a New Class of Negartive Regulators Affecting Sporulation-Specific Gene Expression … · 2002. 7....

Copyright 0 1997 by the Genetics Society of America

Identification of a New Class of Negartive Regulators Affecting Sporulation-Specific Gene Expression in Yeast

Mei Li Benni* and Lenore Neigeborn*+*

* Waksman Institute of Microbiology, +Department of Molecular Biology and Biochemistry and $The New Jersey Cancer Institute, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854-8020

Manuscript received February 19, 1997 Accepted for publication July 21, 1997

ABSTRACT We characterized two yeast loci, MDS3 and PMDl, that negatively regulate sporulation. Initiation of

sporulation is mediated by the meiotic actintor IMEl, which relies on MCKl for maximal expression. We isolated the MDS3-1 allele (encoding a truncated form of Mds3p) as a suppressor that restores IMEl expression in mckl mutants. mds3 null mutations confer similar suppression phenotypes as MDS3-I, indicating that Mds3p is a negative regulator of sporulation and the MDS3-1 allele confers a dominant- negative phenotype. PMDl is predicted to encode a protein sharing significant similarity with Mds3p. mds3 pndl double mutants are better suppressors of mckl than is either single mutant, indicating that Mds3p and Pmdlp function synergistically. Northern blot analysis revealed that suppression is due to increased IMEl transcript accumulation. The roles of Mds3p and Pmdlp are not restricted to the MCKl pathway because mds3 pmdl mutations also suppress IMEl expression defects associated with MCKl- independent sporulation mutants. Furthermore, mds3 pmdl mutants express significant levels of IMEl even in vegetative cells and this unscheduled expression results in premature sporulation. These phenotypes and interactions with RAS2-Val19 suggest that unscheduled derepression of IMEl is probably due , - -_ to a defect in recognition of nutritional status.

T HE Saccharomyces cermisiae sporulation pathway consists of a complex series of events, the execution

of which is cell-type dependent (restricted to a / a cells) and subject to specific environmental cues. The process initiates with premeiotic DNA synthesis followed by meiosis, which is characterized by high levels of genetic recombination and reduction in chromosome content to generate four haploid nuclei. Continued progression through the developmental program is characterized by morphogeneses that result in packaging of the four meiotic products into prospores and enclosure of the prospores into a mature ascus resistant to a variety of harsh environmental conditions (ESPOSITO and KLAP- HOLTZ 1981; MITCHELL 1988; MALONE 1990; HONIG BERG et al. 1993). Governing the execution of this pathway are a variety of regulatory mechanisms, including alterations in transcriptional gene expression patterns (CLANCY et al. 1983; HOLAWAY et al. 1987; SMITH and MITCHELL 1989; STRICH et al. 1989; KIHARA et al. 1991; VERSHON et al. 1992; BURNS et al. 1994; COE et al. 1994; MITCHELL 1994; BOWDISH, et al. 1995; HEPWORTH et al. 1995; FRIESEN et al. 1997), post-transcriptional control at the level of splicing and mRNA stability (ENGEBRECHT et al. 1991; MENEES et al. 1992; SUROSKY and ESPOSITO 1992; OGAWA et al. 1995) and the involvement of MAP kinase-mediated signal transmission (s) ( COSTIGAN and

Cmresponding author: Lenore Neigeborn, Waksman Institute of Mi- crobiology, Rutgers, the State University of New Jersey, 190 Freling- huysen Rd., Piscataway, NJ 08854-8020. E-mail [email protected]

Genetics 147: 1351-1366 (November, 1997)

SNYDER 1994; FRIESEN et al. 1994; KRISAK et al. 1994; HERSKOWITZ 1995). Thus, this model system represents an ideal tool for investigation of the control of develop mentally regulated cellular events.

A combination of two concurrent signals is required for yeast to initiate the sporulation program: establishment of the a / a cell type and nutrient deprivation (KAS SIR and SIMCHEN 1976; &NE et al. 1981; TATCHELL et al. 1985; MITCHELL and HERSKOWITZ 1986; GOUTTE and JOHNSON 1988; HERSKOWITZ 1988; DRANGINIS 1990; MATSUURA et al. 1990), Both signals converge on stimulation of the meiotic-activator ZMEl (inducer of =io- sis) (KASSIR et al. 1988; SMITH and MITCHELL 1989; MAT- SUURA et al. 1990; MITCHELL et al. 1990). ZMEl is essen- tial for sporulation-specific gene (SSG) expression and, hence, is required for all meiotic and subsequent sporulation events (KASSIR et al. 1988; SMITH and MITCHELL 1989; MITCHELL et al. 1990; SMITH et al. 1990). Thus, control of ZMEl expression is a vital determining factor in triggering the sporulation pathway. Despite the critical role played by ZMEl in the control of meiosis and sporulation, little is known about the molecular mechanisms mediating the signals governing its expression. Indeed, several modulators of ZMEl expression have been identified genetically, but only one, R M E l , has been shown to act directly at the ZMEl promoter (see below); the specific molecular roles of the others are still unclear.

The cell type signal is established via a gene expression regulation pathway under control of the master

1352 M. Li Benni and L. Neigeborn

regulatory locus, MAT (KASSIR and SIMCHEN 1976; HER- SKOWITZ 1988). Yeast heterozygous at the MAT locus (MATa/MATa diploids) express the negative regulator a l a 2 , which represses expression of haploid specific genes, among them being RMEl (regulator of meiosis) (MITCHELL and HERSKOWITZ 1986; G o u m and JOHN- SON 1988; DRANGINIS 1990). h e l p is expressed only in haploid cell types ( a and a cells) and functions directly as a negative regulator of IMEl expression (COV- ITZ et al. 1991; COVITZ and MITCHELL 1993). Hence, the a / a cell type is a positive regulator of sporulation because it leads to repression of a repressor of IMEI. The a / a cell type also activates I&%# expression and RES1 (-@ME1 escape) activity, which also promote the expression of IMEI, but in an RMEl-independent manner (SHAH and CLANCY 1992). From a biological per- spective, the a / a cell type is characteristic of the diploid phase of the yeast life cycle; thus this signal ensures that only diploid cells undergo meiosis, which would be lethal to haploid a and a cell types.

The response to nutrient deprivation represents a fundamental difference between the haploid and d i p loid cell types. Both reproduce vegetatively when nutrients are plentiful, however, nitrogen starvation, in the presence of nonfermentable carbon sources, leads to the cessation of cell growth and normal mitotic division (HERSKOWITZ 1988). Following starvation, a and a cells (haploids) enter a quiescent phase of the cell cycle considered to be analogous to Go (ESPOSITO and KIA- PHOLTZ 1981). In contrast, a / a (diploid) cells activate IMEl expression and initiate the sporulation program (ESPOSITO and KLAPHOLTZ 1981; SMITH et aZ. 1990). The starvation signal triggering meiosis is partially trans- duced through decreased CAMP-dependent protein kinase (PJSA) activity (TATCHELL et aZ. 1985), which results in mitotic growth arrest that can lead to induction of IMEl expression (SMITH and MITCHELL 1989; MATSU- URA et al. 1990). However, this cannot be the only mechanism, because CAMP levels alone do not appear to influence sporulation ability (MATSUMOTO et aZ. 1983; OLEMPSKA-BEER and FREESE 1987; CAMERON et aZ. 1988).

The MCKl (meiosis and centromere regulatory b- nase) locus encodes a protein kinase that mediates the decision to initiate the sporulation program by functioning as a positive regulator of IMEl (NEIGEBORN and MITCHELL 1991). An mckl deficiency results in delayed and reduced levels of sporulation; excess MCKI gene dosage has a stimulatory effect. These phenotypes result as a direct consequence of altered gene expression of IMEI. MCKl is required for maximal IMEl transcript accumulation, as well as the expression of other SSGS. The mechanism controlling Mcklp activity has not been determined, however, it is clear that Mcklp does not contribute to cell type regulation. Its role in transmitting the starvation signal or another, as yet undeter- mined, signal remains to be elucidated. In addition, MCKl also promotes efficient ascus maturation and

centromere activity, but not via its role as an activator of IMEl expression (NEIGEBORN and MITCHELL 1991; SHERO and HIETER 1991; JIANG et al. 1995).

The MCKl gene encodes a protein kinase that is constitutively expressed (DAILEY et al. 1990; NEIGEBORN and MITCHELL 1991). In vitro, Mcklp can autophosphory- late at tyrosine and serine, but phosphorylates exogenous substrates at only serine and threonine (LIM et al. 1993; L. NEIGEBORN, unpublished results). Since it is unlikely that Mcklp controls IMEl expression directly, we suggest that Mcklp activates an IMEI-specific trans- activator, either directly or indirectly, via a phosphorylation cascade. Identification of the factors functioning downstream of Mcklp is complicated by the fact that an mcklA deficiency produces only a partial defect in activation of IMEI. This observation suggests that there may be other pathways sharing functional specificity with Mcklp. Indeed, genetic studies reveal that the RTMI, 8, 9, 13 pathway is one example of such a redundant pathway (LI and MITCHELL 1997; Su and MITCH-

To identify IMEl regulatory components functioning downstream of or parallel to Mcklp we screened for dosagedependent suppressors of an m k l deficiency. We obtained a clone harboring an unusual dominant- negative allele of a previously uncharacterized locus that we denote MDS3 (Zck l dosage suppressor). We show here that MDS3 and its paralog PMDl (Earalog of mS3) represent synergistic negative regulatory activities that operate downstream of both the mckl and riml pathways to curb IMEl expression.

ELL 1993a,b).

MATERZALS AND METHODS

Strains, media, genetic methods and assays: All of the yeast strains used in this study are isogenic and derived from the SKI genetic background (ME and ROW 1974). Rele- vant genotypes and sources are shown in Table 1. The mcklA4::TWl, imelA13-HIS3, rmelAS::LEU2, and riml mutations, as well as the arg6 and met4 auxotrophic markers and the I"lacZ::URA? reporter alleles have been described previously (SMITH and MITCHELL 1989; NEICEBORN and MITCH- ELL 1991; su and MITCHELL 1993a,b).

Yeast strains were grown at 30". Complete (YEPD and YEPAc), synthetic (minimal and dropout), and sporulation media were prepared as described by SHERMAN et al. (1986) ; carbon sources, as indicated (glucose or potassium acetate), were added to a final concentration of 2%.

Standard genetic procedures were used for mating, diploid selection, sporulation, and tetrad analysis (SHERMAN et ul. 1986). Yeast transformations were performed by the lithium acetate procedure (IT0 et al. 1983). Sporulation efficiency was assessed in quadruplicate from plate cultures unless otherwise indicated: strains were first grown overnight on YEPD plates and then replica plated to sporulation plates and allowed to incubate at 30" for 48 hr. Quantitation of total asci formed was determined by microscopic examination; no less than 200 cells were counted per sample. The values are the average of four independent assays for each strain.

