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Temperature sensitive cells have been isolated from Syrian and Chinese hamster cells using a method based on selective detachment from a glass substrate. The Syrian hamster isolates occurred at a high frequency (about 1 in 10") and reverted rapidly; polyoma virus transformation conferred on cells the ability to grow, perhaps abnormally, in agar suspension. A slightly modified isolation technique was applied to Chinese hamster cultures and resulted in the isolation of' at least one mutant (from a starting population of 5 X 108 cells) with a spontaneous reversion rate of less than one in 6 X 10'. Treatment of the mutant with ethyl methane sulphonate induced reversion. It was concluded that selective detachment provided a useful method for the isolation of conditional lethal mutants of mammalian cells.

Viability, DNA synthesis and mitosis have been followed in the temperature sensitive Chinese hamster cell mutant K12 under permissive and non-permissive conditions. On incubation a t 40°C cells retained their ability to form colonies at 3 3°C for 15 to 20 hours, but viability was lost gradually during the following 20 hours. When random cultures of K12 were shifted to 40°C the rate of DNA synthesis was normal for three to four hours but then decreased markedly, reaching 95% inhibition after 24 hours. Under the same conditions mitosis was inhibited after 15 hours. If cultures which had been incubated at 40°C for 16 hours were placed at 33°C the rate of DNA synthesis increased five hours after the shift down and mitosis 18 hours after. These results can be interpreted on the assumption that K12 at 40°C is unable to complete a step in the cell cycle which is essential for DNA synthesis and which occurs three to four hours before the start of S at 33°C.The preceding paper described the isolation of a temperature sensitive mutant K12 derived from the Chinese hamster cell line Wg-1A (Roscoe, Read and Robinson, '73). K12 was selected from a large population because it detached relatively easily from a glass substrate at the nonpermissive temperature. It was found to have a very low spontaneous reversion rate (less than 1 in 6 X lo7) and over a period of 48 hours at 40°C K12 cells stopped growing and became fully rounded in contrast to a normal spread configuration and good growth at 33°C. In this paper experiments with K12 and Wg-1A are reported which describe the effect of temperature shifts between 33 O C and 40 O C on viability, DNA synthesis and mitosis. MATERIALS AND METHODS CellsWg-lA, the isolation of K12 and the growth medium used have been described previously (Roscoe, Read and Robinson, '73). Stocks of cells were maintained at 31 "C with transfers at weekly intervals. Experiments were carried out at 33°C or 40°C in plastic bottles (Falcon Plastics) or Petri dishes (NUNCLON).For continuous observation of cells at 33°C or 40°C micrographs of cultures were taken at intervals of 90 or 120 seconds using 16 mm cine film. When cells were shifted from low to high temperature or vice-versa the constant temperature cabinet and microscope equilibrated within one hour. The resulting films were analysed using a cine projector fitted with a frame counter. Measurement of D N A synthesis: (i) By thymidine incorporationThe method used was based on that described by Bachetti and Whitmore ('69). All the Petri dishes (50 mm diameter) for a given experiment were inoculated from one cell suspension with about 5 X 104 cells; they were then incubated for 24 hours at 3 3°C before a n y required temperature shifts. In these experiments operations at 40°C were performed in a constant temperature room. For each point at which the rate of DNA synthesis was to be measured the medium was carefully removed and replaced with fresh growth medium containing 0.5 pC/ml "H-thymidine (TdR; 5.25 C per mmole). In some experiments "H-TdR was a...