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A molecular biological study on Campylobacter pylori
C. pylori have been shown to be associated with gastritis and peptic ulceration, but the mechanism of their pathogenicity is unknown. Since a number of virulence factors are known to be plasmid mediated, it was decided to study the plasmids of C. pylori. A variety of techniques were used to establis...
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| Format: | Thesis |
| Language: | English |
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Division of Medical Microbiology
2017
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| _version_ | 1867614519632592896 |
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| access_status_str | Open Access |
| author | Penfold, Sonya |
| author2 | Lastovica, A J |
| author_browse | Lastovica, A J Penfold, Sonya |
| author_facet | Lastovica, A J Penfold, Sonya |
| author_sort | Penfold, Sonya |
| collection | Thesis |
| description | C. pylori have been shown to be associated with gastritis and peptic ulceration, but the mechanism of their pathogenicity is unknown. Since a number of virulence factors are known to be plasmid mediated, it was decided to study the plasmids of C. pylori. A variety of techniques were used to establish the best method of plasmid extraction from C. pylori. The method of alkaline lysis as described by Birnboim and Doly was shown to give the most consistent results and the greatest plasmid yield. Plasmid DNA was found in 54% (26 out of 48) of the isolates examined and the plasmids varied in size from 3,4kb to greater than 137kb. The majority (21 out of 26) of isolates had unique plasmid profiles, but 5 isolates showed common ones. Three of these 5 isolates were studied in more detail. The evidence presented here suggests that all 3 plasmid bands visible in these three isolates were different conformations of the same plasmid which has a molecular weight of 6, 2 kilo bases. The plasmids appeared labile and covalently closed circular DNA was rarely isolated. Restriction enzyme digestion was done with a variety of enzymes, but only 3 of the enzymes used digested the DNA. EcoRI and HindIII partially digested the DNA, while Sau3A digested the plasmids completely, generating 2 fragments of 2,2kb and 2,4kb, and a number of smaller fragments. The DNA was shown to be methylated and the fragments generated by Sau3A digestion suggest that the plasmids may contain a repetitive element. Chromosomal DNA was also isolated and digested with a variety of enzymes. The chromosomal DNA restriction pattern was shown to be affected by methylation, which may be important when using restriction enzyme patterns to differentiate between strains. Plasmid restriction fragments were end-labelled to detect bands which were poorly visible by ethidium bromide staining. This technique was shown to be more sensitive than ethidium bromide staining of DNA, but the inability to obtain complete digestion of C. pylori DNA made it impossible to construct a restriction enzyme map of the plasmids. Hybridization experiments showed the plasmids of C. pylori to be related and was also used to detect bands which were not easily visible after ethidium bromide staining. Attempts were made to clone C. pylori DNA into pUC18 and pUC19, but no recombinant plasmids containing C. pylori DNA were obtained. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/25731 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:53:20.321Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Division of Medical Microbiology |
| publisherStr | Division of Medical Microbiology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/25731 A molecular biological study on Campylobacter pylori Penfold, Sonya Lastovica, A J Steyn, Lafras M Campylobacter pylori infections Stomach - Diseases - Microbiology Campylobacter Campylobacter Infections - microbiology Medical Microbiology C. pylori have been shown to be associated with gastritis and peptic ulceration, but the mechanism of their pathogenicity is unknown. Since a number of virulence factors are known to be plasmid mediated, it was decided to study the plasmids of C. pylori. A variety of techniques were used to establish the best method of plasmid extraction from C. pylori. The method of alkaline lysis as described by Birnboim and Doly was shown to give the most consistent results and the greatest plasmid yield. Plasmid DNA was found in 54% (26 out of 48) of the isolates examined and the plasmids varied in size from 3,4kb to greater than 137kb. The majority (21 out of 26) of isolates had unique plasmid profiles, but 5 isolates showed common ones. Three of these 5 isolates were studied in more detail. The evidence presented here suggests that all 3 plasmid bands visible in these three isolates were different conformations of the same plasmid which has a molecular weight of 6, 2 kilo bases. The plasmids appeared labile and covalently closed circular DNA was rarely isolated. Restriction enzyme digestion was done with a variety of enzymes, but only 3 of the enzymes used digested the DNA. EcoRI and HindIII partially digested the DNA, while Sau3A digested the plasmids completely, generating 2 fragments of 2,2kb and 2,4kb, and a number of smaller fragments. The DNA was shown to be methylated and the fragments generated by Sau3A digestion suggest that the plasmids may contain a repetitive element. Chromosomal DNA was also isolated and digested with a variety of enzymes. The chromosomal DNA restriction pattern was shown to be affected by methylation, which may be important when using restriction enzyme patterns to differentiate between strains. Plasmid restriction fragments were end-labelled to detect bands which were poorly visible by ethidium bromide staining. This technique was shown to be more sensitive than ethidium bromide staining of DNA, but the inability to obtain complete digestion of C. pylori DNA made it impossible to construct a restriction enzyme map of the plasmids. Hybridization experiments showed the plasmids of C. pylori to be related and was also used to detect bands which were not easily visible after ethidium bromide staining. Attempts were made to clone C. pylori DNA into pUC18 and pUC19, but no recombinant plasmids containing C. pylori DNA were obtained. 2017-10-23T08:13:56Z 2017-10-23T08:13:56Z 1989 2017-07-20T13:57:21Z Master Thesis Masters MSc (Med) http://hdl.handle.net/11427/25731 eng application/pdf Division of Medical Microbiology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Campylobacter pylori infections Stomach - Diseases - Microbiology Campylobacter Campylobacter Infections - microbiology Medical Microbiology Penfold, Sonya A molecular biological study on Campylobacter pylori |
| thesis_degree_str | Master's |
| title | A molecular biological study on Campylobacter pylori |
| title_full | A molecular biological study on Campylobacter pylori |
| title_fullStr | A molecular biological study on Campylobacter pylori |
| title_full_unstemmed | A molecular biological study on Campylobacter pylori |
| title_short | A molecular biological study on Campylobacter pylori |
| title_sort | molecular biological study on campylobacter pylori |
| topic | Campylobacter pylori infections Stomach - Diseases - Microbiology Campylobacter Campylobacter Infections - microbiology Medical Microbiology |
| url | http://hdl.handle.net/11427/25731 |
| work_keys_str_mv | AT penfoldsonya amolecularbiologicalstudyoncampylobacterpylori AT penfoldsonya molecularbiologicalstudyoncampylobacterpylori |