Analysis of Trypanosome mRNA Synthesis by Gene Transfer

描述（申请人提供）：锥虫病和利什曼病是世界上发展中国家的主要疾病。在这些地区，目前有10万人感染锥虫。人类疾病包括非洲锥虫病（“昏睡病”）、恰加斯病（南美洲和中美洲地区流行病）和皮肤、粘膜皮肤和内脏利什曼病（中东和印度次大陆部分地区流行病）。非洲锥虫是通过携带布氏锥虫亚种的采采蝇叮咬传播给人类的。据世界卫生组织估计，疾病负担为200万残疾调整生命年。治疗费用很高，未经治疗的感染通常导致死亡。然而，锥虫在它们之间共享许多代谢途径，这些代谢途径与人类途径足够不同，以用于开发比目前可用的治疗剂更有效的治疗剂。 一种可能的治疗方法是通过观察锥虫具有惊人的不寻常的mRNA生物合成途径而提出的。这个提议的目的是了解SL RNA和mRNA在锥虫中是如何转录的。在锥虫中似乎有广泛的转录，但我们对mRNA如何产生的信息很少。因此，在一个生命周期形式中存在于不同mRNA之间的mRNA成熟差异可能部分地受到差异mRNA合成的调节。我们已经取得了重大进展，在该领域的锥虫基因表达的鉴定和开始表征细单胞菌seymouri PBP-1和PBP-2。这些蛋白是锥虫中鉴定的第一批双链序列特异性DNA结合蛋白。它们似乎作为调节SL RNA表达的基础转录因子发挥作用;它们在前mRNA合成中的作用尚不清楚。我们建议通过对T.布氏杆菌PBP-1和PBP-2。在确定锥虫SL RNA基因启动子为RNAP II依赖性后，我们假设PBP-1和/或PBP-2可能在其他RNAP II依赖性合成中起作用，特别是前mRNA合成。我们将直接测试这种可能性，并通过分析T细胞中心区域的前RNA合成来鉴定参与RNAP II依赖性RNA合成的其他转录因子。布鲁氏菌2号染色体。我们最近开发了一个preinitiation复合物形成试验，染色质免疫沉淀试验和酵母双杂交系统，我们的转录解剖的相关工具。

DESCRIPTION (provided by applicant): Trypanosomiasis and Leishmaniasis are major diseases in developing countries throughout the world. In these areas 100,000 people are currently infected with trypanosomes. Human illness includes African trypanosomiasis ("sleeping sickness"), Chagas' disease (endemic in regions of South and Central America) and cutaneous, mucocutaneous and visceral Leishmaniasis (endemic in parts of the Middle East and the Indian subcontinent). African trypanosomiasis is spread to humans by the bite of tsetse flies that harbor the Trypanosoma brucei subspecies. The disease burden is estimated by the World Health Organization to be two million Disability Adjusted Life Years. The cost of treatment is high, and untreated infection usually results in death. However, trypanosomatids share many metabolic pathways among themselves that differ enough from human pathways to be exploited in the development of therapeutics that are more efficacious than those currently available. One possible therapeutic approach is suggested by the observation that trypanosomatids have strikingly unusual mRNA biosynthetic pathways. The purpose of this proposal is to gain an understanding of how SL RNA and mRNAs are transcribed in trypanosomes. There appears to be extensive transcription in trypanosomes, but we have little information as to how mRNAs are produced. Therefore, the differences in mRNA maturation that exist among different mRNAs in one life cycle form may be regulated, in part, by differential mRNA synthesis. We have made major headway in the field of trypanosome gene expression by identifying and beginning to characterize Leptomonas seymouri PBP-1 and PBP-2. These proteins are among the first double stranded sequence-specific DNA binding proteins identified in trypanosomatids. They appear to function as basal transcription factors that regulate SL RNA expression; their role in pre-mRNA synthesis is unknown. We propose to understand to the structure and function of these proteins by characterizing in biochemical and genetic detail T. brucei PBP-1 and PBP-2. Having identified the trypanosomal SL RNA gene promoter as RNAP II-dependent, we hypothesize that PBP-1 and/or PBP-2 may function in other RNAP II-dependent synthesis, specifically pre-mRNA synthesis. We will directly test this possibility and identify additional transcription factors involved in RNAP II-dependent RNA synthesis by analyzing pre-RNA synthesis from a central region of T. brucei chromosome 2. We have recently developed a preinitiation complex formation assay, a Chromatin Immunoprecipation assay and a yeast two-hybrid system that are pertinent tools for our transcriptional dissections.