UBC Theses and Dissertations
Cross reactivation and partial replication of bacteriophage T7 DNA Burck, Kathy Louise Bauman
Cross reactivation is a process whereby genetic markers are rescued from a UV-irradiated phage by a coinfecting mutant phage. The probability of rescue of a specific marker during T7 infection depends on the map position of the marker. This phenomenon is observed also with bacteriophage T4. During T7 infection, markers from the left end of the genome are rescued with high efficiency. Recombination is necessary for marker rescue. Photoreactivation and the E.coli pyrimidine dimer specific endonuclease enhance rescue efficiency. It is found that markers that are rescued efficiently are in segments of the DNA which replicate 32 efficiently, ie ³²P-labeled progeny DNA synthesized after infection by a UV-irradiated T7 phage hybridizes predominantly with restriction fragments of T7 DNA from the left end of the genome. The effect is dependent on the UV dose: the higher the dose to the T7 phage, the fewer are the markers which are rescued efficiently and the smaller is the area of the genome which replicates. Under the electron microscope, partially replicated, UV-irradiated T7 DNA has double stranded internal duplications (bubbles) or branches. The distribution of partially replicated regions determined by EM correlates very well with regions which have replicated as determined by hybridization analysis and both results correlate with the distribution of marker rescue frequencies along the T7 genome. The results are consistent with the basic idea of the partial replica hypothesis which postulates that replication of a UV-irradiated genome starts from a specific origin and proceeds bidirectionally to a UV lesion. UV lesions block further replication and those regions which are replicated efficiently are rescued efficiently by a coinfecting, genetically marked phage. During the course of this investigation, results were obtained which indicated that during T7 infection, primary infecting phage prevent infection of the same cell by secondary or superinfecting phage. This process is termed superinfection exclusion. Preincubation of cells in chloramphenicol (CM) at 100 μg/ml for 5 minutes prior to primary infection allows production of infective centers by superinfecting phage. Without CM, recombination between a primary and secondary phage decreases as the time between primary and secondary infection is increased. After a 5 minute preincubation in CM, secondary phage added as late as 8 minutes after primary infection are able to recombine with primary phage at a frequency not significantly different from that found after simultaneous infection. Superinfection exclusion occurs at the injection stage. In the absence of superinfection exclusion, CsCl gradient analysis reveals that many parental phage are able to inject their DNA into a single cell. However, only very small amounts of parental (conservative) DNA are found among the progeny phage if unattached parental phage are removed by differential sedimentation.
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