Qin M, Lee E, Zankel T, Ow D W
Plant Gene Expression Center, US Department of Agriculture, Albany, CA 94710, USA.
Nucleic Acids Res. 1995 Jun 11;23(11):1923-7. doi: 10.1093/nar/23.11.1923.
Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation. Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system. Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe. In vitro recombination between chromosomal lox sites and exogenously provided lox oligonucleotides 'cleaved' the chromosome at the defined lox sequences. Site-specific cleavage of lox sites in the tobacco genome was also demonstrated. This recombination-based cleavage method provides a novel approach for structural and functional analyses of eukaryotic chromosomes as it allows direct isolation of chromosome regions that correspond to phenotypes revealed through Cre-lox mediated chromosome rearrangements in vivo. Moreover, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the long range mapping of chromosomes.
位点特异性重组系统是体内染色体工程的有用工具，而位点特异性DNA切割方法在基因组分析和基因分离中具有应用价值。在此，我们报告一种利用Cre-lox位点特异性重组系统在体外切割染色体的新方法。两个lox位点被靶向导入粟酒裂殖酵母5.7 Mb的I号染色体中。染色体lox位点与外源提供的lox寡核苷酸之间的体外重组在特定的lox序列处“切割”染色体。烟草基因组中lox位点的位点特异性切割也得到了证实。这种基于重组的切割方法为真核染色体的结构和功能分析提供了一种新方法，因为它允许直接分离与体内Cre-lox介导的染色体重排所揭示的表型相对应的染色体区域。此外，与末端标记的lox寡核苷酸进行重组将允许对染色体片段进行特异性末端标记，以促进染色体的长距离作图。