Growth rates in YEPAc were monitored by growing each strain to saturation in liquid YEPD followed by dilution into 50 ml YEPAc to a final ODm0 = 0.05. Growth was assessed by

Negative Regulators of Sporulation 1353

determining the ODm of the culture every 2 hr until station- ary phase was attained.

For P-galactosidase assays, cells were grown to early log phase in YEPAc medium (0 time), washed and shifted to an equal volume of liquid sporulation medium (2% KAc), and incubated for 6 hr. Samples for assay were harvested by cen- trifugation, washed in water, resuspended in Zbuffer, perme- abilized by addition of SDS and chloroform, and assayed as described (STERN et al. 1984). Values are the average of at least four independent assays.

Selection for multicopy suppressors of mcklA: LNY266 (mcklA4::TRPl, imelAl3-HIS3) was transformed with a YEp24 genomic library (CARLSON and BOTSTEIN 1982) and 2400 independent Ura+ transformants were picked and screened for acquistion of the ability to grow in the absence of histidine. Twelve transformants were found to express plas- middependent histidine prototrophy. The plasmids were recovered via passage through Escherichia coli and their activity was verified by retransformation into LNY266 followed by test- ing for growth in the absence of histidine. A combination of restriction mapping and Southern analyses revealed the presence of 11 different plasmids: two of the plasmids contained the genomic HIS3 locus but none contained the genomic MCKl locus. Control experiments demonstrate that MCKl clones do confer the His+ phenotype in our screen, however, Southern analysis of library DNA revealed that the genomic MCKl locus is underrepresented as compared to other unique loci (data not shown). The remaining nine plasmids define five different genomic regions (two regions were represented by overlapping clones). Given the relatively low number of transformants screened and the fact that several loci were recovered only once and MCKl was not recovered at all, we conclude that the screen was not saturated.

Plasmid constructions: pMCH8 (&tigopy suppressor conferring His+, no. 8) is the original clone from the YEp24 library (CARLSON and BOTSTEIN 1982) that confers suppression of the mcklA defect in PIMEI-HIS3 expression; it contains an 8.5-kilobase (kb) genomic insert (see Figure 1A). The following plasmids contain the indicated subcloned fragments derived from pMCH8: pMLl contains the 6kb CluI-BumHI fragment cloned into the polylinker of pRS426 (SIKORSKI and HIETER 1989; CHRISTIANSON et ul. 1992); pML2: contains the 3.3-kb EcoRI fragment cloned into pRS426; pML3: contains the 6.1-kb N h I fragment cloned into the SpeI site of pRS426; pML5: contains a 1.4kb NheI fragment spanning the yeast DNA/vector junction cloned into the SpeI site of pRS426; pMLlO: contains the 3.4kb NcoI fragment, which was made blunt ended by filling in the 3’ recessed ends using Klenow enzyme and then cloned into the SmuI site of pRS426; pMLl1 contains a 2.9-kb LEUkontaining fragment cloned into the BgnI site of pMLl. pML51: contains a 4kb Hind111 fragment spanning the yeast DNA/vector junction cloned into pRS426.

We used PCR to obtain a 2.3-kb fragment containing the amino terminus of the genomic MDS3 locus. The following primers were used with genomic DNA as template: 5’-CTG ACTTCCGATI’CCAACGAG3’ and 5”TATAGAGGACCTTA GAAG3’. This PCR product was a used as a probe to identify a genomic library clone carrying the amino terminus of MDS3 via colony hybridization in E. coli (NEIGEBORN and M ~ C H E L L 1991). We used this method, as opposed to directly obtaining the region via PCR, to avoid inadvertent PCR-mediated mum- tion of MDS3. One positive library clone, pML41, was retained and confirmed by restriction analysis to contain the amino terminus of MDs3 as well as several kilobases of upstream DNA. pML43 was constructed by cloning the 4.6kb Sua-NheI fragment from pML41 (including 1.9 kb of upstream DNA and the amino-terminal 2506 nucleotides of AfDS3) into pML10, thus creating a clone containing full-length MDS3

under &e control of its own promoter. The integrity of this clone was confirmed by sequence analysis.

The vector pADC416 contains the constitutive yeast ADcl promoter (including the transcriptional start site) cloned into the XhoI-Hind111 sites within the polylinker of pRs416 (SIKOR- SKI and HIETER 1989; CHRISTIANSON et al. 1992). This plasmid was constructed by subcloning the 1.42-kb %I-Hind111 fragment containing the ADCl promoter from pPC97 (CHEWY and NATHANS 1992) into the XhoI-Hind111 sites of pRs416. pML46 was constructed by cloning a PCRgenerated MDS3 fragment extending from the initiator ATG through the ter- mination codon (containing S m d sites at both ends) into the S m d site of pADC416, thus creating an MDS3 allele driven by the ADCl promoter. To avoid PCR-mediated introduction of mutations into this construct, we then swapped everything from the CluI site (at nucleotide +1241 from the translational start) to beyond the end of the clone with the analogous non-PCRderived MDS3 DNA. pML47, pML48, pML49, and pML50 were constructed in the same way, except that each clone initiates from sequentially carboxy-terminal in-frame ATGs (see Figure 1B; pML48 and pML49 contain the same insert DNA, except in opposite orientations relative to the ADCl promoter). For pML48 and pML49, non-PCRderived wild-type DNA was swapped from the xbd site to the end of the clone; in pML50 non-PCRderived wild-type DNA was swapped from the Me1 site to the end of the clone. PCR- derived sequences retained on all of these clones were verified by sequence analysis not to contain any mutations. The following PCR primers were used in these constructions: 5‘46 5’- T A T T C C C G G G A T G C C T C A - 3 ’ ; 5’47: 5’-TAT TCCCGGGATGCATGTAAAGAACGAMAT-3‘; 5’48: 5‘-ACC CCCGGGATGTCTTCTATCCCT-3’; 5’50: 5”ACCCCC GGGATGAGCATCCCGTCTGGT-3‘; and 3’46-50: 5”ACC CCCGGGGGTTTCATI’AGCCTITGGGAA-3’. Construction of the mds32::LEU2 and mdr3P3::W al-

leles: A 2.9-kb genomic fragment including the LEU2 selectable marker was inserted at the BgllI site within the MLlS3- 1 coding region of pMLl to generate the allele mds3-2::LEU2 in plasmid pMLl1. A fragment containing the mds3-2::LEU2 allele was cleaved from the plasmid by digestion with NcoI and used to perform a one-step gene replacement (ROTH- STEIN 1983) by transforming the diploid strain LNY357 and selecting for leucine prototrophy. Integrity of the mutation was confirmed by Southern blot analysis.

To confirm that mds3-2::LEU2 confers the null phenotype, we also constructed the mds3A3::UM3 null allele that lacks most of the coding sequence. The 2.6kb CZuI-B@I fragment of pML43 was deleted and replaced by a 1.5kb ClaI-Bud1 fragment containing the cTRA3 gene to generate plasmid pML45 containing the mds3A3::UM3 allele. pML45 was then digested with Sac1 and PVuI to liberate linearized mds3A3::URA3, which was purified and used to perform a one-step gene replacement by transforming LNY357 to uracil prototrophy. The integrity of the mutation was confirmed by Southern blot analysis (data not shown). The phenotypes associated with this allele were indistinguishable from those associated with mds3-2::LEU2, thus, we conclude that mds3- 2::LEU2 confers a null phenotype (data not shown). Construction of the pndlAl::LEU2 allele: PMDl (OW

YERl32c) was discovered by searching the yeast genome database for paralogs of MDs3 (ORFYGL197w) using the BLAST program available within the database (CHERRY et al. 1996). We used PCR to obtain a 1-kb fragment from the genomic PMDl locus. The following primers were used with genomic DNA as template: 5”GTl”ITCACCTCATACAGCCT-3’ and 5’-GTGAGATACAGCCGTAGTCTT-3‘. This PCR product was used as a probe to identify a genomic library clone (pML28) carrying the 5’ flanking and amino terminal portion of the


TABLE 1

Yeast strains

LNY266 LNY339

LNY342

LNY357

LNY746

LNY747

LNY748

LNY749

LNY763

LNY764

LNY765

LNY766

LNY767

LNY768

LNY807

LNY808

LNY809

LNY810

LNY890

LNY891

LM1897

LNY899

LNY1007

LNYl008

MATa arg6-2032S mlA5::LEU2 imelAl3-HIS3 mcklA4::TRPl MATa/MATa a@2032S/ARG6 met4-44S/MET4 imelA13-HIS3/IMEl

M A T d M A T a arg62032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl

MATa/MATa ar@2032S/ARG6 met444S/MET4 imelAl3-HIS3/IMEl

MATa/MATa arg6-2032S/ARG6 met4-44S/MET4 mds3-2::LEU2/MDS3

MATa/MATa arg62032S/ARG6 met4-44S/MET4 mds3-2::LEU2/MDS3

MATa/MATa arg6-2032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl

MATa/MATa arg6-2032S/ARG6 met4-44S/MET4 imelA13-HIS3/IMEl

MATa/MATa arg6-2032S/ARG6 met444S/MET4 imelAl3-HIS3/IMEl

mcklA4::TRPl/MCKl mds4Al::LEU2/mds4Al::LEU2

mcklA4::TRPl/mklA4::TRPl mds4Al::LEU2/mds4Al::LEU2

mcklA4::TRpI/MCKl

imelAl3-HIS3/IMEl mcklA4::TRPl/MCKl

mcklA4::TRPl/mcklA4::TRPl imelAl3-HIS3/IMEl

m&3-2::LEU2/ mds3-2::LEU2 mckl A4::TWl/ MCKl

mcklA4::TRPl/mcklA4::TRPl mds3-2::LEU2/mds3-2::LEU2

mds3-2::LEU2/ mds3-2::LEU2 mds4A2::LEU2/mds4A2::LEU2 mcklA4::TRPI/mcklA4::TRPl

mds3-2::LEU2/ mds3-2::LEUL’ mds4A2::LEU2/ mds4A2::LEU2 mcklA4::TRPI/MCKl

rncklA4::TRPl/MCKl mds3-2::LEU2/MDS3 IME2-5-lacZ::URA3/

MATa/MATa arg62032S/ARG6 met4-44/MET4 imelAl3-HIS3/IMEl

MATa/MATa ar&-2032S/ARG6 met4-44S/MET4 imelA13-HIS3/IMEl

MATa/MATa arg62032S/ARG6 met4-44S/MET4 imelAl3-HISjl/IMEl

IME2-5-lacZ::URA3

mcklA4::TRPI/mcklA4::TRPl IME2-5”lacZ::URA3/IME2-5- lacZ::URA3 mds3-2::LEU2/MDS3

mds3-2::LEU2/ mds3-2::LEU2 IME2-5-lacZ::URA3/ IME2-5-lacZ:: URA3 mcklA4::TRPl/MCKl

