Printed in Sweden Copyright © 1977by Academic Press. Inc. All rights of reproduction in anyform reserved ISSN 0014-4827
Experimental Cell Research 108 (1977) 259-268
NUCLEOSIDE
PHOSPHOTRANSFERASE
ACTIVITY THROUGH
THE GROWTH AND CELL CYCLE OF
TETRAHYMENA P YRIFORMIS GL-I N. C. BOLS 1 and A. M. ZIMMERMAN Department o f Zoology, University o f Toronto, Toronto, Ontario, Canada M5S 1A1
SUMMARY Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside
phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.
The patterns of enzyme synthesis during the course of the cell cycle have received considerable attention in recent years [1]. Generally, the patterns of synthesis can be divided into two broad groups [1]. In one group enzyme synthesis occurs continuously during the cell cycle while in the other group synthesis takes place only during a discrete period of the cycle. However, the pattern of synthesis for a particular enzyme can depend on the method used to obtain a synchronous culture [2]. Two basic formats, induction synchrony and selection synchrony, are available for synchronizing cells [1]. In induction synchrony a treat-
1 Present address: Institute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institutet, S-104 01 Stockholm 60, Sweden.
ment is applied to an asynchronous culture and enforces all the cells to divide synchronously while in selection synchrony a population of cells at a particular stage of the cell cycle are selected out of a non-synchronous population. A comparison of an enzyme's pattern of synthesis through cell cycles obtained by different techniques can point out correlations between different synthetic, physiological, or morphological events of the cell cycle. Through the course of the cell cycle of several organisms the activity of thymidine kinase (EC271.75), which uses nucleoside triphosphates to phosphorylate thymidine [3], has been found to increase at about the time DNA synthesis begins and to decrease during late G2 and mitosis [4-12]. This correlation between thymidine kinase Exp Cell Res 108 (1977)
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Bols and Zimmerman
activity and DNA synthesis is observed in cells synchronized both by selection and induction techniques. The periodic pattern of activity has been shown to reflect periodic synthesis of the enzyme [5]. As a result of the close temporal relationship between the synthesis of thymidine kinase and DNA, the suggestion has been made that this enzyme is involved in the regulation of DNA synthesis (for a review that is critical of this point of view see reference [13] page 21). The importance of this correlation might be better evaluated if the relationship between DNA synthesis and other thymidine phosphorylating enzymes was known. Nucleoside phosphotransferase (EC 2.7.1.77), which utilizes nucleoside monophosphates to phosphorylate a wide range of nucleosides including thymidine [ 14], has not been measured through the cell cycle. Recently, Tetrahymena pyriformis has been shown to contain a nucleoside phosphotransferase but no thymidine kinase [15, 16]. In as much as Tetrahymena can be synchronized by a variety of methods [17], this appeared to be an ideal organism to study nucleoside phosphotransferase activity through the cell cycle. We report that in Tetrahymena synchronized by both selection and physical induction techniques nucleoside phosphotransferase is synthesized continuously. In cells that are starved and then refed, the enzyme is synthesized discontinuously but shows no coupling to DNA synthesis.
METHODS AND MATERIALS
Culturing of organism Tetrahymena pyriformis, strain GL-I, were maintained in 2 % protease peptone (Difco Chemicals, British Drug Houses, Toronto, Ont.) supplemented with 0.1% liver fraction L (Nutritional Biochemical Corp., Cleveland, Ohio). This growth media was referred to as PPL.
Exp CellRes 108 (1977)
Synchronization by starvation-refeeding The starvation-refeeding synchronization procedure was performed as described by Cameron & Jeter [18]. An inoculum of stationary cells was used to initiate cell growth. The cells were grown without shaking at 28°C in PPL to cell densities of between 55 000 and 90000 cells/ml. At this time the cells were collected, washed in starvation buffer (0.6 g KH2PO4, 0.15 g K2PO4, and 0.25 g MgSO4, per litre with the pH adjusted to 6.5 with NaOH), and placed into a 2.5 litre low form culture flask which contained 160 ml of the same buffer. The initial starvation cell density was between 15 000-40 000 cells/ml. The cells were starved for 24 h during which time cell number increased approx. 50 %. Cells were refed by the addition of 200 ml of double-strength PPL. During the starvation period and after refeeding the cells were grown without shaking at 28°C.
Synchronization by one heat shock per generation Cells were also synchronized by the one heat shock per generation system developed by Zeuthen [19]. The experiment was started by the inoculation of either 3 ml of a l-day-old culture or 2 ml of a 2-day-old culture into 200 ml of PPL in a 2.5 litre low form culture flask. The cells were grown for 157 min at 28°C and then for 30 min at 34°C. This cycle of events was repeated six times. These changes in temperature were controlled automatically. The cells were shaken gently throughout this treatment. At the end of six heat shocks there were approx. 30 000-50 000 cells/ml.
Synchronization by differential density labeling Tetrahymena were synchronized by the differential density labeling technique of Wolfe [20]. The cells were grown at 28°C with shaking to either 1000015 000 cells/ml or 35 000-40 000 cells/ml, except for the cultures in the 10000 to 15000 cells/ml range, the cells were fed tantalum particles (type SGQ, Norton Metals Division, Newton, Mass.) at these cell densities. The culture at the lowest cell density was first concentrated to approx. 100000 cells/ml before being fed tantalum particles. This was necessary because future steps were limited by the volume of culture that could be rapidly centrifuged. Particles were added to a concentration of 1-2 mg/ml and were kept suspended by swirling the culture. After 5 min of feeding, the culture was centrifuged at 1200 rpm for 2 min. Most of the cell free supernatant was aspirated except for approx. 5 ml. The light dividing cells were now separated from the heavy non-dividing cells on a Ficoll gradient as described by Wolfe [20]. The portion of the gradient which contained the unlabeled cells was placed in sterilized PPL. The percentage of cells containing heavy particles in this fraction was between 0 and 3 %.
