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Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli
Author(s) -
Raynal Alain,
Tuphile Karine,
Gerbaud Claude,
Luther Tatjana,
Guérineau Michel,
Pernodet JeanLuc
Publication year - 1998
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1998.00799.x
Subject(s) - biology , plasmid , escherichia coli , genetics , recombination , gene , insertion sequence , inverted repeat , chromosome , microbiology and biotechnology , lambda phage , sequence analysis , genome , transposable element , bacteriophage
The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site‐specific recombination between the element ( att P) and the chromosomal ( att B) sites. These regions share an identity segment of 58 bp extending from the anti‐codon loop through the 3′ end of a tRNA Pro gene. To facilitate the study of the att B site, the int and xis genes, expressed from an inducible promoter, and att P from pSAM2 were cloned on plasmids in Escherichia coli . Compatible plasmids carrying the different att B 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 att B 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 att B; shorter sequences allowed integration with lower efficiencies. By comparing the 26‐bp‐long att B with att P, 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.