Summary
We cloned the MET17 gene of Saccharomyces cerevisiae by functional complementation after transformation of a yeast met17 mutant. Restriction mapping and nucleotide sequencing of the MET17 clones revealed that these were from the same genomic region as clones isolated previously and shown to contain the MET25 gene encoding the enzyme O-acetylhomoserine, O-acetylserine sulphydrylase (OAH-OAS sulphydrylase). Transformation studies with MET25 clones showed that the MET17 and MET25 functions were both endoced in a single transcription unit. We conclude that met17 and met25 are both mutations in the structural gene for the OAH-OAS sulphydrylase subunit and that each affects a different fuctional domain of the enzyme allowing subunit complementation in the met17xmet25 diploid. Enzyme assays indicated that the diploid, although not requiring methionine, had a low OAH-OAS sulphydrylase activity (10% of wild type). This is consistent with MET17 and MET25 being the same gene. We found that both met17 and met25 mutants were devoid of 3′ phospho-adenosine 5′ phospho-sulphite (PAPS) reductase activity and that this activity was fully restored in the met17xmet25 diploid. The possible interactions between OAH-OAS sulphydrylase and PAPS reductase are discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcombe DT, Struhl K, Davis RW (1979) Sterile host yeasts (SHY): A eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17–24
Breton A, Surdin-Kerjan Y (1977) Sulphate uptake in Saccharomyces cerevisiae: Biochemical and genetic study. J Bacteriol 132:224–232
Carlson M, Botstein D (1982) Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase. Cell 28:145–154
Cherest H, Eichler F, de Robichon-Szulmajster H (1969) Genetic and regulatory aspects of methionine biosynthesis in Saccharomyces cerevisiae. J Bacteriol 97:328–336
Cherest H, Surdin-Kerjan Y, Antoniewski J, de Robichon-Szulmajster H (1973) S-adenosyl methionine mediated repression of methionine biosynthetic enzymes in Saccharomyces cerevisiae. J Bacteriol 144:928–933
Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 227–230
Grant WM (1947) Colorimetric determination of sulfur dioxyde. Anal Chem 9:345–346
Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933
Jones EW, Fink GR (1982) Regulation of amino acid and nucleotide biosynthesis in yeast. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces: Metabolism and gene expression. Cold Spring Harbor Laboratory, New York, pp 181–299
Kerjan P, Cherest H, Surdin-Kerjan Y (1986) Nucleotide sequence of the Saccharomyces cerevisiae MET25 gene. Nucleic Acids Res 14:7861–7871
Kredich NH, Tomkins GM (1966) The enzymatic synthesis of l-cysteine in Escherichia coli and Salmonella typhimurium. J Biol Chem 241:4955–4965
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Masselot M, de Robichon-Szulmajster H (1975) Methionine biosynthesis in Saccharomyces cerevisiae I. Genetical analysis of auxotrophic mutants. Mol Gen Genet 139:121–132
Masselot M, Surdin-Kerjan Y (1977) Methionine biosynthesis in Saccharomyces cerevisiae II. Gene-enzyme relationships in the sulphate assimilation pathway. Mol Gen Genet 154:23–30
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
de Robichon-Szulmajster H, Cherest H (1967) Regulation of homoserine-O-transacetylase, first step in methionine biosynthesis in Saccharomyces cerevisiae. Biochem Biophys Res Commun 28:256–262
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sangsoda S, Cherest H, Surdin-Kerjan Y (1985) The expression of the MET25 gene of Saccharomyces cerevisiae is regulated transcriptionally. Mol Gen Genet 200:407–414
Struhl K (1985) Naturally occuring poly (dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc Natl Acad Sci USA 82:8419–8423
Wiebers JL, Garner MR (1967) Acyl derivatives of homoserine as substrates for homocysteine synthesis in N. crassa, yeast and E. coli. J Biol Chem 242:5644–5649
Yamagata S (1976) O-acetylserine and O-acetylhomoserine sulphydrylase of yeast: Subunit structure. J Biochem (Tokyo) 80:787–797
Yamagata S, Takeshima K, Naiki N (1975) O-acetylserine and O-acetylhomoserine sulphydrylase of yeast: Studies with methionine auxotrophs. J Biochem (Tokyo) 77:1029–1036
Author information
Authors and Affiliations
Additional information
Communicated by W. Gajewski
Rights and permissions
About this article
Cite this article
D'Andrea, R., Surdin-Kerjan, Y., Pure, G. et al. Molecular genetics of met17 and met25 mutants of Saccharomyces cerevisiae: Intragenic complementation between mutations of a single structural gene. Mol Gen Genet 207, 165–170 (1987). https://doi.org/10.1007/BF00331505
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00331505