Nucleoside phosphotransferase activity in Tetrahymena Measurement of cell number and division indices Cell density was determined with a Model Z2 Coulter Counter (Coulter Electronics, Hialeah, Fla.). The division index was determined by counting the percentage of fixed cells showing cytoplasmic furrows on both sides. The division index and per cent increase in cell number was occasionally determined by the drop culture method as described by Cameron & Jeter [18]. Small drops of culture were placed onto disposable plastic Petri dishes by tapping on the surface of the dish with a Pasteur pipette containing a small portion of the culture. A layer of paraffin oil was gently poured over the surface of the drops to prevent evaporation. The cell number and the number of cells showing division furrows in each drop were determined at intervals. Generally, only late dividing cells were scored because this was the only stage which could be scored unequivocally with the low magnification of the dissecting microscope.
[aH]thymidine incorporation through the cell cycle The following method was employed to measure the rate of [3H]thymidine incorporation into acid-insoluble material through the cell cycle. At various times duri n g the cell cycle an aliquot of culture was incubated with [3H]thymidine (spec. act. 20 Ci/mmole) to give either 2 or 10 tzCi/ml. The incubation of this culture was carried out at 28°C without shaking for the starved-refed cell cycle and with shaking for the other cell cycles. The incubation was terminated after either 10 or 20 min by the addition of an equal volume of ice cold 10% trichloroacetic acid (TCA). Extraction was carried out overnight at 4°C. The precipitate was collected by centrifugation (International Clinical centrifuge) at 1500 rpm for 2 min and washed twice with ice-cold 5 % TCA. Finally, the precipitate was collected on a glassfiber filter (grade 934AH, Reeve Angel, Clifton, N.J.) that had been soaked in 5 % TCA containing 1 mg of thymidine per ml. The filter was washed three times with ice-cold 5 % TCA which contained 1 nag of thymidine per ml and further washing and counting was carded out as described previously [21].
Preparation of ceilextracts For the determination of enzyme activity, an aliquot of culture was removed at various times and the cells collected with a hand centrifuge. The pellet of cells was washed once in cold 10 mM Tris (Tris(bydroxymethyl)amino-methan)-HC1 (pH 8.0 at 4°C), and resuspended in the 3 ml of this buffer. Two ml of this was used for Coulter counts. Ten /~1 of 20% Triton X-100 in 10 mM Tris-HC1 (pH 8.0 at 4°C) was added to the remaining 1 ml and the mixture stored at 4°C. Complete cell lysis was observed in 5 rain and the extract was used within 30 rnin for the enzyme assay. In the selection synchrony method where a large aliquot of culture could not be spared, a small aliquot
261
of culture was placed in a 12 ml conical centrifuge tube and brought to 12 ml with 10 mM Tris (pH 8.4 at 4°C). Centrifugation was carried out at 2 200 rpm for 1 rain (International Clinical centrifuge). Eleven ml of supernatant were aspirated. The remaining 1 ml was brought to 12 ml with the same buffer. Centrifugation and aspiration were repeated as above. Half of the remaining 1 ml was used to determine cell number and 5/zl of 20% Triton X-100 in the above buffer was added to the other 0.5 ml of cell suspension. After the cells had lysed this extract was used for enzyme assays. In some cases the media free of cells was assayed for tbymidine phosphorylating activity. An aliquot of culture was centrifuged with a hand centrifuge. The supernatant was collected and centrifuged at 10 000 g for 30 rain at 4°C (Sorvall RC 2). The supernatant from this centrifugation was free of cells and was used for the enzyme assays.
dTMPase assay The dTMPase assay was a slightly modified version of the assay of Conner & Linden [22]. The final reaction mixture contained 5 mM MgCI~, 20 mM Tris-HCl (pH 8.0 at 3&C), and 1 mM [3H]thymidine monophosphate (1,0 tzCi/assay at 200/zl). The final volume was 200 gl. Assay tubes were incubated for 30 min at 30°C and under these conditions the reaction was proportional to the amount of extract added to at least 35 % utilization of substrate. Activity was linear with time for at least 40 min and to at least 25 % utilization of substrate. The reaction was stopped by placing the tubes in boiling water for 2 min. Protein was removed by centrifugation for 2 rain at 3 000 g. The radioactive nucleoside formed was measured by a slight modification of the ion-exchange disc method [23].
Nucleoside phosphotransferase assay Nucleoside phosphotransferase activity was assayed in a reaction mixture similar to that used by Shoup et al. [24]. The final reaction mixture contained in a total volume of 200 /zl: 5 mM AMP, 5 mM MgCI2, 17.5 mM Tris-HC1 (pH 8.0 at 30°C), 0.2 mM [ZH]thymidine (1.0/zCi/assay of 200/zl). A 100/zl of enzyme extract was used. Assay tubes were incubated for 30 min at 30°C. Under these conditions the reaction was linear with time for 35 rain to 19% utilization of substrate and was proportional to the amount of cell extract added to 20 % utilization of substrate. The reaction was terminated by placing the tubes in boiling water for 2 min. After this treatment the tubes were centrifuged for 2 rain at 3 000 g in order to remove coagulated protein. The radioactive nucleotide formed by the nucleoside phosphotransferase reaction was measured by the ionexchange disc method [23].
