Subdivision of the Escherichia coli K-12 genome for sequencing: manipulation and DNA sequence of transposable elements introducing unique restriction sites.
A transposon-based method of introducing unique restriction sites was used for subdivision of the Escherichia coli genome into a contiguous series of large non-overlapping segments spanning 2.5Mb. The segments, sizes ranging from 150 to 250kb, were isolated from the chromosome using the inserted restriction sites and shotgun cloned into an M13 vector for DNA sequencing. These shotgun sizes proved easily manageable, allowing the genomic sequence of E. coli to be completed more efficiently and rapidly than was possible by previously available methods. The 9bp duplication generated during transposition was used as a tag for accurate splicing of the segments; no further sequence redundancy at the junction sites was needed. The system is applicable to larger genomes even if they are not already well-characterized. We present the technology for segment sequencing, results of applying this method to E. coli, and the sequences of the transposon cassettes.
Mahillon
J
J
Laboratoire de Génétique Microbienne, Université Catholique de Louvain, Place Croix du Sud, 5/12, B-1348, Louvaine-la-Neuve, Belgium. mahillon@gene.ucl.ac.be
Kirkpatrick
H A
HA
Kijenski
H L
HL
Bloch
C A
CA
Rode
C K
CK
Mayhew
G F
GF
Rose
D J
DJ
Plunkett
G
G
Burland
V
V
Blattner
F R
FR
D002876:Chromosomes, Bacterial; D004251:DNA Transposable Elements; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D016680:Genome, Bacterial; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA / Q000379:methods*
D004251:DNA Transposable Elements; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific