Characterization of Insect Rpr, Hid, and Grim Homologs

描述(由申请人提供)：细胞凋亡是两种疾病的一个重要过程 脊椎动物和无脊椎动物。分子的功能守恒和进化守恒 凋亡蛋白允许使用无脊椎动物作为哺乳动物模型 促进对癌症生物学和胚胎发生的了解。果蝇 黑素胃泌素促死亡蛋白RPR、HID和GRIM保持其生物学特性 在哺乳动物和无脊椎动物系统中的功能表明同源基因 对这些蛋白质来说，物质是存在的。到目前为止，还没有RPR、HID和GRIM的同源物 甚至在其他昆虫身上也被发现了。南航的初步数据 昆虫基因组DNA杂交表明，至少在其他果蝇中 亚种，与这些死亡蛋白同源的存在。最初，同源基因来自 不同的果蝇亚种将被用作探针来筛选 无脊椎动物、Tribolium和Sppotera的Southern杂交、菌落杂交 和简并的聚合酶链式反应。同源基因将被克隆和测序。蛋白质-蛋白质 同系物的相互作用将通过酵母双杂交分析进行评估 和免疫沉淀。最后，对同系物进行了功能分析 通过蛋白质在无脊椎动物细胞中的过度表达来分析其 在细胞凋亡中的作用，以及RNA干扰(RNAi)将评估功能丧失 表型。

DESCRIPTION (provided by the applicant): Apoptosis is a vital process in both vertebrates and invertebrates. Functional and evolutionary conservation of apoptotic proteins allows the use of invertebrates as mammalian models to expedite the understanding of cancer biology and embryogenesis. The Drosophila melanogaster pro-death proteins RPR, HID and GRIM maintain their biological functions in both mammalian and invertebrate systems indicating that homologs to these proteins mat exist. To date, no homologs of RPR, HID and GRIM have been identified, even in other insects. Preliminary data from Southern hybridizations of insect genomic DNA shows that at least in other Drosophila subspecies, homologs to these death proteins exist. Initially, homologs from different Drosophila subspecies will be used as probes to screen the invertebrates, Tribolium and Sppodoptera by Southern blot, colony hybridization and degenerate PCR. Homologs will be cloned and sequenced. Protein-protein interactions of the homologs will be assessed using yeast-two hybrid analysis and immunoprecipitation. Finally, functional analysis of the homologs will be addresses by protein over-expression in invertebrat cells to analyze their roles in apoptosis, and RNA interference (RNAi) will evaluate loss of function phenotypes.