Molecular cloning and sequence analysis of cDNAs coding for 3-methylcholanthrene-inducible cytochromes P450 in...

Liver microsomes of Xenopus laevis were investigated for specific cytochrome P450s (CYPs) that would be inducible in response to the administration of either 3-methylcholanthrene (3MC) or beta-naphthoflavone (BNF), potent inducers for mammalian CYP1A. When probed with antibodies raised against rat CYP1A1, a 54-kDa protein was detected after administration of polycyclic aromatic hydrocarbons. However, there was no immunoreactive protein in microsomes from untreated frogs. In order to obtain structural information about this CYP1A-like protein, a liver cDNA library of 3MC-treated frog was constructed and screened using a fragment of rat CYP1A2 cDNA under low stringency conditions. We have isolated two cDNA clones (MC1 and MC2) with inherent features of the CYP1A subfamily. The sequence determination revealed that both of them coded for polypeptides composed of 526 amino acid residues, which differed from each other by 30 amino acids. A comparison with other mammalian CYP enzymes demonstrated that both of the sequences share 55 to 63% identity with the sequences of CYP1A family members. Northern blot analysis and RT-PCR results further demonstrated that two discrete transcripts corresponding to clones MC1 and MC2 are indeed inducible in the frog liver by treatment with 3MC or BNF. The names CYP1A6 and CYP1A7 were given to clones MC1 and MC2, respectively.