Schvartzman J B, Van't Hof J
Nucleic Acids Res. 1982 Oct 11;10(19):6191-205. doi: 10.1093/nar/10.19.6191.
Velocity sedimentation in alkaline sucrose gradients, single cell autoradiography and cytophotometry were used to determine if protein synthesis is required for the maturation of nascent replicons to chromosomal-sized molecules in cultured pea-root cells. The results obtained showed that cycloheximide at 5 and 10 microgram/ml, added either before or during labeling with tritiated thymidine, blocked maturation of nascent DNA at an intermediate size of 72-140 X 10(6) daltons single-stranded DNA. To reach this size, nascent replicons - which are 18 X 10(6) daltons single-stranded DNA each - were replicated and groups of 4-8 replicons were joined even though protein synthesis was reduced to 15% of the control. Further maturation of the nascent molecules to chromosomal size, however, was prevented and this resulted in the accumulation of nascent molecules in the 72-140 X 10(6) daltons range. The experiments also showed that the joining of nascent replicons is not an absolute function of late S or G2 phase of the cell cycle, since cells treated with cycloheximide and blocked in mid-S phase had nascent DNA of a size corresponding to 4-8 joined replicons. Finally, the results support the hypothesis that at least one step in the process of nascent DNA maturation may require replication, during late-S phase, of DNA segments that are interspersed within replicon-clusters that replicate early in the S phase.
利用碱性蔗糖梯度中的速度沉降、单细胞放射自显影和细胞光度测定法，来确定在培养的豌豆根细胞中，新生复制子成熟为染色体大小的分子是否需要蛋白质合成。所获得的结果表明，在以氚标记的胸腺嘧啶核苷进行标记之前或期间加入浓度为5和10微克/毫升的环己酰亚胺，会在单链DNA大小为72 - 140×10⁶道尔顿的中间阶段阻断新生DNA的成熟。为了达到这个大小，每个大小为18×10⁶道尔顿的单链DNA的新生复制子进行了复制，并且4 - 8个复制子的组连接在一起，尽管蛋白质合成减少到了对照的15%。然而，新生分子进一步成熟为染色体大小的过程被阻止了，这导致了新生分子在72 - 140×10⁶道尔顿范围内的积累。实验还表明，新生复制子的连接不是细胞周期中S期晚期或G2期的绝对功能，因为用环己酰亚胺处理并在S期中期被阻断的细胞具有与4 - 8个连接的复制子相对应大小的新生DNA。最后，这些结果支持了这样一种假设，即在新生DNA成熟过程中，至少有一个步骤可能需要在S期晚期复制散布在S期早期复制的复制子簇内的DNA片段。
