FUNCTION OF THE KINETOPLAST IN THE HEMOFLAGELLATES

本项目的长期目标是了解动质体原生动物线粒体中尿苷插入/缺失型RNA编辑的分子机制。本项目的具体目的如下：1。线粒体蛋白的分离和RNA编辑相关基因的克隆。酶和结构蛋白都将被研究。这些基因将被克隆并在大肠杆菌中表达，并作为标签蛋白在链乳杆菌中表达，重组蛋白用于产生抗体。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，并在体内和体外测试对编辑的影响。重组蛋白将用于尝试在体外重建编辑活性。2. 双链rna诱导（RNAi）抑制基因功能的机制研究。3. 从布鲁氏锥虫原环细胞中分离功能性20S编辑复合物单个蛋白的纯化和鉴定以及基因的克隆。4. 布鲁氏体和利什曼原虫在生命周期、培养生长和细胞周期中的编辑调控。5. 一种用于RNA编辑研究的瞬时线粒体表达系统和稳定线粒体转化系统的建立。6. 输入性tRNATrp反密码子C34-U34特异性编辑的研究。这些寄生虫是多种人类动物和植物疾病的致病因子，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。u插入/删除RNA编辑这一独特代谢途径的存在，可能为化疗干预提供了一个不影响宿主的选择性处理。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有助于开发能够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.