Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli

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Abstract

Summary

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58 bp extending from the anticodon loop through the 3′ end of a tRNAPro gene. To facilitate the study of the att B site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coli. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long att B with attP, according to the Lambda model, we propose that B and B′, C and C′ core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

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