This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.
Showing the most recent 10 out of 143 publications
