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重要过程， 脊椎动物和无脊椎动物。功能和进化上的保守 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理解。果蝇 黑腹果蝇促死亡蛋白RPR、HID和GRIM维持其生物学活性， 在哺乳动物和无脊椎动物系统中发挥作用，表明同源物 这些蛋白质存在。迄今为止，没有RPR、HID和GRIM的同系物 甚至在其他昆虫中也被发现。南方的初步数据 昆虫基因组DNA的杂交表明，至少在其他果蝇中， 亚种，这些死亡蛋白的同源物存在。最初，同源物来自 不同的果蝇亚种将被用作探针来筛选 无脊椎动物，拟谷盗属和Sppodoptera通过Southern印迹，菌落杂交 简并PCR。同源物将被克隆和测序。蛋白质 同源物的相互作用将使用酵母双杂交分析来评估 和免疫沉淀。最后，将对同系物进行功能分析。 通过蛋白质在无脊椎动物细胞中的过度表达来分析其 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.