268 9

04-Meiosis and sporulation 04-01

A transcriptional cascade during meiosis and sporulation in the fission yeast S c h i z o s a c c h a r o m y c e s p o m b e H. Abe and C. Shimoda Department of Biology, Graduate School of Science, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan. [email protected] We studied a transcriptional cascade operating during meiosis and sporulation in S. pombe. The mei4 + gene encoding a forkhead transcription factor is transcribed prior to the onset of meiosis, depending on the meiotic inducer Mei2. Our Western blotting and GFP-fusion analysis revealed that Mei4p was transiently accumulated and partially phosphorylated during meiosis and localized in nuclei. We found that the mei4 mRNA level was remarkably lower in mei4 mutants than in wild-type cells and expression of the mei4-1acZ gene was stimulated in vegetative cells by ectopic overproduction of Mei4p. These results suggest that a positive feedback mechanism exerts in the mei4 + expression. To identify novel target genes of the Mei4 transcription factor, we searched for the genes in S. pomhe genome sequences (Sanger Centre, UK) which have a Mei4-binding site in their promoter region. These analyses identified three mde (Mei4-dependent expression) genes whose transcription was dependent on Mei4. Mde3 protein proved to be homologous to the meiosis-specific protein kinase Ime2 of S. cerevisiae. The mde3D cells form one, two or threespored asci other than normal four-spored asci, suggesting that mde3 + plays some roles in meiosis.

9

04-02

Possible roles of the meiosis-specific mutS homolog, MSH4, in relieving cellular defects in mitosis and meiosis conferred by mutational alteration of the blml gene of S a c c h a r o m y c e s cerevisiae G. Anyatonwu, E. Garcia, A. Pramanik, C.W. Moore City University of New York Medical School and Sophie Davis School of Biomedical Education The current study describes recombinant plasmids that complement the hypersusceptibility to killing by Neomycin of blml-1 mutant cells of S. cerevisiae. Using a Ycp50-based $288C genomic library, approximately 1500 Ura+ transformants were selected on media lacking uracil. One of the plasmids that rescued the hypersensitivity conferred by blml-1, pPMllS, was selected for further study but was inactivated after recovery from E. coli (HB101). A fragment from the inactive insert was used as a probe to screen the genomic library by in situ hybridization. This resulted in the recovery of a plasmid (pPMll8-4) that partially complemented the blml-1 mutation. By sequence analyses, pPM118-4 was found to have a 5.4 kb overlap with pPM118, and both plasmids mapped to chromosome VI. ORFs found in both plasmids are MSH4 and a series of overlapping transposons (tn[GUU] F, YFLWdelta2, YFLWTy2-1, YFLWdelta3) with unknown roles in the yeast life cycle. Part of MSH4 is missing in pPMll8-4. In further characterization of the blml-1 mutation, tetrads derived from homozygous blml-1/blml-1 diploid cells failed to yield four viable spores. This was in contrast to BLMI-I/BLMI-1 and BLMl-l/blml-1 diploids, from which approximately 60% of the tetrads produced four spores. These results draw a parallel since MSH4 has been implicated in meiosis I. The portion of MSH4 absent in pPM118-4 suggests that region is essential for full functional complementation of blml-l.

