Isolation and Characterization of Saccharomyces cerevisiae mutants defective in Unsaturated Fatty Acid Recognition
Dr. Virginia McDonough, Hope College
Dr. Joseph Stukey, Hope College
When dietary unsaturated fatty acids (UFAs) are present, cells down regulate expression of the enzyme ∆9 desaturase, encoded by OLE1, which is responsible for producing UFAs. A screen was designed to isolate mutants defective in recognition of the presence of UFAs, and the subsequent transcriptional regulation of the ∆9-desaturase. Using a reporter gene construct controlled by the OLE1 promoter, several mutants were identified and one mutant, KH2, was chosen for additional characterization. The mutation that renders KH2 incapable of molecular response to UFAs is a recessive trait due to a single mutation. While the reporter construct demonstrates that transcriptional regulation of OLE1 is defective, fatty acid analysis reveals normal regulation in response to UFA supplements, indicating that post-transcriptional regulation is intact. This implies that the mutation is not responsible for universal response to UFAs, but just transcriptional regulation. Further work to identify genes responsible for regulation of OLE1 was initiated through genomic library screening. Initial results identified a genomic fragment that returned KH2 phenotype to wildtype. However, examination of individual genes on that fragment revealed that one gene may be a suppressor, rather than the responsible for the authentic phenotype due to mutation. Additional work is ongoing to determine the identity of this suppressor. In addition, further library work will attempt to identify the authentic gene responsible for OLE1 regulation.
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