Planta (Berl.) 76, 209--226 (1967)

Induction of Meiosis in Yeast I. Timing of Cytological and Biochemical E v e n t s A. F. C~o~s Department of ]~otany, University, Nijmegen, The Netherlands Received May 31, 1967

Summary. Induction of meiosis in yeast is a complex process starting during the period of premeiotic mitoses. Enlargement of the nucleus is the earliest sign of the actual onset of meiosis. Parallel with this development is an increase of about 10% in the protein content of the cell. Somewhat later there is a sharp rise in cell mass and cell volume. There is only one period of high metabolic activity during sporogenesis and this occurs early during the process. I. Introduction

A thorough knowledge of the physiology of meiosis is necessary for understanding the mechanism of recombination in eukaryotic organisms. As meiosis is considered to have arisen as a deviation from the pattelTa of normal mitotic division, the study of the factors underlying the shift from mitosis to meiosis is of particular interest (TAYLOR, 1967). However, the investigations undertaken to elucidate the mechanism of induction of meiosis have not led to spectacular results (for review, see RHOADES, 1961). I n higher plants the matter is complex because of the unknown interactions between meiocytes and surrounding tissue. When sporogenous tissue is isolated from anthers before the onset of meiosis, the cells do not enter into meiosis, but continue to divide mitotically in tissue culture (HoTTA et al., 1966). I n most lower organisms the occurrence of meiosis results from preceding developments, such as fusion of nuclei or germination of the zygote. However, in a limited number of microorganisms, for example, baker's yeast, meiosis is directly induced b y changes in the environmental conditions. I n yeast meiosis is the first step in sporogenesis which leads to the formation of haploid ascospores from diploid cells (WI~GE, 1935). The vegetative cell is thereby converted into an ascus. A reliable criterium for the actual onset of meiosis is an essential prerequisite for studying the induction as it indicates the end of the inductive phase. A number of events described in literature could provide such a eriterium. These are (1) enlargement of the nucleus (NAG~L, 1946; PO~TE~I~ACT and MrLL~, I962); (2) fragmentation of the vacuole into numerous parts (RwEss, 1870; M I L L ~ et al., 1963 ; Svr~LA et al., 1964) ; (3) accumulation of glycogen and fat (Po~TE~RACT and MILLER, 1962; 15a

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210

A.F. CROES:

M~LLE~ etal., 1963); and (4) increase in cell volume (REESS, 1870; DEYSSON and LAg, 1963). However, none of these features has been exactly timed, so t h a t the sequence in which they occur remains obscure. Therefore these characteristics have to be reinvestigated to find out which of t h e m are reliable and useful criteria for the onset of meiosis. The occurrence of meiosis depends on transfer of the cell from a rich growth medium to a poor sporulation medium (DE SEY~ES, 1868). Sporogenesis in yeast has been intensively studied b y numerous investigators (for review, see MILL]~Ir 1959). These studies were almost exclusively concerned with the composition of the sporulation medium and with other external factors (e.g. aeration, temperature). Almost nothing is known about the physiological and biochemical conditions within the cell. I t is meaningful therefore to investigate the patterns of oxygen consumption and of R N A and protein content during sporogenesis in order to gain information on the course and the extent of metabolic activity. The cells are not considered to have been predetermined for meiosis in a specific way when transferred to sporulation medium. Investigations on the induction of meiosis have indicated t h a t the absence of nitrogen and the presence of a non-fermentable carbon source in the sporulation medium are major inductive factors (MILLER, 1959, 1963). Some doubt remains regarding this view since the presence of a m m o n i u m in the sporulation medium does not prevent a large number of nuclei from entering into the first stage of meiosis (1V[rLLE~, 1963, 1964). However, the possibility remains t h a t meiosis is already induced during the later stages of vegetative growth. I t is interesting to know when the cell prepares itself bioehemically for the subsequent steps in sporogenesis. This m a y be investigated by experiments with an inhibitor. Ethionine is suitable for this purpose. I t strongly affects sporogenesis (MILLER, 1959, 1964); its mode of action is well known (t)A~Ks, 1958; MAw, 1963) ; and the inhibition can be overcome b y methionine (MILLER, 1959; MAW, 1961). The present study deals with the following problems: (1) Which changes in the cell are reliable criteria for the onset of meiosis; (2) which metabolic changes accompany meiosis and aseus formation; and (3) at what time is meiosis induced ? II. Material and Methods a) Organism. Saccharomyces cerevisiaeI:IANS~, strain CBS 5525, was obtained from the Centraal Bureau voor Sehimmelcultures, Yeast Division, Delft, The Netherlands. Colonies originating from single cells were obtained from this strain by conventional plating technique. These clones were tested for constant, high sportflation frequency together with rapid ascus development. Clone No. 10 which best satisfied these requirements was selected for the present study.

