problems of repression and what we would have
to require of any model to account for repres-
sion. We have to be able to explain differential
gene effect, the problem of epigenetic control
and differentiation. How is it that the pea coty-
ledon can synthesize globulin, and yet pea buds
cannot synthesize any detectable amount of
globulin? We have to involve in this model the
fact that a substantial volume of histones does
not turn over at all in the lifetime of a given
DNA molecule. We have to explain the fact that
some do turn over. We have to be able to ex-
plain induction of enzyme formation occurring
at the genetic level. (This will have to account
for hormonal induction). We have to demon-
strate that if we induce a system and then re-
TABLE V
Hormone Binding by Chromatin
Treatment
PfrCent Solubilised
EtOH
102
CHCI3
89
EtjO
92
0.2 M HaSO^
65
2.3M GuCI
>50
o.rsNaCl
<\0
2.0M NaCl
<IO
BANDING OF H^-ESTRADIOL-CONTAINING CHROMATIN
* Recent studies suggest a value of about 2500 mole-
cules of estradiol per nucleus.
TUBE NUMBER
Fig. 18.
move the inducer that we have a reversal to
repression. We have to demonstrate a gene
specificity. This has always been a tremendous
question mark with histone experiments. It has
long been a problem to understand how to
selectively repress a gene with simple electro-
static interactions.
John Frenster, of the Rockefeller Institute,
produced only recently in Nature (15) a model
for the repression and specific gene action.
What is proposed is that you have the whole
genome entirely wrapped up with histones and
repressed. He invokes histones as repressors.
He then requires a specific derepressor of RNA
that finds the section of the genome with which
it is base complementary. It associates with
143
the DNA at this point and displaces histone.
This leaves free just one strand to code for
messenger RNA. That's fine, but I find it dif-
ficult to understand how this process is reversed;
and also, it wasn't explained how he thought
hormones would stimulate this removal process.
Also he doesn't explain differential gene action
or epigenetic control.
So, just for fun. Dr. Maurer and I present
another model. It is shown in Fig. 19. This has
at least the advantage, I think, of concentrating
thought on a dynamic model rather than a static
one, and I feel that it's a dynamic process that's
involved in repression. Now, firstof all, wehave
the genome of a differentiated tissue in which
some specific areas of this genome are com-
pletely unavailable for genetic transcription.
In the in vitro reconstitution experiments of
Bonner and Huang it was found that the histones
were virtually 100% efficient in cutting off
DNA- dependent RNA synthesis. So, we have
areas which are cut off with those histones;
they're not allowed to be transcribed, and, dur-
ing the lifetime of a particular DNA molecule,
there will be no RNA synthesis in these genes.
We have another basic protein; we haven't
specified any more than to say it's basic, and
based on the results of Huang and Bonner (14)
we postualte a basic protein-RNA linkage. Now,
if one wants to think of proteins linked to a
very special RNA like this, it would seem to
require a covalent linkage. Again we're specu-
lating a little; we don't know that it is covalent,
but the evidence is beginning to mount up
toward that possibility. If that's so, these have
to be linked, presumably, through a series of
enzymic reactions. This material which we call
a functional repressor has the ability to recog-
nize a specific gene because of this RNA. It
is then possible to conceive of a dynamic
equilibrium in a section of the genome. We
suggest thinking along the lines of some sort
of dynamic equilibrium with the repressor going
on and the repressor falling off and being de-
graded. Now, if we postulate this dynamic sys-
tem, then it's possible to conceive of induction
acting by somehow inhibiting the linkage of
these two component parts in the formation of
the functional repressor. In this case, the re-
pressor is not formed, so the equilibrium will
shift away from the genome. Protein synthesis
malfunction would also give rise to a decrease
in the concentration of the repressor, as would
inhibition of RNA synthesis. Perhaps we can
begin the discussion with this model that we
have proposed.
GROSS: How do you get the stable histones
which are not turning over? How is their speci-
ficity compared to the one which does?
CHALKELY: Their synthesis would require
both spatial and apparently temporal specificity -
whether this synthesis is directed by messenger
RNA or by other methods is an interesting
problem.
DYNAMIC MODEL FOR INDUCTION AND REPRESSION
PROTEINSYNTHESIS
RNA-SYNTHESIS
c
D
BASIC PROTEIN
I FORMATION OF REPRESSOR
RNA
INHIBITION OF FORMATION OF THE REPRESSOR
FUNCTIONAL
REPRESSOR
= INDUCTION
I _Ji
I FUNCTION OF REPRESSOR] i I REPRESSION]
Z^OOOOOOOC
DEGRADATION
OF REPRESSOR
HISTONE NOT TURNING OVER
GENOME OF DIFFERENTIATED TISSUE
a+ CID + CZD+ ■
DEGRADATION PRODUCTS
Fig. 19.
144
GROSS: It imposes an analytical require-
ment on the model. If you' re going to have recog-
nition, then you have to have sufficient length of
RNA to recognize the gene or the cistron.
CHALKLEY: How big is an operator gene?
GROSS: Well, it's a fraction or a few per
cent of an operon. However, what I'm suggesting
is that isolated chromatin ought to have a lot of
RNA,
CHALKLEY: It has some RNA. When you
isolate chromatin and nucleohistone it has not
got a great deal of RNA; nonetheless a residual
quantity of RNA is always found. This RNA is
very difficult to remove. It's resistant to RNase,
unless you pre-treat it with DNase or unless you
heat it to 60°.
KAHN: I'm curious. Where do you postulate
that the RNA for the functional repressors is
synthesized? It seems to me that the most likely
spot would be the very same portion of the DNA
template that it will later repress.
CHALKLEY: I think that is, in fact, very
reasonable.
PERSON: Are you going to have repression
by the RNA or by the protein?
CHALKLEY: I imagine it's the protein
that's involved in the repression. So far it has
been shown in our group that you can put cationic
polypeptides on the template and get a repres-
sion of the DNA-dependent RNA synthesis. It's
a fact you can work on anyhow.
TS'O: I would like to make a statement about
this. The most difficult problem in setting up the
hypothesis for histone or any proteins to be the
genetic repressor is that we know nothing about
how the proteins and the nucleic acids interact
specifically. How do proteins recognize the base
sequences or the base composition of the nucleic
acids? The present status of our biochemical
knowledge does not give any model in molecular
terms which would lead to this kind of specificity.
For instance, in protein synthesis, even though
the transfer RNA can form base pairs with the
messenger RNA, the translation of the genetic
codes is dependent upon the recognition process
between the transfer RNA and the amino acid
activating enzyme. What you are trying to do
here is almost the same thing, because you are
trying to use the RNA to recognize the DNA
through the accepted based pairing mechanism.
However, specific base proteins have to be able
to recognize and be attached to the specific RNA.
CHALKLEY: Now you're putting a require-
ment in which I don't think is necessary. All we
have to say is that there has to be an enzyme
or enzymes to link those two and this enzyme
has to be able to recognize the basic protein.
Now, surely one protein can recognize another
protein?
POLLARD: I feel you're missing one very
important question. I think your model is very
good, but there has to be something that really
rips the RNA off. It can't just come off; it has
to be torn off. Now, if you were to take just
the soluble RNA and quietly put histone on the
end of the RNA, you would block the tear-off
mechanism. The rate at which that stuff comes
off is impressive. It's linked in complementary
links of base pairing. Nevertheless, it comes
off. No one has been able to observe the time it
takes.
CHALKLEY: That's true.
POLLARD: Also, if you were going to have
as small a section of the DNA in the bacterial
cell as you indicated, then the length of the RNA
being torn off must be 1 5 times as long.
CHALKLEY: Yes, this was an arbitrary
length for purposes of discussion.
POLLARD: I suspect you may be on the
right track with this, but the RNA just doesn't
do anything you like.
MAURER: It should be pointed out that this
is a kind of equilibrium and therefore it is sen-
sitive to all kinds of factors which influence
this equilibrium. If one enhances the degrada-
tion, for example, it would certainly have an
effect on it, probably by shifting the rate of
reactions. Furthermore, I would like to stress,
in respect to the hormone studies so far done,
that it isn't yet completely clear whether there
is an RNA polymerase increase due to new
formation of the RNA polymerase or to an acti-
vation of the enzyme as far as RNA template
synthesis is concerned. However, in these ex-
periments which Dr. Chalkley has presented,
it was shown that there is an increase in tem-
plate activity since we put in high excess of
RNA polymerase. This is the main point. It
really looks like the hormones are, by some
means, removing some kind of repressor and
not just increasing the RNA polymerase. We
suggest that hormones inhibit the formation of
some functional repressor and in this way alter
the dynamics.
PERSON: Wouldn't you than want them con-
nected to the things that are being permanently
repressed in the model, so that they could open
up new regions?
CHALKLEY: Well, if you do that, you have
to specifically involve proteins other than his-
tones in this sort of specific permanent gene
repression and this may be quite possible. There
145
may well be proteins other than histones in-
volved, but I was basing it on the results in
which we've come to the conclusion that the hor-
mone hasn't got as one of its functions to bind
histone, in any sort of binding we could detect.
So, one of the original reasons we developed
this idea was because we had to explain an
increase in template activity. So, we had to
postulate some sort of a dynamic equilibrium
and determine if we could interfere, not by
reaction with histone, but by reaction with
another protein. The suggestion here is that
the protein interacts with a hormone.
The point I really wanted to make and really
wanted to stress is that we would like to approach
the problem in terms of a dynamic situation and
then see if we can disturb the equilibrium in
some way at some point; and this may give rise
to derepression. If you remove the inhibitor,
everything flows back and you set up the original
equilibrium.
EPEL : I'd like to try to integrate your think-
ing with Dr. Wright's stimulating paper this
morning. In your evidence regarding the hor-
monal action you claim that 95% of the hormone
is in the cytoplasm - is that right?
CHALKLEY: We've found large percent-
ages in the cytoplasm but not large percentages
bound in the cytoplasm.
EPEL: Well, here is what I want to bring
out. In our discussion here, we know that these
hormones, such as estradiol or leutenizing hor-
mones, can fantastically affect intermediary
metabolism. These are very fast effects. Let's
take ACTH in the adrenal cortex; it will acti-
vate cyclic AMP formation and thence glycogen
phosphorylase. There are also a number of
papers showing direct cyclic AMP stimulation
of enzymes involved in steroid synthesis and
there is, also, activation of protein synthesis
via pre-existing messenger RNA. So, isn't it
conceivable that you have a system here in
which numerous changes are triggered as a
natural consequence of the hormonal action,
not through the hormone directly but through
the changing levels of various intermediates?
This, then could act on the nucleus?
CHALKLEY: Yes, I would say that is en-
tirely possible.
EPEL: I'm just trying to integrate these
two very interesting lines of reasoning.
CHALKLEY: Well, some of these hor-
mones, of course, repress and activate pre-
existing enzymes. This is well catalogued; and
we know that they bind slightly into the cyto-
plasmic fraction. But on the other hand there is
a body of evidence available which suggests that
an early biochemical effect of hormone adminis-
tration is increased RNA and protein synthesis.
It is entirely possible that the nuclear binding and
the increased RNA synthesis are related.
EPEL: The hormone can be many steps re-
moved from its final action. It' s at least 3 or 4
steps back, for instance, in initiating cyclic
AMP formation.
MAURER: Yes, but why do we always find
a lot of hormone in the nuclei? In the case of
ecdysone, for example, we find hormone about
20 minutes after injection into the larvae of
Calliphora erythrocephala in the nuclei; and the
earliest time it's been found is about 10 minutes
after injection. However, you are quite right;
it's still not ruled out that there isn't some kind
of reaction with the cytoplasm.
CHALKLEY: There is one thing I possibly
didn't bring out fully enough. If we do this hor-
mone incorporation experiment, a great deal of
the hormone goes into the cytoplasm, but if you
look at the amount bound there is a much greater
degree of binding occurring in the nucleus.
EPEL: Are these experiments in the range
of physiological concentration? Perhaps the
experiment to do would be to use an extremely
small amount of hormone, and then see if it
appears in the nucleus.
MAURER: Actually this has been done with
physiological 10-^ M concentration. So, it' s con-
ceivable although not completely proved.
GROSS: What were you saying about ecdy-
sone? When we studied this system, although it
is binding to the ribosomal particles, the amount
of binding in the nucleus is not substantial.
CHALKLEY: I was recalling the results
with binding in the mitochondrial and ribosomal
fractions, because it has been shown that binding
occurs in these particular fractions.
GROSS: Where does the hormone exert its
first recognizable characteristic effect? In this
case isn't the first characteristic response in
the genome?
MAURER: I treated the insect larvae with
ecdysone and I found the highest activity in the
nuclei and not in the mitochondria. I found some
activity in the mitochondria and even in the
microsomes, but the highest activity after one
hours was in the nuclei. This was then followed
by a decreasing level; so it looks suspiciously
as though the hormone first migrates into the
nuclei and then by some process is depleted.
SCHRAER: You keep using the word hor-
mone. Do you mean labeled hormone?
CHALKLEY: Yes.
146
KOHNE: Could there be membrane material
in this chromatin preparation? There have been
reports that even purified DNA has some mem-
brane in it.
CHALKLEY: We've never treated with de-
oxycholine. This might be worth doing, though I
suspect it would dissociate some histone from
the DNA.
PAPACONSTANTINOUS: You know, there's
one question that hasn't come up yet, and I don't
know that there's any answer. That is, how does
your system fit in with the question "Is the
genome read when the cell is replicating or is
it not read?" K the genome isn't read during
replication, then you've got to fit up all your
repressors for replication and then pull them
back off again.
CHALKLEY: I don't think I have an answer
to that.
PAPACONSTANTINOU: Well, I don't know
whether there is an answer to that very ques-
tion of whether it's being read or not.
References
1. R. C. C. Huang and J. Bonner. Proc. Natl,
Acad. Sci. U.S. 48, 1216 (1962).
2. M. Nicolson. In preparation.
3. H. Busch. "Histones and Other Nuclear
Proteins," (Academic Press, New York,
1965).
4. C. F. Crampton, R. Lipshitz and E. Char-
gaff. J. Biol. Chem. 206, 499 (1954).
5. K. Murray. In "The Nucleohistones, " J.
Bonner and P. O. P. Ts'o, eds. (Holden-
Day, Inc., San Francisco, 1964), 21.
6. K. Marushiga and J. Bonner. J. Mol. Biol.
15, 160 (1966).
7. W. G. Flamm and M. L. Birnstiel. In "The
Nucleohistones," J. Bonner and P. O. P.
Ts'o, eds. (Holden-Day, Inc., San Francisco,
1964), 230.
8. B. M. Olivera, R. C. C.Huang and N. David-
son. Ber. der Bunsenges.JUr phys. Chem.
68, 802 (1964).
9. D. Y. H. Tuan and J. Bonner. Plant Physiol.
39, 768 (1964).
10. M. Dahmus and J. Bonner. Proc. Natl. Acad.
Sci. U.S. 54, 1370 (1965).
11. F, F. Kenney and F. J. Kull. Proc. Natl.
Acad. Sci. U.S. 50, 493 (1963); M. Feigel-
son, P. R. Gross and P. Feigelson. Biochim.
Biophys. Acta 55, 495 (1962).
12. H, R. Maurer, D. E. Rounds and C. W.
Raibom. In press.
13. H. R. Maurer and G. R. Chalkley. In press.
14. R. C. C. Huang and J. Bonner. Proc. Natl.
Acad. Sci. U.S. 54, 960 (1965).
15. J. Frenster. Nature 206, 1269 (1965).
16. R. Chalkley and R. Maurer. Proc. Natl.
Acad. Sci. U.S. 54, 498 (1965).
147
DYNAMICS OF THE POINT OF NO RETURN DURING
DIFFERENTIATION IN BLASTOCLADIELLA
EMERSONII
Edward C. Cantino
Department of Botany, Michigan State University,
East Lansing, Michigan
I left East Lansing a few days ago knowing
that cell differentiation and morphogenesis were
riddles which had served admirably for many
years as focal points for honorable speculation.
I will leave Penn State, today, with the strong
suspicion that solutions to these problems are
far from just around the corner. 1 trust, there-
fore, that I will be forgiven if, during the short
time we have left this morning, I add some haze
of my own to this generally smoggy area.
Almost twenty years ago, in a fresh-water
pond behind old MacFarlane Hall on the campus
of the University of Pennsylvania, 1 discovered,
isolated in pure culture, and subsequently chris-
tened as a new species, the Phycomycete known
as Blastocladiella emersonii 1 put this specific
epithet upon it because of fond memories of my
first real teacher. Professor Ralph Emerson,
who but a few years before had introduced me
to the fascinating antics of these ubiquitous
aquatic fungi commonly known among mycolo-
gists as the water molds.
I shall spend perhaps half of my time, this
morning, developing Blastocladiella' s back-
ground. This will serve a dual purpose, because
Dr. Lovett, who follows me on the program, will
also be discussing his studies of B, emersonii.
Let me begin, therefore, by showing you more or
less what 1 saw in 1948 when 1 took the first
spore of B. emersonii ever to be rendered cap-
tive and put it on a slab of nutrient agar medium
in a Petri dish (Fig. 1). The spore germinated
and developed into a little plant with root-like
rhizoids. At maturity, almost (but not quite) all
of this thallus was converted into a spore-bear-
ing sac, the sporangium. The first figure shows
a cross section through a spore sac in which
the protoplast has been cleaved up into spores.
Subsequently, these spores were liberated
through exit pores in the sporangia! wall and
settled on the surface of the agar immediately
around the parent plant. The latter, thus depleted
of its protoplasm and now an empty shell, col-
lapsed. The newly liberated spores, however,
SPORE
J-
FIRST GEN
DC CELL
FIRST GEN
SPORES
^
CLONE OF
2 ND. GEN CELLS
(1) DC CELL WITH 3 RD
GEN IN SITU
(2) 00 CELL, NON VIABLE
(3) OC CELL DISCHARGING
3RD. GEN , 5 X VIABLE
(4) OC CELL DISCHARGING
3RD. GEN, 0 % VIABLE
(5) 00 CELL DISCHARGING
3 RD. GEN, 100% VIABLE
(6) ORANGE CELL
(7) BROWN RS CELL
Fig. 1.
Schematic representation of the totlpotency of 2nd and
3rd generation clones derived from a single spore of
Blastocladiella emersonii.
149
began to develop into a clone of second genera-
tion Blastocladiellas , and therein the totipo-
tency of this organism began to manifest itself.
There appeared, gradually, big cells and small
cells, brown, colorless and orange cells, cells
with thick walls - some sculptured and some
not - and cells with thin ones, dead cells and
living cells, cells which discharged yet another
generation of spores among which no spores
were viable, or in which some or all were
viable. This, then, will suffice to show the gen-
eral nature of the problem as it appeared at
the outset in the late 1940's.
During the past two decades, some -but by
no means all- aspects of this picture have been
unscrambled and clarified. Let us deal with
one of these, now, by way of Fig. 2. We know
that the spore with which B. emersonii begins
its life history is motile, uniflagellate, uninucle-
ate and organized in a very crafty and intriguing
fashion; its internal structure is unique. Indeed,
its architecture has a direct and important bear-
ing upon the story I will try to develop. However,
inasmuch as Dr. Lovett will devote all of his
talk to the spore and its activities, I will by-pass
it without further comment. In synchronized
single generation cultures, some 99% of the
germlings of B. emersonii can develop along
either of two major morphogenetic pathways.
Along both of these paths, the thallus increases
at an exponential rate in dry weight, volume and
other features. A point is reached, however.
OC pathway
motile
spore
r
I S^o-^Q-^O ^''a°ow"th'' differentiation sporangium
^ J^ stage "°^' germinationrj^
germination /:i^^^. /^ Miv^"^
"°^^ I RS pathway
Fig. 2.
The two major developmental pathways taken by Blasto-
cladiella emersonii. (Fig. 2, Cantino and Lovett, In "Adv.
In Morphogenesis," III, 1964; reproduced with permis-
sion of Academic Press.)
when this exponential phase in development
ceases; at this point, the plant embarks upon its
second developmental stage, i.e., cell differen-
tiation. The first visible evidence that this is
about to happen is the formation of the septum,
which delimits the thallus into a large upper cell
and a small lower cell with root-like rhizoidal
appendages. At this point, it is important to
call attention to the fact that at maturity, the
lower cell is (as far as is known) devoid of con-
tents. The upper cell possesses all the cyto-
plasm and all the nuclei - hundreds to thousands
of them, depending upon the environmental con-
ditions selected - which are embedded therein.
Thus, it should be recognized at the start that
we are dealing here with the development of a
single cell, for we are starting with a uninu-
cleate spore and ending with a multinucleate
coenocyte. Finally, however, at the very end of
this organism's life history, these many nuclei
embedded in a common cytoplasm do become
delimited from one another by the formation of
cross walls - or, perhaps more appropriately,
cross "membranes." In any case, each nucleus
functions as a focal point for the formation of a
spore. Each nucleus inherits a tail, a mitochond-
rion and a number of other organelles, and the
lot becomes surrounded by the flexible spore
wall. This population of nuclei is then liberated
from the parent cell in the form of a population
of spores, and we are back where we began.
