Cross reactivation of ultraviolet light irradiated bacteriophage T4
Cohen, Paul Sidney
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Cross reactivation (CR) is defined as the rescue of genetic markers from inactive bacteriophage particles by viable bacteriophage particles. UV was used as the inactivating agent in the experiments reported in this dissertation. Escherichia coli BB was used as the host bacterium and the bacteriophage used was T4. E. coli BB was infected half with UV irradiated T4 and half with normal, unirradiated T4. Under the conditions of the experiment lysis of the infected culture did not take place until 75 minutes after infection. At specific times after infection, infected cells were broken open and the number of intracellular phage per infected cell was determined. The results indicate that a normal size replicating pool of phage molecules is reached as quickly under the conditions of this experiment as when cells are infected with live phage particles. Moreover, this experiment provides further evidence that UV lesions do not replicate. E. coli BB was infected with two different T4 rII mutants. One of these mutants had been UV irradiated (50 lethal hits per phage) and its concentration was adjusted so that no cell received more than one of these particles. The other mutant was unirradiated and each cell received 2 to 3 of these particles. The dose of irradiation chosen was such that essentially all the irradiated particles had at least one lesion between the two loci. Until 22 minutes after infection, infected cells were plated on streptomycin medium to break them open and on a normal medium to allow natural lysis to occur. A comparison of counts on the two types of media showed the fraction of cells undergoing CR which had done so by that time. The results of this experiment show that CR is completed at the end of one normal latent period, i.e., before normal lysis. This is so despite the fact that at the dose of UV employed, only 4.8 per cent of the cells infected with both mutants showed CR. The cells which did show CR did not show an increase in the frequency of CR recombinants upon extension of the latent period. An increase in this frequency would have been expected if many copies of unirradiated portions of irradiated phage genomes had been present in the phage replicating pool. The results of these last two sets of experiments are interpretable as a failure of unhit portions of UV irradiated phage genomes to replicate normally within the phage replicating pool. Somehow these pieces of genome are removed from the pool faster than they are synthesized. It is noteworthy that these results are also compatible with the hypothesis that at the dose of UV employed, unirradiated portions of irradiated phage genomes do not replicate. A model of CR is presented which is consistent with present ideas of bacteriophage T4 genetic recombination, as well as with CR data from other sources. Moreover, this model requires no replication of unirradiated portions of irradiated phage DNA genomes. Finally, the phenomenon of lysis inhibition, as manifested by extension of the latent period by super infection of infected cells under appropriate conditions has been examined. It was found that more and more of these infected cells which had been almost completely deprived of a constant source of superinfection lysed after having been lysis inhibited only once. These results show that continuous reinfection is required to maintain lysis inhibition.
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