We propose to investigate the genetic and evolutionary mechanisms which account for the existence of multiple isozymes of the microsomal mono-oxygenase, cytochrome P-450, with different but overlapping substrate specificities as well as the molecular mechanisms which operate to regulate the expression of the genes encoding these enzymes during development, in different tissues and after treatment of animals with specific inducing agents. These studies will involve a detailed structural and functional characterization of several members of a family of genes which contains the major phenobarbital-induced cytochrome P-450 of rat liver which we have already cloned as well as the isolation and similar characterization of the genes for two major constitutive cytochrome P-450 isozymes of rat liver. One of these is responsible for the testosterone 16 Alpha-hydroxylase found only in the livers of male rats and its expression in adult life is determined by """"""""androgenic imprinting"""""""" in early postnatal life of hypothalamic centers which appear to regulate the pituitary control of sex-dependent hepatic steroid metabolism. The structural studies on the P-450 genes are directed toward elucidating their interrelationships and the structural similarities of their encoded proteins. The regulatory studies deal primarily with the role of chromatin structure and DNA methylation in modulating P-450 gene expression and the identification, using DNA mediated transfection experiments with chimeric genes, of cis-acting regulatory elements involved in controlling expression of the various genes.