Now, let me retrace my steps for just one
moment. Long ago, we labeled the uppermost
cell in the top pathway (Fig. 2) an ordinary
colorless cell, or an "OC" cell for short. The
uppermost cell in the lower pathway is a re-
sistant sporangial cell, or an "RS" cell for short.
These two cell types differ from one another in
many ways, some very obvious but others de-
ceptively subtle. The more obvious differences
include the following: the OC cell wall is
chitinous and thin, while the RS cell wall, also
chitinous, is much thicker; the OC cell wall is
essentially colorless, while the RS cell wall is
brown, being impregnated with melanin. The
protoplast of the OC cell contains no detectable
colored carotenoids, while that of the RS cell
contains gamma-carotene. There are many,
many other differences, but these are too num-
erous to mention now.
Finally, we also learned that these develop-
mental pathways could be controlled at will by
the simple expedient of providing the organism
with a little bit of baking soda. In the presence
of a suitable amount of exogenous bicarbonate,
essentially all spores developed along the RS
150
pathway; in the absence of bicarbonate, they
developed along the OC path. Thus, when this
work began, we set out to learn by what means
this bicarbonate trigger mechanism brought
about the de novo synthesis of melanin and
carotene, the increased rate of synthesis of
chitin, and the numerous other characteristics
associated with the structure and function of the
resistant sporangial cell.
In the late 1950's, mainly through the skill-
ful and conscientious efforts of my former
students - Drs. James Lovett, HowardMcCurdy,
Evelyn Horenstein and others - methods were
envolved for growing submerged, uniformly
distributed cells of the fungus along one path-
way or the other in massive, synchronized,
single-generation cultures. By starting with
several hundred million or a billion spores, all
of them of exactly known age, such spores were
made to go through their acts in nearly perfect
synchrony along either path. Thus, it becomes
immediately evident that, by this means, one
can harvest almost any number of OC cells all
of which have arrived at, let us say, 30% of
their exponential period of growth; or OC cells
all of which are tooling up to manufacture a
second generation of spores; or a billion RS
cells all of which are in the process of laying
down the cross wall which will delimit the
resistant sporangium from the rest of the thallus,
and so on. Here, then, was an elegant system for
studying the relations between biochemical and
morphological differentiation, and we began tc
exploit it.
TILL: Have you got the time scale on this?
CANTINO: The time scale for the OC path-
way depends upon the nature of the medium and
other factors. Under the conditions we have used,
it ranges from 12 to 17 hours, depending on popu-
lation density. For the RS pathway, our standard
procedure yields a generation time of 84 hours.
TILL: The other point I'd like to ask about
is, are those spores the same?
CANTINO: Morphologically, they appear to
be identical. (Note added in proofs: It should be
pointed out that all of our work with synchro-
nized liquid cultures is done with spores derived
from OC cells. The reason for this is that it is
a simple matter to prepare massive suspension
of such spores, but a much more difficult one
to prepare them from RS cells. There is some
evidence, however, that the developmental po-
tentials of - i.e., the kinds of progeny produced
by - RS-spores and OC-spores are not identi-
cal (D).
Now, let us come to grips with the nature
of this bicarbonate trigger mechanism. This
has been under investigation in my laboratory
for a long time. Therefore, let me simply pre-
sent a condensed recapitulation of the essential
biochemical event which we think is operative;
then, we can use this as a convenient point of
departure. At the metabolic level, the focal point
appears to be as follows. In homogenates of OC
cells of various ages, the enzymatic activities
of the tricarboxylic acid cycle are detectable;
thus, the cycle is at least potentially functional.
In spores and germlings of such OC cells, the
in vivo evidence suggests that the Krebs cycle
is operating. In Fig. 3, on the left, only one of
the steps in the cycle is shown - the step medi-
ated by a TPN-specific isocitric dehydrogenase.
But, when bicarbonate is added to a spore or
developing germling, it quickly induces a mul-
tiple set of enzymatic lesions in the tricarboxylic
acid cycle. However, the isocitric dehydro-
genase remains functional, and it now begins to
operate in reverse, mediating reductive car-
boxylation of ketoglutarate back to isocitrate
(Fig. 3, right). At the same time, bicarbonate
also induces the formationofisocitratase, which
cleaves the isocitrate to succinate and glyoxy-
late and thus prevents its accumulation. Finally,
a constitutive glycine-alanine transaminase in
the organism helps to keep the chain of reac-
tions on the move by amination of glyoxylate to
glycine at the expense of alanine.
Let me illustrate, by way of a few examples,
some of the kinds of information obtainable
«n
f
OC Plortt
PYH.
iLYCI
(t
■'ALANINE
GLYOX.
+
sue.
V
/,
GLYOX
sue
'>
■•■^
. ISOCITR.
y
TPNH
Fig. 3.
The bicarbonate trigger mechanism in Blastocladiella
emersonii. (Fig. 6, Cantlno, In "11th Symp. of the Soc.
for Gen. Microbiol.", 1961; reproduced with permission
of the Society for General Microbiology.)
151
with - and, I think, only obtainable with - syn-
chronized single generation cultures, which sup-
port this general picture that I have been trying
to create.
One of the first changes induced by bicar-
bonate, and detectable in vivo immediately after
spore germination, has to do with gas exchange
(Fig. 4). The upper curve reveals the course of
oxygen consumption by an OC cell growing in
the absence of bicarbonate. The lower curve
shows what happens when bicarbonate is present
in the medium. After the spore has germinated,
there occurs an immediate and precipitous drop
in oxygen consumption. Simultaneously, the ex-
ponential growth rate is reduced to 46% of that
of the cell growing in the absence of bicarbonate.
Also, bicarbonate causes the exponential rate of
synthesis of the cell's pool of a soluble poly-
saccharide (made up solely of glucose) to double
relative to the cell's exponential rate of growth
in mass. These facts do not, of course, prove
that lesions have developed in the tricarboxylic
acid cycle as outlined above; they are, however,
consistent with this interpretation.
The available quantitative data which deal
with the enzymes themselves have a direct
bearing at this point in our discussion. For ex-
ample, cells growing along both developmental
pathways have been assayed at various stages
in ontogeny for isocitric dehydrogenase and
ketoglutaric dehydrogenase activities. When the
data are plotted, not as specific activities but
rather as total units of enzyme activity per
cell, it turns out that the exponential rate at
which isocitric dehydrogenase accumulates in
the cell during its exponential growth phase is
about seven times higher than the rate at which
the ketoglutaric dehydrogenase complex does
SIMULTANEOUSLY 1
EXPONENTIAL GROWTH RATE
IS REDUCED BY 45%
AND
EXPONENTIAL SYNTHESIS OF
POLYSACCHARIDE / DRY
WEIGHT INCREASED 100%
SO (Fig. 5). Furthermore, as will be seen
shortly, the net accumulation of this latter
enzyme system in the cell levels off and ceases
long before that of isocitric dehydrogenase.
What does this observation have to do with the
question of oxygen consumption? A glance back
at the previous slide will show that during the
exponential growth of a developing RS cell, oxy-
gen consumption decreases to about one-tenth
of its startinglevel(Qo2 = ^^- 100) in the spore.
If oxygen consumption by the growing cell were
totally and exclusively dependent upon the opera-
tion of the tricarboxylic acid cycle, one might
expect that the rate of turnover of the cycle
would also drop to one-tenth of its starting rate
at zero time. The quantitative data associated
with Fig. 5 are consistent with this thought. From
spore stage to end of exponential growth, the
total units of isocitric dehydrogenase per cell
increase 6,500 times, but the total units of
10-
UJ
o
(/) 3.
z
0
'a KETOGLUTARIC
DEHYDROGENASE
40 60
% GENERATION TIME
100
HOURS
Fig. 5.
24
Fig. 4.
Oxygen consumption by OC and RS cells.
A comparison of the exponential rates of synthesis of
Isocitric and a -ketoglutaric dehydrogenase per RS cell
during exponential growth.
152
ketoglutaric dehydrogenase increase only 6t)0
timesi Thus, the 90% decrease in oxygen con-
sumption goes hand in hand with the 90% de-
crease in the intracellular accumulation of
ketoglutaric dehydrogenase relative to the iso-
citric dehydrogenase which immediately pre-
cedes it in the Krebs cycle. It appears, there-
fore, as if bicarbonate causes the ketoglutaric
dehydrogenase system to become a bottleneck
in the cycle, that it begins to do so early in
ontogeny, and that this soon brings the activity
of the tricarboxylic acid cycle to a halt.
What about the other critical enzyme in the
scheme - the isocitratase? Some years ago. Dr.
McCurdy purified it and established its proper-
ties. Assays with synchronized cultures show
how it, too, is involved. Figure 6 reveals what
happens to the total units of isocitratase per
cell during development in the presence and
absence of bicarbonate. The intracellular quan-
tity of this enzyme in the spore is shown on the
vertical axis, i.e., at zero time. As the spore
gives rise to a germling, and it in turn develops
exponentially into a young OC plant in the ab-
sence of bicarbonate (bottom curve), there is
no net synthesis of this enzyme. It seems as if
the original amount of isocitratase in the spore
is simply diluted out as the growing cell in-
creases in size. Only when the OC cell has
reached about half of its generation time does
synthesis of isocitratase begin. However, when
spores are germinated in bicarbonate media,
exponential synthesis of isocitratase apparently
begins immediately (upper curve). In summary,
while bicarbonate brings about a lesion in the
tricarboxylic acid cycle by creating a bottleneck
at the locus of ketoglutaric dehydrogenase, it
provides relief for the damage done by inducing,
simultaneously, synthesis of isocitratase. Thus,
in the bicarbonate-induced RS cell, isocitrate
leads to succinate and glyoxylate (and thence to
glycine), whereas in the bicarbonate -independent
OC cell, it leads to ketoglutarate (and thence
to succinate) and CO2 .
Finally, let me present one last set of data
which bear upon this mechanism. If the pro-
posed scheme is correct, in vivo uptake of
CO 2 and/or bicarbonate by a developing RS
cell should reach its peak at that point in
ontogeny where the cell's complement of iso-
citric dehydrogenase is maximum relative to
its complement of the bottleneck enzyme, keto-
glutaric dehydrogenase (this point in ontogeny,
as will be seen in a subsequent figure, occurs
at about 36 hours, i.e., 43% of the RS cell's
generation time). To test this notion, RS cells
were grown in synchronized culture and then,
at 30 hours, provided with a dose of H^^COs
and allowed to continue growing for 6 hours.
During this latter period, cells were sampled
and assayed for total ^''C fixed, and the medium
assayed for total i^C which had disappeared.
A similar experiment was done in which 38 hour
cells were fed the H^'^COs. The results were
combined to yield one graphs as shown in Fig. 7.
You will note that uptake of H^^COg per cell
increases as the cell passes through 36 hours
of age, and that uptake decreases again after
39-40 hours. Since the ratio of total units of
ioscitric dehydrogenase to units of ketoglutaric
dehydrogenase is maximum at 36 hours, and then
decreases once again beyond this point, these
data provide further evidence that the bicarbon-
ate trigger mechanism operates as proposed
above.
Let us move on, now, to consider the transi-
tion period between exponential growth of the
RS cell and its subsequent differentiation; the
data I wish to present in this connection also
have a direct bearing upon the biochemical
mechanism we have been discussing. The photo-
graphs which are shown in Fig, 8 were taken
by Dr. Lovett when he was working in my lab,
V, Gen. Time
Fig. 6.
Synthesis of isocitratase during growth of OC and RS
cells. (Fig. 5, Cantino, In "11th Symp. of theSoc.for Gen,
Microbiol,," 1961; reproduced with permission of the
Society for General Microbiology.)
153
and they reveal the microscopic appearance of
RS cells at various stages in their development
in a synchronized culture. During ontogeny, a
point is reached beyond which the cell becomes
irreversibly committed to RS formation. This
is the morphogenetic point of no return. It is
figuratively represented by the point of
dichotomy of the two arrows, and it amounts to
43% of the RS generation time - chronologically,
36 hours under the conditions we use for growth.
Before this point is reached, the cell's morpho-
logical potential displays an inherent plasticity;
if the bicarbonate is removed from its environ-
ment, it reverses "direction" and embarks upon
the alternate morphogenetic pathway. In other
words, functionally it turns into an OC cell.
However, beyond this point of no return, removal
of bicarbonate does not cause morphogenetic
reversal; the cell continues on its way toward
the RS type whether or not the bicarbonate is
still present.
This feature further emphasizes the fact
that a synchronized culture of Blastocladiella
emersonii represents an easily exploitable sys-
tem for experimental studies of morphogenesis.
Indeed, it is for this reason that I and my associ-
ates, past and present, have been trying to track
the various events - intracellular and extracel-
lular - which are associated with the genesis of
a.
o
20--
30--
Fig. 7.
Uptake of h'^COJ by an RS cell as it approaches and
passes the morphological point of no return.
a resistant sporangium as it approaches, passes
through and then departs again from its point of
no return. Many of these events have been fol-
lowed on a per-cell basis, and a superficial
digest of the results is seen in Fig. 9. You will
note that many things begin to increase at an
exponential rate - albeit not all of them at
identical rates - after spore germination, and
cease to do so at the point of no return. Others,
glucose uptake for example, begin much later
but still end at this same point of no return.
However, other features, such as weight, lipid,
total nitrogen, chitin, polysaccharide, RNA,
melanin, etc., continue to increase to different
stages in ontogeny beyond the point of no return.
Then again, there are still other events which
commence only at or beyond the point of no
return. Clearly, then, it was of interest to find
out which, if any, of the qualities associated with
an RS cell before its point of no return would
change to a new state more characteristic of an
OC cell if morphological reversal were induced.
We have done tests of this sort, and I would
like to show you the results that were obtained
from one such experiment that Dr. Lovett and I
did some years ago. Figure 10 shows what hap-
pens to isocitric dehydrogenase and ketoglutaric
dehydrogenase during development along the RS
path up to a stage well beyond the point of no
return. For convenience in making comparisons,
the total units per cell at the spore stage were
set at one for both enzymes, and all other
values were then related to this and plotted
accordingly. When bicarbonate was removed
from RS cells a few hours before the point
of no return, thus inducing morphological re-
versal, the total units/cell of isocitric dehydro-
genase dropped sharply (whereas without re-
versal, it continued to rise) and the total units/
cell of ketoglutaric dehydrogenase rose sharply
(whereas without reversal, it did not do so).
These results are represented by the dotted
lines in Fig. 10. When this same kind of experi-
ment is done with cells which have gone beyond
the point of no return - and which, therefore,
have lost the capacity for morphological re-
versal - the total units/cell of these two en-
zymes is not influenced by removal of bicarbon-
ate. In summary, before the point of no return,
morphological plasticity is associated with a
corresponding plasticity of two key enzyme sys-
tems thought to be directly involved in RS
formation; after the point of no return, this
plasticity is lost. Analyses of this sort have
thus provided additional direct evidence for
the biochemical nature of the bicarbonate trigger
154
Fig. 8,
The morphological point of no return during RS development in Blastocladiella emersonii.
155
%GEN TIME.RS CELL
EXPONENTIAL GROWTH
increase/cell:
VOL, DNA,SOL-PROT
GA-SYNTH.G6P-DEI >"
TRANS, I, I- DE J
4 3
WT, LIPID, TOT-n}
chit, polysac]—
tot-rnaI
-[gluc|}>
— [mel}
Dl FFERENTIATION
change /cell :
incr.g6p-de,
lacl decrL^
polysac j
.FdECR. R- , CMP, A a1
[iNCR. ORG-P J
.DECR. c<KG/|-DE 90 ^_^ |— (PROT,RNA TURNOVER] »
DECR. 02 1 90 >r
Fig. 9.
A digest of some of the events which have been quanti-
fied during exponential growth and differentiation of an
RS ceU.
POINT OF NO RETURN
< Q.
'H 3000
ISOCITKIC OCKVOROCEKASC
AGE (MRS)
mechanism. This system is currently being
exploited further, and in greater depth.
Recently, we have also turned some of our
attention to further exploration of the source
and intracellular localization of the reducing
power necessary for driving the reductive
carboxylation of ketoglutarate to isocitrate.
Some ten years ago. Dr. Horenstein and I found
that RS cells of B. emersonii possessed a poly-
phenol oxidase system which, in crude cell-free
preparations, mediated electron transfer from
tyrosine to either oxygen or TPN (but notDPN).
As was to be expected, this system could be
coupled in vitro with isocitric dehydrogenase to
drive reductive carboxylation of ketoglutarate
to isocitrate (Fig. 11). This tyrosinase, which
is not formed by the OC cell, thus constitutes
one source of reducingpower for the bicarbonate
trigger mechanism in RS morphogenesis. Un-
fortunately, the enzyme is firmly bound to the
RS wall and difficult to solubilize; thus, little
more has been done with it so far.
A second source of reduced TPN in Blasto-
cladiella is glucose-6-phosphate dehydrogenase
(G6PDH). However, unlike the tyrosinase, which
ISOCITRATE
y\
SUCCINATE ^ GLYOXYLATE
Fig. 10.
Enzymatic reversals associated with morphological re-
versals in Blastocladiella emersonii. (Fig. 1, Lovett and
Cantlno, /. Gen. Microbiol. 24, 1961; reproduced with per-
mission of Cambridge University Press.)
Fig. U.
The two metabolic processes presumably Involved in the
generation of reducing power for carboxylation of a -keto-
glutarate.
156
is induced to form de novo by bicarbonate, the
G6PDH is present in both OC and RS cells. We
have had some reasons to suspect, however, that
bicarbonate induction of morphogenesis is as-
sociated with a bicarbonate-induced compart-
mentation of G6PDH within the cell. We have set
up the hypothesis (Fig. 12) that (a) during the
development of an OC cell, intracellular G6PDH
is soluble, but that (b) during the exponential
development of an RS cell, bicarbonate induces
differential distribution and/or differential syn-
thesis of this enzyme in such a way that it
becomes localized on or near the cell wall or
the membranes associated with it, and that
(c) after the point of no return in RS develop-
ment, soluble enzyme once again appears in-
side the cell (either via release of wall-bound
enzyme into the soluble pool, or destruction of
the wall-bound enzyme and concomitant de novo
synthesis of soluble G6PDH, or some combina-
tion of these two). Some of the evidence follows.
To begin with, if the notion has validity,
one might expect that during RS development the
exponential rate of synthesis of total intracel-
lular G6PDH would reflect (or at least be more
nearly similar to) the exponential rate of
deposition of the surface area of the cell rather
than its weight or volume. Conversely, for the
OC cell, one might expect the opposite to hold
true. The data available suggest that this is,
indeed, the case (Fig. 13). Thus, the in vivo evi-
dence, although it does not prove the point, is
consistent with the notion expressed in Fig. 12.
With these results sufficiently suggestive.
Dr. Prem Pandhi and I have begun m vitro
studies of Blastocladiella's G6PDH. Although
attempts to purify it by conventional means
(fractionations with ammonium sulfate, acetone,
DEAE-cellulose, etc.) have only led, thus far,
to several-fold increases in specific activity,
experiments designed to test the hypothesis
in Fig. 12 are yielding evidence in its favor.
For example, when 36 hour RS cells are
homogenized in 0.005 M TRIS-HCl buffer con-
taining 0.001 M EDTA and then centrifuged at
112,000 x G, about 98% of alltheG6PDH activity
is in a soluble form (HSS in Fig. 14). When the
pellet is extracted three times in succession
with 0.005 M TRIS-HCl buffer, the remaining
1-2% of the G6PDH activity comes out - most of
it in the first wash (IW in Fig. 14); this is the
amount one would expect to find if it had simply
been trapped in the fluid volume held back by
the pellet. Only traces of activity are found in
the second and third washes (2W and 3W in Fig.
14). A final extraction of the pellet with 1 M TRIS-
HCl (IM in Fig. 14) yields insignificant activity.
However, similar analyses of RS cells
undergoing exponential growth - in this case 24
hour cells - yield quite different results (Fig.
14). Only about 40% of the total G6PDH activity
is directly soluble in 112,000 x G supernatants.
The first wash yields about half as much again
of the enzyme, a great deal more than one would
expect if it had simply been trapped in the pellet.
The second and third washes yield additional
quantities of G6PDH activity, and the final ex-
traction with 1.0 M TRIS-HCl yields another
20%. Note, too, that the specific activities of the
enzyme in the washes do not vary greatly from
one another (labeled "S.A." in the figure). Thus,
as seen in the insert in Fig. 14, essentially all
of the G6PDH in a 36 hour RS cell is soluble.
But in a 24 hour RS cell which is growing expo-
nentially and has not reached its point of no
return, less than half of the G6PDH is soluble;
more than half of it appears to be "bound" -
albeit loosely "bound," since it behaves as if it
were partitioning between two phases during
successive extractions.
