MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2809-2819 0270-7306/90/062809-11$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 6

Complementary Transcripts from Two Genes Necessary for Normal Meiosis in the Yeast Saccharomyces cerevisiae MICHAEL J. MALAVASICt AND ROBERT T. ELDER* Department of Molecular Genetics and Cell Biology, The University of Chicago, 1103 East 57th Street, Chicago, Illinois 60637 Received 27 November 1989/Accepted 25 February 1990

The SP012 gene, which is required for meiosis I chromosome division during sporulation of the yeast Saccharomyces cerevisiae, has been isolated. DNA sequencing has identified an open reading frame of 173 codons that encodes the putative SP012 protein and has no significant sequence similarities to known genes. The last 15 amino acids of this putative protein have a high negative charge, which appears to be required for function. A second sporulation-specific gene, designated SP016, was found adjacent to SP012 and shown to be necessary for efficient spore formation. The two genes are encoded on opposite DNA strands with only 103 nucleotides between the termination codons. Up to 700 nucleotides of the SP012 and SP016 transcripts are complementary, and the 3' untranslated region of the longest SP016 transcript is complementary to all or nearly all of the SP012 mRNA. A strain homozygous for an insertion which removes the complementarity between the SP012 and SP016 mRNAs has an efficiency of sporulation, number of spores per ascus, and spore viability identical to those of a wild-type strain. The complementarity therefore has either no function or only a subtle function in meiosis and sporulation. Genetic analysis of the yeast Saccharomyces cerevisiae has identified a large number of mutations affecting meiosis and sporulation (reviewed in references 6, 8, and 46). Since meiosis is coupled to sporulation in S. cerevisiae, the four haploid products of meiosis are packaged as spores in an single ascus. This process is initiated in S. cerevisiae by transferring a vegetatively growing culture of diploid cells to a sporulation medium containing a nonfermentable carbon source, usually acetate, but lacking a nitrogen source. Meiosis ordinarily occurs only in diploid cells, but techniques have been developed to isolate recessive mutations affecting sporulation. Many genes have roles in both meiosis and the mitotic cycle, but SPO genes function specifically in meiosis and sporulation. They are defined by mutations which affect sporulation but which do not noticeably affect vegetatively growing cells. On the basis of isolation of multiple alleles, 50 to 200 SPO genes are estimated to be required for normal meiosis and sporulation. SP012 and SP013 have particularly interesting roles in meiosis (20, 21). A diploid homozygous for either of the recessive mutations spol2-1 or spo13-1 has an altered meiosis that results in only two diploid spores in an ascus rather than the four haploid spores seen in the wild type. The single chromosome division which occurs in spol2-1 or spol3-1 strains usually has the characteristics of the second meiotic division. Therefore, the meiosis I chromosome division appears to be skipped in the mutant strains, but earlier and later meiotic events occur normally. Although spol2-1 and spol3-1 share this phenotype, their properties differ in other situations. In a haploid strain designed to allow meiosis and sporulation, a spol3-1 haploid undergoes sporulation to produce asci containing two haploid spores, but a similar spol2-1 haploid does not produce spores (47). This and other experiments (8, 27, 47) indicate that a spol3-1 strain is able

to complete meiosis and sporulation even when pairing of homologous chromosomes does not occur, but that pairing

of homologous chromosomes is necessary for completion of sporulation in a spol2-1 strain. A program of transcriptional regulation is an important part of the sporulation process in S. cerevisiae. Expression of SP013 is not seen in vegetatively growing cells, but increases at least 70-fold during meiosis and sporulation, with peak levels around the time of the meiosis I chromosome segregation (48). Similar results have been found for SPOIl, a gene necessary for meiotic recombination (23). SPOIl RNA levels increase about 70-fold during sporulation, peaking at the same time as SP013 RNA levels (2). The SPSI gene, which is required for spore formation, and SPS100, which is required to form ether-resistant spores at the normal time, also are highly expressed only in sporulating cells (25, 36). However, the relationship between the role of a gene in sporulation and sporulation-specific expression is not always straightforward. For example, SPOS is required for meiosis, plays no obvious role in the mitotic cycle, but is expressed at about the same level in mitotic and meiotic cells (53). Transcriptional regulation during meiosis may also be unrelated to function in some cases, since a number of genes whose transcription is increased during sporulation appear to have no role in meiosis and sporulation (9, 11, 18, 25, 36, 51; for a review, see reference 26). To gain further insight into the program of transcriptional regulation operating during sporulation, to compare the properties of the SP012 and SP013 genes, and to initiate studies on the molecular function of SP012, we have isolated the gene. A second SPO gene not previously characterized is found adjacent to SP012. The 3' portion of transcripts from this SP016 gene are complementary to SP012 mRNA, but removal of this complementarity has no obvious effect on meiosis and sporulation.

* Corresponding author. t Present address: Department of Microbiology, Georgetown University Schools of Medicine and Dentistry, Washington, DC 20007.

MATERIALS AND METHODS Strains, media, and plasmids. Escherichia coli LE392 and HB101 were used for most experiments with pBR322-de2809

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MALAVASIC AND ELDER

rived plasmids. M13 subclones for DNA sequencing and generation of single-stranded DNA probes were grown in JM103, JM105, or JM107 (32). Recombinant DNA procedures were carried out basically as described by Maniatis et al. (28). The library used to isolate the SUF8 gene was constructed by inserting DNA fragments greater than 4 kilobase pairs from S. cerevisiae YRE110 (MATa his4-713 lys2-20 SUF8-1; obtained from M. Culbertson), created by partial digestion with Sau3A, into the BamHI site of YCp19 (42). The library contained a total of about 8,000 plasmids. Yeast media are described by Klapholz and Esposito (22). Transformation was carried out on yeast spheroplasts (15). Sporulation was carried out at 30°C either on SP-III plates as described previously (20) or in liquid sporulation medium consisting of 2% potassium acetate (pH 7.0) and 75 ,ug of adenine sulfate and the amino acids His, Leu, Lys, Met, Try, and Tyr per ml. Cultures were grown in 1% yeast extract-2% peptone-2% potassium acetate-75 pg of adenine sulfate per ml to densities of about 107 cells per ml before being transferred to liquid sporulation medium at a cell density of about 5 x 107/ml. At least 200 cells were scored to calculate the frequency of asci and of three- or four-spored asci. The relevant genotypes of most yeast strains are given in Results. The three MA TotMATa strains used for preparation of sporulation RNA are Z270 and SK1 (48) and YRE92, which is heterozygous for spol2-1 and carries a number of auxotrophic mutations. The genotypes for the two strains used for the transplacements are YRE71 (MATot ho ade2 ade5 canjR CYH2S his7-2 leul-12 lys2-1 metl3-d SP012 trp5-d tyrl-l ura3-3,13) and YRE79 (MATa ho ade2 ADE5 CAN] s cyh2R his7-1 leul -c lys2-2 metl3-c SP012 trpS-c tyrl-2 ura3-1). To avoid any possible effect of the Ura+ versus Ura- phenotype on sporulation characteristics, transplacement with the 1.1-kilobase HindlIl fragment of URA3 (38) converted YRE71 and YRE79 into URA3 strains. The diploid resulting from crossing these two strains is the wild-type strain, with which the URA3 transplacements in the SP0121SP016 region (see Fig. 5) are compared. The structures of all transplacements were confirmed by hybridization analyses of gel transfers after digestion with the appropriate restriction endonucleases. The 1.1-kilobase HindIll URA3 fragment (38) was used for all three transplacements. For spol6-D2, EcoRI linkers (GGAATTCC) were added to the URA3 fragment. For spol2-DI, the EcoRI URA3 fragment was inserted in the EcoRI site of pBR322 and removed as a ClaI-EcoRI fragment before insertion into the C2-E3 sites (Fig. 1). For SP012-l3, XhoI linkers (CCTC GAGG) were added to the URA3 fragment and the RsaI site at positions 1036 to 1039 (Fig. 2). DNA sequence. DNA sequencing was carried out by dideoxy-chain termination (40) with either the Klenow fragment of DNA polymerase I or avian myeloblastosis virus reverse transcriptase on templates prepared from fragments inserted into M13mp8, M13mp9, M13mp18, or M13mp19 (32). Both strands of DNA were sequenced, and all restriction sites used to clone fragments into the M13 vectors were covered by a sequencing reaction. The data banks searched for sequence similarities included EMBL/GenBank Genetic Sequence Database (1986), GenBank (Bolt, Beranek and Newman Laboratories, Cambridge, Mass.) tape release 46; EMBL/GenBank Genetic Sequence Database (1986), EMBL (European Molecular Biology Laboratories, Heidelberg, Federal Republic of Germany), tape release 9; and the Protein Identification Resource (1986) Protein Sequence Data Base (National Biomedical Research Foundation,

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Complementary transcripts from two genes necessary for normal meiosis in the yeast Saccharomyces cerevisiae.

The SPO12 gene, which is required for meiosis I chromosome division during sporulation of the yeast Saccharomyces cerevisiae, has been isolated. DNA s...
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