Identification and quantitation of a 2.0-kilobase messenger ribonucleic acid coding for 3-methylcholanthrene-induced cytochrome P-450 using cloned cytochrome P-450 complementary deoxyribonucleic acid

Abstract

A plasmid containing DNA complementary to 1 of the 2 size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450 from rat liver was used to characterize and quantitate that mRNA. The plasmid used was constructed and identified as follows: total poly(A+) RNA from 3-methylcholanthrene-induced liver was used as a template for c[complementary]DNA synthesis. Double-stranded cDNA was inserted into plasmid pBR322 by the G-C tailing procedure. Recombinants were screened by colony hybridization using as probe [32P] cDNA prepared from partially purified cytochrome P-450 mRNA. A differential screening approach was used in which duplicate filters were hybridized with probe from either 3-methylcholanthrene treated or untreated rats. Plasmid p23 was strongly positive by colony hybridization. It was conclusively shown to contain cytochrome P-450 cDNA sequences by demonstrating that the mRNA which specifically hybridized to nitrocellulose-bound plasmid p23 could be translated in vitro into peptides that were immunoprecipitable with monoclonal antibodies specific for 3-methylcholanthrene-induced cytochrome P-450. The size and quantity of the mRNA complementary to plasmid p23 were determined by hybridization of the 32P-labeled plasmid to rat liver RNA that had been fractionated by electrophoresis under fully denaturing conditions and transferred to diazobenzyloxymethyl-paper. Plasmid p23 hybridized strictly to a single size of mRNA that was .apprx. 2000 nucleotides in length, the smaller of the 2 size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450. The 2 size classes of 3-methylcholanthrene-induced cytochrome P-450 mRNA at least within the region of the mRNA contained within the insert of plasmid p23, were very different in sequence. The mRNA complementary to plasmid p23 was increased .apprx. 4-fold after in vivo administration of 3-methylcholanthrene under conditions that result in maximal induction of 3-methylcholanthrene-induced cytochrome P-450 enzymatic activity. This increase in cytochrome P-450 mRNA parallels the increase in cytochrome P-450 enzymatic activity observed after 3-methylcholanthrene administration. The regulation of mRNA levels is an important point of control of cytochrome P-450 gene expression.