ANALYSIS OF THE TRYPANOSOMA BRUCEI GENOME USING TILING ARRAYS

描述（由申请人提供）：本申请关注非洲锥虫，非洲锥虫病的病原体。世界卫生组织保守估计，非洲锥虫病或昏睡病的疾病负担约为200万残疾调整生命年。治疗费用很高，除非治疗，这种疾病总是致命的。由于抗药性锥虫的出现，没有疫苗，现有的少数治疗药物具有严重的副作用，疗效也在下降。我们对预防和治疗寄生虫感染的兴趣集中在理解和最终利用对寄生虫生命周期的所有阶段都至关重要的遗传机制，但在人类宿主中要么不存在，要么根本不同。布氏锥虫、克氏锥虫和硕大利什曼原虫三种锥虫基因组的完成，开启了对这些基因组的编码潜力和进化的深入了解。下一个挑战将是获得对基因组信息的完整理解，这将大大提高我们对各种生物过程的理解。这一过程的第一步是以全面和公正的方式生成基因组中所有转录区域的图谱。传统上，DNA微阵列用于调查基因表达的全球模式和变化，依赖于与已知或预测基因互补的探针。最近的开创性实验拓宽了微阵列的应用，包括大规模染色体功能的方面。在所谓的基因组平铺微阵列实验中，包含染色体或基因组的所有非重复DNA以低至25个碱基对的序列分辨率表示。由于平铺阵列的设计没有咨询现有的基因注释，他们提供了一个公正的询问基因组的机会。我们建议将这项技术应用于T。布鲁氏菌基因组具有以下特定目的：1.我们将确定完整的T。通过杂交整个非重复T.用从两个不同发育阶段获得的cDNA在NimbleGen基因组拼接微阵列上显示布鲁氏菌基因组;和2.我们将在T.布鲁氏菌基因组拟议的项目将开发锥虫基因组镶嵌微阵列工具，并生成转录和调节序列的全面目录;这两者对于研究界来说都将是极其宝贵的资源。这些基因组镶嵌微阵列的潜在未来应用将是系统地鉴定可能参与转录后基因调控的各个方面的RNA结合蛋白的转录物靶点，以及检查核小体占用和组蛋白修饰状态。 项目叙述：寄生原生动物是全球传染病的主要原因，因此是对公共卫生的最严重威胁之一。其中包括非洲锥虫，它是非洲锥虫病或人类昏睡病的病原体，也是牛、家猪和其他农场动物的一种消耗性和致命性疾病（Nagana），对非洲大陆大部分地区的经济造成深远影响。除非得到治疗，非洲昏睡病总是致命的;没有任何疫苗得到批准，而且药物库非常有限，普遍存在严重缺陷，如毒性高和正在出现耐药性。

DESCRIPTION (provided by applicant): This application focuses on African trypanosomes, the causative agents of African trypanosomiasis. The World Health Organization conservatively estimates that the disease burden of human African trypanosomiasis or sleeping sickness is about two million Disability Adjusted Life Years. The cost of treatment is high and unless treated, the disease is always fatal. There are no vaccines and the few available therapeutic drugs have serious side effects and decreasing efficacy in light of the emergence of drug-resistant trypanosomes. Our interest in preventing and curing parasite infections is focused on understanding and ultimately exploiting genetic mechanisms that are essential for all stages of the parasite life cycle, but are either absent or fundamentally different in the human host. The completion of the genomes of three trypanosomatids, namely Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, has opened insights into the coding potential and evolution of these genomes. The next challenge will be to gain a complete understanding of the genome's information, which will dramatically improve our understanding of various biological processes. One of the first steps in this process is the generation of a map of all transcribed regions in the genome in a comprehensive and unbiased way. Traditionally, DNA microarrays are used to survey global patterns and changes in gene expression, relying on probes complementary to known or predicted genes. Recent pioneering experiments have broadened microarray applications to include aspects of large-scale chromosome function. In so-called genomic tiling microarray experiments, all non-repetitive DNA comprising a chromosome or genome is represented at sequence resolutions as low as 25 base-pairs. Since tiling arrays are designed without consultation of existing gene annotation, they offer the opportunity for an unbiased interrogation of a genome. We propose to apply this technology to the T. brucei genome with the following specific aims: 1. We will determine the complete T. brucei transcriptome by hybridizing the entire non-repetitive T. brucei genome on NimbleGen genome-tiling micro-arrays with cDNA obtained from two different developmental stages; and 2. We will perform an unbiased mapping of transcription factor binding sites in the T. brucei genome. The proposed project will develop trypanosome genome-tiling microarray tools and generate comprehensive catalogues of transcribed and regulatory sequences; both will be extremely valuable resources for the research community. Potential future application of these genomic tiling microarrays will be the systematic identification of the transcript targets of RNA-binding proteins that are likely involved in various aspects of post-transcriptional gene regulation, and an examination of nucleosome occupancy and histone modification status. PROJECT NARRATIVE: Parasitic protozoa are a major cause of global infectious diseases and thus, represent one of the most serious threats to public health. Among these are African trypanosomes, the causative agents of African trypanosomiasis or sleeping sickness in humans and a wasting and fatal disease (Nagana) in cattle, domestic pigs and other farm animals causing a profound effect on the economy of much of the continent. Unless treated, African sleeping sickness is always fatal; no vaccine has been approved and there is a very limited arsenal of drugs with generally severe shortcomings, such as high toxicity and emerging resistance.