Cloning and Characterization of the 5′-Flanking Region of the Human Growth Hormone-releasing Hormone Receptor Gene*
Cloning and Characterization of the 5′-Flanking Region of the Human Growth Hormone-releasing Hormone Receptor Gene*
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人类生长激素释放激素受体基因 5-侧翼区域的克隆和表征*
DOI：
10.1074/jbc.274.17.12108
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
K. Chihara
中科院分区：
文献类型：
--
作者：
G. Iguchi;Y. Okimura;Tetsuya Takahashi;I. Mizuno;M. Fumoto;Yutaka Takahashi;H. Kaji;H. Abe;K. Chihara
我们克隆了人类生长激素释放激素受体（GHRH-R）基因的5 '侧区，并确定了翻译起始位点上游2.7千碱基的核苷酸序列。RNase保护分析表明，主要的转录起始位点位于翻译起始位点上游122个碱基对处。cDNA 5′-快速扩增的最长产物的5′端靠近该位点。没有典型的TATA同源物，但有几个假定的调控元件，包括pit -1结合位点样元件。荧光素酶报告基因的瞬时转染研究表明，5 ' -侧翼区域在GH3细胞（来源于大鼠垂体瘤）中具有启动子活性，而在非垂体细胞、BeWo和HeLa细胞中没有。然而，共转染Pit-1表达载体可提高BeWo细胞的荧光素酶活性。缺失研究表明，−310 ~−130和−130 ~−120区域对GHRH-R基因在GH3细胞中的表达很重要，但后者对GHRH-R基因的表达贡献较小。在共转染了Pit-1表达载体的BeWo细胞中，- 310至- 130区域是GHRH-R基因Pit-1依赖性表达所必需的区域。在−310 ~−120区域有两个假定的pit -1结合位点P1和P2，分别位于−129 ~−123和−171 ~−160之间。迁移率转移试验和dna - i足迹分析表明，P2具有比P1更高的Pit-1结合亲和力。P2突变降低GH3细胞中GHRH-R基因的表达。这些发现与−310 ~−130区域是GHRH-R基因pit -1依赖性表达的重要元件的结果一致。
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 kilobases upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site. The 5′-end of the longest product of 5′-rapid amplification of cDNA ends was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-1-binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated that 5′-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in nonpituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed that 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 contributed less to the 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 sites, P1 and P2, located from −129 to −123 and from −171 to −160, respectively. Both mobility shift assay and DNase-I footprint analysis showed that P2 had much higher Pit-1 binding affinity than P1. Mutation of P2 decreased GHRH-R gene expression in GH3 cells. These findings were consistent with the results that the region from −310 to −130 is an important element for Pit-1-dependent expression of GHRH-R gene.
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组成型启动子指导神经生长因子受体基因的表达。
影响因子：
5.3
作者：
Sehgal,A;Patil,N;Chao,M
通讯作者：
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DOI：
10.1210/endo.137.6.8641220
发表时间：
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期刊：
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影响因子：
--
作者：
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影响因子：
4.8
作者：
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影响因子：
4.5
作者：
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影响因子：
56.9
作者：
CASTRILLO, JL;THEILL, LE;KARIN, M
通讯作者：
KARIN, M