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http://hdl.handle.net/11375/5999
Title: | Construction and Characterization of Insertion and Deletion Mutations in the Transforming Genes of Human Adenovirus Type 5 |
Authors: | McKinnon, Donald Randy |
Advisor: | Graham, Frank Lawson |
Department: | Biology |
Keywords: | Biology;Biology |
Publication Date: | Apr-1984 |
Abstract: | <p>The process whereby a normal cell becomes malignant, termed transformation, can be initiated by a number of agents that have been implicated in carcinogenesis. Understanding the mechanism of oncogenic transformation is a necessary prelude to developing a rational approach to therapeutic and preventative treatment of this disease. This thesis examines the molecular mechanism off cell transformation caused by a member of a family of DNA tumor viruses, the human adenoviruses. The experimental approach taken in this work was to use recombinant DNA techniques to isolate the oncogenes encoded in the genome of adenovires serotype 5 (Ad5). Defined mutations were constructed in these Ad5 oncogenes, and mutated plasmids were assayed for their transforming activity on primary rat and on primary hamster kidney cells in order to identify which Ad5 genes were necessary for primary cell transformation.</p> <p>This study reports the construction and characterization of a library of recombinant bacterial plasmids, containing DNA restriction endonuclease fragments representing the entire Ad5 genome. This library of cloned viral DNA fragments has served as a useful source of reagents for both biological and biochemical studies on the molecular biology of adenovirus.</p> <p>This study employs the prokaryotic transposable element Tn5 as an insertional mutagen for cloned Ad5 sequences. The results demonstrate the usefulness of bacterial transposable elements for gene mapping experiments with cloned eukaryotic genes. A number of insertion mutations located in the Ad5 oncogenes were constructed and were characterized by DNA sequence analysis as a prelude to studies on their transforming activity. The results of studies on the specificity of target DNA sequences chosen for Tn5 insertion suggest that transposition is influenced by transcriptional activity in target DNA.</p> <p>The results of morphological transformation experiments with primary rodent cells using plasmids containing the adenovirus oncogenes (early regions E1A and E1B), and with plasmids containing defined insertion and deletion mutations in these oncogenes, have demonstrated the following. First, viral genes located exclusively in the rightward transcribed DNA strand of E1A are essential for transformation. Secondly, the viral gene located in the promoter proximal region of early region E1B, encoding a Mr=21,000 product, is required for transformation. Third, the requirement for this E1B product could be replaced with serum supplements to cells transformed by only region E1A. Finally, it was demonstrated that not all primary cells require two cooperating oncogenes for cell transformation since insertion mutations located in E1A, which eliminate transformation on primary rat kidney cells, did not eliminate transformation on hamster kidney cells.</p> <p>These results have helped to define the adenovirus genes encoding functions that are essential for cell transformation. Results presented also suggest possible roles for these viral functions in maintaining the transformed state. It will be of considerable interest to determine the biological properties of these gene products which allow them to bring about the process of cell transformation.</p> |
URI: | http://hdl.handle.net/11375/5999 |
Identifier: | opendissertations/1336 2362 1294819 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Size | Format | |
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fulltext.pdf | 4.33 MB | Adobe PDF | View/Open |
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