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Studies on the immortalisation of rodent embryo fibroblasts by simian virus 40 large tumour antigen

Powell, Andrew Jonathan; (1995) Studies on the immortalisation of rodent embryo fibroblasts by simian virus 40 large tumour antigen. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Introduction of simian virus 40 large tumour antigen (T antigen) into primary rodent fibroblasts in vitro results in them acquiring an infinite proliferative potential, i.e. becoming immortal. To study the mechanism by which T antigen immortalises rodent embryo fibroblasts I assayed mutants of T antigen for their ability to complement the growth defect of a rodent embryo fibroblast cell line that had been conditionally immortalised with a temperature sensitive mutant (tsA58) of T antigen. I also assayed the same T antigen mutants for their ability to immortalise secondary rodent embryo fibroblasts. I identified several functional domains of T antigen which are required for both the immortalisation of secondary rodent embryo fibroblasts and the maintenance of immortalisation in the T antigen-dependent cell line at the non-permissive temperature. Interestingly, an amino-terminal deletion mutant, dll l35, which was negative for immortalisation readily complemented the growth defect of the conditionally immortal cells. I found that the inability of the dll 135 mutant to immortalise could be overcome by cotransfection with either of two carboxy-terminal point mutants (5031 or 5041), both of which themselves are negative for immortalisation. To clarify the role of these mutants in this trans-complementation I constructed temperature sensitive mutants of dll 135, 5031 and 5041 by introducing the tsA58 point mutation into them. Using combinations of these mutants in immortalisation assays at both the permissive and non-permissive temperature I have demonstrated the presence of a functional domain of T antigen, lacked by dl1 135, which is required transiently for the initiation of immortalisation. Once immortal cell lines have been established this functional domain is no longer required and the growth of these cell lines can be maintained by the dl1 135 T antigen. I have thereby demonstrated that the initiation and maintenance of immortalisation by T antigen are functionally separable. I have also attempted to isolate novel immortalising cDNAs from a newt limb blastema cDNA expression library by transfecting the library into rodent embryo fibroblasts and selecting for immortal cell lines. I describe the cloning of a cDNA fragment which, although it is unable to encode a protein, is clearly able to stimulate cellular proliferation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Studies on the immortalisation of rodent embryo fibroblasts by simian virus 40 large tumour antigen
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Biological sciences; Immortalisation of rodent embryo fibroblasts; Simian virus 40
URI: https://discovery.ucl.ac.uk/id/eprint/10102724
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