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Title: Studies on transduction by bacteriophage P1
Author: Huang, Haomin
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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The original aim of this thesis was to address the origin of the abortive transduction protein (TPA), which appears to join the ends of transduced DNA and render it refractory to degradation by host nuclease and recombination with the chromosome. We planned to isolate abortively transduced DNA (ATD) from transduced cells, which should have TPA attached, in order to characterize it. To increase the packaging of transducing DNA and TPA, a pac site was inserted into the “donor” chromosome which was labelled with BU to make it heavy. This method proved technically difficult and I did not succeed in purifying sufficient ATD-TPA to characterize. I decided instead to study P1 packaging of transducing DNA further. The argB gene was replaced with the P1 EcoRI-20 fragment, which contains pac. Transduction and UV irradiation showed that transduction frequencies of chromosomal markers close to pac were increased several hundred fold and could be further increased by UV irradiation, indicating that TPA was likely to be bound as normal. Southern blotting demonstrated that packaging is unidirectional; that increase in transduction frequency decreases with distance from pac and that the packaging of markers up to 30min from pac was increased. 4 further pac sites were placed at intervals on the chromosome. Strains containing different numbers of pac sites were analyzed by transduction, titration, and hybridization. Some novel phenotypes were observed. First, the time when cells start to lyse after P1 infection is delayed in multi-pac-containing strains, suggesting that in WT strains P1 starts packaging viral DNA earlier than in multi-pac strains, perhaps because the pac sites on the chromosome compete for recognition and cleavage by Pacase. Furthermore, strains which have more than 2-pac sites give very poor plate lysates, whose titers are 1000-fold lower than WT (or one-pac-containing strain).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available