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Mol Cell Biol. Jan 2000; 20(2): 478–487.
PMCID: PMC85107

CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast

Abstract

Xbp1, a transcriptional repressor of Saccharomyces cerevisiae with homology to Swi4 and Mbp1, is induced by stress and starvation during the mitotic cycle. It is also induced late in the meiotic cycle. Using RNA differential display, we find that genes encoding three cyclins (CLN1, CLN3, and CLB2), CYS3, and SMF2 are downregulated when Xbp1 is overexpressed and that Xbp1 can bind to sequences in their promoters. During meiosis, XBP1 is highly induced and its mRNA appears at the same time as DIT1 mRNA, but its expression remains high for up to 24 h. As such, it represents a new class of meiosis-specific genes. Xbp1-deficient cells are capable of forming viable gametes, although ascus formation is delayed by several hours. Furthermore, Xbp1 target genes are normally repressed late in meiosis, and loss of XBP1 results in their derepression. Interestingly, we find that a deletion of CLN1 also reduces the efficiency of sporulation and delays the meiotic program but that sporulation in a Δcln1 Δxbp1 strain is not further delayed. Thus, CLN1 may be Xbp1's primary target in meiotic cells. We hypothesize that CLN1 plays a role early in the meiotic program but must be repressed, by Xbp1, at later stages to promote efficient sporulation.

The absence of a fermentable carbon source combined with the limitation for nitrogen elicits a developmental switch in the yeast Saccharomyces cerevisiae from mitotic growth to meiosis and gametogenesis. The progress through this pathway, which ultimately results in the formation of an ascus containing four haploid spores, is thought to be regulated mainly at the level of transcription. Based on their temporal sequence of expression, meiotic genes can be divided into at least four classes: early, middle, mid-late, and late (reviewed in reference 24). Entry into the meiotic program is triggered by nutritional and mating type signals which lead to the activation of the transcription factor Ime1 (21, 44). In response to phosphorylation by the Rim11 kinase, the activation domain of Ime1 binds to the DNA-binding protein Ume6 and converts it from a repressor to an activator (40). This complex induces the transcription of early genes which have URS1 elements in their promoters (5, 6). Among these early genes is Ime2, a protein kinase homolog involved in downregulating Ime1 and necessary for the expression of other early and middle genes (19, 31, 43). The gene for the DNA-binding protein Ndt80 is transcribed in the middle of the transcriptional program of meiosis and activates its own expression as well as that of other middle and mid-late genes which are required for meiotic division (e.g., genes encoding B-type cyclins [Clbs]) and spore formation (e.g., SPS1) (11). Ndt80 was shown to bind in vitro to MSEs (mid-sporulation elements) which are found in many meiotically induced genes (11). The regulatory cascade which activates mid-late and late genes is undefined. There is evidence that the protein kinases Sps1, a Ste20 homolog, and Smk1, a mitogen-activated protein kinase homolog, stimulate these late genes (17, 23). It is also possible that the completion of meiotic DNA synthesis generates a signal required for the transcription of later sporulation-specific genes (30).

The cyclin-dependent kinase Cdc28 is essential for progress through mitosis and meiosis (33, 42). During the mitotic cell cycle there are four bursts of cyclin transcription, and each produces cyclins with distinct roles in mitotic progression. The first wave produces Cln3, which is key for inducing the second wave of cyclins, Cln1,2 and Clb5,6. These pairs of cyclins play redundant roles in inducing budding and DNA replication. Then Clb3,4 and later Clb1,2 are made. These, too, have overlapping functions in spindle assembly and mitosis, with Clb2 having a predominant role (for a review see reference 27). All Clbs are also expressed during meiosis (11, 18). The meiotic expression of the six Clbs, with the exception of Clb2, is controlled by the meiosis-specific transcription factor Ndt80, which is expressed during the same time interval (11). Clb1, Clb4, Clb5, and Clb6 have been shown to be the most important meiotic cyclins, and the absence of one or several of them causes a drastic drop in sporulation efficiency (13, 15, 18, 45). In contrast, Clb2, which is of primary importance in mitosis, plays a lesser role in meiosis (13, 18).

Recent experiments have suggested that the G1 cyclins play a negative role in sporulation (12), but this has not been thoroughly investigated. The absence of any one of three G1-specific cyclins Cln1, -2, and -3 does not eliminate spore formation (15), and deletion of CLN3 appears to speed up the meiotic program (12). In addition, overproduction of G1 cyclins inhibits sporulation, probably by transcriptional repression of Ime1, a key regulator for entering meiosis (12). Interestingly, the combination of CLN3 downregulation and ectopic expression of IME1 is sufficient to cause cells to enter meiosis in rich media (12).

The XBP1 gene of S. cerevisiae is of interest because it shares homology to the DNA-binding domain of Swi4 and Mbp1 and binds a related sequence (29). XBP1 mRNA is induced by a variety of stress conditions, including heat shock, high osmolarity, oxidative stress, DNA damage, and glucose starvation. Interestingly, Xbp1 expression is dramatically downregulated when wild-type cells evolve for 250 generations under glucose-limiting conditions (16). Consistent with this observation, overexpression of XBP1 causes slow growth and repression of the G1 cyclins Cln1, Cln2, and Cln3 (29). When Xbp1 is fused to the LexA DNA-binding domain, it acts as a transcriptional repressor (29). In order to use a more global approach to identify targets of this putative repressor, we used the method of RNA differential display and monitored the effect of loss of Xbp1 on the meiotic cycle.

