CLONING OF BACILLUS SUBTILIS DNA: EXPRESSION IN B. SUBTILIS AND ESCHERICHIA COLI.

Persistent Link:
http://hdl.handle.net/10150/183784
Title:
CLONING OF BACILLUS SUBTILIS DNA: EXPRESSION IN B. SUBTILIS AND ESCHERICHIA COLI.
Author:
ZUKOWSKI, MARK MICHAEL.
Issue Date:
1982
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Bacillus subtilis DNA was cloned by ligating restriction endonuclease-generated fragments to plasmid vectors. The plasmid pUB110 was the vehicle in the construction of eight recombinant plasmids, pNM1 through pNM8. Each bears one or more EcoRI fragment(s) of B. subtilis chromosomal DNA. Recovery of the plasmids from host cells demonstrated that recombinant plasmids that bear some homology to the B, subtilis chromosome may be maintained outside of the chromosome in recombination-proficient hosts. The mean size of cloned fragments was 0.78 megadaltons (Mdal). The recombinant plasmid pNM1 interferes with the mechanism that blocks chromosomal recombination in B. subtilis cells that carry the recE4 mutation. Low-level chromosomal recombination at several loci was demonstrated when chromosomal DNA was accompanied by pNM1 in the transformation of recE4 recipient cells. The recombinant plasmid does not appear to code for recE gene products nor does it produce novel proteins when assayed in minicells of B. subtilis. An alternative approach to cloning B. subtilis DNA was successfully accomplished with the vector plasmid pHV33. The vector functions in both B. subtilis and E. coli hosts. B. subtilis chromosomal DNA was digested with Bg1II, then ligated to the unique BamHI site of pHV33. Ligation products were introduced into E. coli by transformation. Plasmid DNAs were isolated from transformants, pooled into several lots, then used to transform auxotrophic B. subtilis recipient cells. The procedure resulted in the construction of two new recombinant plasmids, pNM1055 and pNM1326. B. subtilis cells with the aroD120 mutation restored their ability to synthesize aromatic amino acids when pNM1055 was introduced. The same effect was observed in E. coli recipient cells that had the equivalent mutation. E. coli cells that carried pNM1326 produced granular colonies characteristic of the extraordinary filamentous growth exhibited by individual cells. The pNM1326 plasmid coded for a 16,000 dalton polypeptide produced in abundant quantities in E. coli hosts. A deletion derivative of pNM1326 did not produce the polypeptide, nor was filamentous growth of host cells exhibited. A plasmid-borne fragment of B. subtilis DNA affects cells growth and division of E. coli hosts.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Recombinant DNA.; Molecular cloning.; Bacillus subtilis.; Escherichia coli.; Plasmids.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Genetics; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Mendelson, Neil

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleCLONING OF BACILLUS SUBTILIS DNA: EXPRESSION IN B. SUBTILIS AND ESCHERICHIA COLI.en_US
dc.creatorZUKOWSKI, MARK MICHAEL.en_US
dc.contributor.authorZUKOWSKI, MARK MICHAEL.en_US
dc.date.issued1982en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractBacillus subtilis DNA was cloned by ligating restriction endonuclease-generated fragments to plasmid vectors. The plasmid pUB110 was the vehicle in the construction of eight recombinant plasmids, pNM1 through pNM8. Each bears one or more EcoRI fragment(s) of B. subtilis chromosomal DNA. Recovery of the plasmids from host cells demonstrated that recombinant plasmids that bear some homology to the B, subtilis chromosome may be maintained outside of the chromosome in recombination-proficient hosts. The mean size of cloned fragments was 0.78 megadaltons (Mdal). The recombinant plasmid pNM1 interferes with the mechanism that blocks chromosomal recombination in B. subtilis cells that carry the recE4 mutation. Low-level chromosomal recombination at several loci was demonstrated when chromosomal DNA was accompanied by pNM1 in the transformation of recE4 recipient cells. The recombinant plasmid does not appear to code for recE gene products nor does it produce novel proteins when assayed in minicells of B. subtilis. An alternative approach to cloning B. subtilis DNA was successfully accomplished with the vector plasmid pHV33. The vector functions in both B. subtilis and E. coli hosts. B. subtilis chromosomal DNA was digested with Bg1II, then ligated to the unique BamHI site of pHV33. Ligation products were introduced into E. coli by transformation. Plasmid DNAs were isolated from transformants, pooled into several lots, then used to transform auxotrophic B. subtilis recipient cells. The procedure resulted in the construction of two new recombinant plasmids, pNM1055 and pNM1326. B. subtilis cells with the aroD120 mutation restored their ability to synthesize aromatic amino acids when pNM1055 was introduced. The same effect was observed in E. coli recipient cells that had the equivalent mutation. E. coli cells that carried pNM1326 produced granular colonies characteristic of the extraordinary filamentous growth exhibited by individual cells. The pNM1326 plasmid coded for a 16,000 dalton polypeptide produced in abundant quantities in E. coli hosts. A deletion derivative of pNM1326 did not produce the polypeptide, nor was filamentous growth of host cells exhibited. A plasmid-borne fragment of B. subtilis DNA affects cells growth and division of E. coli hosts.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectRecombinant DNA.en_US
dc.subjectMolecular cloning.en_US
dc.subjectBacillus subtilis.en_US
dc.subjectEscherichia coli.en_US
dc.subjectPlasmids.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGeneticsen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMendelson, Neilen_US
dc.contributor.committeememberBernstein, Harrisen_US
dc.contributor.committeememberMcReynolds, Larryen_US
dc.contributor.committeememberMount, Daviden_US
dc.identifier.proquest8217490en_US
dc.identifier.oclc681978874en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.