Cell. Vol 52. 853-862, March 25, 1988, CopyrIght (c) 1988 by Cell Press

IME7, a Positive Regulator Gene of Meiosis in S. cerevisiae

Yona Kassir, David Granot, and Giora Simchen Department of Genetics The Hebrew University Jerusalem, 91904 Israel

Summary

/ME1 (Inducer of MEiosis) was cloned due to its high copy number effect: it enabled MAT insufficient strains to undergo meiosis. Disruption of /ME1 results in a recessive Spa- phenotype. Diploids homozygous for the two mutations imel-0, rmel-7 are also meiosis deficient. We conclude that /ME1 is a positive regula- tor of meiosis that normally is repressed by RME7. RME1 is repressed by a complex of MATal and MATa gene products. /ME7 is also regulated by the environ- ment: no transcripts could be detected in glucose growing cells, in contrast to acetate growing cells. Starvation for nitrogen further induced (6- to 8-fold) transcription of IME7, but, as expected, the induction was found only in MATaIMATu or rmel-llrmel-7 dip- loids. Furthermore, the /ME7 multicopy plasmids pro- moted sporulation in rich media.

Introduction

Diploid cells of the yeast Saccharomyces cerevisiae em- bark on meiosis and sporulation upon exposure to star- vation conditions. Diploidy and starvation are the two necessary conditions without which meiosis normally does not take place (Roman and Sands, 1953; Shilo et al., 1978). The diploidy of these cells is mediated through the mating-type gene system. The products of both MATa and MATa are required for meiosrs. Thus, MATa and MATu haploids, as well as diploids of the genotypes MATa/ MATa, MATuIMATu (Roman and Sands, 1953) matal/ MATu (Kassir and Simchen, 1976), MATa/mata2 (MacKay and Manney, 1974; Strathern et al., 1981), and matal/ mata (Kassir, 1982), are meiosis deficient. This require- ment IS relieved by the rmel mutation, and the above listed diplotds undergo meiosis if they are homozygous for the recessive rmel-7 allele (Kassir and Simchen, 1976). The simplest model for the interaction between MAT and RMEI is that the products of MATa and MATu2 together repress the RME7 gene. RME7 is thought to be a repressor of meiosis, or of meiosis-specific genes (Kassrr and Srm- then, 1976; Rune et al., 1981). A lo-fold reduction in the level of RMEl mRNA was indeed found in a MATa/MATu dip- loid, compared to an isogenic MATdMATu strain (Mitchell and Herskowitz, 1986) suggesting that at least part of the repression of RME7 is transcriptionally regulated.

Starvation seems to induce meiosis in S. cerevisiae via the adenylate cyclase and CAMP-dependent protern kinase system. Mutations in the cyclase gene, CYR7 (CDC3.5) (Matsumoto et al., 1983), and in two of its effecters, CDC2.5

(Broek et al., 1987; Robinson et al., 1987) and RAS2 (Toda et al., 1985), result in low levels of cellular CAMP, and con- sequently, meiosis and sporulatron take place In nrtrogen- rich medium (Shilo et al., 1978; Matsumoto et al., 1983; Tatchell et al., 1985). Mutations In the gene BCY7 (SRAI; Matsumoto et al., 1983), which codes for the regulatory unit of the CAMP-dependent protein kinase, suppress the mutations cyrl, c&25, and ras2 by making the protein ki- nase Independent of CAMP The recessive mutation bcyl (or sral), when homozygous, results in diploids being sporulationlmeiosis deficient. This implies (Matsumoto et al., 1983) that high actrvrty of thus protein kinase inhrbrts meiosis, and that its reduced actrvity mtght be an inter- mediate step In the regulation of meiosis by starvahon.

In this paper, we describe the cloning and initial charac- terization of a new gene that is a positive regulator of mero- sis; the gene was designated /ME7 for Inducer of MEiosis. IMEl overrides the steps controlled by MAT and RME7. The gene /ME7 is also regulated by starvation, thus it may be a key gene through which the regulatory pathways of both diploidy and starvatton reach the meiosis specific genes.

Results

Cloning and Subcloning A gene coding for a positive regulator may be identrfred and cloned by its high expression when present in the cell on a multicopy plasmid. Thus, In order to clone a positrve regulator of meiosis, a strain deficient in the mating-type system was constructed, from whrch meiosis-proficient derivatives could be selected genetically (strain YD164). The strain was transformed with a yeast DNA library In the high copy number plasmrd YEp24 (which carries the gene URAB, and the 2trm origin of replication); the library was constructed from DNA of a MATu strain (Carlson and Bot- stein, 1982). Ura’ transformants were replicated onto SPO plates (sporulation medrum) from which the colonies were replicated 4 days later onto plates containing both cycloheximide and canavanine Only colonies that un- derwent meiosis could grow on this medium due to hap- loidization and expression of the recessrve resistance markers.

A haploid yeast cell contains three copies of matrng- type information in three different loci on chromosome Ill. The information at MAT IS normally expressed, whereas the other two genes at HMR and HML are not expressed because of the dominant action of four genes, SIR7, SIR2, SIR3, and SIR4 (Herskowitz and Oshima, 1981). In order to avoid the Isolation of plasmids that cause sporulaticn by activating the silent a information that normally resides at HMR, strain YD164 was constructed to Include only (I informatron at both HMR and HML (Table 4). The genotype of strain YD164 was thus HMLo matal HMRoIHMLo MATo HMRo. ura3-521ura3-52, canl-77ICAN7, and its pheno- type was r,-mater Spo Ura

Upon transformation with the gene library, we obtained

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Table 1. Percent Sporulation of Dlplold Strains with and was independent of the information at HMR. (Strain High-Copy Number Plasmids YD164 is homozygous for HMRa.)

