Characterization of Trypanosome telomere complex

锥虫端粒复合物的表征

• 批准号: 7849189
• 负责人: Bibo Li
• 金额: $ 1.77万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-19 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7849189
• 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; Proteins; RNA Interference; Role; Structure; Trypanosoma; Trypanosoma brucei brucei; Yeasts; cDNA Library; in vivo; mutant; nagana; overexpression; protein complex; 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.

布鲁氏弓形虫是一种寄生虫，可导致撒哈拉以南非洲地区人类和牛的昏睡病。