Induction of Meiosis in Yeast. I

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b) Culture Techniques. The presporulation medium of PONTEFRAOTand MILLER (1962), slightly modified, was used for growing the yeast. I t consisted of 2% glucose, 0.67% Yeast Nitrogen Base (Difco) and 0.1% Yeast Extract (Difco) in 0.05M phthalate buffer of p H 5. The yeast strain was maintained on slants of this medium solidified with 1.5% agar (Merck) and subcultured one day before starting an experiment and otherwise every 2 or 3 days. The modified sporulation medium of McCLAI~:r et al. (1959) was used in sporulation experiments. I t consisted of 0.175 M potassium acetate and 0.1% yeast extract in distilled water. All culture liquids were sterilized by autoclaving for 30 rain at 110 ~ All experiments were begun by suspending yeast cells from a slant in the liquid growth medium at a density of 5 • 104 cells/ml. The culture was aerated with moistened air and shaken on a reciprocating shaker at 30 =t=1~ Unless stated otherwise, the cells were centrifuged down 18 hours after inoculation and washed twice with sterile water. The pellet was resuspended in the sporulation medium at a density of 3 • 107 cells/ml. The culture was incubated under the same conditions as during growth. c) Counting Methods. The percentage of asci was determined 24 hours later by examining 500 randomly selected cells. In suspensions designed for inoculation tile number of cells per ml was estimated with a Biirker-Tiirk counting chamber. All other determinations of cell densities were performed with an electronic particle counter (Coulter, Model A, Medical). The cells were suspended in 0.6% NaC1 for counting. The aperture diameter of the measuring tube was 100 ~m. This instrument was also used for size distribution studies according to D~.Ysso~l and L x v (1963). d) Staining Procedures. For staining of the nucleus the procedure of PO~E~RACT and MILL~I~ (1962) was employed, with slight modifications. The yeast cells were applied to cover slips with a thin layer of Haupt's adhesive and fixed for 20 min with the fixative of Helly prepared by the formula of RoBn~ow (1961). After washing with 70 % ethanol and several changes of distilled water, the cover slips were immersed in 1% NaC1 at 60 ~ for 2 hours (GA~ESA~Iand SWXMII~ATHA~I,1958). Thereafter, they were transferred without washing into 1 N HC1 at 60 ~ for 7 min. Following this hydrolysis the cover glasses were washed 10 min in running tap water. The material was then stained in a liquid containing 10 drops of Giemsa solution (Merck) per 10 ml of Gurr's buffer, p H 6.8. The cover slips were kept in this liquid for 1 hour and afterwards washed rapidly in buffer to remove the stain from the cytoplasm. Finally they were mounted on slides and sealed with paraffin. Photomicrographs were made with a Leitz Orthomat automatic camera using a planapochromatie oil immersion objective, N.A. 1.32. e) Extraction Procedures. Nucleic acids and proteins were extracted from the cells according to the following procedures. The material was centrifuged down in the cold and washed once with ice-cold, distilled water. Then the acid-soluble pool was extracted according to WAS~KA(1962) with 0.2 N perchlorie acid (PCA) in 50% ethanol at room temperature. An extraction period of 40 min proved to be sufficient. Extraction for longer periods, up to 80 rain, did not remove any more UV-absorbing material. When, however, the extraction was continued for as long as 100 or 120 min, both the optical density (O.D.) at 260 nm and the ratio between the O.D. at 260 and 230 n m started again to rise. Presumably this is due to onset of hydrolysis of RNA. After the 40-rain extraction period the material was centrifuged down and washed once with the ethanol-PCA mixture. Then the lipid iraetion was removed by gently boiling the material in a mixture of ethanol and ether, 3:1 (v/v). Samples to be analysed for ribonucleic acid (I~NA) or protein were treated twice in this way, for 3 rain each. During these short periods no R N A and protein are lost ( M t I ~ o 15"

212

A . F . CReEs:

