Development of a novel luc based S. cerevisiae biosensor

Research presented in this thesis describes the development of a novel eukaryotic genotoxicity and cytotoxicity assay based on the expression of the modified firefly luciferase gene (luc+) in the brewer’s yeast Saccharomyces cerevisiae. In this biosensor, expression of the luc+ reporter, regulated by the RAD54 DNA damage-inducible promoter, is induced by the presence of genotoxic damage. Inhibition of cell proliferation during the assay is used as a measure of the cytotoxic potential of test compounds. Extensive testing with the S. cerevisiae pGEN –luc+ biosensor demonstrated that it responded appropriately to a wide range of genotoxic compounds and that expression of the reporter was unaffected by non-genotoxic agents. This biosensor has the potential to be developed into a pre-regulator screening assay for the pharmaceutical industry as it provides a rapid, sensitive and inexpensive means with which the genotoxic and cytotoxic potential of compounds can be assessed. Additional research carried out in this study attempted to further improve the biosensor. Proteomic analysis successfully identified a number of proteins that were significantly more abundant in S. cerevisiae cells exposed to genotoxic and hepatotoxic agents. One particular protein (Pst2p) was up-regulated in the presence of all the toxins tested. The function of this protein is unclear but it is believed to be involved in the cell’s response to oxidative stress. Incorporation of the promoter that regulates PST2 expression could allow the S. cerevisiae biosensor to detect chemicals that induce oxidative stress in eukaryotes. Such an assay could prove invaluable in the drug discovery where it could be used to identify compounds with hepatotoxic and/or genotoxic properties.EThOS - Electronic Theses Online ServiceGBUnited Kingdo