Developing a Sensitive Means by Which the Yeast [PSI+] Phenotype May Be Used to Study Amyloid Curing Compounds
Abstract
[PSI+]yeast expressing the Sup35 protein as an amyloid aggregate has been a useful system for the study of prion and amyloid biology associated with diseases like, Fatal Familial Insomnia (FFI) and Alzheimer's Disease (AD), respectively. One potential cure for these diseases is an agent that directly blocks the formation of amyloids. In yeast, the C-terminal domain of Sup35 acts as eRF3 in translational termination, whereas its N-terminal domain contains the prion-forming domain. As a non-Mendelian trait, [PSI+] can be cytoplasmically inherited in daughter cells for an indefinite number of cell generations [strong PSI+] or for many generations [weak PSI+] in its amyloid form, which is detected via suppression of eRF3's ability to terminate translation at a nonsense codon in ade1-14.In a cell-free system our lab has recently shown that a cinnamon extract can block and reverse the formation of the Sup35 prion in vitro. Our current efforts are directed toward replicating this result in growing yeast cells (in vivo). To further sensitize yeast strains we have transformed them with various plasmids; yeast were made competent for transformation by a lithium acetate treatment. Four yeast strains were transformed with nine different galactose-inducible plasmids bearing a selectable auxotrophic marker. Transformants were selected on complete synthetic media missing a key nutrient (Uracil, Histidine, and Leucine drop-out plates). Following transformation, strains were screened, stored, and used in initial experiments to develop the bioassay. The goal of this work is to develop a bioassay to rapidly identify candidate curing compounds.
Developing a Sensitive Means by Which the Yeast [PSI+] Phenotype May Be Used to Study Amyloid Curing Compounds
HSB 207
[PSI+]yeast expressing the Sup35 protein as an amyloid aggregate has been a useful system for the study of prion and amyloid biology associated with diseases like, Fatal Familial Insomnia (FFI) and Alzheimer's Disease (AD), respectively. One potential cure for these diseases is an agent that directly blocks the formation of amyloids. In yeast, the C-terminal domain of Sup35 acts as eRF3 in translational termination, whereas its N-terminal domain contains the prion-forming domain. As a non-Mendelian trait, [PSI+] can be cytoplasmically inherited in daughter cells for an indefinite number of cell generations [strong PSI+] or for many generations [weak PSI+] in its amyloid form, which is detected via suppression of eRF3's ability to terminate translation at a nonsense codon in ade1-14.In a cell-free system our lab has recently shown that a cinnamon extract can block and reverse the formation of the Sup35 prion in vitro. Our current efforts are directed toward replicating this result in growing yeast cells (in vivo). To further sensitize yeast strains we have transformed them with various plasmids; yeast were made competent for transformation by a lithium acetate treatment. Four yeast strains were transformed with nine different galactose-inducible plasmids bearing a selectable auxotrophic marker. Transformants were selected on complete synthetic media missing a key nutrient (Uracil, Histidine, and Leucine drop-out plates). Following transformation, strains were screened, stored, and used in initial experiments to develop the bioassay. The goal of this work is to develop a bioassay to rapidly identify candidate curing compounds.