ID II I I I [0 3EaQOOOQSQSQQQQQE3E Marine Biological Laboratory Library Woods Hole, Mass. Presented by ID [0 I The Estate of D Dr. Arthur K. Parpart a Jan. 1966 K 2 0] 3E5QL3E3QOQQQQE3QBQE3E ^■^ ru AMERICAN SMENT!ST RECEIVED FOR REVIEW CO" a : rn RECENT DEVELOPMENTS IN CELL PHYSIOLOGY t ^ e RECENT DEVELOPMENTS IN CELL PHYSIOLOGY Edited by J. A. KITCHING Proceedings of the Seventh Symposium of the Colston Research Society held in the University of Bristol March 2gth — April ist, ig^4 NEW YORK ACADEMIC PRESS INC., PUBLISHERS LONDON BUTTERWORTHS SCIENTIFIC PUBLICATIONS J954 BUTTERWORTHS PUBLICATIONS LTD. 88 KINGSWAY, LONDON, W.C.2 U.S.A. Edition published by ACADEMIC PRESS INC., PUBLISHERS 125 EAST 23RD STREET NEW YORK 10, NEW YORK September 1954 This book is Volume VII of the Colston Papers. Permission must be obtained from the Colston Research Society, 12 Small Street, Bristol, England, and from the Publishers, before any part of the contents can be reproduced. Previous volumes in the Colston Papers are: Vol. I. 1948 Cosmic Radiation — Editor, Professor F. C. Frank Vol. II. 1949 Engineering Structures — Editor, Professor A. G. Pugsley. Vol. III. 1950 Colonial Administration — Editor, Professor C. M. Maclnnes. Vol. IV. 1 95 1 The Universities and the Theatre — Editor, Professor D. G. James. Vol. V. 1952 The Suprarenal Cortex — Editor, Professor J. M. Yoffey. Vol. VI. 1953 Insecticides and Colonial Agricultural Development — Editors, Professor T. Wallace and Dr. J. T. Martin. There will be no Colston Symposium in 1955, owing to the visit to Bristol of the British Association. The series will be resumed in 1 956. Made and Printed in Great Britain by J. W. Arrowsmith Ltd., Bristol Foreword It was in 1899 that a group of public-spirited Bristol citizens established the 'University College Colston Society', whose chief aim was to assist the then young and struggling University College. The Society was named after the noted seven- teenth-century philanthropist and educationalist, Edward Colston. The Annual Dinner of the Society soon came to be regarded as a function of considerable importance in the life both of the University and of the community as a whole. It was at the Society's dinner in 1908 that the public announcement was made of the gift of £100,000 by H. O. Wills to the University. The period of expansion which was ushered in by this gift resulted finally in the granting of a Charter, and the attainment by the University College of full university status. At this time too the Society changed its name, became the 'Colston Research Society', and decided to direct all its energies to the promotion of research. For twenty years it collected annually an average sum of over £600 which was devoted to this end. In the decade from 1929 to 1939 the activities and resources of the Society underwent considerable expansion, and it not only continued to make research grants to University depart- ments, but it also financed at considerable cost a social survey of Bristol. However, with the further rapid growth of the University in the post-war period it became clear that the financial contribution of the Society was becoming less and less important in relation to the very large funds which were needed by University departments for their research work. Accordingly the Society decided once again on a radical change of policy and resolved to devote the major part of its funds to the promotion of an annual symposium. The rapid growth in popularity of the symposium as a means for the advancement and stimulation of knowledge is one of the remarkable features of the intellectual life of recent years. For this development there have been a number of interesting and compelling reasons, all of which the Society carefully considered before embark- ing on its new policy. This policy has already achieved a remarkable measure of success; it has been a pioneer effort among the universities of Great Britain, and represents a distinctive contribution on the part of Bristol University to the cultural life of the country as a whole. A list of the subjects of the six previous symposia appears on the opposite page. It will be seen that in arranging these symposia it is intended that they shall be free to cover all fields of learning, provided that they are not too highly specialized, but possess a reasonably wide appeal, and are at a sufficiently interesting stage of development to make it likely that they will benefit by symposium treatment. As President of the Society for the year 1953-54 it was my privilege to preside over the seventh symposium, on 'Recent Developments in Cell Physiology'. H. C. I. Rogers. Preface Cell physiology to-day is the common meeting-ground of the botanist and zoologist, of the biochemist and biophysicist, of the geneticist and embryologist. In spite of the ambitious field which we have attempted to cover in fifteen short papers, no excuse is needed for bringing together students from so wide a range of disciplines to present some of their latest work, to discuss their separate and common interests and to speculate on the future of this fascinating subject. How far this aim has been success- ful is only partly to be judged on the contents of this present volume; the many informal groups collected between the official meetings are not the least valuable feature of any symposium of this kind. For financial and other reasons, the geographical range of the speakers was more restricted than their academic one. The Society's guests from overseas included only- workers from Denmark and Belgium, both countries which have made great contri- butions to cell physiology in recent years. In order to assist the discussion, papers were roughly grouped so that each session dealt with a similar general topic. The opening session was concerned with the exchange of material between the •cell and its environment, with particular reference to the mechanisms of active transport. This topic was continued on the second day by papers on membrane structure and on the ionic permeability of the nerve fibre. The metabolism of the cell and certain special problems of nucleic acid synthesis were represented by three papers, and the study of the nucleus was then broadened to include its role in the metabolism of the cell and morphogenesis of the organism. A particularly interesting session on the external synchronization of cell division was followed by a final meeting in which the control of differentiation and of cell division were considered as well as some new physical properties of the cell surface in Protozoa. The contributors to the symposium are particularly to be congratulated on the broad treatment of their subject-matter, which stimulated discussion and speculation in the friendly and informal atmosphere which was so characteristic of the whole meeting. To this atmosphere our Danish and Belgian guests brought a spontaneity and good fellowship which was only equalled by their amazing facility in the English language. My own position as Director of the symposium has been an unexacting one of privilege without responsibility. Dr. J. A. Kitching has taken the whole burden of editing the manuscripts and the discussion, and thanks to him and to the co-operation of all the participants, this volume has been produced in a surprisingly short time. Our indebtedness to the printers and publishers is equally obvious, and gives me the opportunity of paying tribute to the help of Mr. R. H. Brown, who, in his other capacity as Secretary of the Colston Research Society, has been as invaluable as on vii Preface earlier occasions. The administrative arrangements for the meeting were in the capable hands of Dr. H. P. Whiting, who, with Miss Morgan, Warden of Manor Hall, and her staff, was in no small part responsible for the success of the symposium. Finally, the separation of the foreword from this preface gives me a very welcome opportunity, on behalf of all my colleagues, to pay our tribute to the President,, Mr. H. C. I. Rogers and to the Colston Research Society for sponsoring this sym- posium, and for all they have done, and continue to do, for the University of Bristol. J. E. Harris. Bristol, 1954. vm Contents Foreword H. C. I. Rogers Preface J. E. Harris The present position in the field of facilitated diffusion and selective active transport J. F. Danielli Cholinesterase and active transport of sodium chloride through the isolated gills of the crab Eriocheir sinensis (M.Edw.) H. J. Koch Discussion on papers by Danielli and Koch Membrane structure as revealed by permeability studies H. H. Ussing Discussion The ionic permeability of nerve membranes R. D. Keynes Discussion Cellular oxidations and the syntheses of amino-acids and amides in plants E. W. Vemm Discussion The biosynthesis of pentoses and their incorporation into mononucleotides H. Klenow Discussion Deoxynucleic acid in some gametes and embryos E. Hoff-Jorgensen Discussion Nuclear control of enzymatic activities . . J. Bracket Discussion The cell physiology of early development C. H. Waddington Page v vn ^5 27 33 41 43 49 51 64 67 78 79 88 9i 102 105 IX CONTENTS Page The time-graded regeneration field in planarians and some of its cyto- physiological implications . . . . . . . . . . . . . . 121 H. V. Brmdsted Discussion on papers by Waddington and Brondsted . . . . . . 139 Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis, as induced by temperature changes . . . . . . . . 141 E. ^euthen and 0. Scherbaum Discussion . . . . . . . . . . . . . . . . . . 157 A study of bacterial populations in which nuclear and cellular divisions are induced by means of temperature shifts .. .. .. .. ..159 0. Maalee and K. G. Lark Discussion . . . . . . . . . . . . . . . . . . 169 Environmental and genetic control of differentiation in Neurospora . . . . 171 M. Westergaard and H. Hirsch Discussion . . . . . . . . . . . . . . . . . . 183 The control of cell division .. .. .. .. .. .. ..185 M. M. Swann Discussion . . . . . . . . . . . . . . . . . . 195 On suction in Suctoria . . . . . . . . . . . . . . . . 197 J. A. Kitching Discussion . . . . . . . . . . . . • . . . • . 203 List of Members . . 205 The present position in the field of facilitated diffusion and selective active transport by J. F. DANIELLI Z°°l°g.y Department, Kings College, London DEFINITION AND CHARACTERIZATION OF PROCESSES It is desirable to distinguish, as accurately as is possible at the present time, between several processes: diffusion, facilitated diffusion, and selective active transport. This contribution is concerned with selective active transport, i.e. active transport which is selective for a limited range of molecular species. It is not concerned with unselective active transport; for instance a process whereby environmental fluid is accumulated unchanged in a vacuole on one side of a membrane, and discharged unchanged from the vacuole on the other side of the membrane, would be active transport, but un- selective and therefore not of significance in this discussion. Diffusion is brought about by the driving force of thermal agitation. In a homo- geneous fluid the rate at which a given molecular species diffuses may be calculated, at constant temperature and pressure, if the viscosity of the fluid and the molecular weight of the diffusing species are known*. As a result of a diffusion, the free energy of the system is lowered, and there is usually a decline in gradients of chemical poten- tial! ^ no force other than that of thermal agitation is acting upon the molecules (i.e. if gravitational, electrical and other forces have no significant effect upon the final distribution of molecules). Thus diffusion is selective in terms of molecular weight (or linear dimensions j-), but is unselective in terms of structural and steric factors. Facilitated diffusion also occurs under the driving force of thermal agitation, but differs from diffusion in that the rate at which molecules diffuse is strongly influenced by structural and steric factors. It is a process commonly found in studies of the permeability of plasma membranes, and in the past has usually been included in the category of active transport. But it is better separated as a special type of diffusion, since the equilibria attained by facilitated diffusion are the same as those achieved by diffusion. The difference between the two processes is essentially that, by facilitated diffusion, some molecular species may reach diffusion equilibrium much more rapidly than would be possible by non-facilitated diffusion. * This is true for molecules of a molecular weight of up to about 1,000. When the molecules diffusing are very large compared with the solvent molecules, the rate of diffusion is more accurately calculable from the linear dimensions than from the molecular weight. t This is not always so, for if the diffusion of two species is linked, as in Osterhout's well-known guaiacol model for K+ accumulation, there may be an increase in the chemical potential gradient of one species achieved at the expense of a decline in the gradient of another species. DANIELLI Active transport involves the movement of molecules by forces additional to those of thermal agitation. The result of active transport may (if the free-energy change of the active process is ignored) be an increase in free energy of the system, and an increase in chemical potential gradients. Of these three processes we know least about active transport, and most about diffusion. As we learn more about the nature of active transport, it may prove that a number of distinct types of process are involved which can be defined separately, just as it has recently proved possible to separate facilitated diffusion from active transport. CLASSIFICATION OF TECHNIQUES A variety of techniques may be employed to differentiate between diffusion, facili- tated diffusion and active transport, and to approach the mechanisms underlying individual processes of facilitated diffusion and active transport. These techniques can be roughly divided into six groups. ( i ) Morphological studies. The examination of the structure and ultrastructure of secretory organs and cells is of outstanding importance, and too frequently under- rated. In many cases the limits of analysis using the light-microscope are far from exhausted. The exploitation of electron-microscope studies should be of the greatest value, as is suggested by the recent publication of Sjostrand's (1953) work on the free and other cell borders, nuclear membranes and mitochondrial membranes of kidney proximal tubule cells. It is probable that a full understanding of the details revealed by electron-microscopy must await the development of cytochemical methods for use with electron-microscopy, but even to have available the structural detail, without chemical detail, of the membranes concerned will be highly stimulat- ing. (2) Kinetic studies. The examination of the rates of penetration of substances through membranes, and of the effect of variation in concentration, temperature, ionic strength and other environmental conditions on these rates, are included in this heading. Included also is the effect of variation in molecular structure and stereo- chemistry, and the use of isotopes. Isotopes are particularly useful in the determina- tion of total transfer, as opposed to net transfer. (3) Metabolic studies. Although studies of metabolism are unlikely to yield much useful information about the actual mechanism of transfer, they are often useful in showing that transfer is in some way dependent on metabolism. This permits distinc- tion between diffusion on the one hand, and facilitated diffusion and active transfer on the other. It does not necessarily permit distinction between facilitated diffusion and active transfer, since in the former case, although no energy contribution is required from the cell for transfer, energy may be required to maintain the membrane in an active state permitting facilitated diffusion. The effect of metabolism is usually best studied by depriving the cells of metabolites (e.g. glucose) or by use of poisons (e.g. cyanide, dinitrophenol). (4) Cytochemical studies. These are at present sharply limited by the lack of a suffici- ent variety of reliable cytochemical techniques. A few observations of major import- ance have been made, including: (a) the high concentration of alkaline phosphatase at the secretory surfaces of many secretory cells (Danielli, 1953); (b) the high The present position in the field of facilitated diffusion and selective active transport concentration of cholinesterase associated with the membrane of motor end plates (Holt, 1954); the high concentration of periodate-oxidizable carbohydrate at the surface of secretory cells (Ruyter, 1953; Bell, unpublished). (5) Direct activators and inhibitors. It is thought probable, on somewhat slender grounds, that a number of substances act directly on transfer mechanisms, e.g. phosphate esters and acetylcholine (Danielli, 1953), insulin and anterior pituitary hormone (Cori, 1945), phloridzin (Rosenberg and Wilbrandt, 1952), dinitrofluoro- benzene (Bowyer, 1954) and possibly some of the oestrogens (Bullough, 1953). It is to be hoped that more substantial evidence bearing on these interactions will soon become available. (6) Potential studies. Where movement of ions is involved, the selective movement of any one species will result in formation of an electrical potential difference. Such potential differences must be compatible with, and quantitatively explained by, the movements of the individual ions. Consequently, potential measurements have a valuable place in transfer studies although, as Gasser (1933) stated, 'you cannot determine a process from a potential'. EXAMPLES ILLUSTRATING THE PRESENT PROBLEM There have been several recent reviews,, e.g. Rosenberg and Wilbrandt (1952), Goldacre (1952), Danielli (1953), Stadie (1954) and the recent S.E.B. Symposium (Volume VIII, 1954). In this symposium Dr. Koch and Dr. Keynes will be con- cerned with movements of ions: I shall limit myself to non-electrolytes. (A) The penetration of sugars into muscle When a substance is injected into an animal it rapidly becomes distributed through the blood and extracellular spaces, but the extent to which it penetrates into intra- cellular water is determined by ability to pass through cell plasma membranes. Table I summarizes some of the main results obtained by Levine and his colleagues (1950, 1953a, b). These results were obtained on animals which had been eviscerated and nephrectomized, so that side effects due to metabolism and excretion might be minimized. The data contained in the table effectively outline the problem as it presents itself in mammals. Urea, which readily enters most mammalian cells, is distributed in a volume of water equivalent to 70 per cent, of the body weight (i.e. practically all the body water), whereas the non-penetrating substance sucrose is distributed in an equivalent of 45 per cent, weight. Insulin has no effect on the distribution of either sucrose or urea, (/-glucose is initially distributed in 45 per cent., and there is an increase on adding insulin. But results with glucose are complicated by metabolism, and the insulin effect is better seen with (/-galactose, /-arabinose and ^/-xylose, with which a distribution-weight of 45 per cent, is raised to 70 per cent, by insulin, /-rhamnose and (/-arabinose are not metabolized, and show no insulin effect, and (/-fructose, (/-mannose and /-sorbose, though metabolized, show very much less effect of insulin than does glucose. Thus insulin enables some substances to penetrate readily into a volume of body water into which they move with great difficulty, in the absence of insulin. The insulin action is structurally and sterically specific — e.g. is positive for /-arabinose and negligible for (/-arabinose. Also it is not F. DANIELLI Table I The distribution of injected substances in the water of eviscerated nephrectomized animals. The figures are for the equilibrium volume of water in which the substances appear to be distributed, expressed as percentages of body weight. It is assumed that at equilibrium the concentrations of substances in all water into which they penetrate are the same as the concentrations in the blood. Substance Whether metabolized Percentage of body weight occupied no insulin with insulin urea sucrose — 70 45 70 45 ci-glucose ^-galactose /-arabinose rf-xylose + 45* 45 45 45 >45 70 70 70 /-rhamnose ^/-arabinose — 45 45 45 45 55 '3 moles will diffuse out, resulting in a net uptake of o-2 moles. The experimental results were not in agreement with these assumptions, however. The net uptake of water was between three and five times higher than the theoretical value calculated from the heavy-water flux and the difference in osmotic pressure across the skin. The authors concluded that until more became known, diffusion of heavy water could not be used to calculate rates of osmotic uptake. In 1944 Visscher et al. made a study of the water movements between gut and blood of the dog, determining both the net water transfer and the rate of DaO diffusion. Their theoretical assumptions were essentially the same as those of Hevesy et al. (I.e.), except that Visscher and collaborators assumed the rate of diffusion of water to be 33 HANS H. USSING proportional to the activity rather than to the concentration of the water. Even in this case the net transfers of water were much larger than predicted from the water activities, whether the gut contents were hypotonic or hypertonic with respect to the blood. Visscher took this as evidence that the water movements across the intestinal wall are due largely to active processes rather than to simple diffusion. A few years ago in the Zoophysiological Laboratory of Copenhagen we resumed the study of water movements across the amphibian skin. The impetus to this study was a wish to clarify the mechanism underlying the so-called Brunn reaction or water balance reaction of anuran amphibians, which has been extensively studied in recent years (for references compare Heller, 1945, and Jorgensen, 1950). The re- action consists in an increased uptake of water through the skin following the in- jection into the animal of small doses of posterior lobe hormones. The response can also be elicited in the isolated skin of toads (Novelli, 1936) and frogs (Fuhrman and Ussing, 1 95 1, Sawyer, 1951). Since the Brunn reaction is more pronounced in toads than in frogs, skins of the former animal were used. An apparatus was designed which allowed the determina- tion of the net water-transfer rates with an accuracy of ± 10 jul. and, simultaneously, the measurement of the water-diffusion rate, using 5 per cent, heavy water as a tracer. As inside medium ordinary Ringer solution was used, whereas the outside medium was 1/10 Ringer. Some typical results are shown in Table I (Koefoed-Johnsen and Ussing, 1952). The heavy-water diffusion figures are calculated as total influx values {Min) ex- pressed as the amount that would pass through unit area in unit time if the heavy- water concentration were maintained at 100 per cent, in the outside compartment and at zero in the inside compartment. The net water flux, Aw, as well as the influx, is given in /xl./cm.2/hr. The results confirm in every respect those of Hevesy, Hofer and Krogh (I.e.) on live frogs. For the sake of argument, let us assume that the water uptake is due to simple osmosis and that the net uptake is the difference between two diffusion streams. The permeability coefficient as calculated from heavy-water diffusion, namely Pdiff, is defined by the equation Mfa = "diff cw(o) For Mm = 532 /zl./hr. Pdm works out to be 1 48 x io~4 cm./sec. In the same experi- ment Aw was 30 /u,l./hr. Now, for Aw we have Aw = M-m — Mont = PosmCw(o) POSmCw(i) = ° osm(Cw(o) — Cw(i)) Remembering that Aw and (cw(o) — cw{i)) should be expressed in the same units, we get ^osm =2 32 x 10-3 or nearly 1 6 times the figure for Pdiff. It is seen that the influx changes only slightly on the addition to the inside solution of posterior lobe extract. The flux may even go down. But the net flux always in- creases violently, often by more than 100 per cent. In the beginning we took this finding as an indication that the hormone evokes an active transport of water, a view 34 Membrane structure as revealed by permeability studies which was also taken by Capraro et al. (1952) who obtained similar results with isolated frog skins. There was, however, something mysterious about this apparant active transport. It could take place only if there was an osmotic gradient to help it. With isotonic sucrose in the outside compartment and Ringer solution inside, the net water transfer was nil. This is in keeping with the observation made by Krogh years ago that frogs placed in isotonic sucrose will not take up water and do not form Table I Effect of neurohypophysial hormone on influx and net flux of water through toad skin. Inside solution, Ringer solution; outside solution, 1/10 Ringer (Koefoed-Johnsen and Ussing, 1952) Min = influx of water (jul./cm.2/hr.) Aw = net flux of water (/zl./cm.2/hr.) Date Control periods 1 hr. and 2 hr. periods after addition of neurohypo- physial powder a b c d Min Aw M- in K Mm K Mm K 24/1 441 120 460 130 532 30 0 55i 36 0 28/1 305 67 3*9 50 292 10 8 310 77 30/1 Aw 343 97 370 7'4 334 16 0 404 170 3i/i 326 n-7 287 80 344 21 0 369 25 0 any urine. It is true that the isolated skin with Ringer solution on both sides performs a transfer of water from the outside medium to that inside (Huf, 1936), but this is probably connected with the active transport of sodium ions through the skin. The fact that the apparently active water transport needs an osmotic gradient to help it started us wondering whether or not our basic concepts of the nature of os- motic water transfer were correct. Does the net water transfer indeed arise as the difference between the amount of water diffusing in and that diffusing out ? At first sight even the asking of the question seemed to us preposterous, but on second thoughts we realized that the problem needed reconsideration. 35 HANS H. USSING In order to make the point clear, let us consider a model system which, in an exag- gerated form, illustrates the problem. The system consists of two compartments, / and 0, which are separated by a 'membrane'. The 'membrane' is largely imper- meable, communication of solvent between / and 0 being possible only through a number of pores which have the shape of small osmometers with the semi-permeable membrane facing towards / and the long narrow stem opening into 0. Compartment / contains sucrose dissolved in heavy water, whereas the outside medium is pure ordinary water. Owing to the osmotic effect of the sucrose, water will be sucked through the semi-permeable membrane and water will be replenished via the stems of the osmometers. If the area of the semi-permeable membrane is large and the diameter of the stem is small, the linear rate of water flow in the stems may easily exceed the diffusion rate of water. Consequently, although water can easily pass from 0 to /, the heavy water of the inside solution can never reach 0 although it passes easily enough through the semi-permeable membrane. This model system, as already mentioned, represents an exaggerated picture of something that will always occur in pore membranes. Let us now consider a simple pore membrane which is impermeable, for instance, to sucrose. At the boundary of the membrane adjoining the sugar solution events are governed by the ideal law. The net flux arises as the difference between the water diffusing out of the sugar solution and that diffusing into it, and we can write MJMout = flw(o)/flw(i); but, since the water phase filling the pores is pure water, it will only flow to replenish that lost by osmotic suction in so far as a difference of hydrostatic pressure is built up between the ends of the pore. In other words, that part of the water transfer process concerned in overcoming the internal friction in the membrane phase is governed by the laws of laminar flow and not by the laws of diffusion. Now these laws are of a very different nature. For a pore of given length the amount of water which can diffuse through in a given time under steady-state conditions depends on the area, or in other words, on the radius to the second power. Laminar streaming through a cylindrical pore, according to Poiseuille's law, is proportional to the radius to the fourth power. We can put this a little differently and say that for a given area diffusion is independent of the number of pores in which this area is divided up, whereas the flow of water is proportional to the second power of the pore radius. It is quite easy to express these considerations in mathematical terms. I shall not take your time by developing the expressions, but shall confine myself to presenting a few of the resulting expressions. It turns out that the following expression is gener- ally valid for a semi-permeable membrane: M A [Xo i ln-^=-^ -Ax .... (i* Mout AJo a * Footnote : In , . '° , indicating the ' one-sidedness ' of the process, has the dimension of a potential. J« is a ■M0ut I rxa i m . ' current strength ', whereas -=— / -.dx is the diffusion ' resistance '. Thus the whole expression is analogous Dvi J o A to Ohm's law. 36 Membrane structure as revealed by permeability studies The meanings of Min, Mout and Aw have been defined above. Z)w is the diffusion coefficient for water diffusing in water, A is the fraction of the total area of the membrane which is available to water diffusion, x is the distance from the inside boundary, and x0 is the total thickness of the membrane. Evidently the flux ratio for water may vary profoundly, and depends on the shape of the pores inside the membrane. In the case of the action of posterior lobe on the toad skin, in which the net water flux increased by more than ioo per cent, without the influx's changing by more than a few per cent., it turns out that the equation is satisfied if the diffusion area remains constant while, at the same time, a larger number of narrow pores is replaced by a smaller number of large pores. The results therefore do not necessarily indicate an active transport of water. But the alternative to the active transport hypothesis is the acceptance of pores in the membrane. In order to see what the pore hypothesis means in terms of pore dimensions it may be useful to consider an 'equivalent' membrane with uniform cylindrical pores. Furthermore it is assumed that the only force available for the transfer of water is the difference of osmotic pressure across the membrane. We then get the following simple expression : ^/^out=(^))GW/'W (2) \<2w(i) / where G'w is the frictional coefficient for water diffusing in water, and is equal to RTjD^. Dw has been determined by Orr & Butler (1935) and more precisely by Rogener (1941). At 17-5° C. the numerical value of Gw is 1 36 x io15. The term g'w represents the frictional coefficient for water flowing through the membrane. It is a function of the pore diameter, and works out as _ 14477 S w ^2 where 77 is the viscosity of water. At 17-5° C. we have g' =-5. 5 w r2 It is seen that Gw becomes equal to g'w for r = 3 5 x io-8 cm. Since this is less than the average distance between water molecules, we must conclude that at all real pore sizes water flow takes place with a lower resistance than water diffusion. As one might expect, the difference between the two frictional coefficients vanishes when one gets down to molecular dimensions, and one obtains the classical equation as applied by Hevesy et al. (I.e.) and Visscher et al. (I.e.). With increasing pore size, however, the frictional resistance for flow gradually becomes insignificant as compared with that for diffusion. Inserting the numerical values for Gw and g'w in equation (2), we obtain: log (MJMout) = 0-9 x io%2 x log(flw(o)/aw(i)) ... (3) In one of the toad-skin experiments mentioned above, after the hormone had been added, the water influx was 532 /xl./hr. and the net flux 30 /xl. Taking the water activities to be equal to the water concentrations we had ^w(i) = 55'3 mol./l. and 358-362. Nachmansohn, D., Coates, C. W., Rothenberg, M. A. and Brown, M. V. (1946). On the energy source of the action potential in the electric organ of Electrophorus electricus. J. biol. Chem. 165, 223-231. 48 The ionic permeability of nerve membranes Nastuk, W. L. and Hodgkin, A. L. (1950). The electrical activity of single muscle fibres. J. cell. comp. Physiol. 35, 39-73. Shanes, A. M. (1951). Factors in nerve functioning. Fed. Proc. 10, 61 1-62 1. Ussing, H. H. and Zerahn, K. (1951). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiol. scand. 23, 1 10-127. Discussion Chairman: J. Bracket jV. Myant. What ionic movements occur across the membrane between the nodes in a myelinated mammalian nerve? R. D. Keynes. The experiments of Huxley and Staempfli (1949: J. Physiol. 108, 315-339) showed that there was only a small outward current, probably carried by the K+ ions, through the myelin sheath. In contrast to the larger currents flowing in and out at the nodes, this could be explained as a purely passive current due to the potential change acting on a resistance and capacity in parallel. R. J. Goldacre. Has any attempt been made to follow visually the course of active transport of ions in nerve by the use of cationic dyes ? Although the emphasis is on the specificity of these pumps, it is difficult to think that a dye like neutral red would not be taken up by nerve to an extent which would perhaps be sufficient, in the case of a giant axon, for its course to be followed under the microscope. R. D. Keynes. We have never seriously investigated the penetration of dyes into giant axons. Dyes injected into giant axons seem to diffuse as far as the membrane and no further. J. E. Harris. Is there any connexion between the phenomena you have just described and the very active uptake by nerves of methylene blue ? R. D. Keynes. I do not know of any physico-chemical connexion between the activity in a nerve and the uptake of methylene blue; but I suppose that it is conceivable that the dye might enter at the nerve terminals during the non-specific increase in permeability which is thought to occur as a result of liberation of acetylcholine. 49 Cellular oxidations and the synthesis of amino-acids and amides in plants by E. W. YEMM Botany Department, University of Bristol INTRODUCTION The biochemical mechanisms engaged in the synthesis of amino-acids and proteins in the cell have recently been extensively studied. There is now much evidence that amino-acids are directly involved in the biosynthesis of proteins, but two distinct hypotheses have been put forward with regard to the way in which specific peptide structures are built up. The first of these, the so-called 'template' hypothesis, was advanced primarily to account for the reduplication of protein structures; it suggests that amino-acids are orientated on specific surfaces in the cell and are there con- densed en bloc in a single-step reaction. In a review of this mechanism, Dounce (1952) considers that transphosphorylations, mediated by nucleic acids, may provide the energy coupling necessary to promote the reaction. The second hypothesis, developed mainly by Fruton (1952) and Waelsch (1952), suggests that a preliminary synthesis of amino-acid amides or simple peptides takes place, followed by a conversion to proteins by transamidation and transpeptidation reactions controlled by specific transferring enzymes in the cell. As distinct from the 'template' hypothesis, this transamidation mechanism implies an active formation of amides and simple pep- tides and a close coupling between these syntheses and the exergonic reactions of cell respiration. It is the main objective of this paper to consider some further evidence, which has recently been obtained, bearing on this point. An attempt has been made to trace some of the stages by which simple inorganic forms of nitrogen are assimi- lated by plant cells. Under favourable conditions a rapid formation of amino-acids and amides from ammonium salts or nitrates takes place, and affords an opportunity of examining the relation between these syntheses and the breakdown of carbohy- drates in cellular oxidations. CELLULAR RESPIRATION AND THE ASSIMILATION OF NITROGEN It is well established that the rate of respiration of plants and micro-organisms may be greatly increased during the assimilation of nitrogen. Kellner (1874) first showed that pea seedlings respired more rapidly when supplied with nitrates, and his obser- vations have been confirmed and extended to other species by Hamner (1936), Hoagland (1944), Woodford and Gregory (1948), Humphries (1951), and Syrett (I953)> Our work in this direction has been carried out mainly with young seedlings 51 W. YEMM of barley and with food yeast, Torulopsis utilis. These materials were chosen because, despite wide differences in general nutrition, they both have a high capacity for assimilating nitrogen and synthesizing proteins from simple inorganic compounds of nitrogen. For example, cultures of food yeast, supplied with ammonium salts under favourable conditions, will double their protein content within 2-3 hours. High rates of assimilation and protein synthesis also obtain in the early stages of development of barley seedlings. The conditions which favour a rapid uptake of ammonium salts 100- £ in O 50- ■'i\ — 1 ' 1 — Yeast 1 o.oimJnh*h*po< • \na H2P04 1 \ \ \ - \ \ t - \ \ l4^ t \. 1 i \ *^NH H,PO A X 4 2. 4 1 X / k ?\ 9\5 --0 1 . 1 -'-«- NaH2PQ 4^7 1 0 Hours. Figure 1 . The effect of ammonium ions on the rate of oxygen uptake of yeast. Replicate samples, containing 60 mg. of fresh yeast, were treated at the time indicated with either ammonium or sodium phosphate. Oxygen uptake was measured by conventional manometric methods. or nitrate were investigated in a series of preliminary experiments and an account of these has already been given (Folkes, Willis and Yemm, 1952; Yemm and Folkes, 1954). The chief results may be briefly recorded. An essential condition for rapid assimilation is a high level of readily available carbohydrates in the cells. With both organisms this requirement can be met by growing them for a short preliminary period under conditions of high carbohydrate supply, but deprived of nitrogen. The seedlings were therefore grown at high light intensities in a nutrient solution deficient in nitrogen. A similar effect is achieved 52 Cellular oxidations and the syntheses of amino-acids and amides in plants with yeast by treating the cultures in aerated solutions containing sugars and other mineral nutrients but no nitrogen. In this way a high level of soluble sugars or poly- saccharides is built up in the cells, and their ability to assimilate nitrogen in the absence of external supplies of carbohydrate is greatly increased. A further point, shown by the experiments with seedlings, was that the primary reactions, associated with the assimilation of nitrates or ammonia, occur mainly in the root system. On this account most of the work considered here has been carried out with roots immediately after their excision from the growing seedling. Experiments both with yeast and root tissues have shown consistently that the Barley Roots ^ 2 4 12 Hours 24 Figure 2. The effect of ammonium phosphate on carbon dioxide production of excised barley roots. Samples of 40 root systems , freshly cut from the seedlings, were treated in aerated culture solutions with ammonium or sodium phos- phate. CO 2 output was measured by the Pettenkofer method. rate of oxygen uptake increases rapidly when ammonium salts are supplied under favourable conditions. The rate of carbon dioxide production or oxygen consumption is commonly more than doubled within a short time of supplying the ammonia, as illustrated by typical results in Figures 1 and 2. The highest rates of respiration are maintained for only a short time and it is very probable that depletion of the limited carbohydrate reserves in the cells is an important factor causing the secondary decline in rate. No external supply of sugar was provided in these experiments, and analytical data, which are considered in a later section, show that a rapid breakdown of carbohydrates is associated with intense respiratory activities during the assimila- tion of nitrogen. 