a n d D e w , I n , 1964), as could be inferred from U V readings. Samples designed for determinations of deoxyribonucleic acid (DNA) require a more prolonged ethanolether treatment, since f a t t y substances, which are not completely removed during this step, m a y afterwards cause t u r b i d i t y in the DNA-containing extracts. The material was, therefore, suspended three times, 5 min each, in the boiling ethanolether mixture. No traces of DNA were found in a n y of the supernatants obtained Mter centrifugation, After removal of the lipids, the residues were trea~ed in different ways. DNA was hych'olysed in 0.5 N PCA a~ 70 ~ for 1 h o u r (OGvg et al., 1952). I~NA was extracted b y suspending the material in 0.5 N PCA a t 45 ~ according to WANKA (1962). A 2-hour period proved to be sufficient to ensure complete hydrolysis of RNA. The reliability of this procedure was checked with the alkaline extraction method of SCm~IDT a n d T~A~gAVS~R (1945), using 0.3 N KOH. Starting from equal amounts of yeast, extracts were prepared b y b o t h procedures a n d the U V absorptions were compared. I~o difference was found in the O.D. a t 260 nm. However, the alkaline extraction produced a far higher O.D. a t 230 n m t h a n the acid hydrolysis. I t was felt t h a t for our material WA~KA'S procedure is superior to the Schmidt-Thannhauser method. Proteins were extracted in 1 N N a O H b y heating for 4 rain in a boiling water bath. /) Chemical Determinations. D N A was determined b y the diphenylamine reaction using t h e modification of BVRTON (1956). The difference between the O.D. a t 595 n m a n d 650 n m was t a k e n as a measure of the concentration. W h e n the PCA extracts were not completely clear, a parallel series of determinations was r u n using BmCTO~'S reagent without diphenylamine. The readings resulting from this were subtracted from the values obtained with the complete reagent. Herring sperm DNA was used as a standard. The D N A content of the crude preparation was established b y comparing it to highly polymerized calf t h y m u s DNA (Sigma). The R N A concentration was calculated from the difference between the O.D. a t 260 n m a n d 230 nm. There is no need to correct the results for D N A since with the I~NA extraction conditions only 20% of t h e D N A is extracted. Moreover, the yeast cell contains 30 ~imes more R N A t h a n DNA. As a check on this measurement, the orcinol method of CEgmWwI (1955) was used. The color was not intensified by shaking with amyl alcohol. The readings a t 670 n m were corrected for hexose by t h e formula of WAZ~KA on t h e assumption t h a t glucose was the only interfering substance. Commercial yeast 1%NA (Sigma) was used as a standard. Protein concentrations were measured b y the Folin method of L o w R r et al. (1951). Serum albumin (Behringwerke) was used as a standard. Amino acid analyses were performed with a Micro Column Amino Acid Analyzer (Technicon Instruments) b y the procedure of LINSK~NS a n d TvPf: (1966). The O.D. a t 570 n m was read in a 15 m m flow-cell. The preparation of the extracts to be analysed was slightly modified. Samples of yeast were washed twice with distilled water a n d extracted twice with 0.01 M citric acid in 70% ethanol. The combined extracts were vigorously shaken with two volumes of chloroform to remove the ethanol. After phase separation, t h e water phase was pipetted off a n d freeze-dried in vacuo. The residue was t a k e n up in 0.2 M sodium citrate--HC1 buffer, p H 2.2. A sample of 0.25 m] was applied to the column. All extractions were carried out a t 4 ~. Gas exchange studies were performed with a W a r b u r g respirometer using conventional manometric techniques (U~B~]~IT et al., 1964). The temperature of the b a t h was 30 ~ The flasks were allowed to equilibrate for 15 rain. W h e n substrate was present in t h e side arm, it was tipped into the m a i n c o m p a r t m e n t 10 m i n later. Analytical grade chemicals were used throughout.

Induction of Meiosis in Yeast. I

213

III. Results

1. Cytological and Biochemical Events during Sporogenesis When cells of the yeast strain used are transferred from the growth medium to the sporulation medium, growth stops immediately. Meiosis and subsequent ascus formation occur within 24 hours in 69.4 ~_ 4.15 % (standard deviation) of the cells. This figure has been calculated from 25 experiments. a) Nuclear Events. I n order to time the various steps of meiotic development, samples were taken from the sporulation medium at regular intervals and stained by the HCL Giemsa procedure. The convention of French workers on bacterial sporulation was adopted for time indication. T o is the time at which the cells are incubated in the sporulation medium and T~ the time n hours later. Different stages of meiosis are shown in Figs. 1--5. When the T o and T~ stages are compared, it is seen that, at least in a number of cells, the T 2 nuclei are slightly larger (Figs. 1 and 2). This is the first sign of an altered nuclear behavior. At T s the internal structure of the nucleus is coarser, presumably due to progressive contraction of the chromosomes (Fig. 3). This picture is typical for late meiotic prophase I in yeast (McCLARu et M., 1957; GA~nSAN, 1959). The first figures of both nuclear divisions are found at T10. The number of cells t h a t pass through metaphase I increases from t h a t time until T2~ (Fig. 9). The first mature asci appear at T14 when the nuclear divisions are still in full progress. Therefore, a great variety of different stages is present in the stained slides, as shown in Fig. 4. During the next period the number of asci increases at a nearly constant rate until the m a x i m u m is reached at T~4 (Fig. 9). The great majority of non-sporulating cells are buds attached to the mother cells (Fig. 5). Since Giemsa staining showed a nucleus in most of them, they were counted as cells when the percentage of asci was determined. The nuclei of the buds are characterized b y a highly compact chromatin which remains unchanged throughout (Figs. 1--5). The time course of DNA replication has been reported previously (C~oEs, 1966) and is indicated in Figs. 9 and 11 for the sake of comparison. The duration of two developmental steps can be estimated, at least in a rough manner, from the data in Fig. 9. The ~neiocytes pass through metaphase I about 8 hours after Db~A replication. About 31/2 hours later the ascus is mature.

b) Changes in Other Parts o/the Cell, and the Cell Volume. The incubation of yeast in a sporulation medium not only brings about an alteration in nuclear behavior, but also causes characteristic changes in other cell constituents. One of them is the well-known vacuolization of the cytoplasm readily seen by phase optics. In our strain this phenomenon 15b Planta (Berl.),Bd. 76

Figs. 1--8 (~or legends see p. 215)

A. F. CI~oEs: Induction of l~eiosis in Yeast. I

215

was o b s e r v e d in t h e g r e a t m a j o r i t y of cells as e a r l y as T2 (Figs. 7 a n d 8). The large central vacuole conspicuous a t T O seems to fall a p a r t into numerous, small fragments. A n o t h e r k n o w n characteristic of early sporogenesis is t h e extensive a c c u m u l a t i o n of food reserves consisting of glycogen a n d f a t in t h e cell. I n order to t i m e this a c c u m u l a t i o n , t h e increase in cell mass resulting

)i/ IO

T~

lB

~12

Ilg

I20

I2/~

time Fig, 9. BioehemieM and cytologieM characteristics of meiosis and aseus formation.

