CONTROL OF GENE EXPRESSION DURING DROSOPHILA DEVELOPMENT

描述：本提案的目标是了解分子 调控基因的模式化表达的机制， 早期发展。 为此，皮克博士选择将她的注意力集中在 在近端的增强子上 匹克博士 在该增强子中鉴定了323 bp的核心元件， FTZ启动子或连接的异源启动子在7个细胞中的表达 转基因胚胎中的条纹。 其中有两个顺式作用模块， 核心增强子，这两者都是体内表达所需的。 之一 这些位点在核心增强子内重复三次， 四种不同蛋白质的结合位点：Adf-1、TTK、FTZ-F1和fEBP 9。 博士匹克的工作假设是， 与增强子元件结合的多种因子有助于 体内表达模式的显著限制（条纹的形成）。 作为第一个目标，皮克博士将研究四种蛋白质， 323 bp元件的亚区。 Pick博士已经证明， 位点在诱导条带化表达中起着冗余的作用， 报告基因 其中三个因子是以前鉴定的蛋白质， 克隆人已经存在。 皮克博士将分离第四个，fEBP 9，使用 标准生化纯化方法，对肽进行测序并克隆 基于推导的核苷酸序列的基因。 作为第二部分， 第一个目标，Pick博士计划描述Adf-1引起的表型， ttk突变。 ttk的作用将不仅在合子中进行检查， 功能，但也通过生殖系克隆其母体的贡献。 该提案的第二个目的是研究Ftz辅因子如何调节 Ftz蛋白的体内活性。 匹克医生将描述 FTZ和FTZ-F1之间的相互作用，FTZ的一个假定的辅因子。 她将 映射FTZ和FTZ-F1蛋白中的结构域， 互动 与J.B.A.博士合作罗斯，她还将决定 FTZ/FTZ-F1/DNA三元复合物的中间物种。 平衡 将使用分析超离心法鉴别中间体， 荧光各向异性 最后，她将描述新的假定 FTZ的辅因子，称为FIP 2，已在酵母中鉴定 双杂交筛选 她将确定其蛋白质在体内的定位， 表征其作为FTZ DNA结合辅因子的性质。 fip-2基因 被定位在一个富含突变的染色体区域 如果一个 其中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.