The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I.
The synthesis of the human topoisomerase I (Topo I) in Escherichia coli (Ec) results in phenotypes consistent with the appearance of a DNA-relaxing activity. High-level expression was problematic and recloning the gene in a reduced-copy-number plasmid under more stringently regulated control produced a stable plasmid. An Ec strain with an inducible sensitivity to the eukaryotic Topo I poison, camptothecin (CPT), was constructed by introducing a mutation (imp4312) known to enhance the permeability of Ec to a wide variety of compounds. We are able to infer that CPT sensitivity in Ec involves DNA damage by noting elevated sensitivity in a repair-deficient recA strain and by observing the induction of a sulA::lac fusion following exposure to the drug.
Taylor
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ST
Department of Microbial Molecular Biology, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
Menzel
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R
D002166:Camptothecin / Q000494:pharmacology*; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin