Gene, 95 (1990) 65-72 Elsevier

65

GENE 03697

Molecular cloning o f chromosome I D N A from Sacckaromyces cerevisiae: isolation, characterization and regulation o f the S P 0 7 sporulation gene (Recombinant DNA; yeast; meiosis; antimutator)

Wendy Whyte*, Leila H. Keopp *, Jaeque Lamb, Joan C. Crowley * and David B. Kabaek Department of Microbiolo~ and Molecular Genetics. Universityof Medicine and Dentistry of New Jersey-New Jersey Medical School, Graduate School of Biomedical Sciences. Newark, NJ 07103 (U.S.A.) Received by J.A. Gorman: 6 October 1989 Revised: 23 January 1990 Accepted: 22 June 1990

SUMMARY

SPOT is one of several previously identified genes from the yeast Saccharomycescerevisiae that is required for sporulation but not for vegetative growth. The SP07 gene has been cloned by functional complementation and physically mapped 15-16 kb to the left of CENI. Gene-disruption experiments confwmed that the cloned gene was the bona fide SP07 gene. SP07 codes for a 0.95-kb transcript that is expressed at approximately the same level in both vegetative and sporulating cells. The gene was sequenced and has the capacity to encode a 259-amino acid protein that does not appear to be related to other known proteins.

INTRODUCTION

Diploid yeast cells heterozygous for the mating type locus (MATa/MATg) und~T8o premeiotic DNA synthesis, meiosis and ascospore formation whe~ they are deprived of nitrogen and given a nonfermentable carbon source such as acetate (Roth and Halvorsor~, 1969). This process is characterized by a ~porulation-specific program of gene expression. Different genes show distinct temporal patterns Correspondenceto: Dr. D.B. Kaback, Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School MSB-F607, 185 S. Orange Ave., Newark, NJ 07103 (U.S.A.) Tel. (201)456-4192. * Current addresses: (W.W.) Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.) Tel. (212)430-2431; (LH.K.) Department of Mathematics and Science, Bloomfield College, Bloomfield, NJ 07003 (U.S.A.) Tel. (201)748-9000; (J.C.C.) Lederle Laboratories, Pearl River, NY 10965 (U.S.A.) Tel. (914) 732-3460. Abbreviations: aa, amino acid(s); AR$, autonomously replicatingsequences; bp, base pair(s); cM, centiMorgan; EM, electron microscopy; FUN, transcribed regions whose Function is Unknown Now; kb, kilobase(s) or 1000bp; MCS, multiple cloning site; nt, nucleotide(s); ORF, open reading frame; Pollk, Klenow (large) fragment of E. coil DNA polymerase 1; Spo, sporulation: wt, wild type; ::, novel joint (fusion). 0378-1119/90/$03.50© 1990Elsevier SciencePublishers B.V.(BiomedicalDivision)

of transcript accumulation during sporulation (Kaback and Feldberg, 1985). In addition, many genes show greatly induced RNA levels in sporulating cells. Several of these sporulation-induced genes have been cloned and studied (Clancy etal., 1983; Percival-Smith and Segall, 1986; Gottlin-Ninfa and Kaback, 1986). Some ate expressed late during sporulation and have been proposed to be involved in spore-wall formation (Kurtz and Lindquist, 1986; Law and Segall, 1988). However, the functions of most sporulation-induced genes are not known. Approximately 25 genes required for normal meiosis and spore formation but not for vegetative growth have been defined by SPO, MEI, CON, DIS, RED, MER, SPS, IME and HOP mutants (reviewed in Esposito and Klapholz, 1981; Rockmill and Fogel, 1988; Rockmill and Roeder, 1988; Engebrecht and Roeder, 1989; Percival-Smith and Segall, 1986; Kassir et al., 1988; Hollingsworth and Byers, 1989). In addition, several radiation-sensitive (PAD) mutants are sporulation-defective (reviewed in Esposito and Klapholz, 1981). SPOIl, SPOI3 and IMEI have been cloned and their gene expression has been examined (Atcheson et al., 1987; Wang et al., 1987; Kassir et al., 1988). The transcripts from these three genes are either not expressed or expressed at low levels in vegetativecells and

66

are expressed at much higher levels in sporulating cells. IMEI is required early in meiosis, S P O I l is required for meiotic recombination, and SPOI3 is required for meiosis I division. The times of transcript appearance for these three genes are consistent with their suggested role in sporulation. RADSO, a gene required for meiotic recombination, has been cloned (Kupiec and Simchen, 1984). Sequence analysis suggested that the RADSO gene product contains a purine nt-binding domain and may have a role in DNA metabolism (Alani et al., 1989). S P 0 7 was defmed by a Spo- mutant blocked prior to premeiotic DNA replication and was mapped very close to the chromosome I centromere (Esposito and Esposito, 1974; Mortimer and Schild, 1985; Kaback et al., 1989). The precise function of S P 0 7 is not known. However, this gene appears to he required earlier than most of the other known SPO genes (Esposito and Klapholz, 1981). In addition, spo7-1 mutants have an antimutator phenotype that is expressed in vegetative cells; i.e., the frequency of isolating certain suppressor mutations is lowered in spo7.1 mutants (Esposito et al., 1975). To begin to analyze the function of the S P 0 7 gene and its role in meiosis and sporulation, we have cloned and sequenced this gene and examined its expression during both vegetative growth and sporulation.