Dr. Pandhi and I are now in the process of
tracking the soluble and insoluble G6PDH
throughout the ontogeny of RS and OC cells; I
would like to show you some of the things we
HCOJ -
INDEPENDENT
OC PATH
HCO3 -INDUCED
RS PATH
EXPONENTIAL
GROWTH
CELL
DIFFERENTIATION
Fig. 12.
Hypothesis regarding the effect of bicarbonate on glu-
cose-6- phosphate dehydrogenease In Blastocladiella
emersonii.
157
10 60 35 55 75
% GENERATION TIME % 36 HR. GROWTH PERIOD
Fig. 13.
Comparative exponential rates of synthesis of glucose-
6-phosphate dehydrogenase, weight, volume and area by
OC and RS cells.
100- -
HSS IW 2W 3W IM
Fig. 14.
Differential solubilities of glucose-6-phosphate dehydro-
genase In 24 hr and 36 hr RS cells.
have seen so far (Fig. 15). All assays were
done as in the foregoing experiment. The plot
in the figure shows that during the early stages
of exponential growth, most of the enzyme is
insoluble. But as the RS cell approaches the
end of its period of exponential growth, the %
soluble G6PDH gradually increases until, by the
I00--
z
3
<
o
o
o
a.
10
o
80-
60--
40--
20--
HRS. AT 24'>C.
0 12 24 36 48 60 72
c^C
«oO
~i 1 1 1 1 r
MORPH PT.
OF NO. RET
Fig, 15.
Changes In quantity and Isozymic composition of the
soluble G6PDH during ontogeny of an RS cell.
time the point of no return in morphogenesis is
reached, essentially all of it is in soluble form.
This state of affairs persists for many hours
after the point of no return, but some "bound"
enzyme appears again as the RS cell approaches
maturity (i.e., 84 hours). So far, the data gen-
erally support the hypothesis shown in Fig. 12.
In order to obtain more definitive and infor-
mative data about the changes which occur in
this enzyme during cell differentiation, we have
begun to categorize its soluble and "bound"
forms via disc electrophoresis in polyacryl-
amide gel, using a TRIS-HCl-EDT A- Borate
buffer at pH 8.3. Although only a beginning has
been made, the patterns obtained (Fig. 15) for the
soluble enzyme reveal that striking changes in
its composition occur as the RS cell moves
along in its ontogeny. Two major bands (5 and
3) are present at 24 hours during exponential
growth, but as growth continues band 3 gradually
disappears and, by the time the point of no return
is reached, only band 5 remains. After the point
of no return, however, a new complex of bands
of G6PDH activity makes it appearance along
with band 5 which is present throughout develop-
ment. It is too early to speculate as to the role
of these isozymes in the bicarbonate trigger
mechanism, but we have every expectation, now,
that this approach will produce significant re-
sults.
158
GLUCOSE -"C
- *
W'
■777777777777777777Z^777^
„ G6PDH - GLUCOSE -"C
GLUCOSE
+
GLUC0SE-'''C
MORPHOLOGICAL
REVERSAL
-^ I
''RELEASE
INCREASE
IN S.A.
GLUCOSE-'Y
Fig. 16.
Hypothesis regarding effect of morphological reversal on
the specific activity of free glucose in an RS cell.
I30--
IIO--
90--
70--
600--
400--
200-
GLUCOSE - C, SPEC. ACTIV.
I INCREASE IN S.A. OF FREE
1^ GLUCOSE DURING MORPH.
'' REVERSAL
35
CPM(47r SCAN) DUE
TO GLUC0SE-'''C
--I2
) RELEASED INTO
' POOL DURING
I MORPHOLOGICAL
^ REVERSAL
40
45
pM GLUCOSE/ ML.
EXTRACT
■ /
--6 . — . — • — /
RELEASED INTO
POOL DURING
X MORPHOLOGICAL
) REVERSAL
I I I I
_L.
I I I
35 40 35
HOURS
40
Some months ago, I also tried to approach
this problem in another way, arguing as follows.
If the notion outlined in Fig, 12 is valid, the
RS cell could be grown in such a fashion that
an unlabeled glucose pool could be created in it
and detectable some hours before the end of
exponential growth, i.e., before the point of no
return (cf. Fig. 16). At this point, glucose- i**C
could be fed to it, and the RS cell allowed to
continue its normal growth. If G6PDH were
functioning at or near the cell surface, differen-
tial localization of glucose- ^^ C and/or the im-
mediate products of its metabolism might also
occur near the cell surface. Presumably, some
of this labeled glucose (or glucose derivatives)
would also leak into, and mix with, the unlabeled
free pool. If, now, morphological reversal were
induced by removing the bicarbonate before the
point of no return (where, as seen previously, a
shift in isocitric and ketoglutaric dehydrogen-
ases does indeed occur), perhaps the surface
sites to which G6PDH was presumably loosely
bound would also be affected, thus releasing
soluble G6PDH. If this release of enzyme were
also to give rise to release of the glucose (or
derivatives) previously localized at these sites,
Fig. 17.
Effect of morphological reversal on the specific activity
of the free pool of labeled glucose in an RS cell.
specific activity of the free pool of labeled glu-
cose in the cell would increase.
The results of such an experiment are shown
in Fig. 17. Glucose- ^^C was introduced into a
synchronized culture of RS cells and the total
intracellular pool (labeled plus unlabeled) of
free glucose at successive stages in their
ontogeny was determined with the "Glucostat"
reagent. The results are shown by the continuous
line in the lower right hand figure. At 32 hr.
(i.e., before the point of no return) and at 40 hr,
(i.e., after the point of no return) samples of
these labeled cells were also transferred to
water for 3hr.; morphological reversal occurred
in the former but not in the latter. The changes
in the total intracellular pool of free glucose
associated with the successful and unsuccessful
morphological reversals are shown by the
arrows in the same figure.
Samples of the glucose from each pool were
then purified extensively by way of paper and
159
column chromatography to constant specific
activity; the total counts per minute attributable
to this purified glucose are shown in the lower
left hand figure. The two sets of data, when
combined, yielded the specific activities of the
glucose pools in RS cells of different ages - in-
cluding reversals - shown in the upper figure.
By way of summary, morphological reversal
before the point of no return does, indeed, bring
about an increase in the specific activity of the
glucose in the cell's free pool; after the point
of no return, this does not occur. Once again,
although these results could be interpreted in
several ways, they are consistent with the theory
we have been discussing.
Obviously, much more work is needed, and
we are in the process of doing some of it. In
conclusion, Blastocladiella emersonii provides
a very satisfactory system for studying the
relations between biochemical and morphologi-
cal differentiation (2). I am sure that Dr. Lovett,
who now follows me on this program, will illus-
trate in yet another way that this is so.
SCHRAER: What is the ecology of this
organism?
C ANTING: It is found in fairly slow-moving
bodies of water, sometimes streams but more
often ponds and puddles, and in soil. The genus
is ubiquitous, but for this particular species I
cannot say definitely. But what do you mean by
ecology, in particular?
SCHRAER: I was referring to the oxygen
content of the water in which they are found.
CANTINO: It likes to grow in fairly well
aerated bodies of water, but it is unhappy,
apparently, in bodies of water heavily laden with
organic matter. It can be trapped on insect
exoskeleta, fruits of the Rosaceae, etc.
ZIMMERMAN: Does the CO2 content in
these waters vary sufficiently to give you the
either/or type of growth?
CANTINO: I think so, but not directly be-
cause of the CO2 content of the water. Rather,
it is the fact that when this organism grows in
nature, it is often surrounded by microflora
and fauna which tend to localize around it. I feel
certain, although I've never stuck microelec-
trodes therein, that the CO2 concentration with-
in such localized "pockets" must be higher than
on the outside, and that this may be involved in
the induction of RS differentiation in nature.
EPEL: Does isocitrate eliminate CO2?
CANTINO: Isocitrate did not do so in ex-
periments done years ago; we'd like to assume
that this was because it does not get in very
easily. Ketoglutarate does get in, and does the
trick under certain conditions of nutrition.
Presumably, it functions as a substrate for the
backward reaction.
EPEL: Have you been able to use malic
acid to affect CO 2 consumption?
CANTINO: Do you mean the Ochoa "malic"
enzyme? We think it may be operative at some
stages, but I would not care to say "yes" or "no"
because these assays were done about ten years
ago when we were not using synchronized cul-
tures. Therefore, I'mnot sure of the significance
of these old assays.
KAHN: How do you visualize the control of
enzyme levels or activity?
CANTINO: I have no satisfactory basis for
speculation on this point.
CHALKLEY: Is there DNA synthesis at the
point of no return?
CANTINO: Net DNA synthesis ceases at
the point of no return. If we plot the DNA/cell
(using older methods of extraction of some 6-7
years ago) against developmental age, the curve
rises and then levels off almost precisely at
the point of no return. There is no additional net
synthesis of DNA after this point, although net
synthesis of RNA continues. However, during the
period after the point of no return, the composi-
tion (base ratios and physical properties) of the
RNA begins to change, (see subsequent reply by
CANTINO to a question by GROSS).
J. WRIGHT: I gather. Dr. Lovett, that
you're going to discuss this in the next paper?
LOVETT: Yes, but I won't discuss that part
of the life cycle.
KAHN: Is the DNA synthesis synchronous?
CANTINO: If you mean synchronous in
terms of nuclear division, we don't know. For
RNA, we have more data, but I would rather not
go out on a limb even here.
GROSS: Are there mutants of this organism
that are incapable of making the switch?
CANTINO: Yes, during the past 18 years
we've isolated four spontaneous mutants during
essentially day-to-day observations of cultures
growing on plates. So, they occur with a low
frequency. The mutants are incapable of re-
sponding to the bicarbonate trigger mechanism
or, as far as we know, to any other "inducer";
they will not form BS. When some were analyzed
for their enzyme contents, they were found to
lack ketoglutaric dehydrogenase (and aconitase).
GROSS: Do they lack it entirely? They don't
have the characteristic response?
CANTINO: No, they won't respond; they
won't produce RS in response to bicarbonate.
(Note added in proof: The mutants on which
160
these assays were done were lost some years
ago. Their content of ketoglutaric dehydro-
genase activity, based on assays of multiple
generation cultures in 1953, was not absolutely
zero, but about 4% of the level found in the wild
type; see Table I).
J. WRIGHT: They won't respond to any other
system you've tried?
CANTING: That's right. They seemed to
have lost the capacity. We would like to think
this lesion involved some kind of a "master gene"
which exerted pleomorphic effects because so
many, many things (including extreme reduction
in viability) were associated with this loss of
capacity for the formation of an RS cell.
GRUN: In your discussion, you've been
dealing with RS as a unit, as something fairly
constant, but in the beginning slides you were
stressing RS as being highly variant, oranges
ones, light ones, etc. How do you account for
the variability in RS?
CANTING: Well, I didn't stress the varia-
bility of RS at the beginning. I stressed that if
one starts with a population of spores, they have
the capacity to develop along at least four alter-
nate pathways, although I may not have said it
in so many (or rather so few) words. Spores can
develop into RS cells, GC cells, orange cells, or
a type which we call "late-colorless" cells.
Thus, there are four alternate pathways in the
life history of B. emersonii but the ones I have
discussed today are the two major ones.
GRUN: Then was this just one of the changes
that occurs in this system?
CANTING: Let me clarify. (The following
was altered slightly in the proof in order to
clarify the clarification which involved exten-
sive use of the blackboard.) Starting with a pop-
ulation of spores on plates, one obtains two
main cell types in the first generation; either
GC cells or RS cells, depending upon whether
or not bicarbonate is present (cf. Fig. 18).
Between 99 and 100% of the population of spores
will do this. However, depending upon the growth
medium selected, up to 0.5% of the population
of first-generation plants will consist of what
we have called an "G" cell -literally, an orange
cell. The cell is orange because, judging from
evidence obtained with mutant strains, it con-
tains gamma-carotene. Another zero to 0.5% of
the population consists of what we have called
"late-colorless" cells, cells which differ from
GC cells by their much longer generation time.
Does this clarify it? I have been speaking of a
population of spores, not a single spore.
GRUN: Then the orange and the late-color-
TABLE I
Enzyme system assayed Specific Activity
(crude cell-free preparations) in
Wild-type
Mutant
OC cells
cells
Cytochrome C oxidation
111
114
Succinic dehydrogenase
125
126
IsociLric dehydrogenase
160
156
Malic dehydrogenase
220
74
Fumarase
148
70
Aconitase
108
0
Ketoglutaric dehydrogenase
94
4
Fcr derails see reference 5.
less are not triggered by bicarbonate, but by
something else?
CANTING: It gets fuzzier nowl If we start
with an orange cell, harvest all its spores, and
plate them out, the new generation of plants has
essentially the same composition obtained with
the usual population of spores. However, it is not
exactly the same; more nearly 1% of the popula-
tion now consists of orange cells. From the
spores of a late colorless cell, an essentially
normal population is also obtained; but, in this
case, fewer than average numbers of orange
cells are produced. (The reply above was altered
slightly in the proofs for purposes of clarifica-
tion; the reader is referred to the paper by
Cantino and Hyatt cited in the bibliography of
this report for detailed tabulations of the kinds
of progeny produced by the different cell types
of B. emersonii.)
J. WRIGHT: Are these orange or late color-
less cells on the periphery of a culture or are
they different in some way?
CANTING: No, when starting with spores
which have been spread out uniformly on the
surface of a Petri dish so that each one develops
into an individual plant, you find that these vari-
ous cell types are distributed essentially at
random. We have published some evidence to
show that the distribution of a cytoplasmic
particle, which we labeled a "gamma" particle,
may be involved (3).
McCARL: I have a question on theglucose-
6-phosphate dehydrogenase. Do you feel that it's
synthesized on the surface of the membrane?
161
ORANGE PLANT ORDINARY COLORLESS RESISTANT SP0RAN6IAL LATE COLORLESS
(thln-wolled) PLANT.lthIn wolled) PLANT.tbrown.thIcK v»all«d, PLANT,(thln
=7/>:^
7.5
12.5
\ i
0.5
99.0
38 34
I \
No
No
I I
pitted.)
MEDIUM
P •
2 0»C.
%P^
Yes
No
Ava.# gamma"
particles per
spore in plant.
Ave.'/, of first
generation
population.
Ave. generation
time, hours.
Melanin In wall 7
Carotene In
protoplost '
walled)
im
12.5
15.5
I I
less than
0.1
(usually zero)
108
0.5
I
38
I \
Yes
No
I \
Yes No
Fig, 18.
The four alternate pathways of development in Blastocladiella emersonii. (Fig. 2, Cantlno,
In "11th Symp. of the Soc. for Gen. Microbiol.," 1961; reproduced with permission of the
Society for General Microbiology.)
CANTINO: Well, this will be pure guess-
work; my working hypothesis is that it is syn-
thesized on ribosomal particles close to the
membranes near the wall.
FERGUS: To go back to a former question,
if you sample a number of individual sporangia,
would you still get this ratio of the four types
within a single sporangium?
CANTINO: Yes, provided the environment
is favorable. One can alter these ratios quite
easily by modification of the media. For ex-
ample, 60% of the spores in a population can be
induced to produce orange cells if a suitable
concentration of actidione is added (note added
in proof: - and if peptone, which tends to repress
genesis of orange cells, is not present). On the
other hand, addition of diphenylamine eliminates
orange cells from a population.
GROSS: These data that you've been discus-
sing about the population that can result from
germinating spores suggest to me that you're
thinking about the mechanism of this differentia-
tive process, probably, the way Barbara Wright
was thinking about hers. That is this (and tell
me if I'm doing either of you an injustice): I
have the feeling you are thinking of this as a
modulation process. The enzymatic systems
that control the product, giving a physically
different kind of cell, are all there. What's
really critical is that the levels of interaction
162
Percent generation time
I ■■
: : 1
O 60
Pt. of no return
in morDhoaenetis
^'
BNfl.,.,,
o
a. 50
/
y
i
1
o
/
•"^^ .... 1
3
C
<
z^--
./
Y~
o
° 20
/
A-^
HNA^,( -..
•
o
e
4
^ /
V
HNa„„„. -,.. 1
The pattern for net synthesis of different RNA's in an R.S. PLArsrr ouRrNO
DIFFEREmiATION.
T
,
^...^
....GMP
^\
^*
'
Pt. of no return
in Morphoqenesis
Xs^MP
— *
CMP/**^
UMP
"^•^
— ./
30 50 70 a
4
Age, hr
The MOLAR COMPOSITION OF RNAn.Q ►
Values for RNA arc expressed as total /iMotes of aJI nucleotides derived from the RNA by Isolated at different stages during differentiation of an R S plant of B emersonii
KOH hydrolysis.
Fig. 19.
Transformations In the extractablllty and composition of the RNA of Blastocladiella emer-
sonii during RS differentiation. (Figs. 2, 3, Cantino, Phytochemistry 1, 1961; reproduced
with permission of Pergamon Press Limited.)
change in some way so that the system is modu-
lated and the products are different. Now, it
seems to me that in both of these there is a
critical way to determine whether that is the
whole story. That is to find out if at some point
before the point of no return you can disable the
genome. You disable the genes so that no infor-
mation can flow from them. If in that instance
this process continues, it's clear that this is a
modulation. It' s differentiation, to be sure, but
without benefit of gene action. Now, on the other
hand, if, in the condition where we stop the gene
action, this too stops, then you know you've got
to have something additional to the simple modu-
lation of the rates of enzymatic processes and
the way that they interact.
CANTINO: I'm inclined to agree (but I have
no direct evidence which will bear on this point).
However, may I summarize some analytical
results obtained in 1960 which may have an
indirect bearing here? (The following reply is
an expanded version of the original one; the
data can be found in ref. 4). Essentially all of
the RNA in a growing RS cell is soluble in hot
NaCl (= RNANaci-soi ), and it's molar com-
position (CMP:AMP:UMP:GMP = 1.00:1.33:1.08:
1.27) stays constant during the last stages of
exponential growth before the point of no return
is reached. But, after the point of no return, the
quantity of RNAwaci-soi per cell begins to de-
crease again, and simultaneously it undergoes a
sharp change in composition (Fig. 19). Just be-
fore the amount per cell of this NaCl-soluble
RNA reaches its peak, a new RNA appears in the
cell. This new RNA is attached to cell particles
which sediment at 10,000 to 15,000 x G, it is in-
soluble in hot NaCl but detectable by KOH hy-
drolysis to yield its component nucleotides, and
these nucleotides are present in it in almost
exactly equal quantities (CMP:AMP:UMP:GMP =
1.00:1.00:1.00:1.03). This insoluble RNA
(RNA insoi ), as seen in the figure, rises sharply
in the cell immediately after the point of no
return, and it reaches its maximum level when
the cell is about 70 hr. old. The plot for RNA total
in the figure was simply gotten by adding up the
data for RNAinsoi and RNA Naci-soi . Assays
for these two RNA types were also made in RS
cells which had been induced to undergo morpho-
logical reversal. The data (not shown in this
figure) revealed that just before the point of no
return, when RNA insoi first becomes detect-
able, morphogenetic reversal induced a sud-
den loss of about half of this RNA insoi • Similar
experiments performed after the point of no
return did not induce the shift.
DEERING: I have something that might be
relevant to this. I've started some work on the
effects of ultraviolet light on the OC develop-
ment of this organism. Ultraviolet light is be-
163
lieved to act mainly on the nucleic acids, RNA
and DNA in many biological systems. In our
experiments we've watched the morphological
development of the plants after UV irradiation
of the spores. Almost independent of the dose
of ultraviolet light, these plants will go through
the early stages of development to a point
approximately 60% of the way through the
normal life cycle and then they'll essentially
stop. The development up to that point seems
to be quite norm^ except that it proceeds more
slowly, the higher the UV dose. We're currently
starting studies of nuclear counts, and RNA and
DNA content, after exposure of the spores to
ultraviolet light. In the OC form, the point 60%
of the way through the life cycle, at which
growth stops after UV, corresponds to some
of the other changes that Cantino has just talked
about. This suggests that some critical changes,
blocked by earlier UV irradiation, are going on
at this point in the life cycle. This point is
approximately the point of no return from OC
to RS. By that I mean there is a point in the OC
development beyond which you can't add bicar-
bonate and make it go to the RS form.
CANTINO: That's right, it is about the
point of no return for OC cells. I want to add
that this applies only to synchronized plate cul-
tures of OC cells. The point of no return for
OC cells grown in liquid cultures is another
matter.
DEERING: These experiments were on syn-
chronized plate cultures. After the UV, the
development of these plants stops roughly at
the "point of no return." This might indicate
that some damage to the RNA or DNA (or both)
in the spores has stopped them from supplying
information necessary to get them beyond a cer-
tain point in development. This might indirectly
implicate the involvement of nucleic acids in
this change.
