ANALYSIS OF TRYPANOSOME mRNA SYNTHESIS BY GENE TRANSFER

描述（由申请人提供）：锥虫相关疾病对全世界的生产性人类生活造成严重破坏并导致高死亡率。锥虫转录机制似乎与其他经过深入研究的真核生物（酵母和哺乳动物）的更为保守的机制表现出有趣的差异。例如，布氏锥虫和锥虫家族的其他成员含有一种不寻常的 RNA 聚合酶 II 酶和一组似乎被删减的转录因子。此外，前 mRNA 起始位点仍然难以捉摸，转录起始与 mRNA 5' 加帽不协调。这些机制差异为研究锥虫中 RNA 聚合酶 II 依赖性过程的结构和功能提供了令人信服的论据，最终目标是增强我们对这种进化多样性过程的理解，并开发生物体特异性化疗。尽管我们的最终目标是了解 RNA 聚合酶 II 依赖性基因表达的机制，但我们的短期目标将集中于表征已知或怀疑在前 mRNA 合成中起作用的蛋白质和序列。迄今为止，我们已经证明 tSNAPc、tTBP、TFIIB、TFIIA-gamma 和 RNA 聚合酶 II 在 SL RNA 基因启动子处组装，并且是指导 SL RNA 合成所必需的。由于 RNA 聚合酶 II 也是前 mRNA 合成所必需的，因此当前的提案将对这些蛋白质以及我们实验室最近发现的几种新型 RNA 聚合酶 II 相互作用蛋白质的分析扩展到被认为指导前 mRNA 表达的基因组位点。将使用以下两种互补方法来研究前 mRNA 基因表达的机制。首先，纯化的蛋白质因子和 DNA 序列的功能将通过包括我们自己的实验室在内的多个实验室开发的体外转录研究结合蛋白质-蛋白质和蛋白质-DNA 相互作用研究来确定。第二种方法将利用最初由其他人开发的体内研究来评估每种蛋白质在细胞活力和前体 mRNA 表达中的功能。这些研究预计将为我们提供关于真核生物实现基本相似目标的不同机制的有趣的新见解，以及针对仍然广泛传播和致残的病原体的药物治疗的潜在靶点。公共卫生相关性：致病性锥虫是世界范围内主要的公共卫生祸害。在非洲，布氏锥虫亚种。感染人类和家牛，分别引起非洲昏睡病和那加那病。 T. b 亚种对人类的感染。冈比亚和罗得西亚每年造成近20​​0万伤残调整生命年和估计48 000人死亡。在西半球，锥虫感染会导致致命的结肠肿大，以及称为恰加​​斯病的严重心肌病。大约有 1100 万人患有恰加斯病，每年有 45,000 人死亡。在南亚、南美洲和中东，一种密切相关的寄生虫——利什曼原虫，会导致使人衰弱甚至致命的利什曼病。毫不奇怪，这种疾病是驻伊拉克和阿富汗美军的一个问题。回国军人确诊700余例。目前，还没有疫苗可以保护人类免受锥虫感染，并且现有药物有毒、无效且昂贵。我们生物医学工作的目标是定义锥虫生物学的独特特征，然后利用这些特征来设计抗寄生虫化疗。

DESCRIPTION (provided by applicant): Trypanosome-related disease is responsible for significant devastation of productive human lives and high mortality rates throughout the world. The trypanosome transcriptional machinery appears to exhibit intriguing differences from the more conserved machineries of other, better- studied eukaryotes (yeast and mammals). For example, Trypanosoma brucei and other members of the trypanosomatid family contain an unusual RNA polymerase II enzyme and what appears to be an abridged set of transcription factors. Moreover, pre-mRNA start sites remain elusive and transcription initiation is not coordinated with mRNA 5' capping. These mechanistic differences provide a compelling argument for studies into the structure and function of RNA polymerase II- dependent processes in trypanosomes, with the ultimate goals of enhancing our understanding of this evolutionarily diverse process, and developing organism-specific chemotherapies. Although our ultimate objective is to understand the mechanism of RNA polymerase II- dependent gene expression, our short-term goals will focus on characterizing the proteins and sequences that are known, or suspected, to function in pre-mRNA synthesis. To date, we have shown that tSNAPc, tTBP, TFIIB, TFIIA-gamma and RNA polymerase II assemble at the SL RNA gene promoter and are necessary to direct SL RNA synthesis. Because RNA polymerase II is also required for pre-mRNA synthesis, the current proposal will extend analyses of these proteins, as well as several novel RNA polymerase II-interacting proteins recently identified in our lab, to genomic loci thought to direct pre-mRNA expression. The mechanism of pre-mRNA gene expression will be investigated using the following two complementary approaches. First, the function of purified protein factors and DNA sequences will be determined by in vitro transcription studies developed in several laboratories, including our own, in combination with protein-protein and protein-DNA interaction studies. A second approach will exploit in vivo studies, originally developed by others, to assess the function of each protein in cell viability and pre-mRNA expression. These studies are expected to provide us with interesting new insights into the diverse mechanisms by which eukaryotes accomplish fundamentally similar ends, as well as potential targets for drug therapy against what remains a wide-spread and crippling pathogen. PUBLIC HEALTH RELEVANCE: Pathogenic trypanosomes are a major, worldwide public health scourge. In Africa, Trypanosoma brucei ssp. infect humans and domestic cattle, causing African sleeping sickness and Nagana, respectively. Human infection by the subspecies T. b. gambiense and rhodesiense are responsible for almost 2 million disability-adjusted life-years and an estimated 48,000 deaths each year. In the Western hemisphere, trypanosome infection causes fatal enlargement of the colon, as well as serious cardiomyopathy known as Chagas' disease. Approximately 11 million people are afflicted with Chagas' disease and 45,000 people die yearly. In South Asia, South America, and the Middle-East, a closely related group of parasites, the Leishmania, cause debilitating and often fatal Leishmaniasis. Not surprisingly, this disease is a problem for American troops in Iraq and Afghanistan; more than 700 cases have been diagnosed in returning military personnel. At present, there are no vaccines to protect humans from trypanosomal infections and current drugs are toxic, ineffective and expensive. The goal of our biomedical work is to define unique features of trypanosome biology and then to exploit these traits for the design of anti-parasitic chemotherapies.