Dry weight and DNA: percentage of increase above the TO values. Nuclei after met~phase I and mature asei: percentage in 500 randomly selected cells f r o m it was d e t e r m i n e d . A t various times during t h e first 8 hours, samples containing a k n o w n n u m b e r of cells were t a k e n from t h e m e d i u m . The m a t e r i a l was w a s h e d twice a n d d r i e d in an oven for 24 hours a t 105 ~ The m e a n d r y mass per cell is shown in Fig. 9. I t increased during t h e first 8 hours to 146% of the original value. There is a s h a r p rise especially after T~. No e x a c t d a t a are available a b o u t changes of cell volume during early sporogenesis. Therefore, the size d i s t r i b u t i o n in a p o p u l a t i o n of s p o r u l a t i n g cells was d e t e r m i n e d a t several times b y m e a n s of a Coulter Counter. The results are s u m m a r i z e d in Fig. 10. The u p p e r series of h i s t o g r a m s was d e r i v e d from a cell p o p u l a t i o n in a n o r m a l s p o r u l a t i o n m e d i u m . The b o t t o m p a r t of t h e figure was o b t a i n e d from cells in a Figs. 1--8. Figs. 1--5. Stages of meiosis and ascus formation in yeast. The cells were taken from the sporulation medium at To, T2, Ts, T14 and T2t , respectively. Staining: HC1-Giemsa. Fig. 6. T2a cells from a sporulation medium supplemented with 0.5 mM DL-ethionine. Note the single, degenerate nucleus. Staining: HCIGiemsa. Figs. 7 and 8. Living yeast cells showing central and fragmented vacuoles at TO and T2, respectively. Phase opties. Magni/ications: Figs. 1--3, 2500 • Figs. 4--8, 1500 •

216

A.F. Cao~s:

medium containing potassium chloride instead of potassium acetate. In this modified culture liquid, there is neither meiosis nor ascus formation. I t is seen t h a t as early as T2, but far more pronounced at Ts, there is a shift in the normal culture toward classes with larger diameters. Since no apparent change is found in the modified, KCl-containing medium, cell enlargement is obviously coupled with the fh'st stage of sporogenesis, that is, meiosis. By comparing Figs. 9 and 10 maximal cell enlargement is seen to coincide with the sharp increase in the mean cell mass. %

TO

T2

18

T24

TO

T2

T8

T24

40

20

% 40

20

4

8

10

4

10

4

8

10

L,

8 10 diameter (~m}

Fig. 10. Size distributions of sporulating and non-sporulating cells. Upper row: development in normal sporulation medium. Bottom row: control series from a medium containing potassium chloride instead of potassium acetate. No sporogenesis occurs in this medium

c) Pattern o/Nucleic Acids and Protein. I t m a y be expected t h a t the conversion of the vegetative yeast cell into an ascus is accompanied, like other differentiation processes, b y profound changes in I~NA and protein metabolism, resulting in alterations in over-M1 cmnposition of the cell with respect to these macromoleeules. To investigate this possibility, I%NA and protein determinations were carried out at various times during sporogenesis. The results are shown in Fig. 11. During the whole process and particularly during the later stages, there is a steady decline in the R N A content of the cells. At Ted, more than half the original amount has disappeared. The more complex changes in protein content present a striking contrast to the RNA pattern. During the first 2 hours there is a net increase in protein of about 10%. At the onset of D N A synthesis, the

Induction of Meiosis in Yeast. I

217

protein,RNh pg/ce[[ . 20 [xlO~

DNA I

pg/cett [xlO7)

1,25 .......

TO

.

T&

T8

i

T12

f16

I2l,e tim

Fig. 11. Average protein, RNA and DNA content of the yeast cell during meiosis and ascus formation

O.D. 570rLrn

& -Asp_ Ser Glu

Ala

VQI Met lieuLeu

NH 3

Lys His

Ar 9

G) A

h

~.

A

~

A

Fig. 12. Analyses of the pool of free amino acids at To and T4. The extracts were prepared from equal numbers of cells. Asp aspartic acid; Thr threonine; Ser serine; Glu glutamic acid; Ala alanine; Val valine; Met methionine; lleu isoleucine; Leu leucine; Lys lysine; His histidine; Arg arginine

p r o t e i n c o n t e n t decreases to a slightly lower level which is m a i n t a i n e d d u r i n g a long period u p to T12. This depression, t h o u g h small (abouf 4 %), was f o u n d in all e x p e r i m e n t s . A f t e r T12 , when t h e first asei a t t a i n m a t u r i t y , p r o t e i n c o n t e n t begins a s t e a d y decline.

