Raynal A, Tuphile K, Gerbaud C, Luther T, Guérineau M, Pernodet J L
Laboratoire de Biologie et Génétique Moléculaire, Institut de Génétique et Microbiologie, URA CNRS 2225, Université Paris-Sud, Orsay, France.
Mol Microbiol. 1998 Apr;28(2):333-42. doi: 10.1046/j.1365-2958.1998.00799.x.
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 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. 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 attB 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.
来自产二素链霉菌的元件pSAM2通过元件（attP）和染色体（attB）位点之间的位点特异性重组整合到染色体中。这些区域共享一个58bp的同源区段，从反密码子环延伸至tRNA（Pro）基因的3'端。为便于研究attB位点，将从诱导型启动子表达的int和xis基因以及来自pSAM2的attP克隆到大肠杆菌的质粒上。将携带不同待测试attB区域的相容质粒导入这些大肠杆菌菌株中。在这些条件下，单独的Int可促进位点特异性整合；Int和Xis都是位点特异性切除所必需的。该实验系统用于研究attB中高效位点特异性重组所需的序列。一个以反密码子环区域为中心且未完全包含在同源区段中的26bp序列保留了attB的所有功能；较短的序列允许以较低效率进行整合。根据λ模型，通过将26bp长的attB与attP进行比较，我们提出B和B'、C和C'核心型Int结合位点由9bp的不完全反向重复序列组成，中间间隔一个5bp的重叠区域。
