Detection of exonuclease activities in restriction endonuclease preparations using an enforcement plasmid for kanamycin-resistance selection.
A new enforcement (kyosei-) cloning plasmid vector, designated pKF4, was constructed which confers kanamycin resistance (KmR) and enforces streptomycin sensitivity (SmS). Since it is important to employ restriction endonuclease (ENase) preparations free of exonuclease (Exo) activities for effective use of the kyosei-cloning procedure [Hashimoto-Gotoh et al., Gene 137 (1993) 211-216], ENases such as HpaI and SmaI purchased from four different suppliers were examined for possible contamination by exonucleases using pKF4. The plasmid DNA was digested with either ENase, ligated and transformed into Escherichia coli mutants, rpsL, supE, trpR. With pKF4 intact DNA (approx. 8 ng), 2.3 x 10(5) KmR transformant and four KmRSmR transformant colonies were obtained; the efficiency of transformation plating (ETP) of the intact DNA was approx. 2 x 10(-5). On the other hand, the ETP values were significantly higher by one to three orders of magnitude when cut and re-joined DNAs were used under the same conditions in six out of eight ENase samples examined. The results indicate that even commercially supplied ENases, that should have passed their quality control test, could have been contaminated with Exo sufficient to interfere with effective use of the kyosei-cloning method. Therefore, it is advisable to examine ENase samples for possible contamination with Exo activities, in order to choose the right preparations for this method at the beginning of the experiments.
Hashimoto-Gotoh
T
T
Department of Biochemistry and Molecular Genetics, Kyoto Prefectural University of Medicine, Japan.
Tsujimura
A
A
Ogasahara
Y
Y
D003001:Cloning, Molecular / Q000379:methods; D015252:Deoxyribonucleases, Type II Site-Specific / Q000032:analysis*; D004340:Drug Contamination; D029968:Escherichia coli Proteins; D005092:Exonucleases / Q000032:analysis*; D005822:Genetic Vectors; D007613:Kanamycin Resistance; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D000091243:Ribosomal Protein S9; D012641:Selection, Genetic
D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D005092:Exonucleases; C024406:endodeoxyribonuclease HpaI; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific