FUNCTION OF THE KINETOPLAST IN THE HEMOFLAGELLATES

本项目的长期目标是了解发生在动质体原生动物细胞中的尿苷插入/缺失型RNA编辑的分子机制。 本项目的具体目标如下：1.线粒体蛋白的分离和RNA编辑相关基因的克隆。 酶和结构蛋白质都将被研究。 这些基因将在大肠杆菌中克隆和表达。coli中的蛋白质，并在L. tarentolae和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功能敲除来进行，并且在体内和体外测定对编辑的影响。 重组蛋白将用于尝试在体外重建编辑活性。 2.研究双链RNA诱导（RNAi）抑制基因功能的机制。 3.从布氏锥虫原环细胞中分离功能性20 S编辑复合物;纯化和鉴定单个蛋白质并克隆基因。 4. T.布氏杆菌和利什曼原虫在生命周期、培养生长和细胞周期中的作用。 5.开发用于研究RNA编辑的瞬时线粒体表达系统和稳定的线粒体转化系统。 6.对L. tRNATrp反密码子的C34-U34特异性编辑的研究tarentolae这些寄生虫代表了多种人类动物和植物疾病的病原体，例如内脏和皮肤利什曼病、恰加斯病、非洲锥虫病和棕榈腐烂病。 这种独特的代谢途径的存在，如U-插入/缺失RNA编辑，可以提供一个选择性的处理化疗干预，而不影响宿主。 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允许开发可以杀死寄生虫而不影响宿主的药物。

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.