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Several components of the glucose induction pathway, namely the Snf3p glucose sensor and the Rgt1p and Mth1p transcription factors, were shown to be involved in inhibition of sporulation by glucose. The glucose sensors had only a minor role in regulating transcript levels of the two key regulators of meiotic initiation, the Ime1p transcription factor and the Ime2p kinase, but a major role in regulating Ime2p stability. Interestingly, Rgt1p was involved in glucose inhibition of spore formation but not inhibition of Ime2p stability. Thus, the glucose induction pathway may regulate meiosis through both RGT1-dependent and RGT1-independent pathways.
Diploid budding yeasts (Saccharomyces cerevisiae) differentiate to form haploid spores. This process, termed sporulation, consists of two sequential programs: meiosis, which generates haploid nuclei, and spore formation, which encapsulates each haploid nucleus within a spore wall. Sporulation is regulated by specific nutritional signals (reviewed in Honigberg & Purnapatre, 2003). Prominent among these signals is glucose, a potent inhibitor of sporulation.
Glucose inhibits sporulation by repressing two master regulators of meiosis – Ime1p, a transcriptional activator, and Ime2p, a protein kinase (reviewed in Kassir et al., 2003; Honigberg, 2004). This repression occurs at several levels: (1) repression of IME1 transcription (Sagee et al., 1998; Shenhar & Kassir, 2001; Purnapatre et al., 2002), (2) inhibition of the assembly/activation of a transcription factor complex (including Ime1p) that induces transcription of IME2 and other early meiotic genes (EMGs) (Malathi et al., 1997; Vidan & Mitchell, 1997; Colomina et al., 2003; Rubin-Bejerano et al., 2004; Mallory et al., 2007), and (3) destabilization of Ime2p (Donzeau & Bandlow, 1999; Bolte et al., 2002; Purnapatre et al., 2005). These three levels of controls ensure that meiosis does not initiate until glucose is completely exhausted. For example, during late stages of growth, as the concentration of glucose decreases and the concentration of nonfermentable carbon source increases, IME1 is induced to moderate levels, but meiosis does not initiate (Purnapatre et al., 2002). Meiosis is prevented under these conditions in part because even the low concentrations of residual glucose present during late stages of growth are sufficient for the second and third levels of repression described above (Honigberg & Lee, 1998; Purnapatre et al., 2005).
A number of signal transduction pathways mediate the effect of glucose on cellular processes (Rolland et al., 2002; Johnston & Kim, 2005; Belinchon & Gancedo, 2007). Primary among these are the glucose induction pathway, glucose repression pathway, and the Ras2/Gpr1/cAMP pathway. These three pathways cross-regulate one another and also share some of the same targets (Ozcan, 2002; Kaniak et al., 2004; Kim & Johnston, 2006; Palomino et al., 2006; Belinchon & Gancedo, 2007; Ramakrishnan et al., 2007). The latter two pathways have been implicated in regulating meiosis (Simchen & Kassir, 1989; Rubin-Bejerano et al., 1996; Honigberg & Lee, 1998; Reinders et al., 1998; Donzeau & Bandlow, 1999) but whether the glucose induction pathway regulates meiosis is unknown; the known targets of this pathway involve activation of genes required for transport and metabolism of glucose (Kaniak et al., 2004; Palomino et al., 2005; Ronen & Botstein, 2006). The glucose induction pathway is itself activated by binding of glucose to one of two sensors: Rgt2p, which responds to high concentrations of glucose, and Snf3p, which responds to either moderate or high glucose (Ozcan et al., 1996, 1998; Jiang et al., 1997). These sensors activate Yck1p kinase (Moriya & Johnston, 2004), which likely phosphorylates two transcription factors, Std1p and Mth1p, targeting them for ubiquitination by the SCFGrr1p ubiquitin ligase, resulting in their destruction by the proteosome (Flick et al., 2003; Spielewoy et al., 2004; Kim et al., 2006; Pasula et al., 2007). Proteolysis of these transcription factors allows hyperphosphorylation of a third transcription factor, Rgt1p, releasing Rgt1p from the promoters of glucose-inducible genes (GIGs) and allowing the induction of these genes (Lakshmanan et al., 2003; Mosley et al., 2003; Polish et al., 2005).
