Control of major histocompatibility Class I antigen expression in adenovirus transformed cells.

HOLLETT, Tina. (1993). Control of major histocompatibility Class I antigen expression in adenovirus transformed cells. Doctoral, Sheffield Hallam University (United Kingdom)..

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Abstract

Two DNA sequences, termed CRE1 and CRE2, located upstream of the Major Histocompatibility Complex (MHC) class I gene have been shown to play an important role in controlling transcription of this gene. MHC class I expression is down-regulated in cells expressing Adenovirus (Ad) 12 E1A compared to cells expressing E1A from Ad2 or Ad5. The possible involvement of these sequences and the factors that bind to them in this down-regulation was investigated using transient expression assays and in the case of CRE1, also by gel retardation and Western blotting. The transient expression assays did not demonstrate a functional role for these sequences in transcriptional down-regulation. The implications of this result in relation to the experimental design are discussed. Gel retardation assays showed that the levels of CRE1-binding factors detected were dependent on the technique employed to prepare the nuclear extracts. Preparation of nuclear extracts by a technique in which the potential for cytoplasmic contamination and proteolytic degradation was minimized showed that the level of CRE1 factors, likely to represent NF-kappaB (p50/p65 heterodimers) and H2TF1 (p50 homodimers), was higher in cells expressing Ad2 or Ad5 E1A than those expressing Adl2 E1A. Western blotting using an antibody prepared to the p50 subunit of NF-kappaB did not show a difference in levels of this subunit between different Ad-transformed cell lines. These results suggest therefore that the levels of p50 are similar in these cell lines but that the levels of p50 bound to either p65 or another p50 subunit, and capable of binding DNA, are higher in those transformed by Ad2 or Ad5 E1A.The binding site of CRE2-binding proteins was investigated in Ad-transformed cells by DNase I footprinting. The factors RXRbeta/H-2RIIBP which bind to this sequence are members of the ER/TR subfamily of steroid/thyroid hormone receptors. Members of this subfamily have the sequence AGGTCA conserved in their binding sites; this sequence formed part of the area of protection found on the DNase I footprint.The mechanism of nuclear induction of NF-kappaB was investigated in Ad-transformed cells by the use of the free radical producer benzoyl peroxide and other inducers such as tumour necrosis factor and cycloheximide. Benzoyl peroxide was shown to activate transcription and scavengers of free radicals prevented the induction. These results were incorporated into a model of NF-kappaB induction involving hydrogen abstraction.

Item Type: Thesis (Doctoral)
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 1993.
Research Institute, Centre or Group: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:20
Last Modified: 14 May 2018 15:38
URI: http://shura.shu.ac.uk/id/eprint/19814

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