Characterization of gonadal and extra-gonadal forms of the cDNA encoding the Atlantic stingray (Dasyatis sabina) cytochrome P450 aromatase (CYP19)

Cytochrome P450 aromatase (P450arom; CYP19) mediates the conversion of andr ogens to estrogens and its activity has been found in all vertebrates studi ed to date. This study describes the full-length cDNA encoding the ovarian form of P450arom and the differences in the 5'-untranslated region (5'-UTR) of the extra-gonadal P450arom transcript expressed by the Atlantic stingra y (Dasyatis sabina). Elasmobranchs (cartilaginous fishes such as sharks, ra ys and skates) diverged from the other vertebrates more than 350 million ye ars ago, therefore the stingray P450arom cDNA may represent an ancient form of this gene. Northern blot analysis showed that the ovarian follicle expr essed transcripts of 3.1 and 1.7 kb in size which correspond to the clones isolated from a stingray ovarian follicle cDNA library. Both transcripts co nsisted of an identical 1.5 kb coding region and a 41 bp 5'-UTR, however th e 3'-UTRs differed in the use of the most proximate and the most distal of four polyadenylation signals. COS cells transfected with the 1.7 kb cDNA ha d twice the aromatase activity as cells transfected with the 3.1 kb cDNA. T he coding region of the cDNA predicted a 58.5 kDa protein which consisted o f 511 residues. Alignment of the stingray protein indicates that the P450ar om is equally identical (53-59%) to all other vertebrate forms of P450arom characterized to date, thus indicating a common ancestry. The evolutionary relationship of the stingray form of P450arom clearly predates the other fo rms and belongs to a unique lineage. Transcripts of P450arom were expressed in ovarian follicles (of all sizes), the testis, the pituitary, in all sec tions of the brain, and in the kidney. The extra-gonadal transcripts appear to encode a protein identical to the ovarian form, however, the 5'-UTR was 657 bp longer presumably due to the transcription of an untranslated 'firs t exon' as seen in the mammalian form of this gene. (C) 2000 Elsevier Scien ce Ireland Ltd. All rights reserved.