Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria.

Abstract

Construction of physical maps of genomes by pulsed-field gel electrophoresis requires enzymes which cut the genome into an analyzable number of fragments; most produce too many fragments. The enzyme I-Ceu I, encoded by a mobile intron in the chloroplast 23S ribosomal RNA (rrl) gene of Chlamydomonas eugametos, cuts a 26-bp site in the rrl gene. This enzyme digests DNA of Salmonella typhimurium at seven sites, each corresponding to one of the rrl genes of the rrn operons, but at no other site. These seven fragments were located on the previously determined Xba I physical map, and the I-Ceu I sites, and thus the rrn genes of S. typhimurium, were mapped on the 4800-kb chromosome. Eschenchia coli K-12 also yields seven fragments of sizes similar to those of S.

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

“…The enzyme I-CeuI recognizes a highly conserved 26 bp sequence within the rrl genes of most eubacteria (Liu et al, 1993). Therefore, the digestion of L. acidophilus genomic DNA with I-CeuI into four fragments was indicative of the presence of four rrn operons on the chromosome (Fig.…”

Clostridium difficile erm(B)-containing elements and the burden on the in vitro fitness

“…This enzyme specifically targets genes coding for rRNA (Liu et al, 1993). Hybridization patterns indicated that the resistance gene was located on the chromosome in all transconjugants (data not shown).…”

Genetic and Biochemical Characterization of CAD-1, a Chromosomally Encoded New Class A Penicillinase from Carnobacterium divergens

“…Genomic DNA embedded in agarose plugs was digested for 3 h at 37°C with 0.01 U of I-CeuI, an intron-encoded endonuclease specific for rRNA genes (20), or overnight at 27°C with 25 U of SmaI. Fragments were separated on agarose gel (1.2% for I-CeuI and 0.8% for SmaI digestion) with a contour-clamped homogeneous electric field DR III system (Bio-Rad Laboratories, Hercules, CA) under the following conditions: total migration, 24 h for I-CeuI or 27 h for SmaI; initial pulse, 60 s for I-CeuI or 5 s for SmaI; final pulse, 120 s for I-CeuI or 35 s for SmaI; voltage, 6 V/cm; included angle, 120°; and temperature, 14°C.…”