53 E. W. YEMM It may be noted that nitrates, nitrites and, to a less extent, hydroxylamine increase the rate of respiration in barley roots. But here there is evidence of greater complexity compared with the effects of ammonium salts. The respiratory quotient rises con- siderably above unity with nitrates and nitrites, suggesting that they act as hydrogen acceptors in the oxidation mechanism. The action of hydroxylamine is complicated by its toxic effects even at low concentrations. An account of the experiments with barley roots has been given by Willis (1950, *950- THE PRODUCTS OF NITROGEN ASSIMILATION In an attempt to identify some of the reactions associated with the high rates of cellular oxidation, the products formed in the cells during the early phases of assimila- tion have been investigated. For this purpose analyses of the soluble and insoluble nitrogenous constituents were made, so that it is possible to give some account of the changes of amino-acids and proteins. It has been found consistently in experiments with both yeast and root tissues that glutamic acid and its amide, glutamine, are rapidly formed in the early stages of assimilation, corresponding fairly closely in time with the highest rates of cell oxidation. The results of an experiment in which yeast cultures were supplied with ammonium phosphate are given in Figure 3. During the first 30 min. a marked increase of glutamic acid and glutamine occurs with a smaller accumulation of alanine; together these constituents account for about 70 per cent, of the total nitrogen assimilated by the cells over the initial period. Subsequently, they are maintained at a fairly steady or falling level. There is a gra- dual formation of other, as yet unidentified, soluble-N, and a small increase in the tripeptide, glutathione, was observed in some of these experiments (Yemm and Folkes, 1954). The progressive rise in complex insoluble-N in the cells indicates an active synthesis of protein during the course of the experiment. At present, the identification of amino-acids and amides in the yeast rests mainly on separations by paper chromatography, or on the use of specific enzymes for ana- lysis. Most of the estimates of glutamic acid and glutamine were made by means of glutaminase and glutamic decarboxylase, prepared from Clostridium welchii by the method described by Krebs (1948). Analytical data from a similar experiment with barley roots are shown in Figure 4. On a much longer time-scale they have several features in common with the data for yeast. Ammonia-N accumulates temporarily in the roots, but at first the main product of assimilation is glutamine, which makes up about 80 per cent, of the ammonia utilized in the first 12 hours. Asparagine, the other common plant amide, increases at a later stage; together the two amides account for almost all of the free amino-N in the tissues. In some of the experiments with barley roots it has been possible to obtain more decisive evidence of the primary synthesis of amides from ammonia by using isotopic nitrogen to trace the products of assimilation in the cells. Ammonium phosphate, containing about 30 per cent, excess of 15N, was supplied to the roots and its incorpor- ation into the amide and other nitrogen fractions was estimated after varying periods of assimilation. The abundance of the isotope in some of the different fractions is shown in Figure 5. 54 Cellular oxidations and the synthesis of amino-acids and amides in plants 5 - - oL >- z ^3 So.G "Total N Insoluble N J0LU8LE' N Glutamine Glutamic Acid © ALANINE Hours Figure 3. Changes in nitrogenous constituents during the assimilation of ammonia —N by yeast. Total nitrogen, insoluble (protein) and soluble fractions are shown in the upper part of the figure, and the chief amino-acids and amides in the lower part. It is clear that 15N supplied as ammonia is quickly incorporated into glutamine; the abundance in the amide approaches that of the ammonia-N in the tissues after 10I hours, thus providing direct evidence of a primary synthesis. Asparagine amide, on the other hand, has a lower abundance which gradually rises during assimilation ; it is possible that this amide is formed secondarily from glutamine. The protein-N 55 W. YEMM 10 Barley "Roots 0.0025" M NH4H2P04 Glutamine A ASPARAGINE E3 Ammonia -O Other Amino -A 3o Figure 4. Changes of amino-acids and amides in excised barley roots during the assimilation of ammonia-N. Samples of 40 root systems were analysed after varying periods of assimilation in aerated culture solutions containing ammonium phosphate. uj 20- 5 10 Hours. Figure 5. The incorporation of isotopic nitrogen into the nitrogenous constituents of excised barley roots. Ammonium phosphate contain- ing 29-3 atom per cent, excess 15JV was supplied to the roots, and the separated fractions subjected to analysis in a mass spectrometer. 56 Cellular oxidations and the synthesis of amino-acids and amides in plants of the tissues shows a fairly steady rate of incorporation of 15N, which is much greater than can be accounted for by the net synthesis of protein in the roots. It seems prob- able, therefore, that the proteins of the cells are maintained in dynamic equilibrium with soluble nitrogenous constituents by means of exchange or other reactions. Much other work, reviewed by Chibnall (1939), by Steward and Street (1947) and by Virtanen and Rautenen (1952) converges with that discussed above in showing that the amides, asparagine and glutamine, may be readily formed from ammonia in plant cells. The more active role of glutamine in protein metabolism is indicated by earlier experiments of Yemm (1937, 1949, 1950), Steward and Street (1946), Rautenen (1948) with higher plants, and by those of Roine (1947) and Virtanen, Csarky and Rautenen (1949) with yeast. Vickery, Pucher, Schoenheimer and Rittenberg (1940) and Mac Vicar and Burris (1948), using isotopic nitrogen, have shown that glutamic acid and glutamine are highly active in the metabolism of proteins in plants. THE BREAKDOWN OF CARBOHYDRATES IN RELATION TO RESPIRATION AND THE SYNTHESIS OF AMINO-ACIDS As already indicated, a rapid depletion of carbohydrate accompanies the high rate of respiration during the assimilation of nitrogen by the cells. In most of the experi- ments, analytical data were obtained from which it is possible to estimate the losses of readily available carbohydrates. As no external supplies of carbohydrate were provided, these losses can be related to respiration and the synthesis of nitrogenous constituents. For this purpose balance sheets for carbon have been drawn up, in which the production of respiratory COa and the synthesis of amino-acids are balanced against the breakdown of carbohydrates. An example of the data from a typical experiment with barley roots is given in Table I. It is evident that the break- down of carbohydrates, mainly hexoses and sucrose in these tissues, is adequate to Table I Carbon balance sheet for barley roots Roots excised from 10-day-old seedlings and allowed to assimilate for 18 hours in 0*0025 M NH4H2P04 at 22 -5° G. Products mg. c/100 roots (1) Respiratory C02 36 -6 (2) Synthesis of Glutamine 159 (3) Synthesis of Asparagine 2-8 Total (1), (2), (3) 55-3 Loss Carbohydrates 61 7 meet the needs for both the synthesis of amides and the production of C02. The losses of carbohydrates during the eighteen hours of assimilation are in fact slightly greater 57 W. YEMM than the total requirement and there is every indication that the carbon skeletons for the syntheses of the amino-acids and amides are provided in this way. Data obtained from similar experiments with yeast are summarized in Table II. In the yeast a much more active synthesis of amino-acids and proteins occurs, but here again there is evidence that this, together with the respiratory losses, is mainly met by the breakdown of the reserve carbohydrates, glycogen and mannans. Other sources of carbon in the cell are drawn upon to a less extent; measurements of the respiratory quotient suggest that this may be from fat reserves. There are several other points of interest in these records: they show, for example, the very great drain on the reserves associated with nitrogen assimilation so that the diversion of carbon Table II Carbon balance sheet for yeast Aerated cultures allowed to assimilate for 2 hours in o 01 m NH4H2P04 at 250 C. Products mg. C/i gm. Yeast (1) Respiratory COa 59 (2) Syntheses Glutamine 2-2 Glutamic acid 1 • 1 Alanine 05 Other sol. N 19 Protein 6 6 Total (1) and (2) 182 ■S Carbohydrates Other (by difference) 138 44 to the synthesis of amino-acids, amides and proteins is several times greater than that lost as carbon dioxide. To sum up, the chief results of these analytical experiments indicate that the form- ation of glutamic acid and its amide, glutamine, occurs in the first phases of nitrogen assimilation in both yeast and root tissues. The synthesis is associated with high rates of cell oxidation and is sustained by the mobilization of carbohydrate and possibly other reserves in the cells. THE METABOLISM OF GLUTAMINE A close coupling between the formation of glutamine and the metabolism of carbo- hydrates may be inferred from the enzymic mechanisms associated with the synthesis of the amide. It is highly probable that glutamic acid arises in the cell by reductive 58 Cellular oxidations and the synthesis of amino-acids and amides in plants amination of a-ketoglutaric acid, followed by further combination with ammonia to give the y-amide. The course of the synthesis is outlined below. H2CoI Col ATP a-ketoglutarate Transamination +NH, Glutamate ADP +NH2 Alanine (ioo) Aspartic acid (55) Isoleucine (12) Leucine (5) Valine (5) Glycine (1) Transamidation Glutamine Some evidence of the occurrence of these reactions in barley seedlings has been gained by the separation of enzymes from the young embryos. Highly active pre- parations of glutamic acid dehydrogenase, which catalyses the reductive amination of a-ketoglutarate linked with the oxidation of pyridine nucleotide (Col), have been obtained from the seedlings. With yeasts similar preparations of the dehydrogenase, but reacting with coenzyme II, have been obtained by Adler and others (1938), while Elliott (1951) has demonstrated the enzymic synthesis of glutamine coupled with a conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The dependence of amide synthesis on phosphorylation has been further indicated in the present experiments by the action of 2.4-dinitrophenol. This cell poison strongly inhibits the synthesis of glutamine at low concentrations (0-6-2 -5 x io-5 m at pH 55), although at these levels the rate of oxygen uptake is unaffected, or slightly increased. This typical uncoupling action is attributable to the selective action of dinitrophenol on the phosphorylations linked with cellular oxidations (Simon, 1953). The enzymic systems engaged in the synthesis of glutamine may readily account for its close co-ordination with carbohydrate metabolism and respiration. a-Ketoglutaric acid, the organic acid precursor, is an intermediary in the oxidation of carbohydrate by the tricarboxylic acid cycle, while pyridine nucleotides and adenosine triphosphate occupy key positions as electron and phosphate carriers respectively in cell oxidations. These direct links with the exergonic reactions of respiration may form the starting- point in the synthesis of other amino-acids and of peptides by transfer reactions, such as transamination and transamidation, which proceed with relatively little change 59 W. YEMM of free energy. Steward and Street (1946, 1947), Yemm (1949), Fruton (1950), Hanes et al. (1950), and Waelsch (1952) have discussed the potentialities of glut- amine in the canalizing of energy to protein synthesis. Transaminases, which promote the transfer of a-amino groups from glutamic acid to other a-keto acids, are known to be widely distributed in higher plants (Leonard and Burris, 1947) and their presence in food yeast was demonstrated by Roine (1947). A preliminary investigation of these enzymes in young barley seedlings has shown that they provide a mechanism for formation of at least six other amino-acids, as indicated above. Estimates of the relative rates of transamination with the different amino- acids are given in the diagram. It is of interest that the glutamic-alanine and glut- amic-aspartic systems give the highest activities, which may account for the forma- tion of alanine and asparagine during the rapid assimilation of nitrogen. The nature of the transamidation reactions and their significance in the biosyn- thesis of peptides and proteins is at present uncertain. However, Dowmont and Fruton (1952) have found that plant proteinases, such as papain and ficin, catalyse the synthesis of peptide bonds from amides by transamidation, so that, in artificial systems, formation of polypeptide structures occurred. Participation of the y-amide group of glutamine in transfer reactions in the cell is indicated by the occurrence of glutamyl transferase in micro-organisms (Grossowicz, Wainfan, Borek and Waelsch, 1950) and in higher plants, (Stumpf, Loomis and Michelson, 1 951). In this connexion preparations of glutamyl transferase have recently been made from barley seedlings and the activity estimated in model systems by measuring the rate of replacement of the amide group of glutamine by hydroxylamine. The activity of the enzyme in cell- free preparations indicates that it could play a substantial part in peptide synthesis : the rate of transfer of amide groups observed in cell-free preparations is, in fact, adequate to account for the high rates of peptide synthesis which occur in the young embryo. The products of the action of y-glutamyl transferase in the cell are not yet known. The work of Hanes and others (1950, 1952) has suggested that the tripeptide, gluta- thione, which is very widely distributed in living cells, may take part in transpeptida- tions involving the transfer of y-glutamyl groups. But, under the conditions so far tested, the tripeptide is inactive with the glutamyl transferase of barley and, in yeast, the changes of glutathione during assimilation of nitrogen are relatively small, as already indicated. On the other hand, there is some evidence that the formation of glutathione may be correlated with protein synthesis in the early stages of the development of barley embryos. Estimated by means of the nitroprusside reaction of Grunert and Phillips (1951), the peptide increases markedly at a time when syn- thesis of protein is beginning, as shown by the results given in Figure 6. Mainly in the reduced form, glutathione accumulates in the tissues after about two days' germination and at the same time there is an acceleration of protein syn- thesis. Histochemical tests indicate that it occurs mainly in the meristematic regions, which are in all probability very active in the synthesis. However, it is possible that the action of the tripeptide in oxidation-reduction systems of the cell, recently elucidated by the work of Conn and Vennesland (1951) and Mapson and Goddard (1951), may account for this relation. Moreover, glutathione represents only a very small part of the total soluble nitrogen of the embryo, and other analyses suggest the 60 Cellular oxidations and the synthesis of amino-acids and amides in plants -I Protein N. - 1-5- 10 05 Total GSH f m © 0 2 4 DAYS GERMINATION Figure 6. Changes of glutathione during the development of barley embryos. Samples 0/50-100 embryos were extracted with 2-5 per cent, sulphosalicylic acid; glutathione (GSH) was estimated by the nitroprusside reaction, before and after reduction on a mercury cathode. Changes of total insoluble JV (pro- tein) are shown in the upper part of the figure. 8 presence of appreciable quantities of other peptides, which have not as yet been characterized. The study of these peptides, and particularly of their rate of turnover during the assimilation of nitrogen, may provide more decisive evidence concerning the mechanisms of protein synthesis in the cells. 61 W. YEMM CONCLUSION With regard to the wider problems of protein synthesis, the following conclusions may be drawn from the data so far obtained. ( i ) A rapid formation of glutamic acid and glutamine, which occurs in the first stages of nitrogen assimilation in yeast and in barley tissues, is closely coupled with carbohydrate metabolism and the exergonic reactions of cellular oxidations. (2) The primary synthesis of amino and amide groups may be linked with the formation of other amino-acids and of peptides by means of enzymic systems which promote transamination and transamidation in the cell. (3) Some support is therefore given to the hypothesis of peptide-bond formation by transamidation and transpeptidation, but as yet very little is known of the speci- ficity or course of peptide synthesis in living cells. (4) It seems possible from this and other evidence that the action of specific surfaces, visualized in the 'template' hypothesis, operates at a later phase of protein synthesis by affecting the folding and cross-bonding of polypeptide structures. The role of nucleic acids in protein formation may be in this stage, rather than in the direct synthesis of peptide bonds. The experimental work was carried out in collaboration with my colleagues, Dr. Folkes and Dr. Willis; their permission to present some hitherto unpublished results is gratefully acknowledged. REFERENCES Adler, E., Gunther, G. and Everett, J. E. (1938). Uber den enzymatischen Abbau und Aufbau der Glutaminsaure-IV-In Hefe. Hoppe-Seyl. Z- 255> 27- Chibnall, A. C. (1939). Protein Metabolism in the Plant. Yale University Press, New Haven. Conn, E. E. and Vennesland, B. (1951). Glutathione reductase in wheat germ. J. biol. Chem. 192, 17. Dounce, A. L. (1952). Duplicating mechanism for peptide chain and nucleic acid synthesis. Enzymologia 15, 251. Dowmont, Y. P. and Fruton, J. S. (1952). Chromatography of peptides as applied to transamidation reactions. J. biol. Chem. 197, 271. Elliott, W. H. (1951). Studies in the synthesis of glutamine. Biochem. J. 49, 106. Folkes, B. F., Willis, A.J. and Yemm, E. W. (1952). Respiration of barley plants. VII. The metabolism of nitrogen and respiration in seedlings. New Phytol. 51, 317. Fruton, J. S. (1950). The role of proteolytic enzymes in the biosynthesis of peptide bonds. Yale J. Biol. Med. 22, 263. Fruton, J. S. (1952). Synthesis of peptide bonds. Symposium sur la biogenese des proteines, 2e Congres international de Biochimie. Societe d'Edition d'Enseignement Superieur, Paris. Grossowicz, N., Wainfan, E., Borek, E. and Waelsch, H. (1950). The enzymatic formation of hydroxamic acids from glutamine and asparagine. J. biol. Chem. 187, in. 62 Cellular oxidations and the synthesis of amino-acids and amides in plants Grunert, R. R. and Phillips, P. H. (1951). A modification of the nitroprusside method of analysis for glutathione. Arch. Biochem. 30, 217. Hamner, K. C. (1936). Effects of nitrogen supply on rates of photosynthesis and respiration in plants. Bot. Gaz> 97, 744. Hanes, C. S., Hird, F.J. R. and Isherwood, F. A. (1950). Synthesis of peptides in enzymic reactions involving glutathione. Nature, Lond. 166, 288. Hanes, C. S., Hird, F.J. R. and Isherwood, F. A. (1952). Enzymic transpeptida- tion reactions involving y-glutamyl peptides and a-amino acyl peptides. Biochem. J- 51, 25. Hoagland, D. R. (1944). Lectures on the Inorganic Nutrition of Plants. Waltham, Massachusetts : Chronica Botanica Company. Humphries, E. C. (1951). The absorption of ions by excised root systems. II: Observa- tions on roots of barley grown in solutions deficient in phosphorus, nitrogen, or potassium. J. exp. Bot. 2, 419. Kellner, O. (1874). Ueber einige chemische Vorgange bei der Keimung von Pisum sativum. Landwirt. Mersuchs. stat. 17, 408. Krebs, H. A. (1948). Quantitative determination of glutamine and glutamic acid. Biochem. J. 43, 51. Leonard, M.J. K. and Burris, R. H. (1947). A survey of transaminases in plants. J. biol. Chem. 170, 701. MacVicar, R., and Burris, R. H. (1948). Studies on nitrogen metabolism in tomato with use of isotopically labelled ammonium sulphate. J. biol. Chem. 176, 5"- Mapson, L. W. and Goddard, D. R. (1951). The reduction of glutathione by plant tissues. Biochem. J. 49, 592. Rautenen, N. (1948). On the formation of amino-acids and amides in green plants. Acta chem. scand. 2, 127. Roine, P. (1947). On the formation of primary amino-acids in the protein synthesis in yeast. Ann. Acad. Sci.fenn., Ser. A.2. Chem. No. 26. Simon, E. W. (1953). Mechanisms of dinitrophenol toxicity. Biol. Rev. 28, 453. Steward, F. C. and Street, H. E. (1946). The soluble nitrogen fractions of potato tuber, the amides. Plant Physiol. 21, 155. Steward, F. C. and Street, H. E. (1947). The nitrogenous constituents of plants. Ann. Rev. Biochem. 16, 471. Stumpf, P. K., Loomis, W. D. and Michelson, C. (1951). Amide metabolism in higher plants. I. Preparation and properties of a glutamyl transphorase from pumpkin seedlings. Arch. Biochem. 30, 126. Syrett, P. J. (1953). The assimilation of ammonia by nitrogen-starved cells of Chlorella vulgaris. Part I — The correlation of assimilation with respiration. Ann. Bot., Lond. N.S. 17, 1. Vickery, H. B., Pucher, G. W., Schoenheimer, R. and Rittenberg, D. (1940). The assimilation of ammonia nitrogen by tobacco plants: a preliminary study with isotopic nitrogen. J. biol. Chem. 135, 531. Virtanen, A. I., Csarky, T. Z. and Rautenen, N. (1949). On the formation of amino-acids and proteins in Torula utilis in nitrate nutrition. Biochim. Biophys. Acta. 3, 208. 63 E. W. YEMM Virtanen, A. I. and Rautenen, N. (1952). Nitrogen Assimilation. The Enzymes Vol II, Pt. 2, edited by Sumner and Myrbach. Academic Press, New York. Willis, A.J. (1950). Nitrogen assimilation and respiration in barley. Ph.D. Thesis: Uni- versity of Bristol. Willis, A.J. (1951). Synthesis of amino-acids in young roots of barley. Biochem.J. 49, xxvii. Waelsch, H. (1952). Certain aspects of intermediary metabolism of glutamine, asparagine and glutathione. Advances in Enzymology 13, 237. Woodford, E. K. and Gregory, F. G. (1948). Preliminary results obtained with an apparatus for the study of salt uptake and root respiration of whole plants. Ann. Bot., Lond. N.S. 12, 335. Yemm, E. W. (1937). Respiration of barley plants. III. Protein catabolism in starving leaves. Proc. Roy. Soc. B 123, 243. Yemm, E. W. (1949). Glutamine in the metabolism of barley plants. Mew Phytol. 48, 3i5- Yemm, E. W. (1950). Respiration of barley plants. IV. Protein catabolism and the formation of amides in starving leaves. Proc. Roy. Soc. B 136, 632. Yemm, E. W. and Folkes, B. F. (1954). The regulation of respiration during the assimilation of nitrogen in Torulopsis utilis. Biochem. J. 57, 495. Discussion Chairman: J. Bracket G. Pontecorvo. Your results on transamination of glutamic acid to form other amino- acids in the heirarchic order shown are exactly the same as those found by Fincham and others by the less orthodox but more efficient method of using mutants in micro- organisms. This supports your conclusion as to the general occurrence of such pro- cesses. E. W. Yemm. The investigation of transamination in barley embryos is not yet com- plete. The relative activities given are based on comparative measurements in cell- free preparations without addition of pyridoxal phosphate. From the work of Cohen it seems possible that other transaminations may be detectable after reinforcement of the preparations by addition of the coenzyme. W. S. Reith. It is very interesting to see this striking difference in the relative amounts of glutamine and asparagine. We have found in the growing cells of bean roots just the opposite situation. There the amount of asparagine greatly exceeds that of gluta- mine. We interpreted this as an accumulation of asparagine while the glutamine was rapidly depleted owing to its active participation in transaminations. As for protein-nitrogen determinations, I should like to point out that we find that very misleading results can be obtained from trichloracetic acid precipitates. Such protein precipitates can contain a great amount of non-protein nitrogen. 64 Cellular oxidations and the syntheiss of amino-acids and amides in plants E. W. Yemm. The relation between the two amides, glutamine and asparagine, in the metabolism of barley plants has been discussed in an earlier account of our work. In general asparagine accumulates in the cell under conditions of carbohydrate shortage and proteolysis; this seems to hold for root tissues. Under conditions norm- ally obtaining during the growth of the plant glutamine appears to be much more closely related to the metabolism of proteins. The estimates of total insoluble-N (protein) in roots and yeast were usually obtained after extraction with alcohol and water. 0. Maalee. Is it possible, in your system, to follow synthesis of amino-acids, peptides, and protein long enough to observe an equilibrium between the concentrations of low- and high-molecular-weight compounds; if so, can it be estimated what fraction of amino-N, at equilibrium, is in the pool of low-molecular-weight substrates for protein synthesis ? E. W. Yemm. Equilibrium conditions between the nitrogenous constituents do not appear to be established in our experiments; but we have very little knowledge of the nature or amount of peptides present in the cells. W. S. Reith. In the meristematic cell, the amount of peptide nitrogen and amino-acid nitrogen is very small in comparison with the protein nitrogen. B. F. Folkes. The low level of soluble nitrogen other than glutamic acid, glutamine or alanine, indicates the low level of other amino-acids and peptides in the cell. It seems that the low level of these products limits the rate of protein synthesis. L. Rinaldini. The rise in GSH might be connected with the oxygen uptake in view o the respiratory mechanism recently found in plants by Mapson, where GSH acts as a hydrogen carrier between dehydrogenases and ascorbic acid, which in turn reacts with molecular oxygen. E. W. Yemm. I fully agree that glutathione may be active in other processes of cellular metabolism. In addition to transpeptidations and oxidation-reductions it may have a regulating action on -SH enzyme systems. E. Ambrose. With regard to the transpeptidation and template theories of protein synthesis, if the transpeptidation theory is correct, there is a pool of peptides in dynamic equilibrium within the cells, which is increased in concentration by feeding with the source of nitrogen ; this increase may be to some extent independent of other cellular processes. If on the other hand we are dealing with a nucleic acid template, there might be a close correspondence between the concentration of pep- tides and of nucleic acids within the cell. Has a relationship been found between the peptides and nucleic acid concentrations within yeast cells ? E. W. Yemm. We have not yet studied the change of nucleic acids during protein synthesis in food yeast; as far as I am aware no data have been published. Judging from Gale's work with bacteria and Hokin's with animal tissues, substantial synthesis of proteins may occur in cells without appreciate changes in the amount of nucleic acids. J. F. Danielli. The fact that more isotopic nitrogen appears in the proteins than can be accounted for by net synthesis may mean that individual amino-acids or peptides 65 E. W. YEMM are exchanging with the protein. Is there any evidence that this is so, and if so which amino-acids are concerned? E. W. Temm. The distribution of 15N in the proteins of barley roots has not been examined in detail. However, with leaf-tissue proteins we have evidence that the isotope is incorporated to the greatest extent in glutamic, aspartic and amide nitrogen of the protein, although there are appreciable amounts in the monocarboxylic and basic amino-acids. From this and other work it seems probable that the abundance of 15N in the different amino-acids of the tissue proteins reflects the extent to which the amino-acid becomes labelled in the metabolic pool. In both plant and animal tissues, supplied with isotopic ammonia, incorporation is usually greatest in glutamic, aspartic and amide nitrogen, probably owing to the ease with which these are synthesized from ammonia. A. J. Willis. The incorporation of 15N into the protein of barley roots is much more extensive in the amide groups than in the total nitrogen of the protein. This indicates extensive exchange reactions involving these amide groups. J. Bracket. In connexion with Dr. Yemm's suggestion that there might be two differ- ent mechanisms involved in protein synthesis (transpeptidation and template activity), it is worth pointing out that Koritz and Chantrenne recently obtained evidence for such a viewpoint: in reticulocytes, incorporation of labelled amino- acids precedes the peak in RNA synthesis; this peak coincides with the formation of various enzymes, which might be produced by a specific template mechanism. 66 The biosynthesis of pentoses and their incorporation into mononucleotides by HANS KLENOW Universitetets Institutfor Cytofysiologi, Kabenhavn The importance of mononucleotides both as building-blocks of the nucleic acids and as constituents of a number of coenzymes for reactions in intermediary metabolism is generally accepted. An understanding of the mechanism by which the mono- nucleotides are formed might therefore be of significance for the explanation of various biological phenomena. I should like to discuss possible pathways by which mononucleotides may be formed, and also to mention the present evidence for the pathways of the biosynthesis of the sugar part of the nucleotides, i.e. the ribose. In the last few years considerable knowledge has accumulated about enzyme reactions leading to ribose phosphate formation. These new facts have been obtained mainly from experiments on the oxidative breakdown of carbohydrates. By this term we are accustomed to mean the extremely important oxidative cycle of Krebs. The existence of an alternative pathway of carbohydrate oxidation was, however, indi- cated by work of Warburg and Christian (1937), Lipmann (1936) and Dickens (J936). Glucose-6-phosphate 6-Phosphogluconic acid Fructose-6- phosphate -j-Tetrose phosphate D-glyceraldehyde-3-phosphate + Sedoheptulose-7-phosphate Figure 1 . The oxidative cycle. 67 Ribulose-5-phosphate Ribose-5-phosphate HANS KLENOW Recent studies of this alternative pathway have revealed the existence of a new cycle for the oxidative breakdown of carbohydrates. This cyclic mechanism has been established primarily by Horecker and his group, and has been formulated in the following way (Horecker, 1953). In this reaction scheme the oxidation of glucose-6-phosphate to the S-lactone of 6-phosphogluconate is catalysed by Warburg's well-known Zjvischenferment. The further breakdown of 6-phosphogluconate has been found to be an oxidative decarbo- xylation leading to the formation of the five-carbon keto sugar ribulose-5-phosphate. Both of these oxidation steps require triphosphopyridine nucleotide as hydrogen acceptors. To account for the formation of ribulose-5-phosphate it has been postulated that 6-phosphogluconate is first oxidized in the 3-position. A free 3-keto phospho- gluconate has, however, not been isolated as an intermediate, and the possibility exists that both oxidation and decarboxylation are catalysed by the same enzyme as is the case with some other oxidative decarboxylations. The ribulose-5-phosphate can be converted by a pentose phosphate isomerase to ribose-5-phosphate, a reaction which is completely analogous to the interconversion of fructose-6-phosphate and glucose-6-phosphate. These two pentose phosphate esters can now interact, and with a highly purified enzyme the product has been shown in addition to glyceraldehyde- 3-phosphate to be a phosphate ester of the seven-carbon keto sugar, sedoheptulose. This sugar was first isolated from the sedum plant, where it is present in large amounts (La Forge and Hudson, 191 7). Recently Calvin and his group (Benson et al., 1951) have found that sedoheptulose phosphate is one of the earliest products to be formed during photosynthesis, a fact which is a further indication of its importance in the intermediary metabolism. Glyceraldehyde-3-phosphate and sedoheptulose-7-phos- phate can now further interact, and in the presence of the enzyme transaldolase the products are fructose-6-phosphate and a tetrose phosphate. This reaction has been proved to be a transfer of the three first carbons of sedoheptulose-7-phosphate, i.e. the dihydroxyacetone group, to glyceraldehyde-3-phosphate, whereby fructose-6- phosphate is formed by an aldole condensation. Fructose-6-phosphate is then con- verted by hexose phosphate isomerase to glucose-6-phosphate, and we are back at the starting-point of the cycle. Thus, with two turns of the cycle two moles of C02 are evolved, and four moles of triphosphopyridine nucleotide are reduced, which will require two moles of 02 for oxidation. At the same time one mole of glucose-6- phosphate is regenerated, and one mole of tetrose phosphate is formed. This tetrose may, moreover, be further converted to hexose monophosphate by a mechanism not yet completely clarified, whereby the cycle is completed (Horecker, 1953, Horecker et al., 1954). It should furthermore be emphasized that all of the reactions of the cycle have been shown to be reversible. The activity of some of the enzymes involved in this scheme has been investigated in a variety of normal mammalian tissues and in tumours (Glock and McLean, 1954), and the quantitative significance of the oxidative pathway has been investigated with isotopically labelled compounds in several organs (Bloom, Stetten and Stetten, 1953). This system of enzyme reactions then furnishes us with two processes for pentose formation, i.e. the direct oxidation of glucose-6-phosphate to ribulose-5-phosphate and ribose-5-phosphate, and the reaction between one molecule of glyceraldehyde- 3-phosphate and one molecule of sedoheptulose-7-phosphate leading to the formation 68 The biosynthesis of pentoses and their incorporation into mononucleotides of two molecules of pentose phosphate. This latter reaction has been studied with highly purified enzymes from liver, spinach (Horecker et aL, 1953) and yeast (Racker et aL, 1953). Several possibilities obviously exist for mechanisms by which sedoheptu- lose phosphate may be formed from pentose phosphates, but conclusive evidence indicates (de la Haba^a/., 1953; Horecker and Smyrniotis, 1953; Racker et aL, 1953) that it is formed by a condensation between a two-carbon compound and a five- carbon compound, and that it is the ribulose-5-phosphate which is donator of the two-carbon compound. The latter, which would be at the oxidation level of glycol aldehyde, then combines with ribose-5-phosphate to form the sedoheptulose-7- phosphate. Free glycolaldehyde, however, is not active nor does it accumulate in any of these reactions. The sedoheptulose phosphate formation has, therefore, been visualized as an acetoin condensation between an activated form of glycolaldehyde and ribose-5-phosphate. This is consistent with the thiamine pyrophosphate require- ment of the reaction which has been formulated as follows : H.COH 1 C = O HC = O HCOH -4- ThPP-Enzyme ^ HCOH H2COH 1 HCOH 1 H2COP03H, + HC = 0 HXOPO3H, ThPP-Enzyme Ribulose-5-P Transketolase Glyceraldehyde- 3-phosphate 'Active Glycolaldehyde HC = O H2COH H2COH 1 HCOH HC = O 1 C = O 1 HCOH + - HOCH HCOH HCOH + ThPP-Enzyme H2COPOuH2 ThF 'P-Enzyme HCOH 1 HCOH 1 HXOPO3H0 Ribose-5-P Sedoheptulose-7-P Figure 2. The formation of sedoheptulose-'] -phosphate in the transketolase reaction. {From Horecker et al., 1953.) Since the enzyme catalyses the transfer of ketol linkages, it has been named transketolase. Thus, in these reactions the keto sugar esters, ribulose-5-phosphate and sedoheptulose-7-phosphate, serve as donors of 'active glycolaldehyde', and the aldo sugar esters, ribose-5-phosphate and glyceraldehyde-3-phosphate, serve as acceptors of 'active glycolaldehyde'. Also several other compounds can serve as substrates in these reactions. Those known at the present time are listed in Table I, 69 HANS KLENOW and still others may be found. The transketolase reaction then provides us with a system by which pentoses can be formed by condensation between a two-carbon and a three-carbon fragment. A third mechanism for pentose formation is suggested by work of Hough and Jones (1953) who found xylulose phosphate to be formed from triose phosphate and dihydroxymaleic acid in the presence of an enzyme from peas. The details of this mechanism seem, however, not to be entirely clear yet. We have now accounted for some enzyme reactions for pentose formation. But how are the pentoses actually formed in the intact organism ? By which mechanism are the pentoses in the nucleic acids formed ? The best tool for getting such informa- tion is obviously ingestion of isotopically labelled compounds, the fate of which can be followed. In the case of ribose the pattern of labelling of the carbon atoms of the pentose of the nucleic acids obtained in this way may give valuable information. Table I 'Active glycolaldehyde' donors and acceptors 'Active glycolaldehyde' donors 'Active glycolaldehyde' acceptors Ribulose-5-phosphate a,b Z)-Glyceraldehyde-3-phosphate a,b Sedoheptulose-7-phosphate a Ribose-5-phosphate a,b L-Erythrulose a Glycolaldehyde b Hydroxypyruvic acid a,b Z-Glycolaldehyde-3-phosphate a .D-Glyceraldehyde a a Horecker et al. (1953). b Racker et al. (1953). Such experiments have been performed by Bernstein (1953). The concept which led to these experiments was the following: If the pentoses were formed by the oxi- dative breakdown of glucose-6-phosphate by removal of number one carbon or possibly by removal of number six carbon by decarboxylation of a hexuronic acid, the distribution of the tracer in the ribose should be similar to that of the remaining five carbons of the hexose. A deviation from this picture would indicate the involve- ment of some other synthetic mechanism. The liver glycogen and the ribose from the nucleic acids of the internal organs of chicks were therefore isolated after feeding with different 14G-labelled compounds. The glycogen and the ribose were degraded bio- logically and chemically, and the specific activity of each carbon was determined. Assuming that the 14C pattern of glycogen corresponds to that of glucose-6-phosphate during the experiment, and that the 14G pattern of the ribose is not altered when the nucleic acids are formed, Bernstein compared the relative pattern of 14C labelling in the glucose and in the ribose. As can be seen from Table II the pattern of labelling of the ribose does not in any case correspond with that of the 5 carbons in succession of the glucose derived from glycogen. However, pentose formation by a condensation of a two-carbon with a three-carbon compound is consistent with the results obtained. C(3), C(4) and C(5) of the pentose should then arise from the same triose which is the precursor of glycogen. The C(1> and C(2> of the pentose could be derived from C(1) 70 The biosynthesis of pentoses and their incorporation into mononucleotides and C(2) of glycogen. These findings, therefore, are by no means in disagreement with the reaction catalysed by the transketolase. Similar experiments performed with E. coli suggest that the oxidative decarboxylation of 6-phosphogluconic acid is the primary pathway for pentose formation in this organism (Cohen, 1951; Sowden etal, 1954). We have now seen by which possible mechanism ribose may be formed in living organisms. But by which reactions are the ribose phosphates linked to the purines and pyrimidines to form the nucleotides, the building blocks of the nucleic acids? About eight years ago Kalckar (1947) demonstrated the enzymatic synthesis o Table II Relative 14C distribution in ribose and glycogen of chicks fed 14CH2NH2COOH Experiment no Compound analysed Carrier dilution Relative specific activity C(D C(2) C(3) C(4) C(5) C(6) 3 Glycogen 7-4 100 133 16 16 !33 100 (9940) Ribose 170 79 98 27 124 100 (222) The figures in parentheses indicate the level of specific activity (counts per minute per millimole of carbon) at which the determination was actually made for the carbon assigned a value of 100 (From I. A. Bernstein (!953) J- Biol. Chetn. 