Díaz V, Rojo F, Martínez-A C, Alonso J C, Bernad A
Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain.
J Biol Chem. 1999 Mar 5;274(10):6634-40. doi: 10.1074/jbc.274.10.6634.
The development of new strategies for the in vivo modification of eukaryotic genomes has become an important objective of current research. Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes. Here we provide the first evidence that the prokaryotic site-specific recombinase (beta-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments. beta-Recombinase, encoded by the beta gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence. In simian COS-1 cells, transient beta-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate. The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the beta-recombinase protein. In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences. The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.
开发用于真核基因组体内修饰的新策略已成为当前研究的一个重要目标。位点特异性重组已被证明是有用的，因为它允许对小鼠、植物和酵母基因组进行可控操作。在这里，我们提供了首个证据，即仅催化分子内重组的原核位点特异性重组酶（β-重组酶）在真核环境中具有活性。由革兰氏阳性广宿主范围质粒pSM19035的β基因编码的β-重组酶已在真核细胞系中实现功能性表达，显示出对核区室的高亲和力，并且在通过间接免疫荧光检测时形成清晰的斑点状模式。在猴COS-1细胞中，瞬时β-重组酶表达促进了位于两个直接定向的特异性识别/交叉序列（六个位点）之间的DNA片段的缺失，该DNA片段作为染色体外DNA底物存在。在稳定表达β-重组酶蛋白的细胞系中测试的重组依赖性lacZ激活系统中也获得了相同的结果。在稳定的NIH/3T3克隆中，靶序列以不同数量的拷贝整合在不同的染色体位置，瞬时β-重组酶表达也促进了中间DNA的缺失，而与靶序列的插入位置无关。本文讨论了这种用于操纵真核基因组的新型重组工具单独使用或与目前使用的其他重组系统联合使用的效用。
