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UBC Theses and Dissertations

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UBC Theses and Dissertations

Replication and plasmid-bacteriophage recombination Smith, Richard Dana

Abstract

Marker rescue between a plasmid carrying T₇⁺ DNA and a mutant bacteriophage was used to study the role of replication in genetic recombination. The replication of plasmid and phage DNA's could be controlled independently by a mutation in the host bacterium and mutations in T₇ gene 5 (DNA polymerase), respectively. Recombination was monitored by the production of wild-type phage. Results indicated that when only one molecule, i.e. plasmid or phage, could replicate, recombination was decreased slightly. However, if replication was blocked on both molecules, no recombination was detected. Agarose gel electrophoresis, ³H-thymidine incorporation, and copy number analysis showed that plasmid DNA was not degraded after T₇ infection. Density transfer experiments examined recombination between 3 a heavy labeled phage and a H-labeled plasmid molecule. Analysis of such experiments by CsCl density gradients showed that replication was essential for joint molecule formation. Replication was responsible for the displacement of parental DNA which then was assimilated into a recipient DNA molecule. The essential role of the T₇ gene 6 protein (5' exonuclease) during T₇ recombination was determined. This exonuclease creates gaps on T₇ molecules to provide regions where homologous donor DNA can base pair. Recombinant T₇ molecules were found to contain a single stranded insertion of homologous donor DNA. The results are discussed in relation to various models which contain roles for DNA synthesis during recombination. A model for early events during plasmid-phage recombination is presented.

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