Structural analysis of multiple bovine P-450(11β) genes and their promoter activities

A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine. DNA partially digested by EcoRI. Bovine P-450(11β) cDNA, pcP-450(11β)-2 [Morohashi et al. (1987) J. Biochem. 102, 559-568], was used as a probe for screening the genomic library. Ten clones carrying P-450(11β) genomic DNA were isolated from 8×10⁴ colonies and classified into five groups (CB11β-1, CB11β-3, CB11β-7, CB11β-20, and CB11β-21) according to differences in the restriction endonuclease sites. Nucleotide sequences of amino acid coding regions of the five clones were determined by the dideoxy sequencing method using synthetic nucleotides corresponding to various parts of the cDNA as primers. The nucleotide sequences revealed that three clones, CB11β-1, CB11β-3, and CB11β-21, were pseudogenes. Amino acid sequences coded by the other two clones, CB11β-7 and CB11β-20, were identical with that coded by a previously described cDNA, pcP-450(11β)-3 [Kirita et al. (1988) J. Biochem. 104, 683-686]. The promoter regions of the five clones were introduced in front of chloramphenicol acetyltransferase (CAT) gene of pSV00CAT and used to examine P-450(11β) gene regulation in cultured cells. The five recombinant plasmids showed cAMP-responsive CAT activities in Y-1 cells, a cell strain derived from adrenal tumor. The induction rates of the recombinant plasmids carrying the promoters of normal genes, CB11β-7 and -20, were larger than those of pseudogenes, CB11β-1, -3, and -21. CAT activities expressed by the promoter regions of the normal genes in the presence or absence of cAMP in Y-1 cells were almost equal to that by the promoter region of human P-450(SCC) gene. Though the promoter of the P-450(SCC) gene also showed cAMP-responsive CAT activity in I-10 cells, a cell strain derived from Leyding cell tumor, P-450(11β) gene promoter did not express the activity in I-10 cells.