Analysis of regulatory mechanism for gene exprssions of growth hormone-releasing peptide receptor and growth hormone-releasing hormone receptor

我们克隆了人生长激素释放激素受体（GHRH-R）基因的5 '侧翼区，并测定了翻译起始位点上游2.7kb的核苷酸序列。RNA酶保护分析表明，该基因的主要转录起始位点位于翻译起始位点上游122 bp处。最长的初始5 '-RACE产物的5'-末端靠近该位点。没有典型的TATA同源性，但有几个推定的调控元件，包括Pit-1结合位点样元件。使用荧光素酶报告基因的瞬时转染研究表明，5-侧翼区在GH 3细胞（来自大鼠垂体瘤）中具有启动子活性，但在非垂体细胞、BeWo和HeLa细胞中没有。然而，共转染Pit-1表达载体增加了BeWo细胞中的荧光素酶活性。缺失分析表明，GHRH-R基因的-310 ~-130和-130 ~-120区域对GHRH-R基因的表达起重要作用，但后者对GHRH-R基因表达的影响较小。 ...更多信息 基因表达。在共转染Pit- 1表达载体的BeWo细胞中，-310 ~-130区域是Pit-1依赖的GHRH-R基因表达所必需的。从-310到-120的区域具有两个推定的Pit-1结合元件，P1和P2，分别位于从-129到-123和从-171到-160。迁移率变动分析和DNA酶-1足迹分析均表明P2比P1具有更高的Pit-1结合亲和力。此外，我们还克隆了人生长激素促分泌素受体（GHS-R）基因，该基因的5个侧翼区长度为0.6- 2.9kb。用5 'RACE技术分析hGHS-R转录本，推测其转录起始位点位于翻译起始位点上游-453bp处。没有TATA，CAAT或GC盒，但启动子样序列。将5 '侧翼区插入到在GH 3细胞中具有启动子活性的荧光素酶报告载体中。hGHS-R启动子活性似乎为-734至-608。少

We cloned the 5'-flanking region of the human growth hormone-releasing hormone receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 kb upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122bp upstream from the translation start site. The 5'-end of the longest initial 5'-RACE product was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-i binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated 5-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in non-pituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed the regions from -310 to -130 and from -130 to -120 were important for the GHRH-R gene expression in GH3 cells, although the latter less contributied to the … More gene expression. In BeWo cells co-transfected with Pit- 1 expression vector, the region from -310 to -130 was essential for the Pit-1 - dependent expression of GHRH-R gene. The region from -310 to -120 has two putative Pit-1 binding elements, P1 and P2, located from -129 to -123 and from -171 to - 160, respectively. Both mobility shift assay and DNase-l foot print analysis showed that P2 had much higher Pit -1 binding affinity than P1. These findings were consistent with the results that the region from -310 to - 130 is an important element for Pit-i-dependent expression of GHRH-R.In addition, we cloned human growth hormone secretagogue receptor (GHS-R) gene containing the 5-flanking region of 0.6-2.9 kb. Analysis of the hGHS-R transcripts with 5'RACE suggested that putative transcription initiation site was -453 bp upstream from the translation start site. There was no TATA, CAAT, or GC box but an initiator-like sequence sequence. The 5'-flanking region was inserted into a luciferase reporter vector had promoter activity in GH3 cells. The hGHS-R promoter activity appeared to be from -734 to -608. Less