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Title: Transmission, segregation and loss of the 2μm plasmid of the budding yeast Saccharomyces cerevisiae
Author: Hayes, Matthew John
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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The 2μm plasmid encodes a potential plasmid resolution system provided by a trans-acting site specific recombinase FLP and two cis-acting sites, FRTs, arranged in inverted orientation. Recombination between these sites is responsible for allowing amplification of the plasmid to elevated copy numbers. Bacterial low copy number plasmids often encode factors responsible for ensuring their high fidelity segregation between cells at mitosis. The 2μm plasmid encodes such a segregation mechanism provided by two trans-acting factors REP1 and REP2 and a cis-acting site STB. We have investigated the relative contribution of plasmid copy number and plasmid multimerisation to plasmid stability by producing 'strains' of yeast containing different plasmid compliments. In this study plasmid amplification is shown to result in the formation of multimeric forms. Growth of yeast in the absence of FLP recombinase results in accumulation of these forms. Pedigree analysis of individual cells indicates that all forms of the plasmid are segregated accurately between cells at cell division. Plasmid stability is shown to increase with both increased plasmid copy number and increased proportion of plasmid multimers. Plasmid loss is shown to occur from a sub-population of cells with a plasmid copy number below a critical threshold value. This indicates failure of the plasmid segregational machinery at low plasmid copy numbers. Increase in plasmid stability in populations of cells with elevated plasmid copy numbers may be attributed to a reduction in the proportion of cells that contribute to this subpopulaton. Mutagenesis of yeast cells was performed in order to ascertain whether the plasmid stability machinery required components encoded by the chromosomal genome. A number of mutants have been isolated which show unstable maintenance of the 2μm plasmid. This indicates that plasmid segregation requires both plasmid and chromosomally encoded components.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available