04-Meiosis and sporulation 9

269

04-03

Budding yeast Rckl and Rck2 protein kinases interfere with meiotic functions E. Bilsland, A. Ramne, and P. Sunnerhagen Department of Molecular Biology, Lundberg Laboratory, G6teborg University The budding yeast RCKI and RCK2 genes were initially found as suppressors of fission yeast checkpoint mutations (1), and more recently have been shown to cause telomere lengthening when expressed in fission yeast (2). Here, we demonstrate phenotypes in budding yeast indicating a role for these protein kinase genes in meiosis. In diploids with mutations in the meiotic checkpoint genes MEC1 or RAD24, overexpression of RCK1 or RCK2 reduces the meiotic proficiency, most prominently seen for RCK2. Strains with mecl tell double mutations are somewhat more sensitive to this effect than single mecl mutants. The meiotic proficiency of wild-type or radl7 cells is only marginally affected by overexpression of RCK1 or RCK2. Similar effects were found in two different strain backgrounds. These results were corroborated by phenotypes of homozygous disruption mutants. Diploids with a rckl/rckl disruption sporulate in high-glucose medium, and rckl/rckl rck2/rck2 mutants even more readily. In sporulation medium lacking glucose, entry into meiosis is about twice as fast for the rckl/rckl mutants as for the wild type. However, no meiotic cells were found in rich medium, indicating that even though high glucose does not inhibit meiosis in rckl mutants, signaling fi'om high nitrogen is effective enough. The meiotic phenotype ofrck2/rck2 mutants is weaker but otherwise similar to that of rckl/rck I ceils. A possible explanation for the apparent discrepancy between our overexpression results (with a stronger effect for RCK2) and the phenotypes of the disruption mutants (where the rck 1/rckl mutation clearly gives the stronger effect) lies in the fact that the Rck 1 protein fails to accumulate when ectopically overexpressed, indicating instability of the protein product. Rck2 is located diffusely in the cytoplasm in vegetative cells when expressed from a strong promoter. In meiotic cells, its localization shifts to a structure near or surrounding the nucleus. There is a transient shift in the relative abundance of the two RCK2 transcripts in meiotic cells. We propose that one action of Rckl or Rck2 is to counteract entry into meiosis, and that overexpression of these kinases in certain checkpointdeficient backgrounds is sufficient to inhibit the meiotic process, whereas their absence allows meiosis under high glucose conditions. 1. Dahlkvist, A., G. Kanter-Smoler, and P. Sunnerhagen. 1995. The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe. Mol. Gen. Genet. 246:316 326. 2. Dahl~n, M., T. Olsson, G. Kanter-Smoler, A. Ramne, and P. Sunnerhagen. 1998. Regulation of telomere length by checkpoint genes in &'hizosaccharomyces pombe. Mol. Biol. Cell 9:611-621.

9

04-04

Sporulation-specific transcriptional regulation of the yeast genes DITI and DIT2 E. Bogengruber, T. Eichberger, R. Schricker, P. Briza, M. Breitenbach Institut fuer Genetik und Allgemeine Biologie, Universitaet Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria Expression of the yeast genes DIT1 and DIT2 is confined to mid/late sporulation. Transcription of these divergently arranged genes is controlled by a common 900-bp intergenic region. Random mutagenesis of the DITI/DIT2 intergenic region allowed the identification of two point mutations leading to vegetative expression of both genes. Both point mutations reside in a palindromic sequence that we call DRE (DIT repressor element). DRE turned out to be the major negative regulatory site during vegetative growth. Since DRE contains the binding consensus sequence of the transcriptional regulator PacC of Aspergillus nidulans we investigated the role of Rim 101p, the S. cerevisiae homolog of PacC, in yeast sporulation-specific gone regulation. The activated form of Riml01p enhances expression from the DIT1/DIT2 promoter both in vegetative and in sporulating cells. Activation by Riml01p does not seem to involve binding of Riml01p at DRE, since reporter constructs with a deletion of DRE could still be activated by the short form of Riml01p. With the help of band-shift assays and subsequent protein sequencing we currently try to identify the DRE-binding protein. Additionally, we have isolated yeast mutants that ectopically express DIT1 and DIT2 in vegetative cells. Several trans-recessive mutants led to the identification of Sptl0p as a transcriptional regulator necessary for silencing of both genes in vegetative cells. Mutants in Sptl0p exhibit a pleiotropic phenotype including cell shape, growth and ascus formation. We are currently investigating the biochemical role of Sptl0p, which shows sequence similarities to zinc finger-containing transcription factors as well as to histone acetyl transferases.

