Characterization of Trypanosome telomere complex

锥虫端粒复合物的表征

• 批准号: 7335623
• 负责人: Bibo Li
• 金额: $ 30.25万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7335623
• 关键词: Africa South of the Sahara; African Trypanosomiasis; Alleles; Antigenic Variation; Binding; Cattle; Cell Line; Cells; Chromosomes; Cloning; Co-Immunoprecipitations; Complex; Genetic Transcription; Goals; Human; Hybrids; Immune system; Knock-in Mouse; Knock-out; Mass Spectrum Analysis; Membrane Glycoproteins; Monoclonal Antibodies; Mutagenesis; Mutation; Parasites; Pathogenesis; Phenotype; Play; Point Mutation; Polymerase Chain Reaction; Protein Overexpression; Proteins; RNA Interference; Role; Structure; Trypanosoma; Trypanosoma brucei brucei; Yeasts; cDNA Library; in vivo; mutant; nagana; telomere

T. brucei is a parasite that causes sleeping sickness in humans and Nagana in cattle in sub-Saharan Africa. When growing in mammalian host, T. brucei cells regularly switch their major surface glycoprotein, to evade the host immune system - a phenomenon called antigenic variation. These surface glycoproteins are exclusively expressed from one of ~ 20 loci located next to chromosome ends - telomeres. Telomeres in yeasts form a specialized structure that influences transcription of genes located close by. A similar phenomenon has also been observed in T. brucei, and telomeres may play an important role in antigenic variation. The ultimate goal of telomere studies in Trypanosoma brucei is to understand telomere functions in antigenic variation, an essential aspect of T. brucei pathogenesis. The fundamental step would be to identify telomere components and characterize their functions, which is the primary goal of this proposal. Specific Aim 1: To purify tbTRF (a double-stranded telomere DMA binding factor in T. brucei) protein complex, using two approaches: 1) Sequential immunoprecipitate pull-down of FLAG-HA-HA tagged tbTRF using anti-FLAG and anti-HA monoclonal antibodies. Proteins co-immunoprecipitated will be identified by mass spectrometry. 2) Yeast 2-hybrid screen using lexA-tbTRF as bait and a T. brucei GAD-fusion cDNA library. Candidates for tbTRF-interaction factors will be first confirmed for their interaction with tbTRF in vivo by co-immunoprecipitation. Positive clones will be characterized for their roles in antigenic variation and telomere functions by examination of phenotypes in knockout or RNAi knockdown cell lines. Specific Aim 2: Select non-lethal interaction-deficient tbTRF mutations in the tbTRFH domain to better characterize tbTRF's function. Random point mutations or small deletions will be generated in the tbTRFH domain, using PCR cloning. Mutants that lose the interaction with wild-type tbTRF but remain the ability to interact with themselves will be screened using both conventional and reverse 2-hybrid analysis. Cell lines with knock-in or overexpression of these mutant alleles will be characterized for their phenotypes in antigenic variation. The identification of telomere complex components and subsequent characterization of their functions would be a good starting point for studying telomere functions in antigenic variation, an essential aspect of T. brucei pathogenesis.

T. Brucei是一种寄生虫，在撒哈拉以南非洲的牛中引起人类和长纳氏病。 当在哺乳动物宿主中生长时，T。Brucei细胞会定期切换其主要的表面糖蛋白，以逃避 宿主免疫系统 - 一种称为抗原变异的现象。这些表面糖蛋白是 独家从位于染色体末端 - 端粒旁边的20个基因座之一表达。端粒 酵母形成了一种专门的结构，影响位于附近的基因的转录。类似 现象也已经在布鲁氏菌中观察到，端粒可能在抗原中起重要作用 变化。布鲁氏锥虫的端粒研究的最终目标是了解端粒功能 在抗原变异中，是布鲁氏菌发病机理的重要方面。基本步骤是 识别端粒组件并表征其功能，这是该建议的主要目标。 特定目的1：纯化TBTRF（T. brucei中的双链端粒DMA结合因子）蛋白 复合物，使用两种方法：1）flag-ha-ha标记的序列免疫沉淀物下拉 使用抗FLAG和抗HA单克隆抗体。共免疫沉淀的蛋白质将通过 质谱法。 2）使用Lexa-TBTRF作为诱饵和T. brucei Gad-Fusion cDNA的酵母2杂交屏幕 图书馆。首先将确认针对TBTRF相互作用因子的候选者与TBTRF体内的相互作用 通过共免疫沉淀。阳性克隆将以其在抗原变异和 通过检查敲除或RNAi敲除细胞系中表型的端粒功能。 特定目标2：在TBTRFH域中选择非致命相互作用的TBTRF突变以更好 表征TBTRF的功能。 TBTRFH将产生随机点突变或小缺失 域，使用PCR克隆。失去与野生型TBTRF相互作用但仍然能力的突变体 将使用常规和反向2杂交分析筛选与自己的互动。细胞系 这些突变等位基因的敲入或过表达将以其表型为特征 抗原变异。 端粒复合物成分的识别以及随后的功能表征将 成为研究抗原变异中端粒功能的好起点，抗原变异是T的一个基本方面。 Brucei发病机理。