OOCYTE DEVELOPMENT IN DROSOPHILA

描述：这是一项继续研究三种果蝇的建议 基因，山雀，烧焦的和鹌鹑，这是运输必须的 将细胞成分注入发育中的卵母细胞。每种产品的产品 这些基因中有一种可以调节肌动蛋白的细胞骨架：山雀 Quail编码一个Profilin；Quail编码一个Villin；Stered编码一个束蛋白。 库利实验室的表型分析表明，这些突变体 阻止细胞质运输是因为突变体中的哺乳细胞核 不是由肌动蛋白支持系统固定在适当的位置，一旦流动 开始时，细胞核阻止进一步的细胞质流动。 拟议的实验将利用卵子发生的这一方面来 阐明这些肌动蛋白在体内的结构/功能关系 结合蛋白。对于山雀(Profilin)广泛的体外试验 突变和转化实验将探索剖析 可能调节肌动蛋白聚合及其如何调节信号转导 对细胞骨架的影响。对烧焦(法西林)的研究将测试什么 在体外，部分蛋白质是与肌动蛋白结合所必需的。EMS- 诱导后的等位基因会被分离出来，并且是蛋白质阳性的等位基因 将对影响护理细胞中肌动蛋白功能的基因进行测序，并对其 结合和/或捆绑肌动蛋白的能力将在体外进行测试。类似 将对现有的12个EMS诱导的等位基因进行表征， 将对6个蛋白质阳性等位基因进行测序。基因缺失突变体 应移除G和R肌动蛋白结合位点的鹌鹑将接受检测 在体内发挥作用。 对与这些基因产物相互作用的其他蛋白质进行遗传筛选 将会被执行。特定山雀的优势抑制者， 将进行焦烧和鹌鹑突变等位基因检测。 对于每个基因，额外的研究将取决于特定的活动 对基因产物的期望。例如，由于 脊椎动物带有肌动蛋白的绒毛蛋白在体外是钙依赖的，实验 将测试钙水平是否会影响培养鸡蛋中的肌动蛋白捆绑 钱伯斯。 提案的最后一部分探讨了以下两个不同方面 卵子发生。首先，因为出现了Rho家族的GTP酶 为了参与哺乳动物细胞中基于肌动蛋白的细胞形状变化， GAL4系统的改造以表达激活或显性 将开发胚系中特有的阴性Rho蛋白。 第二，由于护士细胞退化具有一些细胞凋亡的特征， 将分析退化的护士细胞的特征体征 细胞凋亡与果蝇收割器基因在卵子发生中的作用 被调查。

DESCRIPTION: This is a proposal to continue study of three Drosophila genes, chickadee, singed and quail, that are necessary for transport of nurse cell components into the developing oocyte. The products of each of these genes act to regulate the actin cytoskeleton: chickadee encodes a profilin; quail encodes a villin; singed encodes a fascin. Phenotypic analysis from the Cooley lab has shown that these mutants block cytoplasmic transport because the nurse cell nuclei in the mutants are not held in position by an actin support system and, once flow begins, the nuclei block further cytoplasmic flow. The proposed experiments will exploit this aspect of oogenesis to elucidate the in vivo structure/function relationships of these actin- binding proteins. For chickadee (profilin) extensive in vitro mutagenesis and transformation experiments will explore how profilin might regulate actin polymerization and how it might mediate signaling effects on the cytoskeleton. Studies on singed (fascin) will test what part of the protein is necessary for binding to actin in vitro. EMS- induced alleles of singed will be isolated and protein-positive alleles that affect actin functioning in nurse cells will be sequenced and their ability to bind and/or bundle actin will be tested in vitro. Similarly for quail (villin) 12 existing EMS-induced alleles will be characterized, and 6 protein-positive alleles will be sequenced. Deletion mutants of quail that should remove G and R actin binding sites will be tested for function in vivo. Genetic screens for other proteins that interact with these gene products will be carried out. Dominant suppressors of particular chickadee, singed and quail mutant alleles will be carried out. For each gene, additional studies will depend on the particular activity expected of the gene product. For instance, because interaction of vertebrate villin with actin is calcium dependent in vitro, experiments will test whether calcium levels affect actin bundling in cultured egg chambers. The final section of the proposal explores two different aspects of oogenesis. First, because members of the rho family of GTPases appear to be involved in actin-based cell shape changes in mammalian cells, a modification of the GAL4 system for expressing activated or dominant negative Rho proteins specifically in the germ line will be developed. Second, because nurse cell regression shares some features of apoptosis, regressing nurse cells will be analyzed for the characteristic signs of apoptosis and the role of the Drosophila reaper gene in oogenesis will be investigated.