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.
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