ANALYSIS OF THE TRYPANOSOMA BRUCEI GENOME USING TILING ARRAYS

描述(申请人提供)：本申请侧重于非洲锥虫，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给人类造成的疾病负担约为200万个残疾调整生命年。治疗费用很高，除非得到治疗，否则这种疾病总是致命的。由于出现了抗药性锥虫，没有疫苗，现有的为数不多的治疗药物具有严重的副作用和降低疗效。我们对预防和治疗寄生虫感染的兴趣集中在了解并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，但这些机制在人类宿主中要么不存在，要么根本不同。三种锥虫，即布氏锥虫、克氏锥虫和大利什曼原虫基因组的完成，使人们对这些基因组的编码潜力和进化有了更深入的了解。下一个挑战将是完全理解基因组的信息，这将极大地提高我们对各种生物过程的理解。这个过程的第一步之一是以一种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微阵列被用来调查基因表达的全球模式和变化，依赖于与已知或预测的基因互补的探针。最近的开创性实验扩大了微阵列的应用范围，包括了大规模染色体功能的各个方面。在所谓的基因组平铺微阵列实验中，所有由染色体或基因组组成的非重复DNA都以低至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，它们提供了对基因组进行无偏见询问的机会。我们建议将这项技术应用于布氏毛滴虫基因组，具体目标如下：1.我们将在NimbleGen基因组平铺微阵列上将整个非重复的布氏毛滴虫基因组与从两个不同发育阶段获得的cDNA杂交来确定完整的布氏毛滴虫转录组；以及2.我们将对布氏毛滴虫基因组中的转录因子结合位点进行无偏见的定位。拟议的项目将开发锥虫基因组拼接微阵列工具，并生成转录和调控序列的全面目录；两者都将是研究界极其宝贵的资源。这些基因组芯片未来的潜在应用将是系统地鉴定可能涉及转录后基因调控的各个方面的RNA结合蛋白的转录靶标，以及检查核小体占据和组蛋白修饰状态。 项目简介：寄生原生动物是全球传染病的主要原因，因此是对公共卫生最严重的威胁之一。其中包括非洲锥虫，这是非洲锥虫病或人类昏睡病的病原体，以及牛、家猪和其他农场动物的一种消瘦和致命疾病(Nagana)，对该大陆大部分地区的经济产生深远影响。除非得到治疗，否则非洲昏睡病总是致命的；没有疫苗被批准，而且具有普遍严重缺点的药物非常有限，例如高毒性和新出现的耐药性。

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.