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Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis
Bibliography: pages 161-175.
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2016
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| _version_ | 1867613919605948416 |
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| access_status_str | Open Access |
| author | Patterton, Danielle |
| author2 | Hapgood, Janet P |
| author_browse | Hapgood, Janet P Patterton, Danielle |
| author_facet | Hapgood, Janet P Patterton, Danielle |
| author_sort | Patterton, Danielle |
| collection | Thesis |
| description | Bibliography: pages 161-175. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/21982 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:43:48.091Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2016 |
| publishDateRange | 2016 |
| publishDateSort | 2016 |
| 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:11427/21982 Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis Patterton, Danielle Hapgood, Janet P Biochemistry Bibliography: pages 161-175. A poly(dG).poly(dC)-binding protein (suGF1) had previously been identified in sea urchin (Parenchinus angulosis) embryonic nuclear extracts (J.P. Hapgood, personal communication). suGF1 may be involved in the regulation of early histone gene expression by interaction with a nudeosome which has been shown to be positioned in vitro over the H1-H4 intergenic region of the early histone gene battery of Psammechinus miliaris (189, H.-G. Patterton & J.P. Hapgood, unpublished). In this investigation suGF1 was purified to near homogeneity by DNA-affinity chromatography. The purification procedure involved a cation exchange step, followed by poly(dG).poly(dC)-affinity chromatography, and finally by affinity chromatography utilizing multimerized specific DNA-binding sites of suGF1. The 59,5 kDa purified protein was identified as suGFl by renaturation of sequence-specific DNA-binding activity from a SDSpAGE gel slice, by Southwestern blotting and by DNase I footprinting. Ultraviolet crosslinking of the nuclear extract and purified suGF1 revealed the presence of the same specific bands on SDS-P AGE. Optimal suGF1 DNA-binding was shown to occur at relatively high ionic strength (175 mM). suGF1 DNA-binding was sensitive to EDTA, implying a requirement for a divalent cation for DNA-binding. The suGF1 DNA-binding interaction was investigated by methylation interference, and DNase I and hydroxyl radical footprinting. The footprinting data was analyzed in difference probability plots, from which a model for the suGFl-DNA complex was inferred. In the model suGFl approaches the DNA helix mainly from one side, and interacts with guanine residues in the major groove. Contacts are made to one of the DNA sugar phosphate backbones abutting this major groove. The data is consistent with the DNA in the complex being curved, and/ or exhibiting a sharp bend at the site of suGFl contact in the major groove. suGFl does not seem to bind to DNA as a rotationally symmetrical dimer. The results of this investigation are discussed in terms of the literature. suGFl may be related to the chicken erythrocyte-specific factor BGPl, which has been shown to bind to 16 contiguous guanines in the βᴬ-globin promoter, both in the naked DNA molecule and wrapped around a histone octamer (37, 139). 2016-09-28T19:05:24Z 2016-09-28T19:05:24Z 1992 Master Thesis Masters MSc http://hdl.handle.net/11427/21982 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Biochemistry Patterton, Danielle Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis |
| thesis_degree_str | Master's |
| title | Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis |
| title_full | Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis |
| title_fullStr | Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis |
| title_full_unstemmed | Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis |
| title_short | Purification and characterization of a poly(dG).poly(dC)-binding protein from Parenchinus angulosis |
| title_sort | purification and characterization of a poly dg poly dc binding protein from parenchinus angulosis |
| topic | Biochemistry |
| url | http://hdl.handle.net/11427/21982 |
| work_keys_str_mv | AT pattertondanielle purificationandcharacterizationofapolydgpolydcbindingproteinfromparenchinusangulosis |