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Date of Award

12-1983

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Dr. Frank L. Graham

Abstract

Adenovirus type 5 is able to oncogenically transform cells growing in culture. These transformed cells show different degrees of transformation. The interest of my studies was to establish the presence and characterize the structure of virus DNA in adenovirus 5 transformed cell lines and to look for a possible correlation between the integrated viral sequences and the phenotype of the cells. For this purpose, cell lines transformed by wild type (virions or DNA) and by host range mutants (virions) were analyzed for their viral DNA content and for their transformed phenotype.

More than the left 8% was always present in virion transformed cells. Cells transformed by host range mutants of complementation Group I generally contained a larger fraction of the genome than did their counterparts transformed by wild type virus. In some host range transformed cells, virtually the entire viral DNA molecule was found colinearly integrated.

In the case of the cells transformed by wild type DNA or virions, it was not possible to correlate any particular phenotype with a specific integration pattern. The same pattern was found in the tumorigenic derivatives of a non oncogenic cell line (293) as in the parental line. Studies with cells transformed by Group I host range mutants showed that partial or complete integration of viral DNA into transformed cells was always associated with the production of some viral proteins and with the induction of a partially transformed, non oncogenic phenotype.

An interesting finding was the limited number of inserts in cell lines isolated from uncloned populations within a small number of passages after transformation. This suggested the possibility of a limited number of sites available for transformation or alternatively, that a few cells rapidly overgrew the rest. In an attempt to answer this question and to obtain more information on the integration process, DNA was extracted from semipermissive rat cells at different times shortly after infection. Adenovirus 5 wild type and host range mutants from Groups I and II were used in individual experiments. The extracted DNA was alnalyzed for the presence and state of viral DNA. New forms of intracellular viral DNA, which might be intermediates for integration, were found. A fragment having the size of both ends of the viral DNA joined together, and which hybridizes with both ends when these are used separately as probes, was detected in Southern blots after digestion of rat cell DNA with different restriction enzymes. This structure was detected earlier after infection with host range 1 (Group I), than after infection/with host range 6 (Group II) or with wild type, and was found also in Hela cells and in two rat cell lines after infection with host range J or wild type. Studies followed to determine the nature of this new arranged viral DNA provided evidence for covalent head to tail joining and for the formation of circular Ad5 DNA molecules.

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