Nudeotide sequence of the Saccharomyces cerevisiae MET

To elucidate further the molecular basis of the specific regulatory mechanism modulating the expression of the genes implicated in methionine metabolism, we have cloned and characterized two genes, MET3 and MET25, and shown that the regulation of their expression is transcriptional ( 1 , 2). The sequence of the cloned yeast MET25 gene which encodes the O-acetyl homoserine O-acetyl serine (OAH-OAS) sulfhydrylase is reported here along with its 5 and 3' flanking regions. The amino acid composition predicted from the DNA sequence is in good agreement with that determined by hydrolysis of the purified enzyme (3). In the 5 flanking region the signal for general amino acid control was not found, corroborating our previous finding that the synthesis of OAH-OAS sulfhydrylase is not submitted to general control. The transcription start points have been determined. The 5 and 3 flanking regions of the MET25 gene suggest initiation and termination signals similar to those associated with other yeast genes. INTRODUCTION The MET25 gene of Saccharomyces cerevisiae encodes the enzyme O-acetyl homoserine O-acetyl serine (OAH-OAS) sulfhydrylase (4, 5). We had shown that the synthesis of this enzyme is submitted to the same regulatory system as other enzymes implicated in methionine metabolism (6, 7). As a f irst step towards elucidating the molecular basis for repression of enzyme synthesis in this pathway, we have already cloned two genes : MET3 encoding ATP sulfurylase (1) and MET25 (2). We have shown that the regulation of MET3 and MET25 expression was transcriptional and, in the case of MET25 we have identified a 120 bp region necessary for regulation to take place. Here we report the complete nucleotide sequence of the MET25 gene with its 5' and 3 flanking regions. MATERIALS AND METHODS Strain and culture conditions The yeast strain used in this work was the wild type strain FL100 © IRL Press Limited, Oxford, England. 7 8 6 1 Nucleic Acids Research (MATa) obtained from Dr. F. Lacroute. Cultures used for the preparation of RNAs were grown to 2x10 cells per ml in YNB medium (0.7% Yeast Nitrogen base without amino acids, 2% glucose). DNA sequencing DNA sequence analysis was performed as described by Maxam and Gilbert (8). End labelling of 5 termini was performed with ( Y 3 2 P A T P ) using polynucleotide kinase as described in Maniatis et al . (9). Preparation of RNAs from S. cerevisiae and SI mapping of the transcripts The RNAs were extracted from cultures of strain FL100, and poly (A) RNAs were isolated on oligo-dT cellulose columns as described previously (1). Mapping experiments with nuclease SI were performed according to standard protocols (9, 10). The 200 pb BamHI-Hpall fragment from pM25-14 (2 and f ig . 3) which includes the 5 end of the MET25 gene was 5 end-labelled using polynucleotide kinase and the two ONA strands were separated on a 5% polyacrylamide sequencing gel. The coding strand was hybridized 4 hours at 44°C to 10 ug of poly(A) RNA In 30 ul of hybridization buffer (40 mM PIPES buffer pH 6.4, 1 mM EDTA, 40 mM NaCI, 80% formamide). The hybrid was then diluted 10 fold in S1 buffer (50 mM sodium acetate pH 4.6, 140 mM NaCI, 2.25 mM ZnS04 and 10 ug/ml of sonicated and denatured thymus DNA) containing 150, 500 and 1500 units of nuclease S I . The SI digestion was carried out for 30 minutes at 37°C. After recovery by ethanol precipitation, the RNA-DNA hybrids were analysed on a 5% polyacrylamide sequencing gel along with the chemical degradation products of the P end-labelled DNA probe used. Computer analysis Analysis of the MET25 sequence data was performed on a GOUPIL 3 microcomputer with programs designed for us by Daniel Kerjan, Computer Department of the Lycee Agricole of Le Rheu (France). RESULTS Nucleotlde sequence analysis of the MET25 gene We had cloned and studied previously the MET25 gene encoding O-acetyl serine O-acetyl homoserine (OAS-OAH) sulfhydrylase of ^ . cerevisiae (2). We had shown that this gene was beared on a 2.4 kb DNA fragment. A restriction map of this region together with the sequencing strategy is shown in Figure 1. We used 5 end labelling and secondary digestion to generate DNA fragments for analysis by the Maxam and Gilbert method. We determined 100% of the sequence on the two strands. The complete sequence of the