A cytochrome P-450 multigene family. Characterization of a gene activated by phenobarbital administration

The capacity of the liver microsomal mixed function oxidase system to metabolize a great variety of exogenous as well as endogenous compounds is thought to reflect the participation of multiple forms of the terminal oxidase, cytochrome P-450, which have different broad, but overlapping, substrate specificities. Several of these isozymes accumulate in the liver after exposure of animals to specific inducing agents. In order to approach the questions of the genetic basis for the existence of multiple cytochrome P-450 isozymes and the molecular mechanisms of the induction process, we have used a cloned cDNA for a major phenobarbital-induced form of cytochrome P-450 to identify and characterize thirteen distinct rat genomic clones containing segments of six different genes. Only three clones, representing overlapping segments of a single gene, hybridized to the cloned cDNA or to phenobarbital-induced mRNA under conditions of high stringency. In vitro transcription studies with isolated rat liver nuclei indicated that only this gene, but not the genes represented by the other genomic clones, appears to be markedly activated by phenobarbital treatment of rats. Although there are a small number of differences, DNA sequence analysis of the eight exons of the gene present in these genomic clones indicates that they encode residues 58 to 491 (the COOH terminus) of cytochrome P-450e, a major phenobarbital-induced isozyme. It appears that the other cloned genes may contain only a small region of very strong homology to the cytochrome P-450e gene, a region which includes the exon encoding a tridecapeptide which is also present in two dissimilar forms of rabbit liver cytochrome P-450, one constitutive and one induced by phenobarbital. Southern blotting analysis of rat liver DNA indicates that the rat genome may contain two additional genes which are very closely related to the cytochrome P-450e gene but which we have not yet isolated from the genomic library. One of these genes is likely to encode cytochrome P-450b, the major phenobarbital induced form of cytochrome P-450 whose mRNA is greater than 95% homologous to that encoding cytochrome P-450e.