Regulation of mouse cytochrome P3-450 by the Ah receptor. Studies with a P3-450 cDNA clone

Abstract

The Ah locus in the C57BL/6N mouse regulates at least 2 cytochrome P-450 gene products, termed in the mouse P1-450 and P3-450; these 2 enzymes are so named because each is responsible for the highest turnover number for the substrates benzo[a]pyrene and acetanilide, respectively. A cDNA library was prepared in pBR322 from sucrose gradient fractionated total liver poly(A+)-enriched RNA (.apprx. 20 S) from 2,3,7,8-tetrachlorodibenzo-p-dioxin- (TCDD) treated C57BL/6N (Ahb/Ahb) mice. Differential colony hybridization screening, with [32P]cDNA [complementary DNA] probes derived from total liver mRNA of both TCDD-treated and control C57BL/6N mice, yielded pP3450-21 (1710 base pair) and pP1450-57 (1770 base pair) cDNA clones. pP1450-57 was found to have 690 base pairs 5''-ward of the original P1-450 cDNA cloned in this laboratory. Restriction maps of pP3450-21 and pP1450-57 are markedly different and clearly are derived from separate genes. By means of hybridization-translation-arrest experiments, anti-(P3-450) precipitates the translation product (MW .simeq. 55,000) of mRNA specifically hybridizing to pP3450-21. Hybridization-translation-arrest experiments using polyclonal antibodies are not specific for proof on a P-450 cDNA clone. pP3450-21 was used to probe liver mRNA from Ahb/Ahb, Ahb/Ahd and Ahd/Ahd mice treated with 3-methylcholanthrene, .beta.-napthoflavone, aroclor 1254, isosafrole, low TCDD, or high TCDD. These genetic data rigorously demonstrate control of the P3-450 (20S) mRNA induction process by the Ah receptor. pP3450-21 fragments hybridized to TCDD-induced C57BL/6N mRNA and to a portion of the cloned 5'' end of the P1-450 gene from a mouse MOPC 41 plasmacytoma library. Fragments of 4 genomic clones having at least parts of the P1-450 gene were probed with pP3450-21 and pP1450-57. The P1-450 (20S) mRNA exhibit a highly homologous region of at least several hundred base pairs in the 5'' portion.