In this report, we describe the identification of CLN1, CLN3, CLB2, CYS3, and SMF2 as target genes of Xbp1. All of these genes have Xbp1 binding sites in their promoters, and evidence is provided that Xbp1 binds these sequences in vitro. Furthermore, we show that XBP1 is expressed late in meiosis and that it represents a new class of meiosis-specific genes whose transcript level increases after meiotic DNA replication is completed and is maintained at a high level throughout gametogenesis. In Δxbp1 cells, ascus formation is delayed and Xbp1 target genes are derepressed late in meiosis. The most dramatic derepression is observed for CLN1. To see if expression of CLN1 late in gametogenesis could be responsible for the delay of spore formation observed in Δxbp1 mutants, we deleted CLN1 in XBP1 and Δxbp1 cells. Deletion of CLN1 alone leads to a delay of spore formation, but deletion of XBP1 leads to no further delay. This finding suggests that CLN1 plays a role early in gametogenesis, but its presence is deleterious later in the process. These findings identify Xbp1 as a novel transcriptional repressor in yeast which facilitates gametogenesis, perhaps by repressing key mitotic cyclins and other genes.

MATERIALS AND METHODS

Yeast strains, growth conditions, and cell analysis.

BY2059 (xbp1::HIS3) transformed with BD2014 (pGAL:XBP1) (29) was used for the differential display screen. Cells were grown at 30°C unless otherwise specified in either YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) or synthetic complete medium supplemented with amino acids as appropriate to select for transformants (2). G418 (200 μg/ml; Gibco BRL) was added to YEPD to select for the presence of the kanamycin resistance gene (kanR); 2% galactose was added to a culture grown in 2% raffinose to induce expression from the GAL1 promoter, and 2% glucose was added to repress expression. Transformation and tetrad analysis were performed as previously described (2).

The yeast strains used in this study are listed in Table Table1.1. SK-1 derivative strains BY1633 (=NKY 278), BY1593, BY1594, BY2386, BY2387, BY2388, BY2673, BY2674, BY2675, BY2676, BY2677, and BY2678 were used to synchronously induce meiosis (1, 20). To determine the role of Xbp1 in the selection of double-strand break (DSB) sites SK-1 derivative strains BY2533 and BY2534 were used. All disruptions of XBP1 were confirmed by PCR using oligonucleotide primers described in reference 29. To sporulate yeasts in liquid medium, cultures were grown in YEPD to an optical density (OD) of 2 to 3, diluted to an OD of 0.1 into YEPA (1% yeast extract, 2% peptone, 2% potassium acetate), and grown overnight with vigorous shaking to an OD of 1 to 1.5. Cells were harvested by centrifugation, washed once with water, resuspended to the same cell density in sporulation medium (1% potassium acetate), and incubated at 30°C with vigorous shaking. At indicated times, aliquots were harvested by centrifugation, and the pellets were stored at −80°C. The time of transfer to sporulation medium is referred to as 0 h.

TABLE 1
Strains used in this study

DNA content was quantitated by fluorescence-activated cell sorting (FACS) analysis using a Becton Dickinson FACScan and CellQuest software as described previously (29). Spore formation was scored by light microscopy (640× magnification; Zeiss, Jena, Germany). The average of at least four fields was calculated.

Detection of DSBs.

Diploid cells homozygous for the rad50S allele do not process or repair the ends of DSBs, and therefore the DSB fragments accumulate as discrete species (1). Cells (200 ml) from rad50S strains carrying either a wild-type or a deleted allele of XBP1 were sporulated, and at each time point 25 ml of cells was removed and mixed with 25 ml of ice-cold 100% ethanol. Chromosomal DNA was prepared as described previously (39) and digested with HindIII. The fragments were separated on a 0.7% agarose gel and blotted to GeneScreen (Dupont, NEN), and hybridizations were done as described elsewhere (29). The probe was a 1,970-bp EcoRI fragment from the 3′ end of the CYS3 gene labeled by random priming with [α-32P]dCTP.

DNA manipulations.

For the expression of full-length Xbp1 in Escherichia coli, an NdeI-XhoI fragment from BD2013 (29) was cloned into pET14b (Novagen) to generate a His6 N-terminal fusion to Xbp1 (BD2207). To create His6-Xbp11-509, BD2207 was cut with SalI, digested for a limited time with Bal 31 nuclease, recut with NdeI, and ligated back into pET14b, generating BD2208.

For construction of the XBP1-kanR disruption cassette, long flanking homology PCR (47) was used. In the first PCR, two separate fragments, corresponding to amino acids 1 to 98 in Xbp1 and 577 to 647 (including 122 nucleotides downstream of XBP1), were generated, using 50 ng genomic DNA of BY2058 (29) as the template; 300 ng of each product from the first PCR together with 1 μM outermost 5′ and 3′ primers (BL152 and BL153) (29) were used to synthesize the 2.1-kb XBP1-kanR disruption cassette from 20 ng of template (pFA-kanMX6 [47]). The PCR fragments were purified through agarose gels, and 100 to 300 ng of fragment was used for each yeast transformation.

Differential display analysis.

Total RNA was prepared from BY2059 that was transformed with the empty expression vector or with pGAL:XBP1, grown in 2% raffinose, and shifted for 80 min to 2% galactose. To remove chromosomal DNA from the RNA preparation, 50 μg of total RNA was incubated with 10 U of RNase inhibitor and 10 U of RNase-free DNase I (Promega) in 10 mM Tris-HCl (pH 8.3)–100 mM KCl–1.5 mM MgCl2 in a total volume of 50 μl. After 30 min at 37°C, the RNA was extracted once with phenol, precipitated with ethanol, and dissolved in 20 μl of H2O. Differential display of Xbp1 overexpression compared to vector control RNA was carried out as described by Liang and Pardee (28).