CANTINO: There is one thing I didn't men-
tion in respect to the data for RNA shown in
Fig. 19. Firstof all, because of the sharp changes
in base ratios and in the quantities of RNA insol
and RNA Naci-soi , the conclusion seems in-
escapable that a large amount of RNA turnover
is occurring after the point of no return in an
RS cell. (If comparable studies had been done
with OC cells, the results might bear directly
upon Dr. Deering's comments). Secondly, at the
point of no return there occurs a sudden and very
fast rise in the free CMP acid pool of the cell,
but this change is not counterbalancedby a com-
parable rise in the pools of AMP or UMP. The
CMP is reutilized, however, as the RS cell
proceeds beyond its point of no return.
References
1. E. C. Cantino and M. T. Hyatt. Leeuwenhoek
ned. Tijdschr. 19, 25 (1953).
2. E. C. Cantino and J. S. Lovett. Adv. in Mor-
phogenesis 3, 33 (1964).
3. E. C. Cantino and E. A. Horenstein. Myco-
logia 48, 433 (1956).
4. E.C. Cantino. Phytochemistry 1,101 (IQ&l).
5. E. C. Cantino and M. T. Hyatt. J. Bacterial.
66, 712 (1953).
6. E. C. Cantino. In "11th Symposium of the
Society for General Microbiology," 1961,
p. 246.
7. J. S. Lovett and E, C. Cantino. J. Gen. Micro-
biol. 24, 90 (1961).
164
NUCLEIC ACID SYNTHESIS DURING DIFFERENTIATION
OF BLASTOCLADIELLA EMERSONII
James S. Lovett
Department of Biological Sciences, Purdue University,
Lafayette, Indiana
Because Dr. Cantino has already presented
an excellent summary of the life cycle of B.
emersonii (1), I can jump right into the material
I want to discuss without further introduction.
This will deal with the formation of zoospores,
which he skipped over very lightly, and their
germination, two phases in the life cycle that
are very close together. Before starting, how-
ever, I would like to mention that most of the
work on spore differentiation was done by a stu-
dent in my laboratory, Sister Mary Nadine
Murphy, now at Mundelein College, and the
electron micrographs I will show were prepared
in cooperation with Dr. A. E. Vatter, at the
University of Colorado Medical School, who
introduced me to the mysteries of electron
microscopy.
We are interested in the formation of spores
and their germination for two reasons: one gen-
eral and one specific. In general, we feel that
the continuous transition from a relatively undif-
ferentiated plant with many nuclei to a large
number of highly differentiated spores and the
germination of these spores to give back tiny,
but nevertheless very similar, plants should
provide an excellent opportunity to study, and
perhaps even discover, some control mechan-
isms for the cellular regulation of differentia-
tion. Our specific reason is an interest in nuclei
acids and, particularly, the spore nuclear cap,
which is shown in the first figure (Fig. 1).*
The zoospore is characterized by the large
"nuclear cap," a structure which has been known
for a long time. The spores in this photograph
*Most of the figures presented here are schematic and
approximate to illustrate the material discussed. The com-
plete curves, micrographs, and experimental details will be
published elsewhere.
were fixed and stained with the basic dye, tolui-
dine blue. In addition to the barely visible
flagellum, one can see the nucleus and a nucle-
olus. All of these structures can be seen better
in Fig. 2, which is an electron micrograph
Fig. 1.
Photomicrograph of a Blastocladiella zoospore, nc,
clear cap; n, nucleus; nu, nucleolus; fl, flagellum.
Fig. 2.
Electron micrograph of a section through a zoospore,
m, mitochondrion; v, vesicle; g, unidentified granule.
165
section through a spore. The micrograph
clearly shows the large basophilic cap with
its finely granular contents; note also that
there is no material of a similar nature outside
the cap. It also shows the nucleus, the nucleolus,
and, although it looks like two, the single large
mitochondrion with the flagellum coming out
through the bottom.
Figure 3 is a tangential section through the
nucleus showing partial sections of the nucleus
and the cap with the small osmiophilic particles
in it. I'm sure you've guessed that these are
ribosomes. That is what we think they are. The
pores in the nuclear membrane are particularly
obvious. K you look carefully at the membrane
of the cap itself, on the other hand, it is seen to
be continuous. There is a single large mito-
chondrion per spore; it is always acentrically
located and specifically associated with lipid
granules and what we suspect may turn out to be
polysaccharide granules. Figure 4 is a section
through the lower end of the spore showing the
centriole, or basal body, at the base of the
flagellum, going through the mitochondrion in
a small channel. Figure 5 is a section taken at
right angles to the last and shows quite clearly
that the flagellum doesn't just pass by the mito-
chondrion; it goes right through a channel in the
middle. Note the direction of the flagellar shaft
with respect to the mitochondrion and the
"rootlets" that extend down through other chan-
nels into the body of the mitochondrion itself.
We don't have much of an idea concerning the
function of these at present. The purpose of this
hasty survey of zoospore morphology has been
to emphasize the highly organized state of the
spores; the nearest approximation I can think
of would be a protozoan or perhaps a sperm
cell. It is obvious that a considerable intra-
cellular transformation must occur in the proc-
ess of forming these spores at the end of the
life cycle.
Several years ago, Dr. Gilbert Turian, in
Switzerland, demonstrated the presence of these
small particles in electron micrographs of
Allomyces nuclear caps (2), but Allomyces is a
filamentous organism and is, therefore, difficult
to grow in synchronized cultures. We have been
interested in looking at the caps in B. emersonii.
partly because of familiarity with the orga-
nism, but partly because, in terms of differentia-
tion, we can get much better synchrony for our
work.
Figure 6 shows that one can readily isolate
the nuclear caps from zoospores. Because the
caps were central to our interests, we wanted
to be sure we could get them out, characterize
the contents, and be certain they were what we
thought they were, that is to say, ribosomes. By
proper procedures, one can gently rupture the
spores, separate the caps, and purify them by
differential centrifugation (3). When we did this,
we found that they were composed of about 40%
RNA, 60% protein, and made up about 70% of
the total RNA of the cell. They also contributed
something like 18% of the total dry weight of
the spore. This was all consistent with a ribo-
somal composition.
Analysis of the purified caps demonstrated
that they matched ribosomes in their chemical
composition. When we purified the caps and then
isolated their particulate contents, we could also
show that they were ribosome-like by examining
them in the analytical ultracentrifuge to obtain
their sedimentation coefficient. They sediment
at about SOS, a value similar to that found for
other fungi. The particles also dissociate in low
magnesium ion concentration and contain a latent
ribonuclease. Thus, they appear to have the
characteristics of ribosomes as determined by
a variety of procedures. They also seem to be
very pure. When the particles are isolated from
caps, one obtains a glassy pellet so transparent
Fig. 3.
Electron micrograph of a section through a zoospore
tangential to the nucleus. Ig, Upid granule; pg, poly-
saccharide; g, granule.
166
o (^"^ y
Fig. 4.
Electron micrograph of flagellar base and rootlet, r,
rootlet; fl, flagellar fibrils; m, mitochondrion.
Fig. 5.
Cross section through the mitochondrion and basal body
of a zoospore, bb, basal body; r, rootlet.
as to be scarcely visible in the bottom of the
Spinco tube. The washed cap ribosomes con-
tain about 63% RNA and 27% protein.
Before launching into some of the experi-
mental work on the formation of the spores, I
would like to discuss very briefly some of our
ideas concerning the structure and function of
the nuclear cap. The observation that all the
cellular ribosomes are packaged in this peculiar
structure surrounded by a membrane raised
some rather obvious questions as to its function.
First, where do the cap ribosomes come from?
One can guess, and I think our original guess,
that they come from the cytoplasm, turned out
to be correct, although it obviously had to be
proved. It is reasonable to expect that the spores
might conserve their ribosomes. However, they
might also be made essentially in situ, at the
time the cap is formed, by degradation of
pre-existing ribosomes followed by resynthesis
in a new location.
A second, and perhaps even more interest-
ing, problem concerns the function of the cap
for the spore. Blastocladiella is not the only
fungus to produce these; they are produced by
a whole series of fungi. But why on earth do
they form such unusual structures? First, it
may be that the cap serves as a storage
Fig. 6.
Isolated zoospore nuclear caps.
reservoir of RNA and protein for early germi-
nation. It is possible that the cell degrades the
cap ribonucleoprotein and uses the products to
make new ribosomes to start growth. Alterna-
tively, it might store the ribosomes during the
non- synthetic zoospore stage. I should em-
phasize that the zoospore is motile and me-
tabolically active, but it doesn't grow. The
167
formation of a cap could be a way of protecting
the ribosomes from degradation during a time
when no synthesis is actually occurring. It also
could be an unusual mechanism for controlling
protein synthesis by isolating the ribosomes
from any one of the many factors involved in
the complete functional synthetic system, such
as ATP from the mitochondria, for example.
It could equally well serve some combination
of these. I am stressing these points because I
think they provide us with some ideas that can
be tested.
For example, if the cap merely conserves
the ribonucleoprotein for synthesis of new
ribosomes, germination, then, it ought to be
associated with or require concomitant ribosome
synthesis at an early stage. K, on the other
hand, the ribosomes are conserved as functional
units and are actually used as ribosomes with-
out alteration, then germination and early pro-
tein synthesis might be quite independent of
ribosomal RNA synthesis but could require
"messenger" RNA synthesis or coding of some
kind. Alternatively, if the ribosomes are func-
tional, the spore could already be precoded and
ready to go; in fact the ribosomes could have
the information stored away with them in such
a manner that only release from the cap would
be necessary. In this case, germination might
be completely independent of early RNA syn-
thesis of any kind,
I think we can test these hypotheses. We
can isolate the caps and look at them in a cell-
free system, for example, to estimate their
functional capacity in vitro. We have been trying
to get a reliable cell-free system to do this,
but we have not yet been successful.
I would now like to talk about differentiation
in terms of the source of the cap ribosomes
and then briefly discuss the process of germina-
tion, which presents additional clues concerning
the particular problem of cap function.
CHALKLEY: Have you looked at these under
the electron microscope to see if there are
polysomes there?
LOVETT: We haven't looked at them in the
electron microscope, but we have tried a few
inconclusive experiments by isolating the caps,
lysing them very gently with detergents, and then
layering them on gradients to look for poly-
somes. This should show us if there are lots of
them. So far, we don't find any. However, we
haven't done enough of this to be sure.
I want to turn to the formation of zoospores,
and RNA synthesis in particular, although I'll
mention a few other things. First, what happens
during the differentiation to form zoospores?
Figure 7 is a summary diagrm illustrating a part
of the life cycle starting with the tiny spore and
extending through the exponential growth phase to
the mature plant containing many nuclei. Under
our conditions, the number of nuclei turns out to
be very close to 256, which is somewhat dif-
ferent from plants grown by Dr. Cantino's
method. Formation of the papillae and subsequent
events lead to the formation of the zoospores.
This graph is a plotof per cent papilla formation
versus culture age to show how sharply the
transition occurs in our system. We grow the
cultures by inoculating zoospores into a rich
medium and then aerating and stirring at
24°C (4). At 15/2 hr. we induce differentiation by
changing the medium; we don't wait for it to occur
normally, although it will do so without induction.
However, we get much better synchrony by
changing the medium to induce differentiation.
This is represented by the fact that the entire
population enters this papilla stage, the first
obvious morphological event, within a span of 1
hr, and 60% of the plants form papillae within a
24 min interval. This is pretty good synchrony
for an organism with a life cycle of approximately
24 hr.
DEERING: How do you induce this?
LOVETT: We just change the medium. We
wash out the growth medium and resuspend the
plants in a dilute inorganic salt solution, the
1/2DS you will see indicated in some of the
figures. Figure 8 summarizes some of the data
obtained for the total protein, RNA and DNA of
cultures. You can see that until 15/2 hr, when
the medium was changed, all these increased
exponentially, but shortly after the change they
began to level off: DNA and RNA at about 16,'4
hr and protein and dry weight at about 17 or
ITA hr.
Figure 9 illustrates a little more dra-
matically, in a nonlogarithmic plot, the pattern
observed for whole- cell RNA. You will notice
that total RNA continues to increase for a while
after the inducation, but then begins to go down-
hill, and does so continuously until the end of
the cycle at 19'^ hr when the spores are dis-
charged. This finally represents a 35% loss in
the total RNA of the plant.
We were interested in these RNA changes,
and Figure 10 shows that not only does RNA
start to disappear after I6/2 hr, but, if one
measures short-term pulse- labeling with C^"*-
uracil, the rate of incorporation also drops very
drastically between 16 and 17 hr. I want to
emphasize the morphological point: the heavy
168
EXPONENTIAL
GROWTH
<
o
ti.
Q.
<
Q.
0 15 17 19
HOURS
Fig. 7.
Papilla formation as a function of culture age.
3
a
E
O
3.
DRY WEIGHT
PROTEIN
DNA
15 17 19
HOURS
Fig. 8.
Semi-log plot of major cell constituents.
UJ
a:
3
3
E
g
<
z
<
O
Q.
OC
O
O
O
LlI
\-
<
OH
Fig. 9.
Total RNA content vs time.
PROTEIN
' V —
1% papillae\
19
Fig. 10.
Incorporation of RNA and protein precursors.
169
o
6
I
I
I
I
•
II
I'
A
1 /
II
20
Q.
U
30 10 30
FRACTION NUMBER
Fig. 11.
Sedimentation profiles of RNA in a pulse-chase experi-
ment at 16 hr.
dashed curve in this figure indicates the pattern
of papilla formation in the same culture. It was
followed in all cultures, but is not always plotted
in the figures. It demonstrates that we have a
good, synchronized culture and that RNA syn-
thesis essentially stops before the appearance
of the first morphological event. I should per-
haps say that gross RNA synthesis has stopped.
The same figure illustrates the pattern of leucine
incorporation. You will notice that this goes up
and continues to rise when RNA synthesis is
dropping. It continues longer and drops more
slowly, although it has reached a low level by
195^ hr.
DEERING: Is this label in the dilute salt
solution that you put in after you wash these?
LOVETT: Every experiment I'm talking
about now is in the dilute salt solution after 155^
hr.
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FRACTION NUMBER
Fig. 12.
Sedimentation profiles of pulse- labeled RNA prepared
at different stages of development.
DEERING: Then the label is added after 15^
hr?
LOVETT: Well, we often check points be-
fore. In this experiment a few points were taken
before 15/^ hr, which is the reason for the drop
and subsequent rise in the RNA incorporation
curve. Changing the medium does modify this
somewhat. We don't, however, attach much
significance to this at present.
The fact that uracil incorporation seemed
to stop so drastically and sharply suggested that
the cells were obviously turning off RNA syn-
thesis and, perhaps, undergoing considerable
degradation and turnover. As I said earlier, we
felt it important to determine where the nuclear
170
cap ribosomes came from. If a great deal of
turnover was going on, it could mean that the
ribosomes were actually being degraded and re-
synthesized and, in this way, being redistributed
from one part of the cell to another.
The results of a pulse-chase experiment at
16 hr are given in Fig. 11 and show that we
could do labeling and density gradient experi-
ments to study RNA synthesis. For this experi-
ment the plants were pulsed with C^"* -uracil
for 2 min and then "chased" with an excess of
"cold" uracil. Zero min indicates the RNA at
the start of the chase. The other graphs show
the pattern of labeling during the "chase." The
first surprising thing we noticed was that
these so-called plants have a very animal- like
characteristic; they produce a rapidly labeled,
heavy RNA that we think, on somewhat indirect
evidence, is actually a precursor of ribosomes.
It labels rapidly and disappears during the
chase as the radioactivity increases under the
ribosomal peaks. Actually, we suggest this on
the basis of comparison with work on animal
tissues where other people have drawn similar
conclusions (5). We haven't, however, identified
it or proven it to be a ribosomal precursor.
To do this it will be necessary to isolate it
and do much more work with it. This experiment
was done at 16 hr, just before all the interesting
events occur.
Figure 12 illustrates the pattern of RNA
synthesis as a function of time. Sixteen hr, 40
min is just before synthesis is apparently shut
down. The experiment is similar to that on the
last slide, except that in this case we only
exposed the plants to C^^-uracilfor 10 min, then
killed them and extracted the RNA for gradient
centrifugation. I only want to point out that this
is a typical pattern for transfer-, ribosomal-,
and heavy- RNA incorporation, but that these
steadily decrease starting at 16 hr until, by
17 hr, you can't detect any label whatsoever in
the RNA fractions. The heavy RNA peak seems
to be the last to disappear.
TS'O: What is the ribonuclease treatment?
LOVETT: The curve for ribonuclease-
treated RNA just shows that if you treat the
RNA's in any of these stages, of which we've
only shown one, with ribonuclease, that all your
material ends up at the top of the gradient. You
don't have any in the region of the O.D. peaks,
as in the untreated samples.
This seemed nicely consistent with our
earlier results on total RNA and post- labeling,
etc., but when we started looking at the pools
in terms of Barbara Wright's work (6), which
HOURS
Fig. 13.
Whole cell uptake vs incorporation Into RNA.
we needed to do, we suddenly discovered that
total uptake versus the amount incorporated into
RNA changed drastically during the same period.
In other words, there was about a 13-fold de-
crease in the ability of uracil to enter the pools
between slightly after 16 hr and 17 hr. This
meant that we had to re- evaluate our previous
interpretation of the labeling patterns as indi-
cating a shut-down in RNA synthesis. Perhaps
what was being changed was our ability to
measure incorporation into RNA rather than the
synthesis of RNA itself.
In order to try to get around this, or at
least get some idea of its significance, we did
an experiment where we added excess label to
a differentiating culture just before 16 hr. The
first points were taken right after 16 hr when
we knew we could get good uracil labeling in
the RNA (Fig. 13). We then followed the dis-
tribution of the label as the culture went through
the period when we no longer could get at it by
pulsing precursors from the outside; thus, we
pre-loaded the pools and then followed what
happened afterwards. As you can see, the pools
were labeled very rapidly. The value for the
pool represents the difference between the total
label and that in RNA. The counts in the pool
increase rapidly, but you will notice that they
go up until 17 hr and then the pool size drops
171
and the total counts decrease. All the loss of
label from the plant can be accounted for by the
label that reappears in the medium. Our inter-
pretation is that part of the problem with uracil
incorporation in our previous experiments was
that we are trying to go against the system. Dur-
ing the stage from 16-17 hr, not only is the plant
degrading RNA, as shown in one of the earlier
figures, but the pools themselves appear to be
actually shrinking at this time. Both factors
would work against uracil penetration.
Figure 14 represents an experiment where
we tried to circumvent this difficulty. I'm not
entirely sure whether we did or not, but I think
we did in part. The experiment was based on an
assumption with which you may not agree and
which we have to prove: that the "heavy" RNA
was predominantly a ribosomal precursor. My
argument will have validity only as far as this
is true. We grew the cells as usual, adding Ci"*-
uracil for 1 hr during the exponential growth to
randomly label the whole- cell RNA. Then, as a
function of time, we pulsed the cells with tritium-
labeled uracil to see how much could enter RNA
from the outside. The pre-existing, randomly
labeled RNA inside the plants should give us
O
Q.
O
I
I
I
I
some idea of the activity within the cells, par-
ticularly at the later stages when we could not
get at it by pulsing from outside. As you can
see (Fig. 15), the pattern of the randomly
labeled RNA remains essentially constant
throughout. However, the pulse labeling with
tritium is very similar to our previous results
with C''* -uracil: rather disperse labeling and a
high-specific activity, heavy peak which com-
pletely disappears by 17-17}^hr. No carbonic
activity appears in this region, even though we
know from all our previous evidence that a con-
siderable amount of this RNA is degraded. If
one accepts the idea that the heavy peak is a
10 30 10
FRACTION NUMBER
Fig. 15.
30
Sedimentation profiles of steady-state labeled (C'^ ) and
pulse- labeled (H^) RNA vs time.
16
HOURS
19
HOURS
Fig. 14.
Incorporation and distribution of C 1* -uracil label vs
dme.
Fig. 16.
Acdnomycin inhibition of uracil Incorporation.
172
precursor for ribosomal RNA, this experiment
can be interpreted to mean that there is very
little, if any, turnover of RNA into new ribo-
somes. It is, however, all based on this particu-
lar argument which we have yet to prove.
I can summarize what I have said by point-
ing out that we did all this work, really, to
prove that our original interpretation was cor-
rect. From what I have just said, as well as
Turian's work with Allomyces (2, 7), it ap-
pears as though the ribosomes actually move
and become aggregated in some way within the
cytoplasm. I haven't any idea about the physical
mechanism for accomplishing this and we are
just starting to look at this stage in the develop-
ment of the spores in the electron microscope.
Our results at this point suggest that new
ribosomal RNA - and, apparently, transfer
RNA - synthesis is not necessary for spore
formation. Can we say anything about other
kinds of RNA? Obviously the question of mes-
senger RNA arises. Does the formation of the
spores require production of messenger RNA
and is there any way that we can show evidence
for it? Our information on this is quite incom-
plete, but I would like to describe what we do
know very briefly.
