The prokaryotic β-recombinase catalyzes site-specific recombination in mammalian cells
The prokaryotic β-recombinase catalyzes site-specific recombination in mammalian cells
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DOI：
10.1074/jbc.274.10.6634
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发表时间：
1999-03-05
影响因子：
4.8
通讯作者：
Bernad, A
中科院分区：
文献类型：
--
作者：
Díaz, V;Rojo, F;Bernad, A
真核生物基因组的体内修饰的新策略的发展已成为当前研究的重要目标。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受控操作。在这里，我们提供了第一个证据，证明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pSM 19035的β基因编码的β-腺苷酸酶已在真核细胞系中功能性表达，当通过间接免疫荧光测定时，其显示出对核区室的高亲合力并形成清晰的斑点图案。在猿猴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/交换序列之间的DNA片段的缺失（6个位点）作为染色体外DNA底物。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活化系统中获得了相同的结果。在携带整合在不同染色体位置的靶序列的不同拷贝数的稳定NIH/3 T3克隆中，瞬时β-重组酶表达也促进了间插DNA的缺失，而与靶序列的插入位置无关。这种新的重组工具的操纵真核生物基因组，单独使用或与目前使用的其他重组系统相结合的效用进行了讨论。
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.