Selective isolation of reversible cold sensitive variants from Chinese hamster ovary cell cultures

Variants of the Chinese hamster ovary cell line CHO-KI (ATTCCCL61)which grow almost normally at 38.5C but very poorly or not at all at 30C were obtained after treatment with mutagens and application of an indirect selection procedure. Two kinds of variants were recovered. In the first of these, the cold sensitive phenotypw is expressed completely only at low cell densities. At higher cell desity, growth continues at the nonpermissive temperature, but at a reduced rate. In the second class, the cold snesitive phenotype is independent of cell density. Two members of the latter class were studied in detail. In both lines, after shift to the nonpermissive temperature, the rateof H-thymidine incorporation declines markedly; the rates of H-uridine and H-phenylalanine uptake are less drastically reduced. Autoradiographs indicate that the decline in thymidine uptake at the nonpermissive temperature is due to an elongation of part of the cell cycle, so that a smaller proportion of the cells lie in the synthetic (S) phase of the cell cycle with a consequent reduction in the fraction labeled cells. The uridine labeling patterns of the mutants appear to rule out a ribsomal lesion. Low temperature growth inhibition of both cell strains was reversible. In one of the cell lines, an apparent stretching of the cells at the low temperature produces substantial alterations in cell shape.