Estimation of PCNA mRNA stability in cell cycle by a serum-deprivation method.
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.
Liu
Y C
YC
Institute of Life Science, National Tsing-Hua University, Hsin-Chu, Taiwan, Republic of China.
Chen
G S
GS
Liu
W L
WL
Wen
S F
SF
D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002453:Cell Cycle; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D046508:Culture Techniques / Q000379:methods; D003609:Dactinomycin / Q000494:pharmacology; D016193:G1 Phase / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D016196:S Phase / Q000235:genetics
D016895:Culture Media, Serum-Free; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D003609:Dactinomycin