Molecular cloning and coding nucleotide sequence of complementary DNA of cytochrome P-450 involved in metabolic...

Complementary (c)DNA clones of phenobarbital-inducible cytochrome P-450 which has a strong activity toward metabolic activation of aflatoxin B1 were identified by a hybridization-arrested translation assay and a positive hybridization-translation assay. The coding nucleotide sequence of the cytochrome P-450 mRNA was determined by analysis of three cloned cDNAs and by primer extension method using a 5' terminal region fragment (62 bp) of a clone (pcP-450 pb-2) as a primer. The deduced amino acid sequence of the cytochrome is composed of 491 amino acid and its predicted molecular weight and amino acid composition concur with those determined with the purified protein. The sequence of one of the three cloned cDNAs is not completely the same as that of the other two. Fourteen nucleotide substitutions occur in their 922 overlapping nucleotides and 7 of them result in 6 amino acid replacements, therefore indicating the presence of at least two similar but distinct mRNAs for phenobarbital-inducible cytochrome P-450. These substitutions occur in a limited portion of the sequence, apparently forming some sort of a "variable regions."