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Title: MAR DNA-binding proteins of the pea nuclear matrix
Author: Grainger, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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The aim of the research described in this thesis was the characterisation of the subcellular localisation and functional properties of two novel matrix attachment region (MAR) DNA-binding proteins, MARBP-1 and MARBP-2, exhibiting strong sequence similarity to each other and to yeast nucleolar box C+D snoRNP proteins Nop56p and Nop58p. MARBP-1 subcellular localisation was examined in onion bulb epidermis and pea leaf cells in a transient expression assay, using fusions of MARBP-1 and the reporters GUS and GFP. These experiments indicated nucleolar localisation of MARBP-1. Deletion analysis demonstrated that nucleolar localisation is conferred within the lysine-rich 98 amino acid C-terminal region. Similarly truncated forms of MARBP-1 were expressed in E. coli, and their ability to bind to a 32P-labelled soybean heat shock (Gmhsp) gene MAR was examined in vitro by filter-based south-western assay. These experiments indicated that MAR-binding capability was also conferred within the 98 amino acid C-terminal region. MAR-binding activity of a soluble form of the 110 amino acid C-terminal region of MARBP-1 was demonstrated by electrophoretic mobility shift analysis, indicating that MAR-binding is not dependent upon prior denaturation of the protein. Disruption by site-directed mutagenesis of seven KKE/D repeats within the C-terminal region did not perturb subcellular localisation or MAR-binding, nor did a 20 amino acid deletion within this region. Disruption of a potential bipartite nuclear localisation sequence also within this region did not perturb subcellular localisation. These experiments suggest that general basic properties of C-terminal residues may be more important than specific sequence motifs for conferring MAR-binding and localisation functions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available