Plasmid

Stram Genotype Y Ep24 Y EpK26-7

419-G MA Ta/MA Ta 56.5 54.0 YD164 matal/MATn 0 3.0 419-Gi MATa/MATa 0 19.5 419.G2 MATalMATn 0 18.5

22 a-mater Spa+ (sporulation proficient) Ura+ transfor- mants (out of 2060 Ura+ transformants). Thus, the plas- mids isolated affected only sporulation and not the mating behavior of the cell. After continuous growth In YEPD medium, the Spa+ phenotype was lost together with the Ura+ phenotype. DNA was isolated from the Ura+ Spa+ colonies, and was used to transform E. coli HE707 to am- picillin resistance. The plasmids obtained were subjected to restriction analysis. In 21 of the plasmids, the restriction pattern was similar to that known for HMRa. We have no explanation for the overrepresentation of HMRa in the genomic DNA library. Other genes (Kassir et al., 1985) were recovered from this gene library with the expected frequency.

One transformant yielded a plasmid (YEpK26) with a new restriction pattern, and with no apparent similarity to the genes MATa or HMRa. Sporulation frequencies of vari- ous strains with plasmid YEpK26-7 (a subclone of YEpK26, see below) are given in Table 1. The plasmid en- abled meiosis and sporulation to occur in matal/MATa as well as in MATalMATa and MATa/MATa diploids. We con- cluded that the insert in YEpK26 relieved the requirement for meiosis of either MATa (the original selection) or MATa

YEpK26

YEpK26-S5

YEpK26-8

YEpK26-7

YEpK26-4

YEpK26-IO

YEpK26- 108

YEpK26-Ill

0.1 kb U

The insert in plasmid YEpK26 (9.2 kb) was character- ized by restriction enzyme mapping and subcloned into the multicopy vector YEp24. The different subclones were examined for their ability to suppress the meiosis defi- ciency of a matal/MATa diploid (strain YD164). The data are summarized in Figure 1. The sporulation-promoting activity was narrowed down to a 2.65 kb region (the insert in YEpK26-7). Furthermore, we inserted Xhol linkers at various sites in this region (see Experimental Procedures) and all of them (Figure 1) suppressed the meiotic defect of a MATa/MATa diploid (strain 419-Gl). Thus, the gene was narrowed further to 1.55 kb.

Southern analysis of genomic DNA digested with differ- ent restriction enzymes, and probed with either the 2.65 kb BamHI-Bglll fragment or the 0.55 kb Hindlll-EcoRI in- ternal fragment (Figure l), showed that the sequence we have cloned was present in the haploid genome as a sin- gle copy (data not shown).

Gene Disruption Generates a Recessive Spa- Allele Disruption of /ME7 was achieved in two ways, as described in Figure 2. The internal fragment integration (Shortle et al., 1982) was called ime7-0 and the disrupted fragment replacement (Rothstein, 1983) was called imel-7. The former disruption was verified by genomic Southern anal- ysis (data not shown), whereas the latter was cloned out of the genome (by gap repair; Orr-Weaver and Szostak, 1983) and its structure verified by restriction enzyme anal- ysis. Neither disruption had an effect on viability or growth characteristics of haploid or diploid strains. Diploids homozygous for one of the two disruptions were meiosis- deficient, but heterozygotes sporulated normally. We thus

‘\ ,,’ \ /’

\ 0.5 Lb

\ /’ H ,

‘G DCEE B /’ xx’

L 1 I 1r I CS G x

I

+ S

G + S

G I + 0

I

B x I I

G - E

cl E I I

Figure 1. RestrictIon Map and Subclones of the /ME7 Gene

YEpK26 is the origlnal /ME7 plasmid. It is drawn with the relevant restriction sites only. The lines below represent the subcloned frag- ments, and their ability to suppress the meiotic defeciency of matal/MATu diplolds. The top line shows a detailed restrictlon map for a re- stricted area Restriction sites are indicated by: B, BarnHI; C. Clal; D, HIndIll; E. EcoRI; G, Bglll, S. Sall; X. Xhol; and XL, Xhol linker insertion.

IME7, a PosItwe Regulator Gene of Meiosis 855

a

Transformation

and integrotion

G D CE B

b. E/C

BIG cut. I

G P E/C Trandorrnation

and replacement

G PC B

I

+ G P E/C B

Figure 2 Gene DIsruptIons of /ME7

(a) DIsruptIon by internal fragment: plasmid YlpK26-106 cut wth Clal was used to transform a MAJa/MAJa dlplold to URA3’. Homologous recombi- natlon resulted in dtsruption of the /ME7 gene. (imel- allele) (b) DIsruption by gene replacement. the JRP7 gene was mserted in the mlddle of /ME7 in plasmid YlpG71. The BamHI-Bglll fragment was Isolated. and was used to transform a dlplold strain to TRPl ‘. Upon homologous recomblnatlon. the wld-type /ME7 gene was replaced by the /me7:JRP7 allele, (fmel-7)

concluded that disruption of /ME7 resulted in a recessive mutation with a Spa- phenotype. The homozygotes for imel-0, the internal fragment disruption, produced an oc- casional ascus (<0.10/o) in sporulation medium, whereas no asci were observed in the homozygotes for the dis- rupted fragment replacement imel-7. We presume that the former may occasionally revert to the original normal gene by looping-out of the plasmid, in reverse fashion to the generation of the disruption (Figure 2a).

