PUI-RESEARCH-MUW-WHITWAM

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。本项目利用功能基因组学研究酿酒酵母（Saccharomyces cerevisiae） [URE3]朊病毒的固化、繁殖和自发形成过程中的基因相互作用。[URE3]朊病毒介导的淀粉样蛋白形成被认为与哺乳动物淀粉样病如痒病、克雅氏病、阿尔茨海默病、帕金森病等的淀粉样蛋白形成具有相似的分子机制（1-3）。该项目的结果将为这些疾病的等效过程提供见解。我们将使用合成基因阵列（SGA）分析对参与[URE3]淀粉样蛋白繁殖和固化/扩散的所有基因进行全基因组调查(4)。这包括将[URE3]测试菌株与完整的单基因缺失菌株文库杂交，并产生所有[URE3]菌株文库，并删除了单个非必需基因。所有这些菌株都将在只有含有朊病毒的[URE3]菌株才能生长的选择性培养基上生长。杂交和选择都将使用南密西西比大学的机器设备来完成。在选择培养基上生长缓慢或不生长的菌株将显示出对朊病毒形成或繁殖至关重要的基因。在选择性培养基上表现出加速生长的菌株将揭示出能够抑制或治愈朊病毒繁殖的基因。所有阳性结果将通过人工交叉进行验证。将验证阳性的基因插入过表达载体中，以确定过表达对朊病毒繁殖的影响。候选基因的各种组合将被删除或串联过表达，以观察它们对朊病毒繁殖或固化的个别影响是否具有加性。作为[URE3]朊病毒的功能形式，Ure2蛋白（Ure2p）已知与转录激活因子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因子隔离在细胞质中，并阻止它们激活细胞核中的基因(6)。我们将进行一系列芯片分析，比较含朊病毒[URE3]菌株与不含等基因朊病毒[ure-o]菌株的基因表达。从来没有人做过这样的比较。目前唯一明确知道受[URE3]影响的基因是DAL5。该基因的差异表达是已知与[URE3]状态相关的唯一表型的背后。确定受Ure2p朊蛋白状态影响的其他基因将有助于确定与[URE3]朊蛋白相关的其他表型。有许多表型来监测，表明这些菌株的朊病毒状态将增加可用的工具库，研究人员使用该系统剖析朊病毒形成背后的分子机制。此外，我们将用微阵列将[URE3]菌株与GLN3基因敲除的等基因[URE3]菌株进行比较。[URE3]与[URE3]在gln3？该菌株将首次明确确定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 amyloidoses as scrapie, Creutzfeld-Jacob disease, Alzheimer¿s disease, Parkinson¿s disease, and others (1-3). 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 (4). 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. 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