205, 317). nucleosides with the nucleoside phosphorylase system. The reaction is the well- known reversible phosphorolytic cleavage of nucleosides to free base and ribose- 1- phosphate. The equilibrium of the reaction: Hypoxanthine riboside -f- orthophosphate ^ hypoxanthine + ribose- 1 -phosphate is in favour of the synthesis of the riboside. High enzyme activity is present in both mammalian organisms and in micro-organisms. The enzyme is active towards a number of different purine nucleosides and also towards purine deoxynucleosides (Friedkin, 1953). Nicotinamide riboside is attacked by an enzyme which is probably identical with the enzyme of Kalckar (Rowen and Kornberg, 1951). Pyrimidine nucleosides, however, can be split by an apparently different phosphorylase (Lampen, 1952). Thus, with these reactions we can account for the formation of the linkage between ribose and several of the nitrogen bases of the nucleic acids. The nucleosides formed in this way might then be phosphorylated to nucleotides by a kinase reaction, i.e. with ATP as phosphate donor. Such reactions have been demonstrated by Kornberg and Pricer (1951). They found that both adenine riboside and 2-amino adenine riboside can be phosphorylated to the corresponding 5/-nucleotides. The enzyme is, however, strictly specific with regard to adenosine and 2-amino adenosine, and other nucleoside kinases have not been found yet. 71 HANS KLENOW A more unspecific reaction for nucleotide formation was found by Brawerman and Chargaff (1953). They showed that some unspecific phosphatases have transferase activity also with nucleosides as acceptors. With phenyl phosphate as phosphate donor they found that all possible nucleotides could be formed from the correspond- ing nucleosides in the presence of prostate phosphatase, while a phosphatase from malt under some conditions catalysed the formation of only 5/-nucleotides. This type of transfer reaction, however, might not play a quantitatively significant role under physiological conditions, at least when growth is involved, as it often requires a high substrate concentration and still gives a fairly low yield. An entirely different pathway for nucleotide formation was suggested by some experiments performed with 14C-labelled formate (Greenberg, 1951). Formic acid is known to be incorporated into the purine ring, and Greenberg was able to isolate labelled hypoxanthine, inosine and inosinic acid from pigeon-liver extracts, which had been incubated with labelled formic acid. But the interesting part of this observa- tion was that the specific activity of inosinic acid was significantly higher than that of inosine and hypoxanthine. In other words, inosine^'-phosphate was probably the primary product, and both the nucleoside and the free base were probably degrada- tion products of the nucleotide. Similarly Leder and Handler (1951), working with nicotinamide nucleotide synthesis in erythrocytes, found evidence for bypassing of the nucleoside stage. Consistent with this concept Buchanan and his group (Williams and Buchanan, 1953) also found evidence for bypassing of inosine in the formation of inosinic acid from hypoxanthine. They furthermore found that synthesis of inosinic acid was considerably activated by addition of ribose-5-phosphate and adenosine triphosphate to pigeon-liver extract. The reaction was shown to be catalysed by at Table III Reactivation of a dialysed extract of pigeon liver Counts/min. in Additions the acid-soluble nucleotides Undialysed extract, 05 ml. 2,150 Dialysed extract, 05 ml. 15 + 3 flM dbose-5-phosphate + I jLtM ATP i,86o + 3 /xm ribose-5-phosphate 82 + 1 /xm ATP 22 + 3 /xm ribose- 1 -phosphate + 1 [xm ATP 2,250 + 3 jjm ribose-^'-phosphate + 1 jum ATP 53 + 5 ju-M deoxyribose- 1 -phosphate + 1 /xu ATP 30 + 5 i^u ribose + 1 fxM ATP 90 + 1 fxM AMP 15 + 1 /xm inosinic acid 15 ■jy — 7,000 counts/min. of 8-carbon-i4-adenine added in every case. (From Saffran, M. and Scarano, E. (1953). Nature, Lond. 172, 949.) 72 The biosynthesis of pentoses and their incorporation into mononucleotides least two enzymes which could be separated by alcohol fractionation. The first enzyme reaction consisted in a reaction between ribose-5-phosphate and adenosine triphosphate to yield an activated ribose phosphate ester. In Dr. Kalckar's laboratory similar observations have been made. Adenine is known to be incorporated into the nucleic acids on a large scale (Brown, 1948), and Goldwasser (1953) found that in pigeon-liver extract 14C-labelled adenine is incorporated into adenosine monophosphate, adenosine diphosphates and adenosine triphosphates at an appreciable rate. Saffran and Scarano (1953) working with the ^265 \ 800 700 600 500 WO 300 200 WO **-*x-x— x. 'X+AZP022/S/V \ ••+, ATP 0.22/jM+R5P3/jM 0 to 20 30 w SO 60 m/n. Figure 3. Phosphorylation of ribose-yphosphate with adenosine-triphosphate. The reference cuvette contained 0-22 /*m adenosine triphosphate in 3 ml., and the spectrophotometer was brought to zero at an optical density of 0*5. The experimental cuvettes contained 0-39 him potassium chloride, 0-03 mM magnes- ium chloride, 0-045 him dipotassium hydrogen phosphate, 5-adenylate kinase 207- of protein per ml., 5-adenylate deaminase 25?- of protein per ml., total volume 3-19 ml., pH 6-8. At o min. no?- of protein per ml. of 5-phospho- ribokinase was added to the experimental cuvettes Abscissa: time in minutes; ordinate: extinction X io3 at 265 rru*. From Scarano (1953)- same system found that here also the presence of ribose-5-phosphate and adenosine triphosphate stimulated the incorporation of adenine into adenosine monophosphate. They found furthermore that in the dialysed extract the incorporation was com- pletely dependent on both of these two compounds (Table III). In this system ribose- 5-phosphate could, however, be replaced by ribose- 1 -phosphate, whereas ribose-2- phosphate and ribose-3-phosphate were inactive. In addition they were able to demonstrate that this reaction also proceeds in at least two steps, the first one being an activation of ribose phosphate with adenosine triphosphate, and the second one being the reaction between this activated compound and adenine to form the 5/-adenylic acid. 73 HANS KLENOW The first enzyme which activates ribose-5-phosphate was found to be fairly heat stable and was partly fractionated. This enzyme fraction was shown among a number of sugars and sugar phosphate esters to utilize adenosine triphosphate in the presence only of ribose-5-phosphate or fructose-6-phosphate (see Figure 3) (Scarano, 1953). All these experiments suggest that the nucleosides may to a great extent be by- passed in the synthesis of the nucleotides, and that a special type of ribose phosphate ester is an intermediate in this synthesis. Here it was natural to consider a ribose- 1, TIME IN MINUTES Figure 4. Phosphoribomutase activity in the presence of different amounts of glucose- 1 ,6-diphosphate. Ribose- 1 -phosphate, 3 X io-3 m; magnesium sulphate, io-3 m; trihydrochloric acid buffer, 2 X io-2 m, pH 7-3; 8-hydroxyquinoline, io-3 m; muscle enzyme, 60/xg. ; protein per ml.; glucose- 1 ,6-diphosphate, synthetic sample. O control; # glucose-i,6-diphosphate2 X io-6 m; A glucose- 1 ,6-diphosphate 3 X io_5m; I I glucose- 1 ,6-diphosphate 8 X io-6 m. The 5-diphosphate, as was already suggested by Leder and Handler (1951). nucleotide formation should then proceed as follows: Ribose- 1, 5-diphosphate + base ^ nucleoside + orthophosphate. The first indication of the existence of this di-ester was obtained from experiments on the enzymatic conversion of ribose- 1 -phosphate to ribose-5-phosphate (Klenow, 1953). This reaction is analogous to the phosphoglucomutase reaction which was shown by Gardini et al. (1949) to require glucose- 1,6-diphosphate as a coenzyme. The mechanism of this reaction was found (Sutherland et al, 1949) to be the transfer of the 1 -phosphate of the coenzyme to the six-position of glucose- 1 -phosphate, where- by a new molecule of coenzyme and the reaction product, glucose-6-phosphate, are formed. Therefore the possibility existed that the phosphoribomutase reaction pro- ceeded in a similar way, i.e. that it required ribose- 1, 5-diphosphate as a coenzyme. 74 The biosynthesis of pentoses and their incorporation into mononucleotides During our study of the phosphoribomutase we found (Klenow, 1953) that the ratio between the activity of this enzyme in muscle extract and that of the phosphogluco- mutase was altered only slightly during preparation of the latter as a crystalline enzyme (Najjar, 1948). Furthermore, it was found that the ribomutase reaction under certain conditions could be activated by glucose- 1,6-diphosphate (see Figure 4). This suggested that the phosphoglucomutase could catalyse the transfer of a phosphate of glucose- 1,6-diphosphate to ribose- 1 -phosphate, whereby a ribose-i,-5-diphosphate might be formed. This presumed that ribose- 1,5-diphosphate might then function as a coenzyme for the phosphoribomutase reaction. Further evidence for the reaction -0.02 W mm Figure 5. Formation of glucose-6-phosphate from ribose- 1 -phosphate and glucose-i ^-diphosphate. Glucose- 1,6-diphosphate: 6 X io-5 m; ribose- 1 -phosphate: 3 x io-4 m; triphosphopyridine nucleotide: i -2 X io-4 m; magnesium chloride: 2 X io-3 m; glycyl-glycine cysteine buffer pH 7-2:1 X io-2 m; crystalline phosphogluco- mutase: 0-015 mg. protein per ml.; £wischenferment : 0-5 mg. protein per ml. ©complete O control without ribose- 1 -phosphate; X control without glucose- 1 ,6-diphosphate. The reaction is measured in a spectrophotometer at 340 rmt. between ribose- 1 -phosphate and glucose- 1,6-diphosphate was obtained with glucose- 6-phosphate dehydrogenase {^wischenferment) and triphosphopyridine nucleotide. With this system it could be demonstrated that glucose-6-phosphate is formed from glucose- 1,6-diphosphate in the presence of ribose- 1 -phosphate and phosphogluco- mutase (Klenow and Emberland, 1954) (see Figure 5). In the same system it could furthermore be shown that not only ribose- 1 -phosphate, but also deoxyribose-i- phosphate and galactose- 1 -phosphate can serve as acceptors of a phosphate from glucose- 1,6-diphosphate (Klenow, 1953). From incubation mixtures of ribose- 1- 75 HANS KLENOW phosphate, glucose- i,6-diphosphate, and phosphoglucomutase a ribose phosphate has been isolated, the properties of which suggest it to be ribose- 1,5-diphosphate. The final proof that this compound is an intermediate in mono-nucleotide forma- tion is, however, still lacking. Most recently a completely new type of pentose phos- phate ester, important for these systems, has been isolated. These important findings were obtained from experiments on 6-carboxy-uracil, also called orotic acid, which is known to be the precursor of the uracil of nucleic acids. Kornberg (1954) found that orotic acid could be incorporated into a nucleotide by an enzyme system which, as in the foregoing cases, involved an activation of ribose-5-phosphate by adenosine triphosphate; he identified the activated form as 5-phospho-ribosyl-i -pyrophosphate. Whether this reaction proceeds in one step, i.e. consisting of a transfer of pyrophos- phate from adenosine triphosphate to ribose-5-phosphate, or in two steps having ribose- 1,5-diphosphate as an intermediate, still has to be seen. With the 5-phospho-ribosyl- 1 -pyrophosphate the formation of the 5 '-mononucleo- tide of adenine has been demonstrated to proceed as follows : 5-phospho-ribosyl- 1 -pyrophosphate -1- adenine % 5/-adenylate + pyrophosphate. Likewise orotic acid gave rise to orotodylic acid with the same reaction mechanism. The enzymes responsible for these reactions have been found in pigeon-liver acetone powder and in yeast. Thus this interesting new reaction for nucleotide formation is a reversible pyrophosphorolytic cleavage of the 5/-nucleotides. The establishment of this reaction might very well lead to the explanation of the enzyme reaction responsible for the synthesis of the imidazole and the pyrimidine rings of the purines. It has been found that in the case of inosinic acid the purine synthesis is completed only after introduction of ribose phosphate into the precursors (Greenberg, 1953). The formation of the nucleotides of these precursors might occur through Kornberg's new ribose phosphate ester as intermediate. In that case it might be possible to synthesize purine precursor ribotides enzymatically and with these to study the reactions which lead to completion of the purine rings. Thus we have now accounted for some enzyme reactions by which ribose may be formed and for pathways for the formation of some ribosides and ribotides from ribose-phosphate esters and the appropriate purines and pyrimidines. How these nucleotides are linked together to form the nucleic acids is obviously a most appealing problem. No experimental evidence on this problem is yet in existence, but extremely stimulating theories have recently been advanced (Kalckar, 1953). REFERENCES Benson, A. A., Bassham, J. A. and Calvin, M. (1951). J. Amer. chem. Soc. 73, 2970. Bernstein, I. A. (1953). J. biol. Chem. 205, 317-329. Bloom, B., Stetten, M. R. and DeWitt Stetten jr. (1953). J. biol. Chem. 204, 681-694. Brawerman, G. and Chargaff, E. (1953). J. Amer. chem. Soc. 75, 41 13. Brown, G. B., Roll, P. M., Plentl, A. A. and Cavalieri, L. F. (1948). J. biol. Chem. 172, 469-484. 76 The biosynthesis of pentoses and their incorporation into mononucleotides Cardini, C. E., Paladini, A. C, Caputto, R., Leloir, L. F. and Trucco, R. E. (1949). Arch. Biochem. 22, 87-100. Cohen, S. S. (1951). Nature Lond. 168, 746. Dickens, F. (1936). Nature Lond. 138, 1057. Friedkin, M. (1953). J. cell. comp. Physiol. 41, suppl. 1, 261-282. Glock, G. E. and McLean, P. (1954). Biochem. J. 56, 1 71-175. Goldwasser, E. (1953). Nature Lond. 171, 126. Greenberg, G. R. (1951). J. biol. Chem. 190, 623. Greenberg, G. R. (1953). Fed. Proc. 12, 651-659. de la Haba, G., Leder, I. G. and Racker, E. (1953). Fed. Proc. 12, 194. Horecker, B. L. (1953). The Brewers Digest 28, no. 1 1, 214-219. Horecker, B. L., Smyrniotis, P. Z. and Klenow, H. (1953). J. biol. Chem. 205, 661-682. Horecker, B. L. and Smyrniotis, P. Z. (1953). J. Amer. chem. Soc. 75, 2021. Horecker, B. L., Gibbs, M., Klenow, H. and Smyrniotis, P. Z. (1954). J. biol. Chem. 207, 393-403. Hough, L. and Jones, J. K. N. (1953). J. chem. Soc. Jan., 342-345. Kalckar, H. M. (1947). J. biol. Chem. 167. 477-486. Kalckar, H. M. (1953). Biochim. Biophys. Acta 12, 250-264. Klenow, H. (1953). Arch. Biochem. Biophys. 46, 186-200. Klenow, H. (1953). Unpublished results. Klenow, H. and Emberland, R. (1954). Unpublished results. Kornberg, A. and Pricer, W. E., jr. (195O.J. biol. Chem. 193, 481-495. Kornberg, A. (1954). Unpublished work, private communications to Dr. H. M. Kalckar. La Forge, F. B. and Hudson, C. S. (1917). J. biol. Chem. 30, 61. Lampen, J. O. (1952) in McElroy and Glass: Phosphorus Metabolism II. Johns Hop- kins Press, Baltimore, 363-384. Leder, I. G. and Handler, P. (1951) in McElroy and Glass: Phosphorus Meta- bolism I. Johns Hopkins Press, Baltimore, 422-427. Lipmann, F. (1936). Nature Lond. 138, 588. Najjar, V. A. (1948). J. biol. Chem. 175, 281-290. Racker, E., de la Haba, G. and Leder, I. G. (1953). J. Amer. chem. Soc. 75, 1010. Rowen, J. W. and Kornberg, A. (1951). J. biol. Chem. 193, 497-507. Saffran, M. and Scarano, E. (1953). Nature Lond. 172, 949. Scarano, E. (1953). Nature Lond. 172, 949. Sowden, J. C., Frankel, S., Moore, B. H. and McClary, J. (1954). J. biol. Chem. 206, 547-552. Sutherland, E. W., Cohn, M., Posternak, T. and Cori, C. F. (1949). J. biol. Chem. 180, 1 285-1 295. Warburg, O. and Christian, W. (1937). Biochem. Z- 292> 287-295. Williams, W.J. and Buchanan, J. M. (1953). J- biol. Chem. 203, 583-593. 77 HANS KLENOW Discussion Chairman: C. H. Waddington J. Brachet. Is there anything known about the intracellular distribution of the various enzymes involved in nucleotide synthesis? H. Klenow. The enzyme system responsible for the incorporation of adenine into AMP in the presence of ribose-5-phosphate and ATP is present in the soluble part of pigeon-liver homogenate. H. V. Bmndsted. Have you any indication that any of the B-vitamins enter into the synthesis of RNA ? I am asking because we have shown that RNA accelerates re- generation in starved planarians, and so does riboflavin. The constituents of RNA given separately but in the right proportion act as a poison. H. Klenow. It is known from the work of Greenberg and of Buchanan that citrovorum factor is significant for the formation of the purine part of inosinic acid. This acid can apparently be formed from 4-amino-5-imidazole-carboxamide ribotide and formic acid. This incorporation of formic acid, which appears in G(2> of the purine ring, seems to require citrovorum factor as a coenzyme. 78 Deoxynucleic acid in some gametes and embryos by E. HOFF-J0RGENSEN Biokemisk Institut, Kobenhavns Universitet The sensitivity and specificity of the known chemical methods for the determination of DNA seem to be insufficient for the estimation of the minute concentration of DNA present in eggs and in embryos during the early stages of development. A microbio- logical assay method, which is very sensitive and highly specific, has been used in the investigations reported in this paper. ASSAY METHOD Principle. The lactic acid bacterium Thermo bacterium acidophilus R 26 Orla Jensen (ATCG 1 1506) requires a deoxynucleoside as an essential growth factor. Neither vitamin B12 nor any other of many substances tested can replace the requirement for a deoxynucleoside. This organism therefore can be used as a test organism for micro- biological assays of deoxynucleosides and also of DNA after depolymerization of the DNA. (Hoff-Jorgensen, 1952). Stock cultures are maintained in the following medium by weekly transfer : o • 1 g. of cysteine and 05 g. of yeast extract (Difco) are dissolved in 100 ml. skimmed milk, at pH 6-8. The milk medium is dispensed in 2 ml. quantities to test-tubes (100 x 10 mm.). About 01 g. of CaC03 is added to each tube. The tubes are plugged with cotton, autoclaved at 1200 C. for 10 min., inoculated with a wire loop, incubated for 24 hr. at 370 C, and stored in a refrigerator. Inoculum medium. 50 ml. of the double-strength basal medium are mixed with 50 ml. of water. The minimum amount of peptone (e.g. about 5 mg. Difco per ml.) which gives maximum growth is added. The medium is dispensed in 5 ml. quantities to 15 ml. centrifuge tubes, each containing a glass bead. The tubes are plugged with cotton, autoclaved at 1200 C. for 10 min., and stored in a refrigerator. Fresh inoculum medium is prepared every month. Inoculum. A small loopful of the stock milk culture is transferred to a tube contain- ing 5 ml. of the inoculum medium. After incubation at 370 G. for 20-24 hr., the cells are centrifuged, washed once with 10 ml. of sterile saline, and resuspended in 10 ml. of sterile saline. One small drop of this suspension is used to inoculate each assay tube. 79 E. HOFF-J0RGENSEN Standard. Stock solution: io~4 g. mol. of a deoxyriboside, e.g. 24 -2 mg. of thymi- dine, is dissolved in 100 ml. of 25 per cent, ethanol. This solution is stable for at least one year. Working standard: 5 X io-9 g. mol. of deoxyriboside per ml. To prepare this, 50 [xl. of the stock solution is diluted to 10 ml. with water. Basal medium, double strength (100 ml.] HCl-hydrolysed casein solution Papain-hydrolysed casein solution Salt A SaltD Tween 80 Cytidylic acid solution Potassium acetate solution Thioglycolic acid solution Adenine-guanine-thymine solution Vitamin solution Glucose Z)Z-Tryptophane Z-Cysteine 30 ml. 10 5 1 1 1 5 1 1 1 3g- 20 mg. 20 „ Dissolve the glucose, tryptophane and cysteine in the previously mixed solutions, adjust the pH to 67 with 1 n KOH, and add water to make 100 ml. Prepare the various solutions as follows : HCl-hydrolysed casein and papain-hydrolysed casein: as described by Hoff- Jorgensen, Moustgaard and Moller (1952). N (00 0.5 W /.5 2.0 2.5 Thymidine JO'9 g. moi./mi Figure 1. Growth curve for Tbm. acidophilus R 26, 37° C, 24 hr. 80 Deoxynucleic acid in some gametes and embryos Salt A: dissolve 20 g. of monobasic potassium phosphate, KH2P04, in water to make 100 ml. Salt D: dissolve 03 g. of Mohr's salt (Fe(NH4)2(S04)2 . 6H20), 02 g. of sodium chloride, 08 g. of manganese sulphate (MnS04 . 4.H20), 4 g. of magnesium sulphate (MgS04 . 7H20), and 2 ml. of 1 n HC1 in water to make 100 ml. Tween 80 solution: dissolve 10 g. of Tween 80 (polyxyethylene sorbiton mono- oleate) in water to make 100 ml. Store in a refrigerator. Cytidylic acid solution: dissolve 05 g. of cytidylic acid in water, adjust the pH to 70 with about 2 m sodium acetate solution, and add water to make 100 ml. Store in a refrigerator. Potassium acetate solution : dissolve 500 g. of potassium acetate in water to make 1,000 ml. Adenine-guanine-thymine solution: dissolve 02 g. each of adenine sulphate, guanine hydrochloride, and thymine with the aid of heat in 10 ml. of 2 n HC1. Add water to 100 ml. Thioglycolic acid solution: dissolve 1 g. of thioglycolic acid in water to make 100 ml. Vitamin solution: dissolve 05 mg. of folic acid and 5 mg. each of/>-aminobenzoic acid, riboflavin, nicotinic acid and calcium pantothenate in 50 ml. of water. Store under a preservative in a refrigerator. Prepare a fresh solution every month. Assay procedure The assay is carried out in lipless uniform test-tubes (100 x 8 mm. i.d.). To each series of tubes the standard vitamin solution is added in the following amounts : 00, 01, 02, 04, 06, 08 and 1 o ml. each with an error of not more than 2 per cent. Each level is set up in duplicate. The extract of the sample to be assayed is similarly added to a series of tubes in the following amounts: 0*2, 0-4, o*6 and o*8 ml., also in duplicate. All tubes are diluted to 1 o ml. with distilled water and i-o ml. of the basal medium is added. The tubes are shaken, covered with glass or aluminium caps, autoclaved at 1200 G. for 5 min., cooled to room temperature, and inoculated with one drop of the immediately previously prepared inoculum suspension. To two of the four tubes containing o ml. of standard no inoculum is added. These tubes are used as blanks in the turbidimetric determination of growth. All tubes are incubated at 370 C. for 24-36 hr. Determination of response The tubes are shaken and the turbidity is read in a photometer (e.g., Lumetron 402 C, Photo volt Corporation, 95 Madison Avenue, New York, 16) at A = c. 650 rmt. The microcuvettes are filled with a pipette and emptied with a piece of plastic tub- ing connected to a suction pump. Calculation of results A standard dose-response curve is prepared by plotting the average of the turbidity values found at each level of the deoxynucleoside standard against the amount of E. HOFF-J0RGENSEN deoxynucleoside present. The deoxynucleoside content of a sample is determined by interpolating the response to the known amount of the test solution onto this standard curve. The deoxynucleoside content per ml. of the test solution is now calculated for each of the duplicate sets of tubes, and the deoxynucleoside content of the sample is calculated from the average of the values. Preparation of samples for assay Deoxynucleosides : a solution containing about 3 m/zmol. (or 05-10 /xg.) deoxynucleoside per ml. is prepared in water, or in a not-more- than o 05 m maleic acid buffer at pH 67. Deoxynucleotides : incubation of a solution of deoxynucleotides with crude intes- tinal phosphatase (Schmidt and Thannhauser, 1943) is without effect on the response; it is therefore concluded that deoxynucleotides give the same response as deoxy- nucleosides on a molar basis. Deoxynucleic acid. Pure DNA has a growth effect which is less than 1 per cent, of the effect of the deoxynucleosides present in the DNA. If, however, the DNA is depolymerized by deoxyribonuclease, (Kunitz, 1950) the growth response is equival- ent to the effect of the calculated content of deoxyribosides in the DNA. Samples of bacteria, yeast or tissue may either be analysed in the wet state or after drying with acetone. For the analysis of bacteria and yeast, the cells should be disintegrated, e.g. in 'the tuning-fork disintegrator' (obtained from H. Mickle, Hampton, Middlesex, England). The sample containing at least 0-2 fxg. P as DNAP is placed in a small test- tube. An exactly measured amount of 0-5 n NaOH solution (e.g. 0-5 ml.) is added, or if the sample is a solution, enough 1 -o n NaOH solution to make the final solution 05 n in NaOH. The tube is placed in a boiling water-bath for 15 min. During this time the tissue is disintegrated with a glass rod. After the incubation at ioo° C. 5 vol. of a solution containing 0-06 g. mol. of maleic acid and o-oi g. mol. of magnesium sulphate per 1. are added for each vol. of 0*5 n sodium hydroxide used above. The pH of the mixture should now be 6-3-7-0. In order to depolymerize the DNA 01 ml. of a solution usually containing 100 /xg. of crystalline deoxynuclease (Worthington, Biochemical Lab., Freehold, New Jersey, U.S.A.) is added and the mixture is incub- ated for 16-20 hr. at 370 G. For each new material assayed, the minimum amount of DNAase which gives maximum response should, however, be found by experi- ments. After incubation the mixture is diluted to contain about 3 mju. mol. deoxy- nucleoside per ml. and assayed (one g. mol. deoxynucleoside — ' 310 g. DMA). Differentiation between purine and pyrimidine deoxynucleosides As the pyrimidine deoxynucleosides are stable towards mild acid hydrolysis, whereas the purine deoxynucleosides are not, it is possible to distinguish between these two types of deoxynucleosides by assaying the depolymerized sample before and after boiling for 5 min. at pH 1 . Before assaying the acid solution must be neutralized. Specificity, sensitivity and accuracy The method seems to be absolutely specific for the deoxyribonucleic linkage and allows the determinations of amounts greater than about 2 jug. of deoxynucleosides, deoxynucleotides or DNA with a standard deviation of about 5 per cent. 82 Deoxynucleic acid in some gametes and embryos DNA IN GAMETES AND EMBRYOS OF PARAC EN TROTU S LIVIDUS Material. The work was carried out at the Stazione Zoologica, Naples. To 600 ml. of an egg suspension containing about 4 x io4 eggs per ml. in sea water there was added 0-5 ml. of a sperm suspension containing about 10 sperms per egg. The egg suspension was placed at 200 G. and continuously mixed by a slow stirrer. To fix the fertilized eggs or embryos for microscopic examination 9 ml. of the suspension were withdrawn and added to one ml. of 40 per cent, formalin. For the DNA determination 40 ml. of the suspension were withdrawn, cooled in ice water and centrifuged at low speed. The embryos were suspended in 40 ml. of acid sea water at pH 36 to remove the jelly capsule and adhering sperms, and again centri- fuged. The washing with acid sea water was repeated once and followed by one washing with 40 ml. of distilled water to remove salts. The embryos were washed twice with acetone and once with ether and then dried in a vacuum desiccator over sulphuric acid. Washing and centrifuging were performed at o° G. with precooled fluids. The treatment described above is without effect on the DNA content of the eggs. Results (1) DNA in sperm and unfertilized eggs: (a) sperm 20 ml. sperm suspension ^ 60 mg. dry matter 1 /xl ,, ,, ~o-5i X 1 o6 sperm 1 mg. dry matter ~ 390 m/xmol. deoxyriboside 390 x 60 x o 310 „ __ . per sperm: — — - — ^ =071 x io-6ug- DNA 051 x io6 x 20 x io3 ^& (b) eggs 40 ml. egg suspension ^ 70 mg. dry matter 1 ml. „ „ ~4-4 X io4 eggs 1 mg. dry matter ■ — ' 1 35 mju,mol. deoxyriboside 1 3S X 70 x 0310 „ „ per egg: — ^ J— 6— = 166 x io"6 ug. DNA 4-4 x io4 x 40 r& DNA per egg 166 DNA per sperm 071 23 Elson and Chargaff (1952), using a microbiological assay of thymine, found about 25 X io-6 jitg. DNA per egg and 1 o x io~6 ^g. DNA per sperm. 83 E. HOFF-J0RGENSEN (2) DNA in embryos during the early stages of development : hr offer fertilization -h # 1'/2 3 t/fe 6 7?2 numder of ce//s per embryo 1 100 /oo 60 ' 31 15 15 15 2 - - to 10 2 1 - 1 — - - 16 22 - - 8 - - - 13 W - - 16 - - - - 20 61 27 >/6 - - - - - 23 58 tO'^g DA/A per embryo 17.5 18.3 17.9 mo 57.5 175 935 fySO * too 4 350 ft $ 300 k 250 % 200 ^ /50 \ 100 § 50 • • i i ' > 1 l i 150 100 -'/2 0 3/y 1*/z 3 1'/2 6 m hours after fertilization Figure 2. Table: Percentage of embryos (Paracentrotus lividus) at differ- ent stages of development. Graph : Content of DMA per embryo or egg. Figure 2 shows that the content of DNA in the embryo is the same as in the unferti- lized eggs until the 16-cell stage. DNA IN EGGS, SPERM AND EMBRYOS OF RANA TEMPORARIA Material. The eggs were fertilized as described by Rugh (1948). The jelly was re- moved with scissors. The eggs and embryos were dried with acetone and ether. In sperm DNA was determined without drying. Results (1) DNA in sperm and unfertilized eggs: (a) sperm 4 ml. sperm suspension ~ 1 4 x io6 sperm ^ 12 -6 /ng. DNA per sperm: 8-6 x io-6 /ng. DNA (b) unfertilized eggs 25 eggs ~ 1 -73 /xg. DNA 25 » ~ I/91 » » 25 » ~ 1-82 „ „ 84 Deoxynucleic acid in some gametes and embryos average per egg: 7 3 X io-2 fig. DNA DNA per egg 73 x 10 -2 •5 X ioJ DNA per sperm 8-6 x io~6 The value found for the DNA content per sperm seems high, but it agrees well with the finding by Mirsky and Ris (1949), that erythrocytes of the frog contain 150 X io~6/xg. DNA per cell and hepatic tissue 157 X io-6 fig. DNA per cell. If we take the average value 15 35 x io~6 fig. as representing the DNA content of the diploid cells of the frog we get : DNA per egsr 73 x io-2 ^ATA F ^ = -L^ r = 475 X io3 DNA per cell 1535 x io-6 * /J which means that the egg contains enough DNA for about 5000 cells. (2) DNA in embryos during the early stages of development (i3°-i7° C.) : h 8/2/6 20 2h 28 hours after ferii/izafion. temp. /f-/7°C. Figure 3. Content of DNA in eggs and embryos of Rana temporaria. The figures above the curve indicate Shumwafs stages of development. Figure 3 shows that the content of DNA in the embryo is the same as in the unfertil- ized egg until 18 hours after fertilization (Shumway's stage 9). At that stage a rapid synthesis of DNA begins. 5,000 cells at that time would correspond to an average generation time of about i\ hours. DNA IN EGGS AND EMBRYOS OF THE DOMESTIC FOWL Material. Fertilized eggs were incubated at 380 C. and 70 per cent, humidity. 2x2 eggs were taken out daily at the same time of day and treated as follows : 2 whole eggs (white, yolk and embryo) were treated in a blender with 500 ml. of 85 E. HOFF-JORGENSEN acetone and 250 ml. of ether for 10 min. at slow speed. After standing for 10 min. the acetone-ether was withdrawn and 300 ml. of ether added. After stirring for 5 min. the suspension was filtered, washed with ether and dried in a desiccator over sul- phuric acid. ?L 36 1 ^700 ^ 500 \ 369-385- Villee, C. A., Lowens, M., Gorden, M., Leonard, E. and Rich, A. (1949). The incorporation of 32P into the nucleoproteins and phosphoproteins of the develop- ing sea-urchin embryo. J. cell. comp. Physiol. 33, 93-1 12. Discussion Chairman: C. H. Waddington J. Brachet. The estimation of DNA in the cytoplasm of eggs depends largely on the specificity of the method used. In the case of the sea-urchin and the frog, the more specific the method, the less is the DNA found in the egg. Using Ceriotti's method, which certainly is less specific than Dr. Hoff-Jorgensen's, I found about four times as much DNA in frog eggs as he did. However, even so, a definite synthesis of DNA was found to occur during cleavage. To explain these discrepancies other methods will have to be tried. I am rather surprised at the conclusion that there is no DNA synthesis in chick embryos until after the third day of incubation, as the embryos at that stage have already undergone considerable morphogenesis. Is it possible that the yolk contains substances interfering with Dr. Hoff-Jorgensen's method? The material giving a positive Ceriotti reaction for desoxypentose is also in the yolk, but it is hard to see how desoxyribonucleosides could be released from yolk platelets, without digestion of the latter, in such a harmonious way that the liberated nucleosides would exactly match the requirement for nuclear multiplication. If yolk constituents interfered 88 Deoxynucleic acid in some gametes arid embryos with Dr. HofF-Jorgensen's method, this would explain the much greater quantity of DNA in a hen's egg than in a sea-urchin egg. It would be interesting to study separ- ately the DNA content of the embryos and of the yolk, and also to study the DNA content of embryos grown on Spratt's synthetic medium. E. Hqff-Jergensen. It is unlikely that other substances are responsible for the growth of the test organism. The exact amount of desoxyriboside found by the assay method can be recovered as DNA from the cells of the test organism. Also the hypothetical substance supposed to interfere with the test would have to be formed as a result of treatment with the highly specific crystalline DNA-ase; little or no growth factor is found without this treatment. C. H. Waddington. It seems peculiar that the egg should not perform any synthesis of DNA until all its reserves are completely exhausted, and should then immediately start to synthesize at full speed. Moreover, at the time when synthesis begins in the chick embryo, the centre of the yolk is still a long distance away from the nearest cells (those of the yolk-sac). Is it possible that there is a continuous destruction of DNA, and that the synthesis at first balances this but that, eventually, as the number of nuclei increases, synthesis greatly surpasses breakdown ? E. Hoff-Jergensen. We are determining the linkage between desoxyribose and a base, and all we can say is that during the first three days of development the number of these linkages is constant. J. E. Harris. If one is prepared to admit that the bacterium cannot synthesize DNA, it is not unreasonable to suggest that the early embryo may not be able to do so either. 0. Maalee. A breakdown of DNA to balance synthesis would have to be taken all the way down — beyond the nucleoside stage — if the degradation products are not to be detected by Dr. Hoff-Jorgensen's method. C. H. Waddington. It would be interesting to try diploid or tetraploid frog embryos, as these would be likely to contain a different amount of DNA per cell. J. Bracket. I have investigated haploid frog embryos produced by irradiation of the sperm with ultra-violet light. These embryos contain more cells than diploid embryos. There is less DNA in early cleavage stages; the DNA content catches up by the late blastula stage; but after gastrulation the haploid embryo lags behind once more in DNA content. M. M. Swann. Early embryos relying on a store of DNA might be immune to in- hibitors of DNA synthesis, such as aminopterin. Have any been tried ? E. Hoff-Jergensen. The inhibitors tried — acting against thiamine, riboflavin, or folic acid — had no visible effect on the embryos. It is of course possible they they do not penetrate the egg membrane. E. jV". Willmer. Embryo extract stimulates the synthesis of nucleoproteins in tissue cultures of chick fibroblasts. One of the immediate effects of the addition of embryo extract is to increase the glucose uptake by the cells. It is therefore interesting to observe, as I did many years ago, that the sea-urchin egg does not pick up glucose 89 E. HOFF-J0RGENSEN during the first day, but picks it up actively on the second day, by which time the synthesis of nucleoprotein is presumably going on. E. Zeuthen. I should like to call attention to a paper by Blanchard (1935, J. biol. Chem. 108, 251) who isolated 1 08 g. deoxynucleic acid from 4,820 g. of (wet) Arbacia eggs. The substance yielded negative biuret reaction, negative tests for pentose, and a positive Feulgen reaction. It contained 16 35 per cent. N and 10-13 per cent. P. Upon hydrolysis with 5 per cent. H2S04 it yielded 1 1 4 per cent, guanine and 9 87 per cent, adenine, all values close to what is reported for DNA from other sources. RNA was demonstrated in amounts about equal to DNA. Hoff-Jorgensen (for unfertilized Arbacia lixula eggs) finds 0*65 per mil. of the dry matter to be DNA. This would seem to compare reasonably well with Blanchard's value o 23 per mil. for wet Arbacia punctulata eggs. The important fact remains that already in 1935 DNA seems to have been isolated from unfertilized sea-urchin eggs in yields which we can now see indicate that the egg holds far more DNA than the spermatozoon. The excess DNA of the egg may be either in the nucleus or in the cytoplasm. With special regard to the sea-urchin egg I have (Pubbl. Staz. zool. Napoli 23, suppl. 