A study of nucleic acid synthesis in mammalian cells infected with herpes simplex virus

Hay, John (1966) A study of nucleic acid synthesis in mammalian cells infected with herpes simplex virus. PhD thesis, University of Glasgow.

Abstract

The aim of this thesis was to investigate the metabolism of DNA and RNA in mammalian cells before and after infection with Herpes Simplex virus. In furtherance of this aim, a method was developed for the extraction of DNA from Herpes Simplex virus, and from its host cell, BHK 21 (C13). This method was also applied successfully to a variety of DNA-viruses and mammalian cells. The isolated viral DNA was demonstrated to be biologically active in certain transformation experiments, to be relatively homogeneous with respect to size and to have the characteristic melting profile of a double-stranded DNA molecule. Suitable conditions for the irreversible separation of the component strands of viral DNA were established. Bouyant density measurements in CsCl and melting temperature determinations on viral and host DNAs indicated that the base compositions of these DNAs are, respectively, 68-69% guanine plus cytosine and 41% guanine plus cytosine. The average size of a representative preparation of viral DNA was shown to be 24S. Attempts were made to separate viral and host DNAs from a mixture of both, by chromatography on methylated alhumin columns. This proved unsuccessful, probably the size heterogeneity of both DNAs under the conditions employed. A method for the harvesting and lysis of Herpes-infected cells prior to RNA isolation was developed, and factors affecting the isolations of undergraded RNA from these infected cells were assessed: this resulted in the use of Bentonite at several points in the isolation schedule and the involvement of redistilled phenol at the appropriate stages. The pattern of synthesis of RNA in BHK 21 (C13) cells was investigated over several short time periods. During a 30 minute pulse of radioactive precursor, RNA synthesised in these cells was consistently shown to comprise a 45S and a 35S component, with smaller amounts of synthesis of 16S and 4S RNA. Longer pulse times served to increase the incorporation into ribosomal RNA and sRNA. The existence of an RNA species of sedimentation doefficient greater than 45S was noted. 45S RNA was shown to be derived from the BHK 21 (C13) cell genome and to be similar in base composition to 27S ribosomal RNA from these cells. The observations presented here, together with analogues information from other laboratories, suggest that 45S (and probably also 35S) RNA contain ribosomal precursor material. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: H M Keir
• Keywords: Biochemistry, Virology
• Date of Award: 1966
• Depositing User: Enlighten Team
• Unique ID: glathesis:1966-72180
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 12:39
• Last Modified: 17 May 2019 12:39
• URI: https://theses.gla.ac.uk/id/eprint/72180