REGULATION OF EQUINE LH/CG BETA SUBUNIT EXPRESSION

LH和CG分别在垂体和胎盘中合成， 对哺乳动物的繁殖至关重要 它们是异二聚体糖蛋白 由一个共同的α-亚单位非共价连接到一个 独特的β亚基与灵长类动物相比，马的LH和CG β- 亚基具有相同的氨基酸序列，并由单个- 复制基因。因此，eLH/CG β基因必须具有 赋予垂体和胎盘特异性表达。 知之甚少 关于调节促性腺激素β- 亚单位。已经证明，eLH/CG β基因的表达是 由近端启动子内的多个区域调控。此外，委员会认为， 448和3000 bp的eLH/CG β 5 '-侧翼序列指导 转基因小鼠垂体的报告基因。因此这些 构建体必须包含垂体所需的调节元件， 具体表达。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 为了更好地理解 调节脑垂体中基因表达。这将在 以下目标： 1)确定指导的要素和因素的身份 马LH/CG β亚基基因的垂体表达。85 bp的区域 eLH/CG β启动子的启动子是基础转录所必需的， 垂体细胞系。 该区域内包含的元素将 鉴定并克隆其同源反式作用因子， 表征了 实验方法包括DNA酶足迹法， 甲基化干扰，UV交联，西南分析， 瞬时转染和识别位点克隆。 2)确定从以下方面确定的元素的生理相关性： 体外研究。转基因小鼠将用于确定时间 和含有3000个的e-β构建体的表达的空间模式 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 也将用于评估元素的生理意义 在Aim l中识别。这些元素在指导正确的 eLH/CG β基因的时空表达模式也将是 考察这些实验将涉及使用转基因小鼠， 将通过进行荧光分析来评估转基因表达 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.