1951) suggested the latter possibility as the only one that would bring into harmony the very different results obtained for the whole egg and (cf. Lison and Pasteels, 1951, Arch. Biol. 62, 1 ) for the nucleus alone. A recent finding by Agrell (1953, Ark. 62. Beale, G. H. (1954). The Genetics of Paramecium. Cambridge University Press. (In press.) Beerman, W. (1952). Chromomerenkonstanz unde spezifische Modifikationen der Ghromosomen struktur in der Entwicklung und Organdifferenzierung von Chironomus tentans. Chromosoma 5, 139. Billingham, R. E. and Medawar, P. B. (1948). Pigment spread and cell heredity in guinea-pig's skin. Heredity 2, 29. Brachet, J. (1944) . Embryologie Chimique. Masson & Cie., Paris (English edition, 1950, Intersci. N.Y.). Brachet, J. (1952). La role des acides nucleiques dans la vie de la cellule et de Vembryon. Actualites biochim. Paris. Caspari, E. (1948). Cytoplasmic inheritance. Advanc. Genet. 2, 1. Delbruck, M. (1949). Discussion to paper by Sonneborn and Beale, in Unites biolo- giques doue'es de continuity genetique, Centre National de la Recherche Scientifique. Paris, p. 33. 118 The cell physiology of early development Ephrussi, B. (1953). Nucleo-cytoplasmic relations in micro-organisms. Oxford University Press. Gloor, H. (1947). Phanokopie Versuche mit Aether an Drosophila. Rev. suisse £ool. 54, 637. Haldane, J. B. S. (1954). The Biochemistry of Genetics. Allen and Unwin. Kostitzin, V. A. (1937). Biologie Mathematique. Colin, Paris. Laven, H. (1953). Reziprokunterschiedliche Kreuzbarkeit von Stechmiicken (Culicidae) und ihre Deutung als plasmatische Vererbung. £. indukt. Abstamm.- u. VererbLehre 85, 118. Lederberg, J. (1952). Cell genetics and hereditary symbiosis. Physiol. Rev. 32, 403. L'Heretier, P. (1948). CO 2 sensitivity in Drosophila. Heredity 2, 300. Lotka, A. J. (1934). Theorie analytique des associations biologiques. Hermann, Paris. Mechelke, F. (1953). Reversible Strukturmodificationen der Speicheldrusenchro- mosomen von Acricotopus lucidus. Chromosoma 5, 511. Needham, J. (1936). Substances promoting cell growth. Brit. med. J. 2, 892. Nieuwkoop, P. D., and others (1952). Activation and organization of the central nervous system in amphibians. J. exp. £ool. 120, 1 . Pavan. B. (1954). Proc. gth int. congr. Genet, (in press). Salaman, R. N. and Le Pelley, R. H. (1930). Para-crinkle; a potato disease of the virus group. Proc. Roy. Soc. B 106, 140. . Seidel, F. (1929). Untersuchungen iiber das Bildungsprinzip der Keimanlage im Ei der Libelle platycnemis pennipes. Arch. EntwMech. Org. 119, 322. Sonneborn, T. M. ( 1 95 1 ) . Some current problems of genetics in the light of investi- gations on Chlamydomonas and Paramecium. Cold Spr. Harb. Symp. quant. Biol. 16, 483. Spemann, H. (1938). Embryonic development and induction. Yale University Press. Spiegelman, S. (1951). The particulate transmission of enzyme-forming capacity in yeast. Cold Spr. Harb. Symp. quant. Biol. 16, 87. Vogt, M. (1946). Zur labilen Determination der Imaginalscheiben bei Drosophila. Z- Naturf. 1, 469. Waddington, C. H. (1948). The genetic control of development. Symp. Soc. exp. Biol. 2, 145. Weisz, P. B. (1951). A general mechanism of differentiation based on morpho- genetic studies in ciliates. Amer. Nat. 85, 293. "9 The time-graded regeneration field in planarians and some of its cy to-physiological implications by H. V. BR0NDSTED Instilutfor Aim. ^oologi, Kobenhavns Universitet THE TIME-GRADED FIELD Planarian regeneration has received much attention because these animals, especi- ally the fresh-water triclads, seem to be 'immortal under the edge of the knife', to use an impressive phrase of Dalyell (1814). Prominent biologists have sharpened their experimental arts and their scientific wit in their efforts to solve some of the deep-rooted riddles immanent in the specta- cular powers of regeneration exhibited by these inconspicuous animals. T. H. Morgan and C. M. Child have been among the foremost in this research work, and some far-reaching hypotheses regarding morphogenesis in general have emanated from this work, such as the well-known gradient hypothesis of Child. The tide of research on planarian regeneration has flowed and ebbed, as in most other scientific disciplines. Now we are in a period of rising water, thanks in part to the brilliant work carried out in Wolff's laboratory in Strasbourg. The two out- standing results are (1) Wolff's and Dubois' (1947) and Dubois' (1949) demonstration that neoblasts, totipotent embryonic cells, form the regeneration blastema and hence are responsible for the rebuilding of the missing parts, and (2) Wolff's and Lender's (1950, 1 951) finding that eye-formation in Polycelis depends on the presence of head ganglia. Other lines of approach have been followed in my laboratory for several years; one of these I should like to outline before you to-day. We know from the earliest experiments, notably those of Morgan (1902), that in some planarian species every part of the body has the power to regenerate a whole animal ; thus every part of these species is able to regenerate a head from an anterior wound. We may therefore ask: why do not many heads regenerate from the anterior wound of a transected animal? Figures 1 and 2 show some instances of heads formed at rather unexpected places; in the second instance regeneration occurs in spite of the fact that the old head was not cut away — which, incidentally, shows that no inhibitory force for head-regenera- tion can travel through adult tissue. The heads at the 'arms' in Figure 1 and the head in the window in Figure 2 are regenerated at a much lower speed than a median head from an anterior transverse cut. This suggested that time was involved in regeneration of only one head from a surface where the potentiality of plural head-regeneration was present. 121 H. V. BRONDSTED Accordingly, several sets of experiments were carried out with the purpose of find- ing out the rate of head-regeneration from various parts of the body. The experiments were made by cutting the animal in pieces according to a certain pattern and noting the time necessary for the pieces to regenerate heads. (Brondsted, 1946; A. and H. V. Brondsted, 1952). In this way it was found that there existed a static time-graded regeneration field, different for each planarian species so far investigated. Figure 3 shows the extent and intensity of the field in Bdellocephala. The field is static in the unwounded animal, and of course only displays itself after a cut has been made. Figure 1 . Bdellocephala punctata after decapitation. The median part has been re- moved; eyes are regenerated at both 'arms'. (Brondsted, 1946.) Figure 2. Bdellocephala punctata. A quadrangular piece has been removed; a head is regenerated at the anteriorly directed wound surface at the posterior side of the 'window'. (Brondsted, 1946.) The significance of such a dynamic structure is apparent. When the rate of re- generation varies in the way shown, every cut will expose a surface in which some place has the highest regeneration rate ; this place is called the high-point. From here regeneration of the head starts, but in doing so it must at the same time inhibit neighbouring parts from exercising their ability to make heads themselves. Thus the time-graded regeneration field, together with an inhibitory action exercised from the high-point towards neighbouring parts, ensures that only one head is formed from the anterior-facing surface of a wound, and so leads to harmonious regeneration. In order to test this hypothesis some experiments were carried out, of which a full account will be published elsewhere. These experiments were designed to demonstrate the inhibitory influence which must pass from the high-point to the 122 The time-graded regeneration field in planarians sides before these have had time to start irreversibly along the head-determining line. A transverse section is cut out of a great number of Dendrocoelum; the cuts must be made at the same level in the time-graded field in all specimens. The segments are divided into five equal lots. In the first lot a lateral third is isolated by a longitudinal Figure 3. Bdellocephala punctata. The time-graded regeneration field; darkest shading represents highest regeneration rate. cut, as shown in Figure 4, 24 hours after the segment has been cut out of the animal. The second lot is handled in the same way after 48 hours, and so on until the fifth lot has had its lateral part cut away after 120 hours. We know that in Dendrocoelum a lateral third, when isolated, takes 7 days to regen- erate a head. If an inhibitory force emanates from the median high-point, the lateral ( ■ft ( lateral 1 lateral Z ZK fh • to '/* □ lateral 3 lateral 4 laterals 7Zh O 96h A fZOh k. Figure 4. Transverse anterior segments of Dendrocoelum. The signs are those used in the curves of Figure 5. Further explanation in text. piece, when isolated after a certain time, should now require more time to regenerate a head. Figure 5 shows the results of some experiments. It is clear that an inhibition actually sets in at some time between 72 and 96 hours after the segments were cut out. Lateral pieces isolated after this time require more time for head regeneration than pieces which were isolated during the first 72 hours. The distance which the inhibitory influence has to traverse is roughly about 500 microns; if the size of a neoblast is about 10 microns, then 50 cells transmit the 123 H. V. BR0NDSTED influence in, say, 80 hours, giving an average of 100 minutes per cell. This may be of future interest. There is, however, a further point which must be considered concerning the isolat- ing experiment. When the median high-point has regenerated a head on the anterior- facing surface of a transverse cut, the time-graded field will be restored in accordance 100 boars 150 ZOO 250 300 350 Figure 5. Eye-formation in lateral pieces of transverse segments q/Dendro- coelum. + median part ; • lateral parts isolated after 24 hours; [J after 48 hours; O after >72 hours; A after 96 hours; A after 120 hours. with the shortened axis of the animal ; therefore, when regeneration has proceeded for a certain time, the lateral parts of the segment just behind the new head will have reached a level in the time-graded field characteristic of the intact animal. Moreover, the regeneration processes have made the animal normal, the new head is no longer a blastema, the differentiation processes are nearly accomplished, and we know that inhibition from a differentiated head does not occur. It follows from these considera- tions that if the above-mentioned experiment were prolonged after 120 hours we arms arms arms arms arms ZW 48 hu 7Zbo 36/? & 720 hi Figure 6. Bdellocephala punctata. The median stippled parts of five lots of animals have been removed after the times indicated. Note that lot 4 at the time of removal already has regenerated eyes. 124 The time-graded regeneration field in planarians should expect a shortening of the time required for head-regeneration in lateral pieces isolated after 144, 168 hours, etc. This experiment was carried out on another species, Bdellocephala punctata, and in a slightly different way. Figure 6 shows the procedure which was adopted in order to see whether a connexion with the main body would make any difference in 100r + 100 hours 150 ZOO Z50 300 350 Figure 7. Eye-formation at ''arms' in Bdellocephala. • 'arms'1 isolated immediately after decapitation ; -f- arms isolated after 24 hours ; [f] after 48 hours; O after 72 hours ; after 96 hours ; A after 120 hours. the inhibitory effect found in Dendrocoelum. Figure 7 shows that it did not. A differ- ence in the time-relations appeared, however, owing to the higher rate of head-re- generation from the median high-point in Bdellocephala. Already after 96 hours eyes 2:. Figure 15. Planaria lugnbris. Two halves were reunited with a slight shift. A transverse cut was made through the fields as indicated in the left figure. The right figure shows the resulting normal animal; only the right eye is slightly bigger. 128 The time- graded regeneration field in planar ians purpose of the experiment is of course to cut across the time-graded field at different levels in the two halves. As a result of this treatment, a blastema was formed all over the anterior transverse wound. Three distinct types of regeneration followed. If the shift was slight a normal symmetrical head would regenerate, although the eye was a little bigger in the part of the blastema belonging to the half where the cut had hit a higher level; this indicates of course a faster regeneration rate (Figure 15). If the shift was severe, head-regeneration only occurred in the half in which the cut had hit a high level of the field; the head in the other half was inhibited (Figures 16 and 17). A Figure 16. Planaria lugubris. Two halves were reunited with a severe shift; the transverse line indicates the cut made after healing. Figure 17. Planaria lugubris. Head-regeneration only occurred in the right half after the opera- tion indicated in Figure 16. In a few instances, namely when the shift was intermediate between'slight and severe, each half of the common blastema regenerated a complete head (Figures 18 and 19) ; this was rather puzzling, but it is in fact as would be expected. Let us consider what we have learnt about inhibiting forces emanating from the high-point, and we shall see that the results of these experiments agree with the in- hibition hypothesis. If the shift has only been slight, the inhibiting force from the right half of the blas- tema will not have time enough to inhibit eye-formation in the left half before this one has carried its differentiation so far that it has not only made its own left eye but also has set up inhibition towards the right half, in which, accordingly, regeneration of a left eye is inhibited. 129 H. V. BR0NDSTED If the shift, however, has been severe, the half blastema with the high regeneration rate will have time enough to regenerate not only its own eye but also the symmetrical one, because no inhibiting force has been set up yet in the other half; this other half is under so strong an inhibitory force that, on account of its very slow regeneration rate, it has no chance whatever to differentiate eyes. Between these two conditions there must be one in which the relations between the two halves of the blastema are such that both of them may get time enough to make A Figure 18. Planaria lugubris. Two halves reunited with moderate shift. A transverse cut was made in the foremost part of the right piece hitting the field at a high level, but in the left half hitting the field at a some- what lower level. Figure 19. Planaria lugubris. The two-headed chimera result- ing from the operation indicated in Figure 18. not only their own eye but also a symmetrical one before the inhibiting influence from the other half has reached them. If an amphibian egg in the two-cell stage is transected into two separate cells, provided the first cleavage furrow has taken place near the median plane, each of them will regenerate the missing part, and twin embryos are formed. It is just the same when a planarian is split longitudinally: two worms will come forth. It is also true that if one of two amphibian blastomeres is killed by cautery, but otherwise left in its old position as a symmetrical part of the cleaved egg, the living blastomere will continue its development into a half embryo. That is to say, the killed blastomere contains inhibitory forces exercising their influence for a long period of time. 130 The time-graded regeneration field in planarians These are the chief facts so far discovered concerning the time-graded regeneration field in planarians. As to the nature of the field I have only a few suggestions. It may be that the struc- tural foundation is the nervous system; I do not think so, however, because there seems to be no correlation between the pattern of the nervous system and that of the field. More probable is the notion that the quantity of neoblasts determines the rate of regeneration. Some observations support this view, others do not. The question can of course only be answered by counting the neoblasts; this counting is being done in my laboratory, but I can as yet give no figures. But even if the quantity of neo- blasts were to determine the rate of regeneration, we are still ignorant of the factors which determine a strict species-specific distribution of neoblasts, which otherwise can easily wander freely about in the planarian body. Here it might be suggested that some diffusable substance from the head ganglia attracts the neoblasts ; such a mechanism might account for a caudally tapering concentration of the neoblast population. The facts so far revealed about the time-graded regeneration field have led me to some considerations concerning three major problems in morphogenesis: polarity, inhibition, and gene-action. POLARITY We perceive polarity in single cells and in whole multicellular organisms. This has led some authors to postulate polarity in every single cell in the organism, but I think without justification. In the intact planarian body the neoblasts cannot be polarized. Neoblasts from a certain region may migrate to an anterior wound and regenerate a head ; or they may migrate to a posterior wound and regenerate a tail- tip; they may also migrate to a lateral wound and regenerate missing side parts of the body. The neoblasts are totipotent. The neoblasts are compelled to start regeneration in a certain main direction; the directive force must come from the remaining part of the body. For instance in Planaria lugubris a blastema formed at an anterior-facing wound surface will always start by making a head, irrespective of the level along the main axis of the body. In the same way, a blastema at a caudal wound surface will always start by making a tail-tip, also irrespective of level. Here polarity displays itself. Exceptions will be dealt with later. What is the nature of the mechanism ? We know that the neoblasts forming the blastema are totipotent. We know that in the intact body they are under control and are inhibited from displaying their potentiality. But in an anterior blastema, for instance, the neoblasts are freed from inhibition by the forepart of the body, which was removed by the cut, but they are not freed from inhibition by the remaining hindpart of the body. The same holds true — mutatis mutandis — for a posterior blastema. The blastema is to be compared with the anterior or posterior half of a blastula not yet determined. Hence the two kinds of blastemata will act accordingly, that is, they will start morphogenesis at opposite ends of the main axis, comparable with the most animal and most vegetal part of the egg. I cannot help thinking of the well- known double-gradient hypothesis of Runnstrom concerning the echinoderm egg. There we have a close resemblance to the phenomenon dealt with here. 131 H. V. BR0NDSTED If we cut out a narrow transverse section from the middle part of the planarian body, we always get a head anteriorly and a tail posteriorly; if we imagine the narrow section made so short as to vanish but still having neoblasts available, we should find that we had a cluster of totipotent cells in competition, the anterior half of them 'animalized', the posterior half 'vegetalized', and then the double gradient of Runn- strom would be at work. Now, every comparison has its weak point, and this one too. In the planarian blastema the polarity is imposed by the remaining adult body; in the egg the polar- ity is imposed on the oocyte in a manner as yet unknown. But the analogy shows us, I think, that the polarity of the planarian body is a structure derived directly from the oocyte. Here the time-graded field enters into the discussion. The Janus-head or 'Janus-tail' has always puzzled morphogenetecists. We know that polarity may be reversed by external stimuli in hydroids : oxygen, pH, light, electric current, etc. But why should very short transverse segments from the forepart of the planarian body often regenerate heads at both anterior and posterior surfaces ; and why should transverse segments from the hindpart often regenerate two tails ? It should be emphasized that only very short segments regenerate Janus-heads or Janus-tails. It should also be pointed out that Janus-heads are made only in the anterior part of the body, Janus-tails only in the posterior part, and neither of them in the mid-part. It must further be borne in mind that in an ordinary transverse segment from the forepart the head-blastema is the first to be differentiated, but in a posterior segment the tail-blastema. Therefore — again as in the echinoderm egg — the forepart is more 'head-minded', the posterior part more 'tail-minded'. This is probably due to the structure of the nervous system. A very narrow transverse segment from the forepart contains a part of the time- graded field with a high head-building rate. The totipotent cells form the anterior and posterior blastemata almost equally fast. I suggest that the speed in the fore- part of the time-graded field is so great that the totipotent neoblasts have had time to direct themselves into head-formation, also at the caudal wounds, before inhibition from the very small remaining part of adult tissue has got a chance to inhibit, the more so as such a narrow strip of tissue is nearly disarranged by the massive migra- tion of the neoblasts. The same may of course be said — mutatis mutandis — of posterior narrow sections giving rise to Janus-tails. In mentioning the manifestation of polarity in the blastema I have not used such words as organization, organization forces, induction and the like. I must admit that I am not very enthusiastic about such notions. It seems strange to me that every level of the body should be able to 'organ- ize' or 'induce' head-formation in an anterior blastema; and moreover that every level should be able to discern whether to make a head or a tail. It seems more prob- able to me that lack of proper inhibition gives the blastema its main direction of determination. INHIBITION Wolff and Lender (1950) and Lender (1951) have shown that the first structures to be regenerated in an anterior blastema are the head ganglia. Ribonucleic acids are conspicuous elements in the nerve cells; RNA is also a conspicuous element in the 132 The time-graded regeneration field in planarians neoblasts; they are strongly stained by pyronine and by Einarson's gallocyanine. It has been shown by my wife and myself that RNA accelerates head-regeneration in starved planarians. (A. and H. V. Brondsted, 1953). All this opens up certain possibilities concerning the mechanism of morphogenesis in the planarian blastema. Now Brachet has proved that RNA accumulates in the dorsal part of the amphibian germ. This led me to suggest that the vertebrate germ may well be regarded as a time-graded morphogenetic field analogous with the regeneration blastema. We know from a great number of experiments that a vast assortment of stimuli may evoke neurulation in the ventral epidermis of the young amphibian gastrulae; moreover, Barth (1941) has shown self-differentiation in explanted ventral ectoderm; this proves that the ventral epidermis has potencies of the same kind as the dorsal one. Could it be that normally it does not reveal these potencies because its rate of cytoplasmic reactivity is too slow, so that inhibition from the dorsal parts suppresses its tendency to differentiate? It may well be, in induction experiments, that contact with presumptive entomesoderm and with various metabolites accelerates its activi- ties so as to reach so high a level of reactivity towards gene-action and hormones produced by gene-activity as to be able to escape inhibitory influences. This hypo- thesis might be tested experimentally in explants. The current hypothesis, worked out by Holtfreter, Brachet and others, is that some sort of blockage exists in the ventral epidermis ; if the blockage is removed then neurulation follows. I think this hypothesis is right, and I only suggest that the block- age is imposed on the ventral epidermis by the inhibitory influence from the far- advanced dorsal epithelium, the 'high-point'. This reasoning leads to a working hypothesis of a serological nature. Long ago I suggested (Brondsted, 1936) a mechanism for the sorting out of cell types in the reconstitution bodies of freshwater sponges pressed through bolting silk. Four main cell types could be discerned in the dissociated cell material. The results were confirmed shortly after by Brien (1937). The problem was to elucidate how the various cell types regained their proper situations so that a working system could be re-established. While watching the movements of the isolated cells at the bottom of the dish I very often saw a curious phenomenon : when two cells met, then one or both might wriggle and send out pseudopodia at a far greater speed than when not in contact; the phenomenon was photographed by the time-lapse technique. Moreover, when two cells touched one another, one of them might throw out a pseudopodium with explosive force, as shown in Figure 20. Here, surely, one might speak of negative cytotaxis. I therefore suggested that such movements, arising from mutual incompatibility between different cell types, might well be conceived as morphogenetic movements leading to reassortment. I regret that I have had no time to pursue this very promising problem. It was therefore with much pleasure that I found that Holtfreter later (1947) adopted similar views. I think that this problem is connected with far-reaching lines of research concern- ing serological differences arising during early embryogenesis. I have not time here to go deeper into the matter. A very comprehensive and clear survey has been given by Woerdeman (1953). 133 H. V. BR0NDSTED I venture to suggest that serological incompatibility is at the root of negative cytotaxis. I further venture to suggest that when a cell or a group of cells during early morphogenesis has started determination in a fixed direction, it builds up a serological equipment characteristic of the cell or group of cells, and thereby exer- cises negative cytotaxis on neighbouring cells which have not yet reached a level enabling them to start differentiation; these neighbouring cells are inhibited from differentiating along the same lines. It should be possible to test this working hypo- thesis by the explantation technique. According to this hypothesis inhibition is a question of serological nature; it is transmitted from cell surface to cell surface. The response of a cell to the stimulus Figure 20. Spongilla lacustris. Photomicro- graph of two cells touching one another; one is giving off a pseudopodium with explosive force (X 1,000). (Brondsted, 1936.) from its neighbour depends upon its reactive state. This in turn is determined by the situation of the cell in the time-graded morphogenetic field. And this considera- tion brings us to the concluding part of my paper. THE RELATION BETWEEN GENE-ACTION AND THE TIME-GRADED MORPHOGENETIC FIELD It has always puzzled embryologists that cells presumed to contain the same genome should differentiate differently. Even for this there is no conclusive proof, so long as it is not possible to cause differentiated cells to dedifferentiate into a totipotent condi- tion from which they might then regenerate all parts of a new organism, including the germ cells. The same problem of differentiation exists both for embryos and for regenerating blastemata. However it is difficult to transfer our notions of evocation and induction from embryogenesis to morphogenesis in the blastema. In an embryo provision is made for the unequal distribution of substances to the blastomeres, so that they are different from the start. On the other hand in the blastema all the cells are at first alike, and any induction or evocation must be initiated after the wound has been inflicted. It 134 The time-graded regeneration field in planarians is very difficult to imagine that head-inducing capacities should exist at several cell levels of the body when the wound faces forwards, but tail-inducing capacities at the same levels when the wound faces backwards. It is not known what force imposes the time-graded field on the blastema, and drives the cells at the high-point to differentiate more quickly. However it is possible that crowding influences their metabolism, perhaps for instance through a deficiency of oxygen. The blastema derives its polarity from the adult tissues. In the words of A. E. Needham (1952) concerning the determination of the blastema, 'There remain a number of morphogenetic difficulties, however, which cannot be explained by Child's simple theory, and will ultimately demand a more sophisticated scheme with more emphasis on qualitative gradients, and on multilateral competition, rather than on unilateral dominance — a democracy rather than a monarchy. The demo- cracy is not one of anarchic equality, but one in which every section normally ensures its appropriate self-expression.' inhibihon Figure 21. Schematic representation of cell clusters in a transverse section of a blastema information. Explanation in text. It is now well established that the effects of genes become apparent — whether morphologically, physiologically or biochemically — successively in time. This has been shown by Hadorn (1948) and Poulson (1945) on lethal genes in Drosophila. In the following working hypothesis, the differentiation of the blastema is inter- preted in terms of successive gene actions and of inhibitions exercised by the median parts over the more peripheral parts of the blastema. Centrally placed neoblasts (1 in Figure 21) differentiate more quickly, that is to say, their cytoplasm more quickly reaches such a state as to respond to brain-determining gene actions. In differentiating they inhibit the neighbouring cluster of cells (group 2) from respond- ing to brain-forming gene actions. The next gene action in time is eye-determining, and to this these adjacent cells now respond. Neoblasts later in the order of differenti- ation are inhibited from forming brain and eye, and so form muscle (group 3), intestine (group 4), or do not differentiate (group 5). These last remain totipotent. This hypothesis is represented schematically in Figure 22. Geneticists are now laying more and more stress upon the conditions in the milieu necessary for the unfolding of gene action. Beadle and others have stressed the import- ance of the cytoplasmic state of single cells, and the work of Bonner (1952) and Sussmam (1952) on the slime mould Dictyostelium suggests the same principle of cytoplasmic reactivity. In this connexion we must clearly consider the conditions in the time-graded morphogenetic field. 135 H. V. BR0NDSTED Various obvious simplifications have been made in this discussion; interaction of genes has been left out of consideration; and those who study such phenomena as eye colour in Drosophila will stress the complexity of gene action and the influence of hormones. However, planarians are very primitive organisms, and are not so far re- moved from cell colonies as to make impossible the idea that in them genes act to a large extent directly upon the cytoplasm of the neoblasts. The time-graded morphogenetic field and the gene actions are delicately balanced, and it is not surprising that irregularities should sometimes occur. For instance, occasionally the cell cluster to the left of the brain (group 3 in Figure 2 1 ) forms eye tissue instead of muscle. Several extra eyes may be produced under the influence of LiCl (Brondsted, 1942). Waddington (1947, p. 47) has pointed out that 'during a / $ . <$ \ brain ^ -^ ^» \ eye 1 V -& ? \ scie\ « testine\ 1 % \ V 1 1 \ \ « eye 0ENE totipotent stage Figure 22. Schematic representation of the fate of a cell lying either in level 1. 2, 3, 4 or 5 of the time- graded field as set forth in Figure 21. period of competence there may be more than two alternatives open to a tissue'. The heteromorphoses reported by Waddington (1947) affecting the antenna of Drosophila and the well-known cases in Crustacea and primitive insects affecting leg, eye-stalk, and antennal regeneration, may be due to a similar lack of adjustment, although in these higher organisms hormones probably play a major role in the pro- cess of differentiation. The postulated time-graded field may also break down in the egg. At least I am strongly inclined to interpret Witschi's (1922, 1952) well-known experiments with teratomes arising from over-ripe eggs as a breakdown of the field. It would be of great interest to grow an isolated blastema, and see what it produces. So far this has not been possible, because it has always cytolysed. In further investigations we are especially interested in biochemical aspects of regeneration, including amino-acids in various parts of the blastema, and the effects 136 The time-graded regeneration field in planarians of vitamins on differentiation, with special reference to the connexion between RNA, amino-acid metabolism, and protein synthesis. There is much evidence, summarized by Needham (1952), that in nerve cells morphogenesis is linked with RNA content and protein synthesis. It might be suggested that gene activity directs protein syn- thesis when this has reached certain levels, different for the different cell clusters in the time-graded blastema. In conclusion, I stress the need for biochemical studies of the kinetics and time relations of enzymatic processes concerned in morphogenesis, and of morphogenetic studies of the time relations of the differentiation of totipotent cells from different parts of the embryo, such as we are trying to make on different parts of the time- graded morphogenetic field. SUMMARY Regeneration experiments with planarians have revealed the existence of a time- graded regeneration field. It has been shown that in the regeneration blastema there exists a median high-point, or rather a right and left one close to the mid-line, which starts earliest in making head structures. It has been shown that inhibiting forces emanating from the high-point prevent lateral parts of the blastema from differentiat- ing into ganglia and eyes, thereby securing a harmonious regeneration. It has been made probable that the problem of bilaterality may be understood on the basis of the time-graded field. It is suggested that polarity is in some way con- nected with inhibition and the time-graded field. It is suggested that inhibition is a chemical mechanism of a serological nature producing inability in neighbouring cells to reach the same cytoplasmic reaction level as that of the cell from which the inhibition emanates. It is suggested that the time course of inhibition is correlated with the temporal succession of gene-activity. It is suggested that RNA plays a major role in the setting up of the time-graded regeneration field and in doing so is the basis of the time-dependent processes in the blastema. Some suggestions are made for experimental procedures to test the working hypo- thesis here set forth. REFERENCES Barth, L. (1941). Neural differentiation without organizer. J. exp. %ool. 87, 371-382. Bonner, J. T. (1952). The pattern of differentiation in amoeboid slime molds. Amer. Nat. 86, 79-89. Brien, P. (1937). La reorganisation de l'eponge apres dissociation, filtration et phenomenes d'involution chez Ephydatia fluviatilis. Arch. Biol., Paris 48, 185-268. Brondsted, A and Brondsted, H. V. (1952). The time-graded regeneration field in Planaria (Dugesia) lugubris. Vidensk. Medd. dansk. ncturh. Foren. Kbh. 114, 443-47. Brondsted, A. and Brondsted, H. V. (1953). The acceleration of regeneration in starved planarians by ribonucleic acid. J. Embryol. exp. Morph. 1, 49-54. Brondsted, H. V. (1936). Entwicklungsphysiologische Studien uber Spongilla lacustris (L). Acta zool., Stockh. 17, 1-98. 137 H. V. BR0NDSTED Brondsted, H. V. (1939). Regeneration in planarians investigated with a new trans- plantation technique. Biol. Medd. Kbh. 15, No. 1, pp. 1-39. Brondsted, H. V. (1942). Experiments with LiCl on the regeneration of planarians. Ark. £ool. B 34, 1-7. Brondsted, H. V. (1946). The existence of a static, potential and graded regenera- tion field in planarians. Biol. Medd. Kbh. 20, 1— 31. Dalyell, J. G. (1814). Observations on some interesting phenomena in animal physiology exhibited by several species of Planariae. Edinburgh. Dubois, F. (1949). Contribution a 1' etude de la migration des cellules de regeneration chez planaires dulcicoles. Bull. biol. 83, 213-83. Hadorn, E. (1948). Gene action in growth and differentiation of lethal mutants of Drosophila. Symp. Soc. exp. Biol. 2, 177-95. Holtfreter, J. (1947). Structure, motility and locomotion in isolated embryonic amphibian cells. J. Morph. 79, 27-62. Lender, T. (1951). Sur les proprietes et l'etendue du champ d'organisation du cerveau dans le regeneration des yeux de la planaire Polycelis nigra. C.R. Soc. Biol., Paris 145, 1 2 1 1 . Morgan, T. H. (1902). Growth and regeneration in Planaria lugubris. Arch. Entw- Mech. Org. 13, 179-212. Needham, A. E. (1952). Regeneration and wound healing. Methuen's monographs. London, pp. 1-152. Poulson, D. F. (1945). Chromosomal control of embryogenesis in Drosophila. Amer. Nat. 79, 340-63. Sussman, M. (1952). An analysis of the aggregation stage in the development of the slime molds, Dictyosteliaceae. Biol. Bull. Woods Hole 103, 446-57. Waddington, C. H. (1947). Organizers and genes. Cambridge University Press. pp. 1-158. Waddington, C. H. (1950). Genetic factors in morphogenesis. Rev. suisse £ool. 27, Suppl. I, 153-68. Witschi, E. (1922). Uberreife der Eier als Kausaler Faktor bei der Entstehung von Mehrfachbildungen und Teratomen. Verh. naturf. Ges. Basel 34, 33-40. Witschi, E. (1952). Overripeness of the egg as a cause of twinning and teratogenesis : a review. Cancer Res. 12, 763-86. Woerdemann, M. W. (1953). Serological methods in the study of morphogenesis. Sym. biochemical and structural basis of morphogenesis, 1952. Arch, ne'erl. %pol. 10, Suppl. 1, 144-62. Wolff, E. and Dubois, F. (1947). Sur une methode d'irradiation localisee permet- tant de mettre en evidence la migration des cellules de regeneration chez les Planaires. C.R. Soc. Biol., Paris 141, 903-06. Wolff, E. and Lender, T. (1950). Sur le role organisateur du cerveau dans la regeneration des yeux chez une Planaire d'eau douce. C.R. Acad. Sci., Paris 230, 2238-39. 