Synchronous cell division

For certain aspects of work with cell cultures, a useful property of tunicamycin is that it can be used to synchronize cell division. After release of the block imposed hv the drug, the cloning efficiency was also higher, and the cloning size more regular, than in the control culture.547... 

Fraser, R.S.S., Studies on messenger and ribosomal RNA synthesis in plant tissue cultures induced to undergo synchronous cell division, Eur. J. Biochem., 50, 529-537, 1975. 

Scherbaum, O., and Zeuthen, E. (1954). Induction of synchronous cell division in mass cultures of Tetrahymena piriformis. Exp. Cell Res. 6, 221-227. 

This was possible to detect because the monoculture of Thalassiosira rotula employed showed partly synchronized cell divisions during exponential growth. Brockmann et al. [137] carried out combined measurements of dissolved amino acids and carbohydrates. Glucose and lysine occurred in highest concentrations. Mague et al., [22] found that extracellular production of free amino acids counted for 7.1% of the of the total extracellular C released in an exponentially growing culture of S. costatum Myklestad et al., [26] measured 10.7% for C. affinis or 3.6% when calculated as percent of total incorporated cell N. In contrast to this Admiraal et al., [139] found that none of three benthic diatoms released more than 0.1 % of the cellular N as free amino acids and concluded that benthic diatoms may act as net consumers of amino acids. Several authors did measure both intracellular and extracellular concentrations of many amino acids [22 140 -142]. The clear difference in relative composition of intracellular and extracellular fractions as pointed out by the first mentioned of these authors, show that the released pool is not just a portion of the intact cells content. 

In conclusion, an inhibitor of thymidylate synthesis and uptake and thus of DNA synthesis has been found. It consists of two components, methotrexate and uridine. It inhibits DNA synthesis to the extent and in ways which lead to blockage of the next cell division. This would be expected if DNA replication itself or events coupled to progression in the S phase are required to take the cell on to division. Inhibition by methotrexate + uridine can be discontinued by addition of excess thymidine, or just by washing of the cells with fresh growth medium, with or without added folate. These agents shall be used as tools in attempts to control DNA replication independently of cell division in populations with temperature-synchronized cell division. 

SYNCHRONIZATION OF DNA REPLICATION IN A SYSTEM WITH INDEPENDENTLY SYNCHRONIZED CELL DIVISION... 

In conclusion the results with methotrexate + uridine shown in Figures 10 and 11 suggest that in cells treated with heat shocks for the purpose of inducing synchronous cell division, the time from the beginning of DNA replication to a midpoint in the first cell division (S + G2 + 1/2 D) is at a minimum average, 130 to 135 minutes, which is close to the duration of these phases in normally multiplying cells. In the heat-treated, but otherwise undisturbed, cell system the actual duration of S + G2 + 1/2 D for individual cells is variable and in many cells is much extended. As referred to on page 134, this reflects variation in the duration of G2. 

Up to now we have assumed that cells in S are immediately stopped by methotrexate + uridine, and that cells not in S when this inhibitor is present continuously collect just before S. It is on this basis that we can assume that the time from the addition of thymidine to synchronous cell division is a measure of the interval S + G2 + 1/2 D. A second tacit assumption is that cells treated with methotrexate + uridine for some time can be made to progress in the cycle at the normal rate when supplied with thymidine. Neither of the two assumptions may be fully correct, but fortunately errors involved are likely to cancel out. Concerning the first assumption, there is in fact evidence that cells treated with methotrexate + uridine may progress perhaps 10 minutes into S before they are arrested (see Table 5 in Zeuthen, 1968) which might fit the observations by Stone et al. (1965) that acid-soluble thymidine compounds are carried over in the macronucleus from one S period to the next and are used for DNA synthesis. In any case, such effects... 

We have referred much to the latest possible placement of the replication phase which precedes and conditions the first synchronous cell division. However, we also pointed out that the actual time from the division-relevant S period to the synchronous... 

This report has indicated that truly synchronous cell division is limited to the major fraction of cells that initiates replication prior to a time point midway between the two last heat shocks. At this time the population lacks the information that the heat treatment will be discontinued after the next heat shock, and cells continue to engage asynchronously in DNA synthesis. Experiments indicate that this occurs until around the time (EH + 40 minutes) when the next heat shock would have occurred. We have argued that cells that replicate late in the program of heat shocks perturb the division synchrony and subsequently perturb the DNA replication synchrony. It is therefore suggested that further work on temperature synchronization of Tetrahymem cell division should be directed toward the goal of preventing new engagement in DNA replication after a critical time in advance of the synchronous division. 

The present experiments are based on the theory that temperate shocks synchronize cell division and that synchronous cell division synchronizes DNA replication. However, they give no new clues about the mechanisms that operate to arrange cell division and DNA replication sequentially, and the synchrony of DNA replication observed autoradio-graphically falls much behind the hopes with which this investigation was started. See addendum, note 4 p. 149. 

Apparently, almost perfect repetitive synchrony of both cell division and of DMA replication is obtained after five shocks when these are separated by a time (in this case 160 minutes at 28 which equals the generation time of the same cells (in this case 158 3 minutes) grown exponentially at constant 28 Cy the optimum temperature. In Figure 25 the duration of both synchronized cell division and synchronized DNA replication are close to what is seen in the normal cell, and the two phases are normally separated by a short G1 period (see legend foi Fig. 25). On the other hand, the S phase and the next cell division are farther apart than is normal. This extension of G2 equals, or at least does not... 

Fig. 24a. Inhibition of synchronized cell divisions (system of Scherbaum and Zeuthen, 1954) with combinations of 5-fluoro>2 deoxyuridine (FUdR), uridine (U), and thymidine (T). b. Same, only 5-fluorouridine (FUR) is used.

Hjelm, K. K., and E. Zeuthen. 1967a. Synchronous DNA synthesis induced by synchronous cell division in Tetrahymena. C. R. Lab. Carlsberg, 36 127-160. 

Plesner, P. 1961. Changes in ribosome structure and function during synchronized cell division. Cold Spring Harbor Symp. Quant. Biol., 26 159-162. 

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