A possible link between the glucose induction pathway and meiosis was suggested by the finding that the SCFGrr1p ubiquitin ligase (Willems et al., 1999) is required for rapid turnover of Ime2p in media containing glucose (Purnapatre et al., 2005). Because SCFGrr1p acts in a variety of signaling pathways (Abdel-Sater et al., 2004; Spielewoy et al., 2004; La Rue et al., 2005; Rue et al., 2005; Benanti et al., 2007), we examined the role of other components of the glucose induction pathway on glucose inhibition of meiosis.
All yeast strains used in this study (Table 1) are isogenic to W303. All experiments compared only strains with identical auxotrophies because auxotrophic markers can affect the transition from mitosis to meiosis. The rgt2Δ URA3, rgt2Δ LEU2, snf3Δ URA3, rgt1Δ URA3, and mth1Δ LEU2 alleles were constructed by PCR-fragment-mediated targeted gene replacement (Baudin et al., 1993; Lorenz et al., 1995). Primers were selected and transformants confirmed by diagnostic PCR reactions as described (Gray & Honigberg, 2001). To construct strains containing more than one URA3-marked deletion allele, strains containing each allele were crossed and dissected; segregants containing deletion alleles were identified by diagnostic PCR.
Except where noted, cultures were inoculated into YPA growth medium (1% yeast extract, 2% peptone, 1% potassium) at 4 × 104 cells mL−1 and grown to late-log phase, 1.3–2.5 × 107 cells mL−1 (c. 16 h), then washed three times in water and resuspended in Sp, Sp+0.5, or Sp+2 medium. Sp contained 2% potassium acetate and 0.17% yeast nitrogen base lacking ammonium sulfate; Sp+0.5 and Sp+2 were identical to Sp except that they also contained 0.5% or 2% glucose, respectively. All Sp media were supplemented to balance auxotrophies. All other media are as described (Kaiser et al., 1994).
The frequency of sporulation in cultures was determined by examining the culture in a light microscope. Measurements of statistical significance were calculated by standard unpaired t-tests. Sporulation in colonies was detected as previously described (Purnapatre & Honigberg, 2002). In brief, after colonies were grown for 8 days on SC-arginine medium plates, the colony and the entire disk of agar medium was overlaid on a fresh agar plate containing canavanine and SC-arginine. Haploid cells around the periphery of the colony developed into satellite colonies or ‘petals’ around the primary colonies.
Cell size profiles were obtained by elutriation as described (Day et al., 2004). Cells were grown as described above, transferred to Sp medium for 2 h, elutriated, counted, and then sized using a Z2 Coulter Counter Channelyzer (Beckman-Coulter). coulter accucomp z2 software (v. 3.01a) was used to analyze cell size. To determine the relationship between cell size and sporulation competence, elutriated fractions were resuspended in the sporulation medium to a final OD600nm of c. 2.0, and the efficiency of ascus formation determined as above.
Yeast RNA was isolated by vortexing 2–4 × 108 yeast cells with glass beads and phenol (Elder et al., 1983). Transcript levels were measured by S1 nuclease protection (Lee & Honigberg, 1996). Templates for the IME1, IME2, and DED1 riboprobes used in S1 nuclease analysis have been described (Honigberg & Lee, 1998; Purnapatre & Honigberg, 2002).
For the analysis of protein expression levels, total protein was precipitated from the extracts with trichloroacetic acid (TCA) (Hann & Walter, 1991). IME2-6XHAp was detected by Western blot using monoclonal antibody to the HA epitope (12CA5, CRP Inc.), HRP-conjugated secondary antibodies (Santa Cruz Biotech), and the ECLplus fluorescent detection system (Amersham). Anti-Cdc2 p34 (PSTAIRE) antibody (Santa Cruz Biotech) was used as a loading control.
To measure glucose inhibition of meiosis, we transferred growing cells to nutrient medium containing acetate, which promotes meiosis, and 0%, 0.5%, or 2% glucose (Sp, Sp+0.5, and Sp+2 medium, respectively). After 72 h, wild-type yeasts sporulated fivefold less efficiently in Sp+0.5 than in Sp, and 50-fold less efficiently in Sp+2 (Fig. 1).
