Search

The incorporation of tritiated thymidine, uridine and leucine, into the acid-precipitable material of DNA. RNA and proteins, respectively, was studied by autoradiography throughout the cell cycle of Amoeba proteus. DNA synthesis occupied the first 17 h of the cycle (57 h long) and 2 peaks between 0.5 and 9.13 h accounted for the majority of the thymidine incorporation. RNA synthesis was represented by a series of peak uridine grain counts, the 3 major peaks occurring at 10, 26-27 and 47-48 h. The incorporation of leucine also followed a pattern of peaks and dips, the main peaks occurring 1-2 h after the major increases in uridine incorporation. The fraction of label present over the nucleus decreased during the cell cycle, and this was probably due to a lowered incorporation of the leucine label by proteins synthesized in the cytoplasm and destined to become nuclear proteins. The incorporation patterns of 6 amino acids (arginine, aspartic acid, leucine, lysine, serine and valine) were studied individually during 3 periods of the cell cycle: 0-10 h (S phase); 20-30 h (early G2); and 40-50 h (mid-late G2). Variations in the intensity and timings of the incorporation maxima of the amino acids were observed, although the timings of increased grain counts of some of the amino acids frequently coincided. "Unique" incorporation peaks (i.e. only observed in one of the amino acids studied) possibly indicate the synthesis of phase-specific proteins. The amino acid and nucleoside incorporation profiles presented in this paper will enable the results obtained from future studies on amoebae to be related to the macromolecular synthesis patterns.

The fine structure of the nuclei of synchronously growing cell population of Amoeba proteus was studied at I-h intervals during the interphase. This study showed that the nuclear helices undergo increases in their number at certain stages during interphase. These changes were found to correlate with ultrastructural changes occurring in the nucleoli.

Tracer kinetic studies have revealed the existence of a variable pattern of 3H-amino acid incorporation into amoeba proteins during the early G2 phase of the cell cycle. Two peaks of incorporation of [3H]leucine were found to occur at 19 and 22 h, whereas a single peak at 17 h was noticed in the amoebae labelled with [3H]lysine. An almost 2-fold increase of the labelled amino acid incorporation occurred during the peak periods, while the other periods showed a more or less steady state of incorporation, suggesting a basal rate of synthesis at these times. In a detailed study involving the peaks and the basal incorporation period of [3H]leucine, it was shown that the removal of the nucleus or Actinomycin D treatment eliminated the peaks but the base line protein synthesis was not affected. This suggests that for the peak synthetic periods, mRNA is probably transcribed concurrently, followed by immediate translation, whereas long-life mRNA accounts for the basal synthetic activity.

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.