Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA.
A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.
D000818:Animals; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004247:DNA; D004790:Enzyme Induction / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D008099:Liver / Q000201:enzymology*; D008297:Male; D009693:Nucleic Acid Hybridization; D010634:Phenobarbital / Q000494:pharmacology*; D011061:Poly A; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D011061:Poly A; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital