Cytosine DNA Methylation and Transcriptional Repression in African Trypanosomes

非洲锥虫中的胞嘧啶 DNA 甲基化和转录抑制

• 批准号: 7660827
• 负责人: KEVIN MILITELLO
• 金额: $ 1.3万
• 依托单位: COLLEGE AT GENESEO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660827
• 关键词: Africa South of the Sahara; African; African Trypanosomiasis; Antibodies; Antigens; Biochemical; Biochemical Genetics; Biochemistry; Bioinformatics; Biology; Blood Circulation; Chromosomes; Code; Complex; Cytosine; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA analysis; Databases; Detection; Development; Differentiation and Growth; Disease; Drug Delivery Systems; Drug usage; Elements; Environment; Escherichia coli; Eukaryota; Eukaryotic Cell; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Goals; Homologous Gene; Human; Immune system; In Vitro; Individual; Infection; Knowledge; Life Cycle Stages; Location; Membrane Glycoproteins; Mitochondrial DNA; Modeling; Modification; Molecular; Northern Blotting; Nuclear; Organism; Parasites; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Proteins; RNA Interference; Reaction; Recombinants; Regulation; Research; Retrotransposon; Role; Site-Directed Mutagenesis; Southern Blotting; Staging; Surface Antigens; System; Technology; Testing; Tetracycline; Tetracyclines; Therapeutic Intervention; Transcript; Transcriptional Regulation; Translations; Trypanosoma; Trypanosoma brucei brucei; Tsetse Flies; Vaccines; Variant; base; fly; gene repression; genome sequencing; improved; in vivo; insight; mRNA Stability; novel; novel therapeutics; prevent; promoter; protein expression; research study; transmission process

DESCRIPTION (provided by applicant): 4.4 Item 6: Project Summary Trypanosoma brucei is the protozoan parasite responsible for Human African Trypanosomiasis (HAT), otherwise known as African Sleeping Sickness. There is an urgent need for new control strategies for this disease, as there is no effective vaccine. Many of the drugs used to treat HAT are toxic and ineffective. A complete understanding of the parasite's life cycle will potentially reveal novel targets for therapeutic intervention. The life cycle of the parasite requires growth and differentiation in both the tsetsfe fly and mammalian host. The ability of the parasites to adapt and replicate in each unique environment is dependent on the ability to regulate the presence of specific proteins throughout the life cycle. Our bioinformatic studies suggest that African trypanosomes contain a modified base called 5-methylcytosine, and this modified base is required for life cycle-dependent protein expression by repressing gene transcription. We will test this hypothesis using a combination of molecular, biochemical and genetic strategies. First, the subcellular localization and developmental regulation of 5-methylcytosine will be determined by probing purified nuclear and mitochondrial DNA from T. brucei parasites with an antibody specific to 5-methylcytosine. Next, we will identify the enzymatic machinery responsible for 5-methycytosine generation in T. brucei. The putative T. brucei cytosine-5 DNA methyltransferase will be produced and purified from E. coli. In vitro DNA methylation reactions will be used to determine if this protein truly has the ability to synthesize 5-methylcytosine, and determine the biochemical requirements for this reaction. Last, we will determine the relationship between 5-methlycytosine and transcriptional repression. Parasites lacking 5- methylcytosine will be constructed using RNA interference technology, and the effect on transcriptional silencing of target genes will be analyzed using northern blots. Our studies will potentially improve control of HAT. We predict that the 5-methylcytosine pathway is required for the parasite's ability to regulate surface antigen expression and evade the human immune system. Thus, drugs targeting this DNA modification will likely compromise parasite transmission or pathogenicity. 4.4 Item 7. Project Narrative There is an urgent need for new therapeutics to prevent and treat Human African Trypanosomiasis, as there is currently no effective vaccine and drug treatments are toxic and unreliable. Our studies of cytosine DNA methylation in Trypanosoma brucei may reveal the mechanism by which the parasites regulate expression of surface antigens and evade the human immune system. Drugs that target cytosine DNA methylation could possibly eliminate parasite immune system evasion or transmission.

描述（由申请人提供）：4.4项目6：项目概述布氏锥虫是一种原生动物寄生虫，可引起人类非洲锥虫病（HAT），也称为非洲昏睡病。由于没有有效的疫苗，迫切需要对这种疾病采取新的控制战略。许多用于治疗HAT的药物是有毒和无效的。对寄生虫生命周期的全面了解将可能揭示治疗干预的新靶点。寄生虫的生命周期需要在采采蝇和哺乳动物宿主中生长和分化。寄生虫在每个独特环境中适应和复制的能力取决于在整个生命周期中调节特定蛋白质存在的能力。我们的生物信息学研究表明，非洲锥虫含有一种称为5-甲基胞嘧啶的修饰碱基，这种修饰碱基是通过抑制基因转录来实现生命周期依赖性蛋白质表达所必需的。我们将使用分子、生物化学和遗传学策略的组合来检验这一假设。首先，5-甲基胞嘧啶的亚细胞定位和发育调控将通过探测纯化的核和线粒体DNA从T。布氏疟原虫与5-甲基胞嘧啶特异性抗体。接下来，我们将确定在T中负责5-甲基胞嘧啶生成的酶机制。布鲁塞。假定的T.将从E.杆菌体外DNA甲基化反应将用于确定该蛋白质是否真的具有合成5-甲基胞嘧啶的能力，并确定该反应的生化要求。最后，我们将确定5-甲基胞嘧啶和转录抑制之间的关系。将使用RNA干扰技术构建缺乏5-甲基胞嘧啶的寄生虫，并使用北方印迹分析对靶基因转录沉默的影响。我们的研究将有可能改善HAT的控制。我们预测，5-甲基胞嘧啶途径所需的寄生虫的能力，调节表面抗原的表达和逃避人类免疫系统。因此，针对这种DNA修饰的药物可能会损害寄生虫的传播或致病性。4.4项目7.项目叙述迫切需要新的疗法来预防和治疗非洲人类锥虫病，因为目前没有有效的疫苗，而且药物治疗有毒且不可靠。我们对布氏锥虫胞嘧啶DNA甲基化的研究可能揭示该寄生虫调节表面抗原表达和逃避人类免疫系统的机制。针对胞嘧啶DNA甲基化的药物可能会消除寄生虫免疫系统的逃避或传播。