Hoy C A, Lewis E D, Schimke R T
Department of Biological Sciences, Stanford University, California 94305.
Mol Cell Biol. 1990 Apr;10(4):1584-92. doi: 10.1128/mcb.10.4.1584-1592.1990.
The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.
在中国仓鼠卵巢细胞中研究了氚标记胸腺嘧啶核苷掺入对DNA复制的影响。用每毫升2微居里的[3H]胸腺嘧啶核苷（200微居里/毫摩尔）标记细胞60分钟后，通过碱性洗脱技术测定，快速洗脱（小）的DNA在大约2至4小时内成熟为不可洗脱的大尺寸。然而，暴露于每毫升10微居里[3H]胸腺嘧啶核苷（66微居里/毫摩尔）的细胞中的DNA最初洗脱更快，并且在随后的孵育过程中未成熟为不可洗脱的尺寸。通过溴脱氧尿苷取代的DNA的氯化铯梯度分析测量的半保留DNA复制也被发现受标记方案中使用的[3H]胸腺嘧啶核苷的最终比活性影响。这些对DNA复制的影响伴随着显著的细胞周期扰动，表明常用于研究DNA代谢的标记方案会产生异常的DNA复制和随后的细胞周期扰动。
