THE CDNA CLONING AND TRANSIENT EXPRESSION OF A CHICKEN GENE ENCODING A 3-BETA-HYDROXYSTEROID DEHYDROGENASE DELTA(5-]4) ISOMERASE UNIQUE TO MAJOR STEROIDOGENIC TISSUES

Two cDNA clones, one containing a 2.7-kb and the other a 1.6-kb insert , were obtained from a chicken adrenal gland cDNA library by screening with a partial chicken cDNA fragment generated by PCR. Primers were u sed that corresponded to conserved regions among several published cDN A sequences of mammalian 3 beta-hydroxysteroid dehydrogenase/Delta(5-- >4) isomerase (3 beta-HSD). Both clones contained the same open readin g frame (ORF) encoding 377 amino acids (aa). The difference in their s izes was due to the use of different polyadenylation sites. The deduce d aa sequence showed 54-57% overall identity with those of the human, macaque, bovine, mouse, rat and rainbow trout 3 beta-HSD. A 9-aa stret ch (FYYISDDTP) is conserved among the chicken, mammalian and rainbow t rout 3 beta-HSD, suggesting that it is essential for enzymatic activit y. Northern blot hybridization demonstrated the presence of two types of mRNA, corresponding to the above two cDNA classes, in the adrenal g land, ovary and testis. Transient expression of the chicken ORF in COS -7 cells demonstrated coupled dehydrogenase and isomerase activities o f 3 beta-HSD on three different substrates; pregnenolone, 17 alpha-hyd roxypregnenolone and dehydroepiandrosterone. Among the K-m values dete rmined for the three substrates, that for dehydroepiandrosterone was t he lowest, which was different from the case for human 3 beta-HSD.