Free running synchrony

Fig. 18. Free-running synchrony after four repetitive heat shocks as a function of shock temperature. In each experiment the height (temperature) of the shocks is fixed, but it is varied from experiment to experiment as indicated.

Fig. 21. Free-running synchrony by four heat shocks (upper continuous curve) and restricted repetitive synchrony by four to five heat shocks (upper continuous curve extended by dashed curve). Results of electronic cell counts and of chemical analyses refer to the restricted system at the end of shock 5 (7.2 x 10 cells/ml) DNA 12.5 RNA 344 protein 3,330 ju/ig/cell. Tetrahymena cells synchronized repetitively and under restricted conditions are of almost the same size as are normally growing cells. This will be apparent from comparison of the figures just presented with those in the legend accompanying Figure 11 in the review by Zeuthen Rasmussen, 1971.

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. 

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... 

Test 3, II. Cultures were synchronized using shock temperatures ranging from 30.8 to 35.8°C. The free-running systems obtained with four shocks are illustrated in Figure 18. The conclusion is that synchrony of good quality is obtained with shock temperatures between 31.8 and 34.3°C. In the following discussions the first chosen shock temperature, 33.8°C, is henceforth fixed (see A). 

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.

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. 

In all free-running synchronous systems there is quick decay of synchrony. In the case of Tetrahymena this pertains to synchrony obtained with multiple heat shocks, with repetitive shocks, and by collection of normal cells in the division stage. In all cases the rate of decay of synchrony is about the same, which immediately tells us that no cure for the special weaknesses in the system obtained with multiple heat shocks (p. 134) can be expected to improve the synchrony greatly. Full synchronization would always be resisted by the existence of a natural spread in cell generation times in the population... 

---