Analysis of Saccharomyces cerevisiae genetic background and mitochondrial DNA polymerase variants on maintenance of the mitochondrial genome.

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dc.contributor.supervisor Court, Deborah A. (Microbiology) en
dc.contributor.author Young, Matthew J.
dc.date.accessioned 2008-09-10T13:41:35Z
dc.date.available 2008-09-10T13:41:35Z
dc.date.issued 2008-09-10T13:41:35Z
dc.identifier.citation Young, M. J., Theriault, S. S., Li, M. and Court, D. A., (2006) Yeast 23: 101-16. en
dc.identifier.citation Young, M. J., Bay, D. C., Hausner, G. and Court, D. A., (2007) BMC Evol Biol 7: 31. en
dc.identifier.uri http://hdl.handle.net/1993/3064
dc.description.abstract The contribution of yeast strain background, specifically auxotrophic markers, to stability and fidelity of mtDNA replication was investigated. In summary, the ade2, his3delta200, and hap1 mutations have complex effects on mitochondrial functions, the severity of which appears to depend on other components in the genetic background of the strain. These results are important as many commonly used laboratory strains are related to the respiratory hampered S288c strain and are used for studies of orthologous human mutations associated with various mitochondrial diseases. These observations have added to our understanding of fungal mtDNA replication and have informed the mitochondrial community of problematic strains that need to be considered when using this model organism. The function of the yeast mitochondrial DNA polymerase (Mip1p) carboxyl-terminal extension (CTE) was investigated both in vivo and in vitro by genetically engineering various truncations of the CTE. The respiratory competence of mip1delta175 and mip1delta205 cells, in which Mip1p lacks the C-terminal 175 and 205 residues respectively, are indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351, Mip1pdelta279, Mip1pdelta241, and Mip1pdelta222 rapidly lose mtDNA. At a low frequency, mip1delta216 cells grow poorly on glycerol. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and rapid loss of mtDNA during fermentative growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. To determine more precisely the defects associated with polymerase truncation variants, these proteins were overexpressed in yeast and used in a novel non-radioactive mtDNA polymerase assay. The threonine-661 and alanine-661 variants, shown by others to be responsible for the increased mtDNA mutability of various laboratory yeast strains at increased temperature, were examined in combination with CTE-truncations. These experiments suggest that exonuclease function is not effected in the alanine-661 variant at 37 degrees Celsius whereas polymerase activity is, and this higher relative level of exonuclease activity could be a contributing factor to mtDNA instability in S288c-related strains. Lastly, isogenic CTE truncation variants all have less DNA polymerase activity than their parental wild-type. Based on these results, several possible roles for the function of the CTE in mtDNA replication are suggested. en
dc.format.extent 11509449 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.rights info:eu-repo/semantics/openAccess
dc.subject mitochondria en
dc.subject yeast en
dc.subject mitochondrial DNA polymerase en
dc.subject S288c en
dc.subject phylogeny en
dc.subject Saccharomyces cerevisiae en
dc.title Analysis of Saccharomyces cerevisiae genetic background and mitochondrial DNA polymerase variants on maintenance of the mitochondrial genome. en
dc.type info:eu-repo/semantics/doctoralThesis
dc.type doctoral thesis en_US
dc.degree.discipline Microbiology en
dc.contributor.examiningcommittee Hausner, Georg (Microbiology) Oresnik, Ivan (Microbiology) Huebner, Erwin (Biological Sciences) Harkness, Troy (External, Anatomy and Cell Biology, College of Medicine, University of Saskatchewan) en
dc.degree.level Doctor of Philosophy (Ph.D.) en
dc.description.note October 2008 en

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