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Title: Mathematical and experimental approaches to the dimer catastrophe theory
Author: Field, Christopher Martyn
ISNI:       0000 0004 2708 3870
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2011
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Multicopy plasmids rely on random distribution for stable inheritance by daughter cells at division. Threats to plasmid copy number increase the probability of plasmid loss, which can be detrimental to both plasmid and host. Plasmid dimers emerge through homologous recombination. Dimers have two independent origins of replication and thus have a replicative advantage and reduced copy number. Models of plasmid behaviour suggest that dimers would overtake a cell population, but that this can be prevented if they impose a small metabolic load, which has been observed in vivo. Plasmid ColE1 also contains a cer site, which allows for dimer resolution by XerCD site-specific recombination. A small RNA, Rcd, is expressed from the cer site in dimers and interacts with tryptophanase to increase the concentration of indole in the cell. It is proposed that, as indole inhibits cell division, Rcd imposes a checkpoint on the cell until plasmid dimers are resolved. In this work, plasmid behaviour in a growing cell population was modelled stochastically in more detail than previous work. A plasmid replication model suggested that dimers replicate to more than half the average copy number of monomers, perhaps accounting for their increased metabolic load. A cell population model suggested that the presence of dimer-only cells decreased the average plasmid stability by less than in previous models, which used a fixed plasmid copy number. The rate of dimer resolution required to affect plasmid stability was unreasonably high, indicating the necessity of the Rcd checkpoint. The model thus suggested that the checkpoint may be an escape route for dimer-only cells rather than an immediate response to the emergence of an initial dimer. The Rcd checkpoint itself was also subject to critical analysis. It was realised that neither inhibition of cell division nor cell growth were sufficient to assist dimer resolution; inhibition of plasmid replication was required. Experiments in vivo found that indole inhibited plasmid replication at a concentration that may be achievable endogenously. DNA gyrase was investigated as a component of the mechanism of this inhibition, and indole was found to inhibit its supercoiling activity in vitro.
Supervisor: Summers, David Sponsor: Medical Research Council (UK)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral