Published online 15 June 2004
Nucleic Acids Research, 2004, Vol. 32, No. 10 E82
© 2004 Nucleic Acids Research, Vol. 32 No. 10 © Oxford University Press 2004; all rights reserved
The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI
Division of Microbiology and Molecular Genetics, Department of Biochemistry and Microbiology, Loma Linda University, Loma Linda, CA 92350, USA and 1 Division of Gene Expression and Regulation, National Institute for Basic Biology, Okazaki, Aichi-ken 444-8585, Japan
* To whom correspondence should be addressed. Tel: +1 909 558 1000/4480; Fax: +1 909 558 4035; E-mail: jryu{at}som.llu.edu
Present addresses: Masumi Hidaka, Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan
Masataka Iida, Department of Surgery, Akita University, Akita, Japan
Received May 4, 2004; Revised and Accepted May 15, 2004
Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).
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