218

A . F . C~o~s:

d) The Amino-Acld Pool. An intriguing question is: W h y doesthe protein synthesis found during early sporogenesis not proceed beyond the level reached at Ts and T~ ? One possibility is t h a t exhaustion of the pool of free amino acids is limiting protein synthesis. To test this idea, analyses of this pool were carried out at T 4 and T4 . To facilitate comparison, the extracts were prepared from equal numbers of cells (Fig. 12). I t is seen t h a t the individual pools of most amino acids are strongly reduced at T4. The fall of alanine is particularly conspicuous. I n contrast, only minor modifications are found at T 4 in the concentrations of some other amino acids, such as arginine ~nd methionine. The concentration of glutamic acid, though reduced, is still high at T 4. I t is inferred tha~ during early sporogenesis the production of a number of amino acids is the limiting factor in protein synthesis. 2. The Process el Sporogenesis a) Acetate Metabolism. Directly after transfer to the sporulation medium the cells start to take up acetate. Due to the replacement of acetate b y bicarbonate and carbonate, the p H value of the medium rises rapidly during the first hours (Fig. 13). The large deposits of glycogen and fat mentioned above arc built up from acetate as reported for Torulopsis utilis ( = Candida utilis) by JAcKso~ and J o H ~ s o ~ (196I). Another portion of the substrate is oxidized to ~neet the energy requirements of the cell. Since there is no fermentation when acetate is the substrafe, the rate of oxidation is a parameter for the general metabolic activity of the cell. Therefore, a sporulation experiment was carried out in a Warburg vessel and the oxygen consumption was followed during several stages of meiosis and ascus formation. The result is shown in Fig. 13. The cells apparently a d a p t to the new substrate before T2, because oxidation occurs then at a m a x i m u m rate. After T2 the rate of oxygen consumption decreases steadily due to impoverishment of the medium. As a consequence, the development of the cell becomes increasingly more independent of the surrounding medium. This is well illustrated by the finding of STANTrAL (1935) t h a t cells incubated for some time in sporulation medium and then transferred to distilled water, complete their development. b) Timing o/ Biosynthetic Processes. The gradual decline in oxygen uptake and the progressive breakdown of I~NA mentioned in sections I I I . 2 . a and I I I . l . c are indicative of a lowering of metabolic activity during the later stages. This is remarkable since the gross morphological changes (formation of the tetrad and especially of the spore walls) occur just during the period between T~ and Tea. However, it m a y be t h a t a number of substances utilized during the later stages are synthesized long before. Such antecedent synthesis is also found in bacterial sporulation

Induction of Meiosis in Yeast. I

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(for review, see SZULlVIAJSTER, 1964). Therefore, the hypothesis is put forward t h a t a number of developmental steps are prepared early in sporogenesis by the production of specific substances (for example, enzymes) during the period of high pH Ooz metabolic activity. The validity of this idea was tested b y experiments with ethionine. A number of preliminary experi2 .... 9 ments were undertaken to study the nature of the ethionine inhibition. The actions of ethionine and methionine on sporogenesis in yeast clone No. 10 are shown in Table 1. Giemsa staining showed in T24 cells from ethioninetreated cultures one intensely col-7 cured, degenerate nucleus contracted 0 t i ~ i I i [ b y clumping of the ehromatin (Fig. 6). 10 1~ 18 112 ~12~ time Thus, meiosis is blocked at a stage Fig. 13. Oxygen uptake, and rise in before anaphase I. To gain information on the mode the pFI vMue of the medium resulting from acetate metabolism during of action of ethionine in the cell, meiosis and aseus formation. Qo2: DNA, t~NA and protein analyses mms 02 taken up by 106 cells in 30 min were carried out in sporulation eultures supplemented with 0.5 mM ethionine (Fig. 14). I t is seen that the I~NA and protein patterns are not essentially altered. As the turnover rates of these compounds have not been determined, the effect of ethicnine on them remains unknown. On the other hand, the apparent inability of ethionine to block net protein synthesis during early sporegenesis suggests that ethionine does not interfere with general protein metabolism. I n contrast, the meiotic DNA synthesis is completely inhibited b y ethionine (Fig. 14; compare Fig. ll). Even if this is the only action of Table 1. Inhibition o/sporogenesis by ethionine and its reversal by methionine Compound added at TO

Concentration

Asci at T24 ( %)

(raM) None (control) DL-ethionine 9L-ethionine + ] DL-methionine ~ DL-methionine

-0.5 0.5 1 1.0 ~ 1.0

62 0 69, 63

220

A.F. C~o~s:

the analog, the effect is sufficient to account for the complete blocking of meiosis and subsequent ascus formation. The question at what time ethionine exerts its action on DNA replication, is pertinent to the hypothesis set up above that some steps of sporogenesis are prepared early in the development b y the production of specific substances. As DNA synthesis starts at T~, it is possible to pr0!ein,RNA pg/ceH 20 [xI06)

DNA pg/ce(I (xlO 7)

x

x --.-.-......._ x __

I% 0,9

-

~

TrO

2

o

o

I

Tr

I

I

T time Fig. 14. Average protein, I~NA and DNA content of the cell in a sporul~tion medium supplemented with 0.5 mM DL-ethionine investigate whether the analog is active during the process or before. Therefore, ethionine was added to sporulation cultures at different times during the first 6 hours. Two hours after addition the inhibitor was rendered ineffective by supplementing the culture with methionine. The pattern of DNA synthesis was followed in these cultures from T 2 until T~o (Fig. 15). The strongest effect of the inhibitor on DNA synthesis is found in the period T~)--T2. The curve starts to rise after a delay of 6 hours and, as will be shown below, probably never reaches the final value of the control. When the inhibitor is active from T~ to Ta, it causes a delay of about 4 hours. Thereafter, synthesis proceeds at about the same rate as in the control. Although no direct data are available, indirect evidence to be presented below shows that the final amount of DNA is not less than in the control. Ethionine is without any effect when present from T4 to To, that is during the period of normal DNA synthesis. Thus, the analog does not interfere with DNA synthesis itself, but rather with its preparation which occurs mainly during the first 2 hours after transfer to sporulation medium.

