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

基本信息

批准号：
7253484
负责人：
KEVIN MILITELLO
金额：
$ 19.34万
依托单位：
COLLEGE AT GENESEO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7253484
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

项目摘要

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：项目摘要Brucei是负责人类非洲锥虫病（HAT）的原生动物寄生虫，也称为非洲睡眠病。由于没有有效的疫苗，因此迫切需要对这种疾病进行新的控制策略。用于治疗帽子的许多药物都是有毒和无效的。对寄生虫的生命周期的完全理解将有可能揭示用于治疗干预的新目标。寄生虫的生命周期需要Tsetsfe Fly和哺乳动物宿主的生长和分化。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的存在的能力。我们的生物信息学研究表明，非洲锥虫含有一个称为5-甲基胞嘧啶的改良碱，而这种改良的碱是通过抑制基因转录来依赖生命周期依赖性蛋白表达所必需的。我们将使用分子，生化和遗传策略的结合来检验这一假设。首先，通过探测来自布鲁氏菌寄生虫的纯化核和线粒体DNA的5-甲基胞菌素的亚细胞定位和发育调节，其具有5-甲基环霉素的抗体。接下来，我们将确定促成Brucei 5-甲基霉素产生的酶促机械。假定的t. brucei cytosine-5 DNA甲基转移酶将从大肠杆菌中产生并纯化。体外DNA甲基化反应将用于确定该蛋白是否确实具有合成5-甲基胞嘧啶的能力，并确定该反应的生化需求。最后，我们将确定5-甲基霉素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，并将使用Northern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对帽子的控制。我们预测，寄生虫调节表面抗原表达和逃避人类免疫系统的能力是必需的5-甲基胞嘧啶途径。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治疗剂来预防和治疗非洲人类锥虫病，因为目前尚无有效的疫苗，药物治疗是有毒和不可靠的。我们对brucei锥虫瘤中胞嘧啶DNA甲基化的研究可能揭示了寄生虫调节表面抗原表达并逃避人类免疫系统的机制。靶向胞质DNA甲基化的药物可能会消除寄生虫免疫系统逃避或传播。