MATa/MATa met4-44S/MET4 a@2032S/arg62032S imelA13-HIS3/ IMEl mcklA4::TRPI/mklA4::TRPl mds3-2::LEU2/mds3-2::LEU2 IME2-5-lacZ::URA3/IME2-5-lacZ::URA3

MATa/MATa arg62032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl mds3-2::LEU2/mds3-2::LEU2 mcklA4::TRPI/mcklA4::TRPl rimlA/ rim1 A

MATa/MATcu arg62032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl mds3-2::LEU2/MDS3 mcklA4::TRP1/MCKl rimlA/rimlA

MATa/MATa ar&-2032S/ARG6 met444S/MET4 imelAl3-HIS3/IMEl mds3-2::LEU2/MDS3 r imlA/r imlA mcklA4::TRPl/mcklA4::TRPl

MATa/MATa a@2032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl mcklA4::TR?’l/MCKl r imlA/r imlA mds3-2::LEU2/mds3-2::LEU2

MATa/MATa a@2032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl pmdlAl::LEU2/pmdlAl::LEU2 mds3-2::LEU2/mds3-2::LEU2 mcklA4::TRPI/MCKl

pmdlAl::LEU2/pmdlAl::LEU2 md.s3-2::LEU2/mds3-2::LEU2 mcklA4::TRPl/mcklA4::TRPl

MATa/MATa arg6-2032S/ARG6 met444S/MET4 imelA13-HIS3/IMEl mcklA4::TRPI/mcklA4::TRPl pmdlAI::LEU2/pmdlAl::LEU2

MATa/MATa ar&2032S/ARG6 met444S/MET4 imelAl3-HIS3/IMEl pmdlAl::LEU2/pmdlAl::LEU2 rncklA4::TRPUMCKl

MATa/MATa arg62032S/ARG6 met444S/MET4 imelAl3-HIS3/lMEI m&3-2::LEU2/MDS3 pmdlAl::LEU2/PMDl mklA4::TRPl/MCKl r imlA/RIMl

pmdlAI::LEU2/pmdlAl::LEU2 mds3-2::LEU2/MDS3



MATa//MATa met4-44S/MET4 imelAl3-HIS3/IMEl rimlA/rimlA

This laboratory This laboratory

This laboratory

This laboratory

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

Negative Regulators of Sporulation

TABLE 1

continued

1355

Strain ~~~ ~

Genotype Source

LNYl009

LNYlOlO

LNYlOll

LNYl102

LNYl104

LNYl105

LNYl106

LNYl119

LNYl121

MATa/MATa a@2032S/ARG6 &444S/MET4 imelA13-HIS3/IMEl prndlAI::LEUZ/pmdlAl::LEU2 mklA4::TRPl/mcklA4::TRPl rimlA/rimlA mds3-2::LEU2/MDS3

MATa/MATa imelAl3-HIS3/IMEl met444S/met444S rimlA/rimlA pmdlAI::LEU2/pmdlAl::LEU2 mds3-2::LEU2/mds3-2::LEUZ mcklA4::TRPl/MCKl

pmdlAl::LEU2/pmdlAl::LEU2 mds3-2::LEU2/mds3-2::LEU2 mklA4::TRPl/mcklA4::TRPl rimlA/rimlA

pmdlAl::LEU2/PMDl mds3A3::URA3/MDS3 mcklA4::TRPl/ MCKl

pmdlAl::LEUZ/PMDl mcklA4::TRPl/MCKl mds3A3::URA3/ mds3A3::URA3

pmdlAl::LEU2/pmdlAl::LEU2 mds3A3::URA3/MDS3 mcklA4::TRPl/MCKl

pmdlAl::LEU2/pmdlAl::LEUZ mds3Ajr::URA3/mds3A3::URA3 mcklA4::TRPl/MCKl

MATa/MATa ar@2032S/ARG6 met444S/MET4 mcklA4::TRPl/ MCKl imelAl3-HIS3/imelAl3-HIS3 pmdlAl::LEU2/ pmdlAl::LEU2 mds3A3::URA3/mds3A3::URA3

MATa/MATa a@2032S/ARG6 met444S/MET4 mklA4::TRPl/ MCKl imelAl3-HIS3/imelAl3-HIS3 pmdlAl::LEU2/PMDl mds3A3::URA3/MDS3

MATa/MATa ar&Z032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl

MATa/MATa ar@2032S/ARG6 met4-44S/MET4 imelA13-HIS3/IMEl

MATa/MATa a@2032S/ARG6 met444S/MET4 imelAl3-HIS3/IMEl

MATa/MATa arg6-2032S/ARG6 met4-44S/MET4 imelAl3-HIS3/IMEl

MATa/MATa arg62032S/ARG6 met444S/MET4 imelAl3-HIS3/IMEl

This study

This study

This study

This study

This study

This study

This study

This study

This study

All yeast strains were derived from the SK1 background and harbor the following markers in addition to ~ ~~

those listed above; ura3SK1, h2-hisG, lys2SK1, ho::LYS2, tql-hisG, and his3.

PMDl locus via colony hybridization in E. coli (NEIGEBORN and MITCHELL 1991). We were unable to obtain a library clone carrying the PMDl locus in its entirety, therefore we used PCR with the following primers to obtain the carboxy terminal half of PMDl: 5’GCGTCAAGhlGGTCTTCTCA’IT- 3’ and 5’-TGCGCATGTAAC’ITCG’ITCA-3’. The resulting PCR product was cut with PVuII and Sac1 and cloned into pML28 generating pML29, which contains the PMDl locus reconstructed in its entirety. A 7.6kb ChI-EcoRI fragment from pML29 containing PMDI was cloned into pRS426 to create pML30. A 4.5-kb BgnI fragment within the PMDl coding sequence was deleted from pML3O and replaced by the LEU2 selectable marker to create the pmdlAl::LEU2 allele in pML31. Digestion of pML31 with SmaI and ApaI released a pmdlA1::LEUkontaining fragment that was used to create the chromosomal pmdlAl::LEU2 allele via the standard one- step gene disruption procedure. The integrity of this disrup tion was confirmed by Southern blot analysis (data not shown).

DNA sequencing, PCR reactions, and oligonucleotide synthesis: Both single- and double-strand sequencing techniques were used. To sequence MDS3-1, Exonuclease 111-generated nested deletions were made from one end of pMLlO using the Erase-a-Base kit as recommended by the manufacturer (Promega). The deletion clones were then sequenced by the dideoxy method using the Sequenase Kit (Amersham) and the universal primer provided by the manufacturer. In addition, single-strand pMLlO was sequenced using appropriate synthetic primers. All other clones containing PCRderived inserts were also sequenced to confirm the integrity of the

inserts. Computer analysis of the sequences was performed using the University of Wisconsin Genetics Computer Group nucleic acids sequence analysis programs (DEVEREUX et al. 1984; GENETICS COMPUTER GROUP 1991) and the Saccharo- myces Genome Database (CHERRY et al. 1996). The following PCR primers were used to obtain the 0.46kb DNA fragment derived from the genomic ACTl locus: ACT974 5’“rTCGA ACAAGAAATGGS’ and ACT1924: B’-AGMCACTTGTG GTG3’.

Northern blots and probes: Cells were cultured overnight to early log phase inYEPAc. For sporulation, cells were shifted from YEPAc when they were in the early log phase to an equal volume of Spo medium; samples were harvested at various time points for RNA isolation (SMITH and MITCHELL 1989). Standard methods were used for Northern blot preparation (SMITH and MITCHELL 1989). The following “P-labeled probes were made by Random-Prime Labeling (Boehringer Mannheim Biochemicals, Indianapolis, IN): a 0.6kb HindIII- EcoRI fragment of IMEl (SMITH and MITCHELL 1989), a 0.4 kb BamHI-EcoRI fragment of IME2 (SMITH and MITCHELL 1989), a 1.0-kb BamHI-Sac1 fragment of HOP1 (HOLLINGS WORTH et al. 1990), a 0.46kb fragment of ACTl corresponding to bases 974-1434 of the coding region (CHERRY et al. 1996), and pC4 used as a loading control (LAW and SEAGALL 1988).

Hybridization was visualized with a Molecular Dynamics Phosphorimager Model 425E using the IPLabGel software provided by the manufacturer. With the exception of bright- ness adjustment no additional manipulations of the images were performed. Images were imported into QuarkExpress for labeling and then printed on a Kodak ColorEase PS


A mcklA S RX HXh RCBg NXh Nh BgR N B BXhHBgXhRBg H Nh SUppreSSiOn

Chrom I I I I I \ \ I / / I I I I I I I / I 1

VI1 ///ps3y////////////+j I I I k b I e

pMCH8 / / / / / I / / / / / / / fl-b + pML3 v////////- pML2 pMLl +

- - pMLll - pMLSI -/////////////- + pMLlO pML5

+/- - pML43 -

FIGURE 1,"Delineation of the mcklA suppressor locus (A4DS3). LNY266 was transformed with the indicated plasmids. The ability of a given plasmid to suppress mcklA was determined by scoring P,,v,;I-HIS3 expression by monitoring growth on SC-His- Ura medium. +, growth in 2 days; +/-, growth in 3 days; -, no growth. B, BnmHI; Bg, BglII; C, CluI; R, EcoRI; H, HindIII; N, NcoI; Nh, NheI; S, SalI; Xb, XbnI; X, XhoI. (A) The open bar is a restriction map of the genomic region surrounding MDSZ the il.IDS3 predicted ORF is indicated by the hatched box. The lower, thick horizontal bars indicate the pMCH8-derived yeast DNA insert carried on the indicated plasmid (hatched areas indicate the il.1DS3 coding regions predicted to be expressed); thin horizontal bars denote that the pMCH8-derived yeast DNA insert carried with it flanking vector sequences derived from the E p 2 4 tetracycline resistance locus (containing fortuitous promoter activity). The arrowhead on pMCHS indicates that the clone contains additional genomic sequences beyond those indicated. (B) Truncated forms of A4DS?were cloned behind the constitutive ADCl promoter (PADCI). The hatched boxes indicate the extent of m S 3 coding sequences present; Mds3p in-frame initiator ATGs are shown. The transcript arising from pML49 is derived from the noncoding strand and probably does not encode a meaningful translation product.

Printer. ZlllE2 quantitation was determined using the IPLab- Gel software; the pC4 signal served as a normalization control.

