The Varkud satellite (VS) plasmid is found in the mitochondria of some strains of Neurospora, and exhibits properties that may allow it to be developed as a genetic transformation vector to study mitochondrial molecular biology. An ideal transformation vector would not confer a phenotype. The overall goal of my thesis was to examine interactions of VS with its Neurospora host to identify possible phenotypes, using biochemical and proteomic approaches. Biochemical experiments provided evidence consistent with the plasmid transcript, VS RNA, being present as a ribonucleoprotein particle that can be separated from ribosomal subunits by sucrose density gradient centrifugation; however, no VS-specific proteins were identified under the purification conditions examined. During the analyses of proteomic data I obtained new insights into the consequences of the statistical methods commonly used to normalize quantitative 2D gel data. However, irrespective of the method used, the fraction of the proteome amenable to 2D gel-based proteomics revealed, at most, subtle effects of VS on the abundance of a few proteins. I also observed no differences in growth rate between strains differing by the presence or absence of VS when grown in the presence of inhibitors and stressors affecting a wide range of mitochondrial and other cellular functions. Overall, despite VS RNA being as abundant as the large and small mitochondrial ribosomal RNAs, my genetic, biochemical and proteomic investigations of the effect of VS on its host strain provides evidence that VS is a phenotypically neutral element.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31801 |
| Date | 10 January 2012 |
| Creators | Keeping, Andrew |
| Contributors | Collins, Rick |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis, Dataset |
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