Isolation and characterization of the cDNA encoding the spiny dogfish shark (Squalus acanthias) form of cytochrome P450c17.
Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This protein possesses two enzymatic activities. The 17 alpha-hydroxylase activity results in the introduction of a hydroxyl group at the 17 alpha-position. The resultant 17 alpha-hydroxylated, C21 pregnene is converted to a C19 androgen by the C17,20-lyase activity. A cDNA library was constructed from poly(A)-enriched mRNA isolated from spiny dogfish shark (Squalus acanthias) testis and ligated into EcoRI-cut lambda arms. The amplified library was screened using a bovine P450c17 cDNA probe and five positive clones were isolated. The described cDNA encompasses 23 bp of the 5'-untranslated region, a 1,527 bp open reading frame, and 414 bp of the 3'-untranslated region. A putative polyadenylation signal (AATAAA) is 18 bp from the poly(A) tail. Northern blot analysis showed a single transcript of 1.9 kb, thus indicating the isolated clone is a full-length cDNA. The deduced amino acid sequence of the shark form of P450c17 is 59% and 57% identical to the rainbow trout and chicken forms, respectively. The shark form is 43% to 46% identical to mammalian forms (rat, human, mouse, bovine, and porcine). There are large regions of extremely high identity shared among all the forms. The deduced shark 17 alpha-hydroxylase protein is 509 residues in length with a predicted weight of 57.2 kDa. Non-steroidogenic COS cells, transfected with the shark P450c17 cDNA, was capable of 17 alpha-hydroxylase and C17,20-lyase activities using both pregnenolone and progesterone as initial substrates.
Trant
J M
JM
Department of Zoology and Physiology, Louisiana State University, Baton Rouge 70803, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004284:Dogfish; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D013737:Testis / Q000378:metabolism
D018076:DNA, Complementary; D013254:Steroid 17-alpha-Hydroxylase