In a total volume of 20 μl, 200 ng of each RNA was reverse transcribed by using 200 U of SuperScript II reverse transcriptase (Gibco BRL) in 0.01 mM dithiothreitol–20 μM deoxynucleoside triphosphates (dNTPs)–1 μM oligo(dT) anchored primer (T12MA, T12MC, T12MG, or T12MT, where M can be A, C, or G). After 50 min of incubation at 37°C, the reverse transcriptase was inactivated by heating the reaction mixture for 5 min to 95°C. Four different cDNA syntheses were performed per RNA, each using a different T12MN oligo(dT) primer. All of the following PCRs were done in duplicate to exclude PCR artifacts. Two microliters of each cDNA reaction mixture was PCR amplified with the same T12MN oligo(dT) primer (1 μM) and an arbitrary decamer primer (0.2 μM) in a total volume of 20 μl containing 1 U of Taq polymerase (Fisher), 2 μM dNTPs, and 1 μCi of [α-33P]dCTP (Dupont, NEN). The arbitrary primers (kit OPA-G) were purchased from Operon Technologies Inc. (Alameda, Calif.); the individual sequences can be retrieved online (http://web712d0.ntx.net/ss2b1/stockproducts/kkitd.html). PCR amplification was carried out in 40 cycles as follows: 94°C for 30 s, 40°C for 2 min, and 72°C for 30 s, followed by a 5-min extension at 72°C. Prior to loading onto a 6% denaturing polyacrylamide gel, a 3.5-μl aliquot of each reaction was mixed with 2 μl of formamide loading buffer and heat denatured for 3 min at 95°C.

The gel was dried onto Whatman 3MM paper without fixation and autoradiographed overnight. The bands of interest were cut out of the gel and soaked in 100 μl of H2O for 10 min at room temperature and 15 min at 100°C. The cDNA in the supernatant was recovered by ethanol precipitation, using 50 μg of glycogen (Boehringer Mannheim) as the carrier. The cDNA was dissolved in 10 μl of H2O, from which 4 μl was used for reamplifications in a total volume of 20 μl, using the same primer combination and PCR conditions except that the dNTP concentration was 20 μM and the isotope was omitted. Most cDNA fragments had to be reamplified twice with 1 μl of the first reamplification as the template. The amplified DNA was purified through an 1.5% agarose gel. The isolated DNAs were used directly as probes for Northern blots or as templates for cycle sequencing using the appropriate decamer primer and around one-fifth of the isolated DNA. For nearly all cDNAs, unambiguous sequences were obtained.

A total of 20 decamer primers were used. Whereas most of the PCR bands were reproducible, only 50 to 60% showed the expected regulation pattern on Northern blots. In general, only fragments between 100 and 500 nucleotides in length were considered.

Gel retardation assay.

Gel retardation assays were performed as described previously (29), using full-length His6-Xbp1. The double-stranded 32P-labeled oligonucleotide probes were the consensus binding site oligonucleotide, a mutant consensus sequence to which Xbp1 does not bind (29), CY3 (5′-TCGACATAAAAATCCTCGAGGAAAAGAA-3′), and CL3 (5′-TCGATCTGTACTTTCCTCGAGCTTTTAATCTTCTT-3′) (only the upper strands are shown). Competition experiments included a 500-fold molar excess of unlabeled competitor DNA over labeled probe.

RNA analyses.

Total yeast RNA was analyzed by Northern blotting as previously described (29). Hybridizations were done simultaneously or sequentially to the following random-labeled probes, which were generated by PCR using yeast GenePairs primer (Research Genetics, Huntsville, Ala.) and yeast genomic DNA: CLB2 (YPR119w), CYS3 (YAL012w), DIT1 (YDR403w), DMC1 (YER179w), NDT80 (YHR124w), SGA1 (YIL099w), SMF2 (YHR050w), SPS100 (YHR139c), and TCM1 (YOR063w). Probes for XBP1, ACT1, CLN1, and CLN3 were generated as described in reference 29.

Antibodies.

To generate polyclonal antibodies directed against Xbp1, His6-tagged full-length Xbp1 or a His6-tagged C-terminal deletion of Xbp1 (His6-Xbp11-509) was expressed in E. coli BL21(DE3)pLysS (Novagen) and purified by nickel chelate chromatography essentially as described previously (29). To further purify Xbp1, the eluate was diluted 1:1 with binding buffer (20 mM Tris [pH 7.5], 100 mM NaCl, 1 mM MgCl2, 2% glycerol, 0.1% NP-40) and loaded on a heparin-Sepharose CL-6B column (Pharmacia). The column was washed with 200 mM NaCl in binding buffer, and bound proteins were eluted with a 200 to 1,000 mM NaCl gradient in binding buffer. Xbp1 eluted at around 500 mM NaCl. These preparations were used to inject rabbits (500 μg per injection) five times with incomplete Freund's adjuvant. Antibodies were stored in aliquots at −80°C.

Immunoprecipitations and Western blot analysis.

