Isolation of a mammalian cell cycle ts mutant, tsJT663, defective in preparation for passing restriction point

A cell cycle ts mutant, tsJT663, isolated from Fischer rat cell line 3Y1, was characterized. The execution point of tsJT663 was approximately 2 h before S phase, starting either from a G0 (quiescent) state or from the previous cell cycle. Progression of the cell cycle other than from G0/G1 to S phase was temperature insensitive. The restriction (R) point was also estimated as about 2 h before S phase. When tsJT663 cells at log phase were transferred to 40 degrees C, the cell number increased and then decreased within 2 days and thereafter the cells were growth arrested stably for more than 4 weeks. When the serum-stimulated cells were cultured at 34 degrees C for 6 h at 40 degrees C for various period of time and then at 34 degrees C up to the end of the experiments at 30 h, the frequency of cells entering S phase declined depending upon the duration of culture at 40 degrees C. Assuming that the decline of the frequency of tsJT663 cells capable of entering S phase corresponded to the decline of a putative material required for cells to pass the R point toward S phase, the half-life of this material at 40 degrees C was 1 h. These results are consistent with a tentative conclusion that tsJT663 is a cell cycle ts mutant which does not prepare at nonpermissive temperature for passing the restriction point toward S phase.