We next tested the role of the Rgt2p and Snf3p glucose sensors in regulating meiosis. For this purpose, we transferred rgt2Δ, snf3Δ, and rgt2Δsnf3Δ strains to Sp, Sp+0.5, and Sp+2 media and determined the proportion of these cultures that formed asci after 72 h (Fig. 1). As expected, all three mutants sporulated to approximately the same level as the wild type in Sp medium. In addition, in Sp+0.5 medium the rgt2Δ mutant sporulated at a frequency comparable to the wild type, consistent with the fact that the Rgt2p sensor only responds to high glucose concentrations. In contrast, the snf3Δ mutant (and the rgt2Δsnf3Δ double mutant) sporulated approximately three times more efficiently than the wild type in Sp+0.5; indeed, these two mutants sporulated nearly as efficiently in Sp+0.5 as in Sp. Thus Snf3p, which senses moderate concentrations of glucose, is required for this concentration of glucose to inhibit sporulation.
At a higher concentration of glucose (Sp+2), spore formation was strongly repressed in all of the above strains. Consistent with the fact that both Rgt2p and Snf3p are activated by high glucose, the rgt2Δsnf3Δ double mutant sporulated to significantly higher levels than the wild type (unpaired t-test, P = 0.046). The snf3Δ mutant may also sporulate to higher levels than the wild type, although the difference was not quite significant (P = 0.06). Because these experiments do not distinguish whether both Rgt2p and Snf3p are required for high glucose to repress spore formation or only Snf3p is required for this repression, most of our subsequent experiments were performed in the rgt2Δsnf3Δ double mutant. Importantly, even in this double mutant, sporulation in Sp+2 is less than one-sixth as efficient as in Sp. Thus, pathways that do not require Rgt2p/Snf3p sensors must also mediate inhibition of meiosis at high glucose concentrations.
The Sp+0.5 medium may mimic conditions encountered during late stages of growth on glucose, when both a low concentration of glucose and a higher concentration of nonfermentable carbon source are present. To measure the role of glucose sensors in repressing meiosis during late stages of growth in colonies, we used a genetic assay that detects meiotic cells on the surface of colonies (Purnapatre & Honigberg, 2002). In brief, colonies grown on the medium containing glucose were transferred together with the underlying nutrient agar medium to a medium containing the drug canavanine. Canavanine, in the strain background used for these experiments, selects specifically for meiotic cells. As a result, meiotic cells on the surface of the original colony give rise to smaller satellite colonies after transfer to the new medium.
Wild-type colonies incubated on SC medium lacking arginine (2% glucose) for 8 days did not yield satellite colonies after transfer and neither did rgt2Δ colonies; in contrast, snf3Δ and snf3Δrgt2Δ colonies grown under the same conditions efficiently form satellite colonies (Fig. 2). As expected, when snf3Δrgt2Δ and wild-type colonies were suspended in water and examined by light microscopy, at least several-fold more asci were observed in the mutant than the wild type (2% vs. < 0.5%, respectively). Thus, the glucose sensors are required to efficiently repress sporulation in colonies grown on SC medium.
We next examined the role of the glucose sensors in regulating sporulation in colonies grown on one of two other carbon sources: acetate and raffinose. Because acetate is a nonfermentable carbon source, the glucose sensors would not be expected to affect growth (Bisson et al., 1987) or sporulation on this medium. Indeed, wild-type and rgt2Δsnf3Δ colonies grew at approximately the same rate on the 2% acetate medium, and these two strains sporulated to almost the same frequency (after 8 days, wild-type colonies yielded 11.6 ± 4.5% asci, whereas rgt2Δsnf3Δ colonies yielded 11.1 ± 2.8% asci). In contrast to acetate, raffinose is a fermentable carbon source. Efficient metabolism of raffinose requires the Rgt2p/Snf3p glucose induction pathay, at least in part, because this pathway mediates the induction of invertase (SUC2), which converts raffinose to glucose (Ozcan et al., 1997). Consistent with earlier results, we found that rgt2Δsnf3Δ colonies grew more slowly than wild-type colonies on raffinose medium (Neigeborn & Carlson, 1984). In addition, rgt2Δsnf3Δ colonies sporulated at much higher levels than wild-type colonies (after 8 days, wild-type colonies yielded 0.1 ± 0.1% asci, whereas rgt2Δsnf3Δ colonies yielded 14.1 ± 1.2% asci). Thus, the glucose sensors repressed meiosis in colonies on carbon sources (glucose and raffinose) that activate the glucose induction pathway but not on a carbon source (acetate) that does not activate this pathway.