RESULTS

Identification of MDS3-1 as a dosage-dependent suppressor of mckl: To identify genetic loci functioning downstream of or parallel to Mcklp in the regulation of sporulation we exploited the role of MCKl in the activation of IMEl expression by using the reporter gene PTMEI-HIS3 (NEIGEBORN and MITCHELL 1991). The PIM;,-HIS3 allele consists of the HIS? coding sequence situated downstream of the IMEl promoter and mRNA start site. PIM;I-HIS3 strains deficient at the chromosomal his? locus are histidine prototrophs only under conditions in which the IMEl promoter is active. Thus, wild-type strains are phenotypically His' while rncklA mutants are His- because the rnckl deficiency inhibits even basal levels of transcription driven by the IMEl promoter (NEIGEBORN and MITCHELL 1991).

We screened a yeast genomic multicopy library for sequences capable of restoring histidine prototrophy to

the rncklA PrMF;,-HIS3 strain LNY266 (NEIGEBORN and MITCHEL~L 1991). We reasoned that increased expression of a substrate or target of Mcklp might be able to restore P,,,-HIS3 expression in an rncklA mutant; likewise, overexpression of positive regulators acting in MCKl-parallel pathways might also suppress an mcklA defect. Among the suppressor clones obtained in this screen was pMCH8, which contains the 8.5-kb yeast genomic insert shown in Figure 1A.

The rnckl-suppressor activity residing on pMCH8 was localized by a combination of subcloning, deletion, and insertion analyses. Plasmid constructs (always containing the high-copy yeast 2p origin of replication) derived from pMCH8 were reintroduced into the rncklA PTM,-HIS? strain and assayed for rncklA suppression activity as defined by restored histidine prototrophy. The results of these assays, shown in Figure lA, suggested that the suppressor activity resides on a 3.6- kb NcoI fragment at the extreme end of the clone adjacent to the plasmid tetracycline resistance locus (defined by pML10). Although this fragment does not show full suppressor activity (as compared to pMCH8), this


result strongly suggests that it contains at least part of the locus.

Sequence analysis of the 3.6-kb NcoI fragment revealed an apparent open reading frame (ORF) of 295 amino acids (see Figure 3) immediately upstream of GCNl on chromosome VI1 (MARTON et al. 1993). Com- parison of this region with that derived by the Saccharo- myces Genome Project revealed that this short reading frame actually represents an amino-terminal truncated portion of the larger ORF YGL197w (CHERRY et al. 1996). In other words, pMCH8 contains only the carboxy-terminal portion of this locus, which we have named MDS3 (Eckl dosage Suppressor).

Surprisingly, the fragment of the gene retained on pMCH8 confers an mckl suppression phenotype despite the absence of -1 kb of coding sequence, the initiator methionine, and a promoter. Two possibilities can explain this result: the activity responsible for mckl suppression is not due to expression of a gene product, but rather to increased dosage of the DNA itself (titration of a regulatory factor, for example) or a truncated version of the gene product is fortuitously expressed under the influence of a cryptic promoter, probably located in adjacent vector sequences. We favor the latter model for several reasons. The region conferring activity contains only protein coding sequence and it is unlikely (albeit, not unprecedented) that DNA control elements are present. More compelling, however, is the observation that a simple insertion at the unique BglrI site within the coding region is sufficient to eliminate suppressor activity even though large fragments spanning this site do not possess activity (Figure lA, compare pMLl and pML3 to pMLl1 and pML43). Finally, “flipping” the relative orientation of the truncated coding region abol- ishes suppressor activity (see Figure lB, compare PML48 and pML49). This is the expected result if a cryptic promoter were driving expression of the gene fragment. Indeed, the plasmid region adjacent to the yeast DNA insert contains the tetracycline resistance locus, which has been previously reported to contain nonspecific fortuitous promoter activity (STRUHL and DAVIS 1981).

Figure 1A shows the restriction map of the full-length MLX3 locus and indicates the “coding” region expected to be expressed in pMCH8derived clones (as- suming that translation of the fortuitous transcript initiates at the first AUG, see below); we denote the allele present on pMCH8 as MDS3-1. To confirm the model that expression of m S 3 - 1 leads to mckl suppression and to determine whether this phenomenon is related to the function of the wild-type allele, we retrieved the full-length locus from a yeast genomic library by colony hybridization using a probe derived from pMCH8 (see M.4TERJAL.S AND METHODS). various constructs, using either the native MDS3 promoter (pML43) or the constitutive ADCl promoter (Figure lB) , were tested for ability to confer mckl suppression. Figure 1B summarizes

TABLE 2

enrpl.eseion suppresses the 4 1 A sporulation defect

Sporulation efficiency

Clone Clone Clone Clone Plasmid 1 2 3 4 Average

Vector 1.6 22 22 22 16.9 YEpMCKl 57 68 48 12 46.3 YEpMDS3-1 48 65 19 50 45.5

Strain LNY747 was transformed with YEp24 (vector), pAM301 (YEpMCKl), or pMCH8 (YEpMDS3-1) and four independent transformants of each (clones 1-4) were trans- ferred to sporulation medium and ascus formation was quantitated after 48 hr.

our findings. Neither the full-length gene under the control of its own promoter (Figure lA, pML43) or the constitutive ADCl promoter (Figure IB, pML46) was capable of restoring Pzml-HIS3 expression to m k l A mutants. In contrast, a construct containing the same MDS3 coding information as that provided by MDS3-1 under the control of the ADCl promoter (pML48) is an efficient suppressor of mcklA, whereas the reverse orientation of this construct is not (pML49). Th’ IS experiment proves that MDS3-1 mediated suppression activity is, indeed, a consequence of expression of a truncated form of the MDS3 gene product. Furthermore, mckl suppression represents a novel, dominant phene type, because expression of the full-length gene does not behave in the same manner as MDs3-1 and MDS3-1 confers its phenotype in an MDS3 genetic background.

MDS3-1 expression suppresses the quautitative sporulation defect associated with ntcR1A: To verify the biological significance of the suppression activity conferred by MDS3-1, we tested the ability of this allele to suppress the sporulation defect conferred by mcklA. A diploid strain homozygous for the mcklA4::TRPl deficiency was transformed with plasmids harboring either m S 3 - 1 , MCKl, or vector alone and subjected to sporulation conditions followed by microscopic quantitation of sporulation efficiency. The results shown in Table 2 demonstrate that MDS3-1 restores sporulation levels to those comparable to that observed in the presence of MCKl (-46% as compared to 17% in the mcklA mutant carrying vector alone). It should be noted that the absolute sporulation level achieved in these strains is somewhat lower than those observed in strains carrying the genuine chromosomal MCKl allele. This is due to the inability to maintain plasmid selection under sporulation (nonvegetative) conditions. The result is an accumulation of a subpopulation of cells that have lost the plasmid and, therefore, lose complementation (or sup pression) activity. Nevertheless, this experiment con- firms that MDS3-1 suppression is not an artifact of the Pzm1-HZS3 reporter gene system and does indeed reflect a relevant biological activity.

1358 M. Li Renni and L. Neigeborn

Relevant GenotvDe PlMEI-HIS3 Exuression Wild-type

mckl

mds3

mds3 mckl

+++

+++ ++

SDorulation 75 %

15 %

75 %

40 %

FIGURE 2.-mds3::IJW2 suppresses the mcklA sporulation defecn. Strains homozygous for the indicated mutations (wild tvpe, LNY746; mckl, LNY747; md.d, LNY748; mckl mds3, LW749) were assayed for their ability to express Pr,\ler-HI.Y3 and for their ability to sporulate. Pr,\II_,-Hl.S3 expression is shown in the photographic insert after 3 days incubation on SCUra medium. +++, growth in 1 or 2 days; ++, growth in 3 days; -, no growth. Sporulation data is an average of three independent trials for the each genotype; SD < 13%.

MDS3 is a negative regulator of sporulation: To un- derstand the relationship between the roles played by the wild-type MIX3 and the MLX3-1 gene products, we created the chromosomal mds3”2::IJ<U2 allele. This allele represents an insertion of the IEU2 selectable marker into the second BgAI site within the MDS3 coding sequence. Although this did not necessarily represent a null mutation, we chose this site for disruption because we had previously found it to be required for MDS3-I suppression activity (see Figure lA, pML11). Therefore, this mutation represent5 a perturbation of the activity defined by the MDS3-1 allele. Furthermore, subsequent experiments with the mds3A3::URA3 null allele (see MATERIALS AND METHODS) indicated that mds3”2::IXU2 does indeed confer the null phenotype. Following replacement of the chromosomal MDS3locus with m(ts3-2::IEU2, standard strain construction methods were used to generate the md~3-2::1fW2 strains listed in Table 1. Both haploid and diploid mds3 mutants are viable and display no prominent growth defects under vegetative conditions.

mds3”2::IEU2 strains were assayed for both Pl,,l,.~l-Hl.T3 expression and sporulation efficiency. If Mds3p is a positive regulator of IMEI expression and sporulation (which dominant suppression of mckl suggests), a loss of function allele should result in decreased Pl,~ll~l-Hl.T3 expression and sporulation efficiency. However, as the data provided in Figure 2 indicate, this is not the case. The results in Figure 2 demonstrate that the mds3- 2::IJW2 allele confers no observable reduction in the ability of Pl.,,l~l-HLS3 strains to grow in the absence of histidine. Furthermore, quantitation of sporulation efficiency of mds3-2::IdXJ2 homozygous diploids also revealed no deficiency (Figure 2). Thus, the mds3”2::LEU2 mutation does not block entry into the sporulation program. There are several possible explanations to ac- count for this observation: mds3-2::LEU2 is not a null mutation; the role played by MDs3 is only significant

in the absence of MCKI, or 1bfD.73 function is redundant. The possibility that rnds3-2::IXU2 is not a null mutation was ruled out by the fact that a deletion of MILT3 displays the same phenotype as mds3”2::IJZJ2 (data not shown).