Expression of enzyme activity The nucleoside phosphotransferase activity has been expressed per 106 cells and per millilitre of culture because this method most clearly indicates the pattern of change through the cell cycle. If the activity is expressed per mg of protein, the pattern of activity
ExpCellRes 108(1977)
Bols and Zimmerman
262
that the results may be expressed per millilitre of culture. This requires that the cell density of the culture be known at the time when the aliquot of culture is taken for enzyme assays. This is not possible in some situations where there is not enough culture to make frequent measurements of cell density. In these cases activity is expressed only on a per cell basis. (
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Fig. 1. Abscissa: time after the inoculation of the culture (hours); ordinate: (far right) nucleoside phosphotransferase activity expressed as nmoles dTMP formed/10 e cells/30 min; (right) nucleoside phosphotransferase activity expressed as nmoles dTMP formed/ml culture/30 min. (left) ceUs/ml. Nucleoside phosphotransferase activity expressed per ml of culture (&---A) or per 106 cells (11-..11) through a growth curve. The cells were grown at 28°C with shaking and cell density ( 0 - - 0 ) and enzyme activity measured. (A) is early logarithmic growth phase which is defined as the period from 10 h after the initiation of growth to the half way mark of the remainder of logarithmic phase. (B) is late logarithmic growth phase which is the period from the midway point of the logarithmic phase to the start of the decelerating phase. (C) is the period between logarithmic growth phase and stationary phase where cell number increases at a reduced rate. (D) is stationary phase. is influenced by the change in total protein through the cell cycle (see reference [1] p. 162). In the present study the enzyme activity is most conveniently expressed per 10e cells. This is an adequate means of expressing the results in situations where there is no change in cell number. However, when the cells divide, the activity on a per cell basis drops due to the distribution o f the enzyme between twice as many cells. Therefore, the changes in enzyme activity through the division period are difficult to visualize. This difficulty is overcome by expressing the activity per millilitre of culture. Since Tetrahymena are motile cells, the recovery of cells from an aliquot of culture is not complete and the per cent recovery is not always constant at all stages of the cell cycle. Therefore, a correction for cell loss must be made in order
Exp Cell Res 108 (1977)
Chemicals Proteose peptone was obtained from Difco Chemicals (British Drug Houses, Toronto, Ont.). Liver fraction L was purchased from Nutritional Biochemical Corporation (Cleveland, Ohio). Thymidine, AMP, cycloheximide, hydroxyurea, Ficoll, and Tris (hydroxymethyl) amino-methane were obtained from the Sigma Chemical Company (St Louis, MO.). Triton X-100, Permablend I, PPO(2,5-diphenyloxazole), and bis-MSE (p-bis-(-Methylstyryl)-Benzene) were bought from Packard Instrument Company, Inc. (Downers Grove, Ill.). For the enzyme assays [3H]thymidine (spec, act. 18-22.4 Ci/mmole) was purchased from the Amersham/Searle Corporation (Don Mills, Ont.) while [3H]thymidine (spec. act. 20 Ci/mmole) was obtained from New England Nuclear (Dorvai, P.Q.) for the incorporation studies.
RESULTS
Nucleoside phosphotransferase activity through the growth curve of Tetrahymena Nucleoside phosphotransferase activity changed over the course of a growth curve (fig. 1). During early logarithmic growth phase nucleoside phosphotransferase activity increased at a greater rate than cell number. For example, between 11.5 h and 17 h after inoculation of a culture, the cell number increased 4 times while nucleoside phosphotransferase activity increased 10 times (fig. 1). On a per cell basis the greatest amount of enzyme activity was found in late logarithmic growth phase. However, during late logarithmic growth phase activity increased at a slower rate than cell number. For example between 17 and 22.5 h, cell number increased 4-fold while activity increased only 2.2 times (fig. 1). During the decelerating and stationary phases of the growth curve nucleoside phosphotransferase activity declined.
Nucleoside phosphotransferase activity in Tetrahymena The change in enzyme activity through the growth curve appeared to reflect changes in enzyme synthesis. The possibility that nucleoside phosphotransferase activity did not increase in stationary phase due to the appearance of an inhibitor or the disappearance of an activator was tested by mixing (1:1) extracts from early logarithmic growth phase with extracts from stationary phase. The mixed extracts gave activities equal to the sum of the activities of the individual extracts, dTMPase activity fluctuated through the growth curve in a manner similar to that of nucleoside phosphotransferase. The possibility that differential stability of the enzyme accounted for the pattern of activity was tested by the addition of 10/zg/ml of cycloheximide during early logarithmic growth and during decelerating growth phase. In an early logarithmic growth phase culture treated with cycloheximide, nucleoside phosphotransferase activity had decreased approx. 15 % at 3 h after the addition of cycloheximide. In a decelerating phase culture treated with cycloheximide, nucleoside phosphotransferase activity had declined approx. 20 % at 3 h after the addition of cycloheximide. The increase in enzyme activity observed in early logarithmic growth phase was blocked by the addition of cycloheximide. Another possibility for the decrease in activity during the decelerating and stationary phases was the release of the enzyme into the medium. However, in the present study nucleoside phosphotransferase activity was not detected in the cell-free media from stationary cultures.
Synchron&ation by the starvationrefeeding technique With the starvation-refeeding technique a low rate of [aH]thymidine incorporation into DNA was observed between 100-220
263
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Fig. 2. Abscissa: time after refeeding starved cells (min); ordinate: (far right) % increase in cell no.; (right) % of cells showing division furrows; (left) [aH]thymidine incorporation into DNA expressed as cpm/105 cells/20 min. Characteristics of a cell cycle obtained upon refeeding starved Tetrahymena. Cells were grown at 28°C without shaking to a density of 55 000 cells/ml and then starved. The % increase in cell number ( t - - - t ) , the % of cells showing division furrows (bars), and the incorporation of [3H]thymidine into DNA ( x - - x ) were recorded at various times after the cells were refed.
min while significant incorporation began around 240 min after refeeding (fig. 2). The early incorporation probably represents mitochondrial and nucleolar DNA synthesis [25]. Dividing cells were observed first between 270 and 300 min after refeeding while the peak division index occurred around 350 min. The division index was never greater than 19 %. The cell number had generally doubled by 430 min after refeeding. Nucleoside phosphotransferase activity increased discontinuously after starved cells were refed (fig. 3). Activity was constant for the first 75 min after refeeding and then increased 9 times from 75 to 180 min. While this increase correlated with the first incorporation of [aH]thymidine into DNA, this increase occurred well before the period of major thymidine incorporation. The addition of cycloheximide at 60 or 180 min after refeeding prevented any subsequent rise in enzyme activity (fig. 3). Enzyme activity was not inhibited by the presence of extracts of 60 min cells in equal Exp Cell Res 108 (1977)
264
Bols and Zimmerman
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Fig. 3. Abscissa: time after refeeding starved cells (rain); ordinate: nucleoside phosphotransferase activity expressed as nmoles dTMP formed/106 cells/30 min. The rise in nucleoside phosphotransferase activity observed after starved cells are refed and the effect of cycloheximide. Activity in the absence (Q---l) and in the presence (O---O) of cycloheximide. Arrows indicate the addition of cycloheximide.
