COOPERATIVITY OF THE MORPHOGENETIC PROTEIN BICOID

这项拨款提案的长期目标是了解 胚胎模式形成和基因表达的机制 果蝇，专注于形态发生蛋白Bicoid，它控制 前部结构的发育。 Bicoid蛋白，分布为 胚胎中的梯度在前部具有最高浓度， 转录激活因子。 Bicoid蛋白的一个靶基因是 hunchback，响应于Bicoid蛋白梯度，表达 均匀分布在胚胎的前半部分，具有尖锐的后边缘。 这项拨款申请旨在研究Bicoid蛋白质梯度 导致靶基因hunchback的这种全或无反应， 着重于DNA协同结合和蛋白质-蛋白质结合方面 互动 缺乏任何生物化学研究，这是必不可少的 对这一基本生物学问题的理解， 大量的蛋白质：各种各样的团体试图 但未能成功地大量分离出完整的Bicoid蛋白。 这个实验室最近成功地制造了大量的Bicoid 使用杆状病毒表达系统在SF-9昆虫细胞中表达蛋白。 这个实验室已经证明重组Bicoid蛋白 协同作用于hunchback增强子元件中的多个位点。 这 基金申请建议进一步研究 Bicoid蛋白在生物化学和果蝇体内协同性 胚胎 这项拨款建议有四个具体目标。 具体 目的1进一步研究Bicoid蛋白与DNA的协同结合。 重组Bicoid蛋白将进一步纯化， 衍生物将被生成，以定义重要的领域， 协同DNA结合 将进行动力学研究，以确定 协同DNA结合的分子机制和 稳定的蛋白质/DNA复合物。 具体目标2将进一步研究 Bicoid分子之间的相互作用机制，一个重要的 Bicoid的特性最近在这个实验室得到了证实。 将生成更多的删除衍生物以缩小区域 对蛋白质相互作用很重要。 这些衍生品将 用来缩小蛋白质-蛋白质 互动 这些衍生品也将用于确定这些 域之间相互作用。 具体目标3建议隔离 蛋白质-蛋白质相互作用缺陷的Bicoid蛋白的特异性突变体 和协同DNA结合。 这些突变体将在果蝇中进行测试 胚胎，以确定其对基因表达和模式的影响 阵 特定目标4将确定共有Bicoid结合位点。 这些合成的Bicoid结合位点将用于确定 体外亲和力和体内活性之间的相关性。 实验 也被提出来解决Bicoid蛋白如何 与明显不同类型的结合位点相互作用。 因为很多 对哺乳动物的发育很重要的蛋白质，比如果蝇， 形态发生蛋白Bicoid含有同源结构域， 这一资助申请将对理解 哺乳动物发育和基因表达。

The long-term goal of this grant proposal is to understand the molecular mechanisms of embryonic pattern formation and gene expression in Drosophila, focusing on the morphogenetic protein Bicoid, which controls the development of anterior structures. The Bicoid protein, distributed as a gradient in the embryo with the highest concentration at the anterior, is a transcriptional activator. One target gene of Bicoid protein is hunchback, which, in response to the Bicoid protein gradient, is expressed uniformly in the anterior half of the embryo with a sharp posterior border. This grant application proposes to study how the Bicoid protein gradient results in such an all-or-none response of the target gene hunchback, focusing on the aspects of cooperative DNA binding and protein-protein interaction. A lack of any biochemical studies which are essential to the understanding of this fundamental biological problem has been due to a lack of large quantities of the protein: Various groups have attempted unsuccessfully to isolate an intact Bicoid protein in large quantities. This laboratory has recently successfully generated large amounts of Bicoid protein in the SF-9 insect cells using the baculovirus expression system. This laboratory has demonstrated that the recombinant Bicoid protein binds cooperatively to multiple sites in the hunchback enhancer element. This grant application proposes to further study the molecular mechanisms of cooperativity of Bicoid protein both biochemically and in Drosophila embryos. There are four specific aims in this grant proposal. Specific Aim 1 proposes to further study cooperative DNA binding of Bicoid protein. The recombinant Bicoid protein will be purified further and other derivatives will be generated to define the domains important for cooperative DNA binding. Kinetic studies will be performed to determine the molecular mechanisms of cooperative DNA binding and the formation of stable protein/DNA complexes. Specific Aim 2 will further study the mechanisms of the interaction between Bicoid molecules, an important property of Bicoid that has recently been demonstrated in this laboratory. More deletion derivatives will be generated to narrow down the regions important for protein-protein interaction. These derivatives will be generated to narrow down the regions important for protein-protein interaction. These derivatives will also be used to determine how these domains interact with each other. Specific Aim 3 proposes to isolate specific mutants of Bicoid protein defective in protein-protein interaction and cooperative DNA binding. These mutants will be tested in Drosophila embryos to determine their effects on gene expression and pattern formation. Specific Aim 4 will determine consensus Bicoid binding sites. These synthetic Bicoid binding sites will be used to determine the correlation between in vitro affinity and in vivo activity. Experiments are also proposed to address the question of how Bicoid protein can interact with apparently different types of binding sites. Because many developmentally important mammalian proteins, like the Drosophila morphogenetic protein Bicoid, contain homeodomains, the studies proposed in this grant application will have great impact on the understanding of mammalian development and gene expression.