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• W2061314148 abstract "Article15 October 1997free access Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle Paul K. Herman Paul K. Herman Search for more papers by this author Jasper Rine Corresponding Author Jasper Rine Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Paul K. Herman Paul K. Herman Search for more papers by this author Jasper Rine Corresponding Author Jasper Rine Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA Search for more papers by this author Author Information Paul K. Herman2 and Jasper Rine 1 1Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA, 94720 USA 2Department of Molecular Genetics, The Ohio State University, Columbus, OH, 43210 USA The EMBO Journal (1997)16:6171-6181https://doi.org/10.1093/emboj/16.20.6171 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression. Introduction Saccharomyces cerevisiae cells respond to specific nutrient deprivations by entering into either of two distinct resting states, stationary phase and the spore (Esposito and Klapholz, 1981; Werner-Washburne et al., 1993). Entry into either of these states is reversible as both cell types will resume mitotic cell division when presented with the appropriate nutritional environment. Yeast spores are formed from vegetative diploid cells that are starved for nitrogen while in the presence of a non-fermentable carbon source. During sporulation, diploid cells proceed through a single round of meiotic division and the four resulting haploid progeny are packaged into individual spores (reviewed in Esposito and Klapholz, 1981; Mitchell, 1994). Spores possess a number of unique structural elaborations that confer an elevated resistance to a variety of environmental stresses, including heat shock and starvation. The most notable spore-specific structure is the specialized, multi-layer coat that provides a physical barrier between the outside environment and the spore within (Briza et al., 1986, 1988). Saccharomyces cerevisiae spores regain the capacity for cell division during a process known as germination. A priori, it is likely that germination involves the recognition of germinant, the extracellular compound that triggers entry into the germination program; the production of an intracellular signal; the transduction of this signal into new transcription and translation; spore coat removal or uncoating; and re-entry into the cell cycle. During germination, the spore loses those attributes specific to this cell type and begins to take on the features associated with the vegetative cell. Therefore, spore germination will likely involve both processes unique to this transformation and others that are shared by similar transitions between quiescence and cell cycle progression, such as the exit from stationary phase in yeast and the G0-to-G1 transition in mammalian cells. Despite the importance of spore germination to the proliferative cycle of the yeast cell, there have been few recent studies of this process. No systematic genetic analysis of germination has ever been performed and hence the genes and precise environmental cues that control this process remain unknown. Early studies of spore germination indicate that this process is controlled by nutrient availability and identify a series of morphological changes that occur to the spore prior to the onset of the first cell cycle (Hashimoto et al., 1958; Nagashima, 1959; Palleroni, 1961; Rousseau and Halvorson, 1973; Savarese, 1974; Kreger-Van Rij, 1978; Sando et al., 1980). In these pioneering studies, germination assays based largely on morphological criteria were used to examine the requirements for spore germination. These assays revealed that germination does not require oxygen and is most efficient when a readily fermentable carbon source, such as glucose or fructose, is present (Palleroni, 1961; Seigel and Miller, 1971; Savarese, 1974; Tingle et al., 1974). In addition, a role for protein synthesis was suggested by the ability of chemical inhibitors of translation to block the above morphological changes that occur during germination (Rousseau and Halvorson, 1973; Choih et al., 1977). However, most of these early studies of germination used assays that measured events occurring relatively late in germination. As a consequence, the identity and requirements of the critical early signaling events regulating spore germination are poorly understood. We have initiated a study of spore germination to further the understanding of those mechanisms regulating the resumption of growth from this resting state. This report describes a novel, quantitative assay for spore germination and an