Vol. 130, No. 2 Printed in U.S.A.
JouRNAL OF BACTBRIOLOGY, May 1977, P. 766-774 Copyright C 1977 American Society for Microbiology
Recovery of Saccharomyces cerevisiae Mating-Type a Cells from Gl Arrest by a Factor RUSSELL K. CHAN'
Department of Genetics (SK-50), University of Washington, Seattle, Washington 98195 Received for publication 11 November 1976
Mating-type a cells of the yeast Saccharomyces cerevisiae that had been specifically arrested in the Gl phase of the cell cycle by a factor, an oligopeptide pheromone made by a cells, recovered and resumed cell division after a period of inhibition which was dependent on the concentration of a factor used. These treated a cells were more resistant to a factor than untreated a cells, but lost their resistance upon further cell division. However, cells arrested for 6 h were no more resistant to a factor than cells arrested for only 2.5 h. Mating-type a strains could inactivate or remove a factor from the culture fluid, but two a sterile (nonmating) mutants and an a/a diploid strain could not. These results suggest that a cells have a mechanism, which may involve uptake or inactivation of a factor, for recovering from a factor arrest. However, the results do not distinguish between a recovery mechanism which is constitutive and one which is induced by a factor. The loss of a factor activity during recovery appeared to be primarily cell contact mediated, although an extracellular, diffusible inhibitor of a factor that is labile or that functions stoichiometrically could not be ruled out.
Haploid cells of the budding yeast Saccharomyces cerevisiae normally reproduce by mitotic cell division. However, when haploid cells of opposite mating type are mixed together, cell and nuclear fusion occur, producing a zygote that buds off diploid daughter cells. The ability to undergo this process, called mating or conjugation, is controlled by a single genetic locus which exists in two allelic forms, a and a (9). Mating will usually occur only between cells carrying different mating-type alleles. Each mating type constitutively secretes a mating factor whose apparent function is to ensure that cells ofthe opposite mating type are arrested at the appropriate stage in the cell cycle for mating to occur. The a factor, made by mating-type a cells, is an oligopeptide (3, 4, 14, 15) which acts specifically to arrest mating-type a cells at the Gl phase of the cell cycle (2). The arrested a cells continue to make ribonucleic acid and protein even though cell division and deoxyribonucleic acid replication are inhibited (16). The a factor, made by mating-type a cells, has similar effects upon mating-type a cells (17). Diploid a/a cells, which are the end product of mating, are not affected by either mating
Cell cycle arrest of a cells by a factor can be reversed by the removal of a factor from the medium (2). Surprisingly, a cells will eventually recover from arrest when a factor is left in the medium (16). Using partially purified a factor, I have studied the recovery of mating-type a cells from a factor arrest by looking at the response to different amounts of a factor, the fate of a factor in the medium, the response of strains insensitive to a factor, and the resistance of recovering cells. In addition, I have looked for the presence of an extracellular, diffusible inhibitor of a factor. My results suggest that a cells have a mechanism, which may involve the uptake or inactivation of a factor, for recovering from a factor arrest.
MATERIALS AND METHODS Strains and media. The following strains of S. cerevisiae were used. X2180-1A agal2 and X2180-1B a gal2 are prototrophs, isogenic except at the mating-type locus (3), from the collection of R. K. Mortimer. C276 is an a/a diploid strain produced by mating X2180-1A a with X2180-1B a (11). VU3 and VY3 are independently isolated sterile (nonmating) mutants derived by Vivian MacKay from XT1177-S47c factor. a ade2-1 his2 lysl-1 trp5-18 gal2 canl (11). Synthetic medium contained (per liter of medium) 1.45 g of 1 Present address: Department of Microbiology, Univer- yeast nitrogen base (Difco) (without amino acids sity of Cincinnati, College of Medicine, Cincinnati, OH and ammonium sulfate), 20 g of glucose, 10 g of 45267. (NH4)2SO4, 10 g of succinic acid, and 6 g of NaOH. 766
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When required, adenine was added at 10 ,ug/ml, lysine at 80 ,ug/ml, and histidine and tryptophan at 40 jug/ml. Partial purification of a factor. a factor was partially purified from a 50-liter culture of X2180-1B a as described by Duntze et al. (4) except that eluate III from the Amberlite CG-50 column was concentrated by evaporation at 4°C under a stream of compressed air and then lyophilized. The residue after lyophilization was dissolved in 50 ml of sterile distilled water and stored at 4°C. The same batch of partially purified a factor was used throughout this study except for the experiments depicted in Fig. 4, 5, and 8, which used different batches. Monitoring cell number and budding. The cell concentration and percentage of unbudded cells was determined as previously described (6). For each point, at least 200 cells were scored for their bud morphology.
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Response to a factor as a function of a factor concentration and cell concentration. When mating-type a cells are exposed to a factor, they arrest in the Gl phase of the cell cycle as single, unbudded cells. This observation provides a sensitive and convenient way to assay the biological activity of a factor. The response of X2180-1A a to varying concentrations of a factor is shown in Fig. 1. At 23°C in synthetic medium, 50 to 60% of the cells in an exponentially growing culture of X2180-1A were unbudded. However, after a factor was added, the percentage of unbudded cells increased as the cells accumulated at the a factor block (Fig. 1B). When the entire population became unbudded, the cell number stopped increasing (Fig. 1A). The cells remained unbudded for a period of time, which depended on the concentration of a factor; higher a factor concentrations resulted in longer periods of arrest (Fig. 1B). All the a cells treated in Fig. 1 recovered and resumed budding. The response of cultures containing different initial cell concentrations to a constant amount of a factor was examined (Fig. 2). When the percentage of unbudded cells is plotted as a function of time, the response curves look very similar to those shown in Fig. 1. These observations show that for a given concentration of a factor, the higher the initial cell concentration, the faster the cells recover. These results suggest that the cells antagonize the a factor activity. Fate of a factor in the medium. A culture of X2180-1A was treated with a factor, and the culture fluid was sampled at various times (Fig. 3A) to see if recovering a cells could remove a factor activity from the culture medium. After the cells were removed by centrifu-
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FIG. 1. Response of mating-type a cells to a factor: the effect of varying the a factor concentration. X2180-1A was grown with shaking in synthetic medium at 23°C to 3 x 106 cells/ml (660-nm absorbancy of 0.2 units) and divided into six parallel cultures. Then a factor was added directly to five of the cultures to give final a factor dilutions of 1:1,000 (a), 1:500 (A), 1:200 (A), 1:100 (O), and 1:50 (). (O) No a factor added. The cultures were shaken at 23°C. At intervals, portions were taken from each culture to determine the cell concentration (A) and the percentage of cells that were unbudded (B).
