A cell mutant of the Chinese hamster ovary line, which is temperature sensitive for protein synthesis, is specifically defective in vivo in its ability to charge tRNA with leucine. Cytoplasmic extracts exhibited temperature-sensitive leucyl-tRNA synthetase activity. It is, therefore, highly likely that the mutant has a structural alteration in leucyl-tRNA synthetase. The lowleakiness and low reversion rate of this mutant, combined with the specificity of the defect in its protein-synthesizing machinery, make it an appealing tool for investigating regulatory mechanisms in animal cells.Our understanding of biological processes at the molecular level has been advanced immeasurably by the judicious combination of genetics with biochemistry. This approach has played a far more important role in microbial systems; systems involving somatic mammalian cells have lagged behind, due in part to the paucity of well-defined mutations. We (2, 4) and others (3) have been developing selection procedures for the isolation of temperature-sensitive (ts) conditional lethal mutant lines of mammalian cells, and various mutant phenotypes isolated from various cell lines are now becoming available for study. Although there is indirect evidence that many ts as well as drug-resistant and nutritional-auxotrophic phenotypes are likely due to true mutations [see review by Thompson and Baker (4)], there have been only a few explicit demonstrations that altered gene products are associated with altered phenotypes (5). It is important to know whether ts mutant lines actually contain proteins (or other gene products) that are temperature sensitive, especially since some workers have proposed that altered phenotypes induced in culture might be explained by nongenetic modifications (6).We describe here a mutant of Chinese hamster ovary cells which has a temperature-sensitive leucyl-tRNA synthetase, the enzyme that adds leucine to tRNALU. A preliminary observation that protein synthesis declined rapidly and profoundly at the nonpermissive temperature attracted us to this mutant, as the protein-synthesizing machinery has been implicated in the control of proliferation of animal cells (7). Elucidation of the molecular basis for the phenotype of this mutant together with its stable genetic character render it very attractive as a device for investigating various molecular regulatory phenomena in animal cells. MATERIALS AND METHODSCells and Culture Conditions. The wild-type cells were a clone of the Chinese hamster ovary line (CHO) (8), which has a stable karyotype consisting of 21 chromosomes with little cell-to-cell variation (9). Cells from frozen stocks of this line and of the mutant tsHl were maintained in exponential growth in suspension culture at 34°. The growth medium was a-minimal essential medium (10) supplemented with antibiotics and 10% fetal-bovine serum (Flow Laboratories). Temperature was monitored with matched precision thermometers and was carefully controlled with water baths with a precision of 40.1'. The thermometers were ...

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Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.

When CHO cells are incubated under conditions of extreme amino acid starvation, effected by withdrawal of an amino acid from the medium together with genetic or chemical interference with the activity of the corresponding aminoacyl-tRNA synthetase, there is a rapid and profound decline in the functional capacity of the protein synthetic machinery. The effect was observed for all amino acids tested including leucine, asparagine, histidine, methionine and glutamine. This decline in protein synthetic potential appears to be due to a progressive permanent inactivation of the specific aminoacyl-tRNA synthetase concerned, as shown by a decline in the amount of cellular, specific aminoacyl-tRNA and a decline in the cell-free enzyme activity, measured after reversal of the starvation conditions. When cells are left for more than several hours under these starvation conditions, they shrink in size, lose viability and eventually disintegrate, with anomalous rapidity. We suggest that the progressive loss of protein synthetic capacity of the cells is the prime cause of these subsequent events. If the starvation conditions are reversed before cell death, regeneration of the protein synthetic potential occurs rapidly but requires protein synthesis itself, implying the existence of strong control mechanisms for cellular aminoacyl-tRNA synthetase activities.