Cyclic AMP regulates expression of the rat gene for steroid 17 alpha-hydroxylase/C17-20 lyase P-450 (CYP17) in rat Leydig cells.
The upstream region of the rat CYP17 gene shows significant homology to the upstream regions of the bovine and human genes, 53 and 60 percent, respectively. The start site of transcription was determined by primer extension and S1 nuclease protection to be 41 base pairs (bp) upstream of the initiating methionine codon. Expression vectors were constructed by ligation of upstream sequences into promoterless chloramphenicol acetyl transferase (CAT) vectors. Transient transfection studies using primary cultures of rat Leydig cells indicate a strong cAMP-responsive element located within the -26/+65 region. Stimulation by cyclic AMP was abolished when sequences upstream of -264 were included in expression vectors. No significant expression was seen in Leydig cells in the absence of dbcAMP nor was there any expression in the presence or absence of dbcAMP in rat skin fibroblasts or in mouse adrenal (Y-1) cells in which CYP17 is not normally expressed. Three possible regulatory elements were found in the 5' upstream region: a CRE/ATF consensus sequence (GACGTCA) starting at position -57; a GRE consensus sequence (TGTTCT) starting at position -501; and a consensus sequence for AP-1 binding (TTAGTCA) starting at position -659. It was concluded that the CRE/ATF at -57 is not responsible for increased transcription in the presence of cyclic AMP.
Nason
T F
TF
Department of Endocrinology, Prince of Wales Hospital, Randwick, Australia.
Han
X G
XG
Hall
P F
PF
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D007985:Leydig Cells / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D001616:beta-Galactosidase / Q000378:metabolism
D004247:DNA; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase