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Investigations into the use of maize streak virus as a gene vector
Bibliography: pages 190-214.
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2016
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| _version_ | 1867613164275761152 |
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| access_status_str | Open Access |
| author | Palmer, Kenneth Edward |
| author2 | Rybicki, Edward P |
| author_browse | Palmer, Kenneth Edward Rybicki, Edward P |
| author_facet | Rybicki, Edward P Palmer, Kenneth Edward |
| author_sort | Palmer, Kenneth Edward |
| collection | Thesis |
| description | Bibliography: pages 190-214. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/22554 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:31:47.142Z |
| 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:11427/22554 Investigations into the use of maize streak virus as a gene vector Palmer, Kenneth Edward Rybicki, Edward P Microbiology Bibliography: pages 190-214. This thesis describes investigations into the potential use of the Subgroup I geminivirus, maize streak virus (MSV), as a gene vector. These involved testing MSV-based replicons in transgenic cell lines, in transient expression assays in maize cells and in an infectious gene expression system in plants. MSV vectors which contained three different versions of a bar (bialaphos resistance) expression cassette in place of the viral movement and coat protein genes were used to generate transformed maize cell lines. A high proportion of these contained MSV-based episomes at high copy number. However, embryogenic maize tissue of the Hill line was not regenerable when an MSV-based replicon was present, possibly due to toxicity of the viral replication associated protein. In non-regenerable Black Mexican sweetcorn cell lines some of the MSV-bar episomes, which ranged in size from 3.15 kb to 4.78 kb, replicated for periods of over two years, and appeared structurally stable. The cellular levels of the bar gene product, phosphinothricin acetyl transferase (PAT), were significantly enhanced in lines where the gene was amplified by linkage to an MSV replicon in comparison with lines where the same gene was not amplified. Northern blot analysis also showed that higher levels of bar mRNA were produced in lines where the gene was amplified. However, the 3- to 5-fold enhancement in gene expression was less than was anticipated, based on results from similar Subgroup ill geminivirus-based transgene amplification systems. Several mutants of the MSV genome were generated to investigate the extent to which genome amplification contributes to the expression of the viral coat protein gene. The introduction of an Ncol restriction site at the start of the coat protein gene facilitated fusion of the gus marker gene with the coat protein upstream transcription and translation regulatory sequences. In one viral construct the plus strand origin of replication was inactivated by insertion of a short oligonucleotide; in another, the viral rep gene was inactivated by a frameshift mutation. These constructs were used to show that the MSV coat protein promoter has low, but measurable constitutive activity in the absence of genome amplification, but that viral replication enhances coat protein expression about 45-fold. I found no evidence for Rep-mediated transactivation of the coat protein promoter. 2016-11-16T13:22:58Z 2016-11-16T13:22:58Z 1997 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/22554 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Microbiology Palmer, Kenneth Edward Investigations into the use of maize streak virus as a gene vector |
| thesis_degree_str | Doctoral |
| title | Investigations into the use of maize streak virus as a gene vector |
| title_full | Investigations into the use of maize streak virus as a gene vector |
| title_fullStr | Investigations into the use of maize streak virus as a gene vector |
| title_full_unstemmed | Investigations into the use of maize streak virus as a gene vector |
| title_short | Investigations into the use of maize streak virus as a gene vector |
| title_sort | investigations into the use of maize streak virus as a gene vector |
| topic | Microbiology |
| url | http://hdl.handle.net/11427/22554 |
| work_keys_str_mv | AT palmerkennethedward investigationsintotheuseofmaizestreakvirusasagenevector |