To address the possibility that the phenotype conferred by mds3-2::141:‘U2 is masked in the presence of a functional MCKI allele (in this scenario, MDS3 would be a component of one of the Mcklpparallel pathways), we analyzed the behavior of mcklA mds3-2::1.1N2 double mutant strains (Figure 2, line 4). If the M I X 3 dependent pathway positively regulates IMI.‘l expression and sporulation, we expect the double mutant to be even more defective in sporulation ability than the mcklA single mutant. However, the result is exactly the opposite: rnrl.s3-2::IJW2 is a recessive supp-pssor of the mck1A defect. This observation is manifested both by restored l’l,~,l.~l-HlS3 expression and nearly threefold increased sporulation efficiency (Figure 2). Thus, bo!h the dominant MDS3-I and recessive rnd~3-2::IJ,‘U2 alleles confer the same phenotype: suppression of mcklA. Since the suppression phenotype conferred by m.dy3- 2::IJW2 is recessive (data not shown), this must represent the loss of function phenotype. Therefore, the wild-type role of 1 W S 3 must be negative regulation of IMEI expression and sporulation, and, MDS3-I represents a dominant-negative allele.

m&3-2::LEU2 leads to increased expression of ZME2 To expand the biological relevance of MdsSp function, we analyzed the expression of the early SSG IME2, whose expression is completely dependent on Imelp function (SMITH and MITCHELL 1989; YosHlnA PI nl. 1990). We followed IME2 expression using the previously described IML2-5-lncZ::URA3 reporter gene (MITCHEI.I. P/ nl. 1990; SMITH P/ 01. 1990; SV and MITCH- ELL 1993b) and assayed 0-galactosidase expression quantitatively (Table 3). Our results reveal that mds3- 2::IJXJ2 mcklA double mutant strains display significantly higher levels of sporulation-specific IME2-lncZex- pression than that observed in mck1A strains; this result is in agreement with the l’l,,ll~l-HlS3 and sporulation data. In addition, this assay uncovered a modest, but consistently reprodltcible, increase in IME2”lncZexpres- sion in mds3-2::I.EU2 mutants as compared to wild type (Table 3, compare md.~3 and wild type). All these data support the model that MdsSp is a negative regulator of Imelpdependent SSG expression.

Suppression by mds3 is not restricted to Mcklpde- pendent pathways: The fact that increased IME2 expression is observed in md.s3-2::IJ<U2 mutants even in the presence of functional MCKI (see Table 3) indicates that Mds3p probably functions independently of the Mcklp pathway and suppression of mcklA is the cumulative result of release from this parallel negative regulation (we note that this is not observed when monitoring IME2 steady-state transcript accumulation directly, see below, which most likely reflects the stablilty

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 p P m d l p

M d s 3 o

1 1

7 7 8 1

1 6 1 1 3 3

2 4 1 2 1 3

3 0 8 2 9 3

3 5 4 3 7 3

4 3 3 4 5 3

4 9 1 5 3 3

5 6 8 6 1 3

6 4 8 6 8 8

7 2 5 7 6 0

7 7 8 8 3 0

8 4 4 9 1 0

9 2 4 9 8 6

1 0 6 6 9 6 6

1 0 4 3 1 1 4 6

1 1 2 3 1 2 2 6

1 2 0 1 1 3 0 6

1 2 7 5 1 3 8 5

1 3 4 8 1 4 6 4

1 4 2 6 1 5 4 1

1 6 2 1

P m d l p 1 7 0 1

FIGURE 3.-Comparison of Mds3p and Pmdlp predicted amino acid sequences. Identical residues are highlighted in black boxes and conserved residues are highlighted in gray boxes. *, the initiator methionine (Mds3p amino acid 517) of the peptide corresponding to the m S 3 - I allele.

of the lacZ gene product). Thus, loss of Mds3pmedi- ated repression results in increased ZMEl, ZME2, and SSG expression. We are unable to see this phenomenon

TABLE 3

mnk3-Z::LEUZ suppresses the m c k l A defect in IMEZ-lac2 expression

Relevant genotype IM2-lacZ expression

Wild type mck 1 mds3 mckl m k 3

90

150 60

6.1

Strains of the indicated genotype (wild type, LNY765; mckl, LNY766 m k 3 , LNY76f; and mckl md.73, LNY768) were assayed for sporulation-specific ,&galactosidase expression as described in MATERIALS AND METHODS. Values are expressed in Miller Units and are the average of at least three independent assays; SDs were < 15%.

when monitoring actual sporulation efficiency because our wild-type strains sporulate with high efficiency. Thus, it is only manifest in strains compromised for sporulation induction, such as mcklA mutants. Accord- ingly, i t is possible that mds3-2::ZXU2can suppress other mutations affecting ZMEI expression that define Mcklpindependent pathways.

RIMI , 8, 9, and 13 (yegulator of m E 2 ) were isolated in a genetic screen aimed at identifying positive regulators of ZME2 expression ( SV and MITCHELL 199Sb). Like m c k l A , mutations at these loci also confer partial defects in sporulation efficiency as a consequence of reduced ZMEI and SSG expression (SU and MITCHELL. 199%). Any pairwise double mutant combination between these rim mutations results in a sporulation defect indistinguishable from that of either parent (SV and MITCHELL. 199%). Thus, all four of the gene products are thought to act in the same pathway. In contrast, the mcklA rimlA double mutant result$ in significantly


A Wild Type mds3A mds3 mckl mcklA mds3 pmdl mckl

Hours in Spo

IME2 - I M E l +

HOPI +

-w

ACT1 +

Control+

B

Relative [ME2 60 Expression 5 0

E -. .o" E

lower sporulation levels than that attributable to either mutation alone (Su and MITCHELL 1993b). Therefore, Mcklp and the Rimlp group are thought to function in separate pathways that share functional redundance (SU and MITCHELL 1993b). A similar situation was revealed for the MDS4 locus (defining a third independent pathway), which was identified in the same screen as MDS3 (L. NEIGERORN, unpublished results).

To determine whether mds3-2::LEU2 can also suppress the I M I expression defects conferred by mds4A and r i m l a , we constructed the appropriate double and triple mutant strains. Table 4 summarizes our findings. As expected, the r i m l A and m d ~ 4 A mutations confer reduced sporulation efficiencies: 30% as compared to

FIGL-RIC 4.-MD.Y3 and I'M111 negatively control early meiotic gene expression. (A) Homozygous diploid strains of the indicated genotype (MT, LNY746; m h 3 , L.NY748; m 1 . d mrltl, LNY749; mrltl, L.NY747; rnh3 p m d l mrkl . LNY891) were grown to early log phase i n WAC medium (time 0) and then shifted to sporulation medium. At the indicated times, samples were re- moved and total RNA was prepared, fraction- ated, and subjected to Northern analysis as described in MATICRIAIS ASD METHOIX A single blot was probed for both IMEI and IME2 simultaneously and then sequentially stripped and reprobed for HOPI, ACTI, and the loading control probe pC4. (R) Quantitation of the data shown in A for IME2.

75% for the wild type. In contrast, both the m,d.s3 riml and mds3 mds4 double mutants demonstrated better sporulation efficiency: 60% and 75%, respectively. In- deed, the md.~3-2::IXU2 mutation could even relieve, to a certain extent, the requirement for both Mcklp and any one of these additional positive regulators ( m d ~ 3 mckl riml and mds3 mckl mds4 triple mutants sporulate much more efficiently than the mckl riml and mckl mds4 double mutants). These results indicate that m d ~ 3 - 2::ZEU2 bypasses the requirement for several independent positive regulators of I M H and sporulation, suggesting that MdsSp functions downstream or independently of all three of these pathways.

MDS3 and its paralog PMDl function synergistically


TABLE 4

nU"2::LEU2 suppression is not specific to the MCKZ pathway

Percent sporulation

Relevant genotype m s 3 mds3

Wild type 75 73 mckl 15 39 riml 30 60 mds4 30 75 mckl riml 0.5 30 mckl mds4 15 40

Strains of the indicated genotype (wild type, LNY746; mckl, LNY747; d 3 , LNY748; riml, LNY808; mds4, LNY339; mckl riml, LNY809; mckl mds4, LNY342; mckl mds3, LNY749; riml mds3, LNY810; mds4 mds3, LNY764; mckl riml mds3, LNY807; and mckl mds4 m h 3 , LNY763) were assayed for sporulation efficiency after 48 hr. Values are the average of at least three independent quantitations; SD were <15%.

to repress SSG expression: A search of the Saccharo- myces Genome Project database revealed that Mds3p shares a considerable degree of homology to the predicted product of OW YER132c (CHERRY et al. 1996), an uncharacterized locus located on chromosome V adjacent to GLC7 (FENG et al. 1991). We named this locus PMDl (Earalog of mS3). Figure 3 shows a comparison of the predicted amino acid sequences of MDS3 and PMDl; they share 47% identity and 63% similarity over their entire lengths. Neither protein shows significant similarities to other proteins in the GenBank or SwissProt databases nor are any significant functionally identified motifs present.

Given the high degree of similarity shared by Mds3p and Pmdlp, it seemed reasonable that PMDl might also play a role in controlling IMEl expression. To gain a better understanding of how PMDl functions, we created the chromosomal pmdlAl::LEU2 mutation. As w a s the case for mds3 mutants, pmdl mutants are viable, express PIMEI-HIS3 normally, and sporulate as efficiently as wild type (Table 5). But, unlike mds3, @dl was unable to suppress the mckl or riml sporulation defects or restore P,MEI-HIS~ expression ( r i m l A data not shown). However, mutants containing both a pmdl and mds3 allele (pmdl mds3 mckl and pmdl mds3 riml triple mutants, for example) displayed even higher sporulation levels than that of their PMDl counterparts, indicating that Pmdlp does play a role in sporulation efficiency when observed under the appropriate conditions (compare Table 5, line 6 with line 8 and Table 4, line 5 with Table 5, line 9). Indeed, sporulation efficiency in the pmdl mds3 mckl riml quadruple mutant strain is restored to a level as high as that of the wild type (75% sporulation, as compared to only 0.5% in the mckl riml double mutant and 30% in the mds3 mckl riml triple mutant). Similar results were also obtained for the com- binatorial effect of ma33 and pmdl on mds4 and mds4

TABLE 5

MDs3 and P m l function synergistically to regdate sporulation efficiency

PImI-HIS3 Percent Relevant genotype expression sporulation

Wild type +++ 75 mckl - 15 mds3 +++ 75 pmdl +++ 75 mds3 pmdl +++ 86 mckl mds3 ++ 40 mckl pmdl - 20 mckl mds3pmdl +++ 85 riml mds3 pmdl ND 75 riml mckl mds3pndl ND 75

Homozygous diploid strains of the indicated genotype (wild type, LNY746; mckl, LNY747; mds3, LNY748; p n d l , LNY899; mds3pmdl LNY890; mckl d 3 , LNY749; mckl W d l , LNY892 mckl m h 3 pmdl , LNY891; riml mds3 p d l , LNYl010; and riml mckl mds3 pmdl , LNYlOll) were scored for P1m1-HIS3 expression by monitoring growth on SGHis-Ura medium. Sporulation efficiency was quantitated after 48 hr as described in MATERIALS AND METHODS. Values are the average of at least three independent quantitations; SD <15%. +++, growth in 1 or 2 days; + +, growth in 3 days; -, no growth.

mckl mutants (data not shown). The observation that coincident mutation of both mds3 and pmdl leads to greater suppression of mckl, riml, and mds4 than that of either single mutation alone suggests that Mds3p and Pmdlp function synergistically to repress sporulation.

Although the effects of mds3 and pmdl on sporulation efficiency and the expression of the PjmI-HIS3 and IME2-lacZ reporter alleles suggests that these regulators exert their effects at the level of transcript accumulation, we confirmed this by monitoring SSG expression via Northern analysis of sporulating cultures. Diploids were grown vegetatively to late log phase, shifted to sporulation medium (Spo) at time 0, and RNA was prepared after varying terms of incubation (Figure 4A). In the wild-type strain, IMEl transcript levels were low in vegetative cells and induced and peaked following 4 hr in Spo. IME2 transcript was undetectable in vegetative cells and appeared after 2 hr in Spo, reaching a peak at 4 hr. In the mckl mutant the patterns of both IMEl and IME2 transcript levels showed delayed kinetics and decreased overall accumulation, as previously observed (NEIGEBORN and MITCHELL 1991). In contrast, the mckl m&3 and mckl mds3 pmdl strains expressed consider- ably increased levels of both IMEl and IME2. In addition, the IMEl and IME2 temporal expression patterns in these mutants is expanded: expression was observed at earlier time points and transcript accumulation per- sisted further into the sporulation program. Thus, the suppression of mckl by mds3 and pmdl is at the level of IMEl and IME2 transcript accumulation. Indeed, IME2 transcript is even present under vegetative conditions in the mckl mds3 pmdl triple mutant, a circumstance

M. Li Benni and L. Neigeborn 1362

A 5

4

0

s3 r3 0

2

1

0

""---"---........""""".."""

.."--.."-..""".__.___.____

""""-.""......._._ .""".""...

.."""""".." """"_""._."".""..

""."".....

0 5 10 15 20 25 30 35

Time in W A C (hours)

B Relevant % Sporulation Genome in Rich Medium

Wild Type 0.2 mds3 1.0 pmdl 0.2 mds3 pmdl 10.0

C

0 5 10 15 20 25 30 35 Time in YPA.c (hours)

FIGURE 5.-mds3 pmdl double mutants are defective in sensing nutrient availability. (A) Homozygous diploids of the indicated genotype were grown to saturation in WD, diluted into WAC to an ODm = 0.05 (time = 0 ) , and monitored for growth over a 3@hr period. Wild type, LNYl102 0; imel, LNYl1210; md.~3,LNYl104m;pmdl,LNYl105 +; mds3pmd1, LNYl106 0; mds3 pmdl imel, LNYl119 .. (B) Homozygous diploids of the indicated genotype were grown to saturation in WD, diluted into WAC to an ODm = 0.05 (time = 0), and incubated for 24 hr. Ascus formation was monitored mi- croscopically. Wild type, LNYl102; mds3, LNYl104; pmdl,

never observed in the wild type. Similar results were obtained for HOPI, another early SSG, whereas no al- teration was observed in actin gene expression, indicating that the effect is specific for SSGs. Mutation of only pmdl had little-or no effect on SSG expression in either MCKl or mckl strains (data not shown). Figure 4B illus- trates the significance of the unscheduled IME2 expression via quantitation of the IME2 data shown in Figure 4A. Here we see that mckl mutants harboring an mds3 mutation express increased levels of IME2 and this expression is accelerated relative to wild type. Further- more, the combination of mds? and pmdl results in the appearance of significant levels of vegetative IME2 expression, an occurrence never oberserved in the wild type (similar results were observed for the HOP1 transcripts; see Figure 4A). Indeed, this result is likely to represent an undersestimate of vegetative early SSG expression since mature asci were already visible at the time RNA samples were prepared, suggesting that early SSG expression might already have been down-regulated (see Figure 5B and discussion below). Thus, in the absence of the Mds3p and Pmdlp gene products, unscheduled expression of early SSGs occurs. There- fore, Mds3p and Pmdlp are bona $de negative regulators of early SSG expression and we conclude that the increase in sporulation observed in mckl mds3 pmdl mutants is a consequence of removal of this negative regulation.

mds3 pndl double mutants are defective in recognition of their nutritional status During the course of our analyses, we observed that mds3 pmdl double mutants displayed a marked growth defect, as compared to mds3, pmdl , or wild-type strains, under specific culture conditions. When grown in rich medium in the presence of glucose as the sole carbon source, the wild-type, mds3, pmdl , and mds3 pmdl strains formed colonies on solid medium at comparable rates (data not shown). However, in rich medium containing a nonfermentable carbon source (acetate), the mds3pmdl double mutants displayed a dramatic growth defect (Figure 5A). This defect was observed in both haploid and a / a diploid mds3 pmdl strains (data shown for a / a diploids only). Microscopic inspection of these cultures revealed that the a / a diploid mds3pmdl strains had not only arrested growth, but a proportion had initiated the sporulation program and formed mature asci (Figure 5B). Wild type and mds3 or pmdl single mutants, in contrast, grew normally and did not sporulate significantly. Thus, the premature and unscheduled vegetative meiotic gene

LNYl105; mds3pmd1, LNYl106. ( C ) Homozygous diploids of the indicated genotypes and harboring plasmids containing either the wild-type RAS2 gene (open symbols) or the RAS2- Vu119 allele (filled symbols) were grown to saturation under selective conditions and then diluted into WAC to a final ODeo0 = 0.05. Growth was monitored over a 33hr period. Plasmid loss was <30% in all strains. Wild type, LNY746 0 and 0; mds3pmd1, LNY890 0 and a.


expression observed in the d 3 pmdl double mutants corresponds to productive execution of the meiotic pathway.

If the cessation of growth in the presence of a nonfermentable carbon source is due to execution of the meiotic pathway in rote response to inappropriate IMEl expression, then we would expect the growth rate to return to normal in imeldeficient m h 3 pmdl strains. However, we found that although imel mds3 pmdl strains do not sporulate in WAC medium (because imd blocks all subsequent meiotic gene expression), they still grow very poorly (Figure 5A). Thus, growth arrest and unscheduled execution of the sporulation program are not simply due to the presence of Imelp. This is not surprising because it has been shown that the mere presence of Imelp is not sufficient for stimulation of meiosis; starvation is still required to activate Imelp and downstream processes (SHERMAN et aZ. 1993). Rather, our results indicate that the mds3 pmdl defect is u p stream of IMEl, at a point when cells make the decision whether to execute another round of the mitotic cell cycle or derepress the meiotic program. Thus, the phenotype of m h 3 pmdl strains suggests that they have become refractive to the presence of nutrients in their environment and, thus, trigger the sporulation pathway because they “believe” they are starved. Because the mds3 pmdl mutants still require a nonfermentable carbon source to initiate the meiotic, pathway, it seems likely that they are defective in the response to nitrogen availability, as opposed to glucose repression.

Components of the RAS-mediated CAMP signal trans- duction pathway have been implicated in the mechanism governing the decision to execute the mitotic vs. the meiotic cell cycles in response to nutrient limitation (MATSUMOTO et al. 1983; MALONE 1990; MATSUURA et aL 1990). Inhibition of this pathway (decreased cAh4P- dependent PKA due to rm2, qrl, or other mutations) results in a phenotype highly reminiscent of the mds3 pmdl phenotype: reduction of vegetative growth and concomitant execution of the sporulation program, even in the absence of starvation. Constitutive activation of the pathway (increased cAMPdependent kinase activity due to RAS2-Vall9, bcyl, or other mutations) results in uncontrolled vegetative growth and a reluctance to sporulate, even in the presence of starvation conditions (MATSUMOTO et al. 1982; BROEK et al. 1985; MA- LONE 1990). We determined whether stimulation of this pathway could suppress the mds3 pmdl growth and premature sporulation defects by introducing into mds3 pmdl strains a single-copy plasmid harboring the dominant, constitutively active RAS2-VaZl9mutation. Indeed, expression of RAS2-Val19 restored a normal vegetative growth pattern to the m h 3 pmdl mutants, whereas a plasmid harboring the wild-type RAS2 gene was unable to suppress the defects (Figure 5C). This result suggests that RAS2 functions downstream of or parallel to &fDs3

and PMDl in governing the decision to continue vegetative growth or undergo sporulation.

DISCUSSION

We have identified and characterized two yeast genes, MDS3 and PMDl, that function synergistically to negatively regulate sporulation. MDS3 was uncovered as a fortuitous dominant-negative amino-terminal trunca- tion allele, &fDS3-l, which confers suppression of the mcklA sporulation defects. Recessive deletion and disruption alleles of mds3 confer the same phenotype as MDs3-1 (suppression of mcklA and stimulation of SSG expression), indicating that the normal role of Mddp is as a negative regulator of the sporulation pathway. The existence of PMDl was revealed by comparison of the Mds3p sequence to that of the recently completed Saccharomyces genome. The two loci share significant sequence homology and some functional redundancy. Although mutation of pmdl alone does not confer noticeable defects, the mh3pmdl double mutant is consid- erably more defective in repression of the sporulation pathway than is the mh3single mutant. Thus, the activities of the two gene products function synergistically to maintain repression of the sporulation pathway.

What is the nature of the MDs3-1 allele and why does it lead to a dominant negative phenotype? One appeal- ing model is that Mds3p functions as one component of a complex, and the stoichiometric ratios of each member of this complex are critical for establishment or function. In this model, MDs3-1 might encode only “binding” or “interaction” domains, thus leading to titration of one or more members of the complex with an accompanying loss of productive complex activity. Titration phenomena have been observed in several processes, including histone assembly, the SPT6 (sup presser of I y ) effect on gene transcription, bacterio- phage morphogenesis and bacterial chemotaxis, and mitotic chromosome transmission (FLOOR 1970; DE- FRANCO and KOSHLAND 1981; MEEKS-WAGNER and HARTWELL 1986; MEEKS-WAGNER et al. 1986; CLARK-AD- AMs and WINSTON 1987; CLARK-ADAMS et al. 1988). In another variation on this theme, Mds3p need not interact with any other proteins; it could be a transcriptional regulator that binds DNA and leads to reduced gene expression. Thus, the mutant phenotype would be a result of the inability of the mutant protein to exert its regulatory effect while blocking access of the wild-type protein to its target sequence. In this respect, we note that neither Mds3p nor Pmdlp contain any previously identified DNA-binding motifs and experiments are currently in progress to determine the biochemical role of these gene products. Preliminary two-hybrid data suggest that both Mds3p and Pmdlp interact with two functionally related protein kinases (M. LI BENNI, unpublished data). Furthermore, these interact specifi- cally with the region of Mds3p present in the MDs3-


1 mutant protein, supporting the interactiodtitration model.

In our studies, the pmdlAl::LEU2 mutation did not confer a noticeable phenotype on its own. However, functional analysis of all of the genes on chromosome V via genetic footprinting revealed that Ty insertions into ORF YER132c, which corresponds to PMDl, result in a severe growth disadvantage but permit mating (SMITH et al. 1997). One simple explanation for this discrepancy is strain background variation: the studies presented here used the SKI genetic background exclu- sively (we also obtained similar results with the W303 background, data not shown), while the genetic fin- gerprint analysis used an S288C strain. Indeed, there were several similar examples of phenotypic variation uncovered in the latter study, including the behavior of mutations in M 8 , GDAl, and MMS21, which had been previously reported as nonessential but were found in the study to be at a substantial growth disadvantage (PRAKASH and PRAKASH 1977; ~ E I J O N et aL 1993; GRAHAM et a1 1995). It is interesting to postulate that an MDSMependent factor might be defective in the S288C background. If this were true, then a pmdl mutation would result in abolition of both A D S 3 and PMDl-dependent events and mimic our rnds3pmdl double mutant. Although this is intriguing, it does not explain the total growth disadvantage observed even in the presence of glucose as a carbon source. Alterna- tively, the Ty insertion mutations created in the genetic fingerprinting study could result in the formation of dominant interfering alleles that confer growth disad- vantages not associated with null mutations. In addition, the authors note that this locus has strong 5' site preferences for Tyl insertion, creating some potential for PCR artifact (SMITH et al. 1997).

We found that MDS3 and PMDl negatively regulate the sporulation process by repressing ZMEl and early SSG. Thus, loss of function mutations at these loci result in increased levels of ZMEl and early meiotic gene expression, which is most readily observable as suppression in strains that are compromised for induction of sporulation, such as mcklA mutants. The fact that mds3 and pmdl mutations suppress the defects conferred by several independently acting positive sporulation regulatory mutants (such as mckl, n'ml, and mds4) suggests that they function at a downstream point in the control pathway. This is consistent with our observation that mds3 and pmdl mutations have an impact on ZMEl and ZME2 transcript accumulation, which are the conver- gent targets of these positive regulatory pathways.

Several regulatory influences mediate induction of the sporulation pathway in yeast, including cell type, carbon source, and nutrient availability. The effects conferred by mds3 and pmdl are independent of cell type and are, thus, unlikely to be transmitting a cell- type signal. However, the carbon source- dependent growth defect, premature sporulation, and unsched-

uled ZMEl expression associated with the mds3 pmdl double mutants is reminiscent of perturbation of the RASmediated nutrient availability control pathway. U1- timately, this pathway regulates PKA, whose activity mediates the decision to commence a new mitotic cell cycle or adopt an alternative fate, such as sporulation (THEVELEIN 1992). Thus, failure to stimulate this pathway results in cessation of vegetative growth accompa- nied by induction of sporulation; indeed, this is what we observe in mds3pmdl double mutants. Furthermore, we found that uncontrolled stimulation of the pathway, achieved via introduction of the constitutively active RAS2-Vall9allele, restores vegetative growth and blocks unscheduled sporulation in our mutants. It is possible that Mds3p and Pmdlp are previously unidentified components of the RAS signaling pathway. If this is the case, they might be expected to function very early in the signal cascade, since they are suppressible by RAS2- Vall9, which is an upstream component. Alternatively, they could function downstream in the pathway as mod- erators of the signal. In this scenario, constitutive stimulation resulting from RAS2-Vall9 activity might simply inundate any downstream fine-tuning and result in suppression. Finally, MDS3 and PMDl might represent a Windependent pathway controlling the decision to undergo mitosis as opposed to meiosis. Suppression of the double mutant by RAS2-Val19 would then be due to compensation by overstimulation of the redundant RAS/cAMP pathway. This notion is not totally incredi- ble since CAMP levels alone cannot explain the sporulation behavior ofyeast cells in response to nutrient deprivation (OLEMPSKA-BEER and FREESE 1987; CAMERON et al. 1988). In addition, the activities of the RAS pathway appear to be more intimately involved in carbon source (glucose) sensing, leaving open the possibility that a primarily nitrogen sensing pathway may also exist (ROSE and BROACH 1991; GROSS et al. 1992). We are currently investigating the subcellular localization, genetic interactions, and biochemical activities associated with Mds3p and Pmdlp and expect that we will soon be in a position to address the specific roles of these gene products.

We thank DAVID NORRIS, DREW VERSHON and TERRI KINZY for many informative discussions and advice. We also thank members of the NEICEBORN lababoratory for help in the preparation and critical reading of the manuscript. This work was supported by the National Institutes of Health, General Medical Sciences grant ROl-GM-50497 (L.N.) and a Charles and Johanna Busch Fellowship (M.L.B.).

LITERATAURE CITED

ABEIJON, C., K. YANAGISAWA, E. C. MANDON, A. HAUSLER, IC MOREMEN et aZ., 1993 Guanosine diphosphatase is required for protein and sphingolipid glycosylationin the Golgi lumen of Saccham myces cermisiae. J. Cell. Biol. 124: 307-323.

BOWDISH, K. S., H. E. YUAN and A. P. MITCHELL, 1995 Positive control of yeast meiotic genes by the negative regulator UME6. Mol. Cell. Biol. 15: 2955-2961.

BROEK, D., N. SAMIY, 0. FASANO, A. FUJIYAMA, F. TAMAN01 et al., 1985


Differential activation of yeast adenylate cyclase by wild type and mutant RAS proteins. Cell 41: 763-769.

BURNS, N., B. GRIMWADE, P. B. ROSSMACDONALD, E. y. CHOI, K. FIN- BERG et al., 1994 Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces c m i s i a e . Genes Dev. 8: 1087-1105.

CAMERON, S., L. LEVIN, M. ZOLLER and M. WIGLER, 1988 CAMP- independent control of sporulation, glycogen metabolism, and heat shock resistance in S. meuisiae. Cell 5 3 555-566.

CARLSON, M., and D. BOTSTEIN, 1982 Two differentially regulated mRNh with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell 28: 145-154.

CHERRY, J. M., C. ADLER, C. BALL, S. DWIGHT, S. CHERVITZ et al., 1996 Saccharomyces Genome Database. http://genome-www. stanford.edu/Saccharomyces/, Stanford University.

CHEVRAY, P. M., and D. NATHANS, 1992 Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc. Natl. Acad. Sci. USA 8 9 5789- 5793.

CHRISTIANSON, T. W., R. S. SIKORSKI, M. D m , J. H. SHERO and P. HIETER, 1992 Multifunctional yeast highcopy-number shuttle vectors. Gene 110: 119-122.

CLANCY, M. J., B. BUTEN-MAGEE, D. J. STRAIGHT, A. L. KENNEDY, R. M. PARTRIDGE et al., 1983 Isolation of genes expressed preferen-

Natl. Acad. Sci. USA 80: 3000-3004. tially during sporulation in the yeast Smcharomyces cermisiae. Proc.

CLARK-ADAMS, C. D., and F. WINSTON, 1987 The SPT6 gene is essen- tial for growth and is required for delta-mediated transcription in Saccharomyces cerevisiae [published erratum appears in Mol Cell Biol 1987 May 7: 20351. Mol. Cell. Biol. 7: 679-686.

CLARK-ADAMS, C. D., D. NORRIS, M. A. OS=, J. S. FASSLER and F. WINSTON, 1988 Changes in histone gene dosage alter transcrip tion in yeast. Genes Dev. 2 150-159.

COE, J. G., L. E. MURRAY and I. W. DAWES, 1994 Identification of a sporulation-specific promoter regulating promoters that control gene expression in Saccharomyces cermisiae. Mol. Gen. Genet. 244:

COSTIGAN, C., and M. SNYDER, 1994 S L K l , a yeast homolog of MAP kinase activators, has a RAS/cAMP-independent role in nutrient sensing. Mol. Gen. Genet. 243 286-296.

COVITZ, P. A., and A. P. MITCHELL, 1993 Repression by the yeast meiotic inhibitor FME1. Genes Dev. 7: 1598-1608.

C O ~ T Z , P. A., I. HERSKOWITZ and A. P. MITCHELL, 1991 The yeast RMEl gene encodes a putative zinc finger protein that is directly repressed by al-alpha 2. Genes Dev. 5: 1982-1989.

et aL, 1990 Novel yeast protein kinase (YPKl gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein- tyrosine kinase activity [published erratum appears in Mol. Cell. Biol. 1991 Apr 11: 23331. Mol. Cell. Biol. 1 0 6244-6256.

DEFRANCO, A. L., and D. KOSHLAND, JR., 1981 Molecular cloning of chemotaxis genes and overproduction of gene products in the bacterial sensing system. J. Bacteriol. 147: 390-400.

DEVEREUX, J., P. HAEVERLI and 0. SMITHIES, 1984 A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 387-395.

DRANGINIS, A. M., 1990 Binding of yeast al and a2 as a heterodlmer to the operator DNA of a haploidspecific gene. Nature 347:

ENGEBRECHT, J. A., K. VOELKEL-MEIMAN and G. S. ROEDER, 1991 Meiosis-specific RNA splicing in yeast. Cell 6 6 1257-1268.

ESPOSITO, R. E., and S . KLAPHOLTZ, 1981 Meiosis and ascospore development, pp. 211-287 in The Molecular Bio@ of the Yeast Saccharomyces: Lve Cycle and Inhedance, edited by J. N. STRATHERN, E. W. JONES and J. R. BROACH. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

FENG, Z. H., S. E. WILSON, Z. Y. PENG, K. K. SCHLENDER, E. M. REI- MANN et al., 1991 The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. J. Biol. Chem. 266 23796-23801.

FLOOR, E., 1970 Interaction of morphogenetic genes of bacterio- phage T4. J. Mol. Biol. 47: 293-306.

FRIESEN, H., R. LUNZ, S. DOYLE and J. SE-, 1994 Mutation of the SPSl-encoded protein kinase of Saccharomyces cerevisiae leads to

661-672.

DAILEY, D., G. L. SCHIEVEN, M.Y. LIM, H. MARQUARDT, T. GILMORE

682-685.

defects in transcription and morphology during spore formation. Genes Dev. 8 2162-2175.

FRIESEN, H., S. R. HEPWORTH and J. SEGALL, 1997 An Ssn6Tupl- dependent negative regulatory element controls sporulation- specific expression of DIT1 and DIT2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 1% 123-134.

GENETICS COMPUTER GROUP, 1991 Program Manual fw the GCG Pack- age, Genetics Computer Group, Madison, WI.

G o u m , C., and A. D. JOHNSON, 1988 a1 protein alters the DNA binding specificity of a 2 repressor. Cell 54: 875-882.

GRAHAM, L. A., K. J. HILL and T. H. STEVENS, 1995 VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces meuisiaevacuolar H( +)- ATPase required for function and assembly of the enzyme complex. J. Biol. Chem. 270 15037-15044.

