Fatty acids are essential structural and functional molecules required for the growth and maintenance of cells. The delta-9 fatty acid desaturase, which catalyzes the first committed step in unsaturated fatty acid synthesis in all eukaryotic cells, is a highly regulated lipid biosynthetic enzyme. It must respond to a diverse array of stimuli in order to meet the requirement for balanced synthesis of membrane lipids, to provide for the maintenance of membrane fluidity, to control toxic products produced by the enzyme and to allow for optimal utilization of exogenous fatty acids. The delta-9 desaturase enzyme in the yeast S. cerevisiae is encoded by the OLE1 gene, which has been cloned and sequenced by this laboratory. Experiments involving the expression of the delta-9 rat desaturase gene in yeast have further demonstrated broad functional homology between eukaryotic desaturases. The finding that OLE1 mRNA levels are strongly repressed by exogenous unsaturated fatty acids has led to the identification of several types of transcriptional regulatory control. Additional controls that appear to affect OLE1 mRNA stability have also been identified. In this application we propose to further study the regulation of the OLE1 gene. The regulation by exogenous fatty acids will be examined in detail since it is a physiologically significant effect that can be clearly defined and identification of its components may yield connections to other modes of desaturase regulation. DNA sequences identified in the OLE1 promoter will be tested to gain information about the size and complexity of the regulatory complex under both derepressed and repressed conditions. Trans-acting regulatory mutants that have been identified using OLE1 promoter-lacZ reporter genes will be used to clone and characterize genes involved in the regulatory response. Regulation of mRNA stability will be confirmed and described by locating the stability determinants within the OLE1 mRNA. 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 desaturase regulation in higher eukaryotes, including the homologous delta-9 enzymes in liver and adipose cells, and the biochemically uncharacterized desaturases that form eicosanoids and other polyunsaturated fatty acids.
Showing the most recent 10 out of 15 publications