Actinomycin D is a very effective inhibitor
of morphogenesis. At 25 //g/ml, the concentra-
tion we have normally used, actinomycin effi-
ciently reduces the incorporation of uracil into
RNA (Fig. 16). In this experiment the actino-
mycin was added at the same time as the uracil
and even without pre- incubation it caused a
/'^.CONTROL
/ \
/ \
HOURS
Fig. 17.
Inhibition of leucine incorporation by protein Inhibitors.
60-80% inhibition of incorporation. It is only
effective on leucine incorporation if the plants
are pre- incubated with the antibiotic. There is
no figure for this, but at least 5 min of pre-
incubation are required for significant inhibition
of leucine incorporation.
Figure 17 shows the effect of two protein
inhibitors on leucine incorporation, puromycin
and p-fluorophenylalanine (PFP). Somewhat to
our surprise, PFP, the lower curve, was much
more effective than puromycin, although this
could be, in part, a concentration effect. PFP,
however, is also much more effective in causing
morphological arrest (Fig. 18).
These graphs require some explanation. The
experimental cultures are on the left while the
control cultures on the right show the morpho-
logical progression of the parent culture. The
top is for actinomycin treatment and the bottom
is for PFP- and puromycin-treated plants.
Samples were removed as a function of time
from a synchronous culture, placed in inhibitor,
100
z
o
100
a:
o
Fig. 18.
Inhibition of development vs time of treatment. Top left,
actinomycin; top right, control. Bottom left, p-fluro-
phenylalanine ( ) and puromycin ( ); bottom
right, control.
173
and allowed time to go through the entire dif-
ferentiation process, in fact, well beyond when
it had ended in the untreated controls. We then
fixed all the samples and examined them to
determine the percentage of cells reaching a
recognizable stage. The curves on the right show
the normal pattern of papilla formation in the
control cultures and the release of the spores
at the end of the experiments.
There was no inhibition of papilla forma-
tion by actinomycin at 17^ hr. That is to say,
there is essentially no effect on the papillae
if added just before the papillae actually ap-
pear, but if the actinomycin was added about
a half-hour earlier, 16-16/^ hr, then no papillae
formed and it gave 100% inhibition. The same
thing was true at a later point for spore for-
mation, the obvious "cleavage" of the protoplast
to form the individual spores. At 11% hr it was
100% effective; by 19 hr it was completely in-
effective. Essentially the same curve was ob-
tained when we used PFP. If you accept the
fact that these are well-synchronized cultures
and notice that the control patterns are super-
imposable, then the experimental curves for
actinomycin D and PFP are virtually super-
imposable also; puromycin is only indicated
here for spore formation because of the
100 /ug/ml concentration used it did not inhibit
papilla formation. It did result in abnormal-
looking papillae. They were long, multiple, and
somewhat twisted in contrast to the short,
single, and symmetrical papilla at the tip of the
normal plant grown under our conditions. We
do not know the reason for this effect. PFP, on
the other hand, completely mimics the behavior
of actinomycin.
GROSS: Do you know that the puromycin is
doing what you want it to do?
LOVETT: No, we don't. This is really the
only positive result that we have with puromycin,
and I am not going to say any more about it.
CHALKLEY: In our work with tobacco cells,
it shows a similar lack of protein inhibition.
If the puromycin does get into the cells, then
one may assume that there is some degradation
of part of the molecule.
LOVETT: I'm glad to hear that, because
we haven't yet tried other concentrations. We
were not sure that we were using an adequate
concentration and thought that it might not be
getting in fast enough.
CHALKLEY: I think the problem is not
unsolvable.
GROSS: I think the pea work is really much
more dramatic.
LOVETT: It is interesting and also most un-
expected.
B. WRIGHT: Is the time of effect of the
actinomycin accompanied by permeability
changes?
LOVETT: It is so effective in inhibiting
uracil incorporation that I think this means
that it is getting into the cells.
B. WRIGHT: Yes, but I mean the time at
which it first starts to inhibit.
LOVETT: No, at least I choose to interpret
it as meaning something about when it is acting.
This is indirect evidence, to be sure. Before we
see obvious morphological changes it stops
everything. When it stops papilla formation, it
has obviously stopped growth. However, the point
is, if you add it later it no longer has any effect,
even on papilla formation. It has no effect on
papilla formation but will stop later develop-
ment.
Returning to the fact that PFP acts like
actinomycin on development, you will note that,
as near as we can tell, it shuts down leucine
incorporation almost immediately. It is an
effective inhibitor. This, plus the fact that it
mimics the actinomycin effects almost iden-
tically in the morphological progression, leads
us to suggest that it may be acting in some
way other than simple incorporation, as has
been shown in E. coli (8), to form "nonsense"
protein. I have heard rumors of a similar
situation in another system. I think that in-
hibition by producing nonsense proteins would
take some time unless there was a critical
protein, and nothing could proceed unless it
was functional. This could be true, but I am
interpreting it somewhat differently.
GROSS: Well, does it shut down RNA?
LOVETT: We haven't done this yet, but I
think it shuts down protein synthesis.
GROSS: It shuts down protein synthesis as
measured by leucine incorporation?
LOVETT: We have only measured leucine
incorporation so far, but on the basis of the
fact that it is so effective on total development
1 think that it shuts down all protein synthesis.
The inhibition results suggest that the
actinomycin effect is on RNA made half an hour
before the papillae are formed, and that the
necessary protein is probably also made at
nearly the same time. This could explain why
PFP mimics the effect of actinomycin. Though
we have much to do before we can really prove
it unequivocally, it is nicely consistent both in
this case and in the case of the spore formation
later on. To me, it suggests that we do not
174
have to invoke long-term messengers. We do
not know what the papilla is composed of, but it
looks as though it might be a polysaccharide
of some kind. It may well be that some kind of
enzyme or enzymes are made that begin syn-
thesis long before one sees the morphological
event itself. We have no reason to say that
there couldn't be other kinds of RNA's having
other functions being made at the same time;
all we can point to is the observable event.
We consider this to be interesting pre-
liminary evidence and it is the kind of problem
we want to pursue with our system. We would
particularly like to be able to characterize
some of the RNA's we think might be produced.
This, I'm sorry to say, just about summarizes
all we know about spore diffferentiation.
Our information on spore germination is
even less definitive. Because of some interesting
parallels with the work discussed by Dr. Gross
on the first day (9) I would like to mention this
in an informal way. The next few plates will
show several stages during the germination of
the spore - a transformation fully as dramatic
as the differentiation of the spores themselves.
The next slide (refer back to Fig. 2) is just to
refresh your memory with regard to the orga-
nization of the spore with its nuclear cap and
the large mitochondrion, which, in certain sec-
tions, extends well up toward the anterior end
of the cell. Germination occurs quite rapidly
under our conditions and the next electron
micrograph (Fig. 19) shows the first stage we
have been able to catch. The spore is rounded
up; it no longer has the very thin, delicate outer
membrane and already looks as though it is
beginning to form wall material. It has retracted
the flagellum, and in this micrograph you can
see a longitudinal section of the flagellum within
the cell. The mitochondrium, instead of being
localized as before, is in several parts of the
cell and appears as more than one. All these
events occur in a matter of a few minutes. Even
at this early stage, there are points where the
originally continuous nuclear cap membrane
has become discontinuous. We do not know how
it occurs, but the ribosomes are beginning to
"leak out". Perhaps this is not the right term,
but that is exactly how it appears.
Figure 20 is a section of a cell a few min-
utes later during germination. It is now a little
round cyst with the ribosomes spread through-
out the cell and no sign of a nuclear cap. There
is the nucleus, a mitochondrion, which we think
may actually have divided by this time, and a
few of the granules from the zoospore. This cell
Fig. 19.
An electron micrograph of an early stage in germination
to show the retracted flagellum and the beginning of nu-
clear cap disorganization.
Fig. 20.
A very young stage to illustrate the complete dispersal
of the cap ribosomes and initiation of the primary rhizoid
(r).
175
Fig. 21.
A young germling plant with a well-established rhizoid.
xs, cross section.
has already started the process normally called
germination; it has begiin to grow out to form
the primary rhizoid.
The next micrograph is a somewhat later
stage (Fig. 21). It is a young germling after
about 30-40 min in culture, which has the
rhizoid well started. It is probably longer than
it appears in the micrograph since a tangential
section makes it taper down, but it could be the
actual size. At this stage the germling looks
like a perfectly normal cell and, in fact, develop-
ment from this point involves mainly increase
in size with branching and multiplicity of the
rhizoids, in the absence of cell division.
DEERING: How big is this germling?
LOVETT: The spore body is about 7x9
microns, and this is roughly the same, about 8
microns in diameter before it starts to grow.
DEERING: What are the long, slender lines
in the cytoplasm?
LOVETT: That is the endoplasmic reticu-
lum. It is fairly prominent here but not at some
other stages.
KAHN: Is that the nucleolus?
LOVETT: Yes, they have a large nucleolus
that is always present. You remember seeing the
spore? It was small and compact in the spore.
I'll have more to say about it in a moment.
DEERING: In published results, Cantino
mentioned that this plant seemed to separate
into two cells.
LOVETT: That is not at this stage; it
occurs later, during the time when spores are
formed at the end of the growth phase.
DEERING: How much later is it? At what
point can you really say that you've got division
into two cells?
LOVETT: There is none at the stage I'm
talking about. That occurs only at the end of the
growth phase.
CANTINO: I didn't speak of cell division,
only nuclear division.
DEERING: I'm talking about the two cells,
the basal one and the one full of nuclei.
LOVETT: That is just before spore are
released and is in the other experiments we
were doing. I didn't discuss it because it is
obscure and hard to see and we haven't really
done anything with it.
We can easily get reasonably well- syn-
chronized cultures of the germinating spores,
at cell densities of about 10^ cells per liter, all
doing very nearly the same thing at the same
time. The synchrony is not, however, quite as
good as we have obtained from zoospore dif-
ferentiation.
The next two figures illustrate the pattern
of synthesis during early stages of germination.
The cells appear to lose some dry weight during
the first hour of germination (Fig. 22). They are
so fragile, however, that we are sure some ma-
terial was lost while trying to collect the cells
and measure their dry weight. Thus, part of the
drop may be artificial. After the first hour, the
dry weight increases linearly for a matter of 6
or 7 hr at least. Figure 22 also shows DNA syn-
thesis during the same stage, and you will notice
that it increases nicely in a step function from
one level to twice as much in a 2 hr period. Nu-
clear division, which still puzzles us, occurs
during about the first hour of the 2 hr period of
DNA synthesis. We are not sure what this
means, but it may indicate that our culture is not
as synchronized as it appears. This, however,
remains to be seen. It does go through this first
nuclear division in a reasonably synchronous
fashion.
Figure 23 roughly illustrates the increase
in total RNA and total protein during germina-
tion and early growth. Net RNA increase is not
apparent for about 20 min, and new protein in-
crease is not detectable until about 40 min.
Figure 24 is a summary diagram which we
176
280
120
MINUTES
Fig. 22.
Dry weight increase and DNA synthesis in synchronous
cultures of young plants.
RNA and protein synthesis in synchronous cultures of
young plants.
Fig. 24.
Stages in the germination and early growth of a zoospore.
Stage 0, zoospore; stage 1, 12-15 mln; stage 2, 20 min;
stage 3, 25 min; stage 4, 120 min; stage 5, 240 min.
177
URACIL-C
0 10 20 30
MINUTES
Fig. 25.
Precursor incorporation by zoospores germinating in an
organic medium.
can use to try and interpret, or to fit into the
morphological sequence, the points I have just
discussed. Zoospore germination begins between
12 and 15 min after inoculating the spores into
the culture medium (Stage 1). It doesn't occur
sooner because the harvested spores are held on
ice in very concentrated suspensions while being
centrifuged and washed. Our germination experi-
ments start when the zoospores are diluted up
in the medium at 24°C. Twelve to 15 min later,
they roimd up and begin to germinate. The next
to last picture in the sequence (Stage 4) is a 2 hr
plant with a well- developed rhizoid.
The previous slide showed that a measur-
able increase in RNA and protein cannot be ob-
served until 20 and 40 min, respectively. The
next figure demonstrates that incorporation of
C^^ -uracil or C^'* -leucine can be detected much
earlier (Fig. 25). Uracil incorporation occurs by
10 min, if not sooner, as does leucine incorpora-
tion, which here looks somewhat slower than
it actually is because it has not been corrected
for its differing specific activity. Such a correc-
tion would move it much nearer to the RNA
curve.
While I have no further data to present, I
would like to mention a few recent and interest-
ing results. First, the pattern of synthesis and
early differentiation of the spores - up to a
stage where they form a tiny, uninucleate plant
with a fairly long, branched rhizoid - will occur
whether you put the spores in the growth medium
or not. The spore appears able to accomplish
this quite well at the expense of whatever it
carries with it. The lipid and polysaccharide
granules may serve as energy supplies for this.
The same is true for precursor incorporation.
Thus, the early events in germination appear to
be nearly independent of the medium. The trig-
gering of the spores to germinate may not be
independent of the medium, but we do not know
what sets the process in motion.
Now to go back to some of the problems I
posed earlier concerning the function of the cap
which we have obviously not really answered. As
I said before, the reappearance of the ribosomes
in the cytoplasm at germination could result
from synthesis of new ribosomes at the expense
of the cap ribonucleoprotein, or from migration
of the pre-existing ribosomes. In the first case,
ribosomal RNA synthesis would be required. The
second might necessitate coding of the ribosomes
for early protein synthesis, or it might require
nothing. This is an area of great interest to us,
and I would like to mention a few preliminary
experiments which are not yet at the stage where
I am ready to put them on a slide.
First of all, zoospore germination will pro-
ceed to between stage 3 and stage 4 (Fig. 24),
when it has just produced a short rhizoidal out-
growth, whether it is in actinomycin D or not.
In other words, if one adds 25-100 |/g/ml of
actinomycin the spore swims normally, settles
down, rounds up, the cap breaks down, and the
primary rhizoid is produced. At this point
further development is completely inhibited.
That is one observation. We know from other
experiments that during the same period,
25 //g/ml of actinomycin is very effective in
inhibiting uracil incorporation. We haven't
looked at the RNA yet.
TS'O: At that stage is there any evidence
that actinomycin is actually going in?
LOVETT: Well, I assume it goes in because
it completely inhibits uracil incorporation which
occurs at the same stage.
TS'O: However, can it still get in the later
part?
LOVETT: It completely inhibits the later
stage, so I assume it's getting in, though I don't
have direct evidence. It is still inhibiting after
that time, at least in control experiments, where
we have had no actinomycin present earlier; by
this I mean that it still inhibits uracil incorpora-
tion at the later stages. The third point is that
actinomycin seems to have practically no effect
on leucine incorporation during the first 30 min;
so far, we haven't followed it much beyond that
point. We have put the spores in 25 /ig/ml of
actinomycin, allowed them to germinate, and
measured leucine incorporation, with C^^-
178
leucine present continuously. There was no dif-
ference between the incorporation in the control
culture and the one with actinomycin.
Because of Paul Gross' results with em-
bryos (9), I'd like to speculate even more: if,
during these stages, we look at pulse-labeled
RNA on sucrose gradients (we are just beginning
to do this), the cells make RNA during the
whole time, but it is not ribosomal RNA. The
new RNA seems to be polydisperse, and in the
spore it is very heterogeneous on gradients. It
looks like a nice, classical bacterial messenger-
RNA pattern. The new RNA does not match the
ribosomal peaks. As soon as germination be-
gins, we also get the same effect that Paul
described for embryos - very "hot" labeling in
the sRNA region. I was originally concerned
that the RNA might be degraded and that some-
thing was wrong. I am only too happy to see that
the same results were obtained with embryos.
It isn't until actinomycin has its effect, at about
30-40 min, that we can show synthesis of ribo-
somal RNA. This suggests that during the first
30 min, the cell is operating with pre-existing
RNA, that the spores, in fact, have been pre-
coded for the earliest events. I say pre-coded
because of the observation, not because we have
direct evidence for it. It is just as mysterious
to me as it is, perhaps, to those who work with
sea urchins. We did look for polysomes and could
not find them. After hearing Paul, I was struck
by the fact that two cell types which, in part at
least, have a similar function (to be non- synthetic
and yet ready to begin synthesis at a certain
time) seem to be so similar in their patterns of
early RNA and protein synthesis.
KAHN: How long can a spore be maintained
before it becomes inviable?
LOVETT: I've never really determined
this, mostly because Tve been interested in just
the reverse. When we harvest our spores, we
try to collect only those that have been dis-
charged over a short interval. We then hold
them on ice until the experiment begins, which
means a matter of half an hour from the time
we isolate them.
KAHN: Then zoospores can probably be
kept a long time?
LOVETT: Well, actually they can swim
almost for days.
CANTDSrO: I can answer that question fairly
positively. Some spores can remain viable for
at least 24 hr, probably 36 hr, as swimmers.
They are getting their energy reserves from
within, not without, because this can happen in
water. We know they have a sizeable polysac-
charide pool.
TS'O: Did I hear correctly? In the begin-
ning you said that you think the ribosomes may
be made in the cytoplasm?
LOVETT: No, what I was suggesting was
that one of the ways the cap might have been
formed was by synthesis of new ribosomes by
turnover and retention of the newly produced
ribosomes just outside the nucleus. Our evi-
dence suggests that the ribosomes were made
before the stage of cap formation. I didn't mean
in the cytoplasm.
KAHN: Let me restate my question. Does
the resistant sporangium, wherein I assume the
nuclear caps (ribosomes) are being formed, re-
main viable for 24 or 36 hours?
LOVETT: I'm not sure of your question.
These spores can't be held very long. They have
a high metabolic rate and they eventually dis-
integrate if not allowed to germinate.
KAHN: Tm talking about prior to release.
K the resistant sporangium is, indeed, "re-
sistant".
LOVETT: The resistant sporangium is re-
sistant, but this isn't the resistant sporangium
we've been working with.
KAHN: I'm aware of that.
CANTINO: There are no spores in the re-
sistant sporangium even when it is mature.
LOVETT: The resistant sporangium is
going to produce spores. Ed Cantino knows a
lot more about the form it is in than I do. I
haven't looked at this.
GROSS: What is the function of this gamete?
Is it just dispersion by swimming or is it stor-
age for a long period of time?
CANTINO: The resistant sporangium is a
thick-walled structure. It appears to tide the
organism over unfavorably environmental con-
ditions in nature. We've had R.S. sitting in the
shelf for almost twenty years, now, practically
dry, and some of them are still viable. As far
as we know, there are no pre-formed spores in
them. Spores are induced to form once the re-
sistant sporangium is put in water. Then the
protoplast cleaves up into spores, the spores
swim out, and to all intents and purposes they
are like the spores Jim Lovett uses.
LOVETT: That takes about 8 hr ; the process
I have described in zoosporangia takes 3 hr.
Our cultures go a little slower than Ed's be-
cause of the way we treat them.
PAPACONSTANTINOU: I'm sorry I didn't
get this. Did you mention where the new ribo-
somal synthesis starts?
LOVETT: New ribosomal- RNA synthesis
179
starts at about 35-40 min and this is the same
time that actinomycin really inhibits develop-
ment. The cells won't go beyond this stage.
However, this doesn't necessarily mean that a
lack of ribosomal-RNA synthesis is the problem.
It could be that pre-coding was exhausted at that
time and the cells would have to make new
messenger. We don't have any idea yet.
PAPACONSTANTINOU: What is the status
of the nucleolus at that stage?
LOVETT: I forgot to mention that. The
spores have a very small, compact nucleolus
always located at the base of the nucleus oppo-
site the flagellum. In plants, the nucleolus may
include almost 60% of the nuclear volume. From
less than 10% of the volume of the nucleus in a
motile spore, it goes to about 50-60% of the
nuclear volume in an actively synthesizing
germling. With actinomycin treatment, they stay
small. However, we have not made any critical
measurements and this is a very rough esti-
mate.
PAPACONSTANTINOU: I was just talking
about the experiment that came out about a
year ago from the Massachusetts General Hos-
pital where they isolated the nucleoli from
B. emersonii I believe.
LOVETT: Well, they said they did. They
didn't prove it by any manner of means.
PAPACONSTANTINOU: I was wondering
about that. Also, they claimed that they isolated
a specific DNA whose base ratio was comple-
mentary to the ribosomal-RNA.
LOVETT: We haven't done anything like
this at all. I'm not able to interpret some of
Dr. Comb's work because he doesn't describe
his methods very completely, and I can't really
evaluate it.
MAURER: Did you try cycloheximide as an
inhibitor of protein synthesis?
LOVETT: No. We did try chloramphenicol
and it had no effect, but it doesn't seem to be
very effective with fungi in general.
McCARL: Was that graph you had on the
board with the 4S peak the labeling?
LOVETT: Yes, this was the labeling from
uracil during a short incubation of 5 min.
McCARL: This shows synthesis?
