Characterization of Trypanosome telomere complex

T.布氏杆菌是一种寄生虫，在撒哈拉以南非洲引起人的昏睡病和牛的Nagana病。 在哺乳动物宿主中生长时，T.布氏细胞有规律地转换其主要表面糖蛋白， 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 仅从位于染色体末端附近的~ 20个位点之一-端粒表达。中的端粒 酵母形成影响附近基因转录的特化结构。类似的 在T.染色体端粒可能在布鲁氏菌的抗原表达中起重要作用 变化量布氏锥虫端粒研究的最终目的是了解端粒的功能 在抗原变异中，T.布氏菌致病性最基本的一步是 确定端粒组成部分并描述其功能，这是本提案的主要目标。 具体目的1：纯化T.布氏杆菌蛋白 1）FLAG-HA-HA标记的tbTRF的顺序免疫沉淀物下拉 使用抗FLAG和抗HA单克隆抗体。免疫共沉淀的蛋白质将通过 质谱分析法来2)以lexA-tbTRF为诱饵进行酵母双杂交筛选，并以T.布鲁氏菌GAD-fusion cDNA 图书馆tbTRF相互作用因子的候选者将首先确认其与tbTRF在体内的相互作用 通过免疫共沉淀。将表征阳性克隆在抗原变异中的作用， 通过检查敲除或RNAi敲低细胞系中的表型来检测端粒功能。 具体目标2：选择tbTRFH结构域中的非致死性相互作用缺陷型tbTRF突变，以更好地 tbTRF的功能。tbTRFH中将产生随机点突变或小缺失， 结构域，使用PCR克隆。失去与野生型tbTRF的相互作用但仍保持与野生型tbTRF相互作用的能力的突变体， 将使用常规和反向双杂交分析来筛选与自身相互作用的细胞。细胞系 这些突变等位基因的敲入或过表达将表征它们的表型， 抗原变异 端粒复合体组分的鉴定及其随后的功能表征将 是一个很好的起点，研究端粒功能的抗原变异，一个重要的方面T。 布氏菌致病性

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.