examination of the genetic and nutritional requirements for this process. We tested whether genes important for regulating the progression through the mitotic cell cycle were also required for spore germination. These experiments revealed a critical role for the Ras protein signaling pathway during the earliest events associated with spore germination. In addition, we identified and defined a commitment point in the germination process. These experiments indicated that the requirements for spore germination differed from those for mitotic division and represented a first step towards understanding yeast spore germination. Results Acquisition of Zymolyase sensitivity as an assay for spore germination Spore germination transforms a non-dividing spore into a vegetative cell competent for mitotic division. During this process, the spore loses those attributes specific to this cell type and takes on the properties of a vegetative cell. In particular, because of a specialized coat structure, yeast spores are more resistant to the cell wall-degrading enzyme preparation, Zymolyase (Ballou et al., 1977; Briza et al., 1988, 1990). The relevant activity in Zymolyase is a β-1,3-glucanase that digests the glucan layer of the cell wall, thus disrupting its structural integrity and resulting in the production of wall-less cells known as spheroplasts (Kitamura et al., 1974; Kitamura, 1982). The cell wall is the primary structure providing osmotic support to the vegetative cell and spheroplasts lyse when placed into a hypotonic environment. In general, spores exhibited a 105-fold higher survival rate than vegetative cells when subjected to a Zymolyase-induced hypotonic lysis regimen (Figure 1A). We therefore evaluated the loss of Zymolyase resistance as a measure of spore germination. Figure 1.Acquisition of Zymolyase sensitivity as a measure of spore germination. (A) Spores exhibit an elevated resistance to Zymolyase-induced hypotonic lysis. Cultures of a sporulating diploid strain [JRY5216; Spo(+)] and a non-sporulating haploid strain [JRY2334; Spo(−)] were incubated for 3 days at 30°C in minimal spore medium. The ability of cells from both cultures to survive hypotonic lysis following treatment with the indicated concentrations of Zymolyase was measured as described in Materials and methods. The numbers for the sporulating culture were corrected for the percentage sporulation in each experiment. The average sporulation efficiency for JRY5216 was 40%. The plots shown represent the average of three independent trials. (B) Germinating spores rapidly acquire sensitivity to Zymolyase-induced hypotonic lysis. Purified wild-type spores, at a concentration of ∼1×107 per ml, were incubated in rich medium containing 3 mg/ml Zymolyase-20T at 30°C. At the indicated times, aliquots of this germination reaction were removed, diluted and then plated to solid rich growth medium to determine the number of survivors. The percentage of survivors relative to the original number of spores is plotted. Download figure Download PowerPoint Purified spores were incubated in a rich growth medium containing Zymolyase for different lengths of time and the number of survivors was determined as described in Materials and methods (Figure 1B). After 4 h, >90% of the spores were sensitive to Zymolyase-induced hypotonic lysis (hereafter referred to simply as Zymolyase sensitivity). The loss in viability did not occur if the spores were incubated in water or if Zymolyase was omitted from the reaction. To test whether this assay was measuring an early or late event in the germination program, the acquisition of Zymolyase sensitivity was timed relative to specific morphological changes that occur during germination, including spore swelling, elongation and bud emergence (Palleroni, 1961; Rousseau and Halvorson, 1973). Bud emergence occurred with much slower kinetics than the acquisition of Zymolyase sensitivity; only 50% of the spores possessed buds after 4-5 h of incubation at 30°C. Since bud emergence serves as an indicator of passage through the G1/S boundary (Pringle and Hartwell, 1981), uncoating occurred several hours before the germinating spore encountered its first G1/S transition. Moreover, Zymolyase sensitivity appeared prior to spore elongation and either before or concomitant with the initial swelling of the spore. Thus, the spore uncoating assay measured an early event in the germination program, and provided the basis of many of the following experiments. For this report, spore germination is operationally defined as the point at which spores acquire sensitivity to Zymolyase-induced hypotonic lysis. Nutritional requirements for spore germination