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Nucleic Acids Research Advance Access published online on June 18, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn056
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Junichi Ryu1 and Edward Rowsell2,*

1Division of Microbiology and Molecular Genetics, Department of Biochemistry and Microbiology and 2Department of Pathology and Laboratory Medicine, School of Medicine, Loma Linda University, Loma Linda, CA92350, USA

*To whom correspondence should be addressed. Email: erowsell{at}llu.edu

Received December 30, 2007. Revised January 24, 2008. Accepted January 25, 2008.

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).

I regret to inform our readers that Dr Junichi Ryu died before publication of this article.


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