138 The cell physiology of early development • The time-graded regeneration field in planarians Discussion OF PAPERS BY (i) C. H. WADDINGTON AND (2) H. V. BR0NDSTED Chairman: E. ^euthen C. D. Darlington. With regard to the classification of genetic particles, it is important to remember that the biological distinctions between virus and plasmagene (gene- initiated or otherwise) break down at the chemical level. The most recent evidence is especially significant. Kenneth Smith (1952, Biol. Rev. 27, 347-357) has found that cell constituents of the sugar-beet at a certain stage of its development propagate themselves without limit in the cow-pea. They behave like a virus, or provirus I should say, as infection is artificial. Thus a particle or a chemical entity determined by heredity can in the course of development become the means of infection. E. W. Temm. The sugar-beet and cow-pea virus described by Professor Darlington seems to imply a transmission of autonomous particles, capable of protein synthesis, from one species to another. In this connexion it is of interest that cellular proteins from leaves of different species are very similar with regard to their constituent amino-acids; we have recently shown that the similarity extends to the cytoplasmic and chloroplastic proteins of the same species. It follows that the amino-acids re- quired for synthesis of cytoplasmic proteins may not vary very much from species to species, and this may in part account for the possibility of interspecific transmis- sion of the type discussed. It is possible that variation of infectivity depends on the activity of the leaf in protein synthesis at different stages of development. C. H. Waddington. The example of the cow-pea virus shows that certain embryonic proteins, when transplanted so as to become associated with a foreign nucleus, can multiply in a more or less uncontrolled manner. I agree with Darlington that this implies that the problems of normal cellular differentiation and of pathological virus-like infections belong to the same general area of developmental-genetical dis- course. But it is to my mind much less clear that there is any very close parallel be- tween the processes which bring about the orderly development of locally differen- tiated regions in an embryo and those which underlie the usually pervasive and un- controlled alterations of cell type caused by a virus infection. L. Rinaldini. The narrowing down of potentialities that accompanies differentiation suggests a progressive loss of synthetic properties coupled with the appearance of new specialized functions. This loss, if genetically determined, may mean that some genes are systematically inhibited during embryogeny. In Professor Waddington's scheme of cyclic reactions the reaction products (P) would on mass active considera- tions be expected to depress the formation of their precursors, but if P were removed from the cell the reaction would be accelerated, and a type of cell would be obtained which would produce more and more P. By cell division more cells with identical metabolism could be produced, but the excess P shed into the surroundings would inhibit cells from developing in the same direction. In this way we have two parallel 139 H. WADDINGTON ' H. V. BR0NDSTED mechanisms combined, one for preserving identity and the other for ensuring diver- sity. By such a mechanism some of the experimental results obtained in planarians by Professor Brondsted, such as double gradients, space-time-graded inhibition, and blockage, might conceivably be explained. It might also offer a new view point on the problem of unspecific inductions, as the unspecific stimulus would simply have the effect of removing or destroying the inhibitor. These views are put forward in a purely tentative way, and no doubt many alternative explanations could be sought. C. H. Waddington. I agree with Dr. Rinaldini that it is quite possible that differentiat- ing cells produce diffusible substances which inhibit neighbouring cells from entering the same path of differentiation. A hypothesis of this kind has recently been proposed by Dr. Merrill Rose. However, although possible, it is not in my opinion very prob- able that such inhibitory substances play an important part in early determination, though they may more probably do so in the growth of already differentiated tissues at later stages. In the early stages of development cells differentiating in one way may stimulate their neighbours to differentiate in the same way (cf. 'homoiogenetic induction'). It may be mentioned that only if the substance P acts autocatalytically, to encourage the production of more P, does one obtain an exaggeration of an initial difference in conditions; this would not happen on ordinary mass action principles. 140 \iu I LIBRA Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis., as induced by temperature changes1 by ERIK ZEUTHEN and OTTO SCHERBAUM fyofysiologisk Laboratorium, Kobenhavns Universitet INTRODUCTION Nature has supplied us with a few cases in which neighbouring cells in a cell com- munity are in phase so that they more or less simultaneously pass through the only recognizable part of the cell cycle, namely cell division. Stimulating examples are presented in d'Arcy Thompson's (191 7 and 1942) book, and in the review of Sonnen- blick (1950) dealing with early embryonic stages in the insect egg. The fertilized insect egg, like many other syncytia and plasmodia, shows a perfect synchronization of nuclear division for many generations, and, as a consequence, the first cell division which cuts out the blastoderm of about 2,000 cells is synchronous in the whole embryo. Apparently, chemical situations identical throughout a syncytium or a Plasmodium induce nuclear and cell division. In cell suspensions cell borders may act as barriers to the diffusion of controlling agents; and in tissues where cells occupy relatively fixed positions in a medium which is not mixed, distance alone would prevent chemical interaction between any considerable number of cells. Whenever synchrony of division is observed in a tissue it is therefore limited to groups of cells all within a short distance of each other. This observation is known to many workers in histology and tissue culture. It may be significant that in spermato- genic tissues where cells line a lumen in which free diffusion and even some mixing may take place, we have the prettiest cases — still limited to a small mass of cells — of synchronous growth and division. THE GOAL AND THE WORKING HYPOTHESIS For the reasons mentioned above we are in a difficult position when it is a question of relating growth to the basic phenomena of synthesis and multiplication in the cell cycle. The goal we had in mind was therefore that of establishing a synchronized system in which the cell cycle could be studied on aliquots representing successive stages in the cycle. Such a system would serve as a useful substitute to the few already known, such as egg material and plant spores (Erickson, 1948). The introductory 1 This work was supported by grants from Rask-0rsted Fondet and from the Eli Lilly Foundation. 141 ERIK ZEUTHEN AND OTTO SCHERBAUM remarks clearly indicate the possibility of inducing synchronous cell divisions by con- trolling the chemistry of the medium. Nevertheless, in view of our present ignorance as to the best way of making such an approach, we decided on changes of temperature as a possible way of changing the relative concentration in the cells of a great number of metabolic intermediaries, which might include one or more agents necessary to enable the cell to pass from one phase of the cell cycle to the next. If in a mass culture of a micro-organism there is a random distribution of all stages in the cell cycle, we shall find that as long as the organism is growing exponen- tially a constant fraction of the cells will be in division. We observe a steady state in which there is an equilibrium between cells entering and cells leaving the division stage. Transitory changes in the number of dividing cells divided by the total num- ber of cells, termed hereafter the 'division index', may indicate either that for some reason the time which the cells spend in division changes relative to the time occupied by the whole cycle, or that cells have accumulated into groups which tend to divide together and possibly pass through one or more full cycles together. In our search for group formation we have accepted the division index as our guide, division indicating all stages from onset of furrowing until separation of the two cells. Decision between the two possible interpretations can only be made if multiplica- tion is estimated by actual cell counts. Let us then accept as a working hypothesis that some phase in the cell cycle has a higher temperature coefficient than any other. Indeed, Ephrussi's work (1926) on dividing sea-urchin eggs had indicated a case where this is so. Such a phase will be more sensitive to temperature changes than any other. With lowering of the tempera- ture cells in this phase will tend to drop behind and unite with cells behind them in the cycle, whereas with increase in temperature they will tend to catch up with cells in front. In both cases we should get group formation, detectable only at the time when the group passes through the division stage. If experiments were to confirm our working hypothesis thus far, the next step would then be to transfer the culture back to the first temperature, after a time shorter than the duration of the cell cycle at the new temperature. When our group was again in the sensitive phase the tem- perature would again be shifted. For every such periodic change in temperature the original synchronized group should become larger, and in the end the whole culture should become synchronized. The first part of our working hypothesis was confirmed by experiment; the second was never put to serious test. It is mentioned here because a number of our experiments might otherwise seem curious. THE EXPERIMENTAL ORGANISM For our experimental organism we chose the ciliate protozoon Tetrahymena pyriformis, because it grows readily on dissolved and fully defined media, as reported by Kidder and Dewey (1951). So far, however, we have grown our cells in 2 per cent, proteose peptone (Difco) plus 1 per mil. liver extract (Wilson Lab.) with salts as in Kidder and Dewey's synthetic medium A. The cultures were shaken and aerated with a flow of air which was passed over them. Each culture contained 150 ml. of medium and the total synchronized population represented about 100 mg. wet weight of cells. 142 Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis Another advantage of Tetrahymena is that the cell cycle is so short (2J-2I hours at the optimum temperature of 28-290 C.) that several cycles can be studied in succes- sion in the course of a day. Also the cell is reasonably big, being 50-70 microns in length. Some will consider it a serious limitation that the cytology is relatively little known and furthermore that it is atypical of cells in general. As in other ciliates there is an amitotic macronuclear and a mitotic micronuclear system. Our cells (the Lwoff strain), like other strains of Tetrahymena which have been grown in pure culture for a long time, do not possess a micronucleus and are therefore amitotic and asexual. Recently, however, mating types of Tetrahymena, all of them having a micronucleus of course, were isolated by Elliott and Gruchy (1952) and were further described by Elliott and Hayes (1953). Chromosomes have been demonstrated in the dividing micronucleus of Tetra- hymena, as in other ciliate micronuclei (Elliott and Hayes, 1953; Faure-Fremiet, 1953; Sonneborn, 1949). In the macronucleus of ciliates a ribonucleic-deoxyribonu- cleic acid system is present, as in other kinds of cells, but morphologically it is organ- ized in a very special way. In the macronucleus of the ciliates clearcut volume changes (Popoff, 1908) and structural changes, simultaneous with the division of the micro- nucleus and thus preceding cell division, can be revealed by the use of nucleic acid stains (Faure-Fremiet, 1953; Sonneborn, 1949). CHARACTERIZATION OF STAGES The successive stages in the cell cycle may also be characterized by other means in addition to the standard cytological ones, for instance by possible differences in the capacity of the system for growth. A single stationary phase Tetrahymena cell was introduced into a microrespirometer and the rate of respiration was accepted as a measure of the respiring mass present at any time. The cell synthesized and multi- plied in the respirometer. The results of a few runs (Zeuthen, 1953a) are shown in Figure 1. From the onset of furrowing in one division until 10-20 minutes before the next division there is a linear increase in mass ( ~ respiratory rate) . In the account which follows this period will be called the 'synthetic phase'. Then follows a period lasting 10-20 minutes with no further increase in mass. This will be called the 'pre- division period'. When division of the cell begins the macronucleus is already some- what stretched. A study of the literature on ciliates in general and on Tetrahymena in particular makes it exceedingly likely that the 'pre-division period' covers or partly coincides with the period of macronuclear swelling and reorganization referred to above. At the end of the pre-division period, or at the beginning of division, synthesis is resumed, and at twice the rate before 'pre-division'. Thus, on the basis of the curves given in Figure 1 , and for purely practical reasons, we have divided the cell cycle in Tetrahymena, growing on our media, into three phases: cell division coincides with the early part of the synthetic phase, and in the pre-division period there is a block to further rise in respiration. In support of this way of dividing the ciliate cell cycle into physiological phases, reference is made to a number of papers in which an attempt is made to follow cell volume from one division to the next. The situation is reviewed by Richards (1941) and by Wichterman (1953). In the present writer's opinion the overall picture given in their papers is very suggestive of that observed 143 ERIK ZEUTHEN AND OTTO SCHERBAUM in Tetrahymena, with rate of respiration used as a measure of mass. The paper by Chalkley ( 1 93 1 ) demonstrates that in Amoeba proteus (which divides by mitosis) the percentage rate of synthesis decreases as the cell approaches division. Therefore, 4 4 0** 3 I Kcells ' I 26&°-26.4°C o J 1 16 Cells £- Tetrahymena piriformis 27°C 5 16 cells J / ,- ^f *^"^ ^-*^_Li t-*-*-N Z7.1'-27.2'C ■-&-,---&-*■ £-3, -A + -a--TA---a-i 0 / § 0 2 6 8/0/2 fit /6 /8 20 22 2V 26 hours Figure 7. The continuous curve represents cell counts ; the broken represents volume of cells per unit volume of culture. curve of treatment multiplication sets in at a rate faster than in the untreated culture. (In this case no attempt was made to resolve multiplication into the steps in which it occurs.) Synthesis continues however at a slower rate than in the untreated culture. After 5 hours the multiplication rate decreases and synthesis speeds up, both to the rate before treatment. The relative position of the two curves 5 hours after end of treatment, as compared with the situation before heat treatment, indicates that nor- mal cell size has become re-established. In the case presented in Figure 7 the culture was diluted after the end of heat treatment. Otherwise it is not possible in one experi- ment to demonstrate these effects, particularly the return to the normal rate of syn- thesis and of multiplication many hours after end of treatment. We want to emphasize that the dip in cell count after dilution is atypical and may be due to an error made in the dilution. If this is correct the absence of synthesis in the first half-hour after dilu- tion may also be erroneous. The inverse relationship between rate of synthesis and 149 ERIK ZEUTHEN AND OTTO SCHERBAUM rate of multiplication in the hours after heat treatment was easily demonstrated even without dilution. Dr. Hoff-Jorgensen kindly made some DXA determinations for us using the method he is describing in this symposium. The data are included in Table I together with Table I Time, Total vol- ume of cells Average DXA fog.) in washed DNA fog.) DNA per hours Cells/ml. fol.) per ml. of medium cell volume (/xl. X IO"5) cells from 1 ml. per fjl. cells* cell fog. X IO"5) 0 32200 0 69 214 0 48 0 70 1 5 i 4 Heat treatment begin s 5i 48000 1 91 3 98 1 86 0 97 3 9 7i 45600 3 18 6 97 2 73 0 86 6 0 7! Heat treatment ends ioi 150000 4 13 2-75 508 1 23 34 12* 279000 7 43 2 66 718 098 26 i3i 558000 8 91 1 60 9 08 1 02 1 6 * DXA fog.) per /il. medium: o 01-0 015. cell counts and measurements of total cell volume per unit volume of the culture. The culture medium itself contains some DXA, but in concentrations of only 1-2 per cent, of what is found in the cells. We consider it most likely that the DXA in the cells represents synthesis and not just accumulation from the medium. (a) normal (b) heat-treated Figure 8. 150 Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis In Table I is given the DXA per total cell volume in the aliquots, the DXA per cell, and also the DXA per unit volume of cells. It will be observed that total DXA follows total cell volume rather closely throughout. This means that while the cells grow bigger during heat treatment there is a more or less proportional increase in the DXA content per cell. After heat treatment multiplication outweighs synthesis, but the volume per cell and the DXA per cell decrease proportionately. The corresponding morphological changes are indicated in Figure 8. Xormal (a) and heat-treated cells (b) were compressed to a standard thickness of 10 microns and photographed through the phase-contrast microscope. The outline of the cell body and of the macronucleus are shown as tracings. It is observed that the relative increase in size is about the same for the macronucleus as for the whole cell. It is therefore reasonable to assume that DXA is accumulating in the macronucleus of the heat-treated cell just in the same way as other specific cell components are accumulating in their natural loci. The situation is comparable to the state of polyploidy in cells dividing by mitosis. RESOLUTION OF GROWTH IN THE SYNCHRONIZED CULTURES INTO STEPS In Figure 7 an insert demonstrates that in all probability growth in cell volume increases more steeply after pre-division than before, thus confirming what is known for single cells of other ciliates cf. p. 143'. Respiration in the synchronized cultures follows a rhythmic pattern f Figure 9, open and full circles). As in the single un- treated cells 'Figure 1), the rate of respiration increases for some time during and after division, but it remains level for some time before division. Only one division [the third in the experiment represented with solid circles] is not in agreement, out of a total of ten divisions observed in the three experiments on synchronized cultures.) If we compare Figures 9 and 1 it seems necessary to conclude that the simple reason for the observed inverse relationship between synthesis and multiplication in the synchronized cultures (Figure 7) is that in the treated cells the synthetic phase is shorter than in normal cells, whereas the non-synthetic pre-division phase is un- changed, or perhaps even somewhat extended. Another fact to be observed in Figure 9 is that there is no clear-cut tendency for the rate of synthesis to accelerate throughout an experiment. This is very different from what was found in cultures started in respirometers with one untreated cell. As discussed before fZeuthen, 1953) the curves of Figure 1 can be interpreted as indicating that in the synthetic phase the rate of growth is controlled by units rather than by the increasing amount of material accumulating in each cell. In the pre-division stage the units ('synthetic centres'; double but they do not at the same time permit synthesis in the whole cell to occur. Synthesis is resumed only after the 'synthetic centres' have doubled, and they then control synthesis at a double rate. Why did we not also find the same situation in the heat-treated cells which divide fast after the end of treatment ? Perhaps because the big cell among other things is overloaded also with 'centres' controlling the rate of synthesis; these 'centres', like the rest of the cell, may increase in numbers but by no means double at even' pre- division. ItI ERIK ZEUTHEN AND OTTO SCHERBAUM 0.5 ■ 10 hours Figure g. Respiration in populations of Tetrahymena cells plotted on arithmetical scales against time in hours. Open and full circles: three experi- ments with synchronized populations. Upper curve: 10 cells initially present, about i oo cells after the experiment. Second curve from above : 1 1 cells initially present. Third curve from above : g cells initially present, 70 in the end. In the three curves frames indicate division periods observed in the divers. All curves are fitted by eye and in each case two broken lines represent ± Zrf2 The fourth curve (triangles) is a control run with 20 cells from an untreated population. A control run with empty diver is represented by crosses. (Experiments by Mr. H. Thormar, method of Zjuthen (1953).) DISCUSSION It is now time to consider what we actually do to the cells, when after growth at optimum or sub-optimum temperature we transfer them for the first time to the cold (70 C.) or to a supra-optimal temperature. In both cases the division index tends to drop after a time which is far shorter than the duration of the cell cycle at the new temperature. This shows that in both cases the rate of entry of cells into division is slower than that of exit from it. Apparently chemical processes occurring in the pre- division stage are exceedingly sensitive to temperature and have a narrower range of optimum temperature, with sharper decline on both sides, than processes under- lying other phases of the cycle. We might also say that they have relatively high tem- perature coefficients below optimum and relatively low above optimum. For this reason the chemical disturbances of cells exposed to cold or to heat might be related to one another, consisting perhaps of changes in the relative concentration of meta- bolites essential for cells to enter a division. Also recovery from exposure to cold or J52 Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis to heat follows the same pattern; cells prevented from dividing enter division with a delay which is much the same when measured at 22-24° C., irrespective of whether the cells have been exposed to low or to high temperatures (cf. Figures 2, 3, 4 and 5). The recovery time (90 ^ 10 min.) at 28-29° C. is three times as long as the interval between the individual heat shocks in our standard treatment. That is why cells never divide during treatment. However, they do synthesize, which they would not have done, or would have done exceedingly slowly, at continuous high temperature. This suggests that a high rate of synthesis is resumed very quickly after return from high to optimum temperature. Thus, the success of heat treatment seems to depend on this different rate of recovery of independent mechanisms for growth and for control of the entrance, first into pre-division, then into division. One more observation needs comment: even though apparently cold and heat block the cell cycle at some point prior to division, after return to constant optimum or room temperature the cells are in a stage which in time is long separated from the next division. This time is only slightly shorter than the duration of the cell cycle in the heat-treated cells (cf. Figure 5). Thus heat treatment, and perhaps also cold, may strike cells in a chemical situation typical of pre-division and leave them in a situation more typical of post-division. One reasonable suggestion is that intermittent heat treatment keeps cells in a chemical condition resembling that in a newly divided cell in which we may imagine that 'precursors of division' are minimal and the capa- city for growth is high. Another possibility is that during heat treatment the cells pass through several incomplete cycles, by-passing the division stage. The latter situation would resemble the one produced by colchicin and radiation in mitotic cells. A problem with which we are confronted and which is open for research with synchronized cultures is this: how do cells which have accumulated in themselves enough material for about 4 cells behave when an artificial block to division is suddenly removed? First of all, with the first division they do not spring directly into 4 cells, although very occasionally this has been observed. They divide in an orderly manner, first into 2, then into 4, and since synthesis occurs, divisions at the high after-treatment rate continue still further before normal cell size is re-established. The rate of synthesis after the end of treatment continues to be lower (about 1/2) than normal until normal cell size has become re-established. Thus, in Tetrahymena, we observe competition between synthesis and cell division. The fertilized egg re- presents an extreme case of such competition. In the ovary there is no division, only synthesis; however, fertilization removes a block to cell division, and divisions then follow each other in extremely rapid succession, while synthesis is completely sup- pressed during cleavage (cf. discussion by Zeuthen (1953b) and by Hoff-Jorgensen in this symposium). Thus, in this competitive interaction between tendencies for synthesis and for division we find the giant Tetrahymena cells produced in heat treatment to be inter- mediate between dividing eggs and log-phase cells. If we include also the ovarian, rapidly synthesizing, non-dividing egg we have a series which represents all situa- tions from complete ascendancy of the tendency to divide to absolute dominance of the synthetic machinery. In its normal cell cycle every growing and dividing cell switches to and fro between these two extremes. 153 ERIK ZEUTHEN AND OTTO SCHERBAUM By this time it will be painfully clear that we are not in the position to look back and point out exactly where we were right and where wrong in our working hypo- theses. The essential thing is that we got some of the results (Scherbaum and Zeuthen, J954) we were hoping for. In the synchronized cultures all those three phases are present into which we divided the cell cycle in normal Tetrahymena cells; only their quantitative relationships are somewhat changed. For bacteria, Hotchkiss (1954) has quite independently and almost at the same time worked out a method of syn- chronization based on very similar arguments and using temperature shifts. We have not made further reference to this work because this will be done by Dr. Maaloe (Maaloe and Lark, 1954), who is going to speak next. REFERENCES Chalkley, H. W. (1931). The chemistry of cell division. II The relation between cell growth and division in Amoeba proteus. Publ. Hlth. Rep., Wash. 46, 1736. Elliott, A. M. and Gruchy, D. F. (1952). The occurrence of mating types in Tetrahymena. Biol. Bull. Woods Hole 103, 301. Elliott, A. M. and Hayes, R. E. (1953). Mating types in Tetrahymena. Biol. Bull. Woods Hole 105, 269. Ephrussi, B. (1926). Sur les coefficients de temperature des differentes phases de la mitose des ceufs d'oursin [Paracentrotus lividus Lk.) et de YAscaris megalocephala. Protoplasma 1, 105. Erickson, R. O. (1948). Cytological and growth correlations in the flower bud and anther of Lilium longiflorum. Amer. J. Bot. 35, 729. Faure-Fremiet, E. (1953). Morphology of Protozoa. Amer. Rev. Microbiol. 7, 1. Hotchkiss, R. D., Cyclical behaviour in pneumococcal growth and transformability occasioned by environmental changes. Proc. nat. Acad. Sci. Wash. (In press.) Kidder, G. W. and Dewey, V. C. (1951). In Biochemistry and Physiology of Protozoa, Part 1, ed. by A. Lwoff. Academic Press Inc., New York. Maaloe, O. and Lark, C. G. (1954). A study of bacterial populations in which nu- clear and cellular divisions are induced by temperature shifts. (This Symposium.) Popoff, M. (1908). Experimented Zellstudien. Arch. exp. £ellforsch. 1, 245. Richards, O. W. (1941). The growth of the Protozoa, in Calkins and Summers: Protozoa in Biological Research. Columbia University Press. Scherbaum, O. and Zeuthen E. (1954). Induction of synchronous cell division in mass cultures of Tetrahymena piriformis. Exp. Cell Res. 6, 221. Sonnenblick, B. P. (1950). In Biology o/Drosophila, ed. by M. Demerec. John Wiley & Sons, Inc., New York. Sonneborn, T. M. (1949)- Ciliated Protozoa: cytogenetics, genetics and evolution, Ann. Rev. Microbiol. 3, 55. Thompson, d'Arcy W. (1917 and 1942). On Growth and Form. Cambridge University Press. Wichterman, R. (1953). The Biology of Paramecium. Blakiston Co., New York. Zeuthen, E. (1953a). Growth as related to the cell cycle in single-cell cultures of Tetrahymena piriformis. J. Embr. exp. Morph. 1, 239. 154 Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis Zeuthen, E. ( i 953^). Biochemistry and metabolism of cleavage in the sea-urchin egg, as resolved into its mitotic steps. Arch. need. £ool. 10, 31. (Presented at the symposium on the biochemical and structural basis of morphogenesis in Utrecht, 1952). 155 ERIK ZEUTHEN AND OTTO SCHERBAUM p ' mm ^ > V ^® 0 w (k ' ^^^ # ^**G 0 0 • S? • >> # fc. i • 'f; ! ft ^ * . < P *''-' 4L* * -.-» i k£« ^dt 100 n Plate i (b) 100 tx P/flfe i (c) Plate i. (a) Untreated Tetrahymena cells from a log-phase culture; (b) after 9 hours of treatment with intermittent heat shocks; (c) 90 minutes after return to constant optimum temperature. 156 Synchronous divisions in mass cultures of the ciliate protozoon Tetrahymena pyriformis Discussion Chairman: J. F. Danielli Muriel Robertson. In the protozoological laboratory at the Lister Institute we have repeated Dr. Zeuthen's work on the synchronization of cultures of the ciliate Tetra- hymena pyriformis (W) with complete success. The variations in temperature must be repeated an adequate number of times, and thereafter the organisms are kept at the optimum of 280 C. rather than at 24° C. The nuclear division of the Tetrahymena is being studied. There is no micronucleus in this strain. The macronucleus does un- doubtedly increase in size as compared with the untreated ciliates, but it does not at present appear that the number of chromosomes is increased. B. F. Folkes. These results suggest a very sharp temperature optimum for the synthesis of some metabolic precursor required for the pre-division stage. This implies not only a high energy of activation for these synthetic reactions but also one of two possi- bilities: (a) an inactivation of the responsible enzymes at temperatures above the optimum, or (b) the presence of some other reaction with an even higher energy of activation which is competing for the same substrates or removing the reaction pro- ducts. If we could distinguish between these possibilities we might be nearer the identification of the metabolic precursor. In the case of reaction (a), holding at high temperatures for increasing lengths of time should result in increasing periods of delay of the pre-division stage on returning the organism to optimum temperature. Have you any evidence of this ? E. £euthen. I think the evidence is that the holding of the organism at high tempera- tures for varying lengths of time does not influence the delay with which cleavage sets in after return to the optimum temperature. I agree that this makes hypothesis (a) less likely, and that therefore the possibility (b), or an even more complicated one, should be favoured. 157 A study of bacterial populations in which nuclear and cellular divisions are induced by means of temperature shifts by OLE MAAL0E and KARL G. LARK* Statens Seruminstitut, Kobenhavn A growing population of micro-organisms is a mixture of cells representing all phases of the division cycle. Studies on such a population, therefore, cannot lead to identification of the successive physiological and cytological states of a dividing cell, and very little can be learned about these states by observing individual cells under- going division. Many problems concerning cell multiplication, which cannot be studied on single cells or on conventional cultures, might be solved by studying samples from a culture in which the cells were induced to divide more or less simultaneously. General considerations of this kind lead us to investigate possible means of producing the desirable 'phasing' or 'synchronization' of cell division in bacterial cultures. Our choice of means was greatly influenced by the recent work of Scherbaum and Zeuthen (1954) and of Hotchkiss (1954), who have used temperature changes to obtain phasing of cultures with respect to certain physiological characters. Our approach is closer to that of Hotchkiss than to that of Zeuthen; and, since Zeuthen's studies are presented elsewhere in this symposium (Zeuthen and Scherbaum, 1954) we may at this point draw attention particularly to Hotchkiss's work (1954). In his experiments, periodic changes in rate of division, as well as in susceptibility to change into a streptomycin-resistant form, were induced by exposing the cultures to a temperature considerably below the normal, and optimal, growth temperature. Hotchkiss mainly used shifts between 37 and 250 G, and the same interval was adopted for our experiments with Salmonella typhimurium. The strain we have employed has properties of particular value for the present investigation: firstly, young cultures of this organism consist of well-isolated cells and cell pairs with no tendency to clumping; secondly, the strain interacts with a certain bacteriophage thereby becoming changed in a characteristic and easily recognizable manner (Boyd, 1950; Lwoff, Kaplan and Ritz, 1954); and, finally, abundant growth can be obtained even in media composed of inorganic salts plus a simple carbon source such as glucose. The first of these properties has permitted * Fellow of the American Cancer Society, upon recommendation of the Committee on Growth, National Research Council. Present address: Statens Seruminstitut, Copenhagen, Denmark. 159 OLE MAAL0E AND KARL G. LARK the growth curves to be determined with satisfactory accuracy by direct colony counts; the second point, the interaction with phage, has been very helpful in that it has revealed periodic changes in susceptibility which, as will be seen later, appear to reflect nuclear doubling. The last property is potentially valuable because bio- chemical work, including tracer studies, can be carried out under well-controlled conditions. We shall first examine the type of growth curve which can be obtained by sub- mitting the culture to cyclic changes of temperature. The technical details of the experiments will be described in a forthcoming publication (Lark and Maaloe, 1954); here a brief mention of the experimental conditions will suffice: aerated broth cultures, not containing more than 20 to 40 million organisms per ml., were used throughout; the temperature shifts were effected almost instantaneously by transferring the cultures to tubes kept at the desired temperature and adding hot or cold broth as required. No loss in viable counts has been observed following the sudden cooling or heating. One temperature cycle, consisting of changing the temperature from 25 to 370 C. and back to 250 C, involves a dilution of the culture by a factor 1 93, which is balanced quite accurately by the increase in colony count during the cycle. It is important to notice that colony counts can give very precise results, even when two large dilution steps are needed between sampling from the culture and spreading on agar plates, provided the experiment is designed in such a way that 300-600 colonies can be counted per sample. Under these conditions and with careful pipetting the standard deviation is about 5 per cent., which is close to the sampling variation to be expected on the assumption that the suspended bacteria are randomly distributed. Figure la shows the step-wise rise in colony count regularly obtained after 2-3 conditioning cycles of temperature shift. In this experiment alternating periods of 30 minutes at 250 C. and 6 minutes at 370 C. were employed. The third, fourth and fifth cycles are represented by points corresponding to individual counts, and a continuous curve below the points shows the growth to be expected if the rate of division was always that characteristic of prolonged growth at the prevailing tem- perature. This lower, theoretical curve is based on control experiments yielding generation times of 45 to 50 minutes for growth at 250 C. and of 18 to 20 minutes for growth at 370 C. Two characteristics of experiments of this type should be em- phasized: the virtual absence of cell division during about 25 minutes of each 250 C period, and the near equality of the experimental and the theoretical genera- tion times. Together these observations show that a considerable degree of phasing or synchronization of cell division has been obtained, and that the temperature regimen employed probably has not impaired the vitality of the cells. It may be added that the steep portions of the experimental curves correspond to a division rate about twice that normally found at 370 C. For convenience, all of the early experiments, including that of Figure \a, were carried out with non-aerated cultures diluted beforehand to such an extent that plating on agar could be made without further dilution. This procedure had to be altered because the bacteriophage experiments as well as the cytological and bio- chemical studies we wanted to carry out on synchronized cultures require densities of at least 10 million organisms per ml. Systematic studies were therefore carried 160 A study of bacterial populations with induced nuclear and cellular divisions out on sufficiently dense cultures, and Figure \b shows the results obtained with a vigorously aerated culture containing about 30 million bacteria per ml. ; under these conditions cycles of 28 minutes at 250 C. and 8 minutes at 37° C. gave the best results. The type of experiment just presented was built up on the basis of preliminary investigations which will be described in more detail elsewhere (Lark and Maaloe, 1954). In this connexion we shall mention only two observations of particular significance. Firstly, it was found that the condition of the culture before temperature \- z o u >- z o _l o -* 1 / / t • 1 > • • • f 100 120 140 TIME IN 160 180 MINUTES 200 1 a. Experiment carried out with a non-aerated broth culture with about 5000 bacteria per ml. Points represent individual colony counts; the intervals between horizontal lines areo-q log units each, corresponding to a doubling of the colony counts. The continuous curve is theoretical; see text on page 160. Figures \a and lb. Growth curves showing step-wise cell divisions. cycling is begun is important. The best results are obtained when a culture, grown to saturation at 370 C, is first diluted one hundred-fold and then aerated lightly for about 75 minutes before the first lowering of temperature takes place. After this preliminary period one ml. of the aerated culture contains 20 to 40 million organisms which have just entered the logarithmic phase of growth. Secondly, a peculiar observation was made concerning the behaviour of a culture when the temperature is raised to 370 C. after the first 250 C. period. If this period is extended to two or more hours, the rise in temperature is followed by a lag of some 20 minutes during which the rate of division is less than or equal to that of the preceding 250 C. period. After the first 20 minutes at 370 C. a sudden rise in colony count is registered before the normal 370 C. growth rate is established. This set of observations is illustrated 161 OLE MAAL0E AND KARL G. LARK by the lower curve in Figure 3. It should be noted that the lag and the subsequent rise may easily be overlooked. As illustrated in Figure 3, the rise causes the linear portions of the growth curve to intersect near the zero-time axis, which means that the lag period will be registered only if a sufficiently large number of counts are made during the first 30 minutes after the temperature increase. The observations just described are, as we shall see later, of great significance for the experiments with bacteriophage and for the cytological studies to which we shall turn next. The phage-sensitive strain of Salmonella typhimurium used for this investigation was obtained from Dr. Lwoff together with the two closely related bacteriophage strains (/) I- z D O u > z o _J o u o o 37° 8' 25° 28' 37° 8' 25° 28' 37° 8' 25° 28' • • s • v'---.