FUNCTION OF THE KINETOPLAST IN THE HEMOFLAGELLATES

血鞭毛虫动质体的功能

• 批准号: 6631662
• 负责人: Larry Simpson
• 金额: $ 45.99万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6631662
• 关键词: Escherichia coli; Leishmania; Trypanosoma; aminohydrolases; cell cycle; cytidine; gene targeting; immunoprecipitation; kinetosome; laboratory rat; life cycle; microarray technology; mitochondria; molecular cloning; molecular genetics; posttranscriptional RNA processing; protein purification; recombinant proteins; transfer RNA; yeast two hybrid system

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.

该项目的长期目标是了解动质体原生动物线粒体中发生的尿苷插入/缺失型 RNA 编辑的分子机制。 该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。 酶蛋白和结构蛋白都将被研究。 这些基因将在大肠杆菌中克隆并表达，并在塔兰托拉菌中作为标记蛋白表达，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 功能性敲除来进行，并在体内和体外测定编辑的效果。 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.从布氏锥虫原循环细胞中分离出功能性20S编辑复合物；单个蛋白质和克隆基因的纯化和鉴定。 4. T. brucei 和利什曼原虫在生命周期、培养物生长和细胞周期期间的编辑调节。 5. 开发用于研究RNA编辑的瞬时线粒体表达系统和稳定线粒体转化系统。 6. L. tarentolae 中输入的 tRNATrp 反密码子的特异性 C34-U34 编辑研究。这些寄生虫是多种人类动植物疾病的病原体，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。 U-插入/删除RNA编辑这样独特的代谢途径的存在可能为化疗干预提供选择性处理而不影响宿主。 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有助于开发能够杀死寄生虫而不影响宿主的药物。