gation, the a factor in the supernatant was assayed by adding back untreated X2180-1A cells (i.e., cells that had not been exposed to a factor) and following the accumulation of unbudded cells (Fig. 3B). The response of the untreated cells shows that the a factor activity began to decline immediately after exposure to a cells; in fact, some a factor activity was lost even from the zero-time point apparently as a result of the exposure of a factor to a cells during the centrifugation of the first sample. By 3 1/e h, the a factor activity in the culture medium had declined about 10-fold, although more than 90% of the cells (see Fig. 3A) remained unbudded for another 3 h (Fig. 3B; the magnitude of the loss in a factor activity in this and subsequent experiments was estimated by comparing the experimental curves with the curves in Fig. 1B where the relative amounts of
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parent was able to cause a 20-fold loss of a factor activity from the culture medium during the same time period (Fig. 5A). Furthermore, C276, an a/a diploid strain, also failed to remove a factor activity from the culture medium (data not shown, but similar to Fig. 5B and C). These observations suggest that the ability to remove a factor activity from the culture medium is physiologically significant and not just a consequence of normal yeast metabolism
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HOURS FIG. 2. Response of mating-type a cells to a factor: the effect of varying the initial cell concentration. X2180-1A was grown to 6 x 106 cellslml in synthetic medium at 23°C and diluted to give four sets of cultures with cell densities of 8 x 105 (0), 1.5 x 106 (O), 3 x 106 (A), and 6 x 106 (A) cellslml. a factor was diluted 1:200 into each culture (B); control cultures (A) did not receive a factor. The cultures were shaken at 23°C and monitored for the percentage of unbudded cells. All the control cultures grew exponentially with a doubling time of about 3.2 h.
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factor are known). Thus, the recovery of mating-type a cells from a factor arrest is preceded by the loss of a factor activity from the culture medium. If the ability to remove a factor activity from the medium is an integral part of the mating process, one might expect it to be affected by genes which affect mating. Therefore, I looked at the ability of an a/a diploid and of two sterile a mutants to remove a factor activity from the culture medium. Diploid a/a strains do not mate and are insensitive to a factor. The two a sterile mutants tested, VU3 and VY3, were isolated for the inability to mate, but are also insensitive to a factor. VY3 still produces a factor, but VU3 does not (11, 12). The results (Fig. 4) confirm that VU3 and VY3 were insensitive to a factor, although their wild-type parent, XT1177-S47c, was fully sensitive. Neither a sterile mutant was able to remove factor activity from the culture medium (Fig. 5B and C), although the wild-type a
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HOURS FIG. 3. Kinetics of the disappearance of a factor activity. (A) A culture of X2180-1A at 3 x 106 cellsl ml was treated with a factor as described in Fig. 1. (0) No a factor; (O) a factor added at a dilution of 1:100. At the times indicated by the arrows, cell-free culture fluid from the a factor-treated culture was prepared by centrifugation and stored at 4°C. (B) After a 16-h storage, the a factor activity in each of these samples was assayed with untreated a cells. A culture of X2180-1A was collected by centrifugation, resuspended at 3 x 106 cellslml in the culture fluid samples, and shaken at 23°C. The percentage of unbudded cells was followed. Symbols: (0) fresh medium; £*) fresh medium containing a factor at 1:100 stored at 4°C for 16 h, (0) 0-h sample from Fig. 3A. (A) 1-h sample from Fig. 3A; (A) 31/e-h sample from Fig. 3A; (O) 6 3/4-h sample from Fig. 3A.
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FIG. 4. The response of sterile mutants to factor. Cultures of two sterile mutants (VU3 and VY3) and their wild-type parent (XT1177-S47c) were grown in synthetic medium at 23°C to 2.5 x 106 cells/ml and treated with a factor at a dilution of 1:100. The cell concentration and the percentage of unbudded cells were followed for 12 h. Symbols: (0) untreated cells; (a) cells treated with 1:100 a factor. a
since that ability is absent in two sterile mutants and absent in an a/a diploid. Resistance of recovering cells. In order to see if treated a cells were more resistant to a factor than untreated a cells, a cells which had recovered from factor arrest were collected by filtration, washed, and resuspended in fresh medium containing a factor. The response of these "conditioned" cells was compared with that of untreated a cells. In the culture of conditioned cells which had been reexposed to a factor at a dilution of 1:100, the percentage of unbudded cells remained at about 60% (Fig. 6A), whereas the percentage of unbudded cells in a culture of previously untreated a cells rose to more than 90% for 3 h when exposed to the same amount of a factor (data not shown, but" similar to Fig. 1B). Thus, the treated a cells appear to be more resistant to a factor than untreated a cells. Cell-free medium from the recovering culture described above was also tested to see if a diffusible inhibitor of a factor was present. No loss of a factor activity was observed when a factor was mixed with conditioned medium and assayed immediately with untreated a cells (Fig. 6B). The resistance of treated cells at various times after recovery from a factor arrest was examined. The results (Fig. 7) show that resistance to a factor arrest was gradually lost after recovery. Cells sampled 4.5 h after recovery (Fig. 7E) were more sensitive to a factor than cells sampled immediately after recovery (Fig. a
7B); however, they were not as sensitive as untreated cells which had never been exposed to a factor (Fig. 7A). The resistance of treated a cells as a function of the length of exposure to a factor was also studied. Cells that had been arrested for 2.5 h with a factor were compared with cells that had been arrested for 6 h. Figure 8 shows that cells that have been arrested for 6 h with a factor appear to be no more resistant to a factor than cells that have been arrested for only 2.5 h. Testing for a diffusible inhibitor of a factor. In the.preceding experiment, no loss of a factor activity was found when a factor was mixed with "conditioned" medium and assayed immediately. However, Hicks and Herskowitz (7) reported that mating-type a cells constitutively secreted a diffusible substance that inhibited or inactivated a factor activity. I, therefore, did a more elaborate version of the experiment described in Fig. 6B to look for a diffusible inhibitor. The sensitivity of the experiment was increased by incubating a factor with the cellfree culture fluid for 14 h to allow more time for a putative inhibitor to act. My results (Fig. 9B) show that little or no a factor activity was lost after incubation for 14 h in medium from an untreated culture of X21801A. Thus, at the concentrations of cells and a factor used in this experiment, no constitutive, diffusible inhibitor of a factor was detected. The experiment for detecting an inducible, diffusible inhibitor of a factor is shown in Fig. 9A, where a factor was incubated for 14 h in
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sulted in about a fourfold loss of a factor activity. This same degree of inactivation was achieved in the presence of a cells by incubation for only 1 h (cf. Fig. 3B). Thus, the amount of inducible, diffusible inhibitor activity observed with cell-free medium (Fig. 9A) is not enough to account for the loss of a factor activity observed in the presence of a cells (Fig. 3B), unless the inducible, diffusible inhibitor activity is labile or acts stoichiometrically.
