Investigations into the role of histone H2A ubiquitination in chromatin

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dc.contributor.advisorLindsey, George Gen_ZA
dc.contributor.advisorBrandt, Wolf Fen_ZA
dc.contributor.advisorPatterton, Hughen_ZA
dc.contributor.authorJason, Laure Jeanine Moniqueen_ZA
dc.date.accessioned2014-11-21T16:09:50Z
dc.date.available2014-11-21T16:09:50Z
dc.date.issued1999en_ZA
dc.descriptionBibliography: leaves 141-150.en_ZA
dc.description.abstractAn 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.identifier.apacitationJason, L. J. M. (1999). <i>Investigations into the role of histone H2A ubiquitination in chromatin</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/9765en_ZA
dc.identifier.chicagocitationJason, Laure Jeanine Monique. <i>"Investigations into the role of histone H2A ubiquitination in chromatin."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1999. http://hdl.handle.net/11427/9765en_ZA
dc.identifier.citationJason, L. 1999. Investigations into the role of histone H2A ubiquitination in chromatin. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Jason, Laure Jeanine Monique AB - 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. DA - 1999 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1999 T1 - Investigations into the role of histone H2A ubiquitination in chromatin TI - Investigations into the role of histone H2A ubiquitination in chromatin UR - http://hdl.handle.net/11427/9765 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/9765
dc.identifier.vancouvercitationJason LJM. Investigations into the role of histone H2A ubiquitination in chromatin. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1999 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/9765en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherBiochemistryen_ZA
dc.titleInvestigations into the role of histone H2A ubiquitination in chromatinen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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