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Title: DNA synthesis in cultured hamster cells and the effect of infection by herpesviruses
Author: Howard, David King
ISNI:       0000 0001 3582 4673
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1974
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The purpose of this project was to investigate the control of DNA metabolism in both 'normal' and virus-infected mammalian cells. The investigation adopted three broad approaches: (i) a study of the phenomenon of serum-stimulation of stationary cultures of BHK 21 cells; (ii) a study of the inhibition of cellular DNA synthesis following infection with psauderabies virus; and (iii) the utilisation of nuclei isolated from both and virus-infected cells to study DNA metabolism in vitro. Although the ultimate aim of this project was to gain a deeper understanding of the processes involved in the control of cellular DNA metabolism, considerable effort has been directed towards an investigation of the inhibition of cellular DNA synthesis by infection with pseudorabies virus. In particular, attempts were made to discover whether this inhibition was caused by the action of a molecule pre-existing in the invading virion or, if not, to identify inhibitory molecules synthesised after infection. Efforts were also made to decide whether initiation and/or propagation of cellular DNA was inhibited. The investigation into the control of DNA metabolism in uninfected cells concentrated mainly on a study of the stimulation of stationery cultures of BHK cells by the addition of fresh serum including the determination of the levels of a number of key enzymes involved in nucleic acid metabolism and of the rates of synthesis of DNA, RNA and protein. This investigation involved the use of autoradiography to determine the percentage of the stationary cell population stimulated to synthesise DNA and the use of isopycnic centrifugation to identify the nature of the DNA synthesised. Use of actinomycin-D showed the presence of two stages n the pre-replicative phase at which the subsequent synthesis of DNA appeared to depend on DNA synthesis, in good agreement with several other similar systems. The development of this technique provided an alternative to exponentially growing cells in which virus inhibition of cellular DNA synthesis could be studied. The use of virus which had been inactivated by ultra-violet light and of inhibitors of protein synthesis has shown that the inhibition of cellular DNA synthesis s not brought about by some pre-existing component of the virus particle and that protein synthesis is necessary before this effect can be observed. It is not possible using cycloheximide, however, to inhibit viral protein synthesis to a sufficient degree to prevent expression of the inhibitory effect on DNA synthesis without at the same time directly inhibiting cellular DNA synthesis. Consequently, it has not been possible to study the proteins produced in normal and in cycloheximide treated virus-infected cells with a view to identifying the inhibitory factor(s) produced after infection, nor has it prove possible to cause an inhibition of cellular DNA synthesis by the addition of crude extracts of infected cells to uninfected cultures. Some initial screening work on DNA synthesis in cells infected with temperature-sensitive mutants of pseudorabies virus has been carried out and further work with these mutants may prove useful as an approach to an understanding of the control of cellular DNA metabolism. Alkaline sucrose velocity centrifugation of DNA isolated from virus-infected cells has indicated that the virus inhibits elongation of cellular DNA which had already been initiated at the time of infection, but it has not been possible to establish whether the initiation of new cellular DNA chains is also affected. The final approach to the problem of control of cellular DNA metabolism has been the development of a system comprising nuclei isolated from control and pseudorabies virus-infected cells and which will incorporate radioactive nuleotides into DNA in vitro. Such DNA synthesis mirrors the DNA synthetic capability of the cells from which the nuclei wore isolated. Some preliminary work has been carried out on the effects of crude 'cytopiesmic' preparations from infected cells on DNA synthesis in the isolated nuclei. A stimulation of DNA synthesis can ho observed in infected cell nuclei when these fractions are added to the incubation mixture, but the effect is not so marked in nuclei from uninfected cells. Synthesis of both cellular and viral DNA is stimulated by these fractions, the effect being more marked on cellular DNA synthesis, and there, are some indications that this stimulation may be rather non-specific and a result of the action of a cytoplasmio DNA polymerase activity on both cellular sad viral DNA. The in vitro nuclear system seems to hold most promise as a suitable system in which to identify controlling factors produced after virus infection and a study of these factors should lead to a better understanding of the processes involved in the control of DNA metabolism both in infected and in uninfected cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available