NEW ULTRARARE RESTRICTION SITE-CARRYING TRANSPOSONS FOR BACTERIAL GENOMICS

Electrophoretic separation of macrorestriction fragments containing a particular genomic interval has until recently depended on fortuitousl y placed native rare restriction sites. We present new IS10-based tran sposons carrying the yeast intron-encoded I-SceI restriction site whic h is absent from most prokaryotic and eukaryotic genomes. Construction of the plasmid vectors containing them is described. Analysis by conv entional or Pulsed Field gel electrophoresis of the DNA fragments gene rated by the I-SceI digestion reveals the physical distance between ge nomic insertions of these transposons: use of the same approach to sub divide the chromosome of Escherichia coli K-12 into equivalently sized contiguous/nonoverlapping I-SceI fragments is demonstrated. Because c oordinates for the loci delimited by their insertions can be readily d etermined in different isolates by either physical or genetic manipula tions, these transposons allow sufficient flexibility for species-wide bacterial genomics.