Martín-Parras L, Hernández P, Martínez-Robles M L, Schvartzman J B
Centro de Investigaciones Biológicas (CIB), Conseja Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
J Mol Biol. 1991 Aug 20;220(4):843-53. doi: 10.1016/0022-2836(91)90357-c.
Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.
二维（2D）琼脂糖凝胶电泳正逐渐取代电子显微镜，成为绘制DNA复制起始和终止位点的首选技术。最初开发了两种不同版本来分析酵母2微米质粒的复制。中性/中性（N/N）二维琼脂糖凝胶电泳随后被用于研究其他真核质粒、病毒和染色体DNA的复制。然而，在某些情况下，结果并不符合预期的二维凝胶模式。为了更好地理解这项技术，我们用它来研究类colE1质粒pBR322的复制。这是首次通过N/N二维琼脂糖凝胶电泳分析单向复制质粒的复制中间体。获得的模式与双向复制的情况有显著不同。我们表明，识别对应于含有内部气泡分子的完整弧不足以区分对称定位的双向起源和不对称定位的单向起源。我们还表明，含有停滞叉的单向复制片段可以产生具有拐点的模式。最后，复制似乎仅在pBR322 DNA每个多聚体中的一些潜在起源处起始。
