MSpace - DSpace at UofM >
Faculty of Graduate Studies (Electronic Theses and Dissertations) >
FGS - Electronic Theses & Dissertations (Public) >

Please use this identifier to cite or link to this item: http://hdl.handle.net/1993/3064

Title: Analysis of Saccharomyces cerevisiae genetic background and mitochondrial DNA polymerase variants on maintenance of the mitochondrial genome.
Authors: Young, Matthew J.
Supervisor: Court, Deborah A. (Microbiology)
Examining Committee: Hausner, Georg (Microbiology) Oresnik, Ivan (Microbiology) Huebner, Erwin (Biological Sciences) Harkness, Troy (External, Anatomy and Cell Biology, College of Medicine, University of Saskatchewan)
Graduation Date: October 2008
Keywords: mitochondria
yeast
mitochondrial DNA polymerase
S288c
phylogeny
Saccharomyces cerevisiae
Issue Date: 10-Sep-2008
Citation: Young, M. J., Theriault, S. S., Li, M. and Court, D. A., (2006) Yeast 23: 101-16.
Young, M. J., Bay, D. C., Hausner, G. and Court, D. A., (2007) BMC Evol Biol 7: 31.
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.
URI: http://hdl.handle.net/1993/3064
Appears in Collection(s):FGS - Electronic Theses & Dissertations (Public)

Files in This Item:

File Description SizeFormat
Matthew Young Thesis.pdf11.24 MBAdobe PDFView/Open
View Statistics

Items in MSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

 

Valid XHTML 1.0! MSpace Software Copyright © 2002-2010  Duraspace - Feedback