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Title: A study of the in vitro replication of adenovirus type 4 DNA
Author: Harris, Mark Peter Gerard
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1987
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The work described in this thesis concerns the development and characterization of an in vitro system for the initiation of adenovirus type 4 DNA replication and the first steps towards the purification and characterization of the individual components of the system. The study was suggested by the differences observed in the minimal DNA sequence requirements for adenovirus type 2 (Ad.2) and adenovirus type 4 (Ad.4) DNA replication in vivo (Hay,1985a,b). The minimal Ad.2 origin of replication consisted of the terminal 45b.p. of the genome containing two important sequence elements: The 10b.p. AT-rich sequence conserved in all the human adenoviruses (Stillman et al, 1982) between nucleotides 9-18 and the consensus binding site for the cellular protein nuclear factor I (NFI) between nucleotides 19-45 (Nagata et al,1983). In contrast the Ad.4 origin of replication comprised only the terminal 18b.p. of the genome, which were identical to the terminal 18b.p. of Ad.2 (Hay,1985b). An in vitro study was seen as the best way to further define the mechanistic differences between Ad.2 and Ad.4 DNA replication. Initially full-length Ad.4 DNA was isolated from virions by a number of treatments leaving it associated with either the terminal protein and core polypeptides (Ad.4 cores), the terminal protein alone (Ad.4 DNA-prot.), or completely protein-free (Ad.4 DNA) and it was shown that these templates could support initiation of DNA replication in vitro. Initiation was defined as the formation of a covalent complex between 5'-dCMP and the 80,000-dalton precursor terminal protein (pTP-dCMP complex). In Ad.2 the formation of this complex is catalysed by the viral DNA polymerase which is tightly associated with the pTP. Use of (a32P)dCTP enabled the detection of this product by SDS-polyacrylamide gel electrophoresis and autoradiography. Subsequent experiments utilized a plasmid containing the terminal 140b.p. of Ad.4 as a template for reasons of stability and reproducibility of results. Formation of a pTP-dCMP complex was shown to be catalysed by either nuclear or cytoplasmic extract of Ad.4 infected cells alone. An inhibitory activity in nuclear extracts meant that cytoplasmic extracts were used exclusively to characterize the reaction. Various parameters of the reaction were studied including the formation of pTP-dCMP complex at different protein concentrations, the time course of pTP-dCMP complex formation and the nucleotide specificity of the reaction. The activity of cytoplasmic extract in forming pTP-dCMP complex was studied at varying concentrations of ATP and divalent cations and in varying conditions of ionic strength. By including in the reaction dATP, dTTP and ddGTP it was shown that the pTP-dCMP complex could be elongated, presumably by the Ad.4 specific DNA polymerase, to the first dG residue in the sequence - the 30th nucleotide. This resulted in the formation of an additional product with an apparent molecular weight of 90,000-daltons. The template requirements for the formation of a pTP-dCMP complex were studied: Both the DNA sequence and template conformation requirements were investigated using both single- and double-stranded DNA. It was shown that the double-stranded DNA sequence requirements were the same as in vivo. Finally the Ad.4 specific DNA polymerase was partially purified from Ad.4 infected HeLa cells and the response of this DNA polymerase to various inhibitors studied. A factor from uninfected HeLa cell nuclei that stimulated pTP-dCMP complex formation by cytoplasmic extract of Ad.4 infected cells was partially purified. This factor was shown to stimulate pTP-dCMP complex formation on a template containing only 18b.p. from the Ad.4 terminus, indicating that it might specifically recognize sequences within that region.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available