Because glucose inhibits transcription of IME1 and IME2, we asked whether the glucose sensors are required for this repression. Wild-type and rgt2Δsnf3Δ strains were grown to midlog phase, transferred to Sp, Sp+0.5, or Sp+2 medium, and then assayed for IME1 and IME2 transcripts at various times (Fig. 3). As expected from the experiments shown in Fig. 1, in Sp medium the rgt2Δsnf3Δ double mutant expressed both the IME1 and the IME2 transcript to the same levels as the wild type (Fig. 3, compare lanes 1–4 to lanes 11–14). Also consistent with Fig. 1, in Sp+2 medium both the wild type and the rgt2Δsnf3Δ mutant expressed IME1 only at the same basal level as in stationary-phase cells and did not express detectable IME2 even after 20 h incubation (Fig. 3, lanes 1, 8–11, and 18–20). Because the rgt2Δsnf3Δ mutant sporulates at detectable (although low) levels after 72 h in Sp+2 (Fig. 1), presumably, IME1 and IME2 are eventually induced in at least some cells in the culture.
As described above, there was no effect of deleting the glucose sensors on IME1 and IME2 transcript levels in either Sp or Sp+2 media; however, there is a slight effect of the deletions on IME2 induction in Sp+0.5. In the wild type, IME1 induction was slightly delayed and IME2 induction clearly delayed in Sp+0.5 relative to Sp; however, eventually the IME2 transcript reached approximately the same high levels in both media (Fig. 3, compare lanes 2–7). The rgt2Δsnf3Δ mutant induced IME2 transcript in Sp+0.5 earlier than in wild type, although not as early as in Sp (Fig. 3, compare lanes 12–14 to lanes 15–17). Thus in Sp+0.5, the glucose sensors have only a modest effect on the timing of IME2 transcript accumulation.
In summary, RGT2 and SNF3 are not required for high glucose to repress IME1 and IME2 and are only partially required for moderate glucose to delay IME2 induction. Thus, although SNF3 efficiently inhibits spore formation when moderate concentrations of glucose are present, it is unlikely that this sensor inhibits spore formation by repressing either IME1 or IME2 transcription.
As described in the Introduction, SCFGrr1p is required both for the glucose induction pathway and for the glucose-stimulated turnover of Ime2p (Purnapatre et al., 2005). To determine whether the glucose sensors also regulate Ime2p turnover, we compared Ime2p-6XHA stability in wild-type and rgt2Δsnf3Δ strains. IME2-6XHA, driven by the tetO promoter, was induced in the growth medium and then repressed by the addition of tetracycline as cells were transferred to Sp or Sp+0.5 medium. As expected from our previous study (Purnapatre et al., 2005), in wild-type cells, Ime2p-6XHA was stable in the Sp medium but rapidly turned over in the Sp+0.5 medium (Fig. 4, compare lanes 2–4 with lanes 5–7). In contrast, in the rgt2Δsnf3Δ mutant, Ime2p-6XHA was stable in both the Sp and Sp+0.5 media. Thus, the glucose sensors are required for glucose-stimulated Ime2p turnover, and it is likely that glucose sensors inhibit sporulation, at least in part, by triggering Ime2p degradation.
Once bound to glucose, the glucose sensors inactivate two transcriptional repressors, Std1p and Mth1p. Of these two repressors, Mth1p is most important in maintaining repression of the GIGs, such as hexose transporters and other genes involved in responding to glucose (Schmidt et al., 1999; Kim et al., 2006). Thus, when glucose is present in the media, Mth1p is inactivated and genes responding to glucose are induced. To determine whether glucose sensors inhibit sporulation by inactivating Mth1p, we measured the efficiency of spore formation in mth1Δ and mth1Δrgt2Δsnf3Δ mutants (Fig. 1). Because Mth1p represses the response to glucose, we did not expect the single mth1Δ mutant to affect sporulation in Sp medium, and as expected, sporulation levels in this mutant were not significantly different from the wild type (P = 0.15). Similarly, sporulation in Sp+0.5 or Sp+2 media was not very different in the mth1Δ mutant compared with the wild type, consistent with Mth1p being repressed by glucose. Furthermore, the mth1Δrgt2Δsnf3Δ triple mutant sporulated with nearly the same efficiency as the rgt2Δsnf3Δ double mutant in Sp medium. However, the triple mutant sporulated much less efficiently than the double mutant in Sp+0.5 or Sp+2 medium. Indeed, in this latter mutant, the triple mutant was inhibited at least as efficiently as the wild type in either medium. This result indicates that at moderate concentrations of glucose, Snf3p likely inhibits meiosis by inactivating Mth1p.
