The eukaryotic 9 fatty acid desaturate, which catalyzes the initial conversion of saturated to unsaturated fatty acids is a highly regulated enzyme. It responds to diverse array of stimuli to meet cellular requirements for balanced synthesis of membrane lipids and the optimal use of nutrients lipids. This laboratory clone and sequenced the Saccharomyces OLE1 gene, which encodes that enzyme. Expression of rat- yeast chimeric desaturates in ole1 gene-disrupted cells demonstrated that there is functional homology between the animal and fungal enzyme systems. OLE1 was found to be regulated by a number of specific nutritional and physiological conditions. Transcriptional controls were identified that are triggered by exogenous fatty acids, by changes in carbon source and by carbon starvation. A second set of controls was found to independently act to regulate OLE1 mRNA stability. These studies also suggested that there are similarities between its expression and the less well defined animal systems. The central working hypothesis of this research is that proteins that regulate desaturate activity also control other genes involved in lipid utilization. These include sterol biosynthetic enzymes and fatty acid chain elongation enzymes. This proposal is to define the regulators of OLE1 expression. Modified regulatory genes will then be used to identify connections that lead to the coordinated control of lipid synthesis and utilization. Regulatory DNA sequences in the OLE1 promoter will be identified and modified to be used to clone and characterize genes encoding members of that regulatory circuit. Regulation of OLE1 mRNA stability will be characterized by locating the stability determinants within the mRNA. That information will be used to identify trans-acting regulators at that level of OLE1 expression. These studies will lead to new information concerning the regulation and cellular functions of unsaturated fatty acids in yeast. They are also expected to be relevant to desaturate regulation in higher eukaryotes, including the homologous 9 enzymes in liver and adipose cells, and the biochemically uncharacterized desaturates that form eicosanoids and other polyunsaturated fatty acids.
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