Isolation And Characterization Of A Novel Cytochrome P-450-like Gene.

Abstract

Cytochrome P-450, a pigment occurring widely in nature, is involved in the oxygenation and other metabolic reactions of a large variety of drugs, carcinogens, and other xenobiotics, as well as naturally occurring substances such as steroids and prostaglandins. A number of P-450 isozymes have already been purified to electrophoretic homogeneity from the rabbit, rat, and mouse, as well as from other species, and the amino acid sequence is known in several instances. Two major classes of P-450 inducers are known, represented by the drug phenobarbital and the chemical carcinogen methylcholanthrene. A rabbit liver genomic library was screened using a cDNA clone coding for a phenobarbital-induced rat P-450. A positive plaque was isolated and used to characterize a novel P-450-like gene. The new gene resembles known genes of the phenobarbital family in having nine exons and in the location of the intron-exon junctions. The predicted sequence of the corresponding protein was calculated to have an overall structural homology of 40% with respect to isozyme 2, the major phenobarbital-inducible P-450 in the rabbit. The homology is particularly evident in three small regions common to the cytochrome P-450 family, including the cysteine-containing peptide believed to provide the sulfur ligand to the heme iron atom. Several lines of evidence, including S(,1) mapping, indicate that the gene is expressed. However, neither phenobarbital nor isosafrole, a typical inducer of the methylcholanthrene type, appear to enhance the expression of the gene in rabbit liver. Of particular interest, the amino acid sequence deduced from the gene has higher homology at the carboxyl-terminal region than at the amino-terminal region as compared with the corresponding sequence of isozyme 2.