Because the glucose sensors are known to target Mth1p for destruction by activating yeast casein kinase 1, we attempted to determine whether this kinase was also required for glucose to inhibit meiosis. Yeast casein kinase 1 is an essential protein encoded by two redundant genes YCK1 and YCK2 (Robinson et al., 1993). A yck1Δyck2ts strain is viable at 25 °C but not at 37 °C. Unfortunately, in the strain background used, even the wild type was defective in sporulation at temperatures > 33 °C, a temperature that is still semi-permissive for growth in the mutant (not shown). Thus we were unable to determine whether yeast casein kinase 1 is involved in glucose repression of sporulation.
Mth1p induces transcription of HXTs and other genes by triggering the dissociation of the Rgt1p transcriptional repressor from the promoters of these genes (see Introduction). To determine whether the glucose sensors repress sporulation through this same pathway, we initially examined sporulation in the rgt1Δ mutant. Surprisingly, this mutant sporulated only half as efficiently as the wild type in Sp medium (i.e. in the absence of glucose), an unexpected result given that Rgt1p is only known to regulate glucose-responsive genes (Fig. 1). However, a possible explanation for this result was suggested by the observation that rgt1Δ cells were on an average 30% smaller than wild-type cells (Fig. 5a). Indeed, a number of mutants that display small cell size also exhibit defects in meiosis (Calvert & Dawes, 1984; Zhang et al., 2002; Day et al., 2004), and cell size may be a principal determinant of the timing of meiosis under optimal conditions (Nachman et al., 2007). To test the idea that the rgt1Δ mutant was defective in meiosis because of its size, an rgt1Δ mutant and a wild-type strain were grown as above, separated by centrifugal elutriation, and cells with different sizes isolated and transferred to a sporulation medium (Fig. 5b). As with wild-type cells, larger rgt1Δ cells sporulated much more efficiently than smaller cells; indeed large rgt1Δ cells sporulated to levels comparable to the wild type. Thus, the sporulation defect of the rgt1Δ mutant is likely attributable to its smaller size.
Because the rgt1Δ mutant has only a partial defect in spore formation, we could still measure the effect of glucose on sporulation in this mutant. As expected, in Sp+2 medium, the rgt1Δ mutant, like the wild type, failed to form spores (<2%) even after 72 h of incubation (Fig. 1). However, the rgt1Δ mutant was somewhat more sensitive to moderate glucose concentrations than was the wild type [(sporulation in Sp)/(sporulation in Sp+0.5) equals 5 for the wild type and 8.8 for the mutant]. This result is consistent with genes required for glucose signaling or transport being released from repression in the rgt1Δ mutant.
As a more definitive test of the role of Rgt1p in regulating meiosis, we measured spore formation in an rgt1Δrgt2Δsnf3Δ triple mutant. Because sporulation in the rgt2Δsnf3Δ mutant was less sensitive to glucose than wild type, whereas sporulation in the rgt1Δ mutant was more sensitive, the rgt1Δrgt2Δsnf3Δ triple mutant was used to determine the epistasis relationship between the rgt1Δ and rgt2Δsnf3Δ mutants. In Sp medium, the triple mutant, like the rgt1Δ single mutant, sporulated approximately twofold less efficiently than the wild type (Fig. 1). More importantly, the triple mutant, again like the rgt1Δ single mutant, was more sensitive to 0.5% glucose than the wild type [(sporulation in Sp)/(sporulation in Sp+0.5) equals 13.3]. Thus, rgt1Δ is epistatic to rgt2Δsnf3Δ with respect to glucose inhibition of sporulation, indicating that the glucose sensors inhibit spore formation at 72 h at least in part by repressing Rgt1p activity.
To determine whether the glucose sensors stimulate Ime2p degradation by repressing Rgt1p, we compared Ime2p turnover in the rgt2Δsnf3Δ double mutant with an rgt1Δrgt2Δsnf3Δ triple mutant (Fig. 4). If glucose-stimulated turnover of Ime2p requires inhibition of Rgt1p, then the triple mutant should restore glucose-stimulated turnover. Surprisingly, we found no difference in Ime2p turnover between the double and triple mutants. Thus, the sensors do not stimulate Ime2p turnover solely by inhibiting Rgt1p function. A corollary of this conclusion is that inhibition of spore formation by moderate glucose, because it does involve Rgt1p, must not depend on destabilizing Ime2p.
