Isolation and characterization of mutations affecting expression of the delta9- fatty acid desaturase gene, OLE1, in Saccharomyces cerevisiae.
Expression of the delta9- fatty acid desaturase gene, OLE1, of Saccharomyces cerevisiae is negatively regulated transcriptionally and post-transcriptionally by unsaturated fatty acids. In order to isolate mutants exhibiting irregulation of OLE1 expression, we constructed an OLE1p-PHO5 fusion gene as a reporter consisting of the PHO5 gene encoding repressible acid phosphatase (rAPase) under the control of the OLE1 promoter (OLE1p). By EMS mutagenesis, we isolated three classes of mutants, pfo1, pfo2 and pfo3 positive regulatory factor for OLE1) mutants, which show decreased rAPase activity under derepression conditions (absence of oleic acid). Analysis of the transcription of OLE1 in these pfo mutants revealed that pfo1 and pfo3 mutants have a defect in the regulation of OLE1 expression at the transcriptional level while pfo2 mutants were suggested to have a mutation affecting OLE1 expression at a post-transcriptional step. In addition, four other classes of mutants, nfo1, nfo2, nfo3 and nfo4 (negative factor for OLE1) mutants that have mutations causing strong expression of the OLE1p-PHO5 fusion gene under repression conditions (presence of oleic acid), were isolated. Results of Northern analysis of OLE1 as well as OLE1p-PHO5 transcripts in nfo mutants suggested that these mutations occurred in genes encoding global repressors. We also demonstrated that TUP1 and SSN6 gene products are required for full repression of OLE1 gene expression, by showing that either tup1 or ssn6 mutations greatly increase the level of the OLE1 transcript.
Fujimori
K
K
Takarazuka Research Institute, Novartis Pharma K.K., Japan.
Anamnart
S
S
Nakagawa
Y
Y
Sugioka
S
S
Ohta
D
D
Oshima
Y
Y
Yamada
Y
Y
Harashima
S
S
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D044943:Fatty Acid Desaturases / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009687:Nuclear Proteins; D019301:Oleic Acid / Q000378:metabolism; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013230:Stearoyl-CoA Desaturase
C065455:CYC8 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; C083348:ROX1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C066502:TUP1 protein, S cerevisiae; D019301:Oleic Acid; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase; D000135:Acid Phosphatase