Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids.
Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).
Division of Microbiology and Molecular Genetics, Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA92350, USA.
Junichi
J
Ryu
Edward
E
Rowsell
D015253:Deoxyribonucleases, Type I Site-Specific / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D010957:Plasmids / Q000737:chemistry / Q000592:standards*; D012015:Reference Standards; D013379:Substrate Specificity
D015253:Deoxyribonucleases, Type I Site-Specific