Protein extracts were prepared by glass bead disruption as described elsewhere (2), using radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 2 mM EDTA, 0.1% sodium dodecyl sulfate [SDS], 1 Triton X-100, 50 mM NaF, 1 μg of leupeptin per ml, 1 μg of pepstatin per ml, 1 mM phenylmethylsulfonyl fluoride). For immunoprecipitations, 400 μg of protein extract was precleared with preimmune serum coupled to protein A-Sepharose beads for 1 h at 4°C. The supernatant was recovered and incubated with 2.5 μl of immune serum coupled to protein A-Sepharose beads for 4 h at 4°C. The beads were washed four times with radioimmunoprecipitation buffer; bound proteins were eluted with SDS sample buffer, boiled, and loaded onto a 6.5% SDS gel. Following electrophoresis, proteins were transferred to nitrocellulose (Micron Separations Inc.), and Western blot analyses were performed, using a polyclonal antibody against Xbp1 at a dilution of 1:5,000 and a protein A-horseradish peroxidase conjugate (Bio-Rad Laboratories) at a dilution of 1:3,000. Enhanced chemiluminescence (Dupont, NEN) was used for detection.

RESULTS

Differential display of mRNAs from Xbp1-overexpressing cells.

The aim of this study was to understand the function of Xbp1. Xbp1 was tentatively classified as a transcriptional repressor because (i) when it is fused to the LexA DNA-binding domain it can repress the expression of a reporter gene and (ii) overexpression of Xbp1 results in the reduction of transcript levels of genes like CLN1, which has Xbp1 binding sites in its promoter. In addition, high levels of Xbp1 are induced under conditions of stress, and this is correlated with reduced transcript levels of CLN1 and other putative target genes. However, elimination of Xbp1 does not lead to derepression of CLN1 during stress (29). Thus, if Xbp1 is a repressor of CLN1 transcription, it cannot be the only one. In hopes of clarifying the role of Xbp1, we used the method of differential display to identify in vivo target genes.

In our first attempt to identify direct or indirect targets of the DNA-binding protein Xbp1, we compared the pattern of genes transcribed in wild-type and Xbp1-deficient cells under stress using the differential display method developed by Liang and Pardee (28). With the number of primer combinations that we used, it should have been possible to detect nearly every expressed gene (3). Nevertheless, we were not able to identify any reproducible differences (data not shown), which suggests that Xbp1-mediated repression has no role in the stress response of yeast or that there are other factors with similar activity. A recent study makes the latter explanation more likely. Mitotic cells were grown continuously for 250 generations under glucose limitation and then subjected to microarray analysis. Xbp1 was one of the 39 known genes that the cells evolved to repress under these conditions (16).

For our second attempt, we induced high-level expression of Xbp1 in Δxbp1 cells. Four different oligo(dT) primer pools were used to reverse transcribe total RNA isolated from cells overexpressing Xbp1 under the control of the GAL1 promoter on pYES2 and from control cells treated equivalently but harboring the pYES2 vector only (see Materials and Methods). The cDNAs were PCR amplified in the presence of [α-33P]dCTP, using 20 different arbitrary decamer primers, and the products were analyzed on denaturing polyacrylamide gels. In this way, 80 primer combinations were used to screen for genes which show changes in expression when Xbp1 is overexpressed.

RNA was collected from the Xbp1-overexpressing cells that had been grown in 2% raffinose and shifted for 80 min to 2% galactose. Although the maximum level of XBP1 mRNA is reached after 30 min (29), a later time point was chosen to allow for the Xbp1 protein to have an effect on potential target genes. This was done because we have observed, in stressed cells and during meiosis, a 20-min to 1-h lag between induction of XBP1 mRNA and appearance of Xbp1 protein (data not shown and Fig. Fig.5).5). Using 80 different primer combinations, we were able to detect four reproducible instances where Xbp1 overproduction reduced gene expression, examples of which are shown in Fig. Fig.1.1. This number of primer combinations should have enabled us to observe expression differences for nearly all yeast genes (3). Since only four reproducible differences were found, we conclude that Xbp1 overexpression does not have global effects on transcription. Rather, Xbp1 appears to affect specific target genes, and in each case it acts to repress transcription.

FIG. 1
Identification of Xbp1-regulated genes by differential display. (A) Differential display using total RNA from control cells (pGAL) and cells overexpressing Xbp1 (pGAL:XBP1). Both strains are Δxbp1. PCRs were done in duplicate, and the products ...
FIG. 5
Expression of XBP1 during meiosis. An SK-1 derivative strain (NKY 278) was shifted at time zero from presporulation medium (YEPA) to sporulation medium. Progression through meiosis was monitored, and aliquots were taken every 2 h for up to 16 h. After ...

Identification of genes downregulated by Xbp1.

To identify the genes corresponding to the differentially regulated messages, the cDNA fragments were isolated from the gel and twice reamplified with the same set of primers. These PCR-amplified products were directly cycle sequenced by using the upstream decamer primer (8). Sequences of the cDNA fragments were compared to those in the Saccharomyces genome database maintained at Stanford University. Downregulated genes were identified as CLB2, CLN3, CYS3, and SMF2, with CYS3 being amplified with several different primer combinations. Clb2 is a G2-specific B-type cyclin (46), Cln3 is a G1 cyclin (38), and Cys3 is a cystathionine gamma-lyase which catalyzes the biosynthesis of cysteine (35). The function of Smf2 is unknown. It was cloned as a high-copy-number suppressor of a lethal mutation in the yeast mitochondrial processing-enhancing protein (48). With the exception of XBP1 in the control sample, none of the differentially regulated bands disappeared completely, suggesting that Xbp1 downregulates these genes but does not eliminate their expression completely. The expression of very few genes was upregulated. Among those, XBP1 itself was isolated with three different primer combinations, as expected.

To confirm Xbp1 regulation of transcripts identified and isolated by differential display, Northern blot analysis was performed with probes comprising the entire coding regions of candidate genes. As shown in Fig. Fig.1B,1B, all identified genes showed a downregulation that is dependent on the overexpression of Xbp1. The G1 cyclin gene CLN1 was included in this analysis as a gene known to be repressed under these conditions from our previous work (29). Consistent with the differential display data (Fig. (Fig.1A),1A), none of the genes are completely shut off, but they are three- to fourfold downregulated.