04-Meiosis and sporulation

270 9

04-05

Systematic analysis of "EUROFAN" deletion strains for sporulation phenotypes M. Breitenbach, E. Bogengruber, A. Thiir, P. Briza Department of Genetics, Salzburg University, Hellbrunnnerstrasse 34, A-5020 Salzburg, Austria We are presenting a testing scheme for sporulation mutants that was developed for the systematic phenotypic analysis of 1000 deletion mutants corresponding to unknown yeast genes deleted in the EU program "eurofan". The amount of LL and DL dityrosine was determined in sporulated homozygous mutant strains. In case of unusual dityrosine data and abnormal microscopic appearance additional tests were performed. Asci were stained with DAPI to detect whether or not the meiotic divisions had been completed. Staining with Calcofluor White showed whether or not the outer spore wall layers were present and intact. Sensitivity of the spores to Glusulase (Boehringer) was tested by observing lysis. In some cases, the wall- and cytoplasmic fraction of the sporulated cultures was analyzed separately for DL- and LL-dityrosine. Five phenotypic classes of sporulation mutants could be clearly recognized, some of which are new and have not been observed before. About 600 homozygous deletion strains have been tested by now. The percentage of deletion mutants that showed any sporulation defect was below 5%. The presentation will concentrate on these new sporulation phenotypes.

9

04-06

The budding yeast MPS1/RPK1 protein kinase is required for regulated expression of sporulation-specific genes and spore wall morphogenesis A. Camasses, F. De Fraipont & R.P. Martin UPR 9005 du CNRS, IBMC, 15 rue Ren~ Descartes, 67084 Strasbourg, France tel: 33 3 88 41 60 01; fax: 33 3 88 41 60 25; e-maih [email protected]

S. cerevisiae MPS1/RPKI encodes an essential dual-specificity protein kinase [1,2] which is required for SPB duplication during the G1 phase of the cell cycle and for a checkpoint which is activated to arrest progression through mitosis when the integrity of the mitotic spindle has been compromised [3, 4]. Results presented here demonstrate that this kinase has a unique role in sporulation: (i) MPS1/RPK1 m R N A levels are sharply increased in the middle period of the sporulation program, with maximal accumulation when cells are completing meiosis II. (ii) A homozygous rpkl temperature-sensitive diploid, although defective in SPB duplication during vegetative growth, can progress through both meiotic divisions at the restrictive temperature. However, the mutant is unable to form mature spores. Electron microscopy revealed that the mutant asci are defective in organizing proper spore wall assembly. (iii) MPS1/RPK1 function is required for normal levels and timely expression of developmentally-regulated genes of the sporulation program. Sporulation-specific genes are retarded and expressed at lower levels in rpkl cells and this effect is the most severe for middle-late and late genes involved in spore wall formation and ascospore maturation. [1] Poch et al. (1994) Mol. Gen. Genet. 243:641 653 [2] Lauz6 et al. (1995) EMBO J. 14:1655 1663 [3] Winey et al. (1991) J. Cell. Biol. 114:745 754 [4] Weiss & Winey (1996) J. Cell. Biol. 132:111 123

04-Meiosis and sporulation 9

271

04-07

Regulation of meiotic nuclear division by M c t l p K.F. Cooper, D.E. Egeland, M.J. Mallory, and R. Strich Fox Chase Cancer Center, Philadelphia PA USA Email kf_cooper~)~fccc.edu The generation of a haploid genome during meiosis requires two sequential nuclear divisions without an intervening S phase. This report describes the isolation and analysis of MCT1, a protein with similarity to a component of the mitotic anaphase promoting complex or APC. MCT1 encodes an intron containing gene that is transcribed and spliced only in meiotic cells. MCT1 splicing is dependent on the previously described MER1 meiotic splicing factor. Cells deleted for the first MCT1 exon completed pro-meiotic S phase but failed to produce spores. Most cells arrest mononucleated indicating a failure in executing either meiotic division. The remainder of the cells contained nuclei that appeared either mis-segregated or fragmented suggesting defects in spindle function. Biochemical studies revealed that Mctlp is required for the transient degradation of Clb lp, a B-type cyclin important for the execution of both meiotic divisions. Similar to other APC components, overexpression of the MCT1 cDNA arrests mitotic cell division with abnormal, elongated buds. These findings suggest that Mct lp is a component of a meiosis-specific APC.

