PUI-RESEARCH-MUW-WHITWAM

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目利用功能基因组学研究酿酒酵母[URE3]蛋白的固化、繁殖和自发形成过程中所涉及的遗传相互作用。[URE3]Prion介导的淀粉样蛋白形成被认为涉及与淀粉样蛋白形成类似的分子机制，淀粉样蛋白形成是哺乳动物蛋白质错误折叠障碍的特征，如瘙痒病、Creutzfeld-Jacob病、阿尔茨海默病、帕金森病等。该项目的结果将提供对这些疾病的同等过程的洞察。 我们将使用合成遗传阵列(SGA)分析，对参与[URE3]淀粉样蛋白的繁殖和治愈/扩散的所有基因进行全基因组调查。这涉及将[URE3]测试菌株与一个完整的单基因缺失菌株文库杂交，并产生一个都是[URE3]且有单个非必需基因缺失的菌株文库。所有这些菌株都将在选择性培养基上生长，只有含有普鲁恩的[URE3]菌株才能在上面生长。交叉和选择都将使用南密西西比大学的设施进行机器人操作。在选择性培养基上生长缓慢或不生长的菌株将揭示基因，当存在时，这些基因对于Pron的形成或繁殖是必不可少的。在选择性培养基上表现出加速生长的菌株将揭示出当存在时能够抑制或治愈普恩病毒繁殖的基因。所有阳性结果将通过手动十字进行验证。被证实为阳性的基因将被插入到过度表达载体中，以确定过度表达对普恩病毒繁殖的影响。候选基因的各种组合将被同时删除或过度表达，以确定它们对普恩病毒繁殖或治愈的单独影响是否是相加的。 我们将研究通过SGA分析确定的基因如何影响酵母中自发的Prion形成。这将包括确定在野生型遗传背景下自发形成[URE3]的行为，然后删除由SGA分析确定的单个基因，并观察该基因的缺失如何改变自发形成[URE3]的速率。 [URE3]Prion的功能形式是Ure2蛋白(Ure2p)，它与转录激活因子Gln3蛋白(Gat1p)和Gat1蛋白(Gat1p)相互作用(5)。Ure2p与Gln3p或Gat1p之间的相互作用将这些转录激活物隔离在细胞质中，并阻止它们激活细胞核中的基因(6)。 我们将进行一系列微阵列分析，比较含有[URE3]基因的菌株和不含[URE3]基因的同基因菌株的基因表达。这样的比较从来没有做过。目前已知的唯一受[URE3]影响表达的基因是DAL5。该基因的差异表达是已知的与[URE3]状态相关的唯一表型背后的原因。识别受Ure2p的Prion状态影响的其他基因将有助于识别与[URE3]Prion相关的其他表型。有一些表型可以监测到这些菌株的Prion状态，这将增加研究人员使用该系统剖析Prion形成背后的分子机制的可用工具的武器库。 此外，我们还将进行微阵列，将[URE3]菌株与具有GLN3基因敲除的同基因[ure-o]菌株进行比较。在gln3菌株中比较[URE3]和[ure-o]的微阵列将首次明确识别Ure2p是否针对任何Gat1p激活的基因。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is using functional genomics to investigate the genetic interactions involved in the curing, propagation, and spontaneous formation of the [URE3] prion of Saccharomyces cerevisiae. [URE3] prion-mediated amyloid formation is believed to involve similar molecular mechanisms as the amyloid formation that is a feature of such mammalian protein misfolding disorders as scrapie, Creutzfeld-Jacob disease, Alzheimer's disease, Parkinson's disease, and others. Results from this project will provide insight into equivalent processes in those diseases. We will do a genome-wide survey of all the genes involved in the propagation and curing/dispersal of the [URE3] amyloid using synthetic genetic array (SGA) analysis. This involves crossing a [URE3] tester strain with a complete library of single-gene deletion strains and producing a library of strains that are all [URE3] and have a single non-essential gene deleted. All of these strains will grown on selective medium on which only prion-containing [URE3] strains can grow. The crosses and selection will all be done robotically using facilities for this purpose at the University of Southern Mississippi. Strains that show slow or no growth on the selective medium will reveal genes that, when present, are essential for prion formation or propagation. Strains that show accelerated growth on the selective medium will reveal genes that, when present, are able to inhibit or cure prion propagation. All positives will be verified by manual crosses. Genes that are verified positive will be inserted into overexpression vectors to determine the effect of overexpression on prion propagation. Various combinations of candidate genes will be deleted or overexpressed in tandem to see if their individual effects on prion propagation or curing are additive. We will investigate how the gene identified by SGA analysis affect spontaneous prion formation in yeast. This will involve determining how spontaneous [URE3] formation behaves in a wildtype genetic background, then deleting individual genes identified by SGA analysis and seeing how rates of spontaneous [URE3] formation are altered by the absence of that gene. The functional form of the [URE3] prion, the Ure2 protein (Ure2p) is known to interact with the transcriptional activators Gln3 protein (Gat1p) and the Gat1 protein (Gat1p) (5). The interaction between Ure2p and Gln3p or Gat1p sequesters these transcription activators in the cytosol and prevents them from activating genes in the nucleus (6). We will do a series of microarray analyses comparing gene expression in prion-containing [URE3] strains to isogenic prion-free [ure-o] strains. Such a comparison has never been made. Currently the only gene whose expression is definitively known to be effected by [URE3] is DAL5. The differential expression of this gene is behind the only phenotypes known to be associated with the [URE3] state. Identifying other genes effected by the prion state of the Ure2p will help identify other phenotypes associated with the [URE3] prion. Having a number of phenotypes to monitor that indicate the prion status of these strains will increase the arsenal of tools available to researchers dissecting the molecular mechanisms behind prion formation using this system. Additionally, we will do microarrays comparing [URE3] strains to isogenic [ure-o] strains that have had the GLN3 gene knocked-out. The microarrays comparing [URE3] to [ure-o] in a gln3 strain will definitively identify for the first time whether Ure2p targets any Gat1p-activated genes.