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題 名 | Protein Binding Activity and Cyclic AMP-responsiveness of a Weak Sp1 Site in Proximal Promoter of Human CYP11A1 Gene |
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作 者 | Guo,Ing-cherng; Chung,Bon-chu; | 書刊名 | Journal of Genetics and Molecular Biology |
卷 期 | 10:1 1999.03[民88.03] |
頁 次 | 頁9-18 |
分類號 | 362.611 |
關鍵詞 | P450scc; Steroidogenesis; Proximal promoter; cAMP; Sp1; Humon CYP11A1 gene; |
語 文 | 英文(English) |
英文摘要 | The cholesterol side-chain cleavage enzyme (P450scc) encoded by CYPIIAI gene catalyzes the conversion of cholesterol into pregnenolone, which is the first step in steroid biosynthesis and regulated by tropic hormones via cAMP-PKA pathway. The regulatory region mediating signal of cAMP on the transcription of human CYPIIAI gene contains upstream cAMP-responsive sequences (U-CRS) and proximal cAMP-responsive sequences (P-CRS). The P-CRS, located in -145 bp, harbors at least three highly conserved sequences including TA.TA box, SFI-binding site and Spl-like sequence. By electrophoretic mobility shift assay, we showed oligonucleotide -117/-94 formed two specific DNA-protein complexes with nuclear extract from adrenocortical Yl, placental JEG-3, or cervical HeLa cells. The oligonucleotide containing Spl consensus sequence formed the same pattern as -117/-94 did. Although Spl was always stronger,-117/-94 and Spl oligonucleotides were effective competitors for each other. These data indicated a weak Spl-binding site located in -117/-94 fragment. To test for its function, the -117/-94 fragment was fused to the TATA box ofOVEC vector, before transfection into Yl or JEG-3 cells. Its transcription activity was measured by RNA analysis after 8-Br-cAMP treatment. The results of primer extension showed -117/-94 directed cAMP-dependent transcription. Based on the observations of DNA-protein interaction assay and transcription activity assay, we conclude that a weak Spl-binding site in the -117/-94 fragment is important for transcription of the CYP11A1 gene by interacting with basal transcription factors. |
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