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Title: DNA replication in herpes simplex virus type 1 infected baby hamster kidney cells (BHK-21/C13)
Author: Baybutt, Herbert N.
ISNI:       0000 0001 3452 0328
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1980
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The project concerned DNA replication in HSV-1 infected BHK cells. This involved a study of DNA-binding proteins, some properties of the HSV-1 induced DMA polymerase, and the effect of an inhibitor of the induced DNA polymerase, phosphonoacetate, on protein and DNA synthesis in the infected cell. The aim was to further the understanding of the mechanisms of HSV replication. HSV-1 infected BHK cells induce more than 50 proteins. Sixteen of these proteins were isolated by DNA-cellulose affinity chromatography, with varying affinities and preferences for native and denatured DNA. Infection with HSV-1 does not immediately inhibit the synthesis of host cell proteins but rather gradually decreases protein synthesis. The HSV-1 induced DNA polymerase was partially purified, and another minor DNA polymerase activity was isolated. This activity did not bind to DNA-cellulose and was less stable than the major HSV-induced DNA polymerase which did bind to DNA-cellulose. All the biochemical properties tested for the two activities were similar, they differed only in their sedimentation values, the minor: DNA polymerase sedimenting with a greater velocity. This activity may be a consequence of protein aggregation. The major HSV-1 DNA polymerase activity had a sedimentation velocity of 6.86S. In the presence of 500mM KC1 the S value decreased to 5.65S. The enzyme possessed biphasic enzyme kinetics giving two Km values with dTTP as the limiting substrate. This DNA polymerase was also shown to be quasi-processive for the length of oligonucleotide synthesised from any one primer using a Poly(dA) oligo(dT)synthetic template. The effects of the polyamine, spermine, on the HSV-induced DNA polymerase are dependent on its concentration. The effects were shown to be due to a change in the number of sites initiated rather than an alteration in the length of the oligonucleotide chains synthesised. Phosphonoacetate inhibits HSV DNA sjoithesis in vivo, but has no effect on the uptake and phosphorylation of thymidine. Phosphonoacetate has very little effect on protein synthesis in HSV-1 infected cells. Indicating that for HSV-1, protein synthesis is not dependent to any large extent on DNA synthesis. A DNA nicking-closing enzyme (topoisomerase) was present during the lytic cycle of HSV-1 at a constant level, but this enzyme is probably of host cell origin. HSV-1 infection of BHK cells induces changes in cell metabolism, especially with regards to DNA synthesis. A model is proposed for the role of dTTP in the control of DNA synthesis in the infected cell.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genetics