LOVETT: I don't know specifically. This is
the same question that Paul has; we don't know
whether it is addition of terminal groups, whether
it represents new synthesis, or what. We have
to get some of it out and look at it.
GROSS: Are you worried about end-
labeling?
LOVETT: No, I can get cytosine very
easily. That's no problem. In fact, Tm sure this
is true.
EPEL: Concerning actinomycin effects,
this could be added during the first 30 min of
germination with no effect?
LOVETT: We can let them germinate in it.
EPEL: Have you pulsed actinomycin?
LOVETT: No, we haven't tried removing it.
We've tried it where they germinate in it and
we've tested it where we take them out of a
normal culture as a function of time and put
them in it. You get the same results either
way; that is, actinomycin doesn't have any
effect until a certain point and then they be-
come sensitive.
EPEL: If you add actinomycin during spore
formation they still form spores, but are these
spores then capable of germinating?
LOVETT: Yes, they seem to be. We haven't
really tested this by growing them, but they seem
to be perfectly normal spores. I think that this
means that all the important events have hap-
pended by that time. It is interesting that the
"packaging" of the ribosomes is one of the very
last events. It does make sense that they are
used for protein synthesis and then packaged
up when practically everything essential is
done. This occurs at about 10 min before 19
hr, and it is very soon after 19 hr that the
spores are actually released.
GRUN: There were some small particles
in your electron micrographs that resembled
amyloplasts in a vague sort of way. Do you know
what I mean?
LOVETT: Are you referring to the little
round ones?
GRUN: There were some little round things
with a darker, fairly homogenous stain.
LOVETT: There are some little cup- shaped
structures in vesicles that are present in ap-
proximately the right numbers to be the particles
that stain with the Nadi reagent, as Ed reported
some years ago. These apparently occur only
in the spore, since we have seen them nowhere
else. I have no idea as to their function.
CANTINO: Their number depends upon the
kind of plant which formed the spores and it can
be modified by environmental changes, (cf.
F. C. Cantino and E. A. Horenstein, Mycologia
48, 443, 1956).
GRUN: They don't contain starch, do they?
LOVETT: I doubt it. I don't know, to be
quite honest with you.
GRUN: They were near the membranes in
the electron micrograph.
LOVETT: They're very characteristic look-
180
ing. They have a single membrane, and are a
little diffuse, but, more often than not, a section
through a cell looks like this.
GRUN: Yes, and there were others that
seemed to have a small drop.
LOVETT: I'm not sure what you mean.
There are other granules.
GRUN: They're similar to amyloplasts.
LOVETT: There are other granules that
are polysaccharide, I'm sure. Ed's done the
work on polysaccharides. We haven't. The other
granules you see in between the lipid granules
in the side body are, I am quite sure, polysac-
charide. They stain with the PAS reagent.
DEERING: I'm still after the answer to a
question that I asked earlier. There seems to
be a conflict in what you said with regard to
the spore development and the later picture
that you drew on the OC sporangium.
LOVETT: Do you mean the basal cell?
CANTESrO: Well, the basal part is just
rhizoids at the germling stage.
DEERING: I'm trying to find out whether
or not it is a separate cell that makes rhizoids.
What about the basal cell and rhizoid formation?
LOVETT: The basal cell occurs about the
same time the papillae form, I would say. Isn't
that right, Ed?
DEERING: It's very late in development?
LOVETT: Yes, and it is so obscure that it
is sometimes almost impossible to see without
very careful microscopic examination.
DEERING: Sometimes if you look at these
in earlier stages, you can see what looks like
a small ridge.
LOVETT: However, you don't see that until
they're really ready to go. Frequently, the basal
cell is so small, if it is there at all, that you
can't see it. You just see the rhizoids.
DEERING: Is this basal "cell" separated
from the rest of it in electron micrographs?
LOVETT: Give me about six or eight
months and I'll be able to tell you. We are
going to look at these stages in the electron
microscope, but we haven't done it yet.
GROSS: Are there any nuclei in that lower
cell?
LOVETT: In the RS form, the lower cell
forms at the time when everything is moving
out. In the OC, I don't think we could say one
way or the other, but probably not. These
plants, after all, are converting all their proto-
plast into spores. When the spores are gone,
there is no reason to leave anything behind, be-
cause the plant is dead. It's an empty hull.
CANTING: That cross wall is laid down,
centripetally, from outward to inward, and as
that happens, what little protoplasm is down
there migrates upward and you're left with
an empty cell; at least, we call it a cell a la
Robert Hooke.
GROSS: That is a bit confusing because it
doesn't sound like a cell at all.
C ANTING: It's a compartment, a separate
compartment.
LOVETT: It is very striking in the resistant
sporangium where it forms a big stalk. You can
almost watch the material move out of the lower
part.
References
1. E. C. Cantino. This symposium, (1966).
2. B. Blondel and G. Turian. J. Biophys. Bio-
chem. Cytol. 7, 127 (1960).
3. J. S. Lovett. J. Bacterial. 85, 1235 (1963).
4. Sister M. N. Murphy and J. S. Lovett. De-
velop. Biol,, in press.
5. R. P. Perry, P. R. Srinivasan and D. E.
Kelly. Science 145, 504 (1964).
6. B. Wright. This symposium (1966).
7. G. Turian. Protoplasma 54, 323 (1962).
8. R. Munier and G. N. Cohen. Biochim. Bio-
phys. Acta 31, 378 (1959).
9. P. R. Gross. This symposium, (1966).
181
THE MOLECULAR ASPECT OF NUCLEIC ACID
INTERACTIONS
Paul 0. P. Ts'o
Department of Radiological Sciences, The Johns Hopkins University,
Baltimore, Maryland
The underlying philosophy and the strategy
of our research is quite different from that
presented in this workshop so far. We are
interested in solving the problem of biology from
the standpoint of chemistry and the approach of
chemistry, especially physical chemistry. The
experimental system usually consists of simple
models. The approach is analytical and quantita-
tive. The conclusion is generally unambiguous
and mechanistic in nature. This is the power or
the characteristic of physical sciences. Our
problems are, however, oversimplification and
unrealism. The conclusion may not be relevant
to the more complex situation in biology in which
we are interested. The major challenge to our
work, therefore, is the meaningfulness of our re-
sults to the central problems of biology. We have
to walk on a tight rope. On one hand, the system
has to be simple enough to be analyzed quantita-
tively from the standpoint of physical sciences.
On the other hand, the system has to be compli-
cated enough and sophisticated enough to contain
the essence of the biological world. I hope to
demonstrate to you how we try to meet both de-
mands in our research.
From the standpoint of chemistry, study of
the biological system can be viewed as the study
of the structures, properties and interactions of
biopolymers, with themselves and with small
molecules. Of all biopolymers, proteins and
nucleic acids appear to be the most important
in terms of the specific interactions which lead
to information transfer.
The significance and some of the general
principles of the interactions of nucleic acids
with one another are well known. These concepts
and this knowledge have served as the foundation
for the development of molecular genetics. We
wish to reexamine quantitatively the basic prin-
ciples and the nature of the forces which govern
the interactions of nucleic acids, and to do so by
physical chemical studies.
The problem is approached at three levels of
complexity: (1) interaction in solution between
the monomeric units of nucleic acid and their
analogs and derivatives; (2) interaction between
the monomeric units and the nucleic acid
polymer; (3) interaction between nucleic acid
polymers. The present review is confined to
investigations at the first two levels.
We concentrate first on interactions of neu-
tral compounds and thus avoid complications due
to the strong electrostatic interactions of
charged molecules. Because both the sugar and
the phosphate moieties are common to all
nucleotide units, specific interactions of nucleic
acids must reside in the purine and pyrimidine
bases. Therefore, the experimental approach
may quite justifiably be focused on the interaction
of uncharged bases and nucleosides and their
interaction with nucleic acid polymers.
The first level of interaction includes the
following problems: Does a solution of mono-
meric units, such as free nucleosides, interact
within itself? To what extent? By what mech-
anism? To answer these questions, three types
of physical- chemical measurements have been
made using, because of solubility problems,
pyrimidine nucleosides and purine.
Vapor pressures of solutions of purine,
uridine, cytidine, 5-bromouridine, and 6-
methylpurine, from 0.1 molal to approximately
0,8 molal have been measured thermoelec-
trically (1, 2). Osmotic coefficients, 0, were
calculated from the data (Table I). Activity
coefficients at 25° were calculated from the
osmotic coefficients by the Gibbs-Diihem rela-
tionship using a computer which performed a
183
TABLE I
Molal Osmotic Coefficients^ of Various Solutes Determined In Water at 25°''
Molal Cone .
Purine
6- Methyl -
purine
Uridine
5-Bromo-
uridtne
Cytidine
0.05
0.917
0.786
0.969
0.948
0.967
0.10
0.849
0.682
0.943
0.894
0'.935
0.15
0.794
0.624
0.921
0.844
0.905
0.20
0.749
0.582
0.901
0.801
0.87 6
0.28
0.714
0.544
0.883
0.766
0.850
0.30
0.685
0.510
0.866
0.738
0.826
0.35
0.662
0.484
0.849
0.715
0.804
0.40
0.643
0.469
0.833
0.693
0.785
0.45
0.627
0.461
0.817
0.763
0.50
0.614
0.456
0.801
0.7 52
0.55
0.601
0.446
0.786
0.738
0.60
0.590
0.427
0.773
0.724
0.65
0.578
0.407
0.762
0.710
0.70
0.567
0.410
0.755
0.695
0.75
0.555
0.80
0.544
0.85
0.532
0.90
0.522
0.95
0.521
1.00
0.505
1.05
0.501
1.10
0.501
'Data from Ts'o, Melvln and Olson, /. Am. aem. Soc. 85, 1289 (1963) and Ts'o and Chan,
ibid. 86, 4176 (1964); reproduced with permission of the American Chemical Society.
These are fitted osmotic coefficients computed from the experimental values.
numerical integration on the fitted polynomials
and related molal concentration to ^ (Table II).
The data clearly indicated that the properties of
these bases and nucleosides in solution are far
from ideal. Values of both osmotic coefficients
and activity coefficients are much below unity.
These results establish the concept that purine
and pyrimidine nucleosides do interact exten-
sively in aqueous solution. Recently we have ex-
tended this type of measurement to other less
soluble purine nucleosides. The osmotic coef-
ficients at 25° at a concentration of 0. 1 molal in
water of 2'-methyladenosine is 0.723, of 2'-
deoxyadenosine, 0.668, and of N^-methyladeno-
sine, 0.548. Evidently, adenine nucleosides do
associate in water even more extensively than
purine and to about the same degree as the
6-methylpurine.
After further analysis for their congruence
to different models for multiple equilibria, the
thermodynamic data were found to be incom-
patible with the model which assumes that only
dimers are formed. Thus, the degree of associa-
tion of these compounds may go beyond the
dimers stage to a higher degree of polymeriza-
tion. Most of the results are consistent with the
model which assumes that the association
process continues through many successive
steps (at least above five steps) with the same
equilibrium constant. Comparison of the equilib-
rium constant and thus the standard free energy
changes is given in Table III (1, 2). This table
shows that the tendency of purine to associate
is much greater than that of pyrimidine nucleo-
sides, which in turn is greater than that of urea.
Now, what isthemodeof association of these
184
TABLE II
Molal Activity Coefficients* at 25° Computed from the Fitted Osmotic Coefficients
6-Methyl-
5-Bronio-
Molal Cone.
Purine
purine
Uridine
uridine
Cvtidine
0.05
0.844
0.626
0.939
0,9U2
0.93O
0.10
0.728
0.469
0.888
0,811
0.878
0.15
0.641
0.385
0.845
0.732
0.824
0.20
0.575
0.329
0.808
0.666
O.77o
0.25
0.522
0.287
0.775
0.613
0.733
0.30
0.480
0.255
0.744
0.569
0,695
0.35
0.446
0.230
0.716
0.533
0.661
0.40
0.418
0.211
0.690
0.502
0.631
0.45
0.394
0.196
0.665
O.o04
0.50
0.374
0.185
0.641
0.580
0.55
0.355
0.173
0.620
0.558
0.60
0.339
0.162
0.600
0,537
0.65
0.324
0.152
0.582
0.518
0.70
0.311
0.146
0.568
0.499
0.75
0.297
0.80
0.286
0.85
0.275
0.90
0.264
0.95
0.255
1.00
0.247
1.05
0.240
1.10
0.235
^Data from Ts'o, Melvin and Olson, /. Am. Chem. Soc 85, 1289 (1963) and Ts'o and Chan,
ibid. 86, 4176 (1964); reproduced with permission of the American Chemical Society.
^ See Table I.
TABLE III
Summary of the Analysesof the Osmotic Data Based on Treatments of Multiple Equilibria*
K
AF°
n
(Molal"
b
(•
-RT In K,
cal.)
(K^ = 0)
Purine
2.1
-440
5 > n > «»«>
6-Methylpurine
6.7
-1120
5 > n > oo
Uridine
0.61
+290
Cytidine
0.87
+80
...
5-Broniour idine
Ki = 1-
0
0
...
K = 2.
9
-630
n = 4
Urea
0.041
+1190
^See Ts'o and Chan, /. Am. Chem. Soc. 86, 4176 (1964); reproduced with permission of
the American Chemical Society.
185
molecules in aqueous solution? Do they associate
with each other vertically through hydrophobic
and stacking interactions, or do they associate
horizontally through hydrogen bonding? These
thermodynamic data do not support the hypo-
thesis of horizontal association through hydrogen
bonding for the following reasons:
1. Methylation and bromination enhance
association.
2. All these bases and nucleosides as-
sociate much more extensively than
urea which is one of the best hydrogen
bonding agents in water.
More direct information about the mode of
association of the bases and nucleosides in
solution can be obtained by the study of nuclear
magnetic resonance. It is well known that
nuclear magnetic shielding is a very sensitive
probe of inter- and intra- molecular interactions.
In this case, vertical stacking interactions are
easily distinguished from hydrogen bonding
interactions and these interactions manifest
themselves differently in the NMR. It is there-
fore hoped that the concentration dependence
of the NMR spectra in aqueous solutions of
purine and nucleosides will shed some light
on the association mechanism. The NMR spectra
of purine have been studied over the concen-
tration range of .05 to 1 molar (3). Chemical
-I lOi
-100
-90
-80
-70
-60
-50 i
-40
0.1 02 0 3 0 4 0,5 0.6 07 08 0.9 1.0
MOLAL CONCENTRATION
Fig. 1.
Concentration dependence of the proton chemical shifts
for purine in aqueous solution at 25° (corrected for bulk
susceptibility); shifts measured from external chloro-
form reference: — o — , experimental values; — X — ,
calculated values from overall average model; — a — ,
calculated values from statistical partial-overlapping
model. (From Chan, Schweizer, Ts'o and Helmkamp,
/. Am. Chem. Soc. 86, 4182, 1964; reproduced with per-
mission of the American Chemical Society.)
shifts of the three protons in purine vs the
concentration are shown in Fig. 1. A pronounced
concentration effect has been observed. Proton
resonances in purine are all shifted to higher
fields as the solute concentration is increased.
Shifts to high fields with concentration are well
known for aromatic systems and are generally
attributed to the magnetic anisotropy associated
with the ring currents in neighboring mole-
cules. Because of the mobile-electrons, a large
diamagnetic current is induced in the plane of
the ring by an external magnetic field when the
field is perpendicular to the plane of the mole-
cule. This ring current gives rise to a small
secondary magnetic field which reinforces the
primary field at the peripheral protons in the
plane of the ring. In the region directly above
and below the molecular plane, the two fields
are opposed, however. As the concentration of
a solution of aromatic molecules is increased,
the average distance between molecules de-
creases and the protons of a given molecule will
feel the secondary magnetic fields produced by
the ring current of neighboring molecules. Since
it is much more probable to find the molecules
somewhere above or below the molecular plane
of another aromatic molecule due to the dish-
shaped nature of the aromatic molecules, this
magnetic anisotropy of the ring current effect
will lead to a high field shift with concentration
or to a low field shift upon dilution. At higher
temperature, or when the purine is dissolved
in organic solvent such as dimethylsulfoxide
and dimethylformamide, such concentration-
dependent chemical shifts for the purine pro-
tons are greatly reduced. Furthermore, when
the purines are protonated by hydrochloride so
they cannot associate because of carrying a
positive charge, such concentration-dependent
chemical shifts are again practically elimi-
nated. These data clearly suggest that the mode
of association of purine is by the vertical stack-
ing of rings in a partial overlapping fashion.
As described above, the osmotic coefficients
and activity coefficients of purine have been
interpreted in terms of multiple equilibria and
on this basis, populations of various associate
species at varying concentrations were com-
puted. Based on these population distributions
of the associated species, we can calculate the
concentration dependence of the chemical shifts
which is also given in Fig. 1 (3). It can be seen
that the calculated value and the experimental
value are in satisfactory agreement. There-
fore, a numerical correlation between the NMR
data and osmotic data has been successful in
186
the sense that they reinforce and support the
interpretations of each other.
Similar results have been obtained from
the purine nucleosides, especially the adenine
nucleosides series. In the case of 2'-0 methyl-
adenosine, 2'-deoxyadenosine, and 6-methyl-
adenosine, the concentration dependence of the
chemical shifts is even larger than that of the
purine. In all these cases, the H-2 proton of the
6 member ring of the adenine is shifted to the
higher field than the H-8 proton in the 5 member
ring. This indicates that the 6 member ring of
the adenine does participate to a greater extent
in the stacks than the 5 member ring of the
adenine nucleosides. The pentose protons of
H-l' are also shifted considerably to higher
fields when concentration is increased while
the pentose protons of the H-5' are hardly
affected. As one proceeds around the pentose
ring from the C-1' to the C-5', there is a
progressive drop or decrease in the magni-
tude of these concentration-dependent chemical
shifts. This indicates that adenine nucleoside
interaction is preferentially localized at the
purine base of the nucleoside so that the ring
current magnetic aniosotropy is principally felt
by the base protons. From this type of study,
therefore, not only can we obtain the general
picture about the mode of association, we can
even get down to the detailed molecular struc-
ture of the stacks.
Currently, we are also working on the as-
sociation of the nucleotides by vapor pressure
osmometry as well as by nuclear magnetic
resonance. In this case we have relaxed our
restriction on the electrostatic effect of the
phosphate group and have included this effect
as a part of our model system with increasing
complexity. Very interesting observations have
been made. For instance, preferential inter-
actions of the phosphate group with certain base
protons of the nucleotides have been observed
which have never been suspected before. Asso-
ciations of base nucleosides and nucleotides
have also been independently studied by
Jardetzky (4).
The experiments detailed above concern
solutions containing only one kind of solute.
They concern, then, the interactions of the
purine or nucleosides with themselves. What
are the interactions between different com-
pounds, for example, between a purine and a
pyrimidine? The increase in solubility of the
sparingly soluble adenine and thymine caused
by the presence of highly soluble purine and
nucleosides was adapted as the method for
investigation of this type of interaction. As
shown in Table IV the solubilities of adenine
are much enhanced by the presence of purine.
The enhancement is moderate in the presence
of cytidine, uridine or pyrimidine and is prac-
tically nil in the presence of cyclohexanol,
adonitol and urea. Similarly, the solubility of
thymine (Table V) is enhanced by the purine
and to a less extent by uridine and cytidine.
These data were also analyzed by the treatment
of multiple equilibria. The assumption in the
treatment is that the bases interact to the
same extent with the free and the associated
forms of the interactants. Equilibrium constants
TABLE IV
Solubility of Adenine in the Presence of Interacting
Compounds *
Concentration,
Solubility S
Compounds Added
molar
molar
X 103"^
(A) 25.5°
None
8.25
+ 0.30''
Purine
0.19
22.8
1.1»
0.39
37.9
1.6
0.58
52.6
2.9
Cytidine
0.18
15.6
1.6
0.36
22.3
1.5
0.54
28.7
1.6
Uridine
0.18
14.5
0.9
0.36
22.3
1.5
0.54
30.9
1.2
Pyrimidine
0.20
11.1
0.23
0.40
15.8
0.58
0.60
19.6
0.52
Phenol
e.20
12.0
0.50
0.40
19.0
0.74
Cyclohexanol
0.20
9.47
0.15
Adonitol
0.60
9.84
0.39
Urea
0.60
(B) 38°
8.88
0.36
None
13.9
0.88*'
Purine
0.193
29.1
1.33
0.386
46.3
1.63
0.58
60.9
1.70
Uridine
0.09
15.9
1.50
0.18
20.1
1.26
0.27
24.3
1.20
0.36
30.5
1.33
0.45
34.9
1.48
0.54
42.7
l.U
Data from Ts'o, Melvin and Olson, /. Am. Ckem. Soc. 85,
1289 (1963); reproduced with permission of the American
Chemical Society.
'' Standard deviation.
