COOPERATIVITY OF THE MORPHOGENETIC PROTEIN BICOID

这项拨款计划的长期目标是理解分子 鸡胚胎型形成及基因表达机制的研究 果蝇，专注于形态发生蛋白类化合物，它控制着 前部结构的发育。双核蛋白，分布为 胚胎在前部的浓度最高的梯度是 转录激活剂。双核蛋白的一个靶基因是 驼背，这是对双核蛋白梯度的反应，表达 均匀地在胚胎的前半部分，具有尖锐的后缘。 这项拨款申请计划研究双核蛋白梯度如何 导致目标基因驼背的这种全有或全无反应， 关注协同DNA结合和蛋白质-蛋白质方面 互动。缺乏任何对生物多样性至关重要的生化研究 对这一基本的生物学问题的理解是由于缺乏 大量的蛋白质：不同的研究小组试图 未能成功地大量分离出完整的双核蛋白。 这个实验室最近成功地产生了大量的双曲面 利用杆状病毒表达系统在SF-9昆虫细胞中表达蛋白质。 本实验室已经证明了重组双核蛋白与 协同到驼背增强子元件中的多个位置。这 格兰特申请建议进一步研究黄曲霉毒素的分子机制 双核蛋白在果蝇体内和生化上的协同作用 胚胎。这项拨款提案有四个具体目标。特定的 目的1提出进一步研究双核蛋白与DNA的协同结合。 重组双核蛋白将进行进一步纯化和其他 将生成派生函数来定义对以下各项重要的域 协同DNA结合。将进行动力学研究以确定 协同DNA结合的分子机制及协同DNA的形成 稳定的蛋白质/DNA复合体。具体目标2将进一步研究 一种重要的双分子间相互作用的机理 最近在这个实验室中证明的双曲面的性质。 将生成更多的删除导数以缩小区域范围 对蛋白质-蛋白质相互作用很重要。这些衍生品将是 产生以缩小对蛋白质-蛋白质重要的区域 互动。这些衍生品也将被用来确定这些 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双核蛋白特异性突变体 和协同DNA结合。这些突变体将在果蝇身上进行测试 胚胎以确定它们对基因表达和模式的影响 队形。特定的目标4将确定共识的双核结合位点。 这些合成的双核体结合位点将被用来确定 体外亲和力与体内活性的相关性。实验 也被提出用来解决双核蛋白如何 与明显不同类型的结合部位相互作用。因为很多人 发育重要的哺乳动物蛋白质，如果蝇 形态发生蛋白类化合物，含有同源结构域，这项研究发表在 这项拨款申请将对理解 哺乳动物的发育和基因表达。

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.