Gene Function and Pathway Analysis Using Systems Level Approaches in Prokaryotes

使用原核生物系统水平方法进行基因功能和通路分析

• 批准号: 8690112
• 负责人: CAROL Anne GROSS
• 金额: $ 45.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-13 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690112
• 关键词: Address; Animal Model; Bacteria; Bacterial Genome; Bacterial Infections; Behavior Control; Biochemical; Biological; Biological Process; Biology; Cell Division Process; Cell division; Cell physiology; Cells; Chemicals; Communities; Complex; Data Analyses; Data Set; Databases; Drug Targeting; Environment; Escherichia coli; Foundations; Funding; Gene Deletion; Genes; Genetic; Genetic Epistasis; Genome; Genomics; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Grant; Growth; Human; Human Microbiome; Industry; Knowledge; Link; Measures; Mediating; Membrane; Metabolism; Metagenomics; Methods; Metric; Microbiology; Molecular; Molecular Genetics; Nature; Organism; Orphan; Pathway Analysis; Pathway interactions; Peptidoglycan; Phenotype; Probability; Process; Prokaryotic Cells; Property; Proteins; Research; Research Personnel; Resources; Series; Social Behavior; Social Controls; Speed; Structure; System; Systems Analysis; Technology; Testing; Work; constriction; cost effective; deletion library; design; fitness; follow-up; gene function; gene interaction; high throughput screening; improved; knowledge base; member; mutant; novel; open source; overexpression; protein protein interaction; prototype; research study; response; tool

DESCRIPTION (provided by applicant): We address a pivotal issue in microbiology: how to decipher the vast reservoir of genomes into a blueprint for the cellular properties of bacteria. Currently, the disparity between speed of acquisition of sequence and functional information impedes utilization of our genomic resources. We have stepped into this gap. We are developing and implementing high throughput phenotyping approaches to accelerate determination of gene functions, pathways and their interconnections. Thus, we function at the interface between systems analysis and mechanistic biology. We have already shown that chemical-genomic profiling (quantitative profiling of the fitness of the complete gene deletion library under many growth conditions) and Epistasis MAPs (E- MAPs; comparison of double vs single mutant phenotypes on a genome level) rapidly accelerates discovery of phenotypes, pathways and pathway interconnections in E. coli. The work proposed in this grant significantly expands our efforts. First, following on our demonstration that chemical genomic profiling provides high correlation associations between orphan (functionally uncharacterized) genes and annotated genes, we will now develop a pipeline for discovery of orphan gene function. We will expand and improve the high correlation associations by profiling more chemical space, assess associations with other, largely non overlapping measures of functional association (e.g. protein-protein interactions) and integrate our multivariate data sets into a single interaction probability score for each potential orphan-gene-to-annotated gene interaction. This compendium will be a powerful resource both for determining orphan gene function, and for assessing which metrics of gene function are most informative and cost-effective for functional characterization. Second, we will investigate the molecular underpinnings of an elusive functional link between cell division and peptidoglycan synthesis identified in our high-throughput screens. Our previous work showed that the PBP1B bifunctional peptidoglycan synthesis machine is partially redundant with Tol-Pal in promoting outer membrane constriction during cell division. We now find that an orphan protein, YbgF, may coordinate both machines, and we will pursue molecular, biochemical and cell biological approaches to explore how coordination is accomplished. Finally, we will expand our high throughput phenotyping approaches to B. subtilis, the key gram-positive model organism and a member of the Firmicutes, one of two major phyla ubiquitously present in the human gut. We will implement chemical-genomic profiling and E-MAP analysis in B. subtilis and use it to dissect gene function and pathway connections. As Gram-positive and negative organisms differ in their envelope structures, social behaviors and control and execution of major cellular processes, including replication and metabolism, our open-source dataset will be rich in novel biology. This work addresses the "phenotype gap" impeding the use of genomic information and demonstrates the combined power of systems analyses and mechanistic studies in establishing gene function and higher-order connections between processes.

描述（由申请人提供）：我们解决了微生物学中的一个关键问题：如何将庞大的基因组库解读为细菌细胞特性的蓝图。目前，序列和功能信息获取速度之间的差异阻碍了我们基因组资源的利用。我们已经填补了这个空白。我们正在开发和实施高通量表型分析方法，以加速基因功能、途径及其相互关系的确定。因此，我们在系统分析和机械生物学之间发挥作用。我们已经表明，化学基因组分析（在许多生长条件下对完整基因缺失文库的适应性进行定量分析）和上位性 MAP（E-MAP；在基因组水平上比较双突变体表型与单突变体表型）可快速加速大肠杆菌表型、通路和通路互连的发现。这笔赠款中提出的工作极大地扩大了我们的努力。首先，在我们证明化学基因组分析提供了孤儿（功能上未表征的）基因和注释基因之间的高度相关性之后，我们现在将开发一个用于发现孤儿基因功能的管道。我们将通过分析更多的化学空间来扩展和改进高相关性关联，评估与其他很大程度上不重叠的功能关联测量（例如蛋白质-蛋白质相互作用）的关联，并将我们的多变量数据集整合到每个潜在的孤儿基因与注释基因相互作用的单个相互作用概率得分中。该纲要将成为确定孤儿基因功能和评估哪些基因功能指标对于功能表征最具信息性和成本效益的强大资源。其次，我们将研究高通量筛选中发现的细胞分裂和肽聚糖合成之间难以捉摸的功能联系的分子基础。我们之前的工作表明，PBP1B 双功能肽聚糖合成机在促进细胞分裂过程中外膜收缩方面与 Tol-Pal 部分冗余。我们现在发现一种孤儿蛋白 YbgF 可以协调这两个机器，我们将采用分子、生化和细胞生物学方法来探索协调是如何完成的。最后，我们将把我们的高通量表型分析方法扩展到枯草芽孢杆菌，它是关键的革兰氏阳性模型生物，也是厚壁菌门的成员，厚壁菌门是人类肠道中普遍存在的两个主要门之一。我们将在枯草芽孢杆菌中实施化学基因组分析和 E-MAP 分析，并用它来剖析基因功能和通路连接。由于革兰氏阳性和阴性生物体的包膜结构、社会行为以及主要细胞过程（包括复制和代谢）的控制和执行不同，我们的开源数据集将富含新颖的生物学知识。这项工作解决了阻碍基因组信息使用的“表型差距”，并展示了系统分析和机制研究在建立基因功能和过程之间的高阶连接方面的综合力量。