Induction of Meiosis in Yeast. I

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The cytological data obtained from the same series of experiments are summarized in Table 2. As could be expected, the strongest inhibition is found when the inhibitor is supplied from T Oto T2. The low number of bi- and tetranucleate cells suggests that the ~g BNA amount of DNA in this culture perce(L never reaches the final value of the IxlOZl control. The numbers of nuclei after meiosis I found at T24 in cultures treated with ethionine during the second and third 2-hour period are normal. From this it may be inferred that the final amount of DNA 12 per cell synthesized under these conditions probably does not differ from the control.

D:

//

A comparison of the numbers of nuclei after meiosis I and the ] ! ! T corresponding numbers of mature T2 T6 TlO time asci shows that there is a second site of ethionine action. I n a consider- Fig. 15. DNA synthesis in sporulation able percentage of the cells, sporo- cultures treated during different 2-hour genesis is apparently blocked some- periods with 0.5 mM DL-ethionine. Periods of inhibition: A, To--T2; where between the completion of the B, T2--Ta; C, Ta--T6; D, control, no first division and the formation of ethionine added the mature ascus. Inspection of the Giemsa-stained slides showed a large number of asci with immature spores in all cultures except the control. On longer incubation, up to T~, this picture remained unaltered, indicating that maturation is blocked and not retarded. A compound essential for spore wall formation seems to be absent in these cells. Table 2. Inhibition o~ sporogenesis by DL-ethionine e//ective during di//erent periods o] 2 hours Period o~ inhibition

Nuclei after Mature asci meiosis I at T~4 at T~ (%) (%)

Control

-38 63 66

To--T~ T2--T 4 T~--T6

65 8 35 45

222

A.F. CROES:

In summary, ethionine interferes with meiotic DNA replication and maturation of the spores. Both processes are only blocked when the inhibitor is present a considerable time before they occur. This result supports the hypothesis that some developmental steps are prepared early in sporogenesis. e) Preparations/or Meiosis during Growth. Turning to the onset of sporogenesis, it appears that a number of early events are insensitive to ethionine inhibition. Acetate metabolism is the same in treated and untreated cultures. MILLER (1964) pointed out that the entering of the nucleus into meiosis is not arrested by ethionine. This is confirmed b y our observation that the behavior of the nucleus in ethionine-treated cells is normal before T5. An extrapolation of the hypothesis mentioned before leads to the idea that the onset of meiosis, though dependent on the exposure of the cells to sporulation medium, is prepared earlier during the last period of growth. To test the validity of this idea, 0.5 mM ethionine was supplied to the growth medium 2 hours before transfer to sporalation medium. Two hours later the cells were transferred to sporulation media with and without 1.0 mM methionine. The results of this experiment are shown in Table 3. The inhibition of growth was determined b y comparing the increases in cell number in treated and untreated cultures. Table 3. Inhibition o] growth and sporogenesis by ethionlne present during the last 2 hours o] growth Ethionine (0.5 raM) in growth medium

Methionine (1.0 raM) in sporulation medium

--

--

0

61

0

--

26

1

99

~-

26

10

~~-

Inhibition of growth ( %)

Asci at T24 ( %)

Inhibition of sporogenesis ( %)

84

I t is seen in this experiment that sporogenesis is severely inhibited whereas the growth rate is reduced b y only 26 %. Methionine supplied in the sporulation medium only slightly reverses the ethionine effect. This excludes the possibility that the inhibition is due to an ethionine pool accumulated during growth and used for protein synthesis during meiosis. The strong action of the inhibitor on meiosis rather than on mitosis in this experiment points to a specific preparation for meiosis during the last phase of growth.

