Characterization of Trypanosome telomere complex

描述（由申请人提供）：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，以逃避宿主免疫系统--这种现象称为抗原变异。这些表面糖蛋白仅从位于染色体末端附近的~ 20个位点之一-端粒表达。酵母中的端粒形成一种特殊的结构，影响附近基因的转录。在T.而端粒可能在抗原变异中起重要作用。布氏锥虫端粒研究的最终目的是了解端粒在抗原变异中的功能，这是布氏锥虫的一个重要方面。布氏菌致病性基本步骤是识别端粒成分并描述其功能，这是本提案的主要目标。具体目的1：纯化T. 1）使用抗FLAG和抗HA单克隆抗体连续免疫沉淀下拉FLAG-HA-HA标记的tbTRF。将通过质谱法鉴定免疫共沉淀的蛋白质。2)以lexA-tbTRF为诱饵进行酵母双杂交筛选，并以T.布鲁氏菌GAD融合cDNA文库。tbTRF相互作用因子的候选物将首先通过免疫共沉淀确认其与tbTRF的体内相互作用。将通过检查敲除或RNAi敲减细胞系中的表型来表征阳性克隆在抗原变异和端粒功能中的作用。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，以更好地表征tbTRF的功能。使用PCR克隆在tbTRFH结构域中产生随机点突变或小缺失。将使用常规和反向双杂交分析来筛选失去与野生型tbTRF的相互作用但保持与它们相互作用的能力的突变体。具有这些突变等位基因的敲入或过表达的细胞系将表征其抗原变异的表型。端粒复合物组分的鉴定及其功能的后续表征将是研究端粒在抗原变异中的功能的一个很好的起点，抗原变异是T。布氏杆菌

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.