FUNCTION OF THE KINETOPLAST IN THE HEMOFLAGELLATES

该项目的长期目标是了解动体原生动物线粒体中尿苷插入/缺失类型的RNA编辑的分子机制。本项目的具体目标如下：1.线粒体蛋白质的分离和RNA编辑相关基因的克隆。将对酶蛋白和结构蛋白进行研究。这些基因将被克隆并在大肠杆菌中表达，并作为标记蛋白在达伦托利亚乳杆菌中表达，以及用于产生抗体的重组蛋白。基因敲除将通过破坏两个等位基因或通过RNAi功能敲除来执行，并在体内和体外分析编辑效果。重组蛋白将被用来尝试在体外重建编辑活动。2.探讨双链RNA(RNAi)抑制基因功能的机制。3.从布氏锥虫原周期细胞中分离出具有功能的20S编辑复合体；纯化和鉴定单个蛋白和克隆基因。4.布氏毛滴虫和利什曼原虫在生活史、培养过程和细胞周期中的编辑调控。5.建立了用于RNA编辑研究的瞬时线粒体表达系统和稳定的线粒体转化系统。6.进口tRNATrp反义密码子的C34-U34编辑研究这些寄生虫代表各种人类动植物疾病的病原体，如内脏和皮肤利什曼病、恰加斯病、非洲锥虫病和棕榈腐病。U-插入/缺失RNA编辑这种独特的代谢途径的存在可能为化疗干预提供一个选择性的句柄，而不会影响宿主。这些细胞的mt代谢需要编辑。对参与编辑的酶和精确的分子途径的详细了解可能会使药物的开发能够杀死寄生虫，而不会影响宿主。

The long term goal of this project is to understand the molecular mechanism of the uridine-insertion/deletion type of RNA editing that occurs in the mitochondrion of kinetoplastid protozoa. The specific aims of this project are as follows: 1. Isolation of mitochondrial proteins and cloning of genes involved in RNA editing. Both enzymatic and structural proteins will be investigated. The genes will be cloned and expressed both in E. coli and as tagged proteins in L. tarentolae, and the recombinant proteins used to generate antibodies. Gene knockouts will be performed either by disruption of both alleles or by RNAi functional knockouts, and the effect on editing assayed both in vivo and in vitro. The recombinant proteins will be used to attempt to reconstitute editing activities in vitro. 2. Investigation of the mechanism of double strand RNA-induced (RNAi) inhibition of gene function. 3. Isolation of a functional 20S editing complex from Trypanosoma brucei procyclic cells; purification and identification of individual proteins and cloning genes. 4. Regulation of editing in T. brucei and Leishmania during life cycle, growth in culture, and during cell cycle. 5. Development of a transient mitochondrial expression system and a stable mitochondrial transformation system for use in studying RNA editing. 6. Investigation of the specific C34-U34 editing of the anticodon of imported tRNATrp in L. tarentolae. These parasites represent the causal agents of a variety of human animal and plant diseases, such as visceral and dermal leishmaniasis, Chagas Disease, African trypanosomiasis, and palm decay diseases. The existence of such a unique metabolic pathway as U-insertion/deletion RNA editing may provide a selective handle for chemotherapeutic intervention without affecting the host. Editing is required for the mt metabolism of these cells. A detailed knowledge of the enzymes involved in editing and the precise molecular pathways may allow the development of drugs which can kill the parasite and not affect the host.