DISCUSSION The conclusions derived from experiments examining the recovery of S. cerevisiae matingtype a cells from Gl arrest by a factor may be summarized. (i) The recovery process was density dependent; if two cultures were treated with identical amounts of a factor, the culture with the higher initial cell concentration recovered faster. This result rules out a recovery
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HOURS FIG. 5. The ability of sterile mutants to remove a factor activity from the culture medium. Samples of the a factor-treated cultures described in Fig. 4 were taken at 0 and 12 h. Cell-free culture fluid was prepared by centrifugation and assayed for a factor activity by using X2180-1A cells as described in Fig. 3B. The a factor activity remaining in the culture fluid is shown in (A) for XT11 77-S47c, (B) for VU3, and (C) for VY3. Symbols: (0) culture medium at 0 h from Fig. 4; (0) culture medium at 12 h from Fig. 4; (A) control medium (never exposed to cells); (A) control medium with a factor at a 1:100 dilution.
medium from which a cells had recovered from a factor arrest. A comparison with Fig. 1B suggests that the 14-h incubation (Fig. 9A) re-
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FIG. 6. Separate testing of the cells and medium from a culture previously arrested by a factor. X2180-1A at 3 x 106 cells/mi was treated with a factor at a dilution of 1:100 as described in Fig. 1 and shaken at 23°C to allow the cells to arrest and recover. By 6 h, 20% of the cells had resumed budding, and the culture was fractionated as described below to separate the cells from the medium. (A) To obtain "conditioned" cells, the a factor-treated culture was collected and washed on a membrane filter (Millipore Corp.). Then the cells were resuspended in fresh medium at a density equivalent to the initial cell concentration and transferred to flasks with no a factor (0) and with a factor added at a dilution of 1:100 (0). The percentage of unbudded cells was followed for 5.5 h at 23°C. (B) "Conditioned" medium was obtained from the supernatant fluid of the a factor-treated culture, which had been centrifuged to remove the cells. Untreated X2180-1A was resuspended at 3 x 106 cellslml in this conditioned medium with no a factor (0) and with a factor added at a dilution of 1:100 (-). As a control to show that our manipulations did not inactivate a factor, X2180-1A was resuspended in cell-free medium, containing a factor at a dilution of 1:100, which had been subjected to the same manipulations as the a factortreated culture (A). The percentage of unbudded cells was followed for 5.5 h at 23°C.
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FIG. 7. Kinetics of loss of resistance to a factor. (A) X2180-1A at 3 x 106 cellslml was treated with a factor at a dilution of 1:100 as described in Fig. 1. The percentage of unbudded cells was followed. Symbols: (0) X2180-lA alone; (0) X2180-lA with a factor added at 1:100. At 6 h, the a factor-treated culture was adjusted by diluting with an equal volume of fresh medium. Samples were taken from this diluted culture at 7.5, 9, 10.5, and 12 h (indicated by the arrows) to determine the response of the culture to additional treatment with a factor. All the samples were diluted to the same absorbance (A6w = 0.33) to make the responses comparable. a factor at a dilution of 1:100 was added to a portion of each sample, and the cultures were shaken at 23°C. The percentage of unbudded cells was followed and is plotted in panels B through E. Symbols: (0) recovering culture without a factor; (a) recovering culture plus additional a factor at 1:100.
mechanism in which the cells become impermeable to a factor, since such a mechanism should be independent of the cell concentration. (ii) Cells that had recovered from a factor arrest were more resistant to a factor than untreated cells; however, the treated cells lost their resistance upon further cell division. Cells that had been arrested by a factor for 6 h were no more resistant to a factor than cells that had been arrested for only 2.5 h. These observations can be explained in two ways. First, the recovery mechanism may be induced by a factor. However, maximum induction must take place by 2.5 h, since cells arrested for 6 h are no more resistant to a factor than cells arrested for 2.5 h. Alternatively, the recovery mechanism may be a constitutive function of a cells. In that case, the resistance of recovering a cells to a factor could be attributed to the synchrony induced by a factor arrest. That is, a partially synchronized population of cells produced by a factor arrest would have an entire cell cycle in which to take up or inactivate a factor before returning to the sensitive Gl period, whereas, an untreated asynchronous culture of a cells would have, on the average, about half a cell cycle in which to take up or inactivate the same amount of a factor. The extra time the synchronized population has to take up or inactivate a
factor would account for the observed resistance. Furthermore, the loss of resistance after recovery could be explained by assuming that the recovering a cells lose their synchrony.
