Kepa J K, Spaulding A J, Jacobsen B M, Fang Z, Xiong X, Radovick S, Wierman M E
Department of Medicine, University of Colorado Health Sciences Center and Research Service, Veterans Affairs Medical Center, Denver 80220, USA.
Nucleic Acids Res. 1996 Sep 15;24(18):3614-20. doi: 10.1093/nar/24.18.3614.
To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.
为评估促性腺激素释放激素（GnRH）潜在的物种特异性表达，克隆了人GnRH启动子远端片段，对其进行特性分析并用于基因转移研究。测定了约3.8 kb的5'侧翼区核苷酸序列。在-551 h（-424 r）与转录起始位点之间的近端启动子区域以及多个远端启动子区域中观察到与人GnRH序列的同源性。相比之下，-1131/-551 h与-1031/-424 r之间的序列几乎没有相似性。构建了5'侧翼人GnRH启动子缺失体，并在产生GnRH的小鼠GT1-7神经元细胞中进行瞬时转染分析。最大的人GnRH启动子构建体（-3832/+5 h）表现出高水平的报告基因活性，与最大的大鼠GnRH构建体（-3026/+116 r）观察到的活性相似。然而，与大鼠基因不同，将人GnRH启动子的远端启动子序列缺失至-1971、-1131或-551并没有导致荧光素酶报告基因活性降低。进一步截短至-350导致荧光素酶活性降低3倍。在神经元细胞中未优先使用假定的上游人GnRH起始位点。DNase I保护分析显示，GT1-7和Gn10神经元细胞的核提取物与人GnRH和大鼠GnRH启动子片段具有独特的保护模式。这些数据表明存在介导物种特异性神经元GnRH表达的不同顺式作用元件和反式作用因子。
