CLONING OF 2 ALTERNATIVELY SPLICED 21-HYDROXYLASE CDNAS FROM RAT ADRENAL

Interest in extra-adrenal corticosteroid synthesis has been revived by technological advances and the quest for answers to clinical problems . The cytochrome P450 21-hydroxylase converts progesterone to deoxycor ticosterone, the obligatory substrate for the production of the main a drenal steroids aldosterone, cortisol and corticosterone. The rat P450 21-hydroxylase was cloned and two constructs, 21OH-5 and 21OH-6, sequ enced. The constructs are similar, except that 21OH-6 has three additi onal major insertions of 64, 70 and 83 bp, a 3 bp deletion, and four e xtra base pairs immediately before the poly-A sequence. The entire cod ing region of 21OH-5 has 87 and 71% homology with the mouse and human 21-hydroxylase cDNA, respectively, whereas the encoded protein has 83 and 65% homology. Reverse transcriptase-polymerase chain reaction (RT- PCR) combined with Southern blot demonstrated expression of both trans cripts in the kidney, aorta, liver, cerebellum, hypothalamus and brain stem, heart and cerebrum, but not the hippocampus, in addition to the adrenal. The entire coding region of 21OH-5 and the corresponding reg ion of 21OH-6 including the three introns were cloned into pCR3 and th e plasmids transiently transfected into COS-7 cells. Only 21OH-5 was t ranslated into active protein, converting approximately 63% of H-3-pro gesterone to deoxycorticosterone in 2 h. (C) 1997 Elsevier Science Ltd .