Division synchrony

D) to break or circumvent the dormancy, the cells enter mitosis (Bennici et al., 1982). The first and second divisions are well synchronized (Serafini-Fracassini et al., 1980) however, with further divisions, synchrony is gradually lost (Yeoman et al., 1965). Likewise, there are marked changes in the timing of the first and second cell cycles with the progression of dormancy (Bennici et al., 1982). 

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. 

We have reached the conclusion that a way to obtain better synchrony in Tetrahymena populations may be to provide conditions such that new engagement in DNA replication does not take place after a critical time prior to the expected division. There may be many possible solutions to this problem, and one may be to arrange synchrony of DNA replication at a suitable time prior to the synchronous division. This approach was dealt with from one aspect in the preceding section. Clearly, the procedure there chosen to synchronize DNA replication has limitations, and neither by itself nor in combination with heat shocks did it result in improvements of the division synchrony. In this section we shall proceed differently and use only temperature changes. 

While the studies here reported have not resulted in any improvement of the division synchrony over what we have had for years (see, however, addendum, note 4, p. 149), they have yielded a repetitively synchronized Tetrahymem system that seems superior to its nearest predecessor (system of Padilla and Cameron, 1964) in that cell division is better synchronized, macronuclear DNA synthesis is synchronized, and it shows good free-running synchrony. An expected advantage of the repetitively synchronized system over the one obtained with multiple heat shocks is that all cell parameters should double in each repetitive cycle (see p. 142 and Fig. 21). The weakness, that in this system the environment and cells are both cyclic, can be controlled by parallel analysis of the free-running system, i.e., under conditions when only the biologic cycle remains. 

The temperature induced division synchrony in Tetrahymena. In Synchrony... 

Since the first publication of the heat shock method of division synchronization of Tetrahymena there has been a steady stream of reports dealing with the system. Many investigators have used the system as first intended, for the chemical analysis of the cell cycle. Others, among them the writer, have taken more interest in problems that concern mechanisms by which the heat shocks force into good synchrony cells that had previously divided over a spread of time equal to the duration of a full cell generation. There is as yet no final explanation of this phenomenon, but there are interpretations based on many experiments, and these are dealt with in this chapter. 

In bacterial and mammalian cell systems in exponential growth new DNA synthesis occurs in synchrony when the blockage of DNA synthesis by selective thymine starvation is released after a period. The same is true in Tetrahymena cultures. This is seen from Figure 7, in which 2 mM thymidine was added after an exponentially multiplying population had been treated with methotrexate + uridine for 3 hours (3 hours is nearly equal to one generation time). Synthesis of DNA began immediately after thymidine was added, and cell division occurred 110 minutes later. 

In most cells the DNA replication that is required for the forthcoming synchronous division 2 begins at the time of synchronous division 1. Most likely, synchrony of this replication phase is obtained in direct response to biologic signals rather than to environmental variables. It will be useful to compare studies directed at this replication period with those just reported for the system with artifically synchronized DNA replication. 

In this section we shall look for evidence that imperfections in the synchrony, first of division then of DNA replication, reflect the fact that, in this system, cells can engage in DNA replication beyond a critical time limit antecedent to the time when most cells show synchronous division. In a preceding section it was reported that asynchronous entry of the cells into DNA replication continued for 40 or so minutes after the end of heat shocks. Also, evidence was presented that there is a time point 35 or so minutes before the shocks are ended, after which no cell that takes part in synchronous division at its peak can have entered into DNA synthesis. What is then the contribution to synchronous division of the fraction of cells that in fact enter DNA synthesis in the critical 1.25-hour interval between the two indicated times These cells will not have had time to finish S and normal G2 before they are supposed to participate in the first synchronous division. We shall see that in fact they contribute negatively to the synchrony, either by dividing later than at the time when synchronous division is at its peak or by not dividing at all. 

We have seen that the cells that initiate DNA replication in a critical interval around the time when the heat shocks come to an end represent a nuisance as far as synchronization of cell division is concerned. We shall now present evidence that these cells also tend to reduce the degree of synchrony of DNA repUcation which is seen after the first synchronous division. 

