CELL SPECIFIC GNRH EXPRESSION

促性腺激素释放激素的细胞特异性表达 对于哺乳动物生殖的协调调节是必不可少的 系统。相对较少的促性腺激素释放激素神经元及其弥散分布 地理位置使得对GnRH的研究变得困难。人类促性腺激素释放激素基因 已被克隆，其转录起始点被定位在 下丘脑和胎盘。在这项提议中，DNA序列中的 人促性腺激素释放激素(HGnRH)基因5‘侧翼区对细胞重要- 具体的表达将被刻画出来。转基因小鼠已经被 使用由各种缺失的基因构成的基因结构生成 HGnRH启动子与荧光素酶报告基因(Luc)融合，产生 GnRH细胞特异性元件的初步定位。其他内容 转基因实验将证明这种元素是否足以 GnRH细胞特异性表达。GnRH细胞特定元素还将 使用表达GnRH的神经细胞系进行体外定位。两个高 广泛表达GnRH mRNA的相关细胞系NLT和Gn11 不同的级别，可用于这一目的。DNA酶I足迹 将用于确定hGnRH启动子的哪些序列 与表达促性腺激素释放激素的细胞提取物中的核蛋白结合。至 识别结合功能重要顺式作用的核蛋白 元素，凝胶位移分析将用定向的抗体进行 在候选蛋白质上。脑2(BRN-2)mRNA已在一个 高表达GnRH mRNA的GnRH细胞株(NLT) 另一个细胞系(Gn11)表达少量的GnRH mRNA，提示 它可能是GnRH基因的细胞特异性激活剂。BRN-2和 其他POU蛋白表达载体将转入Gn11细胞 确定它们是否可以通过其细胞特异性的GnRH启动子激活 元素。最后，GnRH神经元中POU蛋白的存在将 通过原位杂交研究来确定。因此，顺位代理 促性腺激素释放激素的调控元件和转录因子 下丘脑的基因表达将被分离和表征。 这些研究将为控制提供重要的分子见解。 并提示转录因子可能是 在生殖轴的某些病理条件下有缺陷。

The cell-specific expression of gonadotropin-releasing hormone (GnRH) is essential for the coordinate regulation of the mammalian reproductive system. The relatively small numbers of GnRH neurons and their diffuse locations has made the study of GnRH difficult. The human GnRH gene has been cloned and it's transcriptional start site mapped in the hypothalamus and placenta. In this proposal, DNA sequences within the 5' flanking region of the human GnRH (hGnRH) gene important for cell- specific expression will be characterized. Trasgenic mice have been generated using gene constructs consisting of various deletions of the hGnRH promoter fused to the luciferase reporter gene (LUC), yielded a preliminary localization of a GnRH cell-specific element. Additional transgenic experiments will prove whether this element is sufficient for GnRH cell-specific expression. The GnRH cell-specific element will also be mapped in vitro using GnRH-expressing neuronal cell lines. Two high related cell lines NLT and Gn11, which express GnRH mRNA at widely different levels, are available for this purpose. DNAse I footprinting will be used to determine which sequences of the hGnRH promoter are bound by nuclear proteins from GnRH-expressing cellular extracts. To identity nuclear proteins bound to functionally important cis-acting elements, gel-shift analyses will be performed with antibodies directed at candidate proteins. Brain 2(Brn-2) mRNA has been identified in one GnRH cell line (NLT) expressing high amount of GnRH mRNA but not in another cell line (Gn11) expressing low amounts of GnRH mRNA, suggesting it may serve as a cell-specific activator of the GnRH gene. Brn-2 and other POU protein expression vectors will be transfected into Gn11 cells determine if they can activate the GnRH promoter via its cell-specific element. Finally, the presence of POU proteins in the GnRH neuron will be determined using in situ hybridization studies. Thus, cis-acting elements and transcriptional factors responsible for the control of GnRH gene expression in the hypothalamus will be isolated and characterized. These studies will provide important molecular insights into the control of human reproduction and suggest transcription factors which might be defective in certain pathological conditions of the reproductive axis.