Thus, my results can be interpreted as being consistent with either a constitutive recovery mechanism or a recovery mechanism induced by a factor. More information will be required to distinguish between these two possibilities. (iii) The biological activity of a factor disappeared from the culture fluid of recovering a cells; this loss of activity could result either from chemical inactivation of a factor or from uptake of a factor into the cell. (iv) Two sterile (nonmating) mutants derived from an a strain were insensitive to a factor and lacked the ability to remove a factor activity from the culture medium. Furthermore, an a/a diploid, which is normally insensitive to a factor and does not mate, also failed to remove a factor activity from the medium. These results suggest that the ability to remove a factor activity from the medium is physiologically significant and not just a consequence of normal yeast metabolism since that ability is affected by genes, which also affect mating. (v) No a factor activity was lost upon incubation in cell-free medium prepared from an untreated a cell culture. Thus, at the concentra-
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phase or after a factor arrest). Alternatively, a small percentage of the inactivating activity that I find to be cell bound may be extracellular, and this amount may have been sufficient to produce inhibition with the high cell concentrations used by Hicks and Herskowitz (7). Furthermore, the concentration of partially purified a factor used in my experiments might be much higher than the concentration of a factor
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tions of cells and factor used, no constitutive, diffusible inhibitor of a factor was detected. Hicks and Herskowitz (7), however, have provided evidence for a constitutive, diffusible inhibitor of a factor. My failure to detect such a constitutive, diffusible inhibitor could be due to a difference in experimental conditions. For example, Hicks and Herskowitz used dense, concentrated streaks of a cells grown on rich agar plates, whereas I used cultures of low-cell density growing exponentially in liquid synthetic medium. Thus, the diffusible inhibitor might be labile in liquid medium or might be produced only by cells in GI (e.g., in stationary a
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HOURS FIG. 9. The effect of incubating a factor with conditioned medium before assaying for a factor activity. Cell-free medium was prepared by centrifugation and membrane filtration (Millipore Corp.) from three sources: (A) a culture of X2180-1A treated with a factor at a dilution of 1:100 as described in Fig. 1 for 8 h; (B) an untreated culture of X2180-1A (no a factor) grown for 8 h; (C) fresh medium (as a control). Fresh a factor at a dilution of 1:100 was incubated at 23°C for 0 and 14 h with each filtrate. The a factor activity in the 14-h incubation mixture (0) and the zero-time incubation mixture (-) was measured by adding untreated X2180-lA as described in Fig. 3B and following the percentage of unbudded cells. As a control, the a factor activity of each filtrate alone was measured (A).
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produced by the streak of a cells used by Hicks and Herskowitz. In that case the diffusible inhibitor could simply be swamped by the amount of a factor present in my experiments. (vi) A 1-h incubation of a factor with a cells (Fig. 3B) inactivated the same amount of a factor activity as a 14-h incubation of a factor in cell-free medium prepared from an a cell culture which had recovered from a factor arrest (Fig. 9A). This result suggests that the ability to inactivate or take up a factor (I cannot distinguish between the two), a process associated with recovery from a factor arrest, resides primarily in the cell. This may be due to the fact that the major cause of a factor activity loss during recovery is the result of uptake by the cell. On the other hand, if inactivation were the major cause of a factor activity loss during recovery, at least two possible explanations would come to mind. First, most inactivation may take place inside the cell or on the surface, but a small fraction of the inactivation may be due to an inducible, diffusible inhibitor activity (e.g., Fig. 9A). Second, the cell may only serve as a source for an extracellular, diffusible inhibitor that is labile or that acts stoichiometrically. Barrier effect. Hicks and Herskowitz (7) reported that a streak of mating-type a cells could act as a barrier to the diffusion of a factor on an agar slab. They called this property the barrier effect and designated strains possessing it as Bar+. Using this assay, they screened a number of sterile (nonmating) mutants derived from an a strain and found that some of the sterile mutants were still Bar+, but that others had become Bar-. It is tempting to speculate that the ability of a cells to remove a factor activity from the culture medium is responsible for the Bar+ phenotype of a cells. Unfortunately, this hypothesis is not compatible with my observation that VU3, a sterile Bar+ strain, failed to remove a factor activity from the medium. Pheromone inactivation mechanisms in other organisms. My results suggest that mating-type a cells, which are sensitive to a factor, are also capable of inactivating a factor. The idea that the target cells for a pheromone should also possess a means for specifically inactivating or metabolizing that pheromone is not without precedent among the fungi (5). For example, female gametes of the water mold Allomyces secrete a sex pheromone, sirenin, which is a bicyclic sesquiterpenediol that attracts male gametes (sperm) by chemotaxis. Machlis (10) found that sperm were able to remove sirenin activity from the culture fluid
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and that the sirenin activity could not by recovered from the sperm after uptake, suggesting that the spern had inactivated the sirenin. Similarly, female strains of the water mold Achlya secrete a steroid sex pheromone, antheridiol, which induces the formation of antheridial branches in the male strain. Barksdale (1) showed that male strains of Achlya could remove antheridiol activity from the culture fluid. More recently, Musgrave and Nieuwenhuis (12) have shown that male strains of Achlya can metabolize radioactive antheridiol to a biologically inactive compound. In mammals, there is considerable evidence that the target tissues for a number of polypeptide hormones including insulin, glucagon, oxytocin, parathyroid hormone, and calcitonin possess the ability to specifically degrade these hormones (8). This apparent ubiquity of hormone inactivation mechanisms suggests that further study of the a factor inactivation system in Saccharomyces may have broad biological significance. ACKNOWLEDGMENTS I thank Lee Hartwell for his helpful comments and encouragement during the course of this work and during the preparation of this manuscript; Diane Foley and Guy Page, whose experiments with a factor and a factor presaged some of the present work; Jim Hicks, Jeff Strathern, and Ira Herskowitz for sharing information and strains; D. Hawthorne, F. Heffron, H. Klein, D. Livingston, S. Reed, M. Unger, and K. Walsh for their criticisms ofthe manuscript; and Martha Katz, Nancy Ridgway, and Adele Purnell for typing the manuscript. This investigation was aided by grants to L. H. Hartwell from the American Cancer Society, Washington Division (VC-145), and from the Public Health Service, National Institute of General Medical Sciences (GM 17709). I was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. LITERATURE CITED 1. Barksdale, A. W. 1963. The uptake of exogenous hormone A by certain strains of Achyla. Mycologia 55:164-171. 3. Bucking-Throm, E., W. Duntze, L. H. Hartwell, and T. R. Manney. 1973. Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 3. Duntze, W., V. MacKay, and T. R. Manney. 1970. Saccharomyces cerevisiae: a diffusible sex factor. Science 168:1472-1473. 4. Duntze, W., D. Stotzler, E. Bucking-Throm, and S. Kalbitzer. 1973. Purification and partial characterization of a-factor, a mating type specific inhibitor of cell reproduction from Saccharomyces cereuisiae. Eur. J. Biochem. 35:357-365. 5. Gooday, G. W. 1974. Fungal sex hormones. Annu. Rev. Biochem. 43:35-49. 6. Hartwell, L. H. 1970. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bacteriol. 104:1280-1285. 7. Hicks, J. B., and I. Herskowitz. 1976. Evidence for a new diffusible element of mating pheromones in yeast. Nature (London) 260:246-248.
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8. Knights, E. B., S. B. Baylin, and G. V. Foster. 1973. Control of polypeptide hormones by enzymatic degradation. Lancet ii:719-723. 9. Lindegren, C. C., and G. Lindegren. 1943. A new method for hybridizing yeast. Proc. Natl. Acad. Sci. U.S.A. 29:306-308. 10. Machlis, L. 1973. The chemotactic activity of various sirenins and analogues and the uptake of sirenin by the sperm of Allomyces. Plant Physiol. 52:527-530. 11. MacKay, V., and T. R. Manney. 1974. Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of nonmating mutants. Genetics 76:255-271. 12. MacKay, V., and T. R. Manney. 1974. Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. II. Genetic analysis of nonmating mutants. Genetics 76:273-288.
J. BACTERIOL. 13. Musgrave, A., and D. Nieuwenhuis. 1975. Metabolism of radioactive antheridiol by Achyla species. Arch. Microbiol. 105:313-317. 14. St6tzler, D., and W. Duntze. 1973. Charakterisierung eines regulatorischen Peptids aus Saccharomyces cerevisiae. Hoppe-Seyler's Z. Physiol. Chem. 354:1247. 15. Stotzler, D., and W. Duntze. 1976. Isolation and characterization of four related peptides exhibiting a factor activity from Sarccharomyces cerevisiae. Eur. J. Biochem. 65:257-262. 16. Throm, E., and W. Duntze. 1970. Mating-type-dependent inhibition of deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 104:13881390. 17. Wilkinson, L. E., and J. R. Pringle. 1974. Transient Gl arrest of S. cerevisiae cells of mating type a by a factor produced by cells of mating type a. Exp. Cell Res. 89:175-187.
JouRNAL OF BACTBRIOLOGY, May 1977, P. 766-774 Copyright C 1977 American Society for Microbiology
Recovery of Saccharomyces cerevisiae Mating-Type a Cells from Gl Arrest by a Factor RUSSELL K. CHAN'
Department of Genetics (SK-50), University of Washington, Seattle, Washington 98195 Received for publication 11 November 1976
Mating-type a cells of the yeast Saccharomyces cerevisiae that had been specifically arrested in the Gl phase of the cell cycle by a factor, an oligopeptide pheromone made by a cells, recovered and resumed cell division after a period of inhibition which was dependent on the concentration of a factor used. These treated a cells were more resistant to a factor than untreated a cells, but lost their resistance upon further cell division. However, cells arrested for 6 h were no more resistant to a factor than cells arrested for only 2.5 h. Mating-type a strains could inactivate or remove a factor from the culture fluid, but two a sterile (nonmating) mutants and an a/a diploid strain could not. These results suggest that a cells have a mechanism, which may involve uptake or inactivation of a factor, for recovering from a factor arrest. However, the results do not distinguish between a recovery mechanism which is constitutive and one which is induced by a factor. The loss of a factor activity during recovery appeared to be primarily cell contact mediated, although an extracellular, diffusible inhibitor of a factor that is labile or that functions stoichiometrically could not be ruled out.
Haploid cells of the budding yeast Saccharomyces cerevisiae normally reproduce by mitotic cell division. However, when haploid cells of opposite mating type are mixed together, cell and nuclear fusion occur, producing a zygote that buds off diploid daughter cells. The ability to undergo this process, called mating or conjugation, is controlled by a single genetic locus which exists in two allelic forms, a and a (9). Mating will usually occur only between cells carrying different mating-type alleles. Each mating type constitutively secretes a mating factor whose apparent function is to ensure that cells ofthe opposite mating type are arrested at the appropriate stage in the cell cycle for mating to occur. The a factor, made by mating-type a cells, is an oligopeptide (3, 4, 14, 15) which acts specifically to arrest mating-type a cells at the Gl phase of the cell cycle (2). The arrested a cells continue to make ribonucleic acid and protein even though cell division and deoxyribonucleic acid replication are inhibited (16). The a factor, made by mating-type a cells, has similar effects upon mating-type a cells (17). Diploid a/a cells, which are the end product of mating, are not affected by either mating
Cell cycle arrest of a cells by a factor can be reversed by the removal of a factor from the medium (2). Surprisingly, a cells will eventually recover from arrest when a factor is left in the medium (16). Using partially purified a factor, I have studied the recovery of mating-type a cells from a factor arrest by looking at the response to different amounts of a factor, the fate of a factor in the medium, the response of strains insensitive to a factor, and the resistance of recovering cells. In addition, I have looked for the presence of an extracellular, diffusible inhibitor of a factor. My results suggest that a cells have a mechanism, which may involve the uptake or inactivation of a factor, for recovering from a factor arrest.
MATERIALS AND METHODS Strains and media. The following strains of S. cerevisiae were used. X2180-1A agal2 and X2180-1B a gal2 are prototrophs, isogenic except at the mating-type locus (3), from the collection of R. K. Mortimer. C276 is an a/a diploid strain produced by mating X2180-1A a with X2180-1B a (11). VU3 and VY3 are independently isolated sterile (nonmating) mutants derived by Vivian MacKay from XT1177-S47c factor. a ade2-1 his2 lysl-1 trp5-18 gal2 canl (11). Synthetic medium contained (per liter of medium) 1.45 g of 1 Present address: Department of Microbiology, Univer- yeast nitrogen base (Difco) (without amino acids sity of Cincinnati, College of Medicine, Cincinnati, OH and ammonium sulfate), 20 g of glucose, 10 g of 45267. (NH4)2SO4, 10 g of succinic acid, and 6 g of NaOH. 766
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When required, adenine was added at 10 ,ug/ml, lysine at 80 ,ug/ml, and histidine and tryptophan at 40 jug/ml. Partial purification of a factor. a factor was partially purified from a 50-liter culture of X2180-1B a as described by Duntze et al. (4) except that eluate III from the Amberlite CG-50 column was concentrated by evaporation at 4°C under a stream of compressed air and then lyophilized. The residue after lyophilization was dissolved in 50 ml of sterile distilled water and stored at 4°C. The same batch of partially purified a factor was used throughout this study except for the experiments depicted in Fig. 4, 5, and 8, which used different batches. Monitoring cell number and budding. The cell concentration and percentage of unbudded cells was determined as previously described (6). For each point, at least 200 cells were scored for their bud morphology.
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Response to a factor as a function of a factor concentration and cell concentration. When mating-type a cells are exposed to a factor, they arrest in the Gl phase of the cell cycle as single, unbudded cells. This observation provides a sensitive and convenient way to assay the biological activity of a factor. The response of X2180-1A a to varying concentrations of a factor is shown in Fig. 1. At 23°C in synthetic medium, 50 to 60% of the cells in an exponentially growing culture of X2180-1A were unbudded. However, after a factor was added, the percentage of unbudded cells increased as the cells accumulated at the a factor block (Fig. 1B). When the entire population became unbudded, the cell number stopped increasing (Fig. 1A). The cells remained unbudded for a period of time, which depended on the concentration of a factor; higher a factor concentrations resulted in longer periods of arrest (Fig. 1B). All the a cells treated in Fig. 1 recovered and resumed budding. The response of cultures containing different initial cell concentrations to a constant amount of a factor was examined (Fig. 2). When the percentage of unbudded cells is plotted as a function of time, the response curves look very similar to those shown in Fig. 1. These observations show that for a given concentration of a factor, the higher the initial cell concentration, the faster the cells recover. These results suggest that the cells antagonize the a factor activity. Fate of a factor in the medium. A culture of X2180-1A was treated with a factor, and the culture fluid was sampled at various times (Fig. 3A) to see if recovering a cells could remove a factor activity from the culture medium. After the cells were removed by centrifu-
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FIG. 1. Response of mating-type a cells to a factor: the effect of varying the a factor concentration. X2180-1A was grown with shaking in synthetic medium at 23°C to 3 x 106 cells/ml (660-nm absorbancy of 0.2 units) and divided into six parallel cultures. Then a factor was added directly to five of the cultures to give final a factor dilutions of 1:1,000 (a), 1:500 (A), 1:200 (A), 1:100 (O), and 1:50 (). (O) No a factor added. The cultures were shaken at 23°C. At intervals, portions were taken from each culture to determine the cell concentration (A) and the percentage of cells that were unbudded (B).
gation, the a factor in the supernatant was assayed by adding back untreated X2180-1A cells (i.e., cells that had not been exposed to a factor) and following the accumulation of unbudded cells (Fig. 3B). The response of the untreated cells shows that the a factor activity began to decline immediately after exposure to a cells; in fact, some a factor activity was lost even from the zero-time point apparently as a result of the exposure of a factor to a cells during the centrifugation of the first sample. By 3 1/e h, the a factor activity in the culture medium had declined about 10-fold, although more than 90% of the cells (see Fig. 3A) remained unbudded for another 3 h (Fig. 3B; the magnitude of the loss in a factor activity in this and subsequent experiments was estimated by comparing the experimental curves with the curves in Fig. 1B where the relative amounts of
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factor are known). Thus, the recovery of mating-type a cells from a factor arrest is preceded by the loss of a factor activity from the culture medium. If the ability to remove a factor activity from the medium is an integral part of the mating process, one might expect it to be affected by genes which affect mating. Therefore, I looked at the ability of an a/a diploid and of two sterile a mutants to remove a factor activity from the culture medium. Diploid a/a strains do not mate and are insensitive to a factor. The two a sterile mutants tested, VU3 and VY3, were isolated for the inability to mate, but are also insensitive to a factor. VY3 still produces a factor, but VU3 does not (11, 12). The results (Fig. 4) confirm that VU3 and VY3 were insensitive to a factor, although their wild-type parent, XT1177-S47c, was fully sensitive. Neither a sterile mutant was able to remove factor activity from the culture medium (Fig. 5B and C), although the wild-type a
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RECOVERY OF YEAST FROM Gl ARREST BY
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since that ability is absent in two sterile mutants and absent in an a/a diploid. Resistance of recovering cells. In order to see if treated a cells were more resistant to a factor than untreated a cells, a cells which had recovered from factor arrest were collected by filtration, washed, and resuspended in fresh medium containing a factor. The response of these "conditioned" cells was compared with that of untreated a cells. In the culture of conditioned cells which had been reexposed to a factor at a dilution of 1:100, the percentage of unbudded cells remained at about 60% (Fig. 6A), whereas the percentage of unbudded cells in a culture of previously untreated a cells rose to more than 90% for 3 h when exposed to the same amount of a factor (data not shown, but" similar to Fig. 1B). Thus, the treated a cells appear to be more resistant to a factor than untreated a cells. Cell-free medium from the recovering culture described above was also tested to see if a diffusible inhibitor of a factor was present. No loss of a factor activity was observed when a factor was mixed with conditioned medium and assayed immediately with untreated a cells (Fig. 6B). The resistance of treated cells at various times after recovery from a factor arrest was examined. The results (Fig. 7) show that resistance to a factor arrest was gradually lost after recovery. Cells sampled 4.5 h after recovery (Fig. 7E) were more sensitive to a factor than cells sampled immediately after recovery (Fig. a
7B); however, they were not as sensitive as untreated cells which had never been exposed to a factor (Fig. 7A). The resistance of treated a cells as a function of the length of exposure to a factor was also studied. Cells that had been arrested for 2.5 h with a factor were compared with cells that had been arrested for 6 h. Figure 8 shows that cells that have been arrested for 6 h with a factor appear to be no more resistant to a factor than cells that have been arrested for only 2.5 h. Testing for a diffusible inhibitor of a factor. In the.preceding experiment, no loss of a factor activity was found when a factor was mixed with "conditioned" medium and assayed immediately. However, Hicks and Herskowitz (7) reported that mating-type a cells constitutively secreted a diffusible substance that inhibited or inactivated a factor activity. I, therefore, did a more elaborate version of the experiment described in Fig. 6B to look for a diffusible inhibitor. The sensitivity of the experiment was increased by incubating a factor with the cellfree culture fluid for 14 h to allow more time for a putative inhibitor to act. My results (Fig. 9B) show that little or no a factor activity was lost after incubation for 14 h in medium from an untreated culture of X21801A. Thus, at the concentrations of cells and a factor used in this experiment, no constitutive, diffusible inhibitor of a factor was detected. The experiment for detecting an inducible, diffusible inhibitor of a factor is shown in Fig. 9A, where a factor was incubated for 14 h in
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sulted in about a fourfold loss of a factor activity. This same degree of inactivation was achieved in the presence of a cells by incubation for only 1 h (cf. Fig. 3B). Thus, the amount of inducible, diffusible inhibitor activity observed with cell-free medium (Fig. 9A) is not enough to account for the loss of a factor activity observed in the presence of a cells (Fig. 3B), unless the inducible, diffusible inhibitor activity is labile or acts stoichiometrically.