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IV. Discussion a) Onset o] Meiosis. Our observations of nuclear behavior and vacuolization during early sporogenesis confirm the results of earlier investigators ( R ~ s s , 1870; NAG~L, 1946 ; POZqT~F~ACTand MrLLv.~, 1962 ; MInL~R et al., 1963; SVmLA et al., 1964). Moreover, they show that enlargement of the nucleus and fragmentation of the vacuole are the first visible changes in the cell after transfer to the spornlation medium. Enlarged nuclei are visible in only part of the cells at T2. This and the cytological data of Fig. 9 suggest that meiosis does not occur synchronously in all cells. In contrast, vacuolization occurs synchronously since fragmented vacuoles are conspicuous in the great majority of cells at T~. I t follows that a cell may have a vacuolized cytoplasm in the absence of meiosis. Thus the altered nuclear behavior is the best criterium for the onset of meiosis. The synchrony of vaeuolization suggests that exposure to sporulation medium is the predominant factor in this process. On the other hand, nuclear enlargement, which occurs asynchronously, seems to be more dependent on pre-existing conditions which are not the same in all cells. The sharp rises in cell volume and cell mass occur mainly after T~ and cannot be studied in single cells. Consequently, they are less suitable criteria for the onset of meiosis. b) Metabolic Changes. As Mready reported (DgaGAN, 1964), acetate is observed to be oxidized at a maximum rate after an initial lag. The rise in the oxidation rate coincides with the increase in protein content of the cell. This suggests that adaptation to acetate involves enzyme synthesis which may at least partially account for the net protein synthesis found during early sporogenesis. After T2 there is a steady decline in oxidation rate. This and the gradual breakdown of I~NA point to a decrease in metabolic activity. The RNA pattern found is in good agreement with the observation of 1V[uNDKUR (1961) that the number of ribosomes in the cells decreases during sporogenesis. The results indicate that only during early sporegenesis is there a relatively short period of high metabolic activity. As for protein metabolism, the low concentrations of most components of the amino-acid pool at T4 may account for the failure of the protein content of the cells to surpass the levels present at T~ and Ta. The large pool of glutamie acid, still present at Td, is a remarkable feature. Since in yeast the incorporation of NH~ proceeds mainly via glutamie acid (WILT and HoLzER, 1964), it might be expected that this pool is used for replenishing the pools of most other amino acids. Clearly, under the present conditions this occurs, if at all, at an insufficient rate. Acetate is presumably unable to provide the cell with the carbon moieties of several amino acids during sporogenesis.

224

A . F . Cgo~s:

A considerable b r e a k d o w n of p r o t e i n is f o u n d o n l y a f t e r T12. P o s s i b l y this is c o r r e l a t e d w i t h d e g r a d a t i o n of t h e p a r t of t h e c y t o p l a s m t h a t is n o t i n c o r p o r a t e d into t h e spores. A c c o m p a n y i n g this p r o t e i n b r e a k d o w n is t h e release of a m i n o acids i n t o t h e m e d i u m , as o b s e r v e d b y l~AMn~]~z a n d MI~L~g (1964).

c) Time o] Induction. Superficially, meiosis seems to be i n d u c e d b y c o n t a c t w i t h t h e s p o r u l a t i o n m e d i u m . The c h a r a c t e r i s t i c e v e n t s of e a r l y sporogenesis discussed in section I V . a occur n e i t h e r when t h e cells r e m a i n in t h e g r o w t h m e d i u m , n o r when t h e y are s u s p e n d e d in a m o d i f i e d s p o r u l a t i o n m e d i u m l~eking a c e t a t e . Thus, a c e t a t e seems to be t h e real inducer. H o w e v e r , t h e e x p e r i m e n t s w i t h ethionine i n d i c a t e t h a t meiosis is specifically p r e p a r e d for before t r a n s f e r of t h e cells to s p o r u l a t i o n m e d i u m . I f true, a c e t a t e is a trigger r a t h e r t h a n a real i n d u c e r as i t p r o m o t e s a d e v e l o p m e n t a l r e a d y s t a r t e d d u r i n g t h e l a s t phase of growth. As a consequence, t h e i d e a t h a t t h e cells are u n d e t e r m i n e d to meiosis when t r a n s f e r r e d to s p o r u l a t i o n m e d i u m , is incorrect. To e l u c i d a t e t h e real i n d u c t i o n m e c h a n i s m , a t t e n t i o n m u s t be p a i d to t h e p e r i o d of t h e p r e m e i o t i c mitoses. References BURTON, K. : A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J. 62, 315--323 (1956). CE~IOTTI, G. : Determination of nucleic acids in animal tissues. J. biol. Chem. 214, 59--70 (1955). CRo~s, A. F. : Duplication of DNA during meiosis in baker's yeast. Exp. Cell Res. 41, 452--454 (1966). Dv,vsso~, G., et N. T. LAy: Utilisation d'un compteur 6lectronique de particules pour l~ de la croissance de microorganismes: croissance et sporulation de Saccharomycodes Ludwigii ttA~s]~N. Ann. pharm, fran~. 21, 275--285 (1963), DvGGA~, P. F. : Acetate and ethanol oxidation by yeast. Aspects of the metabolism of acetate and ethanol in yeast. Irish J. med. Sci. 457, 19--30 (1964). GAZq~SA_W,A. T. : The cytology of Saccharomyces. C. :[~. Lab. Carlsberg 31, 149--174 (1959). - - , and ~r S. SWAMINATH)-N:Staining the nucleus in yeasts. Stain Teehnol. 33, 115--i21 (1958). HOTTA, Y., M. ITO, and H. S~v,RN: Synthesis of DNA during meiosis. Proc. nat. Acad. Sci. (Wash.) 56, 1184--1191 (1966). JACKSON, W. T., and 1VLJ. J o ~ s o N : Incorporation of acetate and sucrose by Torulopsis utilis. J. Bact. 81, 178--181 (1961). LI~SKE~s, It. F., and J. TvP~: The amino acids pool in the style of selfincompatible strains of Petunia after self- and cross-pollination. Ztichter-Genet. Breed. Res. 86, 151--158 (1966). Lovely, O. It., N . J . ROSXBROVGE,A. L. FAng, and R . J . RA~)ALL: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265--275 (1951).

