Daniel D C, Johnson E M
Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029.
Nucleic Acids Res. 1989 Oct 25;17(20):8343-62. doi: 10.1093/nar/17.20.8343.
A cell-free system using synchronous plasmodial extracts initiates replication selectively on the 60 kb rDNA palindrome of Physarum polycephalum. Preferential labeling of rDNA fragments by nuclear extracts, in which elongation is limited, indicates that initiation occurs at two positions corresponding to in vivo origins of replication estimated by electron microscopy. Both nuclear and whole plasmodial extracts initiate selectively within a plasmid, pPHR21, containing one of these origins. In this plasmid bubbles expand bidirectionally and generate DpnI-resistant DNA. Extracts made at prophase or early S phase, times when the nucleolus is disorganized, are most active in pPHR21 replication. Mapping positions of replication bubbles locates the initiation point in a 3.2 kb BstEII fragment at the upstream border of a series of 31 bp repeats 2.4 kb from the initiation point for ribosomal gene transcription.
使用同步化的黏菌提取物构建的无细胞体系可在多头绒泡菌60 kb的核糖体DNA（rDNA）回文序列上选择性地起始复制。在延伸受限的核提取物中rDNA片段的优先标记表明，起始发生在两个与通过电子显微镜估计的体内复制起点相对应的位置。核提取物和完整的黏菌提取物均可在含有其中一个起点的质粒pPHR21内选择性地起始复制。在该质粒中，泡状结构双向扩展并产生抗DpnI的DNA。在前期或S期早期（此时核仁解体）制备的提取物在pPHR21复制中活性最高。复制泡状结构的定位将起始点定位于一个3.2 kb的BstEII片段中，该片段位于核糖体基因转录起始点上游2.4 kb处一系列31 bp重复序列的上游边界。
