ANALYSIS OF TRYPANOSOME mRNA SYNTHESIS BY GENE TRANSFER

描述（由申请人提供）：锥虫相关疾病在世界各地造成人类生产生命的重大破坏和高死亡率。锥虫的转录机制似乎与其他更保守的真核生物（酵母和哺乳动物）的转录机制表现出有趣的差异。例如，布鲁氏锥虫和其他锥虫家族的成员含有一种不寻常的RNA聚合酶II酶，似乎是一组精简的转录因子。此外，前mRNA起始位点仍然难以捉摸，转录起始与mRNA 5' capping不协调。这些机制差异为研究锥虫中RNA聚合酶II依赖过程的结构和功能提供了令人信服的论据，其最终目标是增强我们对这一进化多样性过程的理解，并开发生物体特异性化疗。虽然我们的最终目标是了解RNA聚合酶II依赖基因表达的机制，但我们的短期目标将集中在描述已知或怀疑在pre-mRNA合成中起作用的蛋白质和序列。到目前为止，我们已经证明tSNAPc， tTBP， TFIIB， tfiia - γ和RNA聚合酶II在SL RNA基因启动子上组装，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，目前的建议将这些蛋白质的分析，以及我们实验室最近发现的几种新的RNA聚合酶II相互作用蛋白，扩展到被认为指导前mrna表达的基因组位点。pre-mRNA基因表达的机制将通过以下两种互补的方法进行研究。首先，纯化蛋白因子和DNA序列的功能将通过几个实验室（包括我们自己的实验室）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mrna前表达中的功能。这些研究有望为我们提供有趣的新见解，了解真核生物实现基本相似目的的不同机制，以及针对广泛传播和致残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，布鲁氏锥虫。感染人类和家畜，分别引起非洲昏睡病和那加纳病。冈比亚锥虫和罗得西亚锥虫亚种的人类感染每年造成近200万残疾调整生命年和估计48 000人死亡。在西半球，锥虫感染会导致致命的结肠肿大，以及被称为恰加斯病的严重心肌病。大约1 100万人患有恰加斯病，每年有45 000人死亡。在南亚、南美和中东，有一种密切相关的寄生虫——利什曼原虫，可引起使人衰弱并往往致命的利什曼病。毫不奇怪，这种疾病对驻伊拉克和阿富汗的美军来说是个问题；回国军人中已确诊700多例。目前，没有疫苗可以保护人类免受锥虫体感染，目前的药物有毒、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.