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Title: Illegitimate recombination in Escherichia coli
Author: Blake, Catherine E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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The factors affecting deletion of long DNA palindromes from high copy number cloning vectors are investigated with particular reference to the mode of deletion and it is shown that the deletion of a 571 bp palindrome deleted from pMS7 using 3 bp repeats. A shorter 109 bp palindrome deleted from a related plasmid using 7 bp direct repeats and the mode of deletion is unaffected by the genotype of the host strain. There also appears to be a bias for the deletion of palindromic sequences on the lagging strand of a replication fork. It is also shown that in a wild-type E. coli strain there is inhibition of plasmid multimerization if the plasmids carry long palindromic sequences. It is proposed that the lack of plasmid multimers in this background is a result of the removal of palindromic sequences form the plasmids by the SbcCD protein of E. coli. In an sbcCD strain, plasmid DNA bearing long palindromes is not detected in a monomeric form, instead the DNA is present in multimeric forms, predominantly dimers. The ability to form plasmid multimers in this background may help to stabilise the palindromic sequences. The behaviour of palindromic sequences carried on plasmids is also investigated in recA sbcCD strains with a view to the correct choice of E. coli strain for the cloning of long palindromic sequences. Finally, the influence of an sbcCD mutation on the formation of araB-lacZ cistron fusion is investigated. The SbcCd proteins are thought to have a role in the processing of secondary structures formed by palindromic sequences. The formation of araB-lacZ fusions occurs via a strand transfer complex involving a complex genome rearrangement and secondary structure. Although an sbcCD mutation did not affect the kinetics or sequences specificity of fusion formation it is possible that SbcCD might have a role in processing the strand transfer complex which is not easily detected.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available