Comparative Analysis of Editosome in Trypanosomatids

描述（由申请人提供）：该项目的母基金是R37 AI 4102，正常和突变锥虫的线粒体DNA。 该项目建立在母基金的研究基础上，该研究确定了布氏锥虫编辑机制（编辑体）的一些组成部分的蛋白质和基因。 亲本资助为这种生物提供了重要的生物学见解，对不同动质体物种中的编辑体进行比较分析将为评估编辑体组成和功能的生理学和进化提供一个难得的机会。 这种分析不仅在识别预测算法未调用的基因方面具有实用价值，而且还为动质体寄生虫中RNA编辑的分子机制提供了宝贵的额外见解。 在本研究中，我们将建立编辑体的组成，确定人类重要的主要动质体寄生虫; brucei、T. cruzi和利什曼原虫。 这将通过利用T.布氏杆菌，用于快速鉴定其他两种主要动质体寄生虫中的编辑体基因和蛋白。 该项目将采用计算机分析（已经在T.布鲁氏菌）和利用亲和标记技术结合串联质谱的直接测定法。 此外，催化活性编辑体的形态将通过电子显微镜（EM）确定，因为可用的数据表明锥虫属和利什曼原虫属物种之间的实质性结构差异。 这项建议的总体目标是确定这三个物种中编辑体的保守和不同的结构和功能特征。 这些分析将有助于理解编辑体及其组分的功能，也有助于开发对所有三种物种都有效的药物。

DESCRIPTION (provided by applicant): The parent grant for this project is R37 AI4102, Mitochondrial DNA of Normal and mutant Trypanosomes. This project builds on studies in the parent grant that identified proteins and genes for some of the components of the editing machinery (editosome) in Trypanosoma brucei. The parent grant provides significant biological insights for this organism, comparative analysis of the editosome in different kinetoplastid species will provide an exceptional opportunity to assess the physiology and evolution of editosome composition and function. This analysis will not only have practical value in the identification of genes not called by predictive algorithms but provides an invaluable additional level of insight on the molecular mechanisms of RNA editing in kinetoplastid parasites. In this proposal we will establish the composition of the editosome, determine the critical major kinetoplastid parasites of humans; T. brucei, T. cruzi, and Leishmania major. This will be accomplished by exploiting the information from T. brucei for rapid identification of editosome genes and proteins in the other two major kinetoplastid parasites. This project will employ a combination of in silico analyses (which have already identified many candidate genes in T. brucei) and direct assays that exploit affinity tag technology combined with tandem mass spectroscopy. Furthermore, the morphology of catalytically active editosomes will be determined by electron microscopy (EM), since the available data indicate substantial structural differences between Trypanosoma and Leishmania species. The overall goal of this proposal is to identify conserved and divergent structural and functional features of the editosome in these three species. These analyses will be useful for understanding the function of the editosome and its components and also for development of drugs that could be effective for all three species.