187
TABLE V
Solubility of Thymine in the Presence of Interacting
Compounds at 25,5° "
Concentration,
Solubl
Lity S
X lO'^
Compounds Added
None
molar
molar
27.4 +
0.70''
Purine
0.095
0.19
33.5
40.2
1.20''
0.87
0.39
49.8
1.10
0.58
56.3
1.03
0.77
64.0
1.50
0.97
70.7
2.10
Uridine
0.18
0.36
33.4
39.7
0.70
0.95
0.54
43.7
1.27
Pyrlmidine
0.10
0.20
29.8
32.6
0.40
0.55
0.40
37.4
0.80
0.60
41.2
1.5U
0.80
44.6
1.67
^ Data from Ts'o, Melvin and Olson, /. Am. Chem. Soc 85,
1289 (1963); reproduced with permission of the American
Chemical Society.
^ Standard deviation.
for such interactions between different com-
pounds have been calculated (Tables IV and V)
and the general conclusions can be summarized
as follows:
Interactions between purine and purine are
stronger than the interactions of purine and
pyrimidine, which are in turn stronger than the
interactions of pyrimidine with pyrimidine.
The cross interaction of the pyrimidine
nucleosides such as cytidine, thymidine and
uridine with purine or other purine nucleosides
can also be studied by nuclear magnetic reso-
nance. In contrast to the large concentration-
dependent chemical shifts previously reported
for the proton resonance of the purine or purine
nucleosides, the concentration dependent-chem-
ical shifts of the pyrimidine nucleosides them-
selves are negligible (5). It is because the
pyrimidine nucleosides are non-aromatic in
nature and therefore do not support ring cur-
rents as do the aromatic purine bases. There-
fore, the self-association of pyrimidine
nucleosides cannot be monitored by proton
magnetic resonance via the effect of the ring
current magnetic anisotropy. However, the
proton resonance of the pyrimidine nucleosides
was found to be greatly affected by the purine
due to cross interaction. Table VI summarizes
the gross purine effect upon the protons of the
pyrimidine nucleosides. A more detailed pres-
entation of the data for the thymidine protons
is given in Fig, 2 (5). Marked upfield shifts are
noticed particularly for the base protons and
anomeric protons H-l'. This effect falls off
progressively as the proton distance from the
ring increases. The direction of the purine-
induced shifts plus their variation with distance
for the respective protons from the apparent
site of interaction suggests that the interaction
is that of vertical ring stacking of the pyrimidine
and purine bases.
The analysis of NMR data on solution
properties of the monomers points the way for
further studies of nucleic acid by this technique.
For instance, an extensive NMR study of the
MOLAL CONCENTRATION OF THYMIDINE'
0.1 02 03 0^<»
■460r
0.1 0.2 03 04 0.5 0.6 0.7 08 0.9
MOLAL CONCENTRATION OF PURINE •-
Fig. 2.
Chemical shift dependence of thymidine protons upon
thymidine ( — a ) and upon purine concentration
( — • — ) at 35° in D2O. Shifts measured from external
SDSS. Magnetic field increases from top to bottom along
ordinate. Spectra obtained at 60 Mc. (From Schweizer,
Chan and Ts'o, /. Am. Chem. Soc. 87, 5241, 1965; repro-
duced with permissionof the American Chemical Society.)
188
TABLE VI
Concentration Dependence of Chemical Shifts for Cytidlne, Thymidine, and Uridine
Protons at 35° ^' "^
Concn . ,
m
H-5
H-6
Chemical shift
H-r
s from SDSS, c.p.s .
(H-2',H-3',H-4')
H-5'
H-5' '
0 ,
0.78^
ail
363.5
359.5
+ 4.0
469.8
467.4
+2.4
A. Cytidine
354.3
352.5
+1.8
252.8
251.3
+1.5
230.0
230.0
0.0
233.8
233.5
+0.3
CH3
H-6
H-r H-2',H-2"
H-3' H-4'
H-5'
H-5"
0.35^
111.8
111.8
^'* 0.0
457.5
456.9
+0.6
376.4
373.9
+2.5
B. Thymidine
141.8
139.8
+2.0
268.3
267.3
+ 1.0
241.2
239.2
+2.0
226.4
224.2
+2.2
228.7
228.7
0.0
H-5
H-b
H-1'
H-2'
H-3'
H-4'
H-5'
H-5"
C.
Uridine
^7^
353.0
469.7
353.8
259.7
253.2
246.9
228.0
233.4
350.1
468.2
350.9
257.6
251.9
245.1
226.7
230.9
A'i +2.9
+1.5
+2.9
+2.1
+1.3
+1.8
+1.3
+2.5
— Solvent- D^O. Numbering of the nucleoside atoms shown, _e .^ . , with cytidine and thymidine.
— ConcentraL ions approaching limits of solubility.
S. From Schweizer, Chan and Ts'o, J. Am. Chera. Soc . 87 , 5241 (1965); reproduced with
permission of the American Chemical Society.
cytidine
thymidine
dinucleotides has been made successfully in
our laboratories. Hopefully, this approach can
be applied to small nucleic acids such as
transfer RNA (6). More complete description
and discussion of these studies can be found
in the original papers published from our lab-
oratory. Nevertheless, sufficient data have been
presented here to indicate the tendency for the
bases and nucleosides in water to stack up
vertically with the heterocyclic rings in a
partial overlapping fashion. We are now ready
to go to the next step of complexity.
The simple system of monomer-monomer
interactions can be studied quantitatively by
thermodynamic and spectroscopic methods.
However, the system does not have the specifi-
city exhibited at the level of polymer-polymer
interaction. Consequently, we turn our attention
to the nucleic acid interaction at the polymer-
monomer level. A model system for this kind
of study should have the following characteris-
tics:
1. The polymer should have a minimal
degree of self-interactions.
2. Solubility of both polymer and monomer
should be sufficiently high.
3. The electrostatic forces shouldbe mini-
mal.
4. Its properties are relevant to those of
a well-characterized polymer-polymer
interaction system.
The above criteria are apparently met by
the system: poly uridylic acid and adenosine
(7). The binding of adenosine to poly Uwas first
189
studied by equilibrium dialysis at 5°. When the
fraction of the occupied poly U binding sites is
plotted as a function of free adenosines (Fig. 3),
the resulting adsorption isotherm shows a very
steep transition. No binding was detectable
until a critical threshold concentration of adeno-
sine was reached. This steep curve of adsorp-
tion isotherm is analyzed by the following
equation derived from lattice statistics based
on the nearest neighbor interaction (8).
ee
din Y,
e-
_ exp (-W/2kT)
4
where d is the fraction of sites occupied, W is
the interaction energy of the nearest neighbor
and Y is the function of absolute activity of the
adsorbate (A= eu/kT)and the partitian function
for a molecule of bound adsorbate, q; Aq at
dilute solution is equivalent to KqM, where Kq
is intrinsic association constant for 1 molecule
of adsorbate with a single site and M is the
molar concentration of free adsorbate. There-
fore:
39
ain M
exp (-W/2kT)
9=%
Estimation from the slope of the curve (Fig. 3,
open circle) yields a value of 30-60. In equation
—I 1 — I 1 M
(1
/
/
-
/
o
./
1 — ' t II
1
10
100
Concen
trotion of AR x 10'' M
Fig. 3.
Adenosine bound per UMP of the poly U (1.5 x 10-2 W)
versus adenosine input concentration at 5°C, 0.4 ,W NaCl,
O.OU/ phosphate (HMP) ( — • ). The fraction of poly
U sites occupied versus free adenosine concentration
under the same condition is given by — o — . (From
Huang and Ts'o, J. Mvt. Biol. 16. 523, 1966; reproduced
with permission of Academic Press.)
2 the W is calculated to be -5 to 6 Kcal/mole
which is the stacking energy of adenosine upon
pairing with 2 U of poly U. This is comparable
to the value of -4.8 Kcal/mole or -7.5 Kcal/mole
calculated for the stacking energy of poly dAT
and poly dI:dBC respectively by Crothers and
Zimm (9).
Similar experiments were also performed
using cytidine or inosine as the dialysable
components. No detectable binding was found
even at input nucleoside concentration as high
as 2 X 10"2 M. Therefore, this interaction has
the same specificity as the system of long
chain polymers, i.e., the base pairing scheme
of Watson-Crick. The stoichiometry of this
binding reaction was studied by the solubility
measurements, and it was found at low tem-
perature that the stoichiometry is 2 U to 1 A,
while at 20° the stoichiometry becomes 1 A to
1 U. The physical properties of this poly U-
adenosine complex were further analyzed by
sedimentation, viscosity, and by optical rotation
measurement.
The formation of poly U- adenosine (AR)
complex can be demonstrated by analytical
ultracentrifugation. Sedimentation coefficients
(S) of poly U in the absence (control) and pres-
ence of nucleosides are given in Table VII and
the patterns are shown in Fig. 4. When N-6-
methyladenosine, cytidine or inosine was mixed
with poly U in equal amounts (1.5 x 10"^ M each)
at 5° and 0.4 M NaCl, no change in either the
pattern or the S value was found. As adenosine
was mixed with poly U under identical condi-
tions, a 33% increase in S value and a sharpen-
ing of the boundary was observed as compared
with the control (Fig. 4). Similar results were
obtained in 0.02 M MgCl2 with the same mixture.
In 0.4 M NaCl, as the temperature was raised,
the percentage change in S value also increased
to 43% at 10° C and 53% at 19° C, but it was
accompanied by decrease in sharpness of the
boundary (Fig. 4). The specific viscosities of
poly U (1.5 X 10-2M) and poly U-AR complex
(1.5 X 10-2 Af of each) in 0.4 M NaCl at 5° were
respectively 0.602 and 1.05. As previously
stated a parallel increase in S value (33%)
has also been observed. The concurrent in-
crease in both specific viscosity and the sedi-
mentation coefficient of the poly U-AR complex
as compared with those of poly U, unambigu-
ously showed that there is a molecular weight
increase in the polymer resulting from the
complex formation.
Optical rotation measurement at 350 mn
was used to determine the conformation and
190
TABLE VII
Sedimentation of Poly U in Nucleoside Solutions *• ''
Nucleosides
Buffer
Temp .
^20
control
^20
complex
%
increase
Adenosine
0.4 M NaCl
5°C
4.68
6.21
33
Adenosine
0.4 M NaCl
10°C
4.03
5.78
43
Adenosine
0.4 M NaCl
19°C
4.00
6.11
53
Adenosine
0.02 M MgClj
5°C
4.70
6.45
37
L-adenosine
0.4 M NaCl
5°C
4.77
6.69
40
N-6-methyl-
adenosine
0.4 M NaCl
5°C
4.77
4.77
0
Cytidine
9.4 M NaCl
5°C
4.06
4.06
0
Inosine
0.4 M NaCl
5°C
4.68
4.68
0
^ Poly U cone, = 1.5 x 10-^ M
Nucleoside cone. = 1.5 x lO-^W
'' From Huang and Ts'o, /. Mol. Biol. 16, 523 (1966); reproduced with permission of the
Academic Press.
CYTIDINE
ADENOSIKE
(O.OZM MgCl^)
L-ADE-\OSlNE
.ADENOSINE
INOSINE
N-6-METHYL-. ADENOSINE
Fig. 4.
Ultracentrifuge patterns of poly U (upper pattern in each
photograph) and poly U-nucleoside mixture (lower), each
in concentration of 1.5 x 10-^ M , at 5°C and 0.4 M NaCl,
HMP unless indicated. (From Huang and Ts'o, J . Mol.
Biol. 16, 523, 1966; reproduced with permission of Aca-
demic Press.)
191
stability of the poly U-AR complex. Poly U ir
0.4 M NaCl gave a small positive rotation at
low temperature (at 1.5 x 10-^ M, the observed
rotation was about 0.2 degree at 5°C). The
rotation decreased with increasing tempera-
ture, finally became temperature insensitive
beyond 12° C as shown in the control curve in
Fig. 5. On the otherhand, l.SxlO-^M adenosine
alone gave an observed rotation of -0.09° cal-
culated from the rotation at 1.2 x lO-^M which
was temperature independent. Nevertheless,
where the two were mixed, a large increase
in positive rotation was observed, +1.03° at
5°C. At the temperature insensitive region,
the rotation of the mixture was the algebraic
sum of its constituents. We took these to mean
that the poly U-AR complex formed an ordered
structure in 0.4 M NaCl and its stability was
reflected by its melting behavior in response
to the temperature variation. In 0.4 M salt, the
optical rotation measurements remained essen-
tially invarient with a temperature range from
0.5° C to 20° C. When poly U is mixed with
cytidine, inosine or methylated adenosines no
complex formation was observed (Fig. 5).
Formations of poly U-AR complex and its
thermostability were highly dependent on adeno-
sine concentration as illustrated in Fig. 6, When
a constant amount of poly U (1.5 x lO-^M) was
allowed to interact with varying amounts of
adenosine ranging from 3 x 10"^M to2x lO'^M,
a saturation phenomenon similar to that ob-
served in the equilibrium dialysis was also
found, i.e., the magnitude of the maximum
rotation and apparent stability remained un-
changed after the ratio of input adenosine per
UMP of poly U (denoted by A/U) reached unity.
Various analogs of adenosine were also
tested for their binding capacity to poly U by
the optical rotation and sedimentation methods
with the expectation of obtaining information
about an involvement of binding sites, and the
role of the sugar moiety. The following com-
pounds were tested: deoxyadenosine,
L-adenosine (the pentose was L-ribose instead
of D-ribose), (9 r-hydroxypropyl) adenine and
(9-hydroxypentyl) adenine, (long-chain alcohols
in replacing the sugar moiety). Complexing
with poly U was found for all these four com-
pounds. When the point of attachment of the
purine ring was changed from the 9 position
to the 3 position as in the case of 3-isoadenosine,
complex formation could still take place. All
these observations indicate that the sugar moiety
of the adenosine does not play an important
role for the binding. Optical rotation studies of
the mixture of poly U with N-6-methyladenosine,
with 1-methyladenosine and with tubercidin
',A pyrrolo 2, 3-d pyrimidine riboside) revealed
that no interaction took place. Therefore, the
N-6-amino group of the adenineand with tuber-
cidin (A pyrrolo 2, 3-d pyrimidine riboside)
revealed that no interaction took place. There-
fore, the N-6-amino group of the adenine ap-
pears to be definitely involved in binding with
poly U. Other possible bonding sites are the
N-1 and N-7 position of the adenine.
The two important aspects of the parti-
cipation of adenosine in the interaction are its
concentration dependence and specificity. The
complex formation is undetectable in low nu-
cleoside concentration. After a threshold con-
centration of adenosine is reached, the binding
-02
20
30
Fig. 5.
Observed rotation of poly U (1.5 x iO M ) - nucleoside
mixtures versus temperature in 0.4 1/ NaCl, HMP. Con-
centrations of the nucleosides are: adenosine (AR),
1.5 X 10-^1/ C — • — ) and 7.3 x W^ M < — • --); L-
adenosine (L-AR), 9.3 x 10"^ W ; deoxyadenosine (dAR)
7.8 X 10-3 ]i ; cytidine (CR), 1.1 x lO-^U; N-6-methyl-
adenosine (M^-AR), 7 x 10-3 1/ ; inosine (HxR), 1.5 x
10-2 i; . (From Huang and Ts'o, /. Mol. Biol. 16.523, 1966;
reproduced with permission of Academic Press.)
192
increases rapidly in a cooperative manner
until saturation. The key to the understanding
of the interaction resides in the properties of
nucleosides in solution of moderate concentra-
tion as detailed in the section of the monomer-
monomer interaction. From these studies, we
know that the stacking of adenosine occurs when
the concentration increases. These stacks be-
have like the oligonucleotides, and therefore have
much greater affinity to poly U than the free
adenosine. At moderate concentrations, these
associated stacks may serve as initiators for
the subsequent binding of the adenosine mole-
cule to poly U by a cooperative mechanism.
In fact, the stability of most completely inter-
acting complexes measured in our experiments
is comparable to that obtainable for the poly U
-trimer or tetramer (oligonucleotides) inter-
action. The forces responsible for stacking
energy are short ranged. Calculation based on
consideration of the nearest neighbor only gave
an estimation of approximately 5 or 6 Kcal/mole
as the free energy of stacking for this poly
U-AR system. The results clearly indicated that
hydrogen bonding cannot be the sole force re-
sponsible for the binding, since in dilute solution
no binding is detected, even though hydrogen
bonding capacity is still present. On the other
hand, hydrophobic stacking forces alone do not
allow the interaction to occur. Inosine, methy-
lated adenosines and other adenine analogs
probably all form stacks, yet they fail to bind
to poly U. It appears, therefore, the hydrogen
bonding and the hydrophobic stacking forces
are both essential, with the former related to
specificity and the latter related to stability.
Recently we have extended our investiga-
tions to the system of poly U-AMP Interaction
as well as to poly U-ATP and poly C-GTP
interaction (10). In all these cases, the polymer
and the monomer form an insoluljle and stoi-
chiometric complex in the presence of mag-
nesium. We hope that the knowledge gained in
this research will not only tell us about the
physical chemical forces responsible for the
structure of nucleic acids, but that it may
also give us some idea about the mechanism
of replication of nucleic acids in the polymerase
system.
In conclusion, we have applied thermo-
dynamic and spectroscopic methods to study
the properties of monomers in solution. This
research gives us knowledge about the exten-
sive stacking interaction of the bases of nucleic
acid in aqueous solution. Subsequently, we have
applied this knowledge to the study of polymer-
monomer interactions. Through this study, we
have obtained certain important parameters
and basic understanding about the forces re-
sponsible for the secondary structure of nucleic
acids. Hopefully, the knowledge about these
forces may also lead us to understand the
mechanism by which nucleic acid replicates
itself. Now, it appears that our chemical ap-
proach has reached the stage which is very
close to being interesting to the biochemists,
and perhaps even of interest to the developmental
biologists.
So far, attention has been focused on the
interactions of nucleic acids with themselves.
Our laboratory is also starting to investigate
the interactions between nucleic acids and pro-
teins. Undoubtedly, research on this interaction
will be of great importance in molecular biol-
ogy and developmental biology. Interactions of
purine with amino acids have already been
o
X
^ — .!^yr:.» — ■
10
20
30
T°C
Fig. 6.
The melting of poly U-AR complex in 0.4 M NaCl, HMP
measured by rotation at 350 mu. The poly U concentra-
tion is constant at 1.5 x 10"^ M . The parentheses indi-
cate the input AR per UMP of poly U(A/U). (From Huang
and Ts'o, /. Wo/. Biol. 16, 523, 1966; reproduced with
permission of Academic Press).
193
published from our laboratory (11), Results
indicated that among all the amino acids, inter-
action of purine with tyrosine is the most
important one. In the future, this approach
may again guide us to the understanding of the
interactions between two different types of
biopolymers, namely, nucleic acids and pro-
teins.
References
1. P. O. P. Ts'o, I. S. Melvin and J. Olson.
J. Am. Chem. Soc. 85, 1289 (1963).
2. P. O. P. Ts'o and S. I. Chan. J. Am. Chem.
Soc. 86, 4176 (1964).
3. S. I. Chan, M. P. Schweizer, P. O. P. Ts'o
and G. K. Helmkamp. J. Am. Chem. Soc.
86, 4182 (1964).
4. O. Jardetzky. Biopolymers Symp., No. 1,
501 (1964).
5. M. P. Schweizer, S. I. Chan and P. O. P.
Ts'o. J. Am. Chem. Soc. 87, 5241 (1965).
6. C. C. McDonald, W. D. Philips and J.
Penswick. Biopolymers 3, 595 (1965).
7. W. M. Huang and P. O. P. Ts'o. J. Mol.
Biol., 16, 523 (1966).
8. T. L. Hill. In "Statistical Thermodynamics"
(Addison-Wesley Co., Reading, Massachu-
setts, 1960), chap. 14.
9. D. M. Crothers and B. H. Zimm. J. Mol.
Biol. 9, 1 (1964).
10. W. M. Huang and P. O. P. Ts'o. Biophys.
Soc. Abstr., Boston, 1966, p. 16.
11. E. O. Akinrimisi and P. O. P. Ts'o.
Biochemistry 3, 619 (1964).
194
THE PROBLEMS AND PROMISES OF RESEARCH
ON THE MOLECULAR ASPECTS OF DEVELOPMENT
(Workshop Summary)
Paul 0. P. Ts'o
Department of Radiological Sciences, The Johns Hopkins University,
Baltimore, Maryland
We have spent three interesting and in-
structive days together. I have been much
benefited not only by the talks presented in the
formal sessions but also by the fruitful discus-
sions with many of the participants in this
■workshop. We share a common feeling that at
this moment we should review and reflect upon
the problems and the progress in the field of
developmental biology. Therefore, I shall pre-
sent to you certain general consensuses and
conclusions which we have reached as a result
of our discussions. These conclusions are
important to all of us for two reasons. The
first is that experimental systems in the field
of developmental biology are highly individual-
istic and specialized. Few investigators in the
field can pick up helpful and specific experi-
mental techniques for their own research by
examining the work and the experience of
others. In most cases each system has its own
characteristics not shown by others. There are,
to be sure, exceptions such as that demon-
strated by the LDH-isozyme story lucidly pre-
sented by Dr. Edward Massaro. Experience
and working knowledge learned from the iso-
zymes' story can certainly be profitably ex-
tended to many other biological systems. The
most important thing we can learn as a group
is' the underlying philosophy and strategy com-
mon to all of our research. The second reason
is that we are continuously confronted by
problems of communication, even as workers
in the same field. There are biologists, chem-
ists, physicists, biophysicists, etc., in this
workshop. This is a very healthy sign reflecting
the vigor and the promise of this scientific
frontier even though it does bring its own prob-
lems. A review of the present status will help
us with this aspect.
