Genomic analysis of iron deficiency and overload in Saccharomyces cerevisiae

Iron is essential micronutrient for virtually all organisms due to its ability to accept and donate electrons. Yet the same property makes it toxic when present in excess. Therefore all organisms have developed well-coordinated mechanism to control intracellular iron level. Iron metabolism in S. cerevisiae has been studied intensively and given us insights into higher eukaryotes. The remarkable correlation in iron metabolism between human and yeast makes yeast an excellent model system. Furthermore, the availability of the entire genome sequence and targeted mutants in many components of iron metabolism are also advantageous in selecting yeast as a model system.; The goals of my dissertation project were to identify novel genes regulated by iron level and to study metabolic reprogramming of iron deficient and iron overload conditions. I utilized both genetic and nutritional models for the purpose. The mac1 knockout yeast represents a cellular copper and iron deficient model and the mrs4 knockout yeast represents cellular iron overload and mitochondrial iron deficient model. Wild-type yeast treated with an iron chelator serves as nutritional iron deficient model. The first part of the dissertation discusses gene expression profiles of the mac1 knockout yeast. Firstly, we proposed several candidate genes as being involved in iron metabolism based on the gene expression studies. Promoter analysis and phenotype analysis complemented gene expression study. We also demonstrated the utility of miniarray analysis as a novel confirmatory method for gene expression studies. Secondly, we identified metabolic reprogramming of yeast during iron deficiency at the transcriptional level. An iron deficient cell changes its metabolism from iron dependent processes to iron independent ones. We also observed down-regulation of genes encoding iron proteins in this mutant.; The second part of the project confirmed that results from the mac1 knockout yeast were due to iron deficiency by using nutritional iron deficient models. We treated wild-type yeast with an iron chelator for up to four hours and examined gene expression patterns. We were able to confirm down-regulation of genes encoding iron proteins using miniarray analysis. We also confirmed metabolic shift from iron requiring pathways to iron independent pathways in the nutritional iron deficient models. In this study, we further developed normalization and statistical method for miniarray analysis. The third part of the dissertation discusses iron overload model using the mrs4 knockout yeast. (Abstract shortened by UMI.)...