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An investigation of the replication of the broad-host-range plasmid pTF-FC2
Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate auto...
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2016
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| _version_ | 1867613158030442496 |
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| access_status_str | Open Access |
| author | Dorrington, Rosemary_Ann |
| author2 | Rawlings , Douglas E |
| author_browse | Dorrington, Rosemary_Ann Rawlings , Douglas E |
| author_facet | Rawlings , Douglas E Dorrington, Rosemary_Ann |
| author_sort | Dorrington, Rosemary_Ann |
| collection | Thesis |
| description | Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/22048 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:31:41.113Z |
| 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:11427/22048 An investigation of the replication of the broad-host-range plasmid pTF-FC2 Dorrington, Rosemary_Ann Rawlings , Douglas E Microbiology Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted. 2016-10-03T04:07:00Z 2016-10-03T04:07:00Z 1990 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/22048 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Microbiology Dorrington, Rosemary_Ann An investigation of the replication of the broad-host-range plasmid pTF-FC2 |
| thesis_degree_str | Doctoral |
| title | An investigation of the replication of the broad-host-range plasmid pTF-FC2 |
| title_full | An investigation of the replication of the broad-host-range plasmid pTF-FC2 |
| title_fullStr | An investigation of the replication of the broad-host-range plasmid pTF-FC2 |
| title_full_unstemmed | An investigation of the replication of the broad-host-range plasmid pTF-FC2 |
| title_short | An investigation of the replication of the broad-host-range plasmid pTF-FC2 |
| title_sort | investigation of the replication of the broad host range plasmid ptf fc2 |
| topic | Microbiology |
| url | http://hdl.handle.net/11427/22048 |
| work_keys_str_mv | AT dorringtonrosemaryann aninvestigationofthereplicationofthebroadhostrangeplasmidptffc2 AT dorringtonrosemaryann investigationofthereplicationofthebroadhostrangeplasmidptffc2 |