The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66

Gene
J FischerJ Altenbuchner

Abstract

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was s...Continue Reading

References

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Citations

Sep 28, 2002·Trends in Genetics : TIG·Carton W ChenRalph Kirby
Mar 4, 1998·Molecular Microbiology·J N Volff, J Altenbuchner
Sep 28, 2005·Journal of Basic Microbiology·Tina StumppJosef Altenbuchner

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