9

04-08

Regulation of mid-sporulation genes: factors that affect M S E function H.E. Dalton, M.J. Straffon, A. Don and I.W. Dawes School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, N.S.W., 2052, Australia Email: z2139074(a student.unsw.edu.au; m.straffon(a~unsw.edu.au; i.dawes(a~unsw.edu.au Detailed analyses of the promoters of the mid expressed genes SPR3 and SPS4 identified the key meiotic regulatory element, the MSE (Mid Sporulation Element) [Oszarac et al., Mol. Cell. Biol. (1997), 17:1152-1159; Hepworth et al., (1994), 15: 3934-3944). The MSE is capable of inducing mid-sporulation-specific expression of a heterologous promoter. The recent genome-wide study of the expression of genes during sporulation [Chu et al. Science (1998), 282: 699-705] revealed that approximately 150 genes are activated during mid-sporulation. Furthermore, 70% of these genes contain an MSE within their promoter regions. In addition to mid genes, many mid-late expressed genes contain the MSE in the promoter regions (e.g. DIT1). This reinforces the importance of the MSE and highlights the requirement for other factors to ensure differential timing and expression levels of mid and mid-late genes. The MSEregulated mid gone SPR3 requires the presence of a neighbouring ABF1 site. This study reveals that the ABF1 site augments sporulation-specific expression by the MSE, without affecting timing. The divergently transcribed genes SPSI8 and SPS19 are regulated by an MSE, however the expression profiles of these two genes are different. Investigation of SPS18/19 auxiliary elements has been undertaken to determine their contribution to the temporal expression profiles and relationship to the MSE.

272 9

04-Meiosis and sporulation 04-09

Characterization o f

YKL165c, a

new essential gene o f the yeast

Saccharomyces cerevisiae

X. Delbecq, T. Godrie, D. Portetelle and M. Vandenbol FUSAGX 6, Avenue Mar~chal Juin, B-5030 Gembloux

Belgium.

E-mail: vandenbol.m(a~!fsagx.ac.be

The YKLI65c gene encodes a 919 amino acids protein which doesn't present any significant sequence homology with any known sequences. Gene inactivation shows a lethal phenotype. In order to determine the protein localization during the S. cerevisiae growing steps, a GFP-Ykl165p fusion protein has been constructed. The protein has been visualized using fluorescent microscopy in diplod cells. Preliminary results show that the Ykl 165p protein is located at the external cell structures with a more accurate localization at one tip of the cell. Analysis of haploid cells is under investigation. In another way, the two hybrid technique was performed to identify yeast proteins interacting potentially with the entire Ykl165p and with the hydrophilic domain of Ykl165p (450 aa at the N-terminus of the polypeptide). No concluant result has been obtained for the entire protein, probably because of inaccessibility of interaction site or due to membrane localization of the protein. The identification of the proteins interacting with the hydrophilic domain of Ykl165p allows us to suggest a possible role of the Ykl165p protein in the organisation of external cellular structures in sporulation or during bud formation, probably in connection with the secretory mechanism.

9

04-10

Biosynthesis of the yeast ascospore wall: identification of a membrane transporter of dityrosine-containing spore wall precursors T. Folder(I), S. Tenreiro(2), I. Sa-Correia(2), M. Breitenbach(1) and P. Briza(l) (l) Inst. f. Genetik u. Allg. Biologie, University of Salzburg, Hellbrunnerstr.34, A-5020 Salzburg, Austria; (2) Biotecnologia, Instituto Superior Tecnico, Av.Rovico Pais, 1049-001 Lisboa, Portugal The surface layer of the spore wall of S. cerevisiae consists of a highly cross-linked macromolecule that contributes to the spores' resistance against environmental stress. The major component of this macromolecule is the sporulationspecific amino acid, dityrosine, a dimer of tyrosine. Low molecular weight precursors of the dityrosine-containing macromolecule (N-formyl tyrosine and N,N'-bisformyl dityrosine) are synthesized in the cytoplasm of the prospore by two sporulation-specific enzymes, Ditlp and Dit2p, a cytochrome P-450. During spore wall maturation, bisformyl dityrosine is transported from the spore cytoplasm to the spore surface and incorporated into the macromolecule. During the systematic analysis of the ~Eurofan' deletion strains, we noted a significant decrease of spore wall dityrosine in a strain with a deletion of YBR18Ow. At the same time dityrosine-containing precursor accumulated in the cytoplasm. YBR180w codes for a membrane spanning protein that belongs to the MFS-MDR family and is expressed mid-late during sporulation. We will present evidence that YBR180w (SPOIOSp) is responsible for the transport of bisformyl dityrosine from the cytoplasm to the maturing spore wall.

04-Meiosis and sporulation 9

273

04-11

Meiosis-specific regulation of post-double strand break recombination complex assembly S.L. Gasior and D.K. Bishop Department of Radiation and Cellular Oncology and Department of Molecular Genetics and Cell Biology, University of Chicago, 5841 S. Maryland Ave. MC1105, Chicago, IL 60637 USA, email = dbishop(~ midway.uchicago.edu Assembly of the strand exchange protein Rad51p at multiple subnuclear sites can be detected during meiotic prophase by immunostaining. These assemblies (loci) are implicated as being functioning recombination complexes. The assembly of Rad51p requires the RAD52, RAD55, and RAD57 genes. Mutations in any one of these three genes prevents Rad51 focus formation in meiosis without corresponding effects on Rad51p expression or nuclear localization. Concurrent biochemical studies, by other groups, have shown that either Rad52p or a heterodimer of Rad55p and Rad57p, can stimulate strand exchange in vitro by overcoming the inhibitory effects of the single strand DNA binding protein RPA suggesting that the stimulation results from promoting assembly of Rad51 on RPA coated ssDNA. The requirements for assembly or Rad51p in vivo following radiation treatment in mitotic cells are different from the requirements in untreated meiotic cells. In mitosis, foci form at normal or elevated frequency in rad52 or rad55 single mutants but at reduced frequency in rad52rad55 double mutants, indicating redundancy of assembly function. The mitotic requirements for Rad51 assembly are consistent with in vitro studies because both Rad52p and Rad55p/Rad57p alone can function alone to stimulate Rad51p-mediated strand exchange in the presence of RPA. The absence of RAD52 -RAD55 redundancy with respect to Rad51p assembly during meiosis suggests assembly of strand exchange complexes is subject to meiosis-specific regulation.

9

04-12

A role for cohesins in the formation of axial elements and sister chromatid cohesion during yeast meiosis F. Klein, P. Mahr, M. Galova, S. Buonomo, M. Tudor, K. Nasmyth Institute for Botany, University of Vienna, institute of Molecular Pathology I.M.P. Sister chromatid cohesion established during DNA replication is crucial for the alignment of sister chromatids during metaphase, whereas its destruction triggers chromosome segregation during anaphase. During meiosis, two rounds of chromosome segregation follow one another without an intervening round of chromosome duplication. Homologous centromeres are segregated away from each other during the first meiotic division, whereas sister centromeres segregate at the second. Meiotic cells use (and lose) sister chromatid cohesion along chromosome arms for meiosis I and that at centromeres for meiosis II. During pachytene when homologous chromosomes are synapsed, chromosomes possess proteinaceous cores, called axial elements, which run along their longitudinal axes. A multisubunit complex called Cohesin containing two SMC proteins, Smclp and Smc3p, and at least two other proteins, Scclp and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scclp, called Rec8p, are required for the formation of axial elements, for recombination between homologues, and for cohesion between sister chromatids. Both RecSp and Smc3p co-localize with axial elements during pachytene, largely disappear from chromosome arms during meiosis I but persist in the neighbourhood of centromeres until the onset of anaphase II.

