148 CELLS, TISSUES, AND ORGANISMS 



philic and micronucleate mating type I, variety I. This strain requires 

 five alternate exposures to 42.6° and 35° C. The cycHng heat catches 

 the cells in the formation of the second mouth. The necessary multipli- 

 cation of the kinetosomes of this region occurs during the cycHng heat, 

 but the organization of the kinetosomes into a new mouth is blocked. 

 The micronucleus is caught in the mitotic anaphase. It is only 40 to 50 

 minutes after the return to constant 35° C. that the micronuclear divi- 

 sion and the stomatogenesis are resumed. The first cell division occurs 

 after 50 to 60 minutes at 35° C; the second comes 65 to 75 minutes 

 later. So, after the heat (.42.6° C), the cells spend perhaps 75 per cent 

 of the time before maximum division in preparing the kinetic phases 

 of the later cell division. The guess can be made ( see also Holz et al., 

 1957) that the cycling temperature blocks or severely slows the forma- 

 tion of specific proteins which play their part in division by supplying, 

 or conditioning the operation of, the kinetic machinery. 



Biochemical mechanisms in the induction of division synchrony 



When the large synchronized cells are returned from the last tem- 

 perature shock to constant temperature conditions, we can assume 

 that the division-promoting metabolic pattern is maximally disor- 

 ganized. We can expect to interfere either with its reorganization or 

 with new products formed by its activity if we let the cells recover in 

 the presence of defined metabolic antagonists. If the normal counter- 

 part of a metabolic analogue is involved in the preparation of the first 

 synchronous division, then this division will be delayed. 



For the present studies we synchronize strain GL the standard 

 way. The cells are then transferred by three washings to an inorganic 

 medium. An extra heat shock is applied so that we can operate from 

 the very moment when the temperature is returned to constant 28° C. 

 and up to the time of the first division maximum. Parallel samples are 

 removed at intervals for incubation at 28° C. or, more simply, at room 

 temperature. To one or more samples an anti-metabolite is added; one 

 sample is kept as a control. The samples are inspected at intervals for 

 the percentage of cells in stages of cytokinesis. Curves through the 

 estimated points allow us to fix in time the point of maximum division 

 activity with an accuracy of about ± 3 minutes. Possible diflFerences 

 between the control and a sample which is incubated with a drug is a 

 measure of the division-delaying effect of the anti-metabolite. Thirty or 

 more samples are easily handled in one day. In this study we consider 

 differences of more than ten minutes between samples significant. 



The synchronized cells are exceedingly sensitive to the presence 

 of analogues to amino acids. Figures 10 and 11 illustrate an experiment 