volume mixture with extracts of 140 min cells, dTMPase activity was monitored after starved cells were refed and unlike nucleoside phosphotransferase, which on the average showed an 18-fold increase in activity from the time of refeeding until cell division began, dTMPase activity increased only 3.5 times. Since the rise in nucleoside phosphotransferase activity began about the same time as the first incorporation of [aH]thymidine into DNA began, the dependence of this rise on prior or ongoing DNA synthesis was tested by blocking DNA synthesis. The addition of 50 mM hydroxyurea at the time of refeeding prevented the incorporation of [aH]thymidine into DNA for at least 280 min. Nucleoside phosphotransferase activity rose despite the absence of DNA synthesis. However, the increase in activity was delayed at least 25 min, was not as rapid, and was not as great as that observed in control cultures. Exp Cell Res 108 (1977)
Synchronization by the one heat shock per generation technique The cell cycle obtained after exposing Tetrahymena to 6 heat shocks was studied (fig. 4). A peak in division index of between 60 and 86% was observed between 80 and 85 min after the 6th heat shock and a second peak of between 38 and 56 % was observed between 190 and 200 min. Peaks of [SH]thymidine incorporation into DNA were observed to begin immediately after the division peaks. The total length of the cell Cycle was between 110 and 120 min with a S phase of approx. 70 min. The cell cycle contained no heat shock and is termed the free running cell cycle [19]. In contrast the cell cycles which contained heat shocks were 187 min in length.
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Fig. 4. Abscissa: time after the 6th heat shock (rain); ordinate: (,4) (right) % of cells showing division furrows; (left) cells/ml x 104; (B) [3H]thymidine incorporation into DNA expressed as cpm x 103/1& cells/10 rain. Characteristics of a free running cell cycle obtained by the one heat shock per generation technique of Zeuthen [19]. Cells were grown at 28°C with shaking for 157 rain at which time they received a heat shock of 34°C for 30 min. This cycle of events was repeated 6 times. From the end of the 6th heat shock cell density (&...&), the % of cells showing division furrows (O----Q), and the incorporation of [aH]thymidine into DNA (11--11) was recorded.
Nucleoside phosphotransferase activity in Tetrahymena
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min with extracts from 80 min after the 6th heat shock resulted in activities equal to the sum of the activities of individual extracts.
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Nucleoside phosphotransferase activity was observed at all stages of this free running cell cycle and increased in a continuous manner through this period (fig. 5). However, activity increased only 1.4 times over this cell cycle. In contrast during a cell cycle which contained a heat shock nucleoside phosphotransferase activity increased 2 times, dTMPase activity fluctuated through the cell cycle in a manner very similar to that observed for nucleoside phosphotransferase. The increase in nucleoside phosphotransferase activity from 5 to 80 min after the 6th heat shock was blocked by the addition of cycloheximide (10 /zg/ ml). The mixing (1:1) of extracts from 5
A cell cycle obtained by the selection of dividing and newly divided cells from late logarithmic phase of the growth curve was studied (fig. 6). A peak in the division index of between 28 and 38 % was observed between 180 and 206 min after selection. The peak incorporation of [SH]thymidine into DNA was observed between 60 and 135 min after selection. Nucleoside phosphotransferase activity was observed at all stages of this cell cycle (fig. 7). The activity increased in a con-
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266
Bols and Zimmerman
tinuous manner through the cell cycle. However, the increase in activity was only 1.5 times so that when the cells divided the activity, expressed on a per cell basis, dropped. However, when cells were selected from an early logarithmic population, enzyme activity increased 2.6 times over one cell cycle time. Unfortunately, the synchrony obtained by selecting cells from an early logarithmic population was very poor and precise pattern of increase could not be determined.
Effect of culture conditions on nucleoside phosphotransferase activity Because the level of nucleoside phosphotransferase activity varied with the different synchronization procedures, shaking, a variable of the culture conditions which changed with the different synchronization procedures, was tested for a possible effect on the level of activity. A culture that was shaken gently during growth gave much more activity on a per cell basis than did a culture that was not shaken (table 1). Previously, Levy [26] has shown that the level of certain enzymes depends on whether a culture is kept static or not.
DISCUSSION The patterns of nucleoside phosphotransferase activity observed in this study appeared to reflect the patterns of nucleoside phosphotransferase synthesis. The increases in activity were blocked by cycloheximide while mixed extract experiments ruled out the possibility that the patterns were due to fluctuations in activators or inhibitors. The patterns of dTMPase activity were similar to those of nucleoside phosphotransferase which ruled out the possibility that the nucleoside phosphoExp Cell Res 108 (1977)
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Fig. 7. Abscissa: time after the selection of cells (rain); ordinate: (A) (right) % of cells showing division furrows; (left) % increase in cell no.; (B) nucleoside phosphotransferase activity expressed as nmoles of dTMP formed/10 e cells/30 min. Nucleoside phosphotransferase activity through a cell cycle obtained by the selection of dividing cells from a logarithmic population. Cells were grown at 28°C with shaking to a density of 36000 cells/ml at which time a population was selected by differential density labeling and gradient centrifugation. The % increase in cell number ( A . . . A ) and the % of cells showing division furrows (O----O) was followed in drop cultures. The bulk of the selected cells was incubated in fresh PPL at 32000 cells/ml at 28°C with shaking. This culture was used to measure nucleoside phosphotransferase activity (11----II) through the cell cycle.
