STRUCTURE OF THE CHROMOSOMAL INSERTION SITE FOR PSAM2 - FUNCTIONAL-ANALYSIS IN ESCHERICHIA-COLI

The element pSAM2 from Streptomyces ambofaciens integrates into the ch romosome through site-specific recombination between the element (attP ) and the chromosomal (attB) sites. These regions share an identity se gment of 58bp extending from the anticodon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int an d xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil, Compatible plasmids carr ying the different attB regions to be tested were introduced in these E. coil 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 re quired in attB for efficient site-specific recombination, A 26 bp sequ ence, centred on the anti-codon loop region and not completely include d in the identity segment, retained all the functionality of attB; sho rter sequences allowed integration with lower efficiencies. By compari ng the 26-bp-long attB with attP, according to the Lambda model, we pr opose 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.