Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation

Abstract

By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16 alpha) encoding mouse liver microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice. mRNA selected by hybridization with clone p-16 alpha translated the P-450(16) alpha apoprotein in vitro. Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B. Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-450(16) alpha and P-450(15) alpha specifically inhibited testosterone 16 alpha-hydroxylase activity in microsomes. The cDNA insert of one recombinant plasmid (clone P-16 alpha-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I. 32P-labeled clone p-16 alpha-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice. This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-450(16) alpha in vitro as did the poly(A)+ RNA from females. Thus, the predominant expression of testosterone 16 alpha-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-450(16) alpha gene.