transferase patterns were due to changes in dTMPase activity. Different patterns of nucleoside phosphotransferase synthesis through the cell cycle were observed with different synchronization techniques. The enzyme was synthesized continuously in cells synchronized by the physical induction technique of heat shocks [19], which resulted in the most synchronous population. The selection technique of differential density centrifugation [20] also gave a continuous pattern. On the other hand, a discontinuous pattern was observed in cells synchronized by the chemical induction technique of starvation-refeeding [18]. The discontinuous
Nucleoside phosphotransferase activity in Tetrahymena
267
Table 1. The effect of culture conditions on nucleoside phosphotransferase activity in log phase cells
synthesis as thymidine kinase synthesis does. However, thymidine kinase synthesis continues also when DNA replication has been blocked with hydroxyurea [5]. Instead nmoles dTMP formed of following the pattern of DNA replication, Culture condition per l0 s cells/30 min nucleoside phosphotransferase synthesis Static 64.4+ 10.4 (n---5) was similar to the pattern of RNA syntheShaken 470.6+72.9 (n---4) sis, which takes place continuously through Cells were grown to a density of between 54000 and the course of the cell cycle [19, 30]. If the 76000 cells/ml when they were collected, lysed, and nucleoside phosphotransferase of Tetraassayed for nucleoside phosphotransferase activity as described in the Methods and Materials. hymena is similar to the nucleoside phosphotransferase of other organisms, this enzyme would be expected to phosphorylate pattern observed with this technique ap- a wide range of nucleosides [14], which peared to be a consequence of the starva- would be utilized for both RNA and DNA tion treatment. Tetrahymena that had been synthesis. Thus the pattern of continuous starved for 24 h had very few polysomes synthesis might reflect the enzyme's inand polysomes only reappeared 40 min after volvement in RNA metabolism. refeeding while incorporation of [aH]uriThe results of this study show that the dine into RNA did not begin until between periodic synthesis of the thymidine phos60 and 90 min after refeeding [27]. Other phorylating enzyme is not an event characworkers have observed that the pattern of teristic of all cell cycles. However, whether synthetic events during the cell cycle are this reflects different regulatory mechaninfluenced by the methods used to obtain a isms, patterns of enzyme synthesis, or nusynchronous culture [2, 28, 29]. cleoside metabolism during the TetrahyThe pattern of nucleoside phosphotrans- mena cell cycle is unclear. Other enzymes ferase synthesis through the Tetrahymena have been reported to be synthesized pericell cycle differs greatly from the pattern odically during the Tetrahymena cell cycle observed for the other thymidine phosphor- [31] and thus both continuous and disconylating enzyme, thymidine kinase, which is tinuous patterns of enzyme synthesis probfound in other organisms. Nucleoside phos- ably exist in this organism as they do in photransferase is synthesized continuously. other organisms. As in other cells, the deIn contrast, thymidine kinase is synthesized oxyribonucleotide pools vary through the discontinuously through many animal cell Tetrahymena cell cycle [32]. cycles and this synthesis begins about the The failure of nucleoside phosphotranstime DNA replication starts [4-12]. In the ferase to double through all cell cycles sugcase in Tetrahymena where nucleoside gests that Tetrahymena are able to synthephosphotransferase synthesis was discon- size some molecules in excess in certain cell tinuous (after starved cells were refed), the cycles and that they are able to go through synthesis began well before macronuclear subsequent cell cycles without synthesizing DNA synthesis and continued when DNA these molecules or synthesizing them at replication was blocked with hydroxyurea. reduced levels. The absence of a doubling Thus nucleoside phosphotransferase syn- in nucleoside phosphotransferase activity thesis does not appear as coupled to DNA through the free running cell cycle obtained Exp Cell Res 108 (1977)
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by the heat shock procedure appears to be a consequence of the synchronization technique. The free running cell cycle is shorter than a normal cell cycle which indicates that cells build up excesses of certain compounds during the cell cycles which contain heat shocks. However, cells selectively synchronized from a late logarithmic population also failed to double their nucleoside phosphotransferase activity over one cell cycle time. This does not appear to be a result of the manipulations required to select these cells as cells selected in the same manner from an early logarithmic population did show a doubling in nucleoside phosphotransferase activity. More importantly, in a study of non-synchronized cells the results indicated that nucleoside phosphotransferase activity increased less rapidly than cell number during late logarithmic phase. Previous workers have presented evidence that the composition of Tetrahymena changes during logarithmic phase [33, 34]. Thus during logarithmic growth no two cell cycles might be the same although certain synthetic events would have to be reproduced faithfully from one cell cycle to the next. This investigation w a s supported by the National Research Council of Canada. N. C.B. was a recipient of a NRC Postgraduate Scholarship.
REFERENCES 1. Mitchison, J M, The biology of the cell cycle. Cambridge University Press, Cambridge (1971). 2. Sissons, C H, Mitchison, J M & Creanor, J, Exp cell res 82 (1973) 63. 3. Okazaki, R & Kornberg, A, J biol chem 239 (1964) 275.
Exp Cell Res 108 (1977)
4. Adams, R L P, Exp cell res 56 (1969) 49. 5. BeUo, L J, Exp cell res 89 (1974) 263. 6. Brent, T P, Butler, J A V & Crathorn, A R, Nature 207 (1%5) 176. 7. Brent, T P, Cell tissue kinet 4 (1971) 297. 8. Harland, J, Jackson, J F & Yeoman, M M, J cell sci 13 (1973) 121. 9. Klevecz, R R, Science 166 (1969) 1536. 10. Littlefield, J W, Biochim biophys acta 114 (1966) 398. 11. Mittermayer, C, Bosselmann, R & Bremerskov, V, Eurj biochem 4 (1%8) 487. 12. Stubblefield, E & Murphree, S, Exp cell res 48 (1%7) 652. 13. Baserga, R, Multiplication and division in mammalian cells. Marcel Dekker, New York (1976). 14. Brunngraber, E F & Chargaff, E, J biol chem 242 (1%7) 4834. 15. Arima, T, Masaka, M, Shiosaka, T, Okuda, H & Fumi, S, Biochim biophys acta 246 (1971) 184. 16. Bols, N C & Zimmerman, A M, Comp biochem physiol 57 (1977) 31. 17. Hill, D L, The biochemistry and physiology of Tetrahymena. Academic Press, New York (1972). 18. Cameron, I L & Jeter, J protozool 17 (1970) 429. 19. Zeuthen, E, Exp cell res 68 (1971) 49. 20. Wolfe, J, Exp cell res 77 (1973) 232. 21. Stocco, D M & Zimmerman, A M, Canj biochem 52 (1974) 310. 22. Conner, R L & Linden, C, J protozool 17 (1970) 659. 23. Ives, D H, Durham, J P & Tucker, V S, Anal biochem 28 (1%9) 192. 24. Shoup, G D, Prescott, D M & Wykes, J R, J cell bio131 (1966) 295. 25. Engberg, J, Nilsson, J R, Pearlman, R E & Leick, V, Proc natl acad sci US 71 (1974) 894. 26. Levy, M R, Biochim biophys acta 304 (1973) 367. 27. Cameron, I L, Griffin, E E & Rudick, M J, Exp cell res 65 (1971) 265. 28. Ley, K D & Murphy, M M, J cell biol 58 (1973) 340. 29. Belfino, F L, J tool bio174 (1973) 223. 30. Prescott, D M, Exp cell res 19 (1960) 228. 31. Zeuthen, E, Cell cycle controls (ed G M Padilla, I L Cameron & A M Zimmerman) p. 1. Academic Press, New York (1974). 32. Next, B A, Biochim biophys acta 378 (1975) 12. 33. Prescott, D M, Exp cell res 12 (1957) 126. 34. Cameron, I L, Biology of Tetrahymena (ed A M Elliot) p. 199. Dowden, Hutchison & Ross, Stroudsburg (1 973). Received February 1, 1977 Revised version received April 4, 1977 Accepted April 28, 1977
Experimental Cell Research 108 (1977) 259-268
NUCLEOSIDE
PHOSPHOTRANSFERASE
ACTIVITY THROUGH
THE GROWTH AND CELL CYCLE OF
TETRAHYMENA P YRIFORMIS GL-I N. C. BOLS 1 and A. M. ZIMMERMAN Department o f Zoology, University o f Toronto, Toronto, Ontario, Canada M5S 1A1
SUMMARY Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside
phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.