Sporulation in liquid was carried out as described previously (Kaback and Feldberg, 1985). Yeast transformations were done by the procedure of Ito et al. (1983) utilizing 0.3 M LiCI. Cells transformed with A R S containing plasmids were tested as previously described for plasmid stability using selective markers on the vectors to monitor plasmid presence (Steensma et al., 1987). One-step gene replacement was carried out by the method of Rothstein (1983). S P 0 7 was scored as described in Table II. To score S P 0 7 in haploids, strains were first crossed to either spo7-1 or spo7::LEU2 testers of opposite mating type. Resultant diploids were selected on appropriate media and tested for their ability to sporulate as described in Table If. (b) Recombinant DNA manipulations

Restriction mapping, DNA ligations, Southern blotting, nick translation, filter hybridization and other DNA manipulations were done by standard procedures (Maniatis et al., 1982). Where necessary, 5' overhanging ends were made blunt using Pollk. Escherichia coil strain HB101 was the host for all plasmids except those used in sequences analysis. Bacteriophage ~ was grown in E. coil strain C600.

32p-labeling by

RESUUfS AND DISCUSSION (n) Molecular cloning of the S P O T gene

MATERIALSAND METHODS

(a) Growth, genetic manipulation and sporulntion of Saccharomyces cere~islae

Yeast strains (Table 1) were grown and analyzed genetically as described previously (Sherman et al., 1973).

$ P 0 7 was mapped 8cM from ,~DEI and only 0.5-2,5 cM to the left of CENI (Mortimer and Schild, 1985; Kaback etal., 1989), Based on its genetic map position, SPOT was expected to be contained within the previously described cloned sequences surrounding C £ N !

TABLE ! Yeast strains Strain

Genotype or cross

Source

R372 DK35?-3A

MATI spo?.lcanl leul ura3.1 pet8 h~2 MATh adel trplleu2.3,112gallO

R, Esposito This study

R372 x DK357-3A

This study This study This study This study This study This study This study This study J. Szostuk This study This study This study This study R. Roth J. Game

LK2 LK2-19D LK2-20D LKI2 LK-8C LK2-14A LK6 LK2-18A DI39-11B LK16(DK366) LKI6tx3-1C LKI6t.x3-1D WWI SKI g716-SA

MATa spoT-I h~2 MATa adel urn3.1 gallO cam h~3-11,15

LK2-19D x LK2-20D MATa spoT.I trpl uro3.1/eu2-3,1128cliO canl MATa spoT-! trpl ura$.! leul

LK2-gc × LK2-14A MATa spoT.l ura$.l trpl lea2-3,112hD$.l!,!$ his2 MATa adel urn3.1 trpl lea2-3,112his.t-I1,15 canl

LK2-18A× DI39-11B MATerspo7-h:LEU2 lea2-3,112trpl ura$.l his2 canl-lO0 MATa $po7-I::LEU2 lea2-3,112trpl urn3-1hD2 canl-100

LKI6tx3-1C x LKI6tx3-1D MATu/MAT~HO/HO MATa hisl.7 honO.lO canl

67 TABLE II

(data not shown). Some of the other plasmids tested gave small increases in~/o sporulation, but the level was no higher than that seen with the YRp? vector alone. This effect could be due to the YRp7 vector or the Trp + phenotype. EM of R-loop containing DNA was used to map transcribed regions on the insert fromplasmid pLF31. The insert contained three transcribed regions expressed at 4 h in sporulating cells. These regions were located at the positions shown in Fig. 2A. R-loops corresponding to the two transcribed regions that mapped on the left side of the plasmid insert were seen infrequently indicating that these transcripts were not abundant. R-loops at the truncated transcribed region on the right side of the insert were seen several times more frequently, suggesting this transcript was more abundant than the other two. Further subcloning and transformation were used to localize the complementing activity to the leftmost intact transcribed region of the pLF31 insert (Fig. 2A). This is the only intact transcribed region on plasmid pLF67 and this plasmid gave the same level of sporulation when transformed into LK6 as plasmid pLF31 (Table 1I). Plasmid pLF62 appears to contain two intact transcribed regions including the one on pLF67. However, plasmid pLF62 did not complement spoT-1. Thus, the central transcribed region from plasmid pLF31 could not possibly be complementing spo?- 1. Since the function of the central transcribed region is not known, we designate it the FUNI4 gene. Since plasmid pLF62 is missing the 0.25-kb EcoR1.Sall fragment from the left, this fragment may contain sequences required for spo?-I complementation. In summary, these results suggest the leftmost intact transcribed region from pLF31 complemented the spoT-1 mutation and show that this region is located 15-16 i~b to the left of C£N1. Restriction map comparisons and hybridization experiments (data not shown) indicated that the truncated transcribed region on the right side of plasmid pLF31 (Fig. 2A) is the SSAI (HSP?O) gene (Craig et ~., 1988). In addition, this plasmid contains the TRNI (tRNApro) gene (Cummins et al., 1985).

Complementationof spoT-i by plasmids Strain a

LKI2 LK6 LK6 LK6 LK6 LK6 LK6 LK6 LK6 LK6

Relevant genotype

Plasmidb

SPOT/spo7-1 spoT-llspoT.l spo7-1/spoT-I spoT-I/spoT-I spoT-l/spoT-! spoT-i/spo7-1 spoT-I/spoT-i spoT-l/spoT-! spoT-l/spo7-1 spo7.1/spoT-i

% sporulationc

Untransformed Untransformed pLF31 pLF32 pLF36 pLF49 pLF34 YRp7 pLF67 pLF62

T~ +

T~-

62 m 43 4

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation, characterization and regulation of the SPO7 sporulation gene.

SPO7 is one of several previously identified genes from the yeast Saccharomyces cerevisiae that is required for sporulation but not for vegetative gro...
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