Orb Gene Function in Translational Regulation

描述(由申请人提供)：本提案的主要目标是更好地了解ORB如何自动调节其自身的表达，以及它如何控制编码因子的现场翻译，这些编码因子对于确定卵母细胞/哺乳细胞的身份和确定卵室和胚胎的极性至关重要。这些问题的部分答案将来自于识别和表征其他对ORB自动调节和/或功能重要的基因。在形成本申请基础的实验中，我们使用了两种互补的方法来确定潜在的“辅助因素”。在第一个实验中，我们产生了一个敏化的球体突变背景，它可以用来筛选与球体基因“相互作用”的第二个位点突变。在第二个实验中，我们使用了一种生化方法来鉴定体内与ORB相关的蛋白质。在之前的资助期间，我们用这些程序发现了许多有趣的“候选”基因。我们还比较详细地描述了这些“候选”中的三个：Squid(Sqd)、Rasputin(Rin)和dFMR1。Sqd和Rin作为正向调节因子，促进ORB的表达/活性，而dFMR1则是负调节因子。在即将到来的资助期间，我们建议在之前研究的基础上，重点了解这些蛋白质如何与其他因素合作来调节ORB的表达/活性。我们的生化实验表明，Orb形成了一系列不同的复合体。由于在这些不同的ORB复合体中发现的许多因子也在我们的遗传筛查中独立地被鉴定，我们知道与ORB相关的蛋白可能对ORB的功能很重要。我们将把我们的初步研究重点放在与ORB：dFMR1和(或)ORB：Rin复合体相关的因素上，并将使用在早期关于Sqd、Rin和dFMR1的工作中开发的方法来表征这些因素。其他感兴趣的候选人也将使用相同的程序进行研究。为了补充对Orb的分析，我们已经开始分析体细胞类Orb基因orb-2。

DESCRIPTION (provided by applicant): The primary goal of the present proposal is to better understand how Orb auto-regulates its own expression and how it controls the on site translation of mRNAs encoding factors critical for establishing oocyte/nurse cell identity and defining the polarity of the egg chamber and embryo. Part of the answer to these questions will come from identifying and characterizing other genes that are important for orb auto-regulation and/or function. In experiments that form the basis of the present application, we have used two complementary approaches to identify potential "co-factors." In the first we generated a sensitized orb mutant background which could be used to screen for second site mutations that "interact" genetically with orb. In the second, we used a biochemical approach to identify proteins that are associated with Orb in vivo. We uncovered many interesting "candidate" genes with these procedures during the previous grant period. We also characterized three of these "candidates", Squid (Sqd), Rasputin (Rin) and dFMR1 in some detail. Sqd and Rin function as positive regulators, promoting Orb expression/ activity, while dFMR1 is a negative regulator. During the upcoming grant period we propose to build on our previous studies, focusing on understanding how each of these proteins collaborates with other factors to regulate Orb expression/ activity. Our biochemical experiments suggest that Orb forms a series of distinct complexes. Since many of the factors found in these different Orb complexes were also identified independently in our genetic screen, we know that the Orb associated proteins are likely to be important for Orb function. We will focus our initial studies on factors associated with Orb:dFMR1 and(or) Orb:Rin complexes and will characterize these factors using approaches that were developed in earlier work on Sqd, Rin and dFMR1. Other interesting candidates will also be studied using the same procedures. To complement this analysis of Orb, we have begun analyzing the somatic Orb-like gene, orb-2.