Characterization of Trypanosome telomere complex

描述（由申请人提供）：布鲁氏绦虫是一种寄生虫，可导致撒哈拉以南非洲地区人类和牛的昏睡病。当在哺乳动物宿主体内生长时，布鲁氏t细胞会有规律地改变其主要的表面糖蛋白，以逃避宿主的免疫系统——这种现象被称为抗原变异。这些表面糖蛋白仅在靠近染色体末端的约20个位点之一——端粒中表达。酵母的端粒形成一种特殊的结构，影响附近基因的转录。类似的现象也在布鲁氏菌中被观察到，端粒可能在抗原性变异中起重要作用。布鲁氏锥虫端粒研究的最终目的是了解端粒在抗原性变异中的功能，这是布鲁氏锥虫发病的一个重要方面。最基本的步骤将是鉴定端粒组分并描述它们的功能，这是本提案的主要目标。特异性目的1：采用两种方法纯化tbTRF（布鲁氏体双链端粒DMA结合因子）蛋白复合物：1)采用抗flag和抗ha单克隆抗体对FLAG-HA-HA标记的tbTRF进行序贯免疫沉淀下拉。免疫共沉淀蛋白将通过质谱法鉴定。2)以lexA-tbTRF为诱饵进行酵母2杂交筛选，获得布鲁氏酵母gad融合cDNA文库。候选tbTRF相互作用因子将首先通过共免疫沉淀法确认其与tbTRF在体内的相互作用。阳性克隆将通过检测敲除或RNAi敲除细胞系的表型来表征其在抗原变异和端粒功能中的作用。特异性目的2：选择tbTRFH结构域的非致死性相互作用缺陷tbTRF突变，以更好地表征tbTRF的功能。使用PCR克隆，将在ttrfh结构域产生随机点突变或小缺失。失去与野生型tbTRF相互作用但仍具有与它们相互作用能力的突变体将使用常规和反向2-杂交分析进行筛选。这些突变等位基因敲入或过表达的细胞系将以其抗原变异的表型为特征。端粒复合物组分的鉴定及其功能的表征将为研究端粒抗原变异的功能提供一个良好的起点，而端粒抗原变异是布鲁氏菌病原体的一个重要方面。

DESCRIPTION (provided by applicant): 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 them 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 pathogen.