Puchta H, Dujon B, Hohn B
Friedrich Miescher-Institut, Basel, Switzerland.
Nucleic Acids Res. 1993 Nov 11;21(22):5034-40. doi: 10.1093/nar/21.22.5034.
Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.
双链断裂（DSBs）的诱导与酵母中的减数分裂和有丝分裂重组相关。我们发现，在高等真核生物中，DSBs的诱导也与重组频率的显著提高直接相关。我们将携带合成I-SceI基因（编码一种高度序列特异性内切酶）的质粒与携带与其同源序列相邻的I-SceI位点的重组底物一起共转染到烟草叶肉原生质体中。我们使用成熟的瞬时β-葡萄糖醛酸酶（GUS）检测法测量了染色体外重组的效率。当转染中包含一个相对于启动子呈正义而非反义方向携带I-SceI基因的质粒时，GUS酶活性显著增加。通过Southern印迹法在重组底物中检测到了体内诱导的DSBs，表明酵母酶在植物细胞中具有功能。在转染的I-SceI基因与I-SceI位点比例较高时，重组底物中的大多数I-SceI位点被切割，这表明DSBs的诱导是所述重组反应中的限速步骤。这些结果表明，在植物基因组中特定位点体内诱导瞬时断裂可以使外源DNA通过同源重组靶向这些位点。
