To define the borders of the domain in polyomavirus middle T antigen required for transformation, mutants of recombinant plasmids encoding the viral oncogene were constructed and tested for their capacity to transform Rat-1 cells. Several different mutations which were predicted to affect the structure of the amino terminus of middle T antigen rendered the DNA incapable of transforming Rat-1 cells as measured by the focus assay. The middle T antigens encoded by these non-transforming mutants were efficiently synthesized but failed to associated with protein kinase activity. By contrast, mutants that were not predicted to affect the structure of the amino terminus of middle T antigen transformed Rat-1 cells efficiently, and their encoded middle T antigens were associated with a kinase activity comparable to that of wt middle T antigen. The data suggest that the amino-terminal 100 amino acid residues of middle T antigen constitute part of a domain that is required for activation of the pp60$ sp{ rm c-src}$ kinase activity and for cellular transformation. / To determine the consequences of simultaneous overexpression of both middle T antigen and pp60$ sp{ rm c-src}$ in the same cells, two recombinant adenoviruses which individually encode middle T antigen and pp60$ sp{ rm c-src}$ were used to co-infect 293 cells. Evidence suggests that the capacity of middle T antigen to activate the intrinsic kinase activity of pp60$ sp{ rm c-src}$ is reduced when pp60$ sp{ rm c-src}$ is overexpressed. / To study the role of small T antigen in cellular transformation by polyomavirus, Rat-1 derived cell lines were established by co-transfection of a cDNA encoding only small T antigen together with a gene encoding resistance to G418. A number of these cell lines exhibited growth in agarose in the presence of a high serum concentration, but failed to induce tumours in isogenic rats. Unlike middle T antigen, small T antigen failed to detectably associate with pp60$ sp{ rm c-src}$ when both proteins were overproduced in 293 cells, suggesting that the mechanisms of action of small T antigen and middle T antigen are different. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.74334 |
| Date | January 1990 |
| Creators | Cook, Donald N. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001232499, proquestno: AAINN63687, Theses scanned by UMI/ProQuest. |
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