Erik Zeuthen

Synchrony in Tetrahymena by heat shocks spaced a normal cell generation apart

Abstract Repetitive synchrony in cultures of Tetrahymena pyriformis, amicronucleate strain GL, grown in proteose peptone, liver extract medium, can be induced and maintained with heat shocks similar to those used in the multi heat-shock procedure of Scherbaum & Zeuthen (34 °C, 1 2 h ) but separated, not by 30–40 min at 28 °C but, by 155–160 min, which is the duration of a cell generation at constant 28 °C. Synchronous division and replication follow each other normally except that G 2 is extended by the duration of the heat shock. In repetitive synchrony each heat shock is initiated each time the population has essentially passed macronuclear S. At this time only few cells have yet undergone transition to insensitivity to heat in terms of capacity to divide. Thus the shocks are placed in early G 2. The sequence of recovery from a shock, followed by division and replication occurs at constant 28 °C, and this may be essential for good replication synchrony. When shocks are discontinued, free running synchrony decays through three divisions. The intervals between free running divisions are slightly short, and this accounts for setback of free running division number two in the repetitive synchrony, and presumably for synchrony itself.

Thymine starvation by inhibition of uptake and synthesis of thymine-compounds in Tetrahymena

Abstract In Tetrahymena populations, which are heat-synchronized in a proteosepeptone, liver medium, addition after the heat-shocks of a folic acid analogue, 0.05 mM methotrexate (M), plus 5 mM uridine (U), inhibit the second synchronous division in most cells. This inhibition can be specifically overcome with thymidine. M slightly stimulates DNA-synthesis as measured by autoradiography with 3H-thymidine. M + U inhibits DNA-synthesis strongly, but only after initial stages of S have been passed. Uridine may interfere with mechanisms which transport thymidine into the cell or into the macronucleus.

Heat shock proteins in Tetrahymena studied under growth conditions

Abstract Heat shocks or cold shocks were found to induce characteristic changes in the pattern of protein synthesis in Tetrahymena pyriformis as analysed on sodium dodecyl sulphate (SDS)-polyacrylamide gels. Within 10 min the temperature shift-up induces increased incorporation of [ 35 S]methionine into 4–5 protein bands, whereas incorporation into other bands is reduced, The heat-induced proteins appear to be metabolically stable, and no change in protein turnover could be detected at the elevated temperature. The protein induction is not observed when RNA synthesis is inhibited by actinomycin D. The temperature shift-down induces a different pattern of protein synthesis and there is no evidence of a common molecular mechanism underlying synchrony induction with heat shocks and cold shocks.

Effects of cytochalasin B on cell division and vacuole formation in Tetrahymena pyriformis GL

Abstract Cytochalasin-B (Cyt-B) was tested for its effect on cell division and vacuole formation in Tetrahymena . Little effect was found on cell division in the synchronous cell system in concentrations up to 37 μg/ml; however, slight delay was caused by 71 μg/ml. As measured by particle uptake, much lower concentrations, 7–8 μg/ml, caused significant inhibition of vacuole formation in exponentially multiplying and in starved cells, 16.6 or 37 μg/ml caused strong inhibition. This effect was immediate and completely reversible. The presence of Cyt-B caused starvation of Tetrahymena . The essential absence of inhibition of cell division by Cyt-B may reflect that the drug can enter the cell only by way of vacuoles.

