Jazwinski S M, Wang J L, Edelman G M
Proc Natl Acad Sci U S A. 1976 Jul;73(7):2231-5. doi: 10.1073/pnas.73.7.2231.
Addition of an extract prepared from a proliferating cell line to nuclei isolated from resting tissues such as frog liver and spleen resulted in the stimulation of DNA synthesis as assayed by [3H]dTTP incorporation. This stimulated incorporation of [3H]dTTP required ATP and depended on Mg2+ and deoxynucleoside triphosphates. Pulse-chase experiments showed that the synthesis of DNA in this system was discontinuous, resulting in the appearance of approximately 4S fragments and their ligation to yield higher molecular weight DNA. In addition, electron microscopic analysis of the DNA molecules from the reaction mixture showed that the frequency of replication "eyes" in the extract-stimulated reaction was 10-fold higher than that observed in controls. All of these results strongly suggest that the extract stimulated initiation of DNA replication in the chromatin of normally resting cells. Preliminary characterization by dialysis, heating, and enzyme treatments indicated that the activity is associated with one or more proteins of high molecular weight (greater than 50,000). Comparison of the levels of stimulatory activity in extracts from various mammalian and avian sources showed that the activity was present in cells proliferating either in vivo or in tissue culture. In contrast, extracts from normally resting tissues and cells had no activity. The level of activity present did not appear to be directly related to the levels of DNA polymerase. These results suggest a use for this system in studying regulation of the initiation of DNA synthesis and control of the various phases of the cell cycle.
将从增殖细胞系制备的提取物添加到从诸如蛙肝和脾等静止组织分离的细胞核中，结果通过[³H]dTTP掺入测定发现DNA合成受到刺激。这种刺激的[³H]dTTP掺入需要ATP，并依赖于Mg²⁺和脱氧核苷三磷酸。脉冲追踪实验表明，该系统中的DNA合成是不连续的，导致出现约4S片段，并将它们连接以产生更高分子量的DNA。此外，对反应混合物中DNA分子的电子显微镜分析表明，提取物刺激反应中复制“眼”的频率比对照中观察到的高10倍。所有这些结果强烈表明，提取物刺激了正常静止细胞染色质中DNA复制的起始。通过透析、加热和酶处理进行的初步表征表明，该活性与一种或多种高分子量（大于50,000）的蛋白质相关。对来自各种哺乳动物和鸟类来源的提取物中刺激活性水平的比较表明，该活性存在于体内或组织培养中增殖的细胞中。相反，来自正常静止组织和细胞的提取物没有活性。存在的活性水平似乎与DNA聚合酶的水平没有直接关系。这些结果表明该系统可用于研究DNA合成起始的调控和细胞周期各阶段的控制。
