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 For sale by the Clearinghouse for Federal Scientific and Technical Information Springfield, Virginia 22151 - Price $3.75 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 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 - ^.«5.'5i■i:•V • % ^»•'•••*• -•^.'• 1' -.r-v^ •jy-:-: km ■K^ •^ Fig. 14. (Fig. 1, Mangan, Miki-Noumura and Gross, Science 147, 1575, 1965; copyright 1965 by the Association for the Advancement of Science.) K. ^ ^^ '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 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-- ATP ADP AMP 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 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 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 References 1. J. Loeb. "Artificial Parthenogenesis and Fertilization" (University of Chicago Press, Chicago, 1913). 2. T. Hultin. Experientia 17, 410 (1961). 3. A. Monroy, R. Maggio and A. Rinaldi. Proc. Natl. Acad. Sci. U.S. 54, 107 (1965). 4. B, Chance, P. Cohen, F. Jobsis and B. Schoener. Science 137, 499 (19Q2). 5. B. Pressman. Proc. Natl. Acad. Sci. U.S. 53, 1076 (1965). 6. D. Epel. Biochem. Biophys. Res. Co mm, 17, 69 (1964). 7. M. Klingenberg and T. Bucher. Anru. Rev. 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Kemi 6, 387 (1953). 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 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 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 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 (a) CONTROL EMBRYOS 10 (b)Mg. STARVED EMBRYOS 20 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 o T) 6000 4500 ? -^3000 1500 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 re plicotion 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. ■ 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 - '^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 (-) \ / \ / \ \* * - 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 (1960). 27. N. O. Kaplan. BrookhavenSympos. Biol. 17, 131 (1964). 28. R. D. Cahn, N. O. Kaplan, L, Levine and E. Zwilling. Science 136, 962 (1962), 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 ^ 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 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 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 >- 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 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 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 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 14 555 223 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 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). 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 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) ' 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 < 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 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* 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 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 ■•■^ . 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 '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 E XPONENTIAL 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. cr 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. 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 re