Construction and identification of a hybrid plasmid containing DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver.

Cytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18S in the sucrose density gradient, accounting for approximately 5% of total mRNA activity on the basis of [3H]leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P]cDNA as a probe, a colony carrying cytochrome P-50 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.