Coppock D L, Pardee A B
Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Mol Cell Biol. 1987 Aug;7(8):2925-32. doi: 10.1128/mcb.7.8.2925-2932.1987.
To investigate the mechanism which controls the onset of DNA synthesis, we examined the regulation of thymidine kinase (TK) and its mRNA in the cell cycle. TK activity provides a useful marker for the onset of the S phase in mammalian cells. The present analysis of regulation of TK mRNA in BALB/c 3T3 cells showed that (i) the increase in TK activity depended on the availability of TK mRNA, (ii) the level of TK mRNA between G0 and S increased more than 20-fold, (iii) the rate of run-on TK transcription increased at most 2- to 4-fold between the G0 and S phases, (iv) the half-life of TK mRNA was greater than 8 to 12 h in the S and M phases and decreased as cells entered quiescence, (v) the TK mRNA increase was fully blocked by inhibition of protein synthesis by only 60%, (vi) this inhibition was completely effective for up to about 10 h following serum addition and progressively much less effective when the drugs were added later. These results suggest that the appearance of TK mRNA at the beginning of the S phase in serum-stimulated 3T3 cells is controlled not only by the rate of gene transcription but importantly also by the decreased rate of mRNA degradation. Similar mechanisms may be involved in regulation of the onset of DNA synthesis and the increase in TK mRNA since both are controlled in a manner consistent with a requirement for a labile protein.
为了研究控制DNA合成起始的机制，我们检测了细胞周期中胸苷激酶（TK）及其mRNA的调控情况。TK活性为哺乳动物细胞中S期的起始提供了一个有用的标志物。目前对BALB/c 3T3细胞中TK mRNA调控的分析表明：（i）TK活性的增加取决于TK mRNA的可利用性；（ii）从G0期到S期，TK mRNA水平增加了20多倍；（iii）在G0期和S期之间，TK转录的连续进行速率最多增加2至4倍；（iv）在S期和M期，TK mRNA的半衰期大于8至12小时，并且随着细胞进入静止期而缩短；（v）蛋白质合成的抑制仅使TK mRNA的增加被完全阻断60%；（vi）血清添加后长达约10小时，这种抑制是完全有效的，而当药物在稍后添加时，其效果逐渐减弱。这些结果表明，在血清刺激的3T3细胞中，S期开始时TK mRNA的出现不仅受基因转录速率的控制，而且重要的是还受mRNA降解速率降低的控制。DNA合成起始的调控和TK mRNA的增加可能涉及类似的机制，因为两者都以与对不稳定蛋白质的需求相一致的方式受到控制。
