Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris

dc.contributor.advisorVon Holt, Clausen_ZA
dc.contributor.authorRetief, Jacques D.en_ZA
dc.date.accessioned2014-09-22T07:53:46Z
dc.date.available2014-09-22T07:53:46Z
dc.date.issued1986en_ZA
dc.descriptionBibliography: leaves 194-217.en_ZA
dc.description.abstractThis thesis investigates whether DNA and histones contain sufficient information to direct nucleosome cores into specific positions. The "in vitro" assembly of nucleosome cores promoted by poly(glutamic acid) has been optimized with respect to rate and yield. This was achieved by paying attention to the purity of the core constituents and in particular by the use of histones in their octameric form. The suitability of a number of octamer purification protocols, to produce pure undenatured histone octamers, has been investigated and the methodology improved. The particles assembled on random DNA have been found to be indistinguishable from native nucleosome cores by the following criteria: Their S value on sucrose gradient centrifugation, resistance to Micrococcal nuclease digestion, DNase I digestion patterns, DNase I digestion kinetics at the susceptible sites, electronmicroscopic appearance, hi stone content and electrophoretic mobility. Cores were also assembled on unique DNA, namely the intact h22 histone quintet of Psammechinus miliaris. Low resolution mapping, by indirect endlabelling of polycores assembled on the quintet, did not reveal any preferred sites of assembly. To investigate the core associated DNA at single base pair resolution, a series of fragments, excised from the H2A-Hl and the Hl-H4 spacer areas, were inserted into pGV403 plasmids. These plasmids can be strand specifically end-labelled with the Klenow fragment at the two different Tth 111 I excision sites utilised to isolate the propagated insert. On the free linearised DNA a complex digestion pattern is produced due to the sequence specificities of Micrococcal nuclease and DNase I. When cores are assembled on this DNA the digestion pattern is changed. This pattern reveals two preferential frames of assembly and indicates that in the remainder of the fragments cores are assembled, randomly, or in a number of overlapping frames. It is concluded that the DNA fragments investigated and the hi stone octamer contain enough structural information to influence the positions occupied by some nucleosome cores. The implications of these findings are discussed.en_ZA
dc.identifier.apacitationRetief, Jacques D. (1986). <i>Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/7596en_ZA
dc.identifier.chicagocitationRetief, Jacques D.. <i>"Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1986. http://hdl.handle.net/11427/7596en_ZA
dc.identifier.citationRetief, Jacques D. 1986. Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris. Thesis. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. http://hdl.handle.net/11427/7596en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Retief, Jacques D. AB - This thesis investigates whether DNA and histones contain sufficient information to direct nucleosome cores into specific positions. The "in vitro" assembly of nucleosome cores promoted by poly(glutamic acid) has been optimized with respect to rate and yield. This was achieved by paying attention to the purity of the core constituents and in particular by the use of histones in their octameric form. The suitability of a number of octamer purification protocols, to produce pure undenatured histone octamers, has been investigated and the methodology improved. The particles assembled on random DNA have been found to be indistinguishable from native nucleosome cores by the following criteria: Their S value on sucrose gradient centrifugation, resistance to Micrococcal nuclease digestion, DNase I digestion patterns, DNase I digestion kinetics at the susceptible sites, electronmicroscopic appearance, hi stone content and electrophoretic mobility. Cores were also assembled on unique DNA, namely the intact h22 histone quintet of Psammechinus miliaris. Low resolution mapping, by indirect endlabelling of polycores assembled on the quintet, did not reveal any preferred sites of assembly. To investigate the core associated DNA at single base pair resolution, a series of fragments, excised from the H2A-Hl and the Hl-H4 spacer areas, were inserted into pGV403 plasmids. These plasmids can be strand specifically end-labelled with the Klenow fragment at the two different Tth 111 I excision sites utilised to isolate the propagated insert. On the free linearised DNA a complex digestion pattern is produced due to the sequence specificities of Micrococcal nuclease and DNase I. When cores are assembled on this DNA the digestion pattern is changed. This pattern reveals two preferential frames of assembly and indicates that in the remainder of the fragments cores are assembled, randomly, or in a number of overlapping frames. It is concluded that the DNA fragments investigated and the hi stone octamer contain enough structural information to influence the positions occupied by some nucleosome cores. The implications of these findings are discussed. DA - 1986 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1986 T1 - Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris TI - Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris UR - http://hdl.handle.net/11427/7596 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/7596
dc.identifier.vancouvercitationRetief Jacques D. Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 1986 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/7596en_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.titlePoly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliarisen_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
Files
Original bundle
Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
thesis_sci_1986_retief_jd (1).pdf
Size:
4.74 MB
Format:
Adobe Portable Document Format
Description:
Collections