274 9

04-Meiosis and sporulation 04-13

Analysis of C o m l / S a e 2 in meiosis and mitosis I. Knezic, F. Klein Institute of Botany, Rennweg 14 A-I030 Vienna

Austria. E-mail: knezic(a:sl.botanik.univie.ac.at

We have isolated the COM1/SAE2 gene which is essential for completion of meiotic recombination. In the absence of the Coml/Sae2 protein meiotic DNA double-strand breaks accumulate in their unresected form, with Spoll the initiating endonuclease blocking the 5' ends of the DNA. Coml/Sae2 is the only known gene, to date, which when completely absent causes the accumulation of these unresected meiotic breaks. Recently, colethality was found between COM1/SAE2 and another gene indicating an essential role for COM1 in mitosis. We have identified a short stretch within the Com l/Sae2 protein sequence with strong homology to the Swi6 interaction domain of Swi4 (Mbpl). We have mutated the site in COM1/SAE2 and the phenotype caused by these mutations in meiotic and vegetative cells will be discussed. We have also constructed a functional MYC-tagged version of Com lp/Sae2p. Com l/Sae2 localizes in the nucleus of very early meiotic cells. The localization of Coml/ Sae2 compared to other cytological landmarks (Mrel 1, Dmcl, Zipl, Msh4), as determined in meiotic nuclei from WT and rad50S cells, will be presented. It will also be shown where in the nucleus Com l/Sae2 protein resides during the cell cycle and after irradiation.

9

04-14

The idiomorphs M A T A and M A TB of the Y a r r o w i a lipolytica mating type locus Cornelia Kurischko I, M.B. Schilhabel1'+, Ingrid Kunzel, Erika Franzl I, R. Rosas Quijano 2, C. Gaillardin 2 1 Lab. Cellular and Molecular Biology, Hans-Kn611-Institut ffir Naturstoff-Forschung e.V., Beutenbergstrasse 11, D-07745 Jena, Germany, email: ckurisch(a~'pmail.hki-jena.de; 2 Lab. Genetique Moleculaire et Cellulaire, INRA-CNRS, Institut National Agronomique, Paris-Grignon, France; +present address: Klinikum der Friedrich-Schiller-Universit~it Jena, Germany The MA TA of Yarrowia lipolytica locus was cloned taking advantage of its ability to induce sporulation in a diploid B/B strain. In addition, it represses the mating capability of this strain. We subcloned a fragment of l 1.7 kb and were able to separate the induction of sporulation and repression of conjugation functions. Sequence analysis revealed one ORF of 119 amino acids (MATAI), which is necessary to induce sporulation. A second ORE of 291 aa (MATA2) is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. MA TA1 shows no homology to other proteins in current available databases and seems to represent a new class of mating-type gene. MA TA2 contains a HMG box with homology to other mating-type genes. By Southern analysis we showed that MATAI and MATA2 are mating-type specific. Probes of flanking regions containing sequences with homology to either SLA2 or a putative ORF, YBB9, of S. cerevisiae hybridised with DNA from cells of either mating type. Using the flanking regions SLA2 and YBB9, the MA TB locus was sequenced. The mating-type specific region starts already 55 bp behind the SLA2 stop codon. The size of the MA TA locus is 2294 bp. The MA TB locus is much larger, 3354 bp. It contains two putative ORFs. MATB1 does not show strong homology to any proteins or protein motifs. MA TB2 contains a homeobox motif with homology to MA T~2 and MA Tal of S. cerevisiae. Differently to MA TA, both OREs of MA TB are oriented in the same direction.

