Identification of genes that induce Diap1 degradation during Drosophila development

果蝇发育过程中诱导 Diap1 降解的基因的鉴定

• 批准号: 15603008
• 负责人: KOBAYASHI Masatomo
• 金额: $ 2.3万
• 依托单位: TSUKUBA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15603008/
• 关键词: Drosophila; Apoptosis; Diap1; Ubiquitin; Proteasome; salivary gland; プロテアソーム; 複眼; アポトーシス; reaper; sickle

Apoptosis or program cell death plays an important role in metazoan development. It is crucial for tissue homeostasis by eliminating unwanted cells and tissues, sculpting developing structure, controlling cell numbers and removing abnormal cells. Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins(IAP), which includes those from viruses as well as their homologues in invertebrates and vertebrates. Drosophila IAP (Diap1) has an important domain, which is RING domain. Diap1 can participate in apoptosis regulation in Fly by polyubiquitylation of target proteins and subsequent degradation by Ubiquitin-Proteasome system. To establish in vivo Diap1 degradation system in fly, GFP-Diap1 was overexpressed in salivary gland by transgenic fly lines, using GAL4-UAS system. In order to screen the genes, which induce Diap1 degradation in vivo, unknown genes were coexpressed with GFP-Diap1 in larval salivary gland. Seven candidate genes were obtained by observing the disappearance of GFP-Diap1. GS lines (Tokyo Metropolitan University) were used in this system. At first screening, about hundred GS lines (University of Tokyo) were picked up by checking the rough eye phenotype when the gene was overexpressed in fly eye among five thousand GS lines. Although seven candidate genes were needed to make sure whether cell death were actually occurred or not in case of GS overexpression, the in vivo system would be more useful for screening the genes that induce Diapl degradation in vivo.

细胞凋亡或程序性细胞死亡在后生动物的发育过程中起着重要的作用。它通过消除不需要的细胞和组织，塑造发育中的结构，控制细胞数量和去除异常细胞，对组织稳态至关重要。细胞凋亡抑制蛋白（IAP）是高等生物中细胞死亡的负调控因子，IAP包括来自病毒的IAP及其在无脊椎动物和脊椎动物中的同源物。果蝇IAP（Diap 1）具有一个重要的结构域，即RING结构域。Diap 1可通过靶蛋白的多聚泛素化和随后的泛素-蛋白酶体系统降解参与果蝇的凋亡调控。为建立Diap 1在果蝇体内的降解体系，利用GAL 4-UAS系统，在转基因果蝇的唾液腺中高效表达GFP-Diap 1。为了筛选体内诱导Diap 1降解的基因，将未知基因与GFP-Diap 1在幼虫唾液腺中共表达。通过观察GFP-Diap 1的消失情况，获得了7个候选基因。在该系统中使用GS系（东京首都大学）。在第一次筛选时，通过检查当该基因在5000个GS系中的蝇眼中过表达时的粗糙眼表型，挑选出约100个GS系（东京大学）。虽然需要七个候选基因来确定在GS过表达的情况下是否实际发生细胞死亡，但体内系统对于筛选诱导Diapl体内降解的基因将更有用。