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The cloning and overproduction of Escherichia coli uracil-DNA glycosylase
Authors:Bruce K. Duncan  Judith A. Chambers
Affiliation:Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111 U.S.A. Tel. (215) 728-2413
Abstract:Escherichia coli cells containing elevated levels of the DNA repair enzyme uracil-DNA glycosylase (the ung gene product) have been constructed by in vitro recombination methods. First, λnadB transducing phages were isolated from two E. coli DNA libraries by selection of nicotinate-independent lysogens. λnadB phage from one of the libraries were also ung+ and carried the ung-nadB genes on an 8.3 kb HindIII restriction fragment. The ung and nadB genes were subcloned into plasmids and a restriction map of the ung region of the E. coli chromosome was constructed. The uracil glycosylase gene was localized to a 1.4-kb restriction fragment by subcloning the gene into pBR322. Uracil glycosylase was overproduced (relative to the specific activity of wild type cells) by about two-fold in λung lysogens and by 15- to 20-fold in cells containing pBR322ung derivatives. When the ung gene and its promoter were placed downstream from the bacteriophage λpL promoter in the plasmid pKC30, uracil glycosylase production was heat-induced to more than 100-fold above the levels of a wild-type cell. By relating the insertion orientation of the λung gene in the plasmid pKC30 to its orientation in λung-nadB transducing phages, the transcription direction of the ung gene on the E. coli linkage map was found to be clockwise.
Keywords:Recombinant DNA  repair  λ transducing phages  plasmid vectors  clone library  ampicillin-resistant  AP  apurinic/apyrimidinic  dUTPase  deoxyuridine triphosphate nucleotidohydrolase  kanamycin-resistant  kb  kilobase pairs  tetracycline-resistant  Uracil-DNA  uracil-containing DNA  Δ  deletion  []  indicates plasmid-carrier state  ()  indicates prophage  ::xyz  indicates an insertion of a fragment “xyz”
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