Comparative Analysis of Editosome in Trypanosomatids

项目描述（由申请人提供）：本项目亲本资助：R37 AI4102，正常和突变锥虫线粒体DNA。本项目建立在鉴定布鲁氏锥虫编辑机制（编辑体）某些组成部分的蛋白质和基因的亲本基金研究的基础上。亲本资助为该生物提供了重要的生物学见解，对不同着丝质体物种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和进化提供一个绝佳的机会。该分析不仅在识别未被预测算法调用的基因方面具有实用价值，而且对着丝质体寄生虫中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，确定人类关键的主要着丝质体寄生虫；布氏体，克氏体，还有利什曼原虫。这将通过利用布鲁氏体的信息来快速鉴定其他两种主要的着丝体寄生虫的编辑体基因和蛋白质来实现。该项目将采用计算机分析（已经在布氏体中发现了许多候选基因）和利用亲和标签技术结合串联质谱的直接分析相结合的方法。此外，催化活性编辑体的形态将通过电子显微镜（EM）来确定，因为现有数据表明锥虫和利什曼原虫物种之间存在实质性的结构差异。本提案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。这些分析将有助于了解编辑体及其组成部分的功能，也有助于开发对这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.