Gene family of male-specific testosterone 16.alpha.-hydroxylase (C-P-45016.alpha.) in mouse liver: cDNA sequences, neonatal imprinting, and reversible regulation by androgen

Abstract

The cDNA clone p16.alpha.-1 for the male-specific isozyme (C-P-45016.alpha.)1 of testosterone 16.alpha.-hydroxylase in livers of 129/J mice [Harada, N., and Negishi, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2024-2028] and two additional full-length cDNAs overlapping with p16.alpha.-1 (p16.alpha.-2 and p16.alpha.-16) were sequenced. p16.alpha.-2 contained a single open reading frame of 1512 nucleotides, consisting of 71 base pairs of the 5''-noncoding region and 63 base pairs of the 3''-noncoding region with an additional poly(A) tract. From this DNA sequence, C-P-45016.alpha. was deduced to contain 504 amino acids with a calculated molecular mass of 56948 daltons. p16.alpha.-1 showed a nucleotide sequence identical with that of p16.alpha.-2 but lacked nine amino acid residues from the N-terminus. Another cDNA clone, p16.alpha.-16, also exhibited the same coding sequence with the exception of a 142 base pair deletion spanning from nucleotide 853 to nucleotide 994 of p16.alpha.-2. This deletion seems to be a whole exon of this gene, resulting in a shift of reading frame and an early termination codon at 10 amino acid residues from the deletion. The expected translation product of this mRNA is calculated to be 294 amino acids and 33,300 daltons. The putative poly(A) addition signal AATAAA is present for all three clones, but there are polymorphisms in the start sites of polyadenylation. Amino acid alignment with P-450b, P-450 M1, and P-450c revealed that P-45016.alpha. is 39.8%, 40.7%, and 31.5% homologous, respectively. Five highly homologous genes were selected from a genomic DNA library of BALB/cJ mice by plaque hybridization to the cDNA and characterized. An oligonucleotide only specific for p16.alpha.-1 and its gene was prepared from p16.alpha.-2 and its gene. The p16.alpha.-1 and the specific oligonucleotide were hybridized with liver poly(A)+ RNA from control and testosterone propionate treated adult 129/J mice that had been castrated at day 1 of birth or at adult age. The results indicated that p16.alpha.-1 recognizes at least two mRNAs in terms of their regulation by androgen; one is reversibly regulated and the other is neonatally imprinted in expression in adult livers. The two differentially regulated C-P-45016.alpha.-dependent testosterone 16.alpha.-hydroxylase activities in the liver microsomes from these mice were also detected and associated with the hybridizable mRNA levels. A hybridization of the specific oligonucleotide probe with the poly(A)+ RNA concluded that p16.alpha.-1 and p16.alpha.-2 encode C-P-45016.alpha., which is reversibly regulated.