Date of Award

Spring 6-24-2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Dr. Rey Sia

Second Advisor

Dr. Michel Pelletier

Third Advisor

Dr. Stuart Tsubota

Abstract

This research involved studying mitochondrial DNA stability using Saccharomyces cerevisiae as a model organism. Gene knockout strains focused on the genes FIS1, DNM1, CLU1 and RTG1. Both FIS1 and DNM1 are involved in mitochondrial fission. The CLU1 gene encodes a protein that may associate with the core complex of eukaryotic translation initiation factor 3 (eIF3) in Saccharomyces cerevisiae. The eIF3 plays a role in initiation of mRNA translation. The specific function of the Clu1p in this process is undefined. The gene knockout of CLU1 does not affect growth or translation initiation. The knockout does however cause defects in mitochondrial distribution and organization. The RTG1 gene encodes a transcription factor (bHLH) involved in interorganelle communication. The protein also contributes to communication between mitochondria, peroxisomes, and the nucleus.

Deletion strains of all four genes were studied for spontaneous respiration loss. This assay reveals if certain nuclear gene products have a role in stabilizing the mitochondrial DNA. The mitochondrial genome encodes proteins that are solely required for respiration. This assay compared the spontaneous respiration loss of a wild type yeast strain to those of the various knockout strains described above. In wild type yeast, a 3.26% spontaneous respiration loss is observed. In the fis1∆, dnm1∆, clu1∆, and rtg1∆ deletion strains, the percentage of spontaneous respiration loss is 13.29%, 16.36%, ~5.55%, and 8.21%, respectively, using dextrose as a carbon source. When raffinose was used as a carbon source, the spontaneous respiration loss was higher for knockout genes involved in morphology. From this result, we can assume that the nuclear genes above are critical in maintaining mitochondrial DNA stability.

Direct repeat-mediated deletion assays were also performed. These assays were performed for both wild-type and fis1Δ strains. Both strains contained nuclear and mitochondrial reporters. Using reporters flanked by homologous repeats we were able to quantify the recombination rates in both nuclear and mitochondrial DNA. The purpose of this assay was to compare nuclear and mitochondrial mutation rates for the wild-type and fis1Δ strains. Homologous recombination rates were not significantly different for fis1Δ strains compared to wild-type in the nuclear genome. There was an increase in the homologous recombination rate in the mitochondrial genome in fis1Δ strains. This result indicates that the FIS1 nuclear gene plays a role in maintaining mitochondrial DNA stability, for without it, mitochondrial DNA recombination rates increase.

Available for download on Wednesday, July 19, 2017

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