CONTROL OF GENE EXPRESSION DURING DROSOPHILA DEVELOPMENT

描述：这项提议的目标是了解分子 控制调控基因模式化表达的机制 早期发展。为此，皮克博士选择将她的注意力集中在 在片段基因FTZ的近端增强子上。皮克博士有 在这个增强子中发现了一个323bp的核心元件，它指导 FTZ启动子或连锁异源启动子在7例初诊患者中的表达 转基因胚胎中的条纹。其中有两个顺应式代理模块 核心增强子，两者都是体内表达所必需的。其中之一 这些位点在核心增强子中重复三次，并包含 四种不同蛋白质的结合位点：ADF-1、TTK、FTZ-F1和fEBP9。 皮克博士的工作假说是，这些物质的组合相互作用 与增强子元件结合的多个因素导致 在体内表达模式(条带的形成)的显著限制。 作为第一个目标，皮克博士将研究四种与三种蛋白质结合的蛋白质 323bP元件的亚区。皮克博士已经证明了这些 位点在诱导条纹状表达中起冗余作用 记者基恩。其中三个因子是先前确定的蛋白质和 克隆人已经存在。皮克博士将分离出第四株fEBP9，使用 标准生化提纯方法，多肽测序及克隆 该基因基于推导出的核苷酸序列。作为第二部分的 第一个目标，皮克博士计划表征ADF-1和ADF-1引起的表型 TTK突变。TTK的作用将不仅仅在受精卵中被检验 不仅是它的功能，也是它通过进行生殖系克隆而做出的母性贡献。 该提案的第二个目的是调查自贸区协同因素是如何调节的 FTZ蛋白的体内活性。皮克博士将描述身体的特征 FTZ与FTZ-F1的相互作用她会的 绘制FTZ和FTZ-F1蛋白中的结构域 互动。与J.B.A.罗斯博士合作，她还将确定 FTZ/FTZ-F1/DNA三元复合体的中间物种。均衡性 中间体将使用分析性超速离心法和 荧光各向异性。最后，她将描述新推定的 FTZ的辅因子，称为FIP2，已在酵母中被鉴定 双混合屏幕。她将确定其在体内的蛋白质定位和 鉴定其作为FTZ DNA结合的辅助因子的性质。FIP-2基因 已经被映射到一个富含现有突变的染色体区域。如果有 其中是FIP-2突变，将进行表型分析。

DESCRIPTION: The goal of this proposal is to understand the molecular mechanisms that direct patterned expression of the regulatory genes during early development. To this end, Dr. Pick has chosen to focus her attention on the proximal enhancer from the segmentation gene ftz. Dr. Pick has identified a core element of 323 bp in this enhancer which directs expression of the ftz promoter or linked heterologous promoters in seven stripes in transgenic embryos. There are two cis-acting modules within this core enhancer, both of which are required for in vivo expression. One of these sites is repeated three times within the core enhancer and contains binding sites for four different proteins: Adf-1, TTK, FTZ-F1, and fEBP9. Dr. Pick's working hypothesis is that combinatorial interactions of these multiple factors that bind to the enhancer element contribute to the striking restriction of expression patterns (formation of stripes) in vivo. As the first aim, Dr. Pick will study four proteins that bind to three subregions of the 323 bp element. Dr. Pick has demonstrated that these sites serve a redundant function in inducing striped expression of the reporter gene. Three of the factors are previously identified proteins and clones already exist. Dr. Pick will isolate the fourth, fEBP9, using the standard biochemical purification method, sequence the peptides and clone the gene based on deduced nucleotide sequences. As the second part of the first aim, Dr. Pick plans to characterize the phenotypes caused by Adf-1 and ttk mutations. The role of ttk will be examined not only for zygotic function but also its maternal contribution by making germ line clones. The second aim of the proposal is to investigate how Ftz cofactors modulate the in vivo activities of Ftz protein. Dr. Pick will characterize physical interactions between FTZ and FTZ-F1, a putative cofactor of FTZ. She will map domains in the FTZ and FTZ-F1 proteins that are required for their interaction. In collaboration with Dr. J.B.A. Ross, she will also determine intermediate species of the FTZ/FTZ-F1/DNA ternary complex. The equilibrium intermediates will be identified using analytical ultracentrifugation and fluorescence anisotropy. Finally, she will characterize the new putative cofactor of FTZ, called FIP2, which has been identified in a yeast two-hybrid screen. She will determine its protein localization in vivo and characterize its property as cofactor of FTZ DNA binding. The fip-2 gene has been mapped to a chromosomal region rich in existing mutations. If one of these is a fip-2 mutation, a phenotypic analysis will be carried out.