FIG. 1.

Temperature-dependent growth of tsFT20 cells. A. Growth curves of FM3A (wild type) and tsFT20 mutant at 33°C, 37°C, 38°C, and 39°C. tsFT20 exhibited significant yet retarded growth at 38°C and no growth at 39°C. Cell density was adjusted to 10⁵/ml at 0 h, and the cultures were continued for 96 h. Cell density was measured at intervals as indicated. B. Cell cycle analysis of FM3A and tsFT20 cells showing logarithmic growth at 33°C and 38°C by FACScan. tsFT20 cells cultured at 38°C showed an increase in the fraction of cells in S phase. The percentages of cells in G₁, S, and G₂+M phases (indicated by brackets above the peak curves) were as follows: 43.2%, 33.5%, and 23.3% for FM3A at 33°C; 49.5%, 29.3%, and 21.2% for FM3A at 38°C; 44.6%, 36.5%, and 18.9% for tsFT20 at 33°C; and 28.7%, 53.8%, and 17.5% for tsFT20 at 38°C. C. tsFT20 cells expressing the wild-type p180 subunit of DNA polymerase α bypassed the slow growth at high temperatures. tsFT20 cells were infected with retroviruses encoding the indicated proteins, and the infected cells were selected by treatment with puromycin for 5 days. The selected cells as well as FM3A cells were used for obtaining growth curves as described for panel A. Mock-infected, vector; wild-type p180 subunit of DNA polymerase α, p180-WT; temperature-sensitive mutant form of p180 derived from tsFT20,p180-tsFT20.