Some new approaches to conventional methods for the study of protein-DNA interactions
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Boissy, Robert James
issued: 2010-02-01T22:00:51Z
issued: 1994
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: Two important experimental techniques used to study eukaryotic, sequence-specific DNA-binding proteins are electrophoretic mobility-shift assays (EMSAs) and sequence-specific DNA-affinity chromatography. This thesis describes some new approaches to these frequently used methods, which were used to study a DNA-binding protein (ceHSBF) with an apparent specificity for a conserved DNA sequence element (the heat-shock element or HSE) that mediates the transcriptional regulation of heat-shock-inducible genes in the soil nematode Caenorlzabditis elegans. Although C. elegans has attracted extensive research interest, a lack of appropriate and reliable experimental methods has severely limited biochemical studies of sequence-specific DNA-binding proteins from this species. As a first step towards addressing this problem, I developed a simple and effective procedure for isolating C. elegans nuclei. I also developed a new class of EMSA electrophoresis buffers and established the running conditions appropriate for their use. The distinctive electrolyte in these buffers is monopotassium hydrogen glutamate (KHG1u), which provides both a physiological concentration of potassium ions (100- 150 mM) and a counterion species (glutamate) that is well-known for its ability to stabilize protein-DNA interactions. Two protein-DNA complexes that appeared to be specific for an HSE-bearing probe were detected using C elegans nuclear extracts and the KHG1u-based EMSA system. These complexes were believed to have been formed by two distinct types of putative ceHSBF activity, which were designated as ceHSBF 0and ceHSBF. The Rf(-.0. 1-0.2) of the protein-HSE complex formed by the putative ceHSBF 0activity agrees with the known Rf of the complex formed by most of the homotrimeric heat-shock factors from other species. The Rf (—0.4-0.5) of the protein-HSE complex formed by the putative ceHSBFm activity suggests that this protein is either a discrete proteolytic degradation product of ceHSBF 0,or t…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/19516
note: Some new approaches to conventional methodsfor the study ofprotein-DNA interactions.byRobert James BoissyB.Sc., The University of British Columbia, 1984.A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment of Biochemistry and Molecular Biology.We accept this thesis as conformingto the required standard:THE UNIVERSITY OF BRITISH COLUMBIANovember, 1994.© Robert James Boissy, 1994.In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives, it is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________Department of 8iot/srA.y ,4Ahf L1Lji9- ‘oYThe University of British ColumbiaVancouver, CanadaDate ‘4/di’DE-6 (2/88)11ABSTRACTTwo important experimental techniques used to study eukaryotic, sequence-specific DNA-bindingproteins are electrophoretic mobility-shift assays (EMSAs) and sequence-specific DNA-affinitychromatography. This thesis describes some new approaches to these frequently used methods, which were usedto study a DNA-binding protein (ceHSBF) with an apparent specificity for a conserved DNA sequence element(the heat-shock element or HSE) that mediates the transcriptional regulation of heat-shock-inducible genes inthe soil nematode Caenorlzabditis elegans.Although C. elegans has attracted extensive research interest, a lack of appropriate and reliableexperimental methods has severely limited biochemical studies of sequence-specific DNA-binding proteins fromthis species. As a first step towards addressing this problem, I developed a simple and effective procedure fo…
has type: Thesis/Dissertation
is shown at: 10.14288/1.0093493
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/19516