ANALYSIS OF THE TRYPANOSOMA BRUCEI GENOME USING TILING ARRAYS

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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.