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Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-110). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9957566.

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Genetic exclusion in phage T4 is the prime responsibility of the imm and sp genes. The map region containing imm does not allow sufficient bps to encode for proteins the size reported for the imm gp. After assaying 30 mutants of the genes adjacent to imm, I found 7 in gene 42 that were defective in the imm phenotype. Upon reverting amNG411(42), the mutant most defective exclusion, for its gene 42 phenotype the exclusion phenotype also changed. When assayed in UGA suppressor hosts, imm+ phage showed a decreased exclusion ability indicating that an opal codon was involved in production of the functional imm gp. I concluded that imm and gene 42 overlap in an out-of-phase orientation with the involvement of an opal readthrough. This overlap has implications in the genetic regulation of this region. This region of T4 also encodes several other genes important in phage intra- and interspecific competition. They are B-gt, 42 and sp. Using recombinant DNA techniques, I precisely located the sp gene to a region between 21.647 and 22.014 kbp on the T4 restriction map and determined its molecular weight as approximately 15 kDa. This same region of T4 was purported to contain gene 40. Complementation and marker rescue experiments with sp+ plasmids indicated that genes sp and 40 are the same. Gene 40 mutants also were found to be defective in sp function. Genes sp and 40 were redesignated gene sp/40 thus linking an early expressing gene with the morphogenic pathway of prohead assembly. Functionally, host enzymes exo III and exo V were found as participants in gp imm mediated exclusion. Presumably gp imm alters the pilot protein of the superinfecting DNA thus exposing it. Gp sp functions by an anti-lysozyme action. But the pleiotrophic effects of sp/40 are best explained by a temperature induced conformational rearrangement hypothesis. This work links molecular genetics to the ecological concept of competition and provides insights into the function and the evolutionary significance of the competition cluster genes. The competition cluster encodes fundamental adaptive strategies found universally in nature.

Recombinational repair is a widespread mechanism for dealing with DNA damage. It is found in both prokaryotes and eukaryotes which implies that it is an ancient process which arose early in the evolutionary history of life on Earth. In addition, it has been implicated as a driving force in the evolution of sexual reproduction. In this dissertation I report experimental results which clarify the role of recombinational repair in bacteriophage (phage) T4. The Luria-Latarjet effect is an increase in resistance to DNA damage by phage T4 during infection. It has often been assumed to involve recombinational repair, but this has never been actually demonstrated. Using eleven phage T4 mutants, I have obtained evidence that the Luria-Latarjet effect is due to three repair pathways--excision repair, post-replication-recombinational-repair (PRRR) and multiplicity reactivation (MR), a form of recombinational repair. My results show that the Luria-Latarjet effect develops in two stages. The first stage starts soon after infection. Damage which occurs during the first stage can be repaired by excision repair or PRRR. The second stage appears to start after the first round of DNA replication is complete. Damage which occurs during this stage can apparently be repaired by MR as well as the other two repair pathways. I have also transferred the yeast RAD50 gene, which is required for recombinational repair, into an E. coli expression vector. After demonstrating expression of the protein, I used this construct to test for complementation by the RAD50 gene of E. coli and phage T4 mutants defective in recombinational repair. I was unable to demonstrate complementation in five different assays. Based on the results discussed above and what is known about the phage T4 life cycle, I propose a model for the Luria-Latarjet effect in phage T4. Further, I propose that recombinational repair has been selected to ensure progeny phage genomes are packaged with minimum damage. Since numerous other viruses also show a Luria-Latarjet effect type resistance to DNA damage, I suggest that the conclusions from this phage T4 study may have wide applicability to other viruses.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 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. / 2031-01-01

Graduation date: 1999

A crucial factor in determining the accuracy of DNA replication is maintenance of a balanced supply of deoxyribonucleoside triphosphates (dNTPs) at replication forks. Perturbation of dNTP biosynthesis can induce dNTP pool imbalance with deleterious genetic consequences, including increased mutagenesis, recombination, chromosomal abnormalities and cell death. Using the T4 bacteriophage system, I investigated the molecular basis of mutations induced by imbalanced dNTP pools in vivo. Two approaches were adopted to disturb dNTP biosynthesis: 1) using mutations which affect the deoxyribonucleotide biosynthesis pathway; 2) exogenously supplying mutagenic deoxyribonucleoside analogs which are then taken up by cells and are metabolized to dNTPs. The levels of dNTPs under different conditions were measured in crude extracts of phage-infected cells, while mutagenic effects were quantitated by analysis of certain rII mutations, thought to revert to wild type along either GC-to-AT or AT-to-GC transition pathways. The mutation pathways stimulated by dNTP pool perturbations were confirmed by direct DNA sequencing after amplification of template by the polymerase chain reaction (PCR). By replacing phage ribonucleotide (rNDP) reductase with the host, Escherichia coli, rNDP reductase, in phage-infected cells, I examined the mechanism of mutation induced by the thymidine analog 5- bromodeoxyuridine (BrdUrd) in vivo. Although both AT-to-GC and GC-to- AT transition mutations were stimulated many hundred-fold when cells were grown in medium containing 100 μM BrdUrd, GC-to-AT transitions were stimulated predominantly when T4 reductase was active, while ATto- GC transitions were stimulated more when E. coli reductase was active. By examining the control by dNTPs on CDP reduction, I found that the T4 rNDP reductase is substantially inhibited by either BrdUTP or dTTP in crude enzyme extracts. These experimental results are consistent with the hypothesis that mutagenic effects of BrdUrd are based on dNTP perturbations, supporting the model that rNDP reductase is a major determinant of BrdUrd mutagenesis. I also studied the mutator phenotype of one temperature-sensitive conditional lethal mutant, T4 ts LB3, which specifies a thermolabile T4 deoxycytidylate (dCMP) hydroxymethylase. At the sites of different rII mutations, I found 8- to 80-fold stimulation of GC-to-AT transitions induced by ts LB3 at a semipermissive temperature (34° C). Sequence analysis of revertants from the most sensitive gene marker, rII SN103, showed that either cytosine within the mutated triplet can undergo change to either thymidine or adenine, supporting a model in which mutagenesis induced by ts LB3 at a semipermissive temperature is based on dNTP pool perturbations. The putative depletion of hydroxymethyldeoxycytidine triphosphate (hm-dCTP) caused by the temperature-labile dCMP hydroxymethylase presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations. However, no significant dNTP pool abnormalities were observed in extracts from ts LB3 phageinfected cells even when cells were grown at the semi-permissive temperature, suggesting that imbalanced dNTP pools occurred only locally, close to replication forks. These results support a model of dNTP "functional compartmentation", in which DNA replication is fed by a small and rapidly depleted pool, with the bulk of measurable dNTP in a cell representing a replication-inactive pool. To further characterize the mutagenic specificity and DNA site specificity induced by T4 ts LB3, I developed a fast forward mutation approach using thymidine kinase as a marker gene. The studies confirmed that the principal mutagenic effect induced by ts LB3 is C-to- T transition, while C-to-A transversion mutagenesis also occurs. Analysis of DNA sequences around each mutation also suggests that local DNA context influences mutation frequency. / Graduation date: 1991

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