Published online 12 August 2004
Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved
Transposon Express, a software application to report the identity of insertions obtained by comprehensive transposon mutagenesis of sequenced genomes: analysis of the preference for in vitro Tn5 transposition into GC-rich DNA
Department of Bioscience, University of Strathclyde, Glasgow, Scotland, UK, 1 Institute of Medical Genetics, University of Wales College of Medicine, Cardiff, Wales, UK, 2 John Innes Centre, Norwich, England, UK and 3 School of Biological Sciences, University of Wales Swansea, Swansea, Wales, UK
* To whom correspondence should be addressed. Tel: +44 0 141 1792 295667; Fax: +44 0 141 1792 295447; Email: p.j.dyson{at}swansea.ac.uk
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received April 23, 2004; Revised July 1, 2004; Accepted July 27, 2004
Comprehensive mutant libraries can be readily constructed by transposon mutagenesis. To systematically mutagenise the genome of the Gram-positive bacterium Streptomyces coelicolor A3(2), we have employed high-throughput shuttle transposon mutagenesis of a cosmid library prepared in Escherichia coli. The location of transposon insertions is determined using automated procedures for cosmid isolation and DNA sequencing. However, a major bottleneck was the subsequent analysis of DNA sequence files. To overcome this limitation, a software application, Transposon Express, was written to allow the rapid location of transposon insertions in a sequenced genome (available at http://www.swan.ac.uk/genetics/dyson/InstallTE). Transposon Express determines the identity both of a disrupted open reading frame (ORF), and the short target site duplication created by transposition. Transposon Express also reports the orientation of the transposon and can therefore predict transcriptional coupling between an upstream promoter and a promoter-less reporter gene carried by the transposon. Analysis of a large dataset of independent insertions created using a Tn5-based transposon revealed an insertional preference for GC-rich streptomycete DNA compared to E.coli vector DNA. In addition to demonstrating the value of Transposon Express as a generic tool supporting genome-wide transposon mutagenesis programs, these data provide insight into target site selection by Tn5.
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