DISCUSSION The conclusions derived from experiments examining the recovery of S. cerevisiae matingtype a cells from Gl arrest by a factor may be summarized. (i) The recovery process was density dependent; if two cultures were treated with identical amounts of a factor, the culture with the higher initial cell concentration recovered faster. This result rules out a recovery
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medium from which a cells had recovered from a factor arrest. A comparison with Fig. 1B suggests that the 14-h incubation (Fig. 9A) re-
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FIG. 6. Separate testing of the cells and medium from a culture previously arrested by a factor. X2180-1A at 3 x 106 cells/mi was treated with a factor at a dilution of 1:100 as described in Fig. 1 and shaken at 23°C to allow the cells to arrest and recover. By 6 h, 20% of the cells had resumed budding, and the culture was fractionated as described below to separate the cells from the medium. (A) To obtain "conditioned" cells, the a factor-treated culture was collected and washed on a membrane filter (Millipore Corp.). Then the cells were resuspended in fresh medium at a density equivalent to the initial cell concentration and transferred to flasks with no a factor (0) and with a factor added at a dilution of 1:100 (0). The percentage of unbudded cells was followed for 5.5 h at 23°C. (B) "Conditioned" medium was obtained from the supernatant fluid of the a factor-treated culture, which had been centrifuged to remove the cells. Untreated X2180-1A was resuspended at 3 x 106 cellslml in this conditioned medium with no a factor (0) and with a factor added at a dilution of 1:100 (-). As a control to show that our manipulations did not inactivate a factor, X2180-1A was resuspended in cell-free medium, containing a factor at a dilution of 1:100, which had been subjected to the same manipulations as the a factortreated culture (A). The percentage of unbudded cells was followed for 5.5 h at 23°C.
VOL. 130, 1977
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FIG. 7. Kinetics of loss of resistance to a factor. (A) X2180-1A at 3 x 106 cellslml was treated with a factor at a dilution of 1:100 as described in Fig. 1. The percentage of unbudded cells was followed. Symbols: (0) X2180-lA alone; (0) X2180-lA with a factor added at 1:100. At 6 h, the a factor-treated culture was adjusted by diluting with an equal volume of fresh medium. Samples were taken from this diluted culture at 7.5, 9, 10.5, and 12 h (indicated by the arrows) to determine the response of the culture to additional treatment with a factor. All the samples were diluted to the same absorbance (A6w = 0.33) to make the responses comparable. a factor at a dilution of 1:100 was added to a portion of each sample, and the cultures were shaken at 23°C. The percentage of unbudded cells was followed and is plotted in panels B through E. Symbols: (0) recovering culture without a factor; (a) recovering culture plus additional a factor at 1:100.
mechanism in which the cells become impermeable to a factor, since such a mechanism should be independent of the cell concentration. (ii) Cells that had recovered from a factor arrest were more resistant to a factor than untreated cells; however, the treated cells lost their resistance upon further cell division. Cells that had been arrested by a factor for 6 h were no more resistant to a factor than cells that had been arrested for only 2.5 h. These observations can be explained in two ways. First, the recovery mechanism may be induced by a factor. However, maximum induction must take place by 2.5 h, since cells arrested for 6 h are no more resistant to a factor than cells arrested for 2.5 h. Alternatively, the recovery mechanism may be a constitutive function of a cells. In that case, the resistance of recovering a cells to a factor could be attributed to the synchrony induced by a factor arrest. That is, a partially synchronized population of cells produced by a factor arrest would have an entire cell cycle in which to take up or inactivate a factor before returning to the sensitive Gl period, whereas, an untreated asynchronous culture of a cells would have, on the average, about half a cell cycle in which to take up or inactivate the same amount of a factor. The extra time the synchronized population has to take up or inactivate a
factor would account for the observed resistance. Furthermore, the loss of resistance after recovery could be explained by assuming that the recovering a cells lose their synchrony.
Thus, my results can be interpreted as being consistent with either a constitutive recovery mechanism or a recovery mechanism induced by a factor. More information will be required to distinguish between these two possibilities. (iii) The biological activity of a factor disappeared from the culture fluid of recovering a cells; this loss of activity could result either from chemical inactivation of a factor or from uptake of a factor into the cell. (iv) Two sterile (nonmating) mutants derived from an a strain were insensitive to a factor and lacked the ability to remove a factor activity from the culture medium. Furthermore, an a/a diploid, which is normally insensitive to a factor and does not mate, also failed to remove a factor activity from the medium. These results suggest that the ability to remove a factor activity from the medium is physiologically significant and not just a consequence of normal yeast metabolism since that ability is affected by genes, which also affect mating. (v) No a factor activity was lost upon incubation in cell-free medium prepared from an untreated a cell culture. Thus, at the concentra-
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phase or after a factor arrest). Alternatively, a small percentage of the inactivating activity that I find to be cell bound may be extracellular, and this amount may have been sufficient to produce inhibition with the high cell concentrations used by Hicks and Herskowitz (7). Furthermore, the concentration of partially purified a factor used in my experiments might be much higher than the concentration of a factor
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tions of cells and factor used, no constitutive, diffusible inhibitor of a factor was detected. Hicks and Herskowitz (7), however, have provided evidence for a constitutive, diffusible inhibitor of a factor. My failure to detect such a constitutive, diffusible inhibitor could be due to a difference in experimental conditions. For example, Hicks and Herskowitz used dense, concentrated streaks of a cells grown on rich agar plates, whereas I used cultures of low-cell density growing exponentially in liquid synthetic medium. Thus, the diffusible inhibitor might be labile in liquid medium or might be produced only by cells in GI (e.g., in stationary a
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RECOVERY OF YEAST FROM Gl ARREST BY a FACTOR
