Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450

M. E. John, M. C. John, P. Ashley, R. J. MacDonald, E. R. Simpson, M. R. Waterman

Research output: Contribution to journalArticle

119 Scopus citations

Abstract

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts ~425 and ~950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450(scc)), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for p-450(scc) with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450(scc) is encoded by mRNA species ~2000 bases long, a majority of which are polyadenylated, P-450(scc) mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450(scc) mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450(scc) gene transcription or acts to stabilize the transcripts. When pBSC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450(scc) may be encoded by a single gene.

Original languageEnglish (US)
Pages (from-to)5628-5632
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number18 I
DOIs
StatePublished - 1984

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