Subdivision of the Escherichia coli K-12 genome for sequencing: manipulation and DNA sequence of transposable elements introducing unique restrictionsites

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 2.5 Mb. The segments, sizes ranging from 150 to 250 kb, were isolated from the chromosome using the inserted r estriction 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 9 bp duplication generated during tran sposition was used as a lag for accurate splicing of the segments; no furth er sequence redundancy at the junction sites was needed. The system is appl icable to larger genomes even if they are not already well-characterized. W e present the technology for segment sequencing, results of applying this m ethod to E. coli, and the sequences of the transposon cassettes. (C) 1998 E lsevier Science B.V. All rights reserved.