Investigations into the role of histone H2A ubiquitination in chromatin

 

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dc.contributor.advisor Lindsey, George G en_ZA
dc.contributor.advisor Brandt, Wolf F en_ZA
dc.contributor.advisor Patterton, Hugh en_ZA
dc.contributor.author Jason, Laure Jeanine Monique en_ZA
dc.date.accessioned 2014-11-21T16:09:50Z
dc.date.available 2014-11-21T16:09:50Z
dc.date.issued 1999 en_ZA
dc.identifier.citation Jason, L. 1999. Investigations into the role of histone H2A ubiquitination in chromatin. University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/9765
dc.description Bibliography: leaves 141-150. en_ZA
dc.description.abstract An in vitro system was used to determine the effect of histone H2A ubiquitination on linker histone binding to mononucleosomes. Hybrid octamers containing either H2A or ubiquitinated H2A (uH2A) were reconstituted onto random sequence 167 bp DNA. The affinity of the resultant nucleosome cores for linker histone H1 was determined from nucleoprotein gel shifts, protein analyses and thermal denaturation. Ubiquitinated H2A did not inhibit linker histone binding to nucleosome cores. The effect of uH2A on nucleosome and chromatosoine positioning on a 208 bp Lytechinus variegatus 5S rDNA fragment was investigated using a combination of micrococcal nuclease digestion and subsequent restriction enzyme digestion of the core particle or chromatosome DNA. Nucleosomes and chromatosomes containing uH2A were found to occupy the same positions on the template DNA as those containing H2A. Chromatin folding of nucleosomal arrays containing either H2A or uH2A was analysed using a quantitative agarose gel electrophoresis system developed by Hansen and co-workers. The extent of folding of nucleosomal arrays containing uH2A was comparable to that of control nucleosomal arrays. A differential centrifugation assay was used to monitor the extent of divalent cation induced oligomerisation of reconstituted nucleosomal arrays. Nucleosomal arrays containing uH2A were found to oligomerise at a lower magnesium concentration than control arrays. As a first step towards studying the effects of H2A ubiquitination in linker histone-bound nucleosomal arrays, a novel method for linker histone reconstitution onto long chromatin stripped of linker histones was developed. The fidelity of linker histone reconstitution was assayed by micrococcal nuclease digestion, thermal denaturation and determination of the orientation of neighbouring linker histone molecules in extended chromatin. In a separate study, the relationship between the observed repeat length of chromatin and the rate of micrococcal nuclease digestion was investigated. The repeat length of the same starting chromatin preparation at equivalent extents of digestion was found to vary according to the rate of digestion. en_ZA
dc.language.iso eng en_ZA
dc.subject.other Biochemistry en_ZA
dc.title Investigations into the role of histone H2A ubiquitination in chromatin en_ZA
dc.type Thesis / Dissertation en_ZA
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Science en_ZA
dc.publisher.department Department of Molecular and Cell Biology en_ZA
dc.type.qualificationlevel Doctoral en_ZA
dc.type.qualificationname PhD en_ZA
uct.type.filetype Text
uct.type.filetype Image


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