-V 4 \ / • < *• • 1 t ', • / e 1 •• • / 100 120 140 160 180 TIME IN MINUTES 200 ib. Experiment carried out with a vigorously aerated broth culture with about 3 X io7 bacteria per ml. For explanation see above. Figures la and lb. Growth curves showing step-wise cell divisions. A and Ac. The ways in which these phage strains interact with the host cells must be briefly reviewed. Infection of a sensitive bacterium with the yl-phage leads to one of the two following results : either, the J-phage multiplies in the cell and this eventually lyses, or the infected cell survives and multiplies, and in that case it and its descend- ants have become immune both to the A- and to the ylc-phage (Lwoff, Kaplan and Ritz, 1954). The surviving cells give rise to pure cultures in which occasionally a cell lyses and liberates a large number of new yl-particles; a culture of this kind is there- fore called lysogenic. To account for the remarkable stability of such lysogenic cultures it must be assumed that the phage particle infecting the parental cell is completely integrated in the cell structure and that, from then on, it multiplies as part of the cell. In the integrated state, the phage particle is referred to as prophage, 162 A study of bacterial populations with induced nuclear and cellular divisions and several independent lines of evidence suggest that this state arises if the infecting phage particle, or part of it, combines successfully with a bacterial nucleus (Murray, 1953; Lederberg, 1953; Jacob and Wollman, 1953; Appleyard, 1953). The prophage state, though very stable, is disrupted occasionally, with the result that unrestricted phage multiplication sets in, and, as mentioned above, the cell eventually lyses. The Ac-pha.ge is a mutant derived from A, and it differs from the parental strain in that it nearly always causes lysis of the cell it infects; this mutant can be used, there- fore, to eliminate non-lysogenic, sensitive bacteria from a mixed population of lyso- genic and non-lysogenic cells (Lwoff, Kaplan and Ritz, 1954). The system just described has been used to study the response of a synchronized culture to infection with the /1-phage. Experiments of the following type were carried out at various times during synchronous division: a sample of the culture was diluted 1:10 into broth containing about 100 million yl-particles per ml.; after exactly one minute a further 1 : 50 dilution was made to stop adsorption of phage particles onto bacteria and a sample of the infected and diluted culture was plated together with a large excess of ^4c-phage. All these operations were carried out at 250 C. The ^4-phage adsorbs very rapidly onto bacteria; under the conditions just described, one minute is sufficient for about 40 per cent, cf the cells to become in- fected. As there are about 100 phage particles per bacterium during this period the percentage of infected cells is independent of such fluctuations in bacterial concen- tration as may be encountered. On the agar plates the excess of Ac -phage eliminates the non-infected and still sensitive bacteria, while permitting the lysogenized and immune cells to form colonies. Figure 2 shows the results obtained by calculating the frequency with which sen- sitive cells become lysogenic when exposed to phage A at various times during cyclic temperature changes. The striking feature of the curve is the rapid increase in frequency of lysogenization, by a factor of approximately two, which regularly occurs some minutes after raising the temperature to 370 C; this is succeeded by a slow decrease which extends over most of the following 250 C. period. Later, when the results of the cytological observations have been presented, the abrupt rise in the lysogenization frequency will be correlated with important changes inside the cells. At this stage we can draw the provisional conclusion, however, that the rises and falls in the frequency curve reflect periodic changes in intracellular conditions which are important in deciding whether a cell is going to lyse or become lysogenic. This can be concluded because control experiments have shown that the percentage of infected cells is constant throughout the division cycle; in other words, the shape of the curve of Figure 2 is the same whether the frequency of lysogenization is expressed as fraction of the total cell count or as fraction of the infected cells only. In Figure 2 is drawn also a broken curve showing the increase in total colony count; it is im- portant to note that the increase in frequency of lysogenization is much steeper than the simultaneously occurring increase in colony count which, as usual, begins some minutes before the temperature is raised. The abrupt increase in frequency of lysogenization, which appears to be induced by raising the temperature to 370 C, is probably a more direct effect of the tempera- ture change than is the increase in the rate of cell division. It therefore seemed likely that changes in the lysogenization frequency might be induced by a treatment which 163 OLE MAAL0E AND KARL G. LARK would not at the same time affect the rate of cell division. To investigate this possi- bility, we returned to the simple system described earlier (p. 160). A culture was grown overnight at 250 C. from a very small inoculum; when a density of about 10 million organisms per ml. had been reached, the culture was aerated for 90 minutes before the temperature was raised to 370 C. During these 90 minutes and the following 60 minutes samples were withdrawn for determination of total colony counts and of lysogenization frequency as described above. Figure 3 shows the results obtained just before and during the period after the temperature shift. For clearness of presentation, the points corresponding to the Z 'o o - — X N Z 5§ ID u 37° 25° 37° 8' 28' 8' 7 - r r 6 - \ r \ 5 1 • • * X» / ,4 V 4 V / +^ ^^^^ • ^1^^ 140 160 160 TIME IN MINUTES Figure 2. Frequency of lysogenization at various times during synchronous cell division. Points show individual frequency deter- minations based on total cell counts and counts of lysogenic colonies. The broken curve shows on an arithmetical scale the increase in total colony count, and thus corresponds to the growth curves in Figures la and lb. counts of lysogenic colonies have been plotted at a convenient distance above the points marking the total cell counts. The lower curve is an ordinary growth curve, representing viable counts, and it exhibits the characteristic lag, followed by an abrupt rise which already has been described (page 161). The upper curve, represent- ing the lysogenic counts, and its relation to the growth curve may be interpreted as follows: before and during the first four to five minutes after raising the temperature to 370 C. the two curves are parallel, which indicates that the lysogenization fre- quency is constant; within the next three to four minutes the lysogenic counts double, and, since the division rate remains very low, this corresponds to approximately a doubling of the lysogenization frequency; then follows a period of 10 to 15 minutes during which the lysogenic counts are almost constant, and during which the lyso- genization frequency gradually decreases as a consequence of cell division; finally, when normal growth has been established at 370 C, the lysogenic counts increase 164 A study of bacterial populations with induced nuclear and cellular divisions at the same rate as the total cell counts and the lysogenization frequency assumes a constant value which is slightly higher than that characteristic of growth at 250 C. The insert in Figure 3 shows the changes in lysogenization frequency during the first 30 minutes; it should be added that the time after the change of temperature when the abrupt rise occurs is very constant from one experiment to another and that the rise is always close to two-fold. 20 40 0 25* O -►37 S *\ 1 S OS • s s V) s 4 H * /» 7 s • ~) s s O „ ^ -o-acH 3 / u / o • > 6°° •• z 0 -1 0 s • 0 1 / • u ?-°z s ** 0 ° 0 0 **0 , -> S*^ ** _l \ -»■ **• jv ^ * J 4%- 0 10 20 30 TIME IN M INUTES Figure 3. Growth and lysogenesis curves as influenced by a single temperature shift. Open circles {and upper curve) show counts of ly so genie colonies ; points [and lower curve) represent total colony counts. The slopes of the early por- tions of the curves are determined by counts made during the 90 minutes preceding the temperature shift. As in Figures la and \b the distance between horizontal lines corresponds tc a doubling of cell counts. The insert in this figure illustrates the changes in the lysogenization frequency during the first 30 minutes of the 370 C. period, and should be compared with the continuous curve of Figure 2. Before attempting to draw a comprehensive picture of the various effects of temperature changes on cell division and lysogenization we shall present the results of cytological studies made on samples taken during an experiment similar to the one presented in Figure 3. First a few words must be said about the fixing and staining techniques employed. Portions of culture were withdrawn and at once mixed with a solution of Os04 in buffered saline to give a final concentration of Os04 of o 2 per cent. ; after centrifugation the pellets were resuspended in small volumes of a o 2 per cent, solution of Os04, and 20 minutes after the first mixing with Os04 loopfuls of the concentrated suspensions were spotted on dry agar surfaces. This somewhat 165 OLE MAAL0E AND KARL G. LARK unorthodox procedure was adopted because we wanted to obtain fixation at a pre- cise time relative to the time of the temperature rise and because it was important that no cytological changes should occur between sampling and the application of the fixing agent. After the process described above, impression prints were made on cover slips, and hydrolysis, staining with a thionine-thionyl chloride solution, and dehydration, were carried out as described by DeLamater (1952). The micrographs obtained in this manner do not compare in beauty with those made by expert cyto- logists using fixation in Os04 vapour; it is not known whether this is due to the fixation process we were forced to employ or to our lack of experience. The micro- graphs do show, however, that simultaneously with the rise in lysogenization fre- quency the number of stained spots, or 'nuclei', per bacterium doubles. We shall continue to talk about these stained regions, in which the bacterial desoxyribosenucleic acid (DNA) is concentrated, as nuclei, remembering that we have no other way of defining a bacterial nucleus and that there are reasons to believe that it does not constitute as well defined an organelle as does the nucleus of cells of higher organisms (Birch-Andersen, Maaloe and Sjostrand, 1953). The distribution of nuclei observed in the micrographs is as follows: while growing at 250 C, most cells have two and a few have four nuclei; during the period between 5 and 10 minutes after raising the temperature to 370 C, while the lysogenization frequency doubles, the fraction of cells containing four nuclei increases rapidly, so that by the time when no further increase in lysogenization frequency is observed the majority of the cells contain four nuclei and very few are found with only two. When the culture has continued growing for a couple of generations at 370 C. the distribution is again much like that observed before the temperature was raised; however, the proportion of cells with four nuclei is a little larger than it was when growth took place at 250 C. Up to now we have presented some rather heterogeneous experiments, one after the other, without attempting to interpret the results. For practical reasons the ex- periments have been described in the order of increasing complexity, ending with the experiment in which cytological studies were included. Fortunately, these studies gave a clear-cut answer which makes it natural now to examine all our findings in the light of the cytological observations. These results may be summarized by stating that upon raising the temperature of a Salmonella typhimurium culture which has grown for some time at 25 to 37 ° C. nuclear division is induced in nearly all the cells. We shall now discuss possible implications of this finding from different points of view: (1) It is natural first to consider what mechanism may be responsible for the induction of nuclear division. The tentative explanation we have in mind is based on general ideas similar to those put forward by Hotchkiss, who writes as follows (Hotchkiss, 1954) : '. . . the biochemical processes of cell growth and division would be disrupted in a systematic fashion by temporary exposure of a growing culture to a temperature well below that at which these systems had achieved a steady-state equilibrium. Certain of the enzyme systems should now be less able than others to transfer an amount of substrate equal to that supplied them, and there would then be a tendency for the metabolism to be selectively slowed or even temporarily halted at certain points.' Concerning the synthesis of DNA which must precede nuclear 166 A study of bacterial populations with induced nuclear and cellular divisions division there is evidence from phage experiments which suggests that this process may be particularly sensitive to temperature changes. We have observed previously that phage synthesis, which to a very large extent means synthesis of DNA, has a temperature coefficient about twice as high as that of the rate of cell division (Bentzon, Maaloe and Rasch, 1952). DNA synthesis, in general, may therefore be selectively slowed down at low temperature; if so, the DNA precursors should accum- ulate when the temperature is lowered and reach a higher concentration in cells grow- ing at 250 C. than in cells growing at 37° C. To account for the observation that nearly all the cells in a culture undergo nuclear division very soon after the tempera- ture has been raised from 25 to 370 C. the following assumptions are made: firstly, that nuclear division is initiated when the precursor concentration reaches a critical level which depends on temperature; and secondly, that cells which recently have undergone nuclear division at 250 G. are left with a precursor concentration so high that a new nuclear division will be initiated if the temperature is raised to 37° C. This hypothesis is tentative, as already stated, but it should be possible to test it by means of biochemical studies using tracers to follow the assimilation from the medium of phosphorus and adenine, for instance. (2) We shall now proceed to consider cell division. In this respect, the most striking observation made on our Salmonella typhimurium strain is that there seems to be an interval of more than half a generation time between nuclear and cellular division. This is most apparent in the experiments in which only one temperature change is involved; here nuclear division occurs in nearly all cells right after the increase in temperature, and the 'burst' of cell divisions corresponding to the nuclear division occurs 12 to 15 minutes later. It looks as if nuclear division actually blocks cell division for a considerable time. The experiments in which synchronous cell division was obtained also suggest that the cells do not divide until a long time after nuclear division has taken place; we shall return to this type of experiment later when discussing the process of lysogenization. Our observations on cell division in Salmonella typhimurium have been made under rather artificial growth conditions as far as temperature is concerned. However, we assume that the time relation between nuclear and cellular division, which we have observed, also applies to cells growing at a constant temperature. In support of this assumption, we may cite the observation, made earlier, that the overall division rate in a synchronized culture is almost as high as might be expected on the basis of the division rates at 25 and 370 C. and the times spent at the two temperatures. The regimen employed to obtain synchronization, therefore, seems not to impair the growth and division processes appreciably; this, we believe, would not be the case if the temperature shifts interfered seriously with the natural sequence of events during the division cycle. As a basis for later discussion, we may point to the striking difference between the division cycle in cells of higher organisms and that observed in Salmonella typhimurium. Most animal and plant cells are uninuclear, which means that, normally, cell division must follow right after nuclear division; in the multinuclear bacterial cells which we have studied, the opposite seems to be normal. (3) Finally, we want to consider the process of lysogenization and its possible relation to nuclear division. It was mentioned earlier that the establishment of the M 167 OLE MAAL0E AND KARL G. LARK prophage state seems to involve an interaction between phage material and a bacter- ial nucleus (Murray, 1953; Lederberg, 1953; Jacob and Wollman, 1953; Appleyard, 1953). If only one phage particle enters the cell the probability of such an interaction occurring before unrestricted phage multiplication begins is rather low; it has been shown, however, that this probability, i.e. the lysogenization frequency, can be in- creased from 15 to 20 per cent, up to about 80 per cent, by increasing the number of phage particles per bacterium (Boyd, 1950; Lwoff, Kaplan and Ritz, 1954). Our experiments show that an increase in lysogenization frequency can be caused also by increasing the number of nuclei per cell. From these observations it appears probable that the lysogenization frequency is determined by the number of en- counters between the entering phage particle, or particles, and the nuclei of the cell, which occur within a limited time after infecting the cell. In our experiments the conditions of infection were such that 80 to 90 per cent, of the infected bacteria re- ceived one phage particle only; and in such experiments nuclear doubling, through- out a culture, seems to cause a doubling of the lysogenization frequency. What remains to be interpreted is the gradual decrease in lysogenization frequency which is observed in the synchronized cultures during the periods while little or no cell division takes place (see Figure 2). We believe that just after the 370 C. period when the lysogenization frequency has reached its maximum, all cells have doubled their nuclei; during the following 20 to 25 minutes the cells prepare for division, presum- ably by developing some kind of internal separation. Gradually all the cells would thus in a functional sense become double cells. In this way we would pass from a situation in which nearly all cells have, say, four nuclei within one functional unit, to a situation in which nearly all cells consist of two functional units, each with two nuclei. In our lysogenization experiments each infected cell unit, including the hypothetical double cells, adsorbed one phage particle only, and in the case of a double cell only one compartment of the cell would therefore be infected. It follows, from what was said above, that the postulated segregation process by which the number of nuclei per functional unit is reduced to one-half should be accompanied by a similar reduction in lysogenization frequency. This analysis of the lysogenization curve of Figure 2 brings us back to the idea that nuclear division is not immediately followed by cell division, which seems to occur only after a long period of preparation. Acknowledgements. We wish to express our thanks to Dr. Lwoff for making available to us the bacterial strains and the phage strains used, and for pointing out some of their great advantages. We also wish to thank Dr. Kauffmann for generous gifts of specific antisera. We are indebted to Dr. Hotchkiss and Dr. Zeuthen for detailed and valuable information about their work prior to its publication. We are grateful to Mr. O.' Rostock for expert assistance in the execution of most of the experiments. REFERENCES Appleyard, R. K. (1953). Cold. Spr. Harb. Symp. Qiiant. Biol. 18, 95. Bentzon, M. W., Maaloe, O. and Rasch, G. (1952). An analysis of the mode of increase in number of intracellular phage particles at different temperatures. Acta path, microbiol. scand. 30, 243. 168 A study of bacterial populations with induced nuclear and cellular divisions Birch-Andersen, A., Maalce, O. and Sjostrand, F. S. (1953). High-resolution electron-micrographs of sections of E. coli. Biochim. Biophys. Acta 12, 395. Boyd, J. S. K. (1950). The symbiotic bacteriophages of Salmonella typhimurium. J. Path. Bad. 62, 501. DeLamater, E. D. (1952). A consideration of the newer methods for the demonstra- tion of nuclear structure in bacteria and other micro-organisms. Mikroskopie 7, 358. Hotchkiss, R. D. (1954). Cyclical behaviour in pneumococcal growth and trans- formability occasioned by environmental changes. Proc. nat. Acad. Sci., 40, 49. Jacob, F. and Wollman, E. (1953). Induction of phage development in lysogenic bacteria. Cold Spr. Harb. Symp. Quant. Biol. 18, 101. Lark, K. G. and Maaloe, O. (1954). Biochim. Biophys. Acta (in press). Lederberg, E. M. and Lederberg, J. (1953). Genetic studies of lysogenicity in Escherichia coli. Genetics 38, 5 1 . Lwoff, A., Kaplan, A. S. and Ritz, E. (1954). Recherches sur la lysogenisation de Salmonella typhimurium. Ann. Inst. Pasteur. 86, 127. Murray, R. G. E. (1953). A cytological study of the lysogenizing process. VI Con- gresso Interna zionale di Microbiologia, Roma 2, 551. Scherbaum, O. and Zeuthen, E. (1954). Induction of synchronized cell division in mass cultures of Tetrahymena piriformis. Exp. Cell. Res., 6, 221. Zeuthen, E. and Scherbaum, O. (1954). Contribution to this Symposium. Discussion Chairman: J. F. Danielli K. E. Cooper. Does the resistance of these cultures to antibiotics change in their differ- ent phases ? We have found a critical time in the development of staphylococci on solid media at about four generations after pouring inoculated plates to test anti- biotic action (1952, J. gen. Microbiol. 7, 1-17). A temporary increase in resistance occurs, and may be related to the time required to form some cellular substance after the act of inoculation. 0. Maaloe. Unfortunately we have no information as yet as to whether bacteria are particularly susceptible to antibiotics at certain points of the division cycle. C. Darlington. With regard to the suggested high temperature coefficient of DNA synthesis, in those flowering plants in which mitosis takes place at freezing-point, a starvation of DNA often occurs at specific points in the chromosomes, the segments of heterochromatin, when the temperature is applied for some time before metaphase. M. M. Swann. Unlike what happens in plants and animals, there is a considerable separation in your organisms between nuclear division and cell division. However, the partition is laid down earlier, and the 'cell division' appears to be a separation of what are already functionally distinct units. Perhaps then we may regard cyto- plasmic division even in this case as essentially following immediately after nuclear division. 169 OLE MAAL0E AND KARL G. LARK K. E. Cooper. Have you tried cell wall stains ? Many bacteria have transverse septa which divide them into multicellular organisms. L. Rinaldini. Has any electron-microscopy been done on these cells during the decline in lysogenesis, to see if the septum develops gradually in each cell or if it appears abruptly and the gradual decline is obtained by more and more cells dividing? 0. Maaloe. Studies by means of sectioning and electron-microscopy are in progress. At the moment we can say only that there is no indication of a morphologically well- defined septum being formed before cell division begins. E. Ambrose. Is there any change in the sensitivity of the bacterial nuclei to phage at various stages of the division cycle ? With larger cells the nuclear membrane dis- appears during metaphase. It seems likely that the phage nucleoprotein, which is of high molecular weight, could combine more easily with the nuclear material in the absence of a membrane. O. Maaloe. The available data seem to indicate that there is no particularly sensitive period during the division cycle. With a high multiplicity of infection as many as 80 per cent, of the bacteria become lysogenic. It is not likely that within the short time during which the decision for or against lysogenization is made, the majority of the cells would pass into any specified phase of the division cycle. Also, if a particularly sensitive phase existed, we might expect the peak in the frequency curve to rise by more than a factor of two. As to the second point, there is some evidence that in the bacteria we have studied there is no well-defined nuclear membrane. E. jV. Willmer. I should like to call attention to some observations, on tissue cultures of chick fibroblasts, which seem to be somewhat parallel to those reported by Dr. Maaloe. These cells, when grown in flasks in a plasma medium, cease to show cell divisions in the outgrowth after about sixty hours; but, if they are then treated with embryo juice for about an hour, they can be caused to divide again for a limited time; such divisions start after about ten hours and cease after about twenty-four hours. Repetition of the dose of embryo juice before the cells start to divide is without effect, but if the second dose is delayed until the cells are dividing, then a second crop of divisions results in due course: thus, the activating agents, possibly with nucleoproteins among them, can only gain limited access to the cells and the latter can become 'saturated' until the situation is changed by the occurrence of the division process. (Jacoby, F., Trowell, O. H. and Willmer, E. N. (1937). J. exp. Biol. 14' 255") Secondly when chick cultures are cooled to a temperature below io° C. divisions temporarily cease, but when the cultures are returned to 370 C. there is a compen- satory excess of cell divisions. (Spear, F. G. (1928). Arch. exp. ^ellforsch. 7, 484.) By combining these two sets of observations a method for obtaining more nearly synchronous mitoses in chick fibroblasts would appear to emerge as a possibility. 0. Maaloe. I am very pleased that a point like this has been raised, because I feel that in our paper it is not emphasized strongly enough that temperature shift is but one means out of a great many which may be tested for their suitability to pro- duce synchronous behaviour in cell populations. 170 Environmental and genetic control of differentiation in Neurospora by M. WESTERGAARD and H. HIRSCH* Universitetets Genetiske Institut, Kobenhavn As is well known, it has been possible in recent years to obtain very precise informa- tion about the biochemistry and physiology of growth and its genetic control in certain fungi and bacteria by means of a very ingenious technique. The 'wild type' strain of the organism is grown on a synthetic 'minimal medium'. Mutants are pro- duced which cannot grow on the minimal substrate but only on a 'complete medium' supplemented with various diffusible growth factors. The mutants lack the capacity to carry out a certain enzymatically controlled metabolic step, necessary for normal growth, and this step can be identified by adding only one growth factor at a time to the minimal medium. Hence the mutation serves the purpose of a very specific enzyme inhibitor, and the advantages of the method have been amply demonstrated in studies of the biosynthesis of various amino-acids and vitamins. This method has almost exclusively been applied to the study of growth, whereas very little work along similar lines has been done on morphogenesis in these organ- isms. It would be interesting, however, to use the technique which has been so suc- cessful for the study of growth to a study of problems of differentiation. This would be possible, (a) if one or several well-defined steps in morphogenesis could be control- led on a synthetic medium, (b) if mutants could then be produced in which normal differentiation was either blocked or modified on the minimal medium, and finally (c) if normal differentiation could be induced again in these mutants by supplement- ing the minimal medium with suitable precursors. The first difficulty is obviously that of selecting a suitable system of differentiation which can be properly controlled on a synthetic minimal medium. The selection of such a system in fungi and other micro-organisms is not too easy, because the morpho- genesis of micro-organisms is far less well defined and far less advanced than that of higher plants and animals. Let us take as an example the mould Neurospora, a well- recognized tool for genetical experiments since the earlier work of Dodge and Linde- gren and the more recent work of Beadle, Tatum and their group. It will be seen from Figure i that Neurospora during development does three different things: (i) it grows; (2) it forms conidia; and (3) it forms, on the haploid mycelium, female * Present address: Div. of Cancer Biology Medical School, University of Minnesota, Minneapolis, U.S.A. 171 M. WESTERGAARD AND H. HIRSCH sex organs, the so-called protoperithecia, which after fertilization develop into perithecia. It might a priori be considered possible to select growth as such for our purpose, because the growth type of Neurospora is easily modified by changes of substrate (Tatum, Barratt and Cutter, 1949) and because a number of morphological mutants of widely different growth type are available (Barratt and Garnjobst, 1949; M. and H. K. Mitchell, 1952; and others). The study of the biochemical genetics of some of these mutants is now in progress (Mitchell, Mitchell and Tissieres, 1953) but it is Conidia Conidia Fertilization j>f protoperit/, v HYPHAL FUSION Figure I. Diagram of the life cycle of Neurospora. (From Beadle in Amer. Scientist, 1947.) still too early to form an opinion of the possibilities of this system for studying prob- lems of differentiation. As is well known a similar system has been studied intensively in yeast by Ephrussi and his group. The implications of their results on general problems of morphogenesis has recently been summarized by Ephrussi (1953). The second differentiation process in Neurospora which might be selected for a study of this kind would be the formation of conidia. This also is strongly influenced by the composition of the substrate, and a number of mutants are available which either form abnormal conidia or fail to form any at all. It is also known that differentiation of conidia is associated with the production of yellow pigments identified as carote- noids (Haxo, 1949; Sheng and Sheng, 1952). However, everybody familiar with work on Neurospora will know that the formation of conidia is a very complicated process involving the production of both macroconidia and uninucleate microconidia, 172 Environmental and genetic control of differentiation in Neurospora and although it might ultimately be possible to work out the biochemical genetics of this process in some detail, the system is certainly not an attractive one. This leaves the formation of protoperithecia as the third alternative which may be suitable for our problem. The formation of these bodies on haploid mycelia is not a very common phenomenon in the fungi, and it is most fortunate that Neurospora belongs to the group of Pyrenomycetes which have them. They had already been reported in the classical paper by Shear and Dodge (1927). However, their function as female sex organs was first described by Dodge and Swift (1933) and by Dodge (1935). Their development was later studied in some detail by Backus (1939). Despite these investigations we are far from having sufficient information about their detailed structure and development. Figure 2. The structure of a protoperithecium o/Neurospora ; asc: ascogonium ; r: receptive hyphae (trichogyne) ; c: conidium which fertilizes the receptive hyphae. (From Backus, 1939.) When Neurospora is grown under suitable conditions to be defined later, the proto- perithecia begin to develop in abundance after 3-4 days, and they can then be ferti- lized. They are shown in Plate la; Figure 2 shows some details of their structure based upon Backus's paper. We have made some preliminary investigations, trying to follow the first steps in their differentiation. First, certain vegetative hyphae begin to curve and form small spirals. These hyphae stain deeply with cotton blue. Next, these spirals seem to attract hyphae from the surrounding mycelium, and the pri- mordium (the ascogonium) becomes surrounded by dense balls of deeply staining hyphae which after 3-4 days are easily recognizable under a low-power microscope. The differentiation of these organs is thus a very well-defined and conspicuous process which takes place within very few days, and which among the three possible systems (growth type, formation of conidia, or protoperithecium formation) seems to be by far the best suited for our purpose. 173 M. WESTERGAARD AND H. HIRSCH Having thus selected the differentiation of the female sex organs as our morpho- genetic system, the next problem is to control this differentiation on a synthetic medium. The minimal medium used in standard work on JVeurospora, the so-called 'Fries-minimal medium', does not allow protoperithecia to develop, and conse- quently sexual reproduction does not take place on this substrate (Table I). Until recently Neurospora was grown on cornmeal agar for sexual reproduction, and this of course is not a synthetic medium. In 1947 Westergaard and H. K. Mitchell worked out a synthetic medium which allows the formation of perithecia and abundant sexual reproduction to take place. This was the first step in the development of a system allowing a study of the biochemical genetics of protoperithecial differentia- tion. This medium, which we call the 'P-minimal medium' (perithecia-promoting medium), differs from the standard Fries-minimal medium in containing no ammonium ions; the pH is adjusted to 65 as compared to 5 5 in the Fries-medium Table I Composition of different media per litre of¥L20. Substrate* Ammonium- tartrate Potassium- tartrate KNO3 NH4N03 PH Fries P-minimal Horowitz 5 g- 638 g- 1 g- 3 g- 1 g- 5-6 6-5 56 * All substrates have in addition: KH2P04 i g., MgS04 0-5 g., CaCl2 o-i g., NaCl o-i g., sucrose 15 g., biotin, and trace elements. The low-sulphur media have 0-0079 g. MgS04 instead of 0-5 g. and in addition 0-4 g. MgCl2. (Table I) . In the same paper it was shown that the development of perithecia depends also upon the carbon/nitrogen ratio, upon pH, and upon temperature. The optimal temperature for perithecium formation is 250 C, whereas no differentiation at all takes place at 35° C. The work of Westergaard and Mitchell was extended by Dr. Herbert Hirsch who worked in our laboratory for two years. His results have recently been published (Hirsch, 1954). He was able to confirm the preliminary observations by Westergaard and Mitchell on perithecium formation, and he extended them to protoperithecium formation ; he made the discovery that the latter system is a reversible one. If slants are incubated for 7-14 days at 350 C. on P-minimal medium (i.e., under conditions where no protoperithecia develop) and then transferred to 250 C, protoperithecia develop after a few days. If, on the other hand, protoperithecia are allowed to develop at 250 C. and the slants are then transferred to 350 C, the protoperithecia can no longer be fertilized. We believe that this reversibility will be of great use in future studies. Hirsch also made the first studies on the biochemical mechanism involved in protoperithecium formation. Starting from the observations that mycelia in which protoperithecia are formed in abundance turn brown or black, that both mature 174 Environmental and genetic control of differentiation in Neurospora protoperithecia, perithecia and ascospores contain a black pigment, and that strains growing under conditions where no protoperithecia develop never turn brown or black, it was assumed that these pigments were melanins and that a causal relation- ship exists between melanin metabolism and protoperithecium formation. The black pigments associated with protoperithecium formation were identified as melanins by various chemical reactions, by studying their absorption spectra, and by the pre- sence of a tyrosinase. No melanins could be demonstrated in mycelia on which proto- perithecia were not found. This observation of course made it important to study tyrosinase activity in greater detail in strains growing under different conditions, favouring or suppressing proto- perithecium formation, as it is well known that tyrosinase converts tyrosine or other Table II Tyrosinase activity of ground suspensions of Neurospora crassa. Strain W 2/49 A grown for 7 days on flter-paper on: (1) P-minimal medium at 250 C; (2) P-minimal medium plus 3,000 mg. ofamino-N (hydrol. casein A) per litre at 250 C. ; (3) P-minimal medium at 350 C. The figures given are net increases {zero time readings deducted) in optical density, multiplied by 100. Time (minutes) 250 C. mycelium 250 C. AA- mycelium 35° C mycelium 0 *5 30 45 60 0 2 5 61 124 192 0 06 1 1 23 37 0 0-2 0 0 04 Dry weight of mycelium (mg. \ per 05 ml. of suspension used) j 76 90 8-2 substrates into melanins via dopa, dopachrome and a number of other intermediates (see Lerner, 1953). Hirsch showed that no tyrosinase activity could be demonstrated in strains grown at 350 C, whereas tyrosinase activity was present in mycelia grown at 250 C. (Table II). As shown in Figure 3, tyrosinase activity on P-minimal medium at 250 C. started on the third day, reached its maximum on the fourth day, and then went down again. It will be noticed from the figure that the highest tyrosinase activity was found in the period when protoperithecium formation was most intense. Table II also includes an experiment in which Neurospora was grown on P-minimal supplemented with 3,000 mg./litre amino-N (the AA-mycelium) . Very little tyrosinase activity was found in mycelium grown on this substrate in 7-day old cultures, and the red pigment did not turn black (melanic), as did the pigment in culture grown 175 M. WESTERGAARD AND H. HIRSCH on P-minimal medium. After 2 weeks, however, when tyrosinase activity in mycelia grown on P-minimal medium went down, the activity was very much increased on the AA-substrate, and now the pigments turned black during the enzymatic test. No protoperithecia developed on the AA-mycelium with 3,000 mg./l., nor was melanin formed during actual growth. At a lower concentration (1,500 mg./l. ), protoperithecia were formed after 2 weeks, but they could not be fertilized. Hirsch made further experiments to test the hypothesis that there is a connexion between tyrosinase and differentiation. A number of tyrosinase inhibitors (^-amino- benzoic acid, sodium-thioglycolic acid, cystein and phenylthiourea) were added to P-minimal medium. They were all able to suppress protoperithecium formation more or less completely, whereas other enzyme inhibitors without specific affinity towards TIME (DAYS) Figure 3. Tyrosinase activity and protoperithecium forma- tion as a function of time in Neurospora crassa strain W 2/49 A, grown on P-minimal medium at 250 C. Curve 1 : tyrosinase activity of ground suspensions ; net in- crease in optical density X 1 00 in 30 minutes. Curve 2 : ditto; net increase in optical density X 100 in 60 minutes. Curve 3: number of protoperithecia. (Hirsch, 1954.) the tyrosinase system (e.g. streptomycin and others) did not interfere with the differ- entiation process. It might be mentioned at this point that the present differentiating system has certain advantages over the growth-system studied by other workers: it is possible to study the metabolism of mycelia in which differentiation is blocked. The same, however, cannot be done with mycelia in which growth is blocked, because the alternative to growth is lethality. Thus, from studying the metabolism of mycelia in which differentiation was blocked by various environmental factors, it has been possible to get information about the biochemistry of differentiation even without bringing mutant strains into the picture. However, the relationship between tyrosinase activity, melanin formation and development of female sex organs is far more complicated than might appear from the data hitherto presented. In recent years tyrosinase activity in Neurospora has been studied by Fox and Gray (1950), and especially by Horowitz and his co-workers (Horowitz and Shen, 1952; Horowitz and Fling, 1953; see also Horowitz, 1951). 