ANALYSIS OF TRYPANOSOME mRNA SYNTHESIS BY GENE TRANSFER

描述(申请人提供)：与锥虫有关的疾病对人类生产生活造成严重破坏，并导致全世界的高死亡率。锥虫的转录机制似乎表现出与其他更保守的、研究得更好的真核生物(酵母和哺乳动物)更保守的机制的有趣区别。例如，布鲁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，以及一组似乎被删减的转录因子。此外，前信使核糖核酸的起始位置仍然难以捉摸，转录启动与信使核糖核酸5‘端的封端不协调。这些机制上的差异为研究锥体中依赖RNA聚合酶II的过程的结构和功能提供了令人信服的论点，最终目的是加强我们对这一进化多样化过程的理解，并开发机体特异性的化疗药物。虽然我们的最终目标是了解依赖RNA聚合酶II的基因表达的机制，但我们的短期目标将集中在表征已知或怀疑在前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-Gamma和RNA聚合酶II组装在SL RNA基因的启动子上，是指导SL RNA合成所必需的。由于RNA聚合酶II也是合成前mRNA所必需的，因此目前的提议将把对这些蛋白质以及我们实验室最近发现的几种新的RNA聚合酶II相互作用蛋白的分析扩展到被认为直接导致前mRNA表达的基因组座位。我们将用以下两种互补的方法来研究Pre-mRNA基因表达的机制。首先，纯化的蛋白质因子和DNA序列的功能将通过几个实验室(包括我们自己)开展的体外转录研究以及蛋白质-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存活和前mRNA表达中的功能。这些研究有望为我们提供有趣的新见解，了解真核生物实现根本相似目的的不同机制，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。与公共卫生相关：致病性锥虫是一种重大的世界性公共卫生祸害。在非洲，布氏锥虫Typanosoma brucei SSP.感染人类和家畜，分别导致非洲昏睡病和纳加纳。冈比亚和罗得西亚亚种的人类感染造成了近200万个经残疾调整的生命年，估计每年有4.8万人死亡。在西半球，锥虫感染会导致致命的结肠增大，以及被称为恰加斯病的严重心肌病。大约有1100万人患有恰加斯氏病，每年有45000人死亡。在南亚、南美和中东，一组密切相关的寄生虫--利什曼原虫--会导致衰弱的、往往是致命的利什曼病。毫不奇怪，这种疾病是驻伊拉克和阿富汗的美军面临的一个问题；在回国的军事人员中，已有700多人被诊断出患有这种疾病。目前还没有保护人类免受锥虫感染的疫苗，目前的药物有毒、无效且价格昂贵。我们的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的化疗药物。

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.