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dc.contributor.authorHeller, Phillip Ferdinand
dc.date.accessioned2012-04-23T16:29:24Z
dc.date.available2012-04-23T16:29:24Z
dc.date.issued1996
dc.identifier.urihttp://hdl.handle.net/10713/1504
dc.descriptionUniversity of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 1996en_US
dc.description.abstractStreptonigrin is a antineoplastic antibiotic whose mode of action is dependent on formation of the hydroxyl radical. A streptonigrin resistance gene was cloned from a partial Sau3A digest of E. coli DNA. One to 10 Kilobase (Kb) fragments were ligated into the vector pUC18 and transformed into E. coli (DH5{dollar}\alpha{dollar}). Once prepared, the library was replica plated and then screened with differing streptonigrin concentrations in a top agar overlay. Clones exhibiting growth into the overlay at the highest STN concentrations (pH191, pH192) were rescued from the overlay and grown for plasmid isolation and for determination of theminimum inhibitory concentration (MIC) for streptonigrin in LB broth, 3 and 1 {dollar}\mu{dollar}g/ml respectively. The isolated plasmids were mapped by restriction endonuclease digestion followed by agarose gel electrophoresis. Restriction sites were assigned based on known location in the multicloning site of the vector, leading to the production of an approximate physical map of the insert in the plasmid. The resistance element (StnR) was localized by deletion studies on pH191. Sequencing and analysis of the deoxynucleotide sequence of pH191 led to the identification of an open reading frame (StnR) with a high homology (97.6%) to the first 185 residues of riboflavin synthase (RibC). The resistance gene was expressed in S. lividans on pIJ702 where it yielded streptonigrin resistance in excess of that found in the wild type producing species, S. flocculus (40 {dollar}\mu{dollar}g/ml). Expression of StnR and RibC in E. coli via the vector pET3C resulted in strains with elevated streptonigrin resistance as compared to the pET3C control in both solid and liquid media. A gene imparting resistance to the hydroxyl radical producing drug streptonigrin has been identified as the riboflavin synthase from E. coli.en_US
dc.language.isoen_USen_US
dc.subjectBiology, Molecularen_US
dc.subjectBiology, Microbiologyen_US
dc.subjectHealth Sciences, Pharmacologyen_US
dc.subjectstreptonigrin resistanceen_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshDrug Resistance, Neoplasm--geneticsen_US
dc.subject.meshStreptonigrinen_US
dc.subject.meshEscherichia colien_US
dc.titleThe cloning and identification of a streptonigrin resistance gene from Escherichia colien_US
dc.typedissertationen_US
dc.contributor.advisorSpeedie, Marilyn K.
dc.identifier.ispublishedYes
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