In order to determine the stage of meiosis at which the imeVime7 diploids arrest, we compared the two closely related strains, D317 (imel-7IIME7) and D320 (imel-7l imel-l), for their behavior in meiosis. The two strains were grown in liquid presporulation medium (PSP2) containing radioactive label (14C uracil) to a titer of 10’ cells/ml, washed in water, and resuspended in sporulation medium (SPM, without label). Three 0.5 ml samples of the culture were taken every 2 hr and 14C label in DNA was determined. At

the same times, cell samples were examined microscopi- cally for percentage of unbudded cells and were spread on -ADE and YEPD plates to determine recombination commitment and viability, respectively (Kassir and Sim- then, 1978). At 24 hr, strain D317 showed 39% asci, and at 48 hr 49%, but no asci were observed in strain D320. Table 2 contains data obtained from two samples: at time 0 and at 48 hr after the transfer to sporulation medium. It shows that cells that are homozygous for ime7-7 arrest early in meiosis, before premeiotic DNA synthesis, and before commitment to recombination. These arrested cells do not lose viability.

The Disruption ime7-0 Is Epistatic to rmel-7 The data presented thus far, namely the suppression by the multicopy /ME7 of the meiotic requirement for MATa and MATa and the recessive Spa- phenotype of the dis- ruption, suggest that /ME7 was indeed a positive regulator

Table 2. The Effect of /ME7 Disruption on Meiosis

Effect

Strain

D317 D320 (a/a imel-7NME7) (a/u ,mel-lhmel-7)

0 hr 48 hr Ohr 48 hr

CPM m DNA (5 x lo6 Cells) 19552 36767 14728 15837 % Budded Cells 59.5 90 .54.7 80 % Viability 100.0 115.8 100 0 121 1 Frequency of Ade’ Colonies (x lo-“) 1.1 83.9 5.6 72

Cell 856

RMEl+ IME l- meiosis

b.

;;I;>,,,, + RMEI --+ meiosis

Frgure 3. Alternative Models for Induction of Merosis

-1 represents negative regulation, > represents positive regu- lation.

of meiosis. The following two simple models can explain our results: /ME7 is an inducer of meiosis, and is normally repressed by RMEl (Figure 3a); IMEl is induced by a com- plex of MATal and MATa2, and its product is a repressor of RME7 (Figure 3b). In the first model, /ME7 on a high copy number plasmid titers out the RME7 repressor and thus leads to a meiosis proficient cell. Without the /ME7 gene product (e.g., when the gene is disrupted), meiosis is not induced, resulting in sporulation deficiency. In the second model (Figure 3b), /ME7 on a high copy number plasmid is sufficiently expressed even without being in- duced by MATal and MATa2, hence, RMEl can be re- pressed and meiosis derepressed. With this model, homozygous disruptions of /ME7 lead to constitutive ex- pression of RMEl and to a Spa- phenotype. In order to chose between the two alternative models, we con- structed double mutants homozygous for both rmel-7 and the disrupted imel-0. The first model (above and Figure 3) predicted that the double homozygotes would be Spa-. whereas the second model predicted they would be Spa+. The double homozygotes (see Experimental Pro- cedures for isolation of double mutants) turned out to be meiosis deficient (Spa-), therefore we chose the first model and concluded that /ME7 was an inducer of meiosis, and probably repressed by RMEl.

Mapping of /ME1 /ME7 was mapped to chromosome X by hybridization of a 3zP-labeled DNA fragment internal to the gene (the 0.55 kb Hindlll-EcoRI fragment in Figure 1) to a chromo- some blot obtained from a chromosome separation gel (Schwartz and Cantor, 1984) of strain F836 (data not shown).

Table 3 presents results from random spore analysis in which the ime7-7 disruption was mapped relative to three markers on the right arm of chromosome X. /ME7 was localized about 14 CM distal to ILV3.

Direction of Transcription of /ME1 RNA was isolated from a MATa/MATa diploid (strain 419- G) growing in acetate vegetative medium. The RNA was subjected to electrophoresis and blotted to a GeneScreen membrane. The blot was hybridized with a 32P-labeled DNA probe that consisted of the 0.55 kb. Hindlll-EcoRI

Table 3. Mapping of /ME7

Markers Number of Examined Recombinants

Total Number of Progeny CM

IMEl - CDC8 30 139 21.6 IMEl - CDCll 31 77 40.0 IMEl - ILV3 27 187 14.4

Strain D300, whrch is Imel-7:TRP7, was mated to the following strains: 355 for cdc8, 339 for cdcll, and 860 for i/v3 (see Table 4).

/ME7 internal fragment. The probe hybridized to a tran- script that was about 1.5 kb long. URA3 or HIS4 RNAs were used as size markers (following hybridization to ap- propriate szP-labeled probes), as well as the ribosomal RNAs on the gels (viewed under UV illumination).

