{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/20905"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/20905","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Plasmid studies in Clostridium perfringens: Plasmid isolation; bacterial conjugation; interstrain mobilization of nonconjugative plasmids","abstract":"Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:47:06Z Item is restricted indefinitely.","abstract_html":"Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:47:06Z Item is restricted indefinitely.","abstract_has_math":false,"creators":["Hampson, Brian Clark"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Food Science and Human Nutrition","degree_department":null,"school":null,"contributors":["Witter, Lloyd D."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T12:52:41Z","date_published":"2011-05-07T12:52:41Z","updated_at":"2026-07-22T22:25:16Z","subjects":["Agriculture, Food Science and Technology","Biology, Genetics","Biology, Microbiology"],"languages":["eng"],"rights":["Copyright 1989 Hampson, Brian Clark"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI8916258","(UMI)AAI8916258"],"render_values":[{"text":"AAI8916258","href":null,"code":true},{"text":"(UMI)AAI8916258","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/20905","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Witter, Lloyd D."]},{"key":"dc:creator","label":"Author","values":["Hampson, Brian Clark"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T12:52:41Z","10000-01-01","1989"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Food Science and Human Nutrition"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Agriculture, Food Science and Technology","Biology, Genetics","Biology, Microbiology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1989 Hampson, Brian Clark"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI8916258","(UMI)AAI8916258","http://hdl.handle.net/2142/20905"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:47:06Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:21:14-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","A procedure for the isolation of both high and low molecular weight DNA from Clostridium perfringens was developed. Cells were washed, subjected to an extended lysozyme treatment and lysed under alkaline conditions (pH 12.4) with a 4% SDS solution. Following neutralization, salt precipitation and protein extractions with organic solvents, the DNA was ammonium acetate/ethanol precipitated up to three times. The procedure may be scaled up or used for mini-prep isolations.","Another procedure, called the rapid boiling method, was also developed. It is a quick, alternative method for plasmid isolation. Cell lysis is achieved by boiling the lysozyme-treated cell suspension for 2 minutes in a 2% SDS solution followed by ultracentrifugation (45,000 rpm for 30 minutes at 4$\\sp\\circ$C) of the lysate to remove cellular debris. Protein extractions using organic solvents, ammonium acetate and ethanol precipitations complete the procedure.","A method for improving the frequency of conjugation between strains of C. perfringens is described. The interstrain plasmid conjugation frequency following filter-mating between a naladixic acid and spectinimycin-resistant C. perfringens 3626B donor strain containing plasmids pIP406 (encoding tetracycline resistance) and pHB101 (caseinase activity) and a rifampicin and streptomycin-resistant, plasmid-free 3624A recipient was increased seven orders of magnitude when compared to the frequency obtained using standard agar plate mating. Adjusting the donor to recipient cell ratio to 1:4 resulted in a conjugation frequency of 1.47 $\\times$ 10$\\sp{-2}$ tetracycline resistant 3624A-based transconjugants per viable post-conjugative donor cell. This improved frequency made possible the mobilization of the 3.2 kb caseinase enzyme activity (Lambda toxin) encoding nonconjugative plasmid designated pHB101. Greater than 39% mobilization of the Lambda toxin phenotype was observed. Southern blotting and hybridization using a radio-labelled pHB101 probe indicated that the transconjugants contained pHB101 in its autonomous form as well as co-integrated with other high molecular weight plasmids. The high frequency of mobilization and the variability in size of plasmids in the transconjugants suggested that the mechanism of mobilization is of the Class II type, as proposed by Kilbane and Malamy (1980). Class II plasmid mobilization involves the recA system, concomitant transfer of the mobilizing plasmid (pIP406), and altered plasmids are yielded at high frequencies.","Made available in DSpace on 2011-05-07T12:52:41Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 8916258.pdf: 4418530 bytes, checksum: e8dd341eaaccc2656c1411bb9a47e446 (MD5) Previous issue date: 1989","ETDs are only available to UIUC Users without author permission","U of I Only"]},{"key":"dc:title","label":"Title","values":["Plasmid studies in Clostridium perfringens: Plasmid isolation; bacterial conjugation; interstrain mobilization of nonconjugative plasmids"]}]}],"canonical_facts":{"dc:contributor":["Witter, Lloyd D."],"dc:creator":["Hampson, Brian Clark"],"dc:date":["2011-05-07T12:52:41Z","10000-01-01","1989"],"dc:description":["Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:47:06Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:21:14-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","A procedure for the isolation of both high and low molecular weight DNA from Clostridium perfringens was developed. Cells were washed, subjected to an extended lysozyme treatment and lysed under alkaline conditions (pH 12.4) with a 4% SDS solution. Following neutralization, salt precipitation and protein extractions with organic solvents, the DNA was ammonium acetate/ethanol precipitated up to three times. The procedure may be scaled up or used for mini-prep isolations.","Another procedure, called the rapid boiling method, was also developed. It is a quick, alternative method for plasmid isolation. Cell lysis is achieved by boiling the lysozyme-treated cell suspension for 2 minutes in a 2% SDS solution followed by ultracentrifugation (45,000 rpm for 30 minutes at 4$\\sp\\circ$C) of the lysate to remove cellular debris. Protein extractions using organic solvents, ammonium acetate and ethanol precipitations complete the procedure.","A method for improving the frequency of conjugation between strains of C. perfringens is described. The interstrain plasmid conjugation frequency following filter-mating between a naladixic acid and spectinimycin-resistant C. perfringens 3626B donor strain containing plasmids pIP406 (encoding tetracycline resistance) and pHB101 (caseinase activity) and a rifampicin and streptomycin-resistant, plasmid-free 3624A recipient was increased seven orders of magnitude when compared to the frequency obtained using standard agar plate mating. Adjusting the donor to recipient cell ratio to 1:4 resulted in a conjugation frequency of 1.47 $\\times$ 10$\\sp{-2}$ tetracycline resistant 3624A-based transconjugants per viable post-conjugative donor cell. This improved frequency made possible the mobilization of the 3.2 kb caseinase enzyme activity (Lambda toxin) encoding nonconjugative plasmid designated pHB101. Greater than 39% mobilization of the Lambda toxin phenotype was observed. Southern blotting and hybridization using a radio-labelled pHB101 probe indicated that the transconjugants contained pHB101 in its autonomous form as well as co-integrated with other high molecular weight plasmids. The high frequency of mobilization and the variability in size of plasmids in the transconjugants suggested that the mechanism of mobilization is of the Class II type, as proposed by Kilbane and Malamy (1980). Class II plasmid mobilization involves the recA system, concomitant transfer of the mobilizing plasmid (pIP406), and altered plasmids are yielded at high frequencies.","Made available in DSpace on 2011-05-07T12:52:41Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 8916258.pdf: 4418530 bytes, checksum: e8dd341eaaccc2656c1411bb9a47e446 (MD5) Previous issue date: 1989","ETDs are only available to UIUC Users without author permission","U of I Only"],"dc:identifier":["AAI8916258","(UMI)AAI8916258","http://hdl.handle.net/2142/20905"],"dc:language":["eng"],"dc:rights":["Copyright 1989 Hampson, Brian Clark"],"dc:subject":["Agriculture, Food Science and Technology","Biology, Genetics","Biology, Microbiology"],"dc:title":["Plasmid studies in Clostridium perfringens: Plasmid isolation; bacterial conjugation; interstrain mobilization of nonconjugative plasmids"],"dc:type":["text"],"thesis:degree_discipline":["Food Science and Human Nutrition"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:16Z"}