PUI-RESEARCH-MILLSAPS-KRAMER

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。子项目及 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。 已经有四种酵母基因被证明可以影响朊病毒的繁殖  HSP 104、SSA 1、SSA 2和YDJ 1。 所有这些都是伴侣蛋白。 当我们正在进行SGA分析以鉴定其他做同样事情的酵母时，我们将研究这些基因对自发性朊病毒形成的影响。 我们将单独敲除这些基因中的每一个，并研究与野生型菌株相比，基因缺失对菌株自发形成[URE 3]朊病毒的能力以及任何自发[URE 3]形成发生的速率的影响。

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. Four yeast genes have already been shown to effect prion propagation  HSP104, SSA1, SSA2, and YDJ1. All of these are chaperone proteins. As we are carrying out the SGA analysis to identify other yeast which do the same thing, we will investigate the effect of these genes on spontaneous prion formation. We will knock out each of these genes individually and investigate the effect of the gene deletion on the ability of the strain to spontaneously form the [URE3] prion and the rates at which any spontaneous [URE3] formation occurs, as compared to wildtype strains.