04-Meiosis and sporulation 9

275

04-15

Analysis of gone expression during gametogenesis and starvation in yeast using DNA micro arrays M. Primig*, R. Williams*, G. Tevzadze, E. Winzelert, A. Conway2, R. DavisI and R. Esposito The University of Chicago, Department of Molecular Genetics and Cell Biology, 920E 58 th Street, Chicago I1 60637 J Stanford University Medical School, Department of Biochemistry, Stanford, CA, 94305-5307 2 Silicon Genetics, 935 Washington Street, San Carlos, CA 94070 * These authors contributed equally to this study, E-mail: rwillia(a)cmgm.stanford.edu, re-esposito(a~uchicago.edu Genes orchestrating the exit from the mitotic cell cycle and the transition through the meiotic developmental pathway are subject to tight transcriptional control in budding yeast. By using high-density oligonucleotide DNA microarray technology we have obtained the expression profiles of ~6200 yeast genes during sporulation. We found almost 700 genes that exhibit a complex pattern of meiotic transcriptional regulation. The majority of these genes are unknown open reading frames. We present a systematic computational analysis of the data from W303 MATa/~ cells compared to the gone expression pattern obtained from W303 MATa/a cells, incapable of executing the meiotic differentiation program. This enables us to distinguish between genes required for the early starvation response and those involved in coordinating nutritional cues with the cascade of transcriptional events that commit cells to meiosis. Finally, we present a molecular link between yeast and mammalian meiosis through sequence homologies.

9

04-16

Specific phosphorylation of the transcriptional activator Imel is required for its recruitment by the DNA-binding protein Ume6 to promoters of early meiosis-specific genes I. Rubin-Bejerano, Y. Kassir Faculty of Biology, Technion, Haifa 32000 Israel Saccharomyces cerevisiae diploid cells can chose between three alternative developmental pathways: mitosis, pseudohyphae growth, and meiosis. Initiation of meiosis depends on nitrogen depletion, the absence of glucose, and the presence of a non-fermentable carbon source such as acetate in the medium. The master regulator that responds to these signals is a transcriptional activator, Imel. Imel activates transcription of early meiosis-specific genes by association with a specific DNA-binding protein, Ume6. Interaction between Imel and Ume6 occurs only under the nutritional conditions that restrict entrance into meiosis, namely, absence of both nitrogen and glucose in the medium. We show that Ime l is a phosphoprotein, and that its pattern of phosphorylation depends on the nutrient signals. By random mutagenesis of IME1 several residues (Proline, Tyrosine and Serine) were identified to be required for the association of lmel with Ume6. Site-directed mutagenesis of one of the Serine residues to Aspartic acid permitted the interaction with Ume6 in vegetative medium, i.e. in the presence of glucose and nitrogen. Since the structure of Aspartic acid resembles that of a phosphorylated Serine, we conclude that phosphorylation of lmel is required for its association with Ume6. We will provide evidence for the role of various kinases, such as the GSK3-beta homologue, Riml 1, in phosphorylation of Imel.