The nutritional requirements for spore germination were assessed using the Zymolyase sensitivity assay. For these measurements, spores were incubated in the relevant medium at 30°C in the presence or absence of Zymolyase. At the indicated times, dilutions of the germination reactions were plated to determine the number of survivors. The sample lacking Zymolyase tested the viability of the strains in each of the media used. The rich growth medium, YPD, served as the best germinant tested (Table II). A defined synthetic complete medium containing yeast nitrogen base and 2% glucose was also an excellent germinant. The rate of germination was slightly slower in this synthetic medium than in the rich medium: 50% of the spores acquired Zymolyase sensitivity after 3 h in the complete minimal medium as opposed to ∼2.2 h for the rich medium. Table 1. Yeast strains used in this study Strain Source JRY5215 MATα his3-11 leu2-3,112 lys2Δ::hisG trp1-1 ura3-1 can1-100 JRY2334 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 JRY3009 MATα ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 JRY5216 diploid JRY2334/JRY3009 JRY5217 diploid JRY2334/JRY5215 STX461-3A MATα prt1-1 ade1 ade2 gal1 tyr1 YGSCha E17 MATa cdc33-1 ade1 ade2 gal1 his7 lys2 tyr1 ura1 YGSCa JRY0891 MATa cdc35-1 ade1 arg4 his2 trp1 ura1 JRY0890 MATa cdc25-1 ade1 ade2 gal1 his7 lys2 tyr1 ura1 JT249 MATα cdc28-4 met tyr ura1 J.Thorner SR672-1 MATa cdc37-1 cyh2 gal2 ura1 A.Mitchell JRY5220 JRY2334 ras2-23 ras1Δ::HIS3 JRY5221 JRY2334 ras2-23 ras1Δ::HIS3 JRY5222 JRY5215 ras2-23 ras1Δ::HIS3 JRY5223 diploid JRY5220/JRY5222 JRY5224 diploid JRY5221/JRY5222 JRY5225 JRY5217 gal1Δ::HIS3/gal1Δ::HIS3 HM56-1A MATa ras2-47 ras1::HIS3 ade8 his3 leu2 trp1 ura3 H.Mitsuzawa HM55-3A MATα ras2-23 ras1::HIS3 his3 leu2 met4 trp1 ura3 H.Mitsuzawa a Yeast Genetics Stock Center, University of California, Berkeley. Table 2. Nutritional requirements for spore germination Medium % Germination YPD >95 Water <5 SC >95 SC no amino acids >95 SC no bases (uracil/adenine) >95 SC no nitrogen source >95 SC no glucose <5 YNB alone <5 2% glucose 95 YP-fructose 91 YP-galactose 51 To determine which components of the media were required for germination, different constituents of the defined synthetic medium were omitted systematically from the germination reactions. Neither the nitrogen source, amino acid nor nitrogenous base (uracil and adenine) supplements were essential for germination as spore uncoating occurred with near wild-type kinetics in media lacking these ingredients. In each case, >95% of the spores were Zymolyase-sensitive after 12 h in the medium (Table II). However, if glucose was omitted from the medium, <5% of the spores germinated, indicating that glucose was necessary for germination. Moreover, a solution of 2% glucose was an efficient germinant allowing for ∼95% germination after 12 h. Therefore, the presence of glucose alone was sufficient to trigger spore germination. Although other carbon sources could substitute for glucose, efficient germination was seen only with readily fermentable sugars, such as glucose and fructose (Table II; see also Palleroni, 1961; Seigel and Miller, 1971; Donnini et al., 1986; Xu and West, 1992). Therefore, the primary requirement for efficient germination was the presence of a fermentable carbon source. Two experiments indicated that the metabolism of the carbon source, and not simply its presence, was required for germination. First, two different non-metabolizable analogs of glucose, 6-deoxyglucose and 2-deoxyglucose, were unable to trigger spore germination. Less than 5% of the spores in yeast minimal medium (SC) with either analog substituting for glucose became sensitive to Zymolyase after 16 h at 30°C (Table III). The presence of these glucose analogs was not inhibitory to germination, as spores germinated with normal kinetics in medium containing both glucose and either non-metabolizable glucose analog. For the second experiment, a yeast gal1 mutant was used to evaluate spore germination in media with either glucose or galactose as the sole carbon source. The GAL1 gene encodes a galactokinase which is required for the first step in the conversion of galactose into glucose prior to the entry into glycolysis (Johnston and Carlson, 1992). gal1 mutant spores were unable to germinate on galactose-containing medium; <5% of the spores became sensitive to Zymolyase after 16 h of incubation (Table III). In contrast, >90% of wild-type spores had germinated in galactose medium after this time. The gal1 spores were not generally defective for germination as they exhibited wild-type germination kinetics in medium containing glucose as the