Radiation-Induced Mitotic Delay in Sea Urchin Eggs Treated with Puromycin and Actinomycin D

Abstract

The inhibition of protein synthesis by puromycin, which functions as an analog of amino-acyl sRNA (1), appears to result from a release of uncompleted protein chains from those ribosomes engaged in protein synthesis (cf. 2-4). Hultin (5) demonstrated that the compound inhibited amino acid incorporation into proteins in fertilized sea urchin eggs. Furthermore, he found that the cells were blocked specifically in the stage of either the first or second mitotic cycle. This mitotic stage corresponds to the time in early prophase when the centrioles divide and migrate to opposite sides of the nucleus, and the fan of astral rays pushes aside particles, forming a clear streak of cytoplasm adjacent to the nucleus (6). Irradiation prolongs the stage (7, 8), and the first division is delayed only if the cells are irradiated before this stage is clearly visible (9, 10). Since both puromycin and radiation affect the same mitotic stage, possible interactions between the two agents were investigated. Actinomycin D binds to DNA and inhibits the synthesis of RNA (11-13). Gross and Cousineau (14, 15) studied the effects of this drug on the development of sea urchin eggs and found little or no mitotic delay when as much as 96 % of the RNA synthesis was inhibited. However, differentiation beyond the ciliated blastula stage did not occur. Their experiments are consistent with the view that the unfertilized sea urchin egg contains a reservoir of inactive messenger RNA, which participates in protein synthesis after fertilization, and that RNA synthesis per se is not necessary for the early cell divisions (see also 16-19). If irradiation delays division by destroying an essential fraction of the existing RNA or by producing a lesion which requires new messenger RNA for its repair, irradiated cells might prove sensitive to actinomycin D. This possibility was investigated.