Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1

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Abstract
Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3-fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats. Differential thermal elution of liver mRNA from DNA-RNA hybrids formed with the two plasmid DNAs followed by in vitro translation indicated that pTF-2 very likely corresponds to the mRNA for P-450 PB-1 whereas pTF-1 corresponds to that encoding a PB-1-related P-450 form not yet identified. On the basis of the available sequence data, it is concluded that the two clones represent members of a gene subfamily which is part of the P-450 PB-4/PB-5 gene family. The complexity of genomic blots obtained by using pTF-1 and pTF-2 as probes indicates that this P-450 PB-1 gene subfamily is likely to include several additional members.