produced by the streak of a cells used by Hicks and Herskowitz. In that case the diffusible inhibitor could simply be swamped by the amount of a factor present in my experiments. (vi) A 1-h incubation of a factor with a cells (Fig. 3B) inactivated the same amount of a factor activity as a 14-h incubation of a factor in cell-free medium prepared from an a cell culture which had recovered from a factor arrest (Fig. 9A). This result suggests that the ability to inactivate or take up a factor (I cannot distinguish between the two), a process associated with recovery from a factor arrest, resides primarily in the cell. This may be due to the fact that the major cause of a factor activity loss during recovery is the result of uptake by the cell. On the other hand, if inactivation were the major cause of a factor activity loss during recovery, at least two possible explanations would come to mind. First, most inactivation may take place inside the cell or on the surface, but a small fraction of the inactivation may be due to an inducible, diffusible inhibitor activity (e.g., Fig. 9A). Second, the cell may only serve as a source for an extracellular, diffusible inhibitor that is labile or that acts stoichiometrically. Barrier effect. Hicks and Herskowitz (7) reported that a streak of mating-type a cells could act as a barrier to the diffusion of a factor on an agar slab. They called this property the barrier effect and designated strains possessing it as Bar+. Using this assay, they screened a number of sterile (nonmating) mutants derived from an a strain and found that some of the sterile mutants were still Bar+, but that others had become Bar-. It is tempting to speculate that the ability of a cells to remove a factor activity from the culture medium is responsible for the Bar+ phenotype of a cells. Unfortunately, this hypothesis is not compatible with my observation that VU3, a sterile Bar+ strain, failed to remove a factor activity from the medium. Pheromone inactivation mechanisms in other organisms. My results suggest that mating-type a cells, which are sensitive to a factor, are also capable of inactivating a factor. The idea that the target cells for a pheromone should also possess a means for specifically inactivating or metabolizing that pheromone is not without precedent among the fungi (5). For example, female gametes of the water mold Allomyces secrete a sex pheromone, sirenin, which is a bicyclic sesquiterpenediol that attracts male gametes (sperm) by chemotaxis. Machlis (10) found that sperm were able to remove sirenin activity from the culture fluid
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and that the sirenin activity could not by recovered from the sperm after uptake, suggesting that the spern had inactivated the sirenin. Similarly, female strains of the water mold Achlya secrete a steroid sex pheromone, antheridiol, which induces the formation of antheridial branches in the male strain. Barksdale (1) showed that male strains of Achlya could remove antheridiol activity from the culture fluid. More recently, Musgrave and Nieuwenhuis (12) have shown that male strains of Achlya can metabolize radioactive antheridiol to a biologically inactive compound. In mammals, there is considerable evidence that the target tissues for a number of polypeptide hormones including insulin, glucagon, oxytocin, parathyroid hormone, and calcitonin possess the ability to specifically degrade these hormones (8). This apparent ubiquity of hormone inactivation mechanisms suggests that further study of the a factor inactivation system in Saccharomyces may have broad biological significance. ACKNOWLEDGMENTS I thank Lee Hartwell for his helpful comments and encouragement during the course of this work and during the preparation of this manuscript; Diane Foley and Guy Page, whose experiments with a factor and a factor presaged some of the present work; Jim Hicks, Jeff Strathern, and Ira Herskowitz for sharing information and strains; D. Hawthorne, F. Heffron, H. Klein, D. Livingston, S. Reed, M. Unger, and K. Walsh for their criticisms ofthe manuscript; and Martha Katz, Nancy Ridgway, and Adele Purnell for typing the manuscript. This investigation was aided by grants to L. H. Hartwell from the American Cancer Society, Washington Division (VC-145), and from the Public Health Service, National Institute of General Medical Sciences (GM 17709). I was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. LITERATURE CITED 1. Barksdale, A. W. 1963. The uptake of exogenous hormone A by certain strains of Achyla. Mycologia 55:164-171. 3. Bucking-Throm, E., W. Duntze, L. H. Hartwell, and T. R. Manney. 1973. Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 3. Duntze, W., V. MacKay, and T. R. Manney. 1970. Saccharomyces cerevisiae: a diffusible sex factor. Science 168:1472-1473. 4. Duntze, W., D. Stotzler, E. Bucking-Throm, and S. Kalbitzer. 1973. Purification and partial characterization of a-factor, a mating type specific inhibitor of cell reproduction from Saccharomyces cereuisiae. Eur. J. Biochem. 35:357-365. 5. Gooday, G. W. 1974. Fungal sex hormones. Annu. Rev. Biochem. 43:35-49. 6. Hartwell, L. H. 1970. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bacteriol. 104:1280-1285. 7. Hicks, J. B., and I. Herskowitz. 1976. Evidence for a new diffusible element of mating pheromones in yeast. Nature (London) 260:246-248.
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8. Knights, E. B., S. B. Baylin, and G. V. Foster. 1973. Control of polypeptide hormones by enzymatic degradation. Lancet ii:719-723. 9. Lindegren, C. C., and G. Lindegren. 1943. A new method for hybridizing yeast. Proc. Natl. Acad. Sci. U.S.A. 29:306-308. 10. Machlis, L. 1973. The chemotactic activity of various sirenins and analogues and the uptake of sirenin by the sperm of Allomyces. Plant Physiol. 52:527-530. 11. MacKay, V., and T. R. Manney. 1974. Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of nonmating mutants. Genetics 76:255-271. 12. MacKay, V., and T. R. Manney. 1974. Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. II. Genetic analysis of nonmating mutants. Genetics 76:273-288.
J. BACTERIOL. 13. Musgrave, A., and D. Nieuwenhuis. 1975. Metabolism of radioactive antheridiol by Achyla species. Arch. Microbiol. 105:313-317. 14. St6tzler, D., and W. Duntze. 1973. Charakterisierung eines regulatorischen Peptids aus Saccharomyces cerevisiae. Hoppe-Seyler's Z. Physiol. Chem. 354:1247. 15. Stotzler, D., and W. Duntze. 1976. Isolation and characterization of four related peptides exhibiting a factor activity from Sarccharomyces cerevisiae. Eur. J. Biochem. 65:257-262. 16. Throm, E., and W. Duntze. 1970. Mating-type-dependent inhibition of deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 104:13881390. 17. Wilkinson, L. E., and J. R. Pringle. 1974. Transient Gl arrest of S. cerevisiae cells of mating type a by a factor produced by cells of mating type a. Exp. Cell Res. 89:175-187.
