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

Abstract

Two overlapping [complementary] cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts .apprxeq. 425 and .apprxeq. 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc), were identified by using 2 differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNA 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, 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, P-450scc is encoded by mRNA species .apprxeq. 2000 bases long, a majority of which are polyadenylylated. P-450scc 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-450scc mRNA within 8 h. ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high MW bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.