Candidate target genes have Xbp1 binding sites in their promoters.

To see if Xbp1 could be directly affecting these target genes, we screened 1,000 bp upstream of the ATG of each candidate gene for the existence of potential Xbp1 binding sites. Indeed, in each of these promoter sequences, at least one sequence closely resembling the Xbp1 consensus binding site was found (29). As shown in Fig. Fig.2,2, the CLN1, CLB2, CYS3, and SMF2 promoters have several potential Xbp1 binding sites, and all of the potential binding sites noted share the 100% conserved core sequence TCGA. The two CLN1 sites are only 17 bp apart and were the first Xbp1 binding sites to be characterized (29).

FIG. 2
Comparison of potential Xbp1 binding sites in the promoters of candidate Xbp1 target genes to the Xbp1 consensus binding site. The sequence 1,000 bp upstream the ATG of each gene was screened for Xbp1 binding sites containing at least the four core bases ...

We also tested the ability of recombinant Xbp1 to bind to two of these sites in gel retardation assays. The binding of Xbp1 to the consensus binding site [GcCTCGA(G/A)G(C/A)g(a/g)] (29) was compared to its ability to bind the potential binding site in the CLN3 promoter (position −408 relative to the ATG) and the first potential binding site in the CYS3 promoter (position −168 relative to the ATG). As shown in Fig. Fig.3,3, Xbp1 was able to bind these sequences. Moreover, these sequences are bound with a higher affinity than the consensus binding site obtained by site selection, as judged by the band intensity of the retarded DNA (Fig. (Fig.3).3). Interestingly, the homologies of these new binding sites to the Xbp1 consensus binding site derived from site selection (29) was limited. The only completely conserved bases were within the central core of the Xbp1 binding site (29), yet those tested (asterisks in Fig. Fig.2)2) bind Xbp1 with higher affinity than does the consensus binding site. This suggests that the central core of the Xbp1 binding site is the most important part of the binding site, but that bases outside of the core are also critical to achieve high-affinity binding. This conclusion warrants the modification of the consensus binding site to [(a/c)CTCGA(g/a)(g/a)(a/g)n (a/g)] (Fig. (Fig.2).2).

FIG. 3
Binding of Xbp1 to CLN3 and CYS3 promoter sequences. Gel retardation experiments used recombinant His6-Xbp1 and the following oligonucleotides as probes and competitors: con (consensus binding site determined by binding site selection), mut (six positions ...

XBP1 is expressed during meiosis.

Chromosomal regions that undergo unusually high levels of recombination during meiosis are termed meiotic recombination hot spots. One of the best-characterized hot spots in S. cerevisiae is the CYS3 locus (9, 34). In the promoter of CYS3 there are two sites where DSBs are observed during meiosis, CYS3-I (−270) and CYS3-II (−160) (34). The CYS3-II site also overlaps exactly the first potential Xbp1 binding site in the CYS3 promoter. Considering the growing number of examples where meiotic recombination and transcription or transcription factor binding are linked (32), we investigated whether XBP1 is expressed during meiosis and therefore could potentially contribute to CYS3-II hot spot activity.

First, we searched the XBP1 promoter for elements known to be involved in regulating gene expression during meiosis. Interestingly, the XBP1 gene is adjacent to and divergently transcribed from the SGA1 gene, a meiosis-specific glucoamylase (22). SGA1 is induced in sporulating cells and repressed by the presence of nutrients (22). Within the 770-bp region between SGA1 and XBP1 there is an upstream activating sequence (UAS) and a negative regulatory element, identified in the region between −620 and −519 relative to XBP1 (22) (Fig. (Fig.4),4), which are able to confer meiosis-specific induction to an heterologous promoter. Overlapping these elements there is a sequence bearing strong homology to the MSE consensus, which is also sufficient to direct sporulation-specific expression (36) (Fig. (Fig.4).4). This sequence was recently shown to be specifically bound by Ndt80 (11). In addition, there are sequences nearer to the XBP1 coding sequence which have homologies to other meiotically regulated elements: UASSPS4 (19) and the T4C element of IME2 (5) (Fig. (Fig.4).4).

FIG. 4
The SGA1-XBP1 intergenic promoter. Previously identified promoter elements responsible for stress-induced expression of XBP1 are shown as gray boxes for stress response elements (STREs), AP1 recognition element (ARE), and heat shock element (HSE) as indicated. ...

To see if these or other sequences modulate XBP1 expression during meiosis, we measured the expression of XBP1 in SK-1 cells (1, 20), which can be induced to go through meiosis and sporulation efficiently and synchronously. After shifting these cells into sporulation medium, we took aliquots every 2 h for up to 16 h. After 24 h, the cells were shifted to rich medium (YEPD) and additional samples were taken for 4 h. RNA was purified from these samples, and the XBP1 transcript and other meiosis-specific transcripts were monitored by Northern blot hybridization (Fig. (Fig.5B).5B). As expected, XBP1 was not expressed during mitotic growth in presporulation medium (29). It was detectable after the culture was shifted to the starvation conditions of sporulation medium, but after 8 h, a very strong induction of XBP1 transcript and protein occurred; this level was maintained for at least 24 h, by which time spore formation was complete (Fig. (Fig.5B5B and C). FACS analyses over this time course indicated that the strong induction of XBP1 occurred well after the completion of meiotic DNA synthesis, which was achieved after 6 h (Fig. (Fig.5A)5A) (30, 37). When the cells were transferred to rich medium, which induces germination and subsequent mitoses, XBP1 RNA and protein dropped to barely detectable levels within 2 h.