The patterns of enzyme synthesis during the course of the cell cycle have received considerable attention in recent years [1]. Generally, the patterns of synthesis can be divided into two broad groups [1]. In one group enzyme synthesis occurs continuously during the cell cycle while in the other group synthesis takes place only during a discrete period of the cycle. However, the pattern of synthesis for a particular enzyme can depend on the method used to obtain a synchronous culture [2]. Two basic formats, induction synchrony and selection synchrony, are available for synchronizing cells [1]. In induction synchrony a treat-
1 Present address: Institute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institutet, S-104 01 Stockholm 60, Sweden.
ment is applied to an asynchronous culture and enforces all the cells to divide synchronously while in selection synchrony a population of cells at a particular stage of the cell cycle are selected out of a non-synchronous population. A comparison of an enzyme's pattern of synthesis through cell cycles obtained by different techniques can point out correlations between different synthetic, physiological, or morphological events of the cell cycle. Through the course of the cell cycle of several organisms the activity of thymidine kinase (EC271.75), which uses nucleoside triphosphates to phosphorylate thymidine [3], has been found to increase at about the time DNA synthesis begins and to decrease during late G2 and mitosis [4-12]. This correlation between thymidine kinase Exp Cell Res 108 (1977)
260
Bols and Zimmerman
activity and DNA synthesis is observed in cells synchronized both by selection and induction techniques. The periodic pattern of activity has been shown to reflect periodic synthesis of the enzyme [5]. As a result of the close temporal relationship between the synthesis of thymidine kinase and DNA, the suggestion has been made that this enzyme is involved in the regulation of DNA synthesis (for a review that is critical of this point of view see reference [13] page 21). The importance of this correlation might be better evaluated if the relationship between DNA synthesis and other thymidine phosphorylating enzymes was known. Nucleoside phosphotransferase (EC 2.7.1.77), which utilizes nucleoside monophosphates to phosphorylate a wide range of nucleosides including thymidine [ 14], has not been measured through the cell cycle. Recently, Tetrahymena pyriformis has been shown to contain a nucleoside phosphotransferase but no thymidine kinase [15, 16]. In as much as Tetrahymena can be synchronized by a variety of methods [17], this appeared to be an ideal organism to study nucleoside phosphotransferase activity through the cell cycle. We report that in Tetrahymena synchronized by both selection and physical induction techniques nucleoside phosphotransferase is synthesized continuously. In cells that are starved and then refed, the enzyme is synthesized discontinuously but shows no coupling to DNA synthesis.
METHODS AND MATERIALS
Culturing of organism Tetrahymena pyriformis, strain GL-I, were maintained in 2 % protease peptone (Difco Chemicals, British Drug Houses, Toronto, Ont.) supplemented with 0.1% liver fraction L (Nutritional Biochemical Corp., Cleveland, Ohio). This growth media was referred to as PPL.
Exp CellRes 108 (1977)
Synchronization by starvation-refeeding The starvation-refeeding synchronization procedure was performed as described by Cameron & Jeter [18]. An inoculum of stationary cells was used to initiate cell growth. The cells were grown without shaking at 28°C in PPL to cell densities of between 55 000 and 90000 cells/ml. At this time the cells were collected, washed in starvation buffer (0.6 g KH2PO4, 0.15 g K2PO4, and 0.25 g MgSO4, per litre with the pH adjusted to 6.5 with NaOH), and placed into a 2.5 litre low form culture flask which contained 160 ml of the same buffer. The initial starvation cell density was between 15 000-40 000 cells/ml. The cells were starved for 24 h during which time cell number increased approx. 50 %. Cells were refed by the addition of 200 ml of double-strength PPL. During the starvation period and after refeeding the cells were grown without shaking at 28°C.
Synchronization by one heat shock per generation Cells were also synchronized by the one heat shock per generation system developed by Zeuthen [19]. The experiment was started by the inoculation of either 3 ml of a l-day-old culture or 2 ml of a 2-day-old culture into 200 ml of PPL in a 2.5 litre low form culture flask. The cells were grown for 157 min at 28°C and then for 30 min at 34°C. This cycle of events was repeated six times. These changes in temperature were controlled automatically. The cells were shaken gently throughout this treatment. At the end of six heat shocks there were approx. 30 000-50 000 cells/ml.
