Quantitative determination of effective nibbling activities contaminating restriction endonuclease preparations.
A simple and sensitive procedure with which to detect residual exonucleolytic nibbling activities contaminating restriction endonuclease preparations is described. The procedure uses the kyosei-plasmid, pKF4, which confers kanamycin resistance and enforces streptomycin sensitivity encoded by the trp promoter/operator-driven rpsL+amber(PO(trp)-rpsL+4(am)) gene onto Escherichia coli streptomycin-resistant, amber-suppressive, trp repressor-negative strains such as TH5. When TH5 cells transformed by pKF4 were selected on agar medium containing kanamycin plus streptomycin, the efficiency of transformation plating was substantially lower than that on agar containing kanamycin alone. However, when pKF4 DNA was digested by restriction enzymes that cut once per molecule within PO(trp)-rpsL+4(am) and relegated, the plating efficiency increased depending on the degree of contamination of exonucleolytic nibbling activities in the enzyme preparations, due to deletion mutation at the ligand junction. Plating efficiency was converted to "effective nibbling activity" corresponding to Bal31 nuclease-equivalent units. Using this procedure, effective nibbling activities were detected in 17 of 34 commercial samples of restriction enzymes tested. The method is simple and more sensitive than the procedures used by the commercial suppliers and it is applicable to the quality control testing of more than 100 restriction enzymes.
Hashimoto-Gotoh
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Department of Biochemistry and Molecular Genetics, Kyoto Prefectural University of Medicine, Japan.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D000091243:Ribosomal Protein S9
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D004262:DNA Restriction Enzymes