carbon source. Therefore, a metabolizable carbon source was both necessary and sufficient to trigger S.cerevisiae spore germination. Table 3. Germination required a metabolizable carbon source Genotype Carbon source % Germination Wild-type glucose >99 Wild-type galactose 91 Wild-type none <5 gal1 glucose >99 gal1 galactose <5 gal1 none <5 Wild-type 6-deoxyglucose <5 Wild-type 2-deoxyglucose <5 Wild-type glucose + 6-deoxyglucose >99 Protein synthesis was required only for early events in the germination program A requirement for protein synthesis during spore germination was tested by using both genetic and pharmaceutical blocks to translation. The addition of the protein synthesis inhibitor cycloheximide to a spore germination reaction resulted in the complete inhibition of germination (Figure 2A). This requirement for protein synthesis was confirmed by analyzing the effects of two temperature-sensitive mutations, prt1-1 and cdc33-1, that block protein translation. The yeast PRT1 gene encodes a component of the translation initiation factor eIF-3 (Naranda et al., 1994; Danaie et al., 1995) and CDC33 encodes the cap-binding protein, eIF-4E (Brenner et al., 1988). Both of these proteins are essential for translation. Neither prt1-1 nor cdc33-1 spores were able to germinate at the non-permissive temperature; in both cases, <5% of the spores became sensitive to Zymolyase. However, both prt1-1 and cdc33-1 mutant spores were able to germinate at the permissive temperature of 23°C (Figure 2A). These data established that protein synthesis was required for yeast spore germination. Figure 2.Protein synthesis requirements during spore germination. (A) Protein synthesis was required for the acquisition of Zymolyase sensitivity by germinating spores. Left columns: wild-type spores were incubated in rich medium containing 3 mg/ml Zymolyase-20T for 6 h at 30°C in the absence (column 1, WT) or presence (column 2, CHX) of 40 μg/ml of cycloheximide. Center columns: either wild-type, prt1-1 or cdc33-1 spores were incubated for 6 h at 37°C in rich medium containing 3 mg/ml Zymolyase-20T. Right columns: same strains incubated for 14 h at 23°C. ‘% Germination’ indicates the percentage of spores that became sensitive to the Zymolyase treatment. Values shown represent the average of three independent trials. In addition, three different diploid strains were examined for each genotype shown. (B) Protein synthesis was required for early steps of the germination program. Wild-type spores were incubated in rich medium containing 3 mg/ml Zymolyase-20T at 30°C. At the indicated time, cycloheximide was added to a final concentration of 40 μg/ml and the 30°C incubation was continued. After 8 h of total incubation, dilutions of the reactions were plated on solid rich medium. ‘% Germination’ represents the percentage of the original spores that became sensitive to Zymolyase-induced lysis after the total 8 h incubation. Download figure Download PowerPoint Broadly, protein synthesis might have one of several roles during spore germination. It might be required only to initiate this transformation, or alternatively, ongoing translation might be necessary for the completion of the germination process. To test whether protein synthesis was required for all, or only a portion of the germination program, cycloheximide was added to a germination reaction at different times after the addition of growth medium to spores (Figure 2B). For these reactions, Zymolyase sensitivity of the spores was assessed at a common point, 8 h following the addition of growth medium. The effects of cycloheximide varied depending upon when this inhibitor was added to the germination reaction. If the drug was added during the first 30 min of incubation, essentially no spores were Zymolyase-sensitive after the 8 h incubation (Figure 2B). However, if the cycloheximide addition took place at 60 min, ∼50% of the spores were Zymolyase-sensitive 7 h later. In contrast, if cycloheximide was added 3 h or later after the addition of medium, there was only a modest effect upon the final level of germination. The inhibitory effects of cycloheximide upon translation at all points of addition were confirmed by removing aliquots of the culture after drug addition and assessing protein synthesis rates by measuring the incorporation of [35S]methionine and [35S]cysteine into protein; in all cases, protein synthesis was decreased to <10% of the uninhibited rate. Taken together, these data indicated that ongoing protein synthesis was required early during the germination process but was not essential for later events. Spore commitment to germination Many biological processes consist of multiple distinct steps, any of whose execution requires the completion of a previous step in the pathway. In some cases, the completion of an early event predisposes the cell to complete later steps, even in the absence of the stimulus that originally started the process. To test for the presence of such a commitment step in the germination pathway, spores were incubated for a variable length of time in rich medium and then transferred to water that contained Zymolyase, as described in Materials and methods. The difference between the original number of spores and the final number of survivors represents the number that became committed to germination (Figure 3). Approximately 60% of the spores became committed to germination after 1 h in the rich growth medium. At this time, <5% of the spores had germinated. In general, spore commitment preceded the acquisition of Zymolyase sensitivity by ∼1.5 h. Therefore, a brief exposure to germinant triggered a series of events that allowed spore uncoating to proceed, even in the absence of the original stimulant. Figure 3.Spores became committed to the germination pathway after a brief exposure to germinant. The percentage of spores committed to the completion of the uncoating reaction (% Commitment) was compared with the percentage already sensitive to Zymolyase-induced lysis (% Germination) at the indicated times. Wild-type spores were incubated at 30°C in rich medium containing 3 mg/ml Zymolyase-20T. At the indicated times, two aliquots of the cultures were removed and processed as follows. To determine the percent germination, one aliquot was plated on solid media and the number of survivors of this Zymolyase treatment was determined. The fraction of spores that had germinated was equal to the fraction of spores killed during the incubation with Zymolyase in rich medium. To determine the percent of the spores committed to the germination program, the second aliquot was pelleted and resuspended in water containing 3 mg/ml Zymolyase-20T. This incubation in water was continued such that the total incubation in rich medium and water was 9 h. After 9 h, the final cultures were plated on solid rich medium and the number of survivors of this commitment regimen was determined. The percentage of spores committed to germination after the indicated time in rich medium was equivalent to the percentage found to be sensitive to Zymolyase after the total 9 h incubation period. Note that spores became committed to the germination pathway ∼1.5 h before they germinated. Download figure Download PowerPoint The commitment period for spore germination was roughly coincident with the interval of essential protein synthesis defined by the cycloheximide experiments described above. This observation suggested that protein synthesis was required for the commitment process, but not for post-commitment events. To test this hypothesis, the effects of adding cycloheximide to either the pre- or post-commitment phases of the uncoating reaction were analyzed. To assess the protein synthetic requirements for commitment, spores were exposed to rich medium containing cycloheximide for 1, 2 or 3 h at 30°C. The spores then were transferred to water that contained Zymolyase, and the incubation was continued such that the total reaction time was 9 h. With protein synthesis inhibited, <5% of the spores committed to the germination program after 3 h of exposure to rich medium (Figure 4). Therefore, protein synthesis was required for spore commitment. Figure 4.Protein synthesis was required for spore commitment. Spores were incubated in rich medium for 1, 2 or 3 h at 30°C in the presence or absence of 2 μg/ml cycloheximide (CHX). Following the rich medium incubation, the cultures were treated as described in Materials and methods to determine the percentage of spores that had committed to the germination process. Download figure Download PowerPoint To evaluate the translational requirements for post-commitment events, we performed the reciprocal experiment and included cycloheximide in the incubation with water and Zymolyase after exposure to rich medium for 1 h. The presence of cycloheximide during the post-commitment period had no effect on the fraction of spores that acquired Zymolyase sensitivity. When cycloheximide was present in the post-commitment reaction, 59% of the spores were found to be Zymolyase-sensitive, as compared with 58% in the control reaction. Similar results were obtained with the prt1-1 mutant; PRT1 gene function was required only during the pre-commitment period and not for later events (data not shown). Taken together, these results indicated that translation was required only for early events in the germination program, including spore commitment. The later post-commitment steps of the germination program, including spore uncoating, proceeded in the absence of ongoing protein synthesis. Many regulators of the mitotic cycle were dispensable for spore germination Spore germination likely involves some events that are unique to this process and others that are shared with a continuing mitotic cycle. Although a large number of genes are known to be essential for yeast cell cycle progression (Pringle and Hartwell, 1981; Norbury and Nurse, 1992; Murray and Hunt, 1993), there has not been a systematic analysis performed to test whether any of these genes also play a role in regulating spore germination. Therefore, we tested whether known regulators of the mitotic cell cycle influenced either the absolute levels or the rate of spore germination. These experiments used conditional mutants that were defective in passage through various phases of the cell cycle. None of the tested genes essential for progress through either the S, G2 or M phases of the mitotic cycle was required for spore germination; the representative cdc mutants defective in these genes exhibited no significant quantitative or kinetic defects in the acquisition of Zymolyase sensitivity (data not shown). These assays included an analysis of two genes whose products act at the G1/S boundary (CDC4 and CDC34), one that acts early in S phase (CDC7) and others (CDC24 and CDC43) that are required for bud emergence (see Pringle and Hartwell, 1981; Murray and Hunt, 1993, and references therein). The other genes tested included CDC2, CDC8, CDC14, CDC15 and CDC46. Our results were consistent with previous analyses of these mutants that had failed to uncover a role for the respective wild-type genes in spore germination using indirect measures of germination. The role of genes that regulate G1 functions was analyzed by measuring germination in yeast mutants defective in the control of ‘Start’. Start identifies a major point of cell cycle regulation that occurs in late G1; once a cell passes this point it is committed to completing the current cell cycle (Pringle and Hartwell, 1981). A key regulator of Start is the cyclin-dependent kinase encoded by the CDC28 gene (Pringle and Hartwell, 1981; Reed, 1992). The role of this kinase in germination was tested by assessing the acquisition of Zymolyase sensitivity by cdc28 mutant spores at the non-permissive temperature of 37°C. cdc28 mutants exhibited germination kinetics identical to that of wild-type spores (Figure 5). After 6 h at 37°C, 95% of the cdc28 spores had germinated. In the 37°C control culture lacking Zymolyase, we observed that >95% of the cdc28 cells were arrested as unbudded cells, indicating that the cdc block had been imposed. This latter result also indicated that, upon germination, spores were in G1 or they would not have arrested at the characteristic cdc28 block. CDC37 is another yeast gene required for the execution of Start and cdc37 and cdc28 mutants exhibit a similar terminal phenotype (Pringle and Hartwell, 1981). CDC37 gene function also was dispensable for spore germination as >95% of the cdc37 spores germinated after 6 h at the non-permissive temperature (Figure 5). Both cdc28 and cdc37 mutant spores underwent the characteristic set of morphological changes associated with germinating spores, such as swelling and elongation. Therefore, several steps of the germination program, including commitment, uncoating and spore shape changes, occurred in the absence of CDC28 and CDC37 gene activity. These results indicated that two primary regulators of exit from the G1 phase of the yeast cell cycle, Cdc28p and Cdc37p, were not required for spore germination. Figure 5.The Ras protein signaling pathway was required for spore germination. Wild-type and the indicated mutant spores were incubated in rich medium containing 3 mg/ml Zymolyase-20T for 6 h at 37°C. The fraction of spores that exhibited sensitivity to Zymolyase-induced lysis after this incubation represented those spores that had undergone the germination process. The results shown represent the average of three experiments with at least two different diploid strains for each mutant analyzed. Download figure Download PowerPoint We also examined the role of two genes, UBC1 and CMK1, reported to play a role in spore ger" @default.
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• W2061314148 date "1997-10-15" @default.
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• W2061314148 title "Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle" @default.
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