Liu Y C, Chen G S, Liu W L, Wen S F
Institute of Life Science, National Tsing-Hua University, Hsin-Chu, Taiwan, Republic of China.
J Cell Biochem. 1995 Apr;57(4):641-6. doi: 10.1002/jcb.240570408.
A simple scheme was developed to study the mRNA stability of the proliferating cell nuclear antigen (PCNA) gene during cellular transition from the G1/S boundary to a quiescent state. By this scheme, CHO.K1 cells were grown to about 80% confluence and then serum-starved for 40 h for synchronization in a quiescent state. The quiescent cells were serum-stimulated for a period of time (between 8 h and 12 h) and then grown in serum-free medium until being harvested for further analyses. The cellular PCNA mRNA level was analyzed by Northern blotting. As compared with that in cells which were continuously incubated in serum-containing medium, the decline of the mRNA level, after reaching the peak, in these serum-deprived cells was virtually devoid of mRNA synthesis. Thus, this mRNA decay was taken for the measurement of mRNA stability. The advantage of the scheme is that, unlike the treatment of transcription inhibitors, it does not prevent the cells from completing the rest of the cell cycle before returning to the resting state, and so the mRNA stability observed is cell cycle dependent. In contrast with the previous report that the stability of PCNA mRNA in quiescent cells is less by severalfold than that in S phase cells, our study shows that the mRNA stability of PCNA remained constant during the cellular transition from G1/S boundary to quiescent state.
我们设计了一个简单的方案，用于研究细胞从G1/S边界过渡到静止状态过程中增殖细胞核抗原（PCNA）基因的mRNA稳定性。按照该方案，将CHO.K1细胞培养至约80%汇合度，然后进行40小时的血清饥饿处理，使其同步进入静止状态。对静止细胞进行一段时间（8至12小时）的血清刺激，然后在无血清培养基中培养，直至收获用于进一步分析。通过Northern印迹法分析细胞中PCNA mRNA水平。与在含血清培养基中持续培养的细胞相比，这些血清剥夺细胞中mRNA水平在达到峰值后下降，且几乎没有mRNA合成。因此，这种mRNA降解被用于测量mRNA稳定性。该方案的优点是，与转录抑制剂处理不同，它不会阻止细胞在恢复静止状态之前完成细胞周期的其余阶段，所以观察到的mRNA稳定性是依赖于细胞周期的。与之前关于静止细胞中PCNA mRNA稳定性比S期细胞低几倍的报道相反，我们的研究表明，在细胞从G1/S边界过渡到静止状态的过程中，PCNA的mRNA稳定性保持恒定。
