Hand R, Gautschi J R
J Cell Biol. 1979 Aug;82(2):485-93. doi: 10.1083/jcb.82.2.485.
We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.
我们使用纤维放射自显影技术来检测在裂解细胞体系中体外合成的DNA产物。用0.01%的Brij - 58处理中国仓鼠卵巢（CHO）细胞，然后将裂解物在30℃下于含有[³H]胸腺嘧啶三磷酸（[³H]TTP）作为放射性示踪剂的完全反应混合物中孵育，用于体外DNA合成。从DNA制备的纤维放射自显影片显示，它是在串联排列的复制单元上合成的，这些复制单元的平均大小为20微米，与在体内发现的单元大小非常相似。复制叉移动的速率是体内速率的25% - 50%。超过80%的复制叉在15分钟时停止运作，95%在60分钟时停止。这表明合成因过早终止而停止。在体外反应中，在前10分钟孵育期间不加入放射性物质，所合成的DNA中带有标记起始点的复制单元为新起始提供了证据。这一点，再加上起始点之间的距离（复制单元大小）没有增加以及过早终止在很大程度上导致合成停止的观察结果，表明在这个体外复制系统中发生了显著的起始过程。