The principal result reported in this study is that a mutant deleted for the Snf3p glucose sensor is defective in glucose inhibition of sporulation. This sensor has a minor role in regulating the transcription of IME1 and IME2, but it is required for glucose to destabilize Ime2p. Snf3p, along with a second sensor, Rgt2p, initiates the glucose induction pathway, and we show that two other components of this pathway, the Rgt1p and Mth1p transcription factors, also mediate repression of sporulation by glucose.
In addition to the Rgt2/Snf3 glucose induction pathway, the Snf1p glucose repression pathway and Ras2p/Gpr1 protein kinase A pathway also mediate glucose inhibition of meiosis (see Introduction). Multiple glucose-signaling pathways may be necessary to respond to a range of glucose concentrations. For example, the glucose induction pathway was required for a moderate concentration of glucose to inhibit spore formation but was largely dispensable when a higher concentration of glucose was present. Thus, at high glucose concentrations other glucose-signaling pathways must be sufficient to inhibit meiosis even when the glucose induction pathway is inactivated by mutation. We propose that the glucose induction pathway may be particularly important to inhibit sporulation during late stages of growth, when glucose concentrations are relatively low and other nutrient concentrations are near optimal for sporulation. Consistent with this interpretation, rgt2Δsnf3Δ colonies grown on glucose or raffinose medium contained at least several-fold more sporulated cells than wild-type colonies.
A consequence of multiple signaling pathways is that different glucose concentrations may regulate different targets. For example, transcription of IME1 and IME2 was completely repressed by high glucose but only slightly delayed by moderate glucose. In contrast, Ime2p is destabilized even by moderate glucose (Purnapatre et al., 2005). The current study indicates that moderate glucose inhibits meiosis primarily through a posttranslational mechanism, and this mechanism requires the glucose sensors. Thus, the glucose induction pathway may allow induction of IME1 and IME2 transcription but destabilize Ime2p kinase during late stages of growth.
The glucose induction pathway may inhibit meiosis through both Rgt1p-independent and Rgt1p-dependent branches. The Rgt1p-dependent branch follows the canonical glucose induction pathway; all known targets of this pathway depend on the release of Rgt1p from GIGs. Because this release leads to inactivation of Snf1p kinase (Kaniak et al., 2004), which is required for late stages of sporulation (Honigberg & Lee, 1998), the canonical glucose induction pathway may block sporulation in moderate glucose by inactivating Snf1p.
An Rgt1p-independent branch of the glucose induction pathway remains hypothetical. Nevertheless, such a branch is implied by our finding that Ime2p turnover is defective in mutants lacking the glucose sensors even when these mutants also lack RGT1. This result is consistent with earlier results suggesting that a key component of the glucose induction pathway, the SCFGrr1 ubiquitin ligase, may directly ubiquitinate a PEST sequence (i.e. a sequence rich in proline, glutamate, serine and threonine) in Ime2p, thereby destabilizing it (Purnapatre et al., 2005). Thus it is possible that the glucose sensors target Ime2p for turnover without inducing GIGs, but the protein modification enzymes that mediate this signal are unknown.
We propose two possible advantages of mediating glucose inhibition of meiosis through multiple signaling pathways. The first such advantage is to increase the fidelity of inhibition; for example, meiosis is not as strongly inhibited in colonies grown on glucose medium in the rgt2Δsnf3Δ mutant as in the wild type. The second advantage is to allow induction of EMGs during late stages of growth without committing these cells to the sporulation program (Purnapatre et al., 2002). Consistent with this hypothesis, transcription of IME1 and IME2 is less sensitive to glucose and less regulated by the glucose sensors than is Ime2p stability or spore formation. In this scenario, transcription of IME1 and IME2 primes cells in late stages of growth for meiosis without committing these cells to a meiotic fate.
We thank Dr Lucy Robinson (LSU-HSC) for strains and advice related to yeast casein kinase 1 and Dr Rita H. Lee (University of Missouri-Kansas City) for initial experiments. This work was supported by NIH grants GM80710 (to S.M.H) and GM77874 (to B.L.S.).
Editor: Ian Dawes