Structural Analysis of Multiple Bovine P-450(11β) Genes and Their Promoter Activities1

Abstract

A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine DNA partially digested by EcoRI. Bovine P-450(11 beta) cDNA, pcP-450(11 beta)-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(11 beta) genomic DNA were isolated from 8 x 10(4) colonies and classified into five groups (CB11 beta-1, CB11 beta-3, CB11 beta-7, CB11 beta-20, and CB11 beta-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, CB11 beta-1, CB11 beta-3, and CB11 beta-21, were pseudogenes. Amino acid sequences coded by the other two clones, CB11 beta-7 and CB11 beta-20, were identical with that coded by a previously described cDNA, pcP-450(11 beta)-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(11 beta) 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, CB11 beta-7 and -20, were larger than those of pseudogenes, CB11 beta-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(11 beta) gene promoter did not express the activity in I-10 cells.

Novel cAMP Regulatory Elements in the Promoter Region of Bovine P-450(11β) Gene11

“…genes in the preceding paper (24). Determinations of the nucleotide sequences revealed that two genes, CB11/5-7 and CB11/?-20, were structurally normal whereas the other three, CB11/5-1, CB11/5-3, and CB11/9-21, were pseudogenes.…”

“…Culture Conditions for Y-l Cells and Transfection with Recombinant Plasmids-Y-l cells were cultured with Ham's nutrient mixture F-10 supplemented with 10% horse serum and 10% fetal bovine serum under a humidified atmosphere of 95% air and 5% CO 2 at 37"C. Plasmids for CAT assay were transfected into Y-l cells by electroporation using a Gene Pulser (Bio Rad Laboratories, Richmond, U.S.A.) as described in the preceding paper (24). Regulators (1 mM BtjcAMP, 25 /*M forskolin, 1//M ACTH, 30ng/ml TPA, 50 nM A23187, and lOOng/ml angiotensin II were added to the culture medium 12 h after the electroporation.…”

“…Regulators (1 mM BtjcAMP, 25 /*M forskolin, 1//M ACTH, 30ng/ml TPA, 50 nM A23187, and lOOng/ml angiotensin II were added to the culture medium 12 h after the electroporation. Cells were scraped off with a rubber policeman at 48 h after the electroporation and CAT assays were carried out as described in the preceding paper (24). Spots of acetylated products were cut from the TLC plates, and their radioactivities were measured with a liquid scintillation counter.…”

Identification of Novel First Exons in Ad4BP/SF-1 (NR5A1) Gene and Their Tissue- and Species-Specific Usage

“…rAd4ECAT2.0K contains an upstream region from the second exon of the rat gene as described (22). These CAT reporter gene constructs were transfected into mouse Y-1 adrenocortical tumor cells (29) to investigate which of the first exons is transcribed. RT-PCR using an RNA prepared from cells transfected with rAd4ECAT2.0K was performed with sets of primers, RAP1 and CAT1, ROP1 and CAT1, and RGP1 and CAT1, to amplify the rat reporter gene transcript containing exon Ia, Io, and Ig, respectively.…”

Human Adrenal Androgens: Regulation of Biosynthesis and Role in Estrogen-Responsive Breast Cancer in a Mouse Model

“…Representative sections of the tissue formed from the cells in animal groups A through F (see Table 1). Magnification x 100. sponsible for the conversion of deoxycorticosterone to aldosterone in other mammals, a specific member of the CYP11B gene family responsible for this conversion has not been described in cattle, and the mechanism of aldosterone biosynthesis in this species is still unresolved (Yanagibashi et al, 1986; Kirita et al, 1990).…”