New ultrarare restriction site-carrying transposons for bacterial genomics |
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| Institution: | 1. Laboratoire de Génétique Microbienne, Université Catholique de Louvain, Place Croix du Sud, 5 Bte 12, B-1348 Louvain-la-Neuve, Belgium;2. Department of Pediatrics, School of Medicine, and Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA;1. Department of Plant Pathology, 4024 Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506, United States;2. Department of Entomology and Plant Pathology, 840 Main Campus Drive, North Carolina State University, Raleigh, NC 27606, United States;1. Biofuels Institute, Jiangsu University, Zhenjiang 212013, China;2. School of Environment & Safety Engineering, Jiangsu University, Zhenjiang 212013, China;3. Department of Zoology & Environmental Biology, University of Nigeria, Nsukka 410001, Nigeria;1. Centre for Ecology and Hydrology, Wallingford, Oxfordshire OX10 8BB, UK;2. Met Office Hadley Centre, FitzRoy Road, Exeter EX1 3PB, UK;3. Oxford University Centre for the Environment, University of Oxford, South Parks Road, Oxford OX1 3QY, UK;4. Atmospheric, Oceanic and Planetary Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU, UK;5. Environmental Change Institute, University of Oxford, South Parks Road, Oxford OX1 3QY, UK;6. Dalton Research Institute, Manchester Metropolitan University, Faculty of Science and Engineering, Chester Street, Manchester M1 5GD, UK;7. Committee on Climate Change, 7 Holbein Place, London SW1W 8NR, UK;8. PBL Netherlands Environmental Assessment Agency, P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands |
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| Abstract: | Electrophoretic separation of macrorestriction fragments containing a particular genomic interval has until recently depended on fortuitously placed native rare restriction sites. We present new IS10-based transposons carrying the yeast intron-encoded I-SceI restriction site which is absent from most prokaryotic and eukaryotic genomes. Construction of the plasmid vectors containing them is described. Analysis by conventional or Pulsed Field gel electrophoresis of the DNA fragments generated by the I-SceI digestion reveals the physical distance between genomic insertions of these transposons: use of the same approach to subdivide the chromosome of Escherichia coli K-12 into equivalently sized contiguous/nonoverlapping I-SceI fragments is demonstrated. Because coordinates for the loci delimited by their insertions can be readily determined in different isolates by either physical or genetic manipulations, these transposons allow sufficient flexibility for species-wide bacterial genomics. |
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