PUI-RESEARCH-MUW-WHITWAM

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目使用功能基因组学来研究酿酒酵母[URE 3]朊病毒的治愈，繁殖和自发形成所涉及的遗传相互作用。 [URE 3]朊病毒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疾病如羊瘙痒病、克-雅二氏病、阿尔茨海默病、帕金森病等的特征。 该项目的结果将为这些疾病的等效过程提供深入了解。 我们将使用合成遗传阵列（SGA）分析对参与[URE 3]淀粉样蛋白繁殖和固化/分散的所有基因进行全基因组调查。 这涉及将[URE 3]试验菌株与完整的单基因缺失菌株文库杂交，并产生全部为[URE 3]且缺失单个非必需基因的菌株文库。 所有这些菌株将在仅含朊病毒[URE 3]菌株可以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人设备来完成。 在选择性培养基上生长缓慢或不生长的菌株将揭示朊病毒形成或繁殖所必需的基因。 在选择性培养基上显示加速生长的菌株将揭示能够抑制或治愈朊病毒繁殖的基因。 所有阳性将通过手动交叉验证。 将证实为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治愈的个体效应是否是累加的。 我们将研究SGA分析鉴定的基因如何影响酵母中朊病毒的自发形成。 这将涉及确定自发[URE 3]形成如何在野生型遗传背景下表现，然后删除通过SGA分析鉴定的单个基因，并观察自发[URE 3]形成的速率如何因该基因的缺失而改变。 已知[URE 3]朊病毒的功能形式Ure 2蛋白（Ure 2 p）与转录激活因子Gln 3蛋白（Gat 1 p）和Gat 1蛋白（Gat 1 p）相互作用（5）。 Ure 2 p和Gln 3 p或Gat 1 p之间的相互作用将这些转录激活因子隔离在胞质溶胶中，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，比较含朊病毒[URE 3]菌株和无朊病毒[ure-o]菌株的基因表达。 这样的比较从来没有做过。 目前唯一明确已知其表达受[URE 3]影响的基因是DAL 5。 该基因的差异表达是已知与[URE 3]状态相关的唯一表型的背后。 鉴定受Ure 2 p朊病毒状态影响的其他基因将有助于鉴定与[URE 3]朊病毒相关的其他表型。 有许多表型监测，表明这些菌株的朊病毒状态将增加可用的工具库，研究人员解剖朊病毒形成背后的分子机制，使用这个系统。 此外，我们将做微阵列比较[URE 3]菌株与已敲除GLN 3基因的同基因[ure-o]菌株。 在gln 3菌株中比较[URE 3]和[ure-o]的微阵列将首次明确确定Ure 2 p是否靶向任何Gat 1 p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.