Studies on the structure and regulation of two highly similar genes in the alcohol-inducible cytochrome P-450 subfamily.

Abstract

Liver microsomal cytochrome P-450s are a group of monooxygenases that are responsible for the metabolism of a wide variety of drugs and other foreign compounds in many species. Of particular biomedical interest is an alcohol-inducible P-450, designated as P-450 3a in rabbit, which catalyzes the oxidation of a number of compounds, including alcohols, acetone, and nitrosamines, and is suspected of playing a role in the conversion of acetaminophen, isoniazid, and some fluorine-containing anesthetics into hepatotoxic intermediates. The study presented in this thesis was concerned with the isolation and characterization of the genes encoding P-450 3a and a previously uncharacterized P-450 3a-like protein. As deduced from the sequence of two overlapping cDNAs, p3a-1 and p3a-2, encoding rabbit P-450 3a, this cytochrome was found to be 492 residues in length with a molecular weight of 56,820. The amino acid sequence of P-450 3a is approximately 50-60% identical to the sequence of rabbit P-450s 1, 2, and 3b and therefore can be grouped with these isozymes into a single family. With the use of 3a cDNA as a hybridization probe, at least two mRNAs differing in size were detected in rabbit liver. Treatment of rabbits with imidazole, which is known to cause a 4.5-fold increase in 3a protein, resulted in only a transient increase in the 3a mRNA level, indicating that the induction of 3a protein results largely from an increase in translational efficiency or in the half-life of the 3a protein. Clones for two distinct genomic loci, genes 1 and 2, were characterized and shown to encode, respectively, P-450 3a and a previously unidentified P-450 that is 97% identical in sequence to 3a. The genes are believed to share a common ancestral origin with one another and with the phenobarbital-inducible P-450 genes since the corresponding nine exons and their splice junctions in all of these genes are arranged identically with respect to the mRNA. Gene 2 mRNA is present in liver only, whereas gene I mRNA occurs in both liver and kidney. The differential distribution of the two gene transcripts may be related to the significant differences found in the 5$\sp\prime$ flanking sequences of genes 1 and 2.