GNRH GENE: TISSUE SPECIFIC CONTROLS

描述（改编自申请人的摘要）：虽然很多是 已知的分泌动力学和翻译后调节， GnRH，很少有关于其机制的信息， 控制其组织特异性表达。 使用两种GnRH 来自转基因小鼠和人的分泌神经元细胞系 使用温度敏感的SV40 T细胞永生化的胎盘细胞系 抗原突变体，申请人提出描绘组织特异性和 GnRH转录调控的种属特异性机制 生产 特异性目的1将阐明特异性顺式作用DNA 介导下丘脑和胎盘GnRH表达的序列。 大鼠和人的基因转移研究及缺失分析 启动子将表征组织所必需的那些DNA区域， 具体表达。 DNA酶足迹和凝胶阻滞测定将 进一步定义了与组织特异性 核蛋白 定点突变将检查 这些区域在瞬时转染中的功能意义 测定。 启动子序列的甲基化干扰测定将在 用于确认与蛋白质的接触。 具体目标2将确定 并表征反式作用核蛋白DNA或蛋白质， 赋予下丘脑和胎盘表达的蛋白质相互作用 促性腺激素释放激素启动子 一旦顺式作用的DNA序列被 严格定义，将采用四种战略来确定和 分离DNA结合蛋白：配体筛选，蛋白纯化， PCR扩增推定的转录因子家族成员，和 差异显示 通过差异显示进行分子克隆将 用于分离发育调节的转录因子， 比较早期（Gn）下丘脑细胞表达的mRNA 10个细胞）和晚期（GT 1 7个细胞）阶段的GnRH神经元迁移， 胎盘细胞来源于早期（SPA29细胞）和晚期（HPA1细胞） 怀孕

DESCRIPTION (Adapted from the applicant's abstract): Although much is known about the secretory dynamics and post translational regulation of GnRH, little information is available concerning the mechanisms which control its tissue specific expression. With the use of two GnRH secreting neuronal cell lines derived from transgenic mice, and human placental cell lines immortalized using a temperature sensitive SV40 T antigen mutant, the applicant proposes to delineate tissue specific and species specific mechanisms of transcriptional regulation of GnRH production. Specific Aim 1 will elucidate the specific cis acting DNA sequences that mediate hypothalamic and placental expression of GnRH. Gene transfer studies with deletion analysis of the rat and human promoter will characterize those DNA regions necessary for tissue specific expression. DNase footprinting and gel retardation assays will further define promoter regions that interact with tissue specific nuclear proteins. Targeted site directed mutagenesis will examine the functional significance of these regions in transient transfection assays. Methylation interference assays of promoter sequences will be used to confirm contacts with proteins. Specific Aim 2 will identify and characterize the trans acting nuclear protein DNA or protein protein interactions that confer hypothalamic and placental expression of the GnRH promoter. Once the cis acting DNA sequences have been rigorously defined, four strategies will be employed to identify and isolate DNA binding proteins: ligand screening, protein purification, PCR amplification of putative transcription factor family members, and differential display. Molecular cloning by differential display will be used to isolate developmentally regulated transcription factors by comparing expressed mRNAs from hypothalamic cells derived at early (Gn 10 cells) and late (GT1 7 cells) stages of GnRH neuronal migration and placental cells derived from early (SPA 2 9 cells) and late (HPA 1) gestation.