1 will limit my comments mainly to the
unicellular organisms and to a unicellular
model. Dr. James Gregg and Dr. Arnold Kahn
have presented to us some very interesting and
relatively simple systems for the study of
multicellular development. In these cases the
importance of intercellular reactions must
certainly be considered. In higher organisms,
hormonal control plays a major role in devel-
opment. Nevertheless, concepts developed from
the unicellular model will provide the basis for
further discussion of more complicated systems.
Differentiation of a cell clearly implies
that the cell is suddenly doing something new,
a diversion from what it was doing before.
The degree of change, of course, depends upon
the experimental system and the monitoring
device. At present, however, it remains largely
a question of semantics and will until we know
more about the ground rules or the basic
mechanisms common to all experimental sys-
tems. We will, in my opinion, soon reach some
agreement that unless the change is sufficiently
qualitative and distinctive we will not honor it
with the name of "differentiation". Assuming
agreement as to what is differentiation, now
we are ready to pose the question, "What
determines that the cell will change from its
present course to a new one?". In this workshop
we have loosely described this act as "the
decision-making process or processes". I shall
try to define and to clarify the concept of "the
decision-making process" so that we can dis-
cuss it without undue confusion.
I would like first to define two decision-
making bodies in the cell. Their existence is
known from cell biology and biochemistry. The
195
first is the genetic-nuclear body shown in
Scheme I. Within this decision-making body,
namely the nucleus, there are many interlock-
ing, interlinking, rate-dependent and rate
limiting-processes. These processes are con-
nected together in clockwise fashion as pictured
in the scheme. The second "decision-making
body" is the cytoplasm. In this "decision-making
body", as well, there are many interlinking,
rate-limiting processes which are shown con-
nected in counter clockwise fashion in the
scheme to indicate that they may be different
from those in the nucleus. We now must set up
a communication system between these two
decision making bodies (Scheme I). Let us
imagine a sensory system for the nuclear body
which receives the cytoplasmic signals. We will
similarly imagine a sensory system for the
cytoplasmic body to receive the signals from
the nucleus. These sensory systems can have
varying degrees of sensitivities to various
signals at a given time so as to allow certain
information to be transmitted with great effi-
ciency while other information is not trans-
mitted at all. Thus, the communication system
between these two decision-making bodies is
controlled by the selectivity in transmission
as well as by the regulation in generation of
these signals. We do not have much information
about the biochemical nature of these signals
or about the sensory systems. This is certainly
one of the most important problems of cell
biology as related to developmental biology, i.e.,
"What is the biochemical nature of the com-
munication network between the nucleus and the
cytoplasm?". Recent research in molecular
biology has indicated that one kind of signal
which goes from nucleus to cytoplasm is the
Schemel
^0'^ Systej,
^^.c^ear. _J^
Decision - making Bodies Inside a Cell
messenger RNA. The production or the trans-
mission of this signal can be blocked by inhibi-
tors such as actinomycin-D. The use of this
inhibitor has provided us with much needed
information about communication via m-RNAas
evidenced in many of the talks given here. As
to the biochemical nature of the cytoplasmic
signals to the nucleus, the following experi-
mental systems may be useful: hormonal con-
trol of protein synthesis in higher organisms;
inductive enzymes formation in bacteria; and
perhaps antibody formation in response to
antigens. Research in these areas is of the
utmost importance.
Because of problems of presentation,
Scheme I is drawn in an awkward manner as a
reminder that the cell has spherical rather
than bilateral symmetry. This has an impor-
tant consequence. An external stimulus can
not enter the nuclear region without passing
through the cytoplasm. Nucleus and cytoplasm
undoubtedly differ in sensitivity to external
stimuli. Some stimuli may be more harmful
to the nucleus than to the cytoplasm even though
the stimulus has first passed through the
cytoplasm. Nevertheless, we should remember
that external effects on the cell always pass
through the cytoplasm and therefore are sub-
ject to possible control from the cytoplasm.
The elegant experiments of Dr. James
Gregg presented in the workshop and the dis-
cussion thereafter, enables us to assume for
the moment that in order for this kind of deci-
sion making process (Scheme I) to take place
no cell division is necessary. Thus, the barrier
and the sensory systems between the nuclear
and the cytoplasmic bodies need not be torn
down in order for the decision to be made.
This, of course, may not be true in all cases
but the assumption will at least simplify dis-
cussion of our scheme. We have a cell which
is going along in a dynamic state. Suddenly, it
receives a new challenge or it reaches a cer-
tain state. It then makes a decision to embark
on a course different from its original one.
Where is the location of the decision making
process and what is its pathway?
I shall try to describe three different
pathways by which the decision making process
might take place. The first I shall call the
genetic-nuclear-determinant path. In this case,
there is only one dominating influence in the
cell. The decision is made in the nucleus in
accordance with preinscribed genetic program
and all the cytoplasm can do is to listen to the
command of the nucleus. The best biological
196
example of this case is the nuclear trans-
plantation experiment donebyDr. Hammerling's
group between Acetabularia mediterraneae and
Acetabularia chronata. These experiments dem-
onstrate very clearly that the morphology of
the algae is governed by the nucleus. Here we
have a direct demonstration of the dominant
role played by the nuclear body in differentia-
tion.
The next pathway, another extreme case,
will be called the cytoplasmic-determinant case.
In this situation the cytoplasm is making all the
decisions that are necessary and requires no
help from the nucleus. Experimental demon-
stration of this situation is to destroy the
nucleus or to block its pathways with an inhibi-
tor such as actinomycin-D. Of this situation
we have two subclasses. The first we shall
call the latent message case. Certain messages
are stored in the cytoplasm which are nuclear
in origin. These messages are latent in the
cytoplasm and will be called upon later when
needed. It appears that this is the case in the
development of sea urchin eggs as described
by Dr. Paul Gross. Perhaps to some extent a
similar mechanism is operating in the system
described by Dr. James Lovett. A detailed
discussion with Dr. Gross revealed that the
result really depends on the monitoring system.
In a superficial examination of the morphologi-
cal appearances or general biochemical data,
the influence of the nucleus may not be detect-
able, but by detailed biochemical analysis, as
Dr. Gross has explained to me, in the case of
protein synthesis the nucleus can be shown to
exert a considerable control over the cytoplasm.
The information originates in the nucleus, but
the cytoplasm does have the power of control
of the expression of this information until the
right time.
The next subclass is the absolute cyto-
plasmic determinant case. The experimental
demonstration of this type is hard to describe
because it is foreign to our thinking on cellular
biology. 1 cannot present any biological example
of it but 1 can describe what the experimental
requirement is in order to demonstrate its
existence. We have a cell which can divide into
two cells or more. Each of these new cells can
make the decision to differentiate into various
cell types as A, B or C shown in Scheme II.
After blocking of the influence of these cells
by direct destruction or by inhibitor, we see
whether the cells can still differentiate into
cell type A, B or C or not. The exciting results
concerned with the stem cells described by
Dr. James Till may provide an experimental
system for testing of this case. It should be
noted that this situation (Scheme II) is very
different from that of the reticulocyte system.
When cells reach the reticulocyte stage, their
fate has been predetermined. Demonstration of
Scheme II has to be done with cells which still
maintain their capacity to choose among vari-
ous paths.
It is not easy to separate these two sub-
classes, i.e., the latent message case from
the absolute cytoplasmic-determinant case. Sui-
cidal experiments with radioactivity decay of
p32 or H3 may be helpful. With proper experi-
mental design, the latent message (if it is RNA)
can be destroyed specifically while the rest of
the cellular machinery is kept intact.
The last type of pathway to be described is
likely to be the most common one, the perturb-
ation-response system. In this case a perturba-
tion, a challenge, arises (most probably) in the
cytoplasm. It receives, for instance, a hormonal
stimulus, it runs out substrates, or it is acti-
vated by an overdose of CO2, or light, etc. In
response, the cytoplasm transmits a signal to
the nucleus as another perturbation. The nucleus,
in response to this signal, picks up the pre-
inscribed genetic program, and issues a new
command for the cell to follow. Under these
circumstances, the answer to the question about
the exact location of the decision making body
is debatable. Research workers who are pri-
marily interested in the function of the genetic-
nuclear apparatus, would say that the decision
making body is in the nucleus, since it is the
nucleus that issues the new command for the
cell to change its course. Workers who are
mainly interested in cytoplasmic events may
claim that the cytoplasm should be called the
r— db
.<Aa
"-Db
Nuclear attack
or inhibition
Experimental requirement for the demonstration of
cytoplasmic determinant case
197
decision-making body since it is the first to
receive the challenge and is also the first one
to send out the request for change. We shall
avoid these opinionated arguments by simply
calling this category of decision-making proc-
esses the nuclear-cytoplasmic interdependent
process.
This analysis brings out certain serious
complications. These workers who have mainly
been interested in the interlocking cycle located
in the cytoplasm have no easy way of studying
the biochemical nature of the cytoplasmic sig-
nals and the mechanism by which they are sent
to the nucleus. This is simply because it requires
a genetic-nuclear apparatus in order to detect
these signals. If such investigators are not
sxofficiently careful they also may not be able
to pick up the nuclear signals. In setting up the
experimental condition to study the cytoplasmic
cycle, the machinery of the cytoplasmic re-
sponse to the nuclear signals may not be kept
functional. Therefore, these workers may have
unknowingly narrowed their point of view to
only the cytoplasmic processes and completely
neglected the important relationships and inter-
dependency between the cytoplasm and the
nucleus. Those workers who, on the contrary,
have been mainly interested in the operation of
the genetic -nuclear apparatus, may not be aware
of the nature of the cytoplasmic signals, the
origin of the cytoplasmic perturbation or the
response of the cytoplasm to the nuclear com-
mand. Furthermore, our biochemical under-
standing of the nuclear events (the clockwise
circle in Scheme I) is comparatively rudimen-
tary. Reliable facts and concepts are few in
this area and they are hard to get. For instance,
we need to have a biochemical preparation of
nuclear apparatus which can respond to cyto-
plasmic signals. Perhaps, such a nuclear prep-
aration should synthesize new types of RNA
when given a dose of hormone. Until we are
sure about the nature of the cytoplasmic signals,
it will, however, be very difficult to prepare
such biochemical machinery responsive to these
signals in an in vitro experiment. When the
experimental result is negative, we don't know
whether the machinery is nonfunctional or
whether we have given the wrong signals. We
are, however, encouraged by the effort and the
results of Professor James Bonner's group in
this direction as presented by Dr. Roger
Chalkley.
In summary, the main theme we have dis-
cussed so far is not much different from the
old idea in biology about a nuclear-cytoplasm
relationship. However, we have redefined it in
a context more adaptable to our time. In doing
so, we have focused our attention on this rela-
tionship as the most important cellular factor
to be considered in developmental biology. We
hope this clarification will reduce the problem
of communication and will provide a proper
perspective about our own research as related
to biology as a whole. Hopefully, this may lead
to successful cooperation and fruitful exchange
of ideas. We have an appropriate example in
the workshop. The work of Dr. Edward Cantino
is more related to the cytoplasmic events of
the interesting water mold, Blastocladiella
emersonii. On the other hand, the work of Dr.
Lovett on this same organism is more con-
centrated on the function of the genetic nuclear
apparatus. In putting the story together from
their work, which undoubtedly they will do, we
may be able to get a more complete picture
about the intriguing mechanism of this creature
in making its decision for differentiation and
development.
Now let us look into the future for the next
five years, say up to 1970. I think that the many
basic problems of developmental biology in
terms of biochemical hardware and mechanism
are solvable to us in the next five years with
sufficient manpower and financial resources.
There is no sign of a shortage in either cate-
gory. What I mean is that we do not need a
technological break-through before we can solve
these problems. For instance, we do not need
to wait for the development of an electron
microscopic movie camera, I think, rather,
that the biggest barrier in fact is educational
or communicational in nature. That is to say,
the chemists working with a system will not
know enough about the biology of the system
and the biologists working on other systems
will not know enough about their chemistry.
Even in the field of biochemistry, those who
work on nuclear events may overlook the cru-
cial points related to cytoplasm and those
who work at the cytoplasmic enzyme level may
neglect the pertinent facts derived from the
nucleus. We do need new ideas and ingenious
approaches in these fields. These inspirations
usually come from an organic synthesis of
various disciplines not brought together before.
This is why workshops of this type are so
valuable.
Let's look ahead further into the coming
ten years, say up to 1975. What will be thinking
about? We saw a glimpse of that in this work-
shop. At that time, hopefully, we will know a
198
lot more about the various pathways. We prob-
ably will start studying the universality of these
pathways as utilized by various organisms.
For instance, we will like to know how generally
the latent message mechanism is being used.
It appears now that it is operative both in the
sea urchin and in the water mold. We will start
asking questions not only about how the genetic
program is to be read (by that time we should
really know what the genetic program is), but we
may also ask, "How did the biological world
derive this kind of program?" In other words,
in the next five years we would like to link
differentiation to molecular biology, and in the
following five years we may wish to link
differentiation to taxonomy and to the study
of evolution. The presentation of Dr. Massaro
about LDH systems in all kinds of fish and
organisms is an early start of this type. By
1975 we may even have some answers from
space study about Martian biology. By com-
paring Earthly biology to Martian biology, we
may start to study developmental biology on
different planets.
POLLARD: I'd like to comment in quite
general terms on the message that may go
from the cytoplasm to the nucleus. You can't
have a message go from the cytoplasm to the
nucleus that is physically too big. Almost
certainly you're going to have to have some-
thing reasonably small which will get into the
nucleus. The cytoplasm is unlike the nucleus,
which has very large molecules moving out of
it. However, to get something out of the cytoplasm
into the nucleus you've got to actually drive it.
It's like the old question of parking and unparking
a car. If you park a car, you've got to put it in
a fairly small place and it's fairly hard to do.
If you unpark a car, you've got the whole world
to go into. You can get big things out, but, I
would suspect, you can only put small things in.
So you've got to move from the cytoplasm to
the nucleus such things as large proteins or
ribosomes if a message is to be passed into
the nucleus from the cytoplasm.
TS'O: Yes, in chemical biology we reach
the same conclusion that you geneticists do:
namely, that this would have a given direction
of movement.
GROSS: I think that it's premature to
decide now what informational macromolecules
pass only in one direction.
POLLARD: Oh no, that's not what I meant.
GROSS: There is accumulating evidence
that proteins as proteins can pass through the
membrane into the nucleus.
POLLARD: Yes, they can pass in both
directions. It is mainly the question as to what
concentration might be necessary and what
probability of passage would be. I would say
that if you want something to diffuse rapidly
and have a high probability of getting inside
the nucleus with a message from the cytoplasm,
while most molecules are moving out, there
must be a high concentration gradient in that
direction.
GROSS: It's feasible, however, that pro-
teins that are present in low concentration in
the cytoplasm as the result of some previous
synthesis, under certain environmental stimuli
might, for one reason or another, be carried
into the nucleus independently of the concen-
tration gradient.
POLLARD: Well, I would call that a
"miracle".
GROSS: Well, it's possible that there are
things that move only by diffusion, to be sure.
However, there may also be carriers which
themselves diffuse down a gradient.
TS'O: I would like to know some of Dr.
Wright' s opinions, especially after hearing her
elegant biochemical experiments. Do you think
such a general scheme is suitable for dis-
cussion?
B. WRIGHT: Well, I don't like the term
"decision- making". I think it's so complicated
that there are many "decision-making" proc-
esses interacting and interlocking. One simpli-
fied way you could look at it is in terms of the
things that I've been concerned with which are
going to be essential in all systems. Then you
can have degrees of less and less criticalness.
I mean, in order to get differentiation, as I said
in my talk, you have to have immediate control
at the level of substrates and enzymes. The
whole small molecule milieu of the cell has to
be keyed just right in order to accomplish this.
Then, with respect to the time of the differen-
tiation process, you have the tendency for less
and less dependency in the criticalness of the
control as you filter back toward the ultimate
message.
TS'O: You would, nevertheless, say there
are cases where the genetic control is a very
predominant matter?
B. WRIGHT: At one time or another it is
always of predominant importance. However,
even when you can show, as with the nuclear
experiment you mentioned, a very striking
effect, all of these other levels of control
have to be perfectly in shape in order to see
199
the effect of the genetic control on differentia-
tion.
TS'O: Of course.
B. WRIGHT: I don't see any way to sim-
plify this picture.
LOVETT: Isn't the important thing the
relationship between how much of it is simple
interaction between pre-existing systems and
how much is neiy? A cell turns out a certain
amount of enzyme as it grows at a certain rate
for a certain amount of time. How much is just
interaction between products in that kind of
pathway and another pathway to which it con-
nects? For instance, perhaps some of these
products are going somewhere else and affect-
ing enzymes because of their actual concentra-
tion level and thus preventing a pathway from
functioning. Even though you might measure it
in vitro and get a certain level, in fact, the
enzyme isn't doing it nearly that fast because
it's been shut off by some kind of a feedback
mechanism. This could occur in the cytoplasm
or by a circuitous path.
B. WRIGHT: This is pure speculation be-
cause there are practically no systems in which
we have knowledge enough to do anything except
point to one little effect somewhere.
LOVETT: Well, specifically, yes. However,
there's certainly good enough precedent that
small molecules having nothing to do with the
function of a specific enzyme can cause things
like inhibition. Why does one have to restrict
it to a previous enzyme in the same pathway?
Why couldn't it be used in a coordinating sense
to regulate more than one pathway?
TS'O: Well, actinomycin is actually a good
demonstration of this. Presumably, actino-
mysin-D doesn't affect enzyme activity,
LOVETT: Yes, but I think the best answer
for that is some other experiments I have done
with another organism where I am really worried
about actinomycin effects: that is, it causes a
lot of turnover of RNA when the cells are not
making any protein.
STROTHER: You're talking about a single
cell situation here, but even so it seems to me
that the geometry must be taken into account.
For instance, the nucleus is surrounded by a
cytoplasm, in general, and the cytoplasm is
surrounded by the external environment. Now,
it would seem to me that what you're really
missing in your overall schematic here is the
interaction that you observe in a multicellular
organism in the very close interaction, even
in the single cell, with the environment sur-
rounding it. I think you're missing a very
important part with regard to your signals.
Also, I don't know of any part of a consistent
theory that doesn't involve statistical analysis
of some sort, and I don't see where that appears
here. Are they buried in the noise?
TS'O: Well, I'd like to take that in two
parts. First, the membrane is of the greatest
importance. Dr. Kahn, Dr. Gregg and myself
have certainly talked a lot on that. We' re very
conscious of the membrane, but at the present
I didn't have enough time to bring in all the
elaborations.
The second point is that, as far as the
noise level is concerned, you really couldn't
argue this question unless you knew more about
the details of this system. You'd have to know
more about the hardware and the mechanics
of doing it, before you could start posing
questions of this kind. You need to know, for
instance, when you make a protein, how much
error you made in the process. Questions like
that are beginning to be approached experi-
mentally by Bob Loftfield and others, questions
as to how often you make a wrong transcrip-
tion. However, I don't think it's germane at this
moment to put that into it. The point is not to
make the system as complicated as possible
but to keep it simple with enough essential
parts to help our own decision-making in con-
ducting our research.
GROSS: I'd like to add to both your re-
sponses. First of all, it's clear what we're
really trying to do is decide what we mean
when we say something is differentiated. Em-
bryologists have really not been able to agree
on that. Eventually this might lead to agree-
ment; it might not. Now there are, in fact,
systems such as this one cell which, in the
absence of other cells, will do what we agree
is differentiation. A single sea urchin egg,
isolated from all other eggs, can be fertilized
and, presumably, it will develop. I suspect that
a single spore ot Blastocladiella will germinate.
So, to this extent, it's reasonable as a first
approximation to talk about this rather simply.
The statistical point is a very good one and
it's precisely there that I would locate all the
considerations that motivate Barbara Wright's
remarks because the noise level in this system
represents the degree of deviation of the over-
all result of metabolism from the norm of the
population as a whole. There's no doubt that in
the cells there are momentary but significant
fluctuations. It's the delicate interplay of all
these separate steps in the pathway that pre-
vents those fluctuations from becoming large
200
scale, rniless a signal arrives from some place
that says now the pattern must change. What
we really want to know is, what are those
signals? How do they work?
TS'O: Very well said.
NASA-Langley, 1967 CR-675 201
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