276 9

04-Meiosis and sporulation 04-17

EME1, a Saccharomyces cerevisiae gene encoding a 130 kda protein interacting with microtubules, is essential for sporulation P. Trachtulcovfi*, I. Janatovfi*, S. D. Kohlwein#, and J. HaSek* * Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, #Institute of Biochemistry and Food Chemistry, Technical University, Graz, Austria The gene EME1 (ORF YOR304 W) of S. cerevisiae encoding a 130 kDa protein recognized by a monoclonal antibody against a mammalian 210 kDa microtubule-interacting protein was characterized. The protein Emel displays a strong similarity to Drosophila nucleosome remodeling factor ISWI. Although produced also in vegetative cells, the protein Emel is not essential for vegetative growth, but is indispensable for sporulation. The emel homozygous deletant diploid strain shifted to the sporulation medium was not able to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle. The overexpression of the Emel protein had the same effect. On the basis of protein sequence similarities and the phenotypic effect of the emel deletion, it is hypothesized that the protein Emel may play a role in chromatin remodelation and/or transcriptional activation of gene(s) required for sporulation. Using the confocal laser scanning microscopy, the intragenously tagged protein Emel was found to partially colocalize with microtubules in diploid cells, both vegetatively growing and incubated in the sporulation medium. This distribution suggests a cooperation between microtubules and the transcriptional apparatus within S. cerevisiae cells. This work was financed by the grants 204/96/1368 GACR to JH, 204/98/P256 GACR to PT, Fonds zur F6rderung der wissenschaftlichen Forschung in sterreich (project F706) to SDK, and the joint grant Kontakt ME222 to JH and SDK.

9

04-18

Regulation of transcription by Ume6: Meiotic and whole-genome studies B. K. Washburn, R. Williams, M. Primig, R. W. DavisI, R. E. Esposito Department of Molecular Genetic and Cell Biology, University of Chicago, 920 E. 58th St., Chicago IL 60637 Email: re-esposito (a~uchicago.edu 1Department of Biochemistry, Stanford University Medical School, Stanford CA, 94305 The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes through interaction with Sin3 (a corepressor) and Rpd3 (a histone deacetylase), and the meiotic activator Imel, respectively. Our finding that a Gal4 activation domain-Ume6 fusion fails to activate in the absence of Imel, and the analysis of ume6 mutations that suppress this failure, indicate that Imel not only provides an activation domain but also relieves Sin3-mediated repression. The suppressing mutations, which lie in a predicted amphipathic alpha helix, disrupt interaction with Sin3 in vivo and in vitro, and identify the Sin3 binding domain in Ume6, the first to be precisely defined in yeast. The mutations also provide evidence that premature expression of Ume6/Sin3-regulated genes does not inhibit meiosis and that Sin3 must play another role in meiosis independent of its interaction with Ume6. Finally, we have used a whole-genome micro-array approach to show that Ume6 plays a crucial role in the regulation of hundreds of genes required for metabolic functions, starvation response, cell-cycle progression and meiosis in S. cerevisiae.

04-Meiosis and sporulation 9

277

04-19

Expression of middle sporulation-specific genes is repressed during vegetative growth by Suml and Hstl, proteins involved in transcriptional silencing E. Winterl, J. Xie2, M. Pierce2, V. Gailus-Durner2, M. Wagnerl, and A.K. Vershon2 Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. USA Waksman Institute, Rutgers University, 190 Frelinghuysen Road, Piscataway, N.J. 08854, USA SMK1 encodes a MAP kinase that is expressed specifically during middle sporulation. The activation of SMK1 transcription requires a DNA element found in many middle promoters termed an MSE (consensus gNCRCAAAA/T) that binds to the NdtS0p transcription factor (Chu and Herskowitz 1988, Mol. Cell., 1:685 96). The MSE in SMK 1 is also required for its transcriptional repression in vegetative cells (Pierce et al. 1998, Mol. Cell. Biol. 18:5970 80). Mutants in 2 complementation groups that specifically derepress an MSE-containing reporter gene in vegetative cells have been isolated. The corresponding genes have been identified as SUM1 and HST1. SUMI has been shown by others to have genetic interactions with silencing (SIR) genes and HST1 is itself 72% identical to SIR2 over a stretch of 375 amino acids. Certain middle genes (including SMK1) are derepressed in suml mutants while others (including NDT80) are not. Heterologous promoters containing MSEs from middle genes that differ at positions within and adjacent to the core consensus show that some repress transcription in a SUM1 dependent fashion while others do not, Sumlp specifically binds to the repressing class of MSE and the core MSE consensus is required for this interaction. These data suggest that the transcriptional cascade during sporulation may be controlled by regulated competition for MSEs by the Sumlp repressor and NdtS0p activator.