Fig. 15. Repetitive synchrony by heat shocks. Functions of the number of shocks applied (abscissa) and of the time in minutes between shocks (see frames). The shock temperature and duration are 33.8°C and 30 minutes. The temperature between shocks is 28°C. This information applies also to Figures 16 through 22 if not otherwise stated. Left ordinates. Percent of cells in division at the time of the first (x) and the second division maximum ( ). Right ordinates. Time in minutes from termination of final shock to the time of the first division maximum (o), or between division number one and two ( ). Note that the right ordinates have been shifted upward in the frame marked 150. Figure 15 through 22, and 24, 25 are from the author s work, and are here published for the first time.

D. Test 2. A -f- C fixed, check of B. (To understand the following the reader is invited to first carefully inspect the upper curves for percent dividers in Fig. 21, p. 143). The system obtained under C is used free running after five shocks. Its quality (see below. Fig. 21) approaches the one previously obtained with multiple shocks. In both systems synchrony decays through three divisions. A sixth (analyzing) heat shock is initiated 150 minutes after the termination of shock 5. This shock comes after the first synchronous division and affects the second free-running synchronous division, which is delayed and at the same time raised in quality to that of the first synchronous division in a free-running system obtained with six shocks. In Figure 16, the... 

Fig. 16. Setback (upper curve, left ordinate) and gain in synchrony (curve II, right ordinate) as functions of the duration (abscissa) of an analyzing heat shock (33.8°C) that affects the second synchronous division in a system that runs freely after five repetitive shocks. The analyzing shock begins 150 minutes after shock five.

Fig. 19. Functions (ordinate) of the duration of the intervals between repetitive heat shocks (abscissa). Repetitive synchrony by four to six heat shocks. Curves II and III show how the time between shocks is split into predivisional (II) and postdivisional time (III) by the synchronous division. Curve I shows the delay of the second free-running division by the next heat shock in the series. Curve IV, The ordinate shows the time between synchronous divisions numbers 1 and 2 in free-running systems.

In Figure 20 four experiments have been combined. They are fitted end to end with some overlap, and together they demonstrate how synchrony of cell divisions (upper curves) develops in the course of three heat shocks (33.8°C, 30-minute) separated by intervals of 150 minutes at 28°. The estimated highest percent cells in the five synchronous divisions which later follow shocks 3 through 7 varies between 70 percent and 90 percent. In fact, the estimates may reveal a periodicity of 9 to 12 hours which has escaped notice up to now. If it exists, this system is not in order yet, but experimentation with parameters is still in progress. The lower curves show the percentage of the cells that incorporates thymidine into nuclear DNA in 15-minute pulses. As is seen, each synchronous division is closely followed by a period in which many cells are in DNA synthesis at the same time, much the same as when synchronization is brought about with multiple heat shocks. The curves also indicate that a next heat shock in the series comes... 

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. 

One observation of interest has been made and is illustrated in Figure 22. The question is what happens if we synchronize repetitively, using four shocks spaced 150 minutes apart, and then shift to the multiple heat shock procedure. We know by now that the first four shocks can start a train of synchronous, free-running cell divisions, each closely followed by synchronous DNA replication. However, when the shift is made as described to the multiple shock procedure, divisions are suppressed but DNA replication continues as a cyclic, though damped, phenomenon occurring with a period of nearly four hours (lower curve, main culture). Samples were removed at the end of each new heat shock. They displayed synchronous division after 1.5 hours at 28 C. The quality of this synchrony (upper curves) bears a distinct relationship to the phase in the DNA replication cycle at the time the sample was removed from the main culture. 

At the time when the removed samples divide they have had a different fate for the past 40 to 50 minutes than the main culture, which in this period received a new heat shock. Nonetheless, the suggestion can be made that the poorest synchrony of division... 

Fig. 22. Synchrony induction by four repetitive 30-minute shocks at intervals of 150 minutes, plus zero to four 20-minute shocks at intervals of 40 minutes. Lower curves. Percentage of nuclei labeled with thymidine in 15-minute pulses. Points are placed at the end of the incorporation intervals. In this case divisions were suppressed for the time covered in the figure, the cells receiving four repetitive and five more shocks (4 + 5 shocks). Upper system of curves, The cells are set free to divide after having received the temperature shocks indicated. Curves represent estimated percentage of dividers at the time of the first synchronous division.

We have suggested that good repetitive synchrony of cell division is obtained when heat shocks are separated by 150 minutes, a condition under which a new heat shock hits the cell population toward the end of S. On the other hand, the synchrony of replication obtained under these conditions is not impressive. 

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... 

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