DNA replication sequence in Tetrahymena is not repeated from generation to generation

Abstract Tetrahymena pyriformis were synchronized by treating the cultures with 30 min heat shocks of 34 °C, separated by 160 min intervals at the optimal growth temperature, 28 °C. In this synchronized system all cells initiate DNA synthesis immediately after cellular division. The progression of DNA replication was followed by BUdR incorporation. After CsCl-gradient centrifugation of DNA isolated at various times of the synchronous S period, the distribution of newly replicated (BUdR labelled) versus non-replicated DNA showed that all DNA of the culture is replicated in one synchronous S period, lasting 75–90 min. This is the duration of S in the single cell (ca 60 min) plus the spread of cell divisions in time (15–30 min). All DNA is replicated once in each synchronized S period, and double replication has not been seen. To study whether the sequence in which DNA molecules replicate in one synchronized S period is repeated in the next, DNA was pulse labelled early or late, respectively, in one S period, and the time course of the next replication of this labelled DNA was followed. The results indicate that the second replication of the labelled DNA took place throughout the whole next S period. Thus the replication sequence in one S period is not repeated in the next.

Synchronization of DNA synthesis in Tetrahymena populations by temporary limitation of access to thymine compounds

Abstract Tetrahymena pyriformis , amicronucleate strain GL, was grown exponentially in 2 % proteose peptone plus 0.1 % liver extract. The synthesis of DNA was inhibited fully and selectively by uridine (20 mM), or thymidine (2 mM). After a generations time (3 h) synthesis was resumed at nearly normal rate. Cell divisions were not affected, so with these agents replication of DNA can be strongly inhibited without disturbing the forthcoming cell division. When combined, uridine and thymidine inhibit DNA synthesis less strongly than each agent separately. When uridine was combined with a folic acid analogue, 0.05 mM methotrexate (4-amino- N 10 -methyl-pteroylglutamic acid), both DNA synthesis and cell divisions were inhibited. DNA synthesis stopped quickly, but cell divisions continued until the system had emptied of cells in division stages, and in G2. Release of these inhibitions could be made with thymidine. When this agent was added to cells incubated with uridine plus methotrexate for nearly a generations time, synchronous DNA synthesis occurred immediately, and synchronous cell division followed.

Synchronization of the Tetrahymena Cell Cycle

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.

Induced reversal of order of cell division and DNA replication in Tetrahymena.

Abstract The sequence of macronuclear DNA replication (S) and cell division (D) in two normal or synchronized cell cycles in (amicronucleate) Tetrahymena can be symbolized S 1 , D 1 , S 2 , D 2 . Using heat shock synchronized cells, the final heat shock (33.8 °C) has been extended from 20 min to 5 h. This leads to an altered sequence: S 1 , S 2 , D 1 , D 2 , characterized by reversal of D 1 and S 2 . S 2 occurs during the extended shock, D 1 comes in standard time after the shock has been discontinued. Thus prolonged stay at elevated temperature can dissociate two cell cycles into a subcycle of DNA replication followed by a subcycle of cell division. S 1 and S 2 charge the cells with four times the amount of DNA in newly divided cells, and D 1 and D 2 partition this DNA to four cells. Synthesis of DNA between D 1 and D 2 is not required, and it occurs in only few cells.

Independent synchronization of DNA synthesis and of cell division in same culture of Tetrahymena cells: Demonstration that heat-shock induced division-synchrony is by differential extension of the G2 phase

Abstract When Tetrahymena populations are treated with heat shocks according to Scherbaum & Zeuthen there is transition from asynchrony to synchrony of both cell multiplication and DNA replication. The transitional interval (blockage of activity considered) is much longer in the first than in the second case, and this is why during the period with heat shocks asynchronous DNA replication can continue in the division-blocked cells. In this study the phase in which divisions are blocked with heat shocks is used for synchronization of DNA synthesis (control of supply of thymidine), so replication and cell division are under separate synchrony controls. The experiments presented with this system lead to the suggestion that always the last replication round in a division-blocked cell conditions the cell for a next division. If this round is separated from the synchronous division by a time which equals or exceeds the time between replication and division in normal cells, then the cell will take part in synchronous division. If the separation is shorter than this, the cell will participate in the synchronous division with some delay, or not take part. The results contribute to an understanding of mechanisms in the induction of synchronous cell division with heat shocks, and they shed light on deficiencies in the division synchronized system.

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

Abstract This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena . Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.