Characterization of Trypanosome telomere complex

布氏毛滴虫是一种寄生虫，在撒哈拉以南非洲地区引起人类昏睡病和牛的纳加纳。 当在哺乳动物宿主中生长时，布氏毛滴虫细胞会定期切换它们的主要表面糖蛋白，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 仅由位于染色体末端附近的~20个基因座中的一个-端粒表达。端粒在 酵母菌形成一种特殊的结构，影响邻近基因的转录。一种类似的 在布氏毛滴虫中也观察到了这种现象，端粒可能在抗原性中发挥重要作用。 变种。布鲁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，布氏毛滴虫致病的一个重要方面。最基本的一步是 鉴定端粒成分并鉴定其功能，这是本提案的主要目标。 特异性目的1：纯化布氏毛滴虫双链端粒DNA结合因子tbTRF蛋白 使用两种方法：1)顺序免疫沉淀FLAG-HA-HA标记的tbTRF的下拉 使用抗FLAG和抗HA的单抗。免疫共沉淀的蛋白质将通过 质谱学。2)以LexA-tbTRF为诱饵与布氏锥虫GAD-融合基因进行酵母双杂交筛选 图书馆。TbTRF-相互作用因子的候选将首先被确认为他们在体内与tbTRF相互作用 通过免疫共沉淀法。阳性克隆的特征是它们在抗原变异和 端粒通过检测基因敲除或RNAi基因敲除细胞系的表型来发挥作用。 具体目标2：选择tbTRFH结构域中的非致命性相互作用缺陷tbTRF突变，以更好地 描述tbTRF的功能。在tbTRFH中会产生随机的点突变或小的缺失 结构域，采用PCR克隆技术。与野生型tbTRF失去相互作用但仍有能力 将使用常规和反向双杂交分析来筛选与其自身的相互作用。细胞系 随着这些突变等位基因的敲入或过度表达，将在 抗原性变异。 鉴定端粒复合体成分并随后鉴定其功能将 这是研究端粒在抗原变异中的功能的一个很好的起点，端粒是T。 布鲁斯病机。

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.