Analysis of Trypanosome mRNA Synthesis by Gene Transfer

描述(申请人提供)：锥虫病和利什曼病是全世界发展中国家的主要疾病。在这些地区，目前有10万人感染了锥虫。人类疾病包括非洲锥虫病(“昏睡病”)、恰加斯病(在南美洲和中美洲地区流行)和皮肤、粘膜皮肤和内脏利什曼病(在中东和印度次大陆部分地区流行)。非洲锥虫病通过携带布氏锥虫亚种的采采蝇的叮咬传播给人类。据世界卫生组织估计，疾病负担为200万个残疾调整寿命年。治疗费用很高，未经治疗的感染通常会导致死亡。然而，锥虫之间有许多共同的代谢途径，这些途径与人类的途径有很大的不同，可以用来开发比目前可用的更有效的治疗方法。 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信使核糖核酸生物合成途径而提出的。这项建议的目的是了解SL RNA和mRNAs是如何在锥虫体内转录的。锥虫体内似乎存在广泛的转录，但关于mRNAs是如何产生的，我们知之甚少。因此，同一生命周期中不同mRNAs之间存在的mRNA成熟差异可能部分是通过不同的mRNA合成来调节的。我们已经在锥虫基因表达领域取得了重大进展，鉴定并开始鉴定塞穆尔细单胞菌PBP-1和PBP-2。这些蛋白是在锥虫中发现的第一批双链序列特异性DNA结合蛋白。它们似乎是调节SL RNA表达的基础转录因子；它们在前mRNA合成中的作用尚不清楚。我们建议通过对布鲁氏锥虫PBP-1和PBP-2的生化和遗传细节的研究来了解这些蛋白的结构和功能。在确定锥虫SL RNA基因启动子是RNAP II依赖的之后，我们假设PBP-1和/或PBP-2可能在其他RNAP II依赖的合成中发挥作用，特别是前mRNA的合成。我们将通过分析布氏毛滴虫2号染色体中心区域的前RNA合成来直接测试这种可能性，并确定参与RNAP II依赖的RNA合成的其他转录因子。我们最近开发了一种预起始复合体形成试验、染色质免疫沉淀试验和酵母双杂交系统，这些都是我们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.