176 Environmental and genetic control of differentiation in Neurospora None of these authors, however, connected their observations with the differentia- tion of the sex organs. Fox and Gray claimed that there is a difference in tyrosinase activity between the two mating types ( + and — ) of Neurospora, an observation which was not confirmed either by Horowitz or by Hirsch. Horowitz and Shen also found, as did Hirsch, that tyrosinase activity in Neurospora depends upon the temperature. Moreover, they made the interesting discovery that, under the conditions and with the strain which they used, an inhibitor was present which suppressed tyrosinase activity. Horowitz used a modified, liquid Fries-medium ('Horowitz-medium'). Its composition is shown in Table I. They showed that this inhibitor disappeared if the mycelium was dialysed. Its presence in non-dialysed mycelium was demonstrated by adding fresh mycelium to a tyrosinase preparation, after which procedure the tyrosinase activity was inhibited. They also found that the tyrosinase inhibitor disappeared when Neurospora was grown on a medium poor in sulphur. This brings the possible connexion between the tyrosinase inhibitor and proto- perithecium formation into the picture. Hirsch found that the inhibitor was present only in mycelia grown in liquid cultures; it was present whether Horowitz or P- minimal medium was used. No protoperithecia were ever formed in liquid cultures. If, however, the mycelia were grown on filter paper in liquid cultures, tyrosinase activity was present in non-dialysed mycelia grown on Horowitz as well as on P- minimal medium, and protoperithecia were formed in both cases. On the other hand, tyrosinase activity was found in non-dialysed mycelia grown on Horowitz low-sulphur medium, but hardly any protoperithecia developed on this substrate. Finally : if the standard P-minimal medium was substituted with a P-minimal low- sulphur medium, protoperithecium formation was almost as good as on standard P-minimal. Evidently until more is known about the nature of the suppressor present in the mycelia grown in liquid media, its pH-dependence, its interaction with the constituents of the different media and variation between different strains, we shall have to leave this problem open. Horowitz and Fling later made a more thorough study of the sensitivity of Neuro- spora tyrosinase to temperature. They found another strain of Neurospora containing a temperature-stable tyrosinase. Unfortunately nothing is said about sexuality in this strain. The tyrosinase from the temperature-unstable strains was found to have a half-life of 3-4 minutes at 590 C, whereas the enzyme from the stable strains had a half-life of at least 30 min. at the same temperature. The difference between the two strains was controlled by a pair of allelic genes designated TsjTL. The absence of tyrosinase activity at 350 C. in temperature-sensitive strains was not due to a sup- pressor (Horowitz and Fling, I.e.; Hirsch, I.e.). We have so far been dealing with only one part of the problem, viz. the environ- mental control of differentiation in Neurospora. As a working hypothesis we want to suggest that there is a causal relationship between tyrosinase activity and melanin formation on one hand and differentiation of female sex organs on the other hand. It also seems as if normal differentiation can be blocked at high temperature because one of the enzymes involved in melanin metabolism is temperature-sensitive, the temperature-dependence of the enzyme being under genie control. Although the evidence is far from conclusive, there may also be a relationship between the forma- tion of a tyrosinase inhibitor on certain substrates and the scarcity or absence of 177 M. WESTERGAARD AND H. HIRSCH protoperithecia on the same substrates. Hence, genes and environment together determine a series of different states, showing different morphological and bio- chemical characteristics. Turning now to the second, or genetical part of the problem, it is evident that the control of differentiation on a synthetic medium has been worked out sufficiently to allow a study of mutants in which protoperithecial development is either blocked or altered on the P-minimal medium. It has not been necessary to produce such mutants experimentally. It is a well-known fact that most Neurospora strains become more or less sterile when they are propagated vegetatively over a long period. From such old strains we have isolated a number of mutants in single- spore cultures which show various degrees of sexual sterility. Some of these mutants never form protoperithecia on P-minimal medium, and they are completely female- sterile (Plate lb). Fortunately, however, most of them are male-fertile so that they can be studied genetically. Other mutants have abnormal-looking protoperithecia which cannot be fertilized, and we have still other mutants which do develop proto- perithecia and after fertilization also normal-looking perithecia, which, however, never contain ascospores. The strains which do not produce protoperithecia do not turn dark, confirming the evidence from the first part of the investigation, that there is a relationship between the formation of protoperithecia and of melanins. We have tested three of these mutants for tyrosinase activity and have found none. On the other hand we have a very interesting group of mutants which produce a very great number of small protoperithecia ; these strains turn completely black in a very short time and they are completely female-sterile (Plate Ic). Apparently both absence of melanin formation and excessive melanin formation interfere with sexual differentiation. Although the formal genetics of the various sterile mutants has not yet been worked out, there is some evidence both from heterokaryons and from other experiments that we are dealing with different, non-allelic mutants. If the standard biochemical genetical technique were to be applied to these mu- tants it would mean feeding the different mutants with different intermediates (precursors) in melanin metabolism and studying the reaction of the mycelia on the supplemented media. (It is a noteworthy fact that none of the sterile mutants become fertile on the standard 'complete' media, which are supplemented with vitamins, casein-hydrolysate, yeast and malt extract). Unfortunately a number of difficulties arise here. For one thing it is unlikely that anything would come out of feeding the melanins directly to the mutants since it is unlikely that such high-molecular com- pounds would penetrate the mycelium. For the same reason it seems unlikely that adding tyrosinase itself to the substrate would have any effect. Unfortunately the various low-molecular intermediates in melanin synthesis have not been available to us (see Lerner, 1953). We have therefore tried a short-cut. Tyrosine + tyrosinase prepared from cultivated mushrooms (Psalliota) were added to sterile mutants which were grown for 3 days on P-minimal medium, in the hope that some of the inter- mediates formed during melanin synthesis might be picked up by the mycelium. Somewhat to our surprise one of the mutants (913/83) gave what might be called a promising reaction to this crude treatment, forming, where substrate + enzyme was added, protoperithecium-like structures (Plate Id). The induction of these structures, although they were far from being normal protoperithecia, certainly suggests that 178 Environmental and genetic control of differentiation in Neurospora tyrosinase-melanin intermediates have a morphogenetic capacity in the protoperi- thecial system. These observations, together with the evidence already presented, suggest that our working hypothesis of a connexion between differentiation of protoperithecia and synthesis of melanin in Neurospora is a sound one. To these observations should be added a rather interesting preliminary observation on a sterile mutant (93/54) which never forms protoperithecia on the standard P-minimal medium, whereas it does form many protoperithecia on a low-sulphur P-minimal medium. This suggests that in this mutant the tyrosinase inhibitor of Horowitz and Shen, which normally is inactive on the standard P-minimal medium, is involved. It is quite obvious that our technique for feeding precursors in this system is much too crude. The various assumed intermediates in melanin metabolism will have to be synthesized and fed individually to the mutants before we can get much further. What we want to present here is a morphogenetic system, namely the formation of protoperithecia, which offers promising opportunities for studying the genetics and biochemistry of morphogenesis on a synthetic substrate; and we hope to have presented convincing evidence for our working hypothesis that at least one of the keys to the biochemistry of this problem is tyrosinase activity and melanin synthesis. Before finishing this presentation it may be worth while to discuss some of the more general aspects of this work. As pointed out already by Dr. Horowitz, melanin problems present one of the few fields where biochemistry, embryology, and genetics meet, and, as Dr. Horowitz has also pointed out, with respect to tyrosinase activity Neurospora behaves like the Siamese cat or the Himalayan rabbit. To this we want to add that in Neurospora this enzyme system is involved in sexuality. We now also have Neurospora mutants lacking tyrosinase and unable to form melanin, thus behav- ing like many albino animals. In other mutants melanin formation is inhibited, just as we have albinos in animals due to epistatic suppressors. We have certain mutant strains in Neurospora where the melanin seems to agglutinate, bringing the dilution gene (D-gene) in rabbits to mind. This investigation might also be discussed in rela- tion to the older work of Goldschmidt on the effect of temperature upon the differ- entiation of the wing pattern in butterflies (see Goldschmidt, 1938). It should also be remembered that we are dealing with the very first system from which biochemical genetics emerged: the work of Garrod on inborn errors of metabolism in man (Garrod 1923; Haldane 1954). Although we may not expect to find Neurospora strains with alcaptonuria, hydroxyphenyluria, etc., we may nevertheless have a system which can be of importance for studying some of the biochemical blocks which in man are so often connected with mental disorders. A second aspect of this investigation is its relationship to the possible occurrence of sex hormones in fungi, a subject recently reviewed by Raper (1952). In Neurospora the earlier claims on the occurrence of diffusible sex hormones (Moreau and Moruzzi, 1 931) have been refuted (Aronescu, 1933; Hirsch, 1954). As far as the higher fungi are concerned, it seems a good idea to look for the intermediates in melanin synthesis in future work. Also the work of Moewus on sexuality in Chlamydomonas which, if taken at its face value, represents a system far better worked out in detail than any other, should be kept in mind. As will be remembered Moewus claims that two quite unrelated groups of chemicals, carotenoids and anthocyanins, play a role in sex 179 M. WESTERGAARD AND H. HIRSCH differentiation in Chlamydomonas (see Moewus, 1950). We may take this as a warning against restricting the biochemical part of this work to the melanins only. The third implication, ?nd still of course a purely speculative one, might also be mentioned here. Melanins are of widespread occurrence among the higher fungi, both in the Ascomycetes and Basidiomycetes; mushrooms like Psalliota and Lactarius are well-known sources of tyrosinase. The pigments seem always to be associated with reproduction. This brings into the picture the so-called Fungi imperfecti — the fungi which only reproduce asexually, the sexual (perfect) stage being unknown. It would be interesting to compare tyrosinase activity in some of the asexual species with that of related sexual species (e.g. Aspergillus niger with A. nidulans, Penicillium chrysogenum with some of the sexual species) and study the reaction of the asexual species to mela- nin precursors. It would have considerable theoretical as well as practical implica- tions if it should prove possible to induce sexuality in some of the asexual fungi, among which we find some of the most important industrial species. REFERENCES Aronescu, Alice (1933). Further studies in Neurospora sitophila. Mycologia 25, 43. Backus, M. P. (1939). The mechanisms of conidial fertilization in Neurospora sitophila. Bull. Torrey hot. CI. 66, 63. Barratt, R. W. and Garnjobst, Laura (1949). Genetics of a colonial microconi- diating strain of Neurospora crassa. Genetics 34, 351. Dodge, B. O. (1935). The mechanisms of sexual reproduction in Neurospora. Myco- logia 27, 418. Dodge, B. O. and Swift, M. E. (1933). A simple way to demonstrate sexual repro- duction in the bakery mould, Neurospora. Torrey a 33, 31. Ephrussi, B. (1953). Nucleo-cytoplasmic relations in micro-organisms. Clarendon Press Oxford. Fox, A. S. and Gray, W. D. (1950). Immunogenetic and biochemical studies of Neurospora crassa. Proc. nat. Acad. Sci., Wash., 36, 538. Garrod, A. E. (1923). Inborn Errors of Metabolism. Oxford University Press. Goldschmidt, R. (1938). Physiological Genetics. McGraw Hill, New York. Haldane, J. B. S. (1954). The Biochemistry of Genetics. Allen and Unwin, London. Haxo, F. (1949). Studies on the carotenoid pigments of Neurospora. I. Arch. Biochem. 20, 400. Hirsch, H. M. (1954). Environmental factors influencing the differentiation of protoperithecia and their relation to tyrosinase and melanin formation in Neuro- spora crassa. Physiol. Plant. 7, 72. Horowitz, N. H. (1951). Genetic and non-genetic factors in the production of enzymes by Neurospora. Growth, Symp. 15, 47. Horowitz, N. H. and Shen, S.-C. (1952). Neurospora tyrosinase. J. biol. Chem. 197, 513- Horowitz, N. H. and Fling, Marguerite (1953). Genetic determination of tyro- sinase thermostability in Neurospora. Genetics 38, 360. Lerner, A. B. (1953). Metabolism of phenylalanine and tyrosine. Advanc. Enzymol. *4, 73- 180 Environmental and genetic control of differentiation in Neurospora Mitchell, Mary B. and H. K. (1952). A case of 'maternal' inheritance in Neurospora crassa. Proc. nat. Acad. Sci., Wash. 38, 442. Mitchell, Mary B. and H. K. and Tissieres, A. (1953). Mendelian and non- mendelian factors affecting the cytochrome system in Neurospora crassa. Proc. nat. Acad. Sci., Wash. 39, 606. Moewus, F. (1950). Die Bedeutung von Farbstoffen bei den Sexualprozessen der Algen und Bliitenpflanzen. £. angew. Chem. 62, 496. Moreau, F. and Moruzzi, C. (193 1). Recherches experimentales sur la formation des peritheces chez les Neurospora. C.R. Acad. Sci., Paris 192, 1475. Raper, J. R. (1952). Chemical regulation of sexual processes in the Thallophytes. Bot. Rev. 18, 447. Shear, C. L. and Dodge, B. O. (1927). Life histories and heterothallism of the red bread mould fungi of the Monilia sitophila group. J. agric. Res. 34, 1019. Sheng, T. C. and Sheng, Ginger (1952). Genetic and non-genetic factors in pig- mentation of Neurospora crassa. Genetics 37, 264. Tatum, E. L., Barratt, R. W. and Cutter, V. M. Jr. (1949). Chemical induction of colonial paramorphs in Neurospora and Synephalastrum. Science 109, 509. Westergaard, M. and Mitchell, H. K. (1947). Neurospora V. A synthetic medium favouring sexual reproduction. Amer. J. Bot. 34, 573. Plate 1 (a) 181 M. WESTERGAARD AND H. HIRSCH Plate i (b) Plate i (c) 182 Environmental and genetic control of differentiation in Neurospora m Plate i (d) Plate I. (a) protoperithecia in normal Neurospora grown on P-minimal ; (b) sterile mutant grown on the same substrate, no protoperithecia are formed; (c) mutant with abnormal protoperithecia, associated with excessive melanin production; (d) ^-day-old culture of a sterile mutant to which tyrosine -+- tyrosinase has been added. (Photos by A. 0ye, linear magnification X 20.) Discussion Chairman: H. V. Bmndsted E. N. Willmer. What do you consider to be the relationship between the formation of melanin and that of carotenoids? In the chromatophores of vertebrates, which are derived from neural crest tissue and are thus among the earliest cells to begin differ- entiation in the body, the formation either of melanin or of carotenoids is of frequent occurrence. Occasionally both products occur together. The other tissues in the body where carotenoids abound are of course those in the reproductive system and in the adrenal cortex. In the latter case it is interesting to consider whether there may be some connexion between the carotenoid pigmentation of the cortex and the tyrosine- adrenaline metabolism of the medulla. It is well known that adrenal cortical disease often leads to abnormalities of melanin formation. M, Westergaard. Wild type Neurospora has both carotenoids and melanins. We may consider the conidia, which have the carotenoids, as part of the male sex system; just as the protoperithecia, which have the melanins, represent the female sex system. Mutation in one system does not affect the other, i.e. strains with no conidia may have perfectly normal protoperithecia and vice versa. Unfortunately the male sex in Neurospora is rather degenerate, because fragments of mycelia can also fertilize the protoperithecia. Probably the biochemistry of the male sex should be studied on a species where it is better differentiated. n 183 The control of cell division by M. M. SWANN Department of Apology, University of Edinburgh introduction: division with growth Cells maintain a roughly constant size*, from which it has been argued that division must be dependent in some way on growth. Lewis (1948), for instance, says that if this were not so, cells might be expected to get smaller and smaller or larger and larger. It must be admitted, however, that in the few cases where serious attempts have been made to measure the size of cells, it seems that they do sometimes get smaller and smaller or larger and larger. In the growth of protozoan cultures for instance, cell size varies within wide limits at different stages. Comparable variations are to be found in the growth of bacteria and yeast in cultures. Even in the case of somatic cells, where the difficulties of measurement are considerably greater, there are scattered references to variations in cell size under different conditions. Nevertheless, to a first approximation, cell size does remain constant. It follows that size plotted against time for a given line of cells must give a relationship of the general type shown in Figure la. The precise form of this relationship has in fact never been settled, because of the extreme difficulty of determining the size of a single cell. There is some evidence from measurements made on Protozoa that the main growth in volume, and perhaps therefore in dry weight, occurs soon after fission, giving a curve of cell size more of the type shown in Figure lb (Calkins and Summers, 1941). This may only be an effect of varying degree of hydration, however, and Zeuthen's work (1953) on respiration in Tetrahymena suggests that synthesis may be more or less continuous. Whatever the exact form of the growth curve between one division and the next, it is clear that the processes of mitosis and cleavage are normally triggered off when the size of the cell is roughly double what it was after the previous division. The idea that it is the total size of the cell which somehow provides this stimulus is an old one. It was first put forward by Hertwig (1903) in the form of the nucleo- cytoplasmic ratio, an idea that has been variously acclaimed and criticized (Wilson, 1925). The importance of the ratio of cell surface to cell volume or cell mass has also been stressed from time to time (Berrill, 1943). In this connexion Rashevsky (1938) has suggested on theoretical grounds that diffusion forces might cause a splitting in * The term 'cell size' is widely used to cover cell volume, total cell mass or weight, and cell dry mass or weight. Such a portmanteau definition may be permissible as a convenience, but it must be emphasized that the most satisfactory measure of individual cell growth is probably dry mass or weight. In certain circum- stances it may be desirable also to measure wet weight or degree of hydration. 185 M. M. SWANN two when the cell reached a certain size, but the idea has not met with much enthusi- asm, perhaps because it seems to neglect the visible machinery of mitosis and cleavage. If growth is responsible for starting off the processes of mitosis and cleavage, any substances which interfere with the syntheses that underlie growth should appear to be inhibitors of division. It is possible for instance that some of the so-called pre- prophase inhibitors of mitosis are in fact inhibitors of growth. If so, it might be expec- ted that the substances in question would not be effective in stopping mitosis in cells where there was no growth, e.g. totally cleaving eggs. Inhibitors that are effective on the one type of cell, however, seem in general to be equally effective in inhibiting Ceil SL3e Tune, 4 Sye V Turrte Figure I. Possible types of cell growth curve; (a) growth at a uniform rate; (b) growth most rapid immediately after division. the other type, though a search of the literature in this field might bring to light some interesting exceptions. From what has been said above, it will be apparent that there are no very solid grounds for supposing that division is triggered off when cell size reaches a certain level. The fact that, for a given cell type, size may vary over a considerable range, and that most if not all inhibitors of division act alike on cells whether or not they are growing, might suggest, on the contrary, that growth and division are not very closely linked together. At present, all too little is known of individual cell growth in relation to division. But two new techniques, namely the measurement of the reduced weight of cells, using the Cartesian diver, and the estimation of cell dry weight by means of the interference microscope, now offer the possibility of great advances. 1 86 The control of cell division DIVISION WITHOUT GROWTH Until we have more definite evidence along these lines, there is much to be said for considering the control of division in cells where growth is not simultaneously in- volved, that is to say in the early stages of totally cleaving eggs. Even here of course it is possible that there is some protein synthesis going on, but it can hardly compare with what occurs during the growth and division of ordinary cells. Mainly because of their convenience as experimental material, sea-urchin eggs especially have been used in the study of division. Much is known about their mech- anisms of mitosis and cleavage, and a certain amount about associated chemical changes. This information is mainly derived from a study of inhibitors. In many cases, of course, the chemical action of the inhibitor is uncertain, or even unknown, and the light thrown on the underlying mechanisms of division is therefore not very great. In some cases, however, the action of the inhibitor has been studied in relation to the cell's general metabolism. A certain amount is known, in consequence, about the relation between respiration, the supply of energy and the division process. This work has been reviewed by Krahl (1950). The processes of mitosis and cleavage involve mechanical work, so it is not sur- prising to find that division is very dependent on a supply of energy from respiration. In the case of the sea-urchin egg at least it appears that anaerobic glycolysis by itself cannot supply sufficient energy for division. The eggs will not enter division in oxy- gen tensions below about 04 per cent., nor in the presence of inhibitors of the cytochrome system (e.g. carbon monoxide, cyanide, azide) or inhibitors of the Krebs cycle (e.g. malonate) . In this respect they differ from various other eggs, and certain somatic cells, no doubt because their powers of glycolysis are only slight*. Again, as might be expected, neither sea-urchin eggs, nor any other type of cell, will enter division in the presence of agents such as dinitrophenol, which interfere with phosphorylation. There is some evidence, however, that once division is under way it is not inhibited by the normal respiratory or glycolytic poisons. The implication would seem to be that the energy for division is stored up beforehand. In spite of this work on the respiratory requirements of division, very little is known about when the energy-producing mechanisms are actually required for the division process. Attempts have of course been made to find variations in respiration during the division cycle, but even the most refined modern techniques of Zeuthen (1950) and Scholander et al. (1952), have shown only minor fluctuations in oxygen consumption, and in many cases, no fluctuations at all. The conclusion seems inescapable that either the energy for division is required in a practically continuous flow, or it absorbs so small a part of the total energy output of the cell as to be scarcely detectable. EXPERIMENTS WITH CARBON MONOXIDE ON DIVISION IN SEA-URCHIN EGGS With a view to finding out rather more about how the energy-producing mechan- isms support cell division, experiments have been carried out on synchronously dividing eggs, using carbon monoxide as an inhibitor of respiration (Swann, 1953). * About 5 per cent, of the sea-urchin egg's ATP supply is derived from glycolysis (Cleland, 1953). 187 M. M. SWANN By taking advantage of the fact that the carbon monoxide-cytochrome oxidase com- plex is only stable in wavelengths of light outside its absorption bands, it is possible, merely by altering the wavelength of the illuminating beam, to switch on and off the inhibition of respiration while actually observing the eggs. In this way, the eggs were inhibited for varying lengths of time at different points of the division cycle, and photographed by time-lapse, so that their average time of cleavage could be worked out. The results show that if inhibition is applied before a certain critical point, which occurs at normal temperatures between about 35 and 40 minutes after fertilization, the first cleavage is delayed by a time roughly equal to the duration of the inhibition. If however the inhibition is applied after this critical point, but before cleavage is complete, then the first cleavage is unaffected, but the ensuing cleavage is delayed, by a period again equal to the duration of the inhibition. The best explanation seems to be in terms of a reservoir mechanism. We may sup- pose this reservoir to be filling steadily as a result of respiration, and to siphon out when it is full, at about 35-40 minutes after fertilization. This starts off the division process, which continues regardless whether the reservoir is then filling or not. In the normal course of events, however, the reservoir would begin refilling at once, and continue filling during division and the next interphase. At about 35-40 minutes after the first emptying we might expect the reservoir to be full once more, and to siphon out again, so starting off the second division. The time relations of the second division suggest that this is what happens. Besides accounting for the observed facts, this scheme explains why the first divi- sion takes longer than the subsequent ones. The reservoir has first to fill up, and the division has then to run through. Subsequent divisions, however, can occur at 35-40 minute intervals, namely the length of time taken for the reservoir to fill up, and in fact they do. The scheme also offers an explanation of why there are no major fluctuations in respiration during the division cycle. The energy from respiration is utilized continuously by the reservoir mechanism, and it is only the siphonings out that are discontinuous. It is perfectly possible therefore for the proportion of the cell's energy supply required for division to be a substantial fraction of the whole. Finally the hypothesis explains such experiments as those of Jacoby, Trowell and Willmer (1937) where it was found that tissue culture cells on a maintenance medium could be brought into division by an application of embryo juice, but could only be made to divide a second time by further applications of embryo juice, if these were made during or after the first mitosis. EXPERIMENTS WITH ETHER ON DIVISION IN SEA-URCHIN EGGS With a view to finding out more about the postulated reservoir from a different angle, a further set of experiments was carried out, in which a narcotic, namely ether, was applied as before to synchronously dividing sea-urchin eggs, at different points in the division cycle, and for varying lengths of time (Swann, 1954). These experiments confirm the conclusions drawn from the carbon monoxide work. They indicate the existence of the same sort of reservoir mechanism, and the same critical point in the division cycle at which the reservoir siphons out. 188 The control of cell division The results are complicated, and the details must be left to the original paper. It is sufficient to say here that ether appears to have two quite distinct effects on the division process. At a concentration of i per cent, it prevents the building up of the mitotic figure, but does not affect the filling of the reservoir. That is to say if it is applied, and then removed, before the critical point, it causes no delay in cleavage. If on the other hand it is removed after the critical point, it causes a delay in cleavage, not equal to the total duration of the inhibition, but only to the duration of the inhi- bition beyond the critical point. The egg in other words remains blocked at the criti- cal point. At higher concentrations not only does ether block the building up of the mitotic figure, but it reduces the rate of filling of the reservoir as well. At a concentra- tion of 2 per cent, for instance, the rate of filling is halved. The first effect of ether can therefore be likened to preventing the siphoning out of the reservoir, or to preventing the normal action of the reservoir contents once they have siphoned out. The second effect resembles that of carbon monoxide in reducing and ultimately stopping the filling of the reservoir. It is of some interest to note that i per cent, ether, by blocking the cell at the critical point, offers a means of bringing batches of eggs fertilized at different times into synchronous division. THE NATURE OF THE RESERVOIR Having arrived, by two quite different routes, at the same hypothesis for the control of division in sea-urchin eggs, it may perhaps be permissible to speculate a little about the proposed mechanism. The compelling question, of course, is the physical and chemical nature of the reservoir. Is it to be regarded as a store of energy, or of some other non-energy-carrying compound ? If the latter, it must follow from the carbon monoxide experiments that division itself can function on glycolytic energy alone. The critical experiment must be to test the effect of a range of inhibitors of glycolysis, when mitosis is already under way. The experiments of Hughes (1950), however, indicate that fluoride, which is amongst other things a glycolytic inhibitor, applied under these circumstances to tissue cells, may slow down division but does not actually stop it. More work is needed, however, to be certain on the point. The evidence would seem to point therefore to the reservoir being a store of energy. This energy might of course be built up in a physical form, such as the ori- ented protoplasmic structure of the sperm aster. The results of the ether experiments, however, make this seem unlikely, for whereas the lower concentrations of ether can be seen under the polarizing microscope to solate the oriented structures of the cell, they are, as was pointed out earlier, without effect on the filling of the reservoir. It is natural then to turn to the idea of a chemical store of energy, perhaps of some organic phosphorus compound. There are difficulties, however, in thinking in terms of the best-known of these substances, namely ATP and phosphagen. The amount of ATP, it is generally agreed, does not seem to vary during the division cycle. The amount of phosphagen does, it is true, appear to increase shortly after fertilization, but the time relations do not fit in at all with the reservoir hypothesis (Chambers and Mende, 1953). Moreover both these compounds appear to be freely available to the cell as a whole, as they decrease during anaerobiosis (Barth and Jaeger, 1947; Cleland, 1953). But the postulated store of energy in the reservoir does not get used 189 M. M. SWANN up during inhibition; if it did, the delays induced by inhibition would always be greater than the duration of the inhibition, in order that the depletion of the reser- voir could be made good. Such objections, however, do not rule out the possibility of other more specific organic phosphorus compounds constituting the postulated store of energy. THE APPLICATION OF THE RESERVOIR HYPOTHESIS TO DIVISION WITH GROWTH We are now in a position to consider the reservoir hypothesis in relation to the vast majority of cells where division is accompanied by growth. Since the reservoir fills up not only throughout interphase, but during the preceding division as well, it must be presumed, unless the reservoir mechanism is peculiar to egg cells, that a similar mechanism also operates during interphase and the preceding division in ordinary cells. How might such a mechanism fit in with the growth process; and is it possible that the two mechanisms could run side by side and independently as suggested in the first section? The issue can perhaps be clarified a little by considering the apportioning of the cell's energy supplies between the various cellular activities. It has long been realized that all activities do not have an equal call on these energy supplies; movement, division and irritability, to mention only three, can all be brought to a halt by partial respiratory inhibition, without affecting the cell's maintenance activities, or at least without affecting them irreparably. There is thus no a priori reason to suppose that division and growth will have an equal call on the energy supplies. In the case of the dividing egg cell, two activities alone presumably absorb all the available supply of energy. The first of these is maintenance : making good the wear and tear on the whole protoplasmic structure, and preserving the salt and water balance of the cell; the second activity is division. It is not difficult to see why the normal processes of selection will lead to maintenance having a prior call over divi- sion on the energy supply. Division can be postponed without the organism perish- ing; maintenance cannot. It is not surprising therefore to find that division is sup- pressed in any tissue by a measure of respiratory inhibition which leaves maintenance more or less unaffected. In the case of the sea-urchin egg, for instance, division is suppressed by a degree of anaerobiosis, which reduces the oxygen consumption to about 30 per cent, of normal (Krahl, 1950). Under these conditions, however, the cell can survive for long periods. It is conceivable, in the light of this figure, that division requires as much as 70 per cent, of the sea-urchin egg's total energy output, i.e. about twice as much as maintenance. When the cell is both dividing and growing, the situation is more complex. Growth must be added to the two activities mentioned above and, in some cases, movement and perhaps other activities as well. For simplicity, however, maintenance, growth and division can be considered alone. Once again it seems likely that main- tenance will have a first call on the energy supply, and as with eggs, there is evidence that cells can withstand for considerable periods a degree of anaerobiosis or respira- tory inhibition that prevents division, and in all probability, growth as well. The difficulty arises over the priority for energy as between growth and division. From the point of view of selection, it would seem that a cell which maintained its growth 190 The control of cell division when the supply of energy was short, but which did not divide, would be in a better position to survive or to withstand further stringency than a cell which continued to divide at the expense of growth. In extremis, for instance, accumulated protein can be used as a source of energy, whereas a division that is past and gone cannot. It must be emphasized here that an argument of this sort is a biological one that sidesteps the biochemical issues. The exact mechanism that results in maintenance, for instance, having a prior claim on energy supplies, is not known; but in the present context it is not necessary to know. It may be that there is a common pool, in which different activities compete for energy; or it may be that different activities are supplied by different metabolic pathways. In the first case it would have to be supposed that the various activities have differing affinities for the available energy. In the second case the various pathways would have to be differentially sensitive to inhibition or anaerobiosis. In either case, however, the end result is the same from the point of view of the cellular economy. This question of the respective claims of division and growth on the cell's energy supply is capable, up to a point, of being settled experimentally. It is in fact highly desirable that the effect of varying degrees of respiratory inhibition in slowing down both growth and division should be investigated. Using either the diver technique or the interference microscope this should not present undue difficulties. Meanwhile, however, it may be worth examining in a little more detail what the possible results of such studies might be. If an actively growing cell is subjected by whatever means to a steadily increasing degree of respiratory inhibition, it would be expected, in the light of what has been said above, that division should first slow up, and then stop. Depending on the extent to which the respective claims of division and growth overlap, this slowing up and ultimate stopping of division should be accompanied by some degree of slowing up of growth. Further inhibition will slow up growth yet more, and finally stop it. By this stage it might be expected that maintenance would begin to be affected, at first reversibly. Further inhibition should affect it more drastically, and in due course, irreversibly. This sequence of events is illustrated in Figure 2a which shows the suggested apportioning of the cell's total energy supply between maintenance, division and growth. As the available energy decreases, the upper regions of this diagram should be imagined as being cut off. At 50 per cent, of the normal energy supply for instance, very little energy is left for division, whereas there is still a certain amount available for growth, and maintenance is quite unaffected. The interesting aspect of this suggested apportioning of the cell's energy supply is that a degree of anaerobiosis or respiratory inhibition, or partial starvation of energy-yielding foodstuffs, will affect division more severely than it will affect growth. Though the cell will therefore grow more slowly, it will grow larger. This is not the only possible scheme of things, though it is perhaps the most likely. An apportioning on the lines of Figure 2b for instance, would give a more extreme effect of the kind described above, since division stops entirely before growth is affected at all. In Figure ic, division and growth are affected equally, and cell size should remain constant under all conditions. In Figure id growth is affected before division, so that the cell should get smaller as the energy supply is reduced. J91 M. M. SWANN Unfortunately there is almost no direct evidence, and very little indirect evidence, about the effect on cell size of reducing the energy supply. In the case of ordinary somatic cells, either in vivo or in vitro, measurements of cell size have, it is true, been made from time to time (reviewed by Hoffman, 1953). But the difficulty, not to say impossibility, of estimating with any accuracy the volume of irregularly shaped cells, together with the considerable uncertainty about their degree of hydration, makes the measurements of little value in the present instance. In the case of micro- organisms the situation is rather better. Volume can be estimated much more 1 D ivision a Growth □ Maintenance Figure 2. Possible arrangements for the apportioning of the cell's energy supply. For explanation, see text, (a) Inhibition leads to increase in cell size; (b) inhibition leads to increase in cell size; (c) inhibition produces no change in cell size; (d) inhibition leads to decrease in cell size. accurately, and what is more valuable, the average dry weight of organisms can be determined. A search of the literature has not brought to light any work on the effect of partial anaerobiosis or respiratory inhibition on cell size in micro-organisms. It has been found, however, by Pace and Ireland (1945), that raising the oxygen tension increases the oxygen consumption but decreases the size of Tetrahymena. Since increased oxygen consumption presumably means an increased energy supply, the decrease in size is what might be expected from an apportioning of energy of the type earlier deduced and illustrated in Figure ia. It must be remembered however that increased oxygen tension is liable to act as a poison of uncertain effect. 