REGULATION OF EQUINE LH/CG BETA SUBUNIT EXPRESSION

黄体生成素和绒毛膜促性腺激素分别在脑垂体和胎盘中合成，并且 哺乳动物繁殖所必需的。它们是异二聚体糖蛋白。 由共同的非共价连接的α-亚基组成的激素 独特的β亚基。与灵长类动物相比，马的黄体生成素和绒毛膜促性腺激素β- 亚基具有相同的氨基酸序列，由一个单一的- 复制基因。因此，Elh/CGbeta基因必须具有 赋予脑下垂体和胎盘特异的表达。鲜为人知 关于调节促性腺激素β-表达的机制- 亚单位。Elh/CGbeta基因在脑垂体中的表达已被证明是 由近端启动子内的多个区域调控。此外， 448和3000bpELH/CGbeta5‘-侧翼序列直接表达 转基因小鼠脑下垂体的报告基因。因此，这些 构建的结构必须包含脑下垂体所需的调节元件- 特定的表达。本提案的总体目标是 阐明这些调控元件及其同源反式作用因子 为了更好地了解生理事件 来调节脑下垂体中的基因表达。这一问题将在 以下目标： 1)确定决定因素和因素的一致性 马黄体生成素/降钙素基因β亚基基因在脑垂体中的表达一个85个BP的区域 ELH/CGbeta启动子的转录是基础转录所必需的 垂体细胞系。此区域中包含的元素将为 鉴定及其同源反式作用因子的克隆和 特色化的。实验方法包括DNA酶足迹， 甲基化干扰，紫外线交联，西南分析， 瞬时转染和识别位点克隆。 2)确定下列元素的生理相关性 在体外研究中。转基因小鼠将被用来确定时间 以及包含3000个e-beta构建体的空间表达模式 或443bp的5‘-侧翼序列。这些研究将开始揭示 正确的时空表达所需的区域。转基因小鼠 也将被用来评估元素的生理意义 在Aim L中确认。这些元素在指导正确的 Elh/CGbeta基因的时空表达模式也将是 检查过了。这些实验将涉及使用转基因小鼠， 将通过进行化学发光来评估转基因表达 检测、X-Gal染色、免疫组织化学和原位杂交。

LH and CG are synthesized in pituitary and placenta, respectively, and are essential to mammalian reproduction. They are heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a unique beta-subunit. In contrast to primates, the equine LH and CG beta- subunits have identical amino acid sequences and are encoded by a single- copy gene. Therefore, the eLH/CGbeta gene must possess elements that confer both pituitary- and placenta-specific expression. Little is known about the mechanisms that regulate expression of gonadotropin beta- subunits. Pituitary expression of the eLH/CGbeta gene has been shown to be regulated by multiple regions within the proximal promoter. Furthermore, 448 and 3000 bp of eLH/CGbeta 5 '-flanking sequence direct expression of a reporter gene to the pituitary of transgenic mice. Therefore, these constructs must contain the regulatory elements required for pituitary- specific expression. The overall goal of the present proposal is to elucidate these regulatory elements and their cognate trans-acting factors in order to develop a better understanding of the physiological events that regulate gene expression in pituitary. This will be addressed in the following aims: 1) Determine the identity of the elements and factors that direct pituitary expression of the equine LH/CGbeta subunit gene. An 85 bp region of the eLH/CGbeta promoter is required for basal transcription in a pituitary cell line. Elements contained within this region will be identified and their cognate trans-acting factors cloned and characterized. Experimental approaches include DNase footprinting, methylation interference, UV crosslinking, southwestern analysis, transient transfections, and recognition site cloning. 2) Determine the physiological relevance of the elements identified from in vitro studies. Transgenic mice will be used to determine the temporal and spatial patterns of expression for e-beta constructs containing 3000 or 443 bp of 5'-flanking sequence. These studies will begin to reveal the regions required for correct spatiotemporal expression. Transgenic mice will also be used to evaluate the physiological significance of elements identified in Aim l. The role these elements play in directing the correct spatiotemporal expression pattern of the eLH/CGbeta gene will also be examined. These experiments will involve the use of transgenic mice which will be evaluated for transgene expression by performing chemiluminescent assays, X-gal staining, immunohistochemistry and in situ hybridization.