The overall goal of this research is to understand the regulated biosynthesis of the rabbit liver microsomal cytochromes P-450. In this proposal the mechanisms of the phenobarbital induction and tissue-specific expression of the P450IIC subfamily will be studied. The P450IIC subfamily contains at least five members, which exhibit striking differences in responses to phenobarbital and different patterns of tissue-specific expression. The general approach will be to try to detect structural alterations in the chromatin that are tissue-specific or that correlate with treatment with phenobarbital. This approach permits an initial """"""""long range"""""""" analysis of the genes to identify potential regulatory regions of the gene and will ultimately result in the isolation and characterization of """"""""trans-acting"""""""" regulatory factors that bind to the gene. The second approach is to assess the effects of in vitro mutagenesis on the expression of the gene after transfection into mammalian cells. This approach should identify """"""""cis-acting"""""""" factors important for transcription. The isolation and characterization of the P450IIC genes will be completed. DNase I hypersensitive regions in liver chromatin from phenobarbital-treated and control rabbits will be detected by Southern blotting using indirect end-labelled probes. Protein binding sites within the hypersensitive regions will be detected by a modified S1 nuclease mapping of DNA from DNase I treated nuclei. Nuclear protein binding sites in the P450IIC genes will also be deteted by in vitro footprinting and the gel mobility shift assay. Expression of the genes will be assayed by transfection into liver-derived cells of the 5' flanking region of the genes fused to the bacterial chloramphenicol acetylase gene. Deletion and site-specific mutagenesis will be used to define regulatory regions and to correlate nuclear protein sites with gene expression. Cytochromes P450 catalyze the oxidation of a large number of drugs, toxic compounds and carcinogens, as well as endogenous compounds such as steroids and prostaglandins. The regulation of cytochrome P-450 levels by xenobiotics, therefore, can modulate the response to a drug or toxic compound. These studies on the expression of the cytochrome P-450 gene should increase our understanding of this important clinical problem.
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