UBC Theses and Dissertations
Understanding cellular response to drugs and toxins with yeast genomics tools Ogbede, Joseph Uchechukwu
Advances in genomics and drug discovery have been accelerated by the introduction of new technologies for screens of increasing complexity. A key test bed for these technologies is yeast (Saccharomyces cerevisiae), a valuable experimental model for understanding the mechanism of action (MOA) of compounds. Unfortunately, some of these screening approaches are, by necessity, biased, while others may not be suitable to address the intended research questions. This thesis used the unbiased yeast chemical genomics and phenomics tools to investigate cellular response to diverse drugs, toxins and mycoparasite-prey interactions. First, I characterized the genome-wide effects of N-nitrosamines and their metabolites, and revealed diverse evolutionarily conserved genes and pathways that mediate their toxicity, including arginine biosynthesis, DNA damage repair, mitochondrial genome integrity and vacuolar protein sorting. I further showed that overexpression of ARG3 (ornithine carbamoyltransferase) confers resistance to N-nitrosamines. In my second project, I identified candidate genes and pathways that could mediate interactions between two yeasts. I observed that resistance and sensitivity to predation by Saccharomycopsis schoenii could be time-dependent, and identified Saccharomyces cerevisiae deletion strains (hits) that were resistant and sensitive to S. schoenii. Genes lacking in the resistant strains are involved in cell wall integrity, arginine/lysine biosynthesis, and oxidative stress response. Conversely, the sensitive strains lack genes involved in endocytosis, vacuolar protein sorting, and cell size regulation. The third project provided data that can help define MOA of anthracycline chemotherapeutics using a multipronged approach. The data showed that some anthracyclines (doxorubicin, daunorubicin and epirubicin) exhibited higher potency in cells grown on glycerol versus glucose media. It further indicated that doxorubicin and daunorubicin MOA may involve mitochondrial processes that are not linked to mitochondrial DNA. I uncovered a spectrum of anthracycline response profiles, and showed that cellular effects of anthracyclines can be distinguished. Using whole genome sequencing, I identified mutations from doxorubicin-resistant clones, and showed that overexpression of ARL1 (ADP ribosylation factor like GTPase 1) and SSL2 (member of RNA polymerase transcription factor TFIIH complex) confers resistance to anthracyclines. Together, my work characterized candidate genes and pathways that could be required in cellular response to chemical perturbation.
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