Search

The amount of radioactivity incorporated into completed polypeptides and into nascent polypeptides bound to polyribosomes was measured for rapidly dividing, cultured hamster embryo cells incubated in the presence of labelled amino acids. Using a mathematical analysis which took into account variations in the specific activity of the intracellular amino acid pool, these measurements yielded a value for the length of time required for the synthesis of a primary polypeptide of average length, the "transit time." Subject to a number of reasonable assumptions in the mathematical analysis which were, however, only approximately verified, the transit time was found to be 23 +/- 2 sec in four independent experiments. Regardless of the absolute accuracy of this measurement, the method is considered a useful measure of translation independent of transcription.

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 low leakiness 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.

Using a new, sensitive and quantitative technique for determining the ribosomal-RNA content of a measured number of cells, the cellular ribosome complement was compared for cultured hamster embryo cells in the stationary growth phase and in the early G1 phase of the cell cycle. Cells from stationary phase cultures were found to contain less than 70% of the ribosome complement of the early G1 phase cells, though the volumes of the two cell types were similar. This would imply that the stationary phase cell is physiologically different from a cell merely arrested at some point in the cell cycle.

The rate of protein synthesis per cell in cultured hamster embryo fibroblasts in the stationary growth phase falls to about one third of the rate in the exponential growth phase. This reduction can be entirely accounted for by the following observations: (1) the average cell in stationary phase contains about one-half the number of ribosomes per cell compared to the average cell in exponential phase; (2) only two thirds of the ribosomes are bound to polysomes in stationary phase, while nearly all of the ribosomes are polysome-bound in exponential phase. In stationary phase, ribosomes which are polysome-bound function with the same efficiency and produce proteins of approximately the same average length as in exponential phase. Experimental findings are presented which suggest that the generation of a higher proportion of free ribosomes in stationary phase in not due to a limitation in messenger RNA, but to a decreased attachment probability of ribosomes to messenger RNA.