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

Shirou Kirita, Toshihide Hashimoto, Masato Kitajima, Shin Ichiro Honda, Ken-Ichirou Morohashi, Tsuneo Omura

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1030-1041
Number of pages12
JournalJournal of Biochemistry
Volume108
Issue number6
DOIs
Publication statusPublished - Jan 1 1990
Externally publishedYes

Fingerprint

Structural analysis
Chloramphenicol O-Acetyltransferase
Genes
Clone Cells
Genetic Promoter Regions
Genomic Library
Nucleotides
Complementary DNA
Pseudogenes
Tumors
Plasmids
Amino Acids
Cosmids
DNA
DNA Restriction Enzymes
Glandular and Epithelial Neoplasms
Gene expression
Screening
Cells
Amino Acid Sequence

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Structural analysis of multiple bovine P-450(11β) genes and their promoter activities. / Kirita, Shirou; Hashimoto, Toshihide; Kitajima, Masato; Honda, Shin Ichiro; Morohashi, Ken-Ichirou; Omura, Tsuneo.

In: Journal of Biochemistry, Vol. 108, No. 6, 01.01.1990, p. 1030-1041.

Research output: Contribution to journalArticle

Kirita, Shirou ; Hashimoto, Toshihide ; Kitajima, Masato ; Honda, Shin Ichiro ; Morohashi, Ken-Ichirou ; Omura, Tsuneo. / Structural analysis of multiple bovine P-450(11β) genes and their promoter activities. In: Journal of Biochemistry. 1990 ; Vol. 108, No. 6. pp. 1030-1041.
@article{810262ba79d340faadb212168f324d34,
title = "Structural analysis of multiple bovine P-450(11β) genes and their promoter activities",
abstract = "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.",
author = "Shirou Kirita and Toshihide Hashimoto and Masato Kitajima and Honda, {Shin Ichiro} and Ken-Ichirou Morohashi and Tsuneo Omura",
year = "1990",
month = "1",
day = "1",
doi = "10.1093/oxfordjournals.jbchem.a123302",
language = "English",
volume = "108",
pages = "1030--1041",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "6",

}

TY - JOUR

T1 - Structural analysis of multiple bovine P-450(11β) genes and their promoter activities

AU - Kirita, Shirou

AU - Hashimoto, Toshihide

AU - Kitajima, Masato

AU - Honda, Shin Ichiro

AU - Morohashi, Ken-Ichirou

AU - Omura, Tsuneo

PY - 1990/1/1

Y1 - 1990/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0025601672&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025601672&partnerID=8YFLogxK

U2 - 10.1093/oxfordjournals.jbchem.a123302

DO - 10.1093/oxfordjournals.jbchem.a123302

M3 - Article

C2 - 1965187

AN - SCOPUS:0025601672

VL - 108

SP - 1030

EP - 1041

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 6

ER -