In order to determine the direction of transcription of IME7, the same 0.55 kb fragment was cloned into the Gem3 transcription vector (Figure 4). Two 3zP-labeled riboprobes were obtained by in vitro transcription using ei- ther T7 polymerase or SP6 polymerase. These riboprobes were used seperately in Northern hybridizations, and a URA3 riboprobe was used as a reference. Each riboprobe consists of transcripts from a single DNA strand, and thus can hybridize only to the opposite direction transcripts. The T7 riboprobe hybridized to a transcript of the same size as the DNA probe, namely 1.5 kb, whereas no hybrid- ization was observed with the SP6 riboprobe. As can be seen in Figure 4, this result shows the direction of /ME7 transcription, namely that the 5’ end of /ME7 is between the Bglll and the Hindlll sites.

Transcription of /ME7 in Vegetative Strains RNA was prepared from various strains growing in acetate or in glucose vegetative liquid media, and equal amounts were used in Northern analysis. The following strains were used: MATa/MATa RMEVRMEl (419-G); MATa/MATa RMEVRMEl (419-Gl); MATalMATa RMEl/RMEl (419-G2); MATalMATa rmel-Vrmel-1 (507); and matal/MATa rmel-Vrmel-7 (515). No transcripts were found to hybrid- ize to the probe when the RNA was prepared from glucose growing cells, whereas clear signals were observed fol- lowing hybridization to RNA from acetate growing cells. RNA samples from the various strains showed similar lev- els of /ME7 transcripts. The level of /ME7 transcripts was found to be from three to seven times higher than the level of URA3 transcripts. (An example, RNA from strain 419-G, is shown in Figure 4.) Since URA3 transcripts are present at about 5-10 copies per cell (Lacroute et al., 1981) we cal- culated that /ME7 RNA is present in acetate growing cells at about 35-70 copies per cell. Another gene, HIS4, was also probed on the same Northern blots, and gave a simi- lar variation in transcript levels between strains. We thus concluded that /ME7 transcription is under glucose re- pression. Furthermore, neither MAT nor RME7 seems to grossly affect the amount of /ME7 message in vegetative growing cells.

Is /ME7 Induced during Meiosis? The levels of /ME7 transcripts at various times during mei- osis were also determined by Northern analysis. The

IME7, a Posltlve Regulator Gene of Melow 857

Alternative RNA polymerase

P3’ labeled probes

Results IMEI

URA3 URA3

Conclusion GDE B

c-----J pYK II

The IMEI gene

Direction of transcription

a

I I 2 4 6 8 IO 12

Figure 4. /ME7 Transcrlptlon in Vegetative Cells. and Its DIrectIon

The wavy lines represent RNA j7P /MEI rlboprobes were made wng either T7 or SP6 RNA polymerase These rlboprobes. as well as a URA3 rlboprobe. were separately hybridized to Northern blots made of RNA Isolated from a MATaIMATu strain grown !n YEPD (a), or YEPA

(W

b matal/MATa rmet-1 /rmel-1

0 I 2 3 4 5 7 8 IO 12

@m*ror--

MATa / MATa 0 2 4 6 810

MATa/MATa RMEl/RMEl

0 2 4 6 75 9.5 I I

*weir *r

Time in SPM

Figure 5. Transcnptlon of /ME7 in Melok Cells

RNA was prepared for Northern blot at different times after exposure to SPM (a) The amount of /ME7 transcript was calculated relative to URA3 RNA, after scanning of the blot. (b) Representative Northern blot hybrlduatlon of /ME7 RNA. Strains used 419-G (MATaIMATu) 419.G2 (MATdMA Trr) ; and 515 (m&l/MA& rmel-l/rmel-7).-----.

above strains were grown in acetate vegetative medium prepare RNA, and equal amounts were loaded nnto a gel (PSP2) to 1 x lO’cells/ml, washed once in sterile distilled for the Northern analysis. Figure 5 shows the relative lev- water, and resuspended in sporulation medium (SPM; els of /ME7 transcripts compared to URA3 transcrrpts at time 0). At different times, 200 ml samples were taken to different times. (Figure 5 contains only representative

Cell 858

data of some of the strains examined.) In strain 419-G (MATa/MATa), the level of /ME7 transcripts increased dur- ing meiosis about 6-fold, reaching a peak 6 hr into meio- sis. A different MATa/MATa strain (nonisogenic to 419-G) gave a sharper peak at 4 hr, and at 6 hr returned to the level of time 0 (data not shown). In both strains, the main increase was already observed at 2 hr. The sharp Increase

in the level of /ME7 transcripts was not simply a response to nitrogen starvation (the transfer from PSP2 to SPM), be- cause strain 419-Gl (MATa/MATa; data not shown) and strain 419-G2 (MATaIMATa; Figure 5), which were both RME7 and meiosis deficient, did not show comparable responses. Thus, the mating-type genes control the ability of the /ME7 gene to respond to the nitrogen starvation signal.

