Quantitative cytophotometric and autoradiographic studies on the rate of protein synthesis during interphase in mouse fibroblasts in vitro

Abstract

An investigation was made of the rate of the overall protein synthesis during interphase growth in mouse fibroblasts in vitro (L-cells). Quantitative microspectrophotometric and microinterferometric methods were used for the determination of the cellular content of DNA, RNA and dry mass. The stage which the cell had reached in its interphase development was determined in two different ways. Firstly, the cell age, expressed in hr after the last cell division, was determined in a direct way by means of time-lapse cinematography. Secondly, the cell age was determined in an indirect way from the relationship between cell mass and relative cell age (the fraction of the total interphase time). This relation was derived from the mass distribution of individual cells in the growing fibroblast population. The amount of 14C-leucine, 3H-leucine and 35S-methionine incorporated into the cell protein after pulse incubation was quantitated by means of autoradiographic methods and used as a measure of the overall rate of protein synthesis in the cell. The absorption of tritium radiation in the cell was taken into account. The rate of incorporation of protein precursors into the growing cell increased over the whole of interphase by a factor of approximately 2. This result is in very strong agreement with previously published interferometric data on L-cells [17], which showed that the cellular dry mass also grew at an increasing rate over the whole of interphase by a factor of 2. The correlations between the number of grains over the cells and their DNA, RNA and mass content were investigated. The grain counts were found to have the best correlation with the cellular content of RNA and mass. These results support the idea that the total capacity for protein synthesis during interphase growth is controlled by the existing number of ribosomes in the cell.