Isolation of the putative cDNA encoding cholesterol side chain cleavage cytochrome P450 (CYP11A) of the southern stingray ( Dasyatis americana)

Abstract

Cholesterol side chain cleavage cytochrome P450 (P450scc; CYP11A) catalyzes the first step in the production of steroid hormones. By utilizing degenerate oligonucleotide primers in a reverse transcriptase-coupled polymerase chain reaction (RT-PCR), a specific 252 bp fragment of the putative P450scc was amplified from RNA of interrenal tissue (the adrenal cortex homolog) from the southern stingray ( Dasyatis americana), blacktip shark ( Carcharhinus limbatus), and the spiny dogfish shark ( Squalus acanthias). The amino-acid sequences predicted by these PCR products were 73–90% identical to each other. Using the homologous PCR-generated probe, five positive clones were isolated from a cDNA library constructed from interrenal mRNA of the southern stingray. The longest clone (4619 bp) contained the 3′-untranslated region, including four putative polyadenylation signals. Northern blot analysis of stingray interrenal RNA revealed a single transcript of 4.2 kb in length. The incomplete amino-acid sequence predicted by the open reading frame of the cDNA (514 residues in length) is 48% homologous to the trout form and 39–40% homologous to mammalian forms. Even though the stingray P450scc contains an amino terminus longer than the other forms of P450scc, no translation initiation signal (ATG) was evident within the open reading frame. This report presents the first sequence of cytochrome P450scc from this evolutionary unique taxon of vertebrates.