GROSS, E., D. GODLBERG and A. LEVITZKI, 1992 Phosphorylation of the S. cereuisiae Cdc25 in response to glucose results in its dissociation from Ras. Nature 360: 762-765.

HEPWORTH, S. R., L. K EBISUZAKI and J. SEGALL, 1995 A 15-base- pair element activates the SPSlgene midway through sporulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 15: 3934-3944.

HERSKOWITZ, I., 1988 Life cycle of the budding yeast Saccharomyces cermisiae. Microblol. Rev. 52: 536-553.

HERSKOWITZ, I . , 1995 MAP kinase pathways in yeast: for mating and more. Cell 80: 187-197.

HOLAWAY, B. L.,G. KAO, M. C. FINNandM. J. CLANm, 1987 Transcrip

210: 449-459. tional regulation of sporulation genes in yeast. Mol. Gen. Genet.

HOLLINGSWORTH, N. M., L. GOETSCH and B. BYERS, 1990 The HOP1 gene encodes a meiosis-specific component of yeast chromw somes. Cell 61: 73-84.

HONIGBERG, S. M., R. M. McCARRoLLand R. E. Es~osrro, 1993 Regu- latory mechanisms in meiosis. Curr. Opin. Cell Biol. 5: 219-225.

ITO, H., Y. FUKUDA, K. MURATA and A. KIMURA, 1983 Transforma- tion of intact yeast cells treated with alkali cations. J. Bacteriol.

JIANG, W., M. Y. LIM, H. J. YOON, J. THORNER, G. S. MARTIN et al., 1995 Overexpression of the yeast MCKl protein kinase suppresses con- ditional mutations in centromere-binding protein genes CBF2 and CBF5. Mol. Gen. Genet. 246: 360-366.

KANE, S., and R. ROTH, 1974 Carbohydrate metabolism during ascospore development in yeast. J. Bacteriol. 118: 8-14.

KASSIR, Y., and G. SIMCHEN, 1976 Regulation of mating and meiosis in yeast by the mating type locus. Genetics 8 2 187-206.

KASSIR, Y., D. GRANOT and G. SIMCHEN, 1988 I W l , a positive regulator gene of meiosis in S. cereuisiae. Cell 5 2 853-862.

KIHARA, K, M. NAKAMURA, R. AKADA and I. YAMASHITA, 1991 Positive and negative elements upstream of the meiosis-specific glucoa- mylase gene in Saccharomyces cereuisiae. Mol. Gen. Genet. 226:

K R I S W L., R. STRICH, R. S. WINTERS, J. P. HALL, M. J. MALLORY et al., 1994 SMKl, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cermisiae. Genes Dev. 8: 2151-2161.

LAW, D. T. S., and J. SEAGALL, 1988 The SPSlOO gene of Saccharo- myces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation. Mol. Cell. Biol. 8: 912- 922.

LI, W., and A. P. MITCHELL, 1997 Proteolytic activation of Rimlp, a positive regulator of yeast sporulation and invasive growth. Genetics 145: 63-73.

LIM, M.Y., D. DAILEY, G. S. MARTIN and J. THORNER, 1993 Yeast MCKl protein kinase autophosphorylates at tyrosine and serine but phosphorylates exogenous substrates at serine and threonine. J. Biol. Chem. 268 21155-21164.

MALONE, R E., 1990 Dual regulation of meiosis in yeast. Cell 61:

MARTON, M. J., D. CROUCH and A. G. HINNEBUSCH, 1993 GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2. Mol. Cell. Biol. 13 3541-3556.

mTSUMOT0, IC, I. UNO, y. OSHIMA and T. ISHIKAWA, 1982 Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMPdependent protein kinase. Pmc. Natl. Acad. Sci. USA 79: 2355-2359.

MATSUMOTO, IC, I. UNO and T. ISHIKAWA, 1983 Initiation of meiosis in yeast mutants defective in adenylate cyclase and CAMPdepen- dent protein kinase. Cell 3 2 417-423.

WTSUURA, A., M. TREININ, H. MITSUZAWA, Y. KASSIR, I. UNO et al.,

153: 163-168.

383-392.

375-378.

1990 The adenylate cyclase/protein kinase cascade regulates entry into meiosis in S. cereuisMethrought the gene Z M E I . EMBO

MEEKS-WAGNER, D., and L. H. HARTWELL, 1986 Normal stoichiome- try of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell 44: 43-52.

MEEKS-WAGNER, D., J. S. WOOD, B. G u m a n d L. H. HARTWELL, 1986 Isolation of two genes that affect mitotic chromosome transmission in S. cerevish. Cell 44: 53-63.

MENEES, T. M., P. B. ROSSMACDONALD and G. S. ROEDER, 1992 M 4 , a meiosis-specific yeast gene required for chromosome synapsis. Mol. Cell. Biol. 12: 1340-1351.

MITCHELL, A. P., 1988 Two switches govern entry into meiosis in yeast. Prog. Clin. Biol. Res. 267: 47-66.

MITCHELL, A. P., 1994 Control of meiotic gene expression in Sac- charomyces cermbiae. Microbiol. Rev. 58: 56-70.

MITCHELL, A. P., and I. HERSKOWITZ, 1986 Activation of meiosis and sporulation by repression of the RMEl product in yeast. Nature

MITCHELL, A. P., S. E. DRISCOLL and H. E. SMITH, 1990 Positive control of sporulationspecific genes by the IMEl and IME2 products in Sacchummyces c e r d i a e . Mol. Cell. Biol. 1 0 2104-2110.

NEIGEBORN, L., and A. P. MITCHELL, 1991 The yeast M a l gene encodes a protein kinase homolog that activates early meiotic gene expression. Genes Dev. 5: 533-548.

OGAWA, H., K JOHZUKA, T. NAKAGAWA, S. H. LEEM and A. H. HAG[- HARA, 1995 Functions of the yeast meiotic recombination genes, MREIl and MRE2. Adv. Biophys. 31: 67-76.

OLEMPSKA-BEER, Z., and E. FREESE, 1987 Initiation of meiosis and sporulation in Saccharomyces meuisiaedoes not require a decrease in cyclic AMP. Mol. Cell. Biol. 7: 2141-2147.

PWH, S., and L. PRAKASH, 1977 Increased spontaneous mitotic segregation in MMS-sensitive mutants of Sacchammyces cerekiae. Genetics 87: 229-236.

&NE, J. D., G. SPRAGUE, F., JR. and I. HERSKOWITZ, 1981 The nnel mutation of Sacchawmyces cerevisiae: map position and bypass of mating type locus control of sporulation. Mol. Cell. Biol. 1: 958- 960.

ROSE, M. D., and J. R BROACH, 1991 Cloning genes by complementation in yeast. Methods Enzymol. 194: 195-230.

ROTHSTEIN, R., 1983 One step gene disruption in yeast. Methods Enzymol. 101: 202-211.

SHAH, J. C., and M. J. CLANCY, 1992 ZMX4, a gene that mediates MATand nutritional control of meiosis in Saccharomyces cerevisiae. Mol. Cell. Biol. If: 1078-1086.

SHERMAN, A., M. SHEFER, S. SAGEE and Y. KASSIR, 1993 Post-transcriptional regulation of Z M E l determines initiation of meiosis in Saccharomyces cmis iae , Mol. Gen. Genet. 237: 375-384.

SHERMAN, F., G. R. FINK and J. B. HICKS, 1986 Labwatq Course Man-

J. 9 3225-3232.

319: 738-742.


ual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

SHERO, J. H., and P. HIETER, 1991 A suppressor of a centromere DNA mutation encodes a putative protein kinase (“1). Genes Dev. 5: 549-560.

SIKORSKI, R. S., and P. HIETER, 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cmeuisiaR Genetics 124: 19-27.

SMITH, H. E., and A. P. MITCHELL, 1989 A transcriptional cascade governs entry into meiosis in Saccharomyces cereuisiae. Mol. Cell. Biol. 9: 2142-2152.

SMITH, H. E., S. S. SU, L. NEIGEBORN, S. E. DRISCOLL and A. P. MITCH- ELL, 1990 Role of hWE1 expression in regulation of meiosis in Sacchamyces cereuisiue. Mol. Cell. Biol. 10: 6103-6113.

SMITH, v., K. N. CHOU, D. LASHKARI, D. BOTSTEIN and P. 0. BROWN, 1997 Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science 274 2069-2074.

STERN, M., R. JENSEN and I. HERSKOWITZ, 1984 Five S W genes are required for expression of the HO gene in yeast. J. Mol. Biol.

S ~ C H , R., M. R. SLATER and R. E . ESPOSITO, 1989 Identification of negative regulatory genes that govern the expression of early meiotic genes in yeast. Proc. Natl. Acad. Sci. USA 8 6 10018- 10022.

STRUHL, K, and R. W. DAVIS, 1981 Position effects in Saccharomyces cereuisiae. J. Mol. Biol. 15P: 569-575.

SU, S. Y., and A. P. MITCHELL, 1993a Molecular characterization of the yeast meiotic regulatory gene RIMl. Nucleic Acids Res. 21:

SU, S. Y., and A. P. MITCHELL, 1993b Identification of functionally related genes that stimulate early meiotic gene expression in yeast. Genetics 133 67-77.

SUROSKY, R. T., and R. E. ESPOSITO, 1992 Early meiotic transcripts are highly unstable in Saccharomyces. Mol. Cell. Biol. 12 3948- 3958.

TATCHELL, IC, L. ROBINSON and M. BREITENBACH, 1985 RAS2 of Saccharomyces cenwisiw is required for gluconeogenic growth and proper response to nutrient limitation. Proc. Natl. Acad. Sci. USA 84: 3785-3789.

THEVELEIN, J. M., 1992 The Madenylate cyclase pathway and cell cycle control in Sacchuwmyces cereuisioe. Antonie Leeuwenhoek

VERSHON, A. K., N. M. HOLLINGSWORTH and A. D. JOHNSON, 1992 Meiotic induction of the yeast HOP2 gene is controlled by positive and negative regulatory sites. Mol. Cell. Biol. 12: 3706-3714.

YOSHIDA, M., H. KAWAGUCHI, Y. SAKATA, K KOMINAMI, M. HIRANO et aL, 1990 Initiation of meiosis and sporulation in Sacchuwmyces

Genet. 221: 176-186. cereuisiae requires a novel protein kinase homologue. Mol. Gen.

98: 503-517.

3789-3797.

6 2 109-130.

Communicating editor: F. WINSTON

a New Class of Negartive Regulators Affecting Sporulation-Specific Gene Expression … · 2002. 7....

Documents

Transcript of a New Class of Negartive Regulators Affecting Sporulation-Specific Gene Expression … · 2002. 7....