Characterization of complementary deoxyribonucleic acid for human adrenocortical 17α-hydroxylase: A probe for analysis of 17α-hydroxylase deficiency

K. D. Bradshaw, M. R. Waterman, R. T. Couch, E. R. Simpson, M. X. Zuber

Research output: Contribution to journalArticle

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Abstract

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17α-hydroxylase (P-450(17α)) activity in adrenal, ovary, and testis as well as human 17α-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450(17α) enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450(17α) cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17αH) was selected for further characterization. pcD-17αH encodes the complete human P-450(17α) protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17αH is compared to the bovine P-450(17α) cDNA sequence. By transient expression of the human P-450(17α) cDNA clone in COS 1 cells, we have demonstrated that the 17α-hydroxylase and 17,20 lyase activities reside within the same human P-450(17α) polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450(17α) gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17α-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.

Original languageEnglish (US)
Pages (from-to)348-354
Number of pages7
JournalMolecular Endocrinology
Volume1
Issue number5
StatePublished - 1987

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Mixed Function Oxygenases
DNA
Complementary DNA
Steroid 17-alpha-Hydroxylase
Clone Cells
COS Cells
Southern Blotting
Gene Library
Cytochrome P-450 Enzyme System
Libraries
Testis
Ovary
Nucleotides
Fibroblasts
Amino Acids
Messenger RNA
Skin
Peptides
Genes
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Characterization of complementary deoxyribonucleic acid for human adrenocortical 17α-hydroxylase : A probe for analysis of 17α-hydroxylase deficiency. / Bradshaw, K. D.; Waterman, M. R.; Couch, R. T.; Simpson, E. R.; Zuber, M. X.

In: Molecular Endocrinology, Vol. 1, No. 5, 1987, p. 348-354.

Research output: Contribution to journalArticle

Bradshaw, K. D. ; Waterman, M. R. ; Couch, R. T. ; Simpson, E. R. ; Zuber, M. X. / Characterization of complementary deoxyribonucleic acid for human adrenocortical 17α-hydroxylase : A probe for analysis of 17α-hydroxylase deficiency. In: Molecular Endocrinology. 1987 ; Vol. 1, No. 5. pp. 348-354.
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