3 FORMS OF RAT CYP11B GENES - 11 BETA-HYDROXYLASE GENE, ALDOSTERONE SYNTHASE GENE, AND A NOVEL GENE

We isolated three genomic clones of rat P-450(11beta) genes (CYP11B). Two of them corresponded to 11beta-hydroxylase gene (CYP11B1) and aldosterone synthase gene (CYP11B2), respectively. The third one was a novel gene resembling both CYP11B1 and CYP11B2, and was named CYP11B3 gene (CYP11B3). CYP11B2 and CYP11B3 are located tandemly in the genome in the same direction approximately 24 kb apart. These three genes were highly homologous in their amino acid coding regions, with 88% (CYP11B1 to CYP11B2), 89% (CYP11B2 to CYP11B3),96% (CYP11B1 to CYP11B3) nucleotide identity. The numbers and the locations of the exons of these three genes also exactly corresponded to each other. However, the nucleotide sequences of the 5' upstream regions of CYP11B1 and CYP11B2 were significantly different, suggesting different transcriptional regulations. CYP11B3 had almost the same sequence as CYP11B1 gene in the 5' upstream region. A putative Ad4 site, a cis-acting element present in the promoter regions of all the steroidogenic P-450s so far reported [Morohashi, K., Honda, S., Inomata, Y., Handa, H., Omura, T. (1992) J. Biol. Chem. 267, 17913-17919], was found in the promoter regions of both CYP11B1 and CYP11B2. Gel retardation analysis showed the binding of Ad4BP purified from bovine adrenal cortex to these two Ad4 sites. We analyzed the relative abundance of the mRNAs corresponding to these three genes by the generation of RT-PCR libraries from rat adrenal total RNAs. In the RT-PCR library from normal-diet rats, the recombinants generated from CYP11B1 were dominant, but the relative abundance of the mRNA corresponding to CYP11B2 was elevated by 12-fold when the rats were treated with low Na+-high K+-diet. The expression of CYP11B3 could not be detected even by the RT-PCR method.