The cloning and overproduction of Escherichia coli uracil-DNA glycosylase.
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, lambdanadB transducing phages were isolated from two E. coli DNA libraries by selection of nicotinate-independent lysogens. lambdanadB 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 lambdaung 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 lambdaPL 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 lambdaung gene in the plasmid pKC30 to its orientation in lambdaung-nadB transducing phages, the transcription direction of the ung gene on the E. coli linkage map was found to be clockwise.
D010582:Bacteriophage lambda / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics; D004586:Electrophoresis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D009699:N-Glycosyl Hydrolases / Q000235:genetics*; D010957:Plasmids; D051981:Uracil-DNA Glycosidase
D004269:DNA, Bacterial; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase