ISOLATION AND CHARACTERIZATION OF MUTATIONS AFFECTING EXPRESSION OF THE DELTA-9-FATTY-ACID DESATURASE GENE, OLE1, IN SACCHAROMYCES-CEREVISIAE

Expression of the Delta 9- fatty acid desaturase gene, OLE1, of Saccha romyces cerevisiae is negatively regulated transcriptionally and post- transcriptionally by unsaturated fatty acids, In order to isolate muta nts exhibiting irregulation of OLE1 expression, we constructed an OLE1 p-PHO5 fusion gene as a reporter consisting of the PHO5 gene encoding repressible acid phosphatase (rAPase) under the control of the OLE1 pr omoter (OLE1p). By EMS mutagenesis, we isolated three classes of mutan ts, pfo1, pfo2 and pfo3 (positive regulatory factor for OLE1) mutants, which show decreased rAPase activity under derepression conditions (a bsence of oleic acid), Analysis of the transcription of OLE1 in these pfo mutants revealed that pfo1 and pfo3 mutants have a defect in the r egulation of OLE1 expression at the transcriptional level while pfo2 m utants were suggested to have a mutation affecting OLE1 expression at a post-transcriptional step. In addition, four other classes of mutant s, nfo1, nfo2, nfo3 and nfo4 (negative factor for OLE1) mutants that h ave mutations causing strong expression of the OLE1p-PHO5 fusion gene under repression conditions (presence of oleic acid), were isolated, R esults of Northern analysis of OLE1 as well as OLE1p-PHO5 transcripts in nfo mutants suggested that these mutations occurred in genes encodi ng global repressors, We also demonstrated that TUP1 and SSN6 gene pro ducts are required for full repression of OLE1 gene expression, by sho wing that either tup1 or ssn6 mutations greatly increase the level of the OLE1 transcript. (C) 1997 Federation of European Biochemical Socie ties.