TRANSCRIPTION FACTOR TARGET OF RAS/RAF-1 PATHWAY

许多生长因子影响细胞分裂或 通过结合特异性受体酪氨酸激酶（RTK）分化， 细胞膜。 已发现RTK的激活通过 一系列常见的中间体，激活MAP（丝裂原激活的 蛋白质）激酶。 不同RTK效应的特异性可能是由于， 至少部分地与转录因子的多样性有关， 这些MAP激酶 很少有转录因子被证明是 直接靶向激活的RTK途径;甚至更少的是知道关于 这些被激活的转录因子调控的基因。 我们的目标是确定和表征转录因子的目标， 一种基因描述良好的RTK。 由躯干（tor）激活的通路 RTK的功能是在早期的两极确定命运， 果蝇胚胎，是唯一适合研究，以确定一个新的 由RTK调节的转录因子。 该途径已在 基因上的细节和它的大部分成分被识别;分阶段的果蝇 可以以适于蛋白质纯化的量收集胚胎。 我们已经鉴定了一个小的（<14 bp）tor应答元件（tor-RE）， tailless（tll）基因的启动子（由tor调控的关键基因 途径）。 tor-RE是不寻常的，因为它作为一个阻遏元件; 其功能在TOR RTK活性的细胞中失活， 允许TLL的转录。 为了鉴定蛋白质（tor-REB） 结合tor-RE，并表征其在tor RTK途径中的功能 并可能在其他途径，我们建议做以下工作：1.）进一步 通过定点诱变确定推定的tor-RE， 转换分析，2.）纯化转录因子（tor-REB） 与tor-RE结合，3.）克隆和测序编码tor的基因， REB，4.）表征tor-REB和组件之间的相互作用 tor系统（特别是mAP激酶），5.）表征tor-REB RNA， 蛋白质在果蝇发育过程中的表达，6.）进行遗传 对tor-REB基因的研究，以证实它在终端功能 途径，并评估其在不同途径和发展中的作用 流程. 7.）鉴定tor-REB的哺乳动物同源物并测试 它是否受到脊椎动物中RTK激活途径的调节。 这些 研究有望为分子生物学提供新的见解， RTK激活途径影响细胞分化的机制， 增殖，并可能确定遗传目标的诊断筛选， 癌

Many growth factors affect the decision of a cell to divide or differentiate by binding to a specific receptor tyrosine kinase (RTK) in the cell membrane. Activation of RTKs has been found to lead, through a series of common intermediates, to activation of MAP (mitogen-activated protein) kinases. The specificity of effect of different RTKs may be due, at least in part, to the diversity of transcription factors modulated by these MAP kinases. Very few transcription factors have been shown to be direct targets of an activated RTK pathway; even less is known about athe genes that these activated transcription factors regulate. Our goal is to identify and characterize the transcription factor target of a genetically well described RTK. The pathway activated by the torso (tor) RTK, which functions to establish fate at the two poles of the early Drosophila embryo, is uniquely suited for studies to identify a novel transcription factor modulated by an RTK. This pathway has been studied in detail genetically and most of its components identified; staged Drosophila embryos can be collected in quantities suitable for protein purification. We have identified a small (<14 bp) tor Response Element (tor-RE) in the promoter of the tailless (tll) gene (the key gene regulated by the tor pathway). The tor-RE is unusual in that it acts as a repressor element; its function is inactivated in cells in which the tor RTK is active, allowing transcription of tll. To identify the protein (the tor-REB) binding to the tor-RE, and characterize its function in the tor RTK pathway and possibly in other pathways, we propose to do the following; 1.) further determine the presumptive tor-RE by site-directed mutagenesis and transformation analysis, 2.) purify the transcription factor (the tor-REB) that binds to the tor-RE, 3.) clone and sequence the gene encoding the tor- REB, 4.) characterize the interaction between the tor-REB and components of the tor system (particularly mAP kinase), 5.) characterize tor-REB RNA and protein expression during development in Drosophila, 6.) carry out genetic studies on the tor-REB gene to confirm that it functions in the terminal pathway, and to assess its role in different pathways and developmental processes. 7.) Identify the mammalian homolog of the tor-REB and test whether it is modulated by RTK activated pathways in vertebrates. These studies are expected to provide novel insights into the molecular mechanisms by which RTK activated pathways affect cell differentiation and proliferation, and may identify genetic targets for diagnostic screening in cancer.