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Analysis of cyclin A expression in cells with a finite mitotic lifespan

Moore, Kathryn Gillian; (1996) Analysis of cyclin A expression in cells with a finite mitotic lifespan. Doctoral thesis (Ph.D.), University College London (United Kingdom). Green open access

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Abstract

Mammalian cells undergo a limited number of divisions before reaching the end of their mitotic lifespan and withdrawing permanently from the cell cycle. Cyclin A is an important regulator of the cell cycle whose expression is down-regulated upon growth arrest in many cell types. I have therefore undertaken to investigate the mechanisms that regulate cyclin A expression during senescence. Since rat embryo fibroblasts undergo asynchronous senescence, I have used a model system in which large numbers of cells can be made to undergo rapid and synchronous growth arrest. Rat embryo fibroblast cell lines were used which have been conditionally immortalised with a temperature sensitive mutant of SV40 large T-antigen. These cell lines proliferate continuously at the permissive temperature but rapidly growth arrest upon inactivation of T-antigen. I have shown that expression of cyclin A is down-regulated at the protein and mRNA levels upon growth arrest in both this model system and in normal rat embryo fibroblasts undergoing senescence. The reduction in mRNA levels is due to a decrease in the level of transcription. I have used the human cyclin A cDNA to screen a rat cDNA library and have cloned two species of rat cyclin A cDNA, corresponding to the 1.6kb and 2.7kb cyclin A mRNA's seen by northern blot analysis and have demonstrated that the difference between these species lies in the 3' untranslated region. I have then used the rat cDNA to clone l.8kb of 5' flanking sequence and have prepared a series of CAT reporter constructs containing 5' flanking region. I have demonstrated transcriptional activity of this series by transient transfection into tsaS cells and 2°REFs. Experiments are presented which show that the promoter is downregulated when tsaS cells have undergone growth arrest at the nonpermissive temperature. Experiments are also presented which demonstrate that SV40 large T- antigen is capable of transactivating this promoter.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Analysis of cyclin A expression in cells with a finite mitotic lifespan
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: (UMI)AAI10106033; Biological sciences; Mitosis
URI: https://discovery.ucl.ac.uk/id/eprint/10098881
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