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The overall goal of this research proposal is to understand the molecular mechanisms involved in the regulation of rat cytochrome P450c27/25 (c27/25) gene expression. P450c27/25 catalyzes two physiologically important reactions in the hepatic mitochondria namely, 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. 27-hydroxylation of cholesterol is believed to play an important role in cholesterol homeostasis by way of regulating some of the early steps of cholesterol biosynthesis, as well as its conversion to bile acids. Similarly, 25-hydroxylation of D3 comprises the first step in its conversion to the active form. The active form of vitamin D, 1-alpha, 25-dihydroxy vitamin D3, is a hormone functioning in maintenance of calcium and phosphate homeostasis.

The specific objective of this proposal is to understand the precise functional and biological significance of the gene overlap between the c27/25 and serine protease inhibitor SPI-1 (SPI) genes in the c27/25 gene expression. The sequence complementarity between the 5' terminal regions of mRNAs for c27/25 and SPI is unique and probably represents the first observation of 5' overlap between two functional mRNAs. The RNA duplex formed between the 5' ends of c27/25 and SPI mRNAs will be identified and how 5' end blocked c27/25 mRNA undergoes translation will be studied in detail. Translation of c27/25 mRNA will be studied in the presence and absence of SPI mRNA by preparing several mutant forms of c27/25 cDNA, using deletion or site directed mutagenesis, and expressing them along with wild type cDNA in COS cells. The structural and functional analyses of the gene locus shared by c27/25 and SPI will be carried out by mapping the transcription start site and identifying the cis-elements necessary for the basal promoter activity. Also growth hormone (GH) response element for the c27/25 gene will be characterized. Rat primary hepatocytes, under conditions shown to maintain GH response, will be used to characterize GH response element. Binding of proteins to GH response element will be studied by DNase I footprinting and gel shift assays and confirmed by transfection in primary hepatocytes and COS cells.
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