Synchronization by differential density labeling Tetrahymena were synchronized by the differential density labeling technique of Wolfe [20]. The cells were grown at 28°C with shaking to either 1000015 000 cells/ml or 35 000-40 000 cells/ml, except for the cultures in the 10000 to 15000 cells/ml range, the cells were fed tantalum particles (type SGQ, Norton Metals Division, Newton, Mass.) at these cell densities. The culture at the lowest cell density was first concentrated to approx. 100000 cells/ml before being fed tantalum particles. This was necessary because future steps were limited by the volume of culture that could be rapidly centrifuged. Particles were added to a concentration of 1-2 mg/ml and were kept suspended by swirling the culture. After 5 min of feeding, the culture was centrifuged at 1200 rpm for 2 min. Most of the cell free supernatant was aspirated except for approx. 5 ml. The light dividing cells were now separated from the heavy non-dividing cells on a Ficoll gradient as described by Wolfe [20]. The portion of the gradient which contained the unlabeled cells was placed in sterilized PPL. The percentage of cells containing heavy particles in this fraction was between 0 and 3 %.
Nucleoside phosphotransferase activity in Tetrahymena Measurement of cell number and division indices Cell density was determined with a Model Z2 Coulter Counter (Coulter Electronics, Hialeah, Fla.). The division index was determined by counting the percentage of fixed cells showing cytoplasmic furrows on both sides. The division index and per cent increase in cell number was occasionally determined by the drop culture method as described by Cameron & Jeter [18]. Small drops of culture were placed onto disposable plastic Petri dishes by tapping on the surface of the dish with a Pasteur pipette containing a small portion of the culture. A layer of paraffin oil was gently poured over the surface of the drops to prevent evaporation. The cell number and the number of cells showing division furrows in each drop were determined at intervals. Generally, only late dividing cells were scored because this was the only stage which could be scored unequivocally with the low magnification of the dissecting microscope.
[aH]thymidine incorporation through the cell cycle The following method was employed to measure the rate of [3H]thymidine incorporation into acid-insoluble material through the cell cycle. At various times duri n g the cell cycle an aliquot of culture was incubated with [3H]thymidine (spec. act. 20 Ci/mmole) to give either 2 or 10 tzCi/ml. The incubation of this culture was carried out at 28°C without shaking for the starved-refed cell cycle and with shaking for the other cell cycles. The incubation was terminated after either 10 or 20 min by the addition of an equal volume of ice cold 10% trichloroacetic acid (TCA). Extraction was carried out overnight at 4°C. The precipitate was collected by centrifugation (International Clinical centrifuge) at 1500 rpm for 2 min and washed twice with ice-cold 5 % TCA. Finally, the precipitate was collected on a glassfiber filter (grade 934AH, Reeve Angel, Clifton, N.J.) that had been soaked in 5 % TCA containing 1 mg of thymidine per ml. The filter was washed three times with ice-cold 5 % TCA which contained 1 nag of thymidine per ml and further washing and counting was carded out as described previously [21].
Preparation of ceilextracts For the determination of enzyme activity, an aliquot of culture was removed at various times and the cells collected with a hand centrifuge. The pellet of cells was washed once in cold 10 mM Tris (Tris(bydroxymethyl)amino-methan)-HC1 (pH 8.0 at 4°C), and resuspended in the 3 ml of this buffer. Two ml of this was used for Coulter counts. Ten /~1 of 20% Triton X-100 in 10 mM Tris-HC1 (pH 8.0 at 4°C) was added to the remaining 1 ml and the mixture stored at 4°C. Complete cell lysis was observed in 5 rain and the extract was used within 30 rnin for the enzyme assay. In the selection synchrony method where a large aliquot of culture could not be spared, a small aliquot
261
of culture was placed in a 12 ml conical centrifuge tube and brought to 12 ml with 10 mM Tris (pH 8.4 at 4°C). Centrifugation was carried out at 2 200 rpm for 1 rain (International Clinical centrifuge). Eleven ml of supernatant were aspirated. The remaining 1 ml was brought to 12 ml with the same buffer. Centrifugation and aspiration were repeated as above. Half of the remaining 1 ml was used to determine cell number and 5/zl of 20% Triton X-100 in the above buffer was added to the other 0.5 ml of cell suspension. After the cells had lysed this extract was used for enzyme assays. In some cases the media free of cells was assayed for tbymidine phosphorylating activity. An aliquot of culture was centrifuged with a hand centrifuge. The supernatant was collected and centrifuged at 10 000 g for 30 rain at 4°C (Sorvall RC 2). The supernatant from this centrifugation was free of cells and was used for the enzyme assays.
dTMPase assay The dTMPase assay was a slightly modified version of the assay of Conner & Linden [22]. The final reaction mixture contained 5 mM MgCI~, 20 mM Tris-HCl (pH 8.0 at 3&C), and 1 mM [3H]thymidine monophosphate (1,0 tzCi/assay at 200/zl). The final volume was 200 gl. Assay tubes were incubated for 30 min at 30°C and under these conditions the reaction was proportional to the amount of extract added to at least 35 % utilization of substrate. Activity was linear with time for at least 40 min and to at least 25 % utilization of substrate. The reaction was stopped by placing the tubes in boiling water for 2 min. Protein was removed by centrifugation for 2 rain at 3 000 g. The radioactive nucleoside formed was measured by a slight modification of the ion-exchange disc method [23].
Nucleoside phosphotransferase assay Nucleoside phosphotransferase activity was assayed in a reaction mixture similar to that used by Shoup et al. [24]. The final reaction mixture contained in a total volume of 200 /zl: 5 mM AMP, 5 mM MgCI2, 17.5 mM Tris-HC1 (pH 8.0 at 30°C), 0.2 mM [ZH]thymidine (1.0/zCi/assay of 200/zl). A 100/zl of enzyme extract was used. Assay tubes were incubated for 30 min at 30°C. Under these conditions the reaction was linear with time for 35 rain to 19% utilization of substrate and was proportional to the amount of cell extract added to 20 % utilization of substrate. The reaction was terminated by placing the tubes in boiling water for 2 min. After this treatment the tubes were centrifuged for 2 rain at 3 000 g in order to remove coagulated protein. The radioactive nucleotide formed by the nucleoside phosphotransferase reaction was measured by the ionexchange disc method [23].
