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Title: Investigation of trinucleotide repeat instability in the Escherichia coli chromosome
Author: Mihaescu, Camelia
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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The expansion of trinucleotide repeat tracts is the cause of nearly twenty genetic disorders. Almost all these diseases are characterised by anticipation, which means an earlier age of onset and an increased severity of the symptoms from one generation to the next. The mechanisms of trinucleotide repeat expansion are not understood. In the course of this project, I have investigated the instability of a trinucleotide repeat array of 43 copies integrated at the attB site of chromosomes of various Escherichia coli mutants. The trinucleotide repeat tract (CTG)43 was integrated into the E. coli chromosome in both possible orientations using an intermediate vector and exploiting site-specific recombination between the attB site of the chromosome and the attP site of the vector. Using this method I have constructed 60 mutation strains of E. coli which contain the trinucleotide repeat tract and are deficient in genes involved in replication, recombination, secondary structure repair or mismatch repair. Techniques for the analysis of the instability of the trinucleotide repeat arrays were developed and used to quantify repeat instability. These included: digestion of chromosomal DNA with a rare-cutting restriction endonuclease and PAGE of the labelled fragments; PCR of the trinucleotide repeat tract, followed by restriction enzyme digestion and PAGE; fluorescent PCR and f-TRAMP (fluorescent trinucleotide amplification which uses just one primer in repeated cycles of linear primer extension): products were separated by capillary electrophoresis and analysed using Gene Scan software. Intensive analyses of different E. coli mutant showed that the trinucleotide repeat arrays integrated into the chromosome are stable. Except in one case, no instability was observed in any mutant deficient in replication, recombination, mismatch repair or secondary structure repair. The only strain, which showed instability, was a mutD mutant (impaired in the proof-reading activity of DNA Polymerase III). Possible explanations for this observation are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available