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Genome Scale Discovery of Mycobacterial Gene Function by Synthetic Genetic Arrays

通过合成基因阵列在基因组规模上发现分枝杆菌基因功能

基本信息

批准号：
8664347
负责人：
KEITH M DERBYSHIRE
金额：
$ 19.44万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-25 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8664347
关键词：
Address Animal Model Antibiotics Bacterial Genome Biochemical Pathway Biological Assay Biology Caenorhabditis elegans Candidate Disease Gene Chronic Collection Complex DNA Data Development Disease Drug Design Drug Targeting Drug resistance Epistatic Gene Escherichia coli Evolution Foundations Genes Genetic Genetic Screening Genome Genomics Genus Mycobacterium Goals Growth HIV Individual Knowledge Libraries Link Methodology Morphologic artifacts Mutagenesis Mutation Mycobacterium smegmatis Mycobacterium tuberculosis Organism Partner in relationship Pathogenesis Pathway interactions Pharmaceutical Preparations Phenotype Process Proteins Proteomics Relative (related person)Research Resources Role Series Surveys System Systems Biology Testing Therapeutic Tuberculosis Vaccines Virulence Work Yeasts base density expectation gene function genetic analysis genetic manipulation genome-wide global health innovation killings meetings mutant mycobacterial next generation novel pathogen public health relevance rapid growth research study resistant strain sex synergism tool tuberculosis treatment

项目摘要

DESCRIPTION (provided by applicant): Mycobacterial disease, primarily tuberculosis, kills nearly two million people annually. Ineffective vaccines, as well as multi-drug and extremely-drug resistant strains of M. tuberculosis, exacerbate this chronic global crisis. Clearly, new efficacious drugs are needed to fill the growing therapeutic void. Rational drug design begins with a foundation of genomic sequence information. Building upon this genomic foundation requires meaningful annotation of gene product activity and, more importantly, how the encoded proteins and the pathways they comprise come together to produce a viable mycobacterium. Screens have been developed in model organisms to connect genes by epistatic interactions on a genomic scale. Two mutations may be viable separately, but lethal when brought together. Such "synthetic lethal" interactions link the respective gene products together in an essential process. The power of synthetic genetic analysis grows exponentially with the number of genes analyzed, so testing many non-essential genes in a synthetic genetic array (SGA) is ideal. Hundreds of thousands of synthetic interactions have been described in yeasts and E. coli, and typically a single gene displays ~30 interacting partners. Large-scale approaches require a genetically tractable organism to easily create individual mutants, a process to efficiently combine individual mutations, and a reliable assay, all amenable to a high-throughput format. Mycobacterium smegmatis meets all of these requirements and will form the basis for the first comprehensive mycobacterial SGA (mSGA). Even though all of the essential components are in place, evaluating the feasibility of the approach and the establishment of a functional pipeline are prerequisites to initiating a full mSGA. We will use conjugal DNA transfer to combine individual mutations in M. smegmatis. An inability of these double mutants to grow will indicate synthetic lethality, linking the two mutant genes. We will perform limited screens progressing from candidate gene pairs with known synthetic lethality, to a more complex system with both known and likely unknown interactions, and finally with representative "hypothetical" genes with no known activity or interaction. This progression will provide the necessary positive controls to show that the system is working as intended, and it provides an opportunity to assess the potential wealth of information that an expanded mSGA could yield. Integration of epistatic information from an expanded mSGA with other meta-data from proteomic and transcriptional studies will provide a systems biology view of a mycobacterium. This application will establish an mSGA pipeline, and will identify a preliminary set of synthetic lethal interactions as proof-of-principle for its potential. Synthetic lethal interactions, identified using an unbiased genome-wid approach, identify essential functions that represent new high-value potential drug targets.

描述（由申请人提供）：分枝杆菌疾病，主要是结核病，每年造成近200万人死亡。无效的疫苗，以及多重耐药和极端耐药的M。结核病加剧了这一慢性全球危机。显然，需要新的有效药物来填补日益增长的治疗空白。合理的药物设计始于基因组序列信息的基础。建立在这个基因组基础上，需要对基因产物活性进行有意义的注释，更重要的是，编码的蛋白质及其组成的途径如何结合在一起产生一个有活力的分枝杆菌。已经在模式生物中开发了通过基因组规模上的上位相互作用连接基因的筛选。两种突变可能单独存在，但结合在一起时是致命的。这种“合成致死”相互作用在一个基本过程中将各自的基因产物连接在一起。合成遗传分析的能力随着分析的基因数量呈指数级增长，因此在合成遗传阵列（SGA）中测试许多非必需基因是理想的。在酵母和大肠杆菌中已经描述了成千上万的合成相互作用。通常一个基因展示约30个相互作用的配偶体。大规模方法需要遗传上易于处理的生物体以容易地产生单个突变体，需要有效地联合收割机组合单个突变的过程，以及需要可靠的测定，所有这些都适合于高通量形式。耻垢分枝杆菌符合所有这些要求，并将成为第一个全面的分枝杆菌SGA（mSGA）的基础。即使所有的基本组成部分都已到位，评估方法的可行性和建立功能管道是启动全面mSGA的先决条件。我们将使用接合DNA转移来联合收割机结合M.恶臭这些双突变体不能生长将表明合成致死性，连接两个突变基因。我们将进行有限的筛选，从具有已知合成致死性的候选基因对，到具有已知和可能未知相互作用的更复杂系统，最后是具有代表性的“假设”基因，没有已知的活性或相互作用。这一进展将提供必要的阳性对照，以表明系统按预期工作，并提供了评估扩展mSGA可能产生的潜在信息量的机会。整合上位性信息从扩大mSGA与其他元数据从蛋白质组学和转录研究将提供一个系统生物学的分枝杆菌。该申请将建立一个mSGA管道，并将确定一组初步的合成致命相互作用，作为其潜力的原理证明。使用无偏全基因组方法鉴定的合成致死相互作用，确定了代表新的高价值潜在药物靶点的基本功能。