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.
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.
Herron
Paul R
PR
Department of Bioscience, University of Strathclyde, Glasgow, Scotland, UK.
Hughes
Gareth
G
Chandra
Govind
G
Fielding
Susan
S
Dyson
Paul J
PJ
D019295:Computational Biology / Q000379:methods; D016384:Consensus Sequence; D003360:Cosmids; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D020862:GC Rich Sequence; D015723:Gene Library; D005822:Genetic Vectors; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D017422:Sequence Analysis, DNA; D012984:Software; D013302:Streptomyces / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial