Isolation of the mouse cytochrome P450J (CYP2E1) cDNA and its reciprocal testosterone regulation in kidney and liver

Abstract

ABSTRACT A hybridization probe that is homologous to the B2 short interspersed repetitive element detects an mRNA in mouse kidney and liver that is regulated by androgen. Administration of testosterone induces this mRNA in kidney and represses it in liver. The mRNA was cloned by first using the B2 probe to select 48 cDNA clones from an androgen-induced kidney library. These clones were then tested for their androgen response by hybridizing them with probes made by reverse transcription of basal and testosterone-treated kidney poly(A)+ RNA. Any homology to the B2 sequence was masked by prehybridizing the filters to an excess of non-radioactive RNA synthesized from a B2 sequence cloned into a riboprobe vector. A unique sequence was subcloned from the largest androgen-responsive cDNA clone. A radioactive riboprobe generated from the unique sequence subclone detected an androgen-responsive mRNA in Northern blots with the same electrophoretic mobility as the predominant androgen-responsive mRNA detected with the B2 homologous riboprobe. The riboprobe also detected a unique sequence in Southern blots of genomic DNA. This subclone was then used as the probe to isolate a full-length cDNA clone from a second androgen-induced kidney library. When sequenced, this full-length cDNA of an androgen-responsive, B2-containing mRNA showed strong homology to the rat and human cytochrome P450J and the rabbit cytochrome P450 3a genes (CYP2E1). It showed only weak homology to the mouse testosterone 15 α-hydroxylase gene (CYP2A3) which is also regulated reciprocally by androgen in kidney and liver. The sequence of mouse P450J is identical to the B2 homologous mRNA previously named B2+mRNAx which is abundant in mouse liver.