Characterization of a cDNA Clone for Mouse Phenobarbital-Inducible Cytochrome P-450b

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Abstract
P40, a mouse 1040-nucleotide cDNA clone encoding a form of phenobarbital-inducible cytochrome P-450b, was selected by probing a cDNA library derived from phenobarbital-treated DBA/2N mouse liver with a rat 1830-nucleotide cDNA probe complementary to P-450b mRNA. When rat and mouse liver poly(A)+-enriched total RNAs are probed with p40, control rat P-450b mRNA levels are at least three times as much as control mouse P-450b levels. A 15-fold increase in rat 19S mRNA and only a 3-fold increase in the corresponding mouse 19S mRNA are found following phenobarbital treatment. An intranuclear 4800-nucleotide mRNA precursor is also detectable during the induction process with phenobarbital. Clofibrate, a hypolipidemic drug, is a potent inducer of P-450. There is no cross-hybridization between clofibrate-induced mRNA and either mouse P-450b cDNA or mouse P1-450 cDNA clones. At the level of hybridization stringency used for the Northern analysis, there is no cross-hybridization between phenobarbital-induced mouse or rat P-450b and the P1-450 cDNA probe or between 3-methylcholanthrene-induced mouse or rat P1-450 and the P-450b cDNA probe. These data indicate that the induction process by clofibrate involves activation of P-450 gene(s) not present in the multigene families inducible by either phenobarbital or polycyclic aromatic compounds, i.e., at least three sets of P-450 genes operate independently of one another. Two previously reported cDNA clones of phenobarbital-inducible rat P-450, R17 (P-450e) and pcP-450pb4 (P-450b), were compared with mouse p40. There are striking similarities in the three restriction maps and 87–96% homology in a 77-nucleotide region corresponding to exon 7 of the P-450e gene. Among the 25 amino acids where p40 could be compared, there is 88–100% homology among the two rat cDNA clones, the mouse p40, and the recently described P-450 protein sequence of rabbit liver microsomal form 2; no such homology is found with the recently reported protein sequence of P-450cam, a Pseudomonas putida drug-metabolizing enzyme. These data illustrate that a highly conserved region exists in the 3′ portion of phenobarbital-inducible genes from mouse, rat, and rabbit.

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