The relative timing of XBP1 transcription was also compared to that for the transcription of other known meiosis-specific genes. For each of the four classes of genes, early, middle, mid-late, and late, we chose one representative and analyzed its expression on Northern blots. As shown in Fig. Fig.5B,5B, DMC1 is an early gene which is expressed between 2 and 8 h, NDT80 shows mid-sporulation expression which peaks between 6 and 10 h, and DIT1 shows a mid-late expression pattern that peaks between 8 and 12 h. SPS100 is expressed even later, between 12 and 24 h. The timely appearance of these genes is as described previously (4, 7, 11, 25). By this analysis, XBP1 defines a new pattern of late sporulation-specific expression, in that it is induced at the same time as mid-late genes like DIT1, but differs from them in that its transcript persists throughout the time course (24 h).

The expression pattern for Xbp1 that we observe is not consistent with that provided by the microarray analysis that has been carried out on sporulating cells (10). However, our Northern blot analysis was repeated three times, and the XBP1 transcription pattern correlates well with our Western blot analysis of Xbp1. The microarray data represent a single experiment in which a low degree of synchrony was achieved and samples were taken for only 11.5 h. The progression of meiotic S phase in our experiment cannot be directly compared to data from the array experiment, but we note that some early and mid-early genes, i.e., DMC1 and NDT80, show a prolonged pattern of expression in the array data compared to ours. Therefore, it is possible that Xbp1 induction occurred after their last time point.

The same blot was hybridized with a probe for SGA1, which is adjacent to XBP1 and is divergently transcribed (Fig. (Fig.4).4). Interestingly, there are clear differences in the timing and strength of expression. SGA1 is expressed at a much higher level (see the legend to Fig. Fig.5B)5B) than XBP1 and is induced at least 2 h before XBP1 is turned on. It is also turned off by 14 to 16 h, when XBP1 mRNA is still high (Fig. (Fig.5B).5B). Although SGA1 has been classified as a late sporulation gene (30), in this strain it is expressed just after NDT80 is turned on and before DIT1 is induced (Fig. (Fig.5B).5B). This timing is more consistent with SGA1 being a mid-late sporulation gene.

The expression of XBP1 during meiosis is roughly correlated with the appearance of DSBs at meiotic recombination hot spots (14). In S. cerevisiae, nearly all meiotic recombination hot spots have been mapped to promoter-containing regions, and for some of them the binding of transcriptional activators has been shown to be necessary (49). In the case of the CYS3 hot spot, which coincides with an Xbp1 binding site, it is possible that the binding of Xbp1 is important. To address this question, we compared the appearance of DSBs at the CYS3 locus. As shown in Fig. Fig.6B,6B, DSBs peak at 8 to 10 h after transfer to sporulation medium. Since deletion of XBP1 does not change the timing or the frequency of DSBs at this locus, we conclude that binding of Xbp1 to sites in the CYS3 promoter is not necessary for the formation of DSBs. Similar observations have been made for the DSBs at the ARG4 locus, where Gcn4 binding sites were identified but a disruption of the GCN4 locus displayed no changes in the levels of ARG4 gene conversion (41). CYS3 is another example where enhanced recombination is coincident with but not dependent on a transcription factor binding site. This inference supports the view that another feature, perhaps chromatin accessibility, is more important for hot spot formation (32).

FIG. 6
Formation of meiotic DSBs at the CYS3 locus in diploid rad50S strains. (A) Map of the CYS3 region indicating positions of the DSBs and the EcoRI probe (gray bar) used to detect the DSBs. (B) rad50S cells wild type for XBP1 (BY2533) or with a deletion ...

Xbp1 represses genes late in meiosis.

The deletion of XBP1 in haploid cells has no obvious phenotype (29). To determine whether XBP1 is required for meiosis and sporulation, we constructed a diploid Xbp1-deficient SK-1 strain and compared its sporulation to that of an isogenic XBP1 strain. These strains showed equivalent timing of DNA replication, as judged by FACS analyses (data not shown), and tetrads dissected after 24 h of sporulation showed high spore viability and normal kinetics of germination (data not shown). The timing of meiosis II in the two strains was also identical as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (DAPI) staining (Fig. (Fig.7).7). In contrast, when the percentage of spore-containing asci was scored, we found that ascus formation was considerably delayed in Δxbp1 cells and was almost 25% less efficient (Fig. (Fig.7).7). These results indicate that Xbp1 is important for events which occur after meiosis and before or during spore formation. DNA synthesis and the meiotic divisions occur with normal kinetics. In wild-type cells, asci form about 2 h later and are 50% complete after 8 h, which is about when Xbp1 expression is induced. When Xbp1 is deleted, half-maximal ascus formation is delayed another 2 to 3 h.

FIG. 7
Spore formation in XBP1 and Δxbp1 strains. The SK-1 derivative strains BY1633 (XBP1) and BY2388 (Δxbp1) were shifted at time zero from presporulation medium (YEPA) to sporulation medium. Aliquots were taken at indicated time points, and ...