The transcription of /ME7 in meiosis may either be directly regulated by the mating-type locus, or as pre- sented in Figure 3a, indirectly via RME7. In order to distin- guish between these two possibilities, RNA samples were prepared from the diploids MATalMATa rmel--l/rmel-1 (strain 507), and matal/MATa rmel-l/rmel-7 (strain 515) at different times in meiosis. Both strains gave 43% and 32% sporulation, and during meiosis, the level of /ME7 transcripts increased as in MATa/MATa strains. (Figure 5 shows the results of Northern hybridization analysis of RNA from strain 515 only.) Although the kinetics of in- duction in the MATa/MATa RME7/RME7 and matal/MATa rmel-l/rmel-7 seems to differ, this does not imply that IME7 might be controlled by both MATa/MATc( and RME7, because the strains we used were nonisogenic, and differ- ent MATa/MATa and rmel-l/rmel-7 strains gave different kinetics. Nevertheless, we can conclude that a strain that is homozygous for rmel-7 is capable of inducing the tran- scription of IME7, without being heteroallelic at MAT. Therefore, the rmel-7 mutation in addition to overriding the requirement of MATa and MATa with respect to mei- osis (Kassir and Simchen, 1976) also does so with respect to /ME7 transcription.

Discussion

In this paper, we have characterized IMEl, a new meiosis- specific gene. Cells homozygous for a gene disruption of /ME7 cannot enter meiosis and arrest as unbudded cells without loosing viability, at an early stage prior to pre- meiotic DNA synthesis and to commitment to meiotic re- combination. From this, it appears that the gene product of /ME7 is required for the initiation of meiosis. Disruption of /ME7 has no effect on mitotic growth, or on induced mi- totic recombination (D. Granot, unpublished data), and therefore, we believe that the gene is specific to meiosis.

The imel disruption alleles are similar to recessive mu- tations in several SPO and ME/ genes, which also arrest meiosis prior to premeiotic DNA replication (Esposito and Klapholz, 1981). These are meil- mei2- mei (double mutant), ~~07-7, ~~08-7, and spo9-7. The SP07gene has been mapped to chromosome I (Mortimer and Schild, 1985) thus this gene is not identical to /MEI. As the map positions of the other genes are not known, and they have

not been cloned, we cannot rule out the possibility that one of them may be allelic to IMEl.

The gene lME7, and the previously described RME7 (Kassir and Simchen, 1976), have opposite effects on mei- osis: a diploid homozygous for imel is Spot, whereas a diploid homozygous for rmel is Spa+. In the absence of one of the normal gene products of MATa and MATa2, RMEl has an inhibitory effect on meiosis. However, a homozygous rmel-7 strain may go into meiosis in spite of this absence (Kassir and Simchen, 1976). On the other hand, the presence of /ME7 on a multicopy plasmid results in a sporulation proficient phenotype, even in MAT insufficient strains, whereas RME7 on a multicopy plas- mid results in sporulation deficiency even in normal MATa/MATa strains (Mitchell and Herskowitz, 1986; J. P. Margolskee and G. Simchen, unpublished data). These phenotypes establish the notion that RME7 is a negative regulator of meiosis, whereas /ME7 is a positive regulator.

The role of /ME7 in the regulatory pathway of meiosis may be deduced from several experiments. First, rmel-7 ime7-0 double mutants exhibit the Ime phenotype, name- ly a recessive Spo The meiotrc deficiency of ime7-0 is epistatic to the effect of the rmel-7 mutation, and there- fore, /ME7 acts either after RME7 or in a parallel pathway. /ME7 on a high copy number plasmid overrtdes the above repression of RME7, and allows meiosis to take place regardless of the information at MAT or RME7. This result strengthens the possibility that /ME7 acts downstream of RME7 rather than in a parallel pathway. The latter is ruled out by the Northern analysis of RNA (see below). Figure 6 shows a model that describes our workmg hypothesis for the initiation of meiosis. We chose the simplest model, although more complicated ones could also explain the results.

Analysis of the levels of transcription of /ME7 in various genotypes has shown that the matmg-type system regu- lates the expression of lME7, and that the above sug- gested regulation is on the level of transcription. This con- clusion is based on the finding that, upon nitrogen starvation, the transcription of /ME7 is derepressed (or in- duced) from 5 to 8-fold in MATa/MAT(x diploids (or in rmel strains; see below). MATa/MATa and MATalMATtx diploids do not show any increase in the level of /ME7 RNA in cells exposed to sporulation conditions. The induction of /ME7 transcripts under starvation conditions may be an essen- tial part of meiosis. This is concluded from the fact that strains carrying the /ME7 gene on a multicopy plasmid by- pass the requirement for MATa and MATu2 for initiation of meiosis. One interpretation of this effect of the mul- ticopy plasmid is that a 50.fold increase of the repressed level of /ME7 is comparable to the derepressed level (see below). Furthermore, as in both matal/MATa rmel-l/rmel-7 and MATdMATu rmel-lhmel-7. the transcription of /ME7 is meiotically induced, and at least part, or possibly all, of the regulation of /ME7 by MATa and MATu2 is via RME7, as suggested in Figure 6.

Sporulation promoted by rmel in MATa/MATa rmel/rmel diploids, or by /ME7 plasmids in MATa/MATa RMEI/RME7 diploids is always lower than sporulation promoted by

/MEI. a Posltlve Regulator Gene of Meiosis 859

MATal MATa 2

RMEl

Nitrogen ond glucose

signal ___2_1 IMEI

meiosis

Flgure 6. Regulation of Meiosis by the Mating-Type Genes and by the Environment

) represents repression, and ~> represents Induction.

