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tsJT16, a cell-cycle ts mutant defective in a function operating soon after growth stimulation, fails to induce a
T Takasuka1, M Sakayama, S Ishibashi
1Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Abstract:
tsJT16 is a temperature-sensitive (ts) mutant of rat fibroblasts that has a ts defect in a function operating soon after the growth stimulation from the G0 phase. After the growth stimulation, the cells express several cell-cycle-dependent genes at both temperatures while they fail at the nonpermissive temperature to synthesize a protein p70 identified on two-dimensional gel electrophoresis. Here we report that 1) synthesis of p70 began within 1 h of stimulation, continued up to the 7th hour and then decreased; 2) the half-life of p70 was shortened after 6 h after the stimulation; 3) p70 was localized in the nuclear fraction; 4) p70 was likely to be a primarily induced protein; 5) mRNA of p70 was supposed to be synthesized exclusively within 2 h of growth stimulation. These and the previous results suggest that p70 is a nuclear protein responsible for the early stage of transition of cells from the G0 toward the S phase and is induced via a different signal transduction sequence from that for the c-fos gene.
Insights
A temperature-sensitive mutant (tsJT16) in rat fibroblasts fails to synthesize nuclear protein p70 at non-permissive temperatures, hindering G0 to S phase transition. This suggests p70 is crucial for early cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The tsJT16 mutant exhibits a temperature-sensitive defect affecting cell cycle progression shortly after growth stimulation from G0 phase.
- While expressing cell-cycle genes, tsJT16 fails to synthesize a specific protein, p70, at non-permissive temperatures.
Purpose of the Study:
- To characterize the synthesis, localization, and regulation of protein p70 in tsJT16 fibroblasts.
- To elucidate the role of p70 in the transition from G0 to S phase.
Main Methods:
- Two-dimensional gel electrophoresis to identify and analyze protein p70.
- Cellular fractionation to determine p70 localization.
- Analysis of p70 synthesis and mRNA expression kinetics post-growth stimulation.
Main Results:
- p70 synthesis initiates within 1 hour of stimulation, peaking by 7 hours, with a decreasing half-life after 6 hours.
- p70 is primarily localized in the nuclear fraction and appears to be an induced protein.
- mRNA for p70 is synthesized exclusively within the first 2 hours of growth stimulation.
Conclusions:
- p70 is a nuclear protein essential for the early G0 to S phase transition in rat fibroblasts.
- p70 induction likely involves a distinct signal transduction pathway compared to c-fos.
- The tsJT16 mutant's defect in p70 synthesis disrupts normal cell cycle progression.