Molecular cloning of a complementary DNA to 3-methylcholanthrene-inducible cytochrome P-450 mRNA from rat liver.

Poly(A)+ RNA was isolated from membrane-bound polysomes of the livers of 3-methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte lysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5% of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli X1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56,000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MC-, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridization with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)