Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation

P. C. White, M. I. New, B. Dupont

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Abstract

We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450(C21)). Serum from rabbits immunized with purified P-450(C21) precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450(C21) on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450(C21) was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC·dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450(C21) serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti P-450(C21) serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, PC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450(C21). The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450(C21) and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Original languageEnglish (US)
Pages (from-to)1986-1990
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number7 I
StatePublished - 1984

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Hydroxylation
Cytochrome P-450 Enzyme System
Organism Cloning
Complementary DNA
Steroids
Plasmids
Messenger RNA
Clone Cells
Serum
Restriction Mapping
Peptides
Staphylococcal Protein A
Phenobarbital
Immunoassay
Base Pairing
Sucrose
Molecular Biology
Polyacrylamide Gel Electrophoresis
Proteins
Swine

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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abstract = "We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450(C21)). Serum from rabbits immunized with purified P-450(C21) precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450(C21) on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450(C21) was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC·dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450(C21) serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti P-450(C21) serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, PC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450(C21). The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450(C21) and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.",
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N2 - We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450(C21)). Serum from rabbits immunized with purified P-450(C21) precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450(C21) on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450(C21) was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC·dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450(C21) serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti P-450(C21) serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, PC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450(C21). The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450(C21) and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

AB - We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450(C21)). Serum from rabbits immunized with purified P-450(C21) precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450(C21) on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450(C21) was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC·dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450(C21) serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti P-450(C21) serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, PC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450(C21). The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450(C21) and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

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