Contursi Patrizia, Pisani Francesca M, Grigoriev Andrei, Cannio Raffaele, Bartolucci Simonetta, Rossi Mosè
Dipartimento di Chimica Biologica, Università degli Studi di Napoli, Via Mezzocannone, 16, 80134, Napoli, Italy.
Extremophiles. 2004 Oct;8(5):385-91. doi: 10.1007/s00792-004-0399-y. Epub 2004 Aug 5.
Here, we describe the identification of a chromosomal DNA replication origin (oriC) from the hyperthermophilic archaeon Sulfolobus solfataricus (subdomain of Crenarchaeota). By means of a cumulative GC-skew analysis of the Sulfolobus genome sequence, a candidate oriC was mapped within a 1.12-kb region located between the two divergently transcribed MCM- and cdc6-like genes. We demonstrated that plasmids containing the Sulfolobus oriC sequence and a hygromycin-resistance selectable marker were maintained in an episomal state in transformed S. solfataricus cells under selective pressure. The proposed location of the origin was confirmed by 2-D gel electrophoresis experiments. This is the first report on the functional cloning of a chromosomal oriC from an archaeon and represents an important step toward the reconstitution of an archaeal in vitro DNA replication system.
在此，我们描述了从嗜热古菌嗜热栖热菌（泉古菌亚域）中鉴定出一个染色体DNA复制起点（oriC）。通过对嗜热栖热菌基因组序列进行累积GC偏斜分析，一个候选oriC被定位在两个反向转录的类MCM和类cdc6基因之间的一个1.12 kb区域内。我们证明，含有嗜热栖热菌oriC序列和潮霉素抗性选择标记的质粒在选择性压力下能在转化的嗜热栖热菌细胞中以附加体状态维持。通过二维凝胶电泳实验证实了所提出的起点位置。这是关于从古菌中功能性克隆染色体oriC的首次报道，代表了朝着重建古菌体外DNA复制系统迈出的重要一步。