192 The control of cell division The other references to variation in cell size almost all relate to the growth of populations of one sort of micro-organism or another. The most detailed work on Protozoa is that of Ormsbee (1942) who worked with Tetrahymena and found that while oxygen consumption was high during the logarithmic growth phase and decreased in the final stationary phase, cell dry weight behaved in the opposite way. This is what would be expected in the light of the argument above. Such studies have not however always given consistent results (see, for instance, references to earlier work given by Ormsbee, 1942; Calkins and Summers, 1941; Adolph, 1931). But this is not surprising, for although there is general agreement that oxygen consumption and hence, no doubt, energy production falls in the stationary phase (Hall, 1953), presumably as the result of a shortage of energy-producing foodstuffs, numerous other substances that are essential specifically for growth may also run short. More detailed knowledge of the factors underlying the onset of the stationary phase would be needed to get much further along these lines. The same is true of the other studies on the growth of micro-organisms. There does, however, seem to be good evidence that cell size in yeast is at a minimum during the logarithmic phase and increases during the stationary phase (Richards, 1934). In bacteria too there is wide agreement that cell size varies, though in a different way, being at a maximum in the late lag and early logarithmic phases (Dubos, 1949). But since Robinow (1949) has shown that the so-called cells of this phase are in reality multiple, it seems more likely that cell dry weight is no greater, and perhaps less, at this stage than in the later phases. The complex conditions of the stationary phase of a culture of micro-organisms are clearly not suited to an investigation of the effect of energy supply on division and growth. If the limiting factor is the energy-supplying substrates, all may be well; but if, as is very likely, other compounds are also lacking, the results will necessarily be confused. Information on the effect of anaerobiosis or respiratory in- hibition on both growth and division is therefore required during the logarithmic phase. Such studies should present no special difficulties given the new techniques mentioned earlier. CONCLUSION AND SUMMARY There is no direct evidence about the way in which division is controlled in cells that are also growing. The tendency has been to think in terms of division being triggered off in some way when the cell has grown to a certain size, but there are a number of objections to such an idea. It would appear from the experiments on sea-urchin eggs with carbon monoxide and ether that division in cells without growth is controlled by a continuously oper- ating reservoir mechanism. The preparations for a division are in fact going ahead not only during the previous interphase, but during the previous division as well. Such a mechanism may of course be peculiar to cells that are not growing. But it is possible at least that a similar mechanism exists in cells that are growing, in which case it would seem that growth and division must be two separate processes function- ing simultaneously and more or less independently. If this were so, the relative constancy of cell size would be due simply to the fact that the two mechanisms oper- ated at more or less constant rates. Size however does not remain completely constant 193 M. M. SWANN for any given cell type, and this could readily be explained as a slowing up of one or other mechanism. In an attempt to follow the implications of this idea, the concept of differing 'priorities' for the cell's supply of energy is introduced. It is suggested on purely bio- logical grounds that the first priority is likely to be for maintenance, with growth second and division third. If so, a reduction in the available supply of energy, whether caused by anaerobiosis, inhibitors, or partial starvation of energy-providing foodstuffs, should lead to a slowing down of division before it leads to a slowing down of growth. Such unfavourable conditions should therefore result in an increase of cell size (dry weight) . Such evidence as is available suggests that this is the case. REFERENCES Adolph, E. F. (1931). The regulation of size. Springfield, Illinois. Barth, L. G. and Jaeger, L. (1947). Phosphorylation in the frog's egg. Physiol. Zool. 20, 133-146. Berrill, N.J. (1943). Malignancy in relation to organization and differentiation. Physiol. Rev. 23, 1 01-123. Calkins, G. N. and Summers, F. M. (1941). Protozoa in Biological Research. Columbia, New York. Chambers, E. L. and Mende, T. (1953). Alterations of the inorganic phosphate and arginine phosphate in sea-urchin eggs following fertilization. Exp. Cell Res. 5, 508-519. Cleland, K. W. (1953). Private communication. Dubos, R.J. (1949). The Bacterial Cell. Cambridge, Mass. Hall, R. P. (1953). Protozoology. Prentice Hall, New York. Hertwig, R. (1903). Uber Korrelation von Zell-und Kerngrosse und ihre Bedeutung fur die geschlechtliche Differenzierung und die Teilung der Zelle. Biol. Zjbl. 23, 49-62, 1 08-1 19. Hoffman, J. G. (1953). The size and growth of tissue cells. Springfield, Illinois. Hughes, A. F. (1950). The effect of inhibitory substances on cell division. A study on living cells in tissue cultures. Quart. J. micr. Sci. 91, 251-278. Jacoby, F., Trowell, O. A. and Willmer, E. N. (1937). Further observations on the manner in which cell division of chick fibroblasts is affected by embryo tissue juice. J. exp. Biol. 14, 255-266. Krahl, M. E. (1950). Metabolic activities and cleavage of eggs of the sea-urchin Arbacia punctulata. A review, 1932-49. Biol. Bull. Woods Hole 98, 175-217. Lewis, W. H. (1948). Mitosis and cell size. Anat. Rec. 100, 247-254. Ormsbee, R. A. (1942). The normal growth and respiration of Tetrahymena geleii. Biol. Bull. Woods Hole 82, 423-437. Pace, D. M. and Ireland, R. L. (1945). The effects of oxygen, carbon dioxide and pressure on growth in Chilomonas Paramecium and Tetrahymena geleii. J. gen. Physiol. 28> 547-557- Rashevsky, N. (1938). Mathematical Biophysics. University Press, Chicago. Richards, O. W. (1934). The analysis of growth as illustrated by yeast. Cold Spr. Harb. Symp. quant. Biol. 2, 157-166. 194 The control of cell division Robinow, C. F. (1949). Addendum in The Bacterial Cell, by R.J. Dubos. Cambridge, Mass. Scholander, P. F., Claff, C. L. and Sveinsson, S. L. (1952). Respiratory studies of single cells III. Oxygen consumption during cell division. Biol. Bull. Woods Hole 102, 185-199. Swann, M. M. (1953). The mechanism of cell division. A study with carbon monoxide on the sea-urchin egg. Quart. J. micr. Sci. 94, 369-379. Swann, M. M. (1954). The mechanism of cell division. Experiments with ether on the sea-urchin egg. Exp. Cell Res. (in press). Wilson, E. B. (1925). The cell in development and heredity. 3rd ed., Macmillan, New York. Zeuthen, E. (1950). Respiration during cell division in the egg of the sea-urchin Psammechinus miliaris. Biol. Bull. Woods Hole 98, 1 44-151. Zeuthen, E. (1953). Growth as related to the cell cycle in single-cell cultures of Tetrahymena piriformis. J. Emhryol. exp. Morphogen. 1, 239 — 249. Discussion Chairman: H: V. Brondsted M. Westergaard. Has it been possible to fix the eggs and see how inhibition affects the differentiation of the chromosomes during mitosis ? It seems a pity that the physio- logical approach to the study of mitosis has a tendency to become detached from microscopic information about the chromosome cycle. M. M. Swann. The sea-urchin egg is not a very satisfactory material for chromosome studies, and I have only made a few observations. Clearly, more ought to be done along these lines. E. ^euthen. I have found that the 32P uptake during mitosis varies cyclically (Zeuthen, 1 95 1, Publ. Staz. zool. jYapoli 23, Supplement, 47-69). This might perhaps be evidence of the building up of a store of some phosphorus compound. M. M. Swann. Agreed. C. D. Darlington. Indications that DNA is an important component of Professor Swann's 'reservoir' are of several kinds. Cells when x-rayed may be forced into mito- sis too soon and produce half-size chromosomes presumably with a half charge of DNA. Their prophases may also be reversed so that when they return to mitosis they have a double set of chromosomes, certainly with double DNA (Darlington and La Cour, 1945, J. Genet. 46, 180-267.) In the differentiation of the bone-marrow, red precursors with a high mitotic rate are marked by a strong DNA charge on the chro- mosomes, high spiralization and a compact and effective spindle. White precursors with a low mitotic rate have a low DNA charge, low spiralization and a hollow and less effective spindle. This DNA-plus-protein contrast is exaggerated with pernicious anaemia, where it leads to the formation of ineffective red cells lacking a balanced chromosome complement (La Cour, 1944, Proc. Roy. Soc. Edin. 62, 73-85). In pollen grains there is normally a differentiation, determined by a cytoplasmic gradient, 195 M. M. SWANN between nuclei which will and will not divide. Again this is correlated with DNA charge and protein supply and can be upset or reversed experimentally (in Sorghum, Darlington and Thomas, 1941, Proc. Roy. Soc. B 130, 127-150; in Tradescantia and Scilla, La Cour, 1944, Heredity 3, 319-337). On the other hand cells can be made to divide with an insufficient DNA charge on the chromosomes which have not re- produced (Beadle, 1933, Cytologia, 5, 118-121, in 2ja mays). There are therefore alternative and competing stimuli. F. J. Ebling. I should like to offer some evidence of a different kind in support of Professor Swann's view that cell growth and cell division are independent. During the oestrous cycle of the female rat there are significant changes in thickness of the stratum germinativum but not in the incidence of cell division. It appears that the rate of keratinization alters independently of the incidence of mitosis. 196 On suction in Suctoria by J. A. KITCHING Department of ^oology, University of Bristol INTRODUCTION The Suctoria are a group of carnivorous Protozoa having tentacles by means of which they hold and apparently suck their prey. I wish to discuss the mechanism of suction. A suctorian tentacle normally consists of a sheath and an inner tube. The sheath is apparently continuous with the surface of the animal, whereas the inner tube usually extends a little way into the body of the animal, where it ends abruptly (Collin, 191 2; Noble, 1932). There is a bulbous or sucker-like expansion at the tip of the tentacle, of a structure not yet convincingly described; in Tokophrya infusionum minute papillae have been detected at the tip of the tentacle with the electron- microscope (Rudzinska and Porter, 1953). Some species of Suctoria are particular as to their food. For instance Podophrya fixa is only known to feed on hypotrich ciliates (Collin, 191 2, p. 253), and Discophrya collini (Root, 19 15, as Podophrya collini) on holotrichs. Others, however, such as Tokophrya infusionum, ingest a wide variety of Protozoa, including both flagellates and ciliates (Iziumov, 1947). In some Suctoria the tentacles contract and extend vigor- ously; in others the movements are extremely slow and for most of the time the tentacles are motionless. When a suitable food organism touches the tip of one or more tentacles, it is held. Sometimes it breaks away, but if it fails to do so within a minute or so it falls victim to the suctorian. Some species of Suctoria appear to paralyse their prey, as though by some toxic agent. The contents of the prey then pass up the tentacles into the suctorian, the ingested material being held in food vacuoles (Noble, 1932; Rudzinska and Porter, 1953), in which it is presumably digested. THE HYDROSTATIC GRADIENT A rough estimate of the force necessary to drive the contents of the prey into the suctorian predator can be derived from the rate of flow up the tentacles by means of Poiseuille's formula. This estimate could be based on the decrease in volume of the prey, but this is difficult to measure accurately. The increase in volume of the suctorian is not by itself a sufficient index of the flow of material up the tentacles, because some of the water so derived is evacuated by extra activity of the contractile vacuole. However, the rate of flow up the tentacles can be estimated from the sum of the increase in volume of the suctorian and the extra vacuolar output. From esti- mates derived in this way for Discophrya piriformis, and on the assumption that 197 KITCHING the diameter of the inner tube of the tentacles is 05^, that three tentacles are active, and that the tentacles are 25^ in length, it was calculated (Kitching, 1952a) that for a viscosity twice that of water a pressure difference of about 1 cm. of water would be needed. If the viscosity of the food material is greater, the pressure required would be proportionately higher. THEORIES OF SUCTION Although the positive hydrostatic pressure within the prey itself might provide the necessary force, it is clear that in the feeding of Suctoria a suction must be exerted. Complete flagellates have been observed inside Tokophrya cyclopum (Collin, 191 2). The ingestion by Tokophrya infusionum of whole ciliates, nucleus, pellicle, and all, has been watched and described by Iziumov (1947). Finally, according to detailed observations by Collin (1912, p. 261), in confirmation of Hartog (1901), the suctorian Choanophrya infundibulifera sucks in particles of crushed Cyclops from a distance, like a miniature vacuum-cleaner. There is no doubt that Suctoria really do suck. To explain suction, two plausible theories have been suggested, as well as some others. Peristaltic waves travelling down the inner tube of the tentacles could drive fluid along the tentacles into the body (Collin, 191 2), or the body surface might actually increase in area and so create a suction (Kitching, 1952a). Waves have actually been seen by Collin (191 2, p. 265) travelling down the inner tube of the tentacles of Tokophrya cyclopum, Discophrya steinii, and Choanophrya infundi- bulifera. On the other hand waves seen by Dragesco and Guilcher (1950) in Disco- phrya piriformis proceeded in both directions and appeared to them to be inadequate to explain the process of feeding. They also failed to find waves in the tentacles of Dendrocometes paradoxus. The possibility of an active increase in the surface area of the body was suggested by some observations made during the course of experiments on the effects of feeding on the activity of the contractile vacuoles of Discophrya piriformis. I noticed that occasionally the body surface of the suctorian became wrinkled soon after the capture of a ciliate, and that later, with the uptake of food into the body, the wrinkles dis- appeared. This wrinkling might be due either to a decrease in volume of the body or to an expansion of the body surface. Photographs showed that, at a stage when just enough food had been taken up almost to cause the wrinkes to disappear, the body was considerably larger than it had been before the wrinkling occurred. It therefore appeared that the wrinkling was due at least in part to an increase in surface area. It is true of course that there might also have been a temporary decrease in body volume, such as might be produced by an injection of material into the prey, but there is no positive evidence in favour of this view, nor will it by itself explain the observations. It was found possible to induce wrinkling regularly in Discophrya piriformis which had been grown in dilute sea water (5 or 10 per cent.) by feeding it on Paramecium in a partially dehydrated state. The Paramecium was transferred from a freshwater culture to the same dilute sea water a few minutes before it were offered to the Discophrya, so that it had lost water by oxosmosis. The Discophrya became wrinkled within a few minutes, and remained so for an hour or more, but ultimately filled up with food so that the wrinkles disappeared. When the wrinkles were about to 198 On suction in Suctoria disappear the body was considerably larger, as seen in profile, than it had been originally. Although again it might be suggested that the wrinkling was due in part to a loss of material to the shrunken Paramecium, there is no positive support for this view, and there is no doubt that the body surface expanded. It seems likely that the attachment of the tentacles to the prey activates the suctorian to an expansion of the body surface which makes room for the uptake of food. EFFECTS OF HIGH HYDROSTATIC PRESSURE The interpretation of the expansion of the body surface has been carried a stage further by a study of the effects of high hydrostatic pressure (Kitching, 1954). Experiments have been carried out with pressures ranging up to 15,000 lb. per sq. inch (1,020 atm.). At pressures of 2,000 lb. persq. inch (136 atm.) and over, the body surface wrinkled. This wrinkling developed in from half a minute to one minute at the lower pressures, but took place within a few seconds at the higher pressures. There was also a tendency for the cuticle to separate from the underlying proto- plasm, and for the protoplasmic surface to become rounded within the cuticle. At the higher pressures this happened at the same time as the wrinkling, but at the lower pressures it was often delayed, and did not always occur. At a pressure of 2,000 lb. per sq. inch (136 atm.) the body surface often became smooth again even while the pressure was still maintained, but at 3,000 lb. per sq. inch (214 atm.) this did not happen. Compression of the contents of the body cannot account for wrinkling. Water is only compressed by about 4 per cent, at a pressure 15,000 lb. per sq. inch, and it is not likely that the presence of proteins or other cell constituents would make any very great difference; nor is there any gas phase in the body. Moreover, the wrinkling took place rather slowly at the lower pressures, and was never reversed immediately on release of the pressure. From photographs taken before and during the application of pressure, it is clear that wrinkling involves an increase in the length of the peri- meter of the organism as seen in a sagittal profile, and this implies an increase in the surface area of the body. This conclusion has received support from the results of recent experiments in which a relatively low pressure (2,750 lb. per sq. inch) was applied to Discophrya piriformis in the process of feeding. This pressure was sufficient to cause wrinkling but not to prevent feeding. By the time enough food had been taken in almost to fill up the wrinkles, the body was considerably larger than it had been before the application of pressure. In those experiments in which the pressure applied was relatively low, the proto- plasm often remained in contact with the expanded cuticle (Kitching, 1954, Plate I), so that it is necessary to conclude that the protoplasmic surface expanded also. The rounding up of the protoplasm which occurred at the higher pressures may be compared with the rounding up of an Amoeba or of a dividing Arbacia egg at similar pressures (Marsland and Brown, 1936; Marsland, 1938). On release of pressure, the protoplasm, if separated from the cuticle, often spread back into contact with the latter within the following few minutes or less. This movement must involve either an increase in volume of the protoplasm or an increase in the wrinkling of the pellicle. It cannot be ascribed to an increase in o 199 J. A. KITCHING Figure I. Effects of hydrostatic pressure on the suctorian Discophyra piriformis*. The specimen shown was subjected to increasing pressures step by step, with from 10 to 20 min. at each step. The drawings illustrate the condition attained near the end of each period of exposure, when any further change was very slow. The drawings have been traced from photographs. #This species is very close to D. collini (Root) and possibly identical with it. 200 On suction in Suctoria volume of the protoplasm due to decompression, as it takes place too slowly. Al- though in some cases there was an indication of a swelling, some time after release of pressure, associated with a prolonged stoppage of the contractile vacuole, it is by no means clear that this accounted for the rather rapid spread of the protoplasmic surface which was sometimes seen. The cuticle remained wrinkled for many hours after the release of pressure. How- ever the wrinkles slowly disappeared and eventually cuticle and protoplasm became rounded in outline. Wrinkles formed at 2,000 lb. per sq. inch were observed to dis- appear even while the pressure was maintained, and in other cases in which limited wrinkling was induced by rather brief exposure to high pressure the wrinkles dis- appeared within a few minutes after release of the pressure. It may therefore be sug- gested that there is a mechanism by which the fit of the pellicle to the protoplasm is constantly subject to adjustment. This mechanism resides in the protoplasmic surface and is likely to be enzymatic. It was noticeable that after release of pressure, if there was some portion of the pellicle which was not brought into contact with the proto- plasmic surface by the spread of the latter, this portion did not become remodelled, but persisted as an excrescence. Mechanisms for the adjustment of pellicular fit must be widespread in the Protozoa. The tendency of the protoplasmic surface to expand when the cuticle allows it to do so is evidently opposed by high pressure, and only reaches its full development after the pressure has been released. FEEDING IN SUCTORIA Let us now return to the process of feeding in Suctoria. There is clearly a process of activation. In Choanophrya infudibulifera suction is not provoked by carmine par- ticles, but is initiated by genuine food (Collin, 191 2). In Discophrya piriformis the expansion of the body surface follows attachment of the tentacles to the prey. The expansion of the cuticle is somewhat reminiscent of the formation of a fertilization membrane, although it is carried out without the elevation characteristic of the latter. The cuticle of Discophrya is probably also made of protein. It is not clear whether expansion of the cuticle merely permits expansion of the protoplasmic surface within it, or whether activation directly stimulates the protoplasmic surface to expand. It is perhaps relevant that, on the application of pressures of 10,000 lb. per sq. inch (680 atm.) or more, there was usually an apparently simultaneous expansion of the cuticle and separation from it of the protoplasmic surface, and that on release of pressure the protoplasmic surface spread, and made contact with the expanded cuticle. This shows that expansion of the cuticle can at least occur indepen- dently, although it may well be brought about by reactions at the protoplasmic surface. So far I have not been able to induce wrinkling in Acineta by the application of high pressure. This suctorian has a very loosely fitting cuticle or case, with plenty of room inside for expansion during feeding, so that presumably a mechanism for the rapid expansion of the cuticle is unnecessary. It is not known whether expansion of the body surface of Discophrya piriformis merely makes room for food material driven in by other means, such as peristalsis of the inner tubes of the tentacles, or whether this expansion actually creates a suction. A more detailed study of the peristaltic movements of the tentacles is 201 J. A. KITCHING needed, in material favourable for this purpose. It is possible that there may be con- siderable specific variations in the part played by this form of activity, and in any case the observation needs repeating. Peristalsis of the tentacles would account for the fact that a feeding suctorian becomes nearly spherical when full, as though pumped up. It would no doubt also help in the passage of lumps of material. On the other hand the expansion of the body surface, if accompanied by a resistance to inward collapse, would very conveniently provide a suction. It might take place in the way suggested by Mitchison (1952) for the surface of dividing sea-urchin eggs. The contractile vacuole plays an important though limited part in the process of feeding (Pestel, 1931 ; Kitching, 1951 and 19526; Rudzinska and Chambers, 1951 ; Hull, 1953). It operates at a much faster rate during feeding, and so serves to remove some of the water brought in as part of the substance of the prey. During a meal the suctorian swells much less than the prey shrinks, and the difference is accounted for by the extra activity of the contractile vacuole, (Kitching, 19526; Hull, 1953). If the body volume were constant, and the surface of the body were rigid, extra activity of the contractile vacuole would presumably produce a suction which might account for the process of feeding. However, the body does in fact get bigger, and this cannot be explained by vacuolar activity. REFERENCES Collin, B. (1912). Etude monographique sur les Acinetiens. II. Morphologie, Physiologie, Systematique. Arch. £ool. exp. gen. 51, 1-457. Dragesco, J. and Guilcher, Y. (1950). Sur la structure et le fonctionnement des tentacles d'Acinetiens. Microscopie 2, 17-24. Hartog, M. (1901). Notes on Suctoria. Arch. Protistenk. 1, 372-374. Hull, R. W. (1953). Observations on Suctoria: contractile vacuole rate changes during feeding and reproduction in Solenophrya micraster Penard 19 14. Proc. Soc. Protozool. 4, 20. Iziumov, G. J. (1947). The digestion process in Tokophrya infusionum. £ool. Jh. 26, 263-268 (in Russian). Kitching, J. A. (1951). The physiology of contractile vacuoles. VII. Osmostic relations in a suctorian, with special reference to the mechanism of control of vacuolar output. J. exp. Biol. 28, 203-214. Kitching, J. A. (1952a). Observations on the mechanism of feeding in the suctorian Podophrya. J. exp. Biol. 29, 255-266. Kitching, J. A. (19526). The physiology of contractile vacuoles. VIII. The water relations of the suctorian Podophrya during feeding. J. exp. Biol. 29, 363-371. Kitching, J. A. (1954). The effects of high hydrostatic pressures on a suctorian. J. exp. Biol. 31, 56-67. Marsland, D. A. (1938). The effects of high hydrostatic pressure upon cell division in Arbacia eggs. J. cell. comp. Physiol. 12, 57-70. Marsland, D. A. and Brown, D. E. S. (1936). Amoeboid movement at high hydro- static pressure. J. cell comp. Physiol. 7, 167-178. 202 On suction in Suctoria Mitchison, J. M. (1952). Cell membranes and cell division. Symp. Soc. exp. Biol. 6, 105-127. Noble, A. E. (1932). On Tokophrya lemnarum Stein (Suctoria) with an account of its budding and conjugation. Univ. Calif. Publ. £ool. 37, 477-520. Pestel, B. (1931). Beitrage zur Morphologie und Biologie des Dendrocometes para- doxus Stein. Arch Protistenk 75, 403-471. Root, F. M. (1915). Reproduction and reactions to food in the suctorian Podophrya collini n.sp. Arch. Protistenk. 35, 164-196. Rudzinska, M. and Chambers, R. (1951). The activity of the contractile vacuole in a suctorian {Tokophrya infusionum) . Biol. Bull. Woods Hole 100, 49-58. Rudzinska, M. and Porter, K. R. (1953). Submicroscopic morphology of structures involved in the feeding of Tokophrya infusionum. Proc. Soc. Protozoal. 4, 9. Discussion Chairman: H. V. Brendsted M. M. Swann. I am interested in the idea of suction generated by expansion in view of my own and Mitchison's work on membrane expansion as the possible mechanism of cell division. In the light of our experiments on the modulus of cell membranes, I think that 1 cm. of water is a possible figure. I should also like to suggest that a study of the flow up the tentacle with different viscosities of protoplasm might help to settle whether suction is caused by expansion or by peristalsis. A very low viscosity would help the first mechanism, but possibly not the second. J. A. Kitching. The spherical shape of the organisms at the end of a good meal supports the idea that they have been filled up, as though by peristalsis of the inner tube of the tentacles. However, both mechanisms may operate. On the rather rare occasions when wrinkling occurs during normal feeding, or when it is induced experimentally by the use of a shrunk ciliate for food, the protoplasmic surface certainly expands together with the pellicle; and the same is true in certain of the pressure experiments in which the pressure used was not very high. In these cases the expansion cannot be attributed to an internal pressure caused by peristalsis of the tentacles. J. F. Danielli. Does the application of pressure stop the inflow of material from the prey? J. A. Kitching. Pressures above about 4,000 lb. per sq. inch stop the uptake of food. The tentacles attached to the prey, shortened during the feeding process, gradually extend, and the prey is imperceptibly released. R. J. Goldacre. Can a comparison be drawn between the expansion of the surface of Discophrya which occurs during feeding and the expansion of the surface of Amoeba caused by fat-solvent anaesthetics? 20J J. A. KITCHING J. A. Kitching. It is likely that the expansion of the surface oiDiscophrya during feeding is secondary to the expansion of the pellicle. It is however interesting that on release of Amoeba from high hydrostatic pressure there is an aggregation of the granular cytoplasm to the centre, and an extrusion of clear liquid beneath the plasmalemma, to give an appearance very like that described by you (Goldacre, 1952, Symp. Soc. exp. Biol. 6, 128-165), for amoebae treated with anaesthetics. 204 List of Members Dr. E. J. Ambrose, Chester Beatty Research Institute, Royal Cancer Hospital, Fulham Road, London, S.W.3. Dr. R. J. Ancill, Department of Anatomy, The University, Bristol 8. Dr. C. A. Ashford, Department of Physiology, The University, Bristol 8. Professor J. L.-A. Brachet, Universite libre de Bruxelles, Brussels. Professor H. V. Brondsted, Institut for Aim. Zoologi, Kobenhavns Universitet, Copen- hagen. Dr. W. G. B. Casselman, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada. Mr. L. Carter, Department of Zoology, The University, Bristol 8. Dr. Anne S. Cole, Department of Physiology, The University, Bristol 8. Professor K. E. Cooper, Professor of Bacteriology, Department of Preventive Medicine, Canynge Hall, Whatley Road, Bristol 8. Professor J. F. Danielli, Department of Zoology, King's College, Strand, London, W.C.2. Professor C. D. Darlington, Professor of Botany, University Department of Botany, South Park Road, Oxford. Mr. D. Davidson, University Department of Botany, South Parks Road, Oxford. Dr. F.J. Ebling, Department of Zoology, The University, Sheffield 10. Dr. B. F. Folkes, Department of Botany, The University, Bristol 8. Dr. S. B. Gertner, National Institute for Medical Research, Mill Hill, London, N.W.7. Dr. R. J. Goldacre, Chester Beatty Research Institute, Fulham Road, London, S.W.3. Dr. J. J. Gordon, 3 Elaine Crescent, Newport, Mon. Dr. L. J. Hale, Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh 9. Dr. A. Haque, University Department of Botany, South Parks Road, Oxford. Professor J. E. Harris, Department of Zoology, The University, Bristol 8. Professor H. Heller, Professor of Pharmacology, The University, Bristol 8. Dr. E. J. Hewitt, A.R.C. Unit of Plant Nutrition (Micro-nutrients), Research Station, Long Ashton, Bristol. Dr. E. Hoff-Jorgensen, Universitetets Biokemiske Institut, Juliane Maries Vej, 30, Copenhagen. Dr. K. W. Keohane, Department of Anatomy, The University, Bristol 8. Dr. R. D. Keynes, Physiological Laboratory, Cambridge. Dr. J. A. Kitching, Department of Zoology, the University, Bristol 8. Dr. H. Klenow, Universitetets Institut for Cytofysiologi, Juliane Maries Vej, 28, Copen- hagen. Professor H. J. A. Koch, Laboratoire de Zoophysiologie de l'Universite, Louvain, Belgium. Dr. G. P. Lewis, National Institute for Medical Research, Mill Hill, London, N.W.7. Dr. O. Maaloe, Statens Seruminstitut, Copenhagen. Miss Margaret J. Manning, Department of Zoology, The University, Bristol 8. Miss Lorna K. Mee, University Department of Radiotherapeutics, Downing Street, Cambridge. Dr. W. K. Metcalf, Department of Anatomy, The University, Bristol 8. Dr. N. Myant, Experimental Radiopathology Research Unit, Hammersmith Hospital, London, W.12. Mr. A. Peters, Department of Zoology, The University, Bristol 8. 205 List of Members Dr. L. E. R. Picken, University Department of Zoology, Downing Street, Cambridge. Dr. G. Pontecorvo, Department of Genetics, the University, Glasgow, W.2. Dr. H. H. R. Reinert, National Institute for Medical Research, Mill Hill, London, N.W.7. Dr. W. S. Reith, Experimental Radiopathology Research Unit, Hammersmith Hospital, Ducane Road, London, W.12. Dr. F. J. Reithel, Chemistry Department, University of Oregon, Oregon, U.S.A. Dr. L. M. Rinaldini, Strangeways Laboratory, Cambridge. Dr. Muriel Robertson, The Lister Institute, Chelsea Bridge Road, London S.VV. 1. Dr. G. de M. Rudolf, Litfield House, Clifton Down, Bristol 8. Dr. C. J. Shepherd, M.R.C. Unit for Chemical Microbiology, School of Biochemistry, Tennis Court Road, Cambridge. Dr. M. Simonsen, Fondation Danoise, Boulevard Jourdan 8, Paris 14*. Mr. C. R. Sladden, Department of Zoology, The University, Bristol 8. Mr. M. A. Sleigh, Department of Zoology, The University, Bristol 8. Dr. H. Sosnowick, Department of Physiology, The University, Bristol 8. Mr. S. P. Spragg, Department of Botany, The University, Bristol 8. Professor M. M. Swann, Department of Zoology, The Ashworth Laboratory, West Mains Road, Edinburgh 9. Dr. P. H. Tuft, Department of Zoology, University of Edinburgh, West Mains Road, Edinburgh 9. Professor H. Ussing, Zoofysiologisk Laboratorium, Kobenhavns Universitet, 36 Juliane Maries Vej, Copenhagen. Dr. T. Vickers, Physiological Laboratory, Cambridge. Professor C. H. Waddington, F.R.S., Institute of Animal Genetics, West Mains Road, Edinburgh 9. Mr. P. M. B. Walker, M.R.C. Biophysics Research Unit, King's College, London. Mr. P. J. Warren, Department of Zoology, The University, Bristol 8. Professor M. Westergaard, Universitetets Genetiske Institut, Universitets-parken 3, Copenhagen. Dr. H. P. Whiting, Department of Zoology, The University, Bristol 8. Dr. E. N. Willmer, Physiological Laboratory, Cambridge. Dr. A. J. Willis, Department of Botany, The University, Bristol 8. Dr. Marion O. P. Wiltshire, Department of Physiology, the University, Bristol 8. Dr. E. W. Yemm, Department of Botany, The University, Bristol. Dr. E. Zeuthen, Zoofysiologisk Laboratorium, Kobenhavns Universitet, 32 Juliane Maries Vej, Copenhagen. 206