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McCLARY,D. 0., W. L. NULTu and G. R. MILLER: Effect of potassium versus sodium in the sporulation of Saccharomyces. J. Butt. 78, 362--368 (1959). --, M. A. W I ~ z , ~ s , and C. C. LI~ID~IGRE:~:Nuclear changes in the life cycle of Saccharomyces. J. Bact. 73, 754--757 (1957). MAw, G. A.. Ability of SMmethyl-L-eysteine to annul the inhibition of yeast growth by L-ethionine and by S-ethyl-L-cysteine. J. gen. Microbiol. 25, 441--449 (1961). - - The uptake of some sulphur-containing amino acids by a brewer's yeast. J. gen. Microbiol. 31, 247--259 (1963). M ~ R , G. R., D. O. M c C o Y , and W. D. BowE~s: Ultraviolet and phase microscopy of sporulating Saccharomyces. J. Bact. 8~, 725--731 (1963). MILLER, J. J.: A comparison of the sporulation physiology of yeast and aerobic bacilli. Wallerstein Lab. Commun. 22, 267--283 (1959). - - Determination by ammonium of the manner of yeast nuclear division. Nature (Lond.) 198, 214--215 (1963). - - A comparison of the effects of several nutrients and inhibitors on yeast meiosis and mitosis. Exp. Cell Res. 88, 46~49 (1964). MV~KUR, B. : Electron microscopical studies of frozen-dried yeast. III. Formation of the tetrad in Saccharomyces. Exp. Cell Res. 25, 24--40 (1961). MV~RO, H. N., and E. D. DOwNI~ : Extraction of protein from tissues during treatment with organic lipid solvents. Arch. Biochcm. 106, 516--524 (1964). NAGEL, L. : A cytological study of yeast (Saccharomyces cerevisiae). Ann. Missouri Bot. Garden 88, 249--289 (1946). OGUR, M., S. MI~C~ER, G. LI~DEaaE~, and C. C. LI~1)EGRE~: The nucleic acids in a polyploid series of Saccharomyces. Arch. Biochem. 40, 175--184 (1952). PARKS, L. W.: S-adenosylethionine and ethionine inhibition. J. biol. Chem. 232, 169--176 (1958). PONTE~AcT, R.D., and J.J.M~LER: The metabolism of yeast sporulation. IV. Cytological and physiological changes in sporulating cells. Canad. J. Microbiol. 8, 573--584 (1962). R~IREZ, C., and J. J. MILLER: The metabolism of yeast sporulation. VI. Changes in amino acid content during sporogenesis. Canad. J. Microbiol. 10, 623--631 (1964). I~EESS, M.: Botanische Untersuchungen fiber die Alkoholg~rnngspilze. Leipzig: Artur Felix 1870. R~OADES, M.M.: Meiosis. In: The cell (BB),C~E~, J., and A.E. MmSKu eds.), vol. 3, p. 1--75. New York and London: Academic Press 1961. ROBINOW, C. F. : Mitosis in the yeast Lipomyces lipo]er. J. biophys, biochem. Cytol. 9, 879--892 (1961). Sc~ID% G., and S. J. Tt~N~-~USER: A method for the determination of desoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. J. biol. Chem. 161, 83--89 (1945). SE:~NES,M. J. ~E: Sur le Mycoderma vini. C.R. Acad. Sei. (Paris) 67, 105--109 (1868). STA~TIX~,~[. : The sporulation o~ yeast: second paper. Trans. roy. Soe. Can., Ser. III, 29, 175--188 (1935). S v ~ A , G., J. L. D~NKo, and F. S t r E a K : Ultraviolet microscopy of the vacuole of Saccharomyces cerevisiae during sporulation. J. Baet. 88, 449--456 (1964). 16 Planta (Berl.), Bd. 76

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SZUL~AJST~a~,J.: Biochimie de la sporogen~se chez Bacillus subtills. Bull. Soc. Chim. biol. (Paris) 46, 443--481 (1964). TABOR, J. H. : Meiosis. In: Encyclopedia of plant physiology (RU/t~AND,W., ed.), voL 18, p. 344--367. Berlin-Heidelberg-New York: Springer 1967. U~rr, W. W., R . H . BURI~IS, and J . F . ST~VF]~ER: Manometric techniques, 4th edn. Minneapolis (Minn.): Burgess 1964. WA~KA, F.: Die Bestimmung der Nueleins~uren in Chlorella.Kulturen. Planta (Berl.) 58, 594--609 (1962). W~G]~, 0.: On haplophase and diplophase in some Saccharomyces. C.I~. Lab. Carlsberg, S6r. Physiol. 21, 77--111 (1935). WIT~r, I., and H. ItOT.Z]~: Hauptweg des NH~-Einbaues in Glucose oxydierender B~ekerhefe. Biochem. Z. 839, 255--265 (1964). A. F. Ci~o~s Department of Botany, University Driehuizerweg 200, Nijmegen, Netherlands