Toward a Complete Genetic Description of the Yeast Actin Cytoskeleton

描述：酿酒葡萄球菌已经成为新的基因组学技术、这些技术的应用以及分析这些数据集所需的生物信息学工具的开发的试验场。此外，一个大型研究团体的重点生物学研究已经对个体基因如何促进基本细胞功能产生了详细的了解。我们建议利用酵母的遗传力量将现有知识与肌动蛋白细胞骨架的功能统一起来，肌动蛋白细胞骨架是细胞系统的主要整合者。肌动蛋白丝由单一蛋白构成，但肌动蛋白功能的多样性是由大量辅助蛋白介导的。对酵母肌动蛋白细胞骨架的研究已经导致了强大的工具和试剂的开发，以研究与肌动蛋白细胞骨架的相互作用如何有助于细胞系统的调节和利用，但许多这些工具和试剂尚未充分利用，以实现酵母肌动蛋白细胞骨架或其影响的细胞系统的完整图像。在这些试剂中，值得注意的是单个基本肌动蛋白基因ACT1的大量突变体，这些突变体专门设计用于破坏蛋白质之间的相互作用。我们建议使用这些试剂以及酵母遗传学和基因组学的力量来揭示影响肌动蛋白细胞骨架或其功能受到肌动蛋白细胞骨架影响的基因的完整或至少接近完整的遗传描述。将使用肌动蛋白的空等位基因对整个酵母基因敲除序列进行复杂的单倍不足筛选，以试图发现对肌动蛋白化学计量减少敏感的基因。然后将针对大量突变的肌动蛋白等位基因重新测试该基因亚网络，以确定哪些遗传相互作用可归因于肌动蛋白功能亚群的减少。为了完成“肌动蛋白相互作用组”，该分析将辅以一组可行的肌动蛋白丙氨酸扫描突变体的合成致死筛选。所有的相互作用都将与肌动蛋白突变所定义的肌动蛋白功能的特定亚群的丧失或减少相关。为了进一步提高数据的一致性，还将检查肌动蛋白相互作用组的基因与所有已知肌动蛋白结合蛋白的基因的遗传相互作用。对这一高度整合的数据集的分析将用于将肌动蛋白等位基因及其相互作用的基因分组，我们假设这些分组将反映共同的缺陷，例如肌动蛋白与特定肌动蛋白结合蛋白之间的结合缺失。与其他数据集的贝叶斯数据集成将用于功能预测和揭示基因集之间的新关系，聚类分析将用于将这些模式与肌动蛋白的结构联系起来。通过开发一个三维版本的网络显示工具鱼鹰，将网络的节点连接回肌动蛋白的结构，将促进网络的可视化。该提案产生的所有数据和数据分析将以免费访问的基于网络的形式保存在多伦多GRID数据库、SGD和PIXIE.Princeton.edu中。

DESCRIPTION: S. cerevsiae has served as a proving ground for new genomics technologies, application of these technologies and the development of the bioinformatics tools required to analyze these datasets. In addition, focused biological investigations by a large research community has resulted detailed understandings of how individual genes contribute to basic cellular functions. We propose to harness the genetic power of yeast to unify existing knowledge with the functions of the actin cytoskeleton, a major integrator of cellular systems. Actin filaments are constructed of a single protein but diversity in actin function is mediated by a large battery of accessory proteins. Investigations on the yeast actin cytoskeleton have led to the development of powerful tools and reagents to study how interactions with the actin cytoskeleton contribute to its regulation and utilization by cellular systems, but many of these tools and reagents have not been utilized to their fullest extent to realize a complete picture of the yeast actin cytoskeleton or the cellular systems it affects. Notable among these reagents is a large collection of mutants in the single essential actin gene ACT1 that were specifically designed to disrupt protein-protein interactions. We propose to use these reagents and the power of yeast genetics and genomics to uncover a complete or at least nearly complete genetic description of those genes that impact, or whose functions are impacted by, the actin cytoskeleton. Complex haploinsufficiency screens will be performed with a null allele of actin against the entire ordered array of yeast gene knock-outs to try and uncover genes sensitive to reductions in actin stoichiometry. This sub- network of genes will then be retested against the large collection of mutant actin alleles to determine which of the genetic interactions can be attributed to reductions in subsets of actin functions. To complete the "actin interactome", this analysis will be complemented by synthetic lethal screening with the set of viable actin alanine scan mutants. All interactions will be correlated back to the loss or reduction in specific subsets of actin functions as defined by the actin mutants. To bring further coherence to the data, genes of the actin interactome will also be examined for genetic interactions with the genes for all known actin-binding proteins. Analysis of this highly integrated data set will be used to arrange both the actin alleles and their interacting genes into groups that we hypothesize will reflect shared defects such as the loss of binding between actin and specific actin binding proteins. Bayesian data integration with other datasets will be used for function prediction and to uncover novel relationships between gene sets and cluster analysis will be used to correlate these patterns back to the structure of actin. Visualization of the network will be facilitated by the development of a three-dimensional version of the network display tool Osprey that will connect nodes of the network back to the structure of actin. All data and data analysis resulting from this proposal will be maintained in a free access, Web-based format within the Toronto GRID database, the SGD, and PIXIE.Princeton.edu.