Since Xbp1 is highly induced during the latter half of gametogenesis, we investigated the effect of loss of Xbp1 on the meiotic expression pattern of the Xbp1 target genes that were identified by Xbp1 overproduction and differential display (reference 29 and Fig. Fig.1).1). As shown in Fig. Fig.8,8, the CLN1 transcript level is very low throughout meiosis and CLN3 is immediately repressed after the shift to sporulation medium. By contrast, in Xbp1-deficient cells, CLN1 mRNA is much higher, particularly after 12 h. CLN3 also shows a moderate derepression at late time points. As previously shown (11), the mRNA for the B-type cyclin gene CLB2 increases transiently after the completion of meiotic S phase (6 to 9 h) (Fig. (Fig.5)5) in wild-type cells. In Δxbp1 cells, CLB2 is induced to the same extent but is maintained at a higher level at the later time points compared to wild-type cells. Another potential Xbp1 target gene, CYS3, is induced early in meiosis, and its mRNA level declines after 6 h. As with CLB2, this decline is absent in Xbp1-deficient cells (Fig. (Fig.8).8). The same behavior was observed for SMF2, whose mRNA declines gradually in wild-type cells but remains high in Xbp1-deficient cells. Thus, it appears that Xbp1 promotes the decline of message levels for all five of its putative target genes during meiosis and has its greatest effect on CLN1.

FIG. 8
Transcriptional effects of XBP1 during meiosis. The SK-1 derivative strains BY1633 (XBP1) and BY2388 (Δxbp1) were treated as for Fig. Fig.5.5. Aliquots were taken at indicated time points, and total RNA was prepared. The expression CLN1 ...

Since spore formation is delayed in Δxbp1 cells, we analyzed the expression of the meiosis-specific genes DMC1, NDT80, SGA1, and DIT1 in the same time course (Fig. (Fig.8).8). The expression patterns of the early and middle genes (DMC1, NDT80, and SGA1) show no difference in timing, although NDT80 is expressed at a higher level in Xbp1-deficient cells (Fig. (Fig.8).8). This is of interest because there is a consensus for Xbp1 binding in the NDT80 promoter region. Xbp1-dependent repression of NDT80 could not have been observed in our differential display screen, as NDT80 is not expressed at all in mitotic cells. Thus, it is possible that Xbp1 exerts some repressive effect upon NDT80, but the fact that the overall pattern of NDT80 expression is not changed indicates that Xbp1 is not the only regulator of this gene. The late gene, DIT1, is expressed for at least twice as long in Δxbp1 cells compared to wild-type cells. This prolonged period of DIT1 expression may be a result of the elevated levels of NDT80 transcript in Δxbp1 cells, since DIT1 is likely to be an Ndt80-regulated gene (10, 11). There are no obvious Xbp1 binding sites in the DIT1 promoter.

CLN1 may be the key target of Xbp1-dependent repression late in gametogenesis.

The G1 cyclin gene CLN1 shows the strongest transcriptional derepression when sporulating wild-type cells are compared to Xbp1-deficient cells. This prompted us to investigate whether this inability to repress CLN1 could be responsible for the defects in spore formation observed in Δxbp1 cells. CLN1 is not required for meiosis in budding yeast (15), but high-level expression of CLN1 has a dramatic deleterious effect upon spore formation (12). Thus, we deleted CLN1 in the Δxbp1 and XBP1 SK-1 strains and quantitatively compared the kinetics of ascus formation in these strains. The first unexpected finding was that loss of CLN1 function caused a 3-h delay and reduced the efficiency of sporulation in SK-1 cells by about 25% (Fig. (Fig.9).9). This indicates a previously unknown requirement for CLN1 function for efficient sporulation. Second, there was no further delay when CLN1 and XBP1 were both deleted. Since Xbp1 represses CLN1 transcription late in sporulation, one possible explanation of these results is that CLN1 is required early in this process, but as the spores are formed, CLN1 must be repressed. This late repression of CLN1 expression may be the critical function of Xbp1 during sporulation, because when CLN1 is deleted, loss of Xbp1 function does not cause a further delay or defect in spore formation.

FIG. 9
Spore formation in the Δcln1 strains. Spore formation in the Δcln1 strains BY2677 (Δcln1) and BY2678 (Δxbp1 Δcln1) was measured as described for Fig. Fig.7.7. For comparison, the percentage of sporulation ...

DISCUSSION

Nutrient limitation starts a transcriptional cascade which triggers meiosis and gametogenesis, or sporulation, in a diploid yeast cell. In budding yeast, 1 of 6 genes (about 1,000) shows significant changes in transcript level during sporulation. Half of these transcripts are induced, and half are repressed (10). Discrete waves of transcriptional induction have been characterized as being early, middle, mid-late, and late in gametogenesis (reviewed in references 24 and 30), but Xbp1 is the first sporulation-specific transcriptional repressor to be identified.

Xbp1 is highly induced during meiosis and defines a new class of sporulation-specific genes. XBP1 message is present at a low level immediately after the shift to starvation conditions; then it undergoes a burst of synthesis at about the same time as the mid-late genes and remains high throughout spore maturation, which occurs long after the mid-late genes are turned off. The low-level expression of XBP1 at the very beginning of the meiotic program is most likely a response to the starvation conditions of these cultures, since XBP1 is induced by limiting for glucose (29). The mid-late burst of XBP1 transcription could be activated through the adjacent UASSPS4 or the other, more distal elements that reside within the XBP1 promoter region, but these elements induce transient expression which typically begins earlier than that of XBP1 (19, 30). Thus, it is plausible that a novel promoter element or an interplay between these known meiotic elements contributes to the pattern of XBP1 transcription. The XBP1 message may also be more stable than other meiotic transcripts, and this could contribute to its unique expression pattern.

