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Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris
Bibliography: leaves 194-217.
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2014
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| _version_ | 1867613181514350592 |
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| access_status_str | Open Access |
| author | Retief, Jacques D. |
| author2 | Von Holt, Claus |
| author_browse | Retief, Jacques D. Von Holt, Claus |
| author_facet | Von Holt, Claus Retief, Jacques D. |
| author_sort | Retief, Jacques D. |
| collection | Thesis |
| description | Bibliography: leaves 194-217. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/7596 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:32:03.909Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2014 |
| publishDateRange | 2014 |
| publishDateSort | 2014 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/7596 Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris Retief, Jacques D. Von Holt, Claus Biochemistry Bibliography: leaves 194-217. 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. 2014-09-22T07:53:46Z 2014-09-22T07:53:46Z 1986 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/7596 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Biochemistry Retief, Jacques D. Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| thesis_degree_str | Doctoral |
| title | Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| title_full | Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| title_fullStr | Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| title_full_unstemmed | Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| title_short | Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| title_sort | poly glutamic acid promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris |
| topic | Biochemistry |
| url | http://hdl.handle.net/11427/7596 |
| work_keys_str_mv | AT retiefjacquesd polyglutamicacidpromotedassemblyofnucleosomecoresonthehistonegenequintetofpsammechinusmiliaris |