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Title: The PML-NB : a stress responsive nuclear organelle
Author: Dovey, C. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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The Promyelocytic Leukaemia Nuclear Body (PML-NB) is a subnuclear structure disrupted in the cells of PML. Identification of proteins that interact with PML in the NB, before and after stress, will help to define the role of this structure in the cellular response to damage. In this study, changes to the PML-NB were investigated after X-ray-induced DNA damage. Initially the reorganisation of the PML-NB was tracked via immunofluorescence and quantified, at time points after exposure to 1-15Gy, in order to identify the optimum dose and time with which to study changes to the PML-NB composition. At the higher doses of X-ray, the PML-NBs dispersed into more numerous, smaller structures. Gel filtration chromatography was conducted to quantify the decrease in size of the PML-NB after DNA damage, which was observed by immunofluorescence. The majority of the cellular PML was shown to be in large complex, of ³ 4MDa in size prior to insult, and exposure to X-ray resulted in a reduction in the size of the PML complexes. Attempts to isolate the entire PML-NB protein complex by large-scale immunoprecipitation were conduced before, and at time points after irradiation, in order to analyse the composition of the endogenous complex. Two novel potential binding proteins were identified from the immunoprecipitates by mass spectroscopy, one of which, Phospholipase C gamma-1, was investigated further. Responses of the PML-NB to other stresses were also investigated in cells expressing intranuclear inclusions of proteins that contain expanded polyglutamine repeats. These inclusion bodies had previously been reported to sequester PML. It was shown in this study that the normal responses of the PML-NB to various stresses were prevented following sequestration to the inclusion bodies, and following sequestration to other non-pathologic inclusion forming proteins. Further, the DNA damage response proteins BRCA1 and Rad51 showed subnormal responses to damage in cells expressing inclusion bodies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available