Sexual dimorphism of testosterone 15 alpha-hydroxylase mRNA levels in mouse liver. cDNA cloning and regulation

Abstract

A cDNA library was constructed from 129/J female mouse liver poly(A)+ RNA immunoenriched for testosterone 15 alpha-hydroxylase (P-450(15)alpha) in the expression vector pUC9. Fifteen clones coding for P-450(15)alpha were identified by duplicate colony hybridization to nick-translated cDNAs synthesized from immunoenriched or depleted poly(A)+ RNA. Recombinant plasmid p 15 alpha-29 contained the largest cDNA (1.6 kilobases) and cross-hybridized strongly with all other clones. cDNAs representing two different transcripts were identified based on restriction map analysis. Clones p 15 alpha-29 and p 15 alpha-15 have identical internal restriction fragments generated by ClaI, BamHI, and StuI digests, but p15 alpha-15 has unique PstI and HindIII sites located within 150 base pairs of each other. This suggests P-450(15)alpha is transcribed from two genes. Northern hybridization of poly(A)+ RNA with nick-translated p15 alpha-29 or p15 alpha-15 showed a single size mRNA of 2.1 kilobases. The level of P-450(15)alpha mRNA was 6.6 times higher in 129/J female than in male mice. The difference between P-450(15)alpha mRNA levels in females and males was positively correlated with P-450(15)alpha protein detected by anti-P-450(15)alpha and testosterone 15 alpha-hydroxylase activity in microsomes. Sexual dimorphism in hepatic testosterone 15 alpha-hydroxylase activity is shown to result from differential mRNA regulation in females and males.