Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
EMBO J. 1988 Feb;7(2):303-8. doi: 10.1002/j.1460-2075.1988.tb02813.x.
In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S-G(2) boundary or early G(2) phase. To determine if the delayed ligation of nascent chains occurs at specific termination sites, we mapped the location of arrested forks in the ribosomal DNA (rDNA) repeats from cells in late S and G(2) phases. Two-dimensional (neutral-alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S-G(2) boundary and early G(2) phase produce alkali-released rDNA fragments of discrete size. The released fragments are from a particular restriction fragment, demonstrating that the replication forks stop non-randomly within the rDNA repeats. Indirect end-labeling with probes homologous to one or the other end of the fork-containing restriction fragment shows that there are two termination regions, T(1) and T(2), where forks stop. T(1) is located in the non-transcribed spacer and T(2) is at the junction between the non-transcribed spacer and the 18S gene. The two termini are separated by 1.3 kb. Replication forks stop at identical sites in both the 8.6- and 9.0-kb rDNA repeat size classes indicating that these sites are sequence determined.
在豌豆(cv. Alaska)同步的根细胞中,新生复制子的连接被延迟到细胞到达 S-G(2)边界或早期 G(2)期。为了确定新生链的延迟连接是否发生在特定的终止位点,我们在核糖体 DNA(rDNA)重复序列中定位了晚期 S 和 G(2)期细胞中停滞的叉。二维(中性-碱性)琼脂糖电泳和特定 rDNA 序列的 Southern 印迹杂交表明,只有位于 S-G(2)边界和早期 G(2)期的细胞才能产生离散大小的碱释放 rDNA 片段。释放的片段来自特定的限制片段,表明复制叉在 rDNA 重复序列中随机停止。用与包含叉的限制片段的一端或另一端同源的探针进行间接末端标记表明,有两个终止区域 T(1)和 T(2),其中叉停止。T(1)位于非转录间隔区,T(2)位于非转录间隔区和 18S 基因之间的连接处。两个末端之间的距离为 1.3kb。复制叉在 8.6kb 和 9.0kb rDNA 重复大小类中相同的位点停止,表明这些位点是由序列决定的。