Discrepancies in mitotic cycle time when measured with tritiated thymidine and colchicine

Abstract

Tritiated thymidine ( 3H-T) and colchicine were compared with respect to their use in measuring the mitotic cycle duration of root meristem cells of Pisum sativum. The mitotic cycle of Pisum root meristem cells at 20 °C ± 1 ° was measured by (1) labeling with 3H-T (spec. act. 5.3 × 10 3 mC/m M, 2 μC/ml) for 30 min and observing the sequential appearance and disappearance of labeled mitotic figures, (2) treatment with 3.76 × 10 −4 M colchicine for 30 min and observing the eventual appearance of tetraploid mitotic figures and (3) simultaneously treating the meristems with 3H-T and colchicine. The latter procedure distinctly marks cells in two different segments of the mitotic cycle. Cells entering mitosis are affected by colchicine in such a manner as to prevent cytokinesis but not karyokinesis; these cells appear as tetraploids in the subsequent mitosis and thereby provide a measurement of the mitotic cycle duration. Since DNA synthesis in Pisum takes place approximately midway in interphase, 3H-T labeled cells appear in mitosis before the tetraploid cells. When the 3H-T labeled cells are in division, the tetraploid cells are in interphase and vice versa. Thus the combined use of 3H-T and colchicine provide a quick and accurate measurement of the interphase period durations. The results indicate that (1) the first mitotic cycle following pulse labeling with 3H-T averages 1.4 times longer than that measured with colchicine, and (2) the increase in the cycle time measured by 3H-T appeared to occur in G 1. The elongated cycle as measured with 3H-T was hypothesized to be either the result of chronic β-irradiation from tritium located in the cellular DNA or the result of different population constituents composing the 3H-T labeled cells and the colchicine-induced tetraploid cells.