MATal and MATa2. This observation suggests that MATal and MATa may regulate the expression of an unknown gene that is essential for good sporulation, as well as of RMEl. Is this second gene IMEl? As discussed above, we have shown that /ME1 expression is regulated by RMEl, but we have not ruled out the possibility that MATal and MATa may play an additional role in /ME1 expression. In order to establish this point, further studies using isogen- ic MATa/MATu RMEVRMEI and MATa/MATa rmel/rmel strains are required. A suggestion that /ME1 may indeed be the second target for MATa/MATa regulation comes from the observation that a matal/matal rmel-Vrmel-1 diploid (strain 2018) containing the YEp24 plasmid gave an average of 3.5% sporulation (12 independent colonies were examined), whereas when the same strain contained the plasmid YEpK26-7 (/ME1 on the multicopy plasmid YEp24), the sporulation improved and the average level was 23.4% (13 independent colonies).

In the pathway leading from the mating-type genes to meiosis, RMEl seems to be the first gene in the cascade of events. RMEl was shown by Mitchell and Herskowitz (1986) to be regulated by the mating-type genes. No con- clusive evidence exists yet as to whether RMEl directly regulates IMEl, or whether the regulation operates in- directly, via another repressor or inducer.

The sporulation promoting activity of the /ME1 mul- ticopy plasmid YEpK26 and its derivatives, can be ex- plained in several ways. First, the low, basal level of IMEl, when multipied 50-100 times, reaches the level required for sporulation. Second, /ME1 on a multicopy plasmid ti- trates out a repressor (e.g., RMEl), which is limited in the cell, and therefore some /ME1 copies escape repression. This postulated repressor may also repress other genes needed for sporulation, which are also relieved from re- pression by the presence of the multicopy /ME1 plasmid.

Third, the /ME1 plasmid does not contain the site(s) on which the repressor acts. Finally, /ME1 is activating meio- sis as a consequence of the plasmid structure, for in- stance, as an abnormal fusion protein or a truncated pro- tein. The last possibility is ruled out by comparisons of various independently cloned /ME1 containing plasmids and the genomic restriction map of /ME1 (data not shown). The second explanation, namely titration of a limited repressor, is ruled out by the observation that a derivative of plasmid YEpK26S5, which contained the TRPl disrup- tion of /ME1 shown in Figure 2b, did not promote sporula- tion of MATa/MATa diploids. Thus, the expression of only the /ME1 gene on these plasmids enabled sporulation to occur in MATa/MATa RMEVRMEl strains. Whether this ex- pression is due to the absence of repression sites or to the multiplication of a low basal level of the cloned IMEl, or both, cannot be determined at the present time.

As we have stated in the Introduction, meiosis is initi- ated only under starvation conditions. /ME1 seems to be the only known specific meiotic gene through which this requirement is manifested. Two results support this con- clusion. First, the level of /ME1 transcripts is elevated upon nitrogen starvation. Second, /ME1 on a multicopy plasmid overrides the requirement for nitrogen starvation in induction of meiosis: in YEPA medium, MATa/MATa dip- loids that contained the plasmids YEpK26 or YEpK26-7 gave about 20% sporulation after 48-72 hr (Granot, 1987). The same strains, with or without the control plasmid YEp24, do not initiate meiosis or sporulate in YEPA.

Mitchell and Herskowitz (1986) showed that MATa/MATa diploids homozygous for the rmel disruption did not sporulate in rich acetate medium, and concluded that the RMEl gene is not regulated by the environment. We there- fore suggest that /ME1 is regulated by the environment in- dependently of RMEl, in addition to being regulated by RMEl.

Formally, IMEl can be either repressed or induced by either pathway. We propose, however, that /ME1 is re- pressed by both pathways (Figure 6). This model is sup- ported by the finding that sequences upstream of IMEl, which are absent in YEpK26, when present on multicopy plasmids, enable sporulation to occur in MATa/MATa RMEV RMEl or in MATalMATa cells as well as in the presence of nutrients (Granot, 1987). The interpretation of these results is that the cloned upstream sequences titrate out the repressors of IMEl.

Northern analysis has not detected any transcripts of /ME1 in glucose grown cells (in YEPD medium), but cells grown on acetate contain a noticable level of transcripts. Glucose has been shown to inhibit meiosis in meiotically induced yeast cells (Shilo et al., 1978). We think that at least part of the effect of glucose on meiosis is manifested via its effect on the expression of IMEl, because the high

copy number plasmids also allow sporulation to take place in the presence of glucose (Granot, 1987). Thus, /ME1 overrides the glucose repression signal.

