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    The cloning and identification of a streptonigrin resistance gene from Escherichia coli

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    Author
    Heller, Phillip Ferdinand
    Advisor
    Speedie, Marilyn K.
    Date
    1996
    Type
    dissertation
    
    Metadata
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    Abstract
    Streptonigrin 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.
    Description
    University of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 1996
    Keyword
    Biology, Molecular
    Biology, Microbiology
    Health Sciences, Pharmacology
    streptonigrin resistance
    Cloning, Molecular
    Drug Resistance, Neoplasm--genetics
    Streptonigrin
    Escherichia coli
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1504
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    Theses and Dissertations All Schools
    Theses and Dissertations School of Pharmacy

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