CELL CYCLE-SPECIFIC REPLICATION OF ESCHERICHIA-COLI MINICHROMOSOMES
CELL CYCLE-SPECIFIC REPLICATION OF ESCHERICHIA-COLI MINICHROMOSOMES
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DOI：
10.1073/pnas.83.14.5101
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发表时间：
1986-07-01
影响因子：
11.1
通讯作者：
HELMSTETTER, CE
中科院分区：
文献类型：
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作者：
LEONARD, AC;HELMSTETTER, CE
利用改进的质粒复制频率研究方法，研究了大肠杆菌小染色体在细胞分裂周期中的复制时间。用[3H]胸苷脉冲标记在葡萄糖/Casamino acids minimal培养基中呈指数增长的培养物，并分析不同年龄细胞的质粒DNA的放射性。在标记期结束时，将细菌结合到硝化纤维素膜过滤器的表面，并通过琼脂糖凝胶电泳定量测定从膜上不断洗脱的新子细胞的放射性。小染色体在细胞分裂周期的一个离散间隔内复制，似乎与染色体复制的开始一致。相比之下，质粒pBR322在整个分裂周期中复制的速率随着细胞年龄的增长而逐渐增加。在携带两种质粒的细胞中，微小染色体和pBR322复制的差异是明显可见的。我们还发现，在分裂周期中，oriC附近的16kD基因不是小染色体复制时间的决定因素。这些结果与小染色体复制频率受控制染色体复制的相同机制控制的结论是一致的。
The timing of Escherichia coli minichromosome replication in the cell division cycle was examined using an improved procedure for studying plasmid replication frequency. Cultures growing exponentially in glucose/Casamino acids minimal medium were pulse-labeled with [3H]thymidine, and the radioactivity incorporated into plasmid DNA in cells of different ages was analyzed. At the end of the labeling period the bacteria were bound to the surface of a nitrocellulose membrane filter, and the radioactivity in new daughter cells, which eluted continuously from the membrane, was quantitated following agarose gel electrophoresis. The minichromosomes replicated during a discrete interval in the cell division cycle that appeared to coincide with initiation of chromosome replication. In contrast, plasmid pBR322 replicated throughout the division cycle at a rate that increased gradually as a function of cell age. The difference in minichromosome and pBR322 replication was clearly discernible in cells harboring both plasmids. It was also found that the 16kD gene adjacent to oriC was not a determinant of the timing of minichromosome replication during the division cycle. The results are consistent with the conclusion that minichromosome replication frequency is governed by the same mechanism that controls chromosome replication.