XBP1 is adjacent to and divergently transcribed from another sporulation-specific gene, SGA1, and yet the SGA1 and XBP1 transcript levels begin to rise at different times, attain very different levels, and persist for different intervals during the sporulation program. These genes are clearly not coordinately expressed, even though they are separated by only 750 bp of DNA which includes several potential promoter elements that are known to be bidirectional. It would be of interest to know how this differential gene expression is achieved.

Xbp1-dependent repression of cyclins during gametogenesis.

Our overexpression studies suggested that three cyclin genes (CLN1, CLN3, and CLB2) and two other genes (CYS3 and SMF2) are likely targets for Xbp1-mediated repression. This is indeed the case, as all five of these transcripts are repressed during the late stages of gametogenesis, and Xbp1 activity is required for this repression. The advantage, if any, of repressing CYS3 and SMF2 is unclear, but it is striking that three cyclins, each of which has a distinct function in the mitotic cell cycle, are actively repressed during the late stages of the meiotic cell cycle.

The role of G1 cyclins in the onset of meiosis in yeast has only recently been addressed (12, 15). CLN3 is highly expressed in presporulation medium, when cells are growing very slowly, but it is immediately downregulated when the cells are transferred to starvation conditions which trigger meiosis. This immediate drop is not Xbp1 dependent, but its radical nature suggests that CLN3 may be actively repressed upon the shift to sporulation conditions. Indeed, Colomina et al. (12) have shown that CLN3 overproduction prevents meiosis, probably by interfering with the activation of Ime1, which is a transcription factor required to start the meiotic cell cycle. Moreover, Δcln3 cells enter premeiotic S phase more rapidly than wild-type cells, and cells in which CLN3 is downregulated and IME1 is induced undergo meiosis spontaneously (12). These results are all consistent with the view that Cln3 kinase activity antagonizes the meiotic program. However, the fact that Δcln3 cells behave differently than CLN3 cells in meiosis indicates that Cln3 is expressed to a significant extent under these circumstances. One possibility is that Cln3 plays a limited role early in the process and then is actively repressed at later times.

The roles of the other two G1 cyclin genes, CLN1 and CLN2 have not been investigated in detail. Strains carrying deletions of either CLN1 or CLN2 still form spores (15), but the timing and efficiency of this process had not been studied previously. We have found that Δcln1 cells display a significant delay and a 25% reduced efficiency of spore formation. This is a rather dramatic phenotype for a gene product which is considered to be largely redundant with Cln2 and clearly indicates a positive role for Cln1 in the sporulation pathway. This is also consistent with the fact that the two primary activators of CLN1 transcription, Swi4 and Swi6, are also present during meiosis and deletion of either gene results in reduced spore viability (26). In agreement with Leem et al. (26), we observe a strong transient induction of Swi4 transcript levels early in the meiotic program and note that the SWI4 promoter contains a consensus URS1 element, which is known to induce many meiotic transcripts (B. Mai and L. Breeden, unpublished data). CLN1 transcript levels appear constant throughout meiosis and spore maturation in wild-type cells. However, in cells with a deletion of XBP1, we detect a strong derepression of CLN1 during the late stages of this process.

CLN1 may be the critical target of Xbp1-dependent repression in meiosis.

In addition to the finding that Xbp1 actively represses a subset of the budding yeast cyclins during gametogenesis, we find that diploids lacking Xbp1 are slower and less efficient in this developmental process. The simplest explanation of this delay is that the inability to repress one or more of the Xbp1 target genes is deleterious to spore formation. The CLN1 transcript is low throughout the meiotic cycle, but in the absence of Xbp1 this transcript is highly derepressed through the late stages of sporulation when Xbp1 is normally expressed. This led us to test whether the delay of sporulation that occurs in Δxbp1 strains could be due to this ectopic expression of Cln1. The results clearly indicate that loss of Xbp1 does not cause a further delay of sporulation in Δcln1 cells. This is consistent with the view that Xbp1's critical function in sporulation is to repress CLN1. However, we also found that deletion of CLN1 itself reduces sporulation efficiency, so we must assume that CLN1 also has a role in this process. The simplest interpretation is that CLN1 has a function in sporulation, but that the high levels of CLN1 that arise late in the absence of Xbp1 is deleterious.

To achieve a maximum efficiency during the process of gametogenesis, Cln1 has to be regulated very tightly at two points during meiosis and spore formation. It has to be expressed at a low level early during meiosis and has to be shut off or at least maintained at that low level later, at a time when spores are formed. If one of these points of regulation is lost, by deleting either CLN1 or XBP1, proper timing of spore formation is abrogated. Thus, we conclude that Xbp1's primary role is to keep CLN1 repressed late in meiosis.

In aggregate, our results suggest that the G1 cyclin Cln1 and its transcriptional repressor Xbp1 play important but nonessential roles in sporulation. We hypothesize that Cln1/Cdc28 kinase activity early in the sporulation program increases the efficiency of this pathway, but the same activity is deleterious if it persists through the late stages of gametogenesis. Since spores are more resistant to unfavorable environmental conditions, the integrity of this developmental process and the speed with which cells can recover from this state and return to the mitotic cycle represent considerable evolutionary advantages. Xbp1 and Cln1 are two of many cellular activities that contribute to this coordination.

ACKNOWLEDGMENTS

We thank Nancy Kleckner, Harvard University, for providing the SK-1 strains and Ron Reeder, Fred Hutchinson Cancer Research Center, for the pFA-kanMX6 plasmid. Special thanks are offered to Ingrid Wolf, Christoph Sachsenmaier, and other members of the laboratory for numerous constructive suggestions and discussions.

This work was supported by NIH grant GM41073 to L.B. and by a Deutsche Forschungsgemeinschaft postdoctoral fellowship to B.M.

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