GNRH GENE: TISSUE SPECIFIC CONTROLS

描述(改编自申请人的摘要)：尽管有很多是 了解神经营养因子的分泌动态和翻译后调节 GnRH，关于这种机制的信息很少 控制其组织特异性表达。使用两个GnRH 从转基因小鼠和人类中分泌神经细胞系 用温度敏感型SV40T使胎盘细胞系永生化 抗原突变体，申请人建议描绘组织特异性和 促性腺激素释放激素转录调控的物种特异性机制 制作。特定目标1将阐明特定顺式作用dna。 调节下丘脑和胎盘GnRH表达的序列。 大鼠和人的基因转移及缺失分析 启动子将描述组织所需的DNA区域 特定的表达。DNA酶足迹和凝胶延迟分析将 进一步定义与组织特异性相互作用的启动子区域 核蛋白。靶点定向突变将检测 这些区域在瞬时转染中的功能意义 化验。启动子序列的甲基化干扰分析将是 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 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：配基筛选、蛋白质纯化、 转录因子家族成员的聚合酶链式反应扩增 差异显示。利用差异显示技术进行分子克隆将 用于分离发育调节转录因子，方法是 比较早期(Gn)下丘脑细胞表达的mRNAs 10细胞)和晚期(GT1 7细胞)的GnRH神经元迁移和 胎盘细胞来源于早期(SPA 29细胞)和晚期(HPA 1) 怀孕了。

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.