How is /ME1 regulated by glucose and nitrogen starva- tion? /ME1 on a multicopy plasmid confers a phenotype similar to the mutations cdc25, cdc35 (cyrl), and ras2

Cd 860

Table 4 Genotypes of Yeast Strains Used r These Studies

Strain Markers

320 17-15 339

355 850.D106

860

J542

F836 D300

D317

D320

YD164

419-G

419.G1 41 g-G2 507 515

2018

MATa. rmel-1, a&2, ura3, leul, canl-11, cyh2.21 matal, rmel-1. ade2. ura3. leul. canl-17, cyh2-21 MATa, cdcll-1. adel. ade2. Ural, /ysZ-2, tyrl-2.

h,s7-1 MATa, cdc8-3. ade2, Ural, hfs7-1. canl-11 MATn. Mel-OtURA3. ade5, hls7-2, /euZ-3.7 12,

trpl-289, ura3-52 MATa. cd&. hfs2. his6. hfs7. adel. ade2. ade6.

lys7, lys9, leul, leu2, ura3. trpl. met2 met74. aro7. argl, arg4, ilv3. asp5. gall, ma/. sue

HMLn matal HMRcr, rmel-1 .w2-1. ade2. ura3. leul, canl. cyh2

MATa. hfs4-15. ade2-1. canl. karl-1 MATn. ade2-101, trpl (de/). ura3-52, GAL’.

hfs4-519 MATa/MATn. Imel-l/IMEl, ade2-RB/adeZ-101.

trpl/trpl, ura3-52/URA3 MATa/MATn. Imel-l/fmel-1. ade2-R8/ade2-1.

rrpl/trpl, ura3-52/URA3 HMLa matal HMRa/HMLn MATn HMLn. ura3-52/

ura3-52, IysZ- 7/L YS2, /euZ-3,11 Z//e@3.7 12. adeWade5. ade2/ADE2, canl-ll/CANl. cyhZ/ CYHP, trp (trp/?/trp7? and/or trp5?/trp5?)

MATa/MATu ura3-52/ura3-52. IeuZ-3,772/ leu2-3.112. ade2/ade2-R8. his7/HIS7. lysZ/LYSZ. canl/CANl, trp5-d/TRP5

MATa/MATa UV Induced in 419-G MATn/MATn UV Induced II- 419-G MATnlMATu, rmel-l/rmel-1, ade2/ade2 matal/MATa. rmel-l/rmel-1. adeZ/adeZ, ura3/

ura3, leul/leul HMLn matal HMRn/HMLn matal HMRn. rmel-ll

rmel-1. ade2/ade2. leul/leul. ura3/ura3. can11 can 1, cyhZ/cyh2

(Shilo et al., 1978; Matsumoto et al., 1983; Tatchell et al., 1985). Diploids homozygous for cdc25 sporulate in a rich acetate medium, and diploids homozygous for cyrl or ras2 also sporulate in such medium as well as in the pres- ence of glucose. We thus assume that glucose and nitro- gen affect the same pathway. It was suggested that nitro- gen starvation regulates meiosis via the CAMP-dependent protein kinase (Matsumoto et al., 1983). Furthermore, Tripp et al. (1986) observed changes in the pattern of phos- phorylation of proteins in meiotic cells compared to vegetative ones. It is possible that a phosphorylated pro- tein represses /ME7 whereas its dephosphorilation derepresses /ME7 transcription, and thus meiosis is in- duced. In order to determine whether the adenylate cy- clase/protein kinase pathway operates on IME7, one could study interactions between mutations and plasmids carry- ing clones of these genes, and the effects of the various mutations on /ME7 transcription. Preliminary experiments along these lines have shown that double mutants imel-7/ ime7-7 cdc25hdc25 are sporulation defictent, as expected from the model suggested above.

A regulatory system of meiosis, somewhat similar to the MAT-RME74ME7 pathway described here, has been re- ported for the yeast Schizosaccharomyces pombe (ltno and Yamamoto, 1985; McLeod and Beach, 1986). Two positive regulators, mei and mei3, and a negative regula- tor, pat7 (ranl), which is a protein kinase, have been de-

scribed. pat7 is epistatic to mei3, whereas mei appears to come afterpat in the pathway. Further characterization of both systems is required, however, before analogies be- tween /ME7 and either mei or mei can be drawn.

Experimental Procedures

Yeast Strains and General Genetic Methods All yeast strains are described in Table 4. Mating, induction of sporula- tlon, tetrad analysis, and mating-type determlnatlon have been de- scribed (Hicks and Herskowltz. 1976; Shilo et al., 1978). Determlnatlon of HMRa versus HMRn was done by Southern analysis (Strathern et al., 1980).

Monitoring Meiotic Events Premelotlc DNA synthesis and commitment to melotlc recomblnatlon were measured as described by Kasslr and Simchen (1978).

Plasmids YEp24 pBR322 + URA3 + 211 (Botsteln et al., 1979). Ylp5: pBR322 + URA3 (Botsteln et al.. 1979). YRp7: pBR322 + ARSI + TRP7

(Tschumpei and Carbon, 1980) YEpK26: YEp24 + a 9 2 kb Sau3A m- sert (at the BamHl site), which contain the /ME1 gene. The subclonlng plasmids (Figure 1) were constructed as follows- YEpK26-4, by sub- cloning the 5.1 kb BamHI-Xhol fragment of YEpK26 in between the BamHI-Sal1 sites of YEp24; YEpK26-7. by subclonmg the 2.65 kb BamHI-Bglll fragment of YEpK26 in the BamHl site of YEp24; YEpK26-8, by subclonlng the 66 kb Bglll-Sal1 fragment of YEpK26 r between the BamHI-Sal1 site of YEp24; YEpK26-10, by subcloling the 4 75 kb Clal fragment of YEpK26 Into the Clal site of YEp24: YEpK26- 108. by subclonlng the 1.1 kb Bglll-EcoRI fragment of YEpK26 in be- tween the BamHI-EcoRI sites of YEp24; and YEpK26-111, by subclon- Ing the 2.1 kb Hlndlll-BamHI fragment of YEpK26-7 in between the HIndIll-BamHI sites of YEp24. The dlsruption plasmlds (Figure 2) were constructed as follows YlpK26-106, by subclonlng the 0.55 kb Htn- dill-EcoRI fragment of YEpK26-7 III between the HIndIll-EcoRI sites ofYlp5. YlpG71, bysubclomng the 1.65 kb BamHI-Clal fragment in be- tween the BamHI-Clal sites of YRp7 and then Dy Inserting the 0.75 kb Pstl-Bglll fragment of YEpK26 in between the Bglll-Pstl (partially cut). YEpK26-S5 by cutting the plasmid YEpK26 with Sal1 and rellgatlng It wIthout the Sal1 fragment: and YpKll. by subclonIng the 0.55 kb HII- dill-EcoRI fragment of YEpK26-7 in between the Hlndlll-EcoRI sites of pGEM3. (Promega Blotec applications guide.)

Media YEPD (rich medium). SPO (for Inductton of sporulatlon on plates). and SD (for scoring or selectIon of nutritional markers) have been de- scribed (Hicks and Herskowltr, 1976) -ADE serves to detect ademne prototrophs (Kasslr and Slmchen. 1978) CAN CYH agar (for scoring resistance to canavanlne and cyclohexlmlde) have been described (Kasslr and Slmchen. 1985). PSPP (mammal acetate vegetative me- dium). YEPA (rich acetate vegetative medium). and SPM (mammal spotulatlon medium) have been described (Shllo el al., 1978) MS (min. imal m&urn for bactettal growth), LB (rich bacterIaI medium). and LB + Amp (for selectton of plasmlds). have been described (Manlatis et

al 1982)

Enzymes RestrIctIon enzymes. T4 DNA Ilgase. and DNA polymerase I were pur- chased from New England Blolab or IBI. All enzymes were used ac- cording to the condltlons suggested by the manufacturers SP6 and T7 RNA polymerases were purchased from Promega RadIoactIve nu- cleotldes were purchased from Amersham

Transformation Yeast transformat!ons were done by one of two methods transforma- tion of yeast spheroplasts (Hlnnen et al 1978). or transformation of cells treated with llthlum acetate (Ito et al 1983) E co11 transformatlon was carried out as described by Mamatis et al (1982)

DNA Preparations PlasmId DNA was extracted from yeast by the method described by Wlnslon et al (1983) and from bacteria as tn Davis et al (1980)

IMEl. a Positive Regulator Gene of Meiosis 861

Xhol Linker Insertion Plasmid YEpK26-7 was partlaIty cut with either Alu or Ftsal restwtlon enzymes Plasmlds that were cut only once were Isolated from a 1% agarose gel and than ligated wth Xhol lmkers (New England Blolab) as described by Manlatls. et al. (1982) FolIowIng Ilgation, the plasmids were cut with Xhol. Ifnear molecules were Isolated from 1% agarose gels, relegated. and transformed mto the E co11 strain MH5 to Ura’ Amp’ (the Ura’ selection enabled us to get rid of molecules with linkers Inserted at the URA3 gene, MH5 contains a mutation in the gene Pyrr that IS complemented by the yeast URA3 gene) PlasmId DNA was Isolated and subjected to restrlction analysis Plasmids that were found to contain a Xhol site within the BamHI-Bglll sites were used to transform a MAJa/MAJa strain in order to check sporulatlon promoting actlwty

Northern Analysis RNA was Isolated from 2 x 10” cells as described by Carlson and Bot- stein (1982). Separation of RNAs on formaldehyde agarose gel, Its transfer to GeneScreen membrane, and hybridization with dextran sul- fate to a DNA probe, were done as described in the instructions manual of GeneScreen (New England Blolab). Preparation of 32P rlboprobes and RNA/RNA hybndlratlon protocols were essentially as described by Promega Blotec appllcatlons guide

Isolation of rrnel-1 ime7-0 Double Mutants Strain 320 (MAJa, rmel-7. ura3, /MEI) was mated to strain 850.D106 (MAJcc, imel-PURA3, RME1. ura3-52) The resultmg dlplold was sporulated, and 35 tetrads were dissected The allele imel- was as- slgned via the Ura’ phenotype. rme7-7 was asslgned by the ability of such segregant to allow sporulaton of matal/MATa or of matal/MAJu dlplolds. Thus, MAJa segregants were mated to strain J542 (HMLu, matal, HMRu, w2, rmel-7, an rt-mater), and MAJu segregants were mated to strain 17-15 (ma&l, rmel-7, an a-mater) We thus Isolated nine MAJcI rmel-7 ime7-tJ.URA3 and SIX MAJa rmel-7 imel-O:URA3 segregants All of the above segregants were mated in all 54 possible combinations to produce 54 MA7WMAJct rmel-l/rmel-7 Imel-Ohmel- dlplolds

Acknowledgments

We thank Dr. M. Kuplec and Ms. M. Trelmn for crItIcal reading of the manuscript This research was supported by a grant from the US-Israel Blnational Science Foundation, Jerusalem, Israel

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertfsemenZ’ in accordance with 18 U.S.C. Section 1734 solely to Indicate this fact

Received May 28, 1987; revised December 16, 1987

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