Expression of enzyme activity The nucleoside phosphotransferase activity has been expressed per 106 cells and per millilitre of culture because this method most clearly indicates the pattern of change through the cell cycle. If the activity is expressed per mg of protein, the pattern of activity
ExpCellRes 108(1977)
Bols and Zimmerman
262
that the results may be expressed per millilitre of culture. This requires that the cell density of the culture be known at the time when the aliquot of culture is taken for enzyme assays. This is not possible in some situations where there is not enough culture to make frequent measurements of cell density. In these cases activity is expressed only on a per cell basis. (
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Fig. 1. Abscissa: time after the inoculation of the culture (hours); ordinate: (far right) nucleoside phosphotransferase activity expressed as nmoles dTMP formed/10 e cells/30 min; (right) nucleoside phosphotransferase activity expressed as nmoles dTMP formed/ml culture/30 min. (left) ceUs/ml. Nucleoside phosphotransferase activity expressed per ml of culture (&---A) or per 106 cells (11-..11) through a growth curve. The cells were grown at 28°C with shaking and cell density ( 0 - - 0 ) and enzyme activity measured. (A) is early logarithmic growth phase which is defined as the period from 10 h after the initiation of growth to the half way mark of the remainder of logarithmic phase. (B) is late logarithmic growth phase which is the period from the midway point of the logarithmic phase to the start of the decelerating phase. (C) is the period between logarithmic growth phase and stationary phase where cell number increases at a reduced rate. (D) is stationary phase. is influenced by the change in total protein through the cell cycle (see reference [1] p. 162). In the present study the enzyme activity is most conveniently expressed per 10e cells. This is an adequate means of expressing the results in situations where there is no change in cell number. However, when the cells divide, the activity on a per cell basis drops due to the distribution o f the enzyme between twice as many cells. Therefore, the changes in enzyme activity through the division period are difficult to visualize. This difficulty is overcome by expressing the activity per millilitre of culture. Since Tetrahymena are motile cells, the recovery of cells from an aliquot of culture is not complete and the per cent recovery is not always constant at all stages of the cell cycle. Therefore, a correction for cell loss must be made in order
Exp Cell Res 108 (1977)
Chemicals Proteose peptone was obtained from Difco Chemicals (British Drug Houses, Toronto, Ont.). Liver fraction L was purchased from Nutritional Biochemical Corporation (Cleveland, Ohio). Thymidine, AMP, cycloheximide, hydroxyurea, Ficoll, and Tris (hydroxymethyl) amino-methane were obtained from the Sigma Chemical Company (St Louis, MO.). Triton X-100, Permablend I, PPO(2,5-diphenyloxazole), and bis-MSE (p-bis-(-Methylstyryl)-Benzene) were bought from Packard Instrument Company, Inc. (Downers Grove, Ill.). For the enzyme assays [3H]thymidine (spec, act. 18-22.4 Ci/mmole) was purchased from the Amersham/Searle Corporation (Don Mills, Ont.) while [3H]thymidine (spec. act. 20 Ci/mmole) was obtained from New England Nuclear (Dorvai, P.Q.) for the incorporation studies.
RESULTS
Nucleoside phosphotransferase activity through the growth curve of Tetrahymena Nucleoside phosphotransferase activity changed over the course of a growth curve (fig. 1). During early logarithmic growth phase nucleoside phosphotransferase activity increased at a greater rate than cell number. For example, between 11.5 h and 17 h after inoculation of a culture, the cell number increased 4 times while nucleoside phosphotransferase activity increased 10 times (fig. 1). On a per cell basis the greatest amount of enzyme activity was found in late logarithmic growth phase. However, during late logarithmic growth phase activity increased at a slower rate than cell number. For example between 17 and 22.5 h, cell number increased 4-fold while activity increased only 2.2 times (fig. 1). During the decelerating and stationary phases of the growth curve nucleoside phosphotransferase activity declined.
Nucleoside phosphotransferase activity in Tetrahymena The change in enzyme activity through the growth curve appeared to reflect changes in enzyme synthesis. The possibility that nucleoside phosphotransferase activity did not increase in stationary phase due to the appearance of an inhibitor or the disappearance of an activator was tested by mixing (1:1) extracts from early logarithmic growth phase with extracts from stationary phase. The mixed extracts gave activities equal to the sum of the activities of the individual extracts, dTMPase activity fluctuated through the growth curve in a manner similar to that of nucleoside phosphotransferase. The possibility that differential stability of the enzyme accounted for the pattern of activity was tested by the addition of 10/zg/ml of cycloheximide during early logarithmic growth and during decelerating growth phase. In an early logarithmic growth phase culture treated with cycloheximide, nucleoside phosphotransferase activity had decreased approx. 15 % at 3 h after the addition of cycloheximide. In a decelerating phase culture treated with cycloheximide, nucleoside phosphotransferase activity had declined approx. 20 % at 3 h after the addition of cycloheximide. The increase in enzyme activity observed in early logarithmic growth phase was blocked by the addition of cycloheximide. Another possibility for the decrease in activity during the decelerating and stationary phases was the release of the enzyme into the medium. However, in the present study nucleoside phosphotransferase activity was not detected in the cell-free media from stationary cultures.
Synchron&ation by the starvationrefeeding technique With the starvation-refeeding technique a low rate of [aH]thymidine incorporation into DNA was observed between 100-220
263
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Fig. 2. Abscissa: time after refeeding starved cells (min); ordinate: (far right) % increase in cell no.; (right) % of cells showing division furrows; (left) [aH]thymidine incorporation into DNA expressed as cpm/105 cells/20 min. Characteristics of a cell cycle obtained upon refeeding starved Tetrahymena. Cells were grown at 28°C without shaking to a density of 55 000 cells/ml and then starved. The % increase in cell number ( t - - - t ) , the % of cells showing division furrows (bars), and the incorporation of [3H]thymidine into DNA ( x - - x ) were recorded at various times after the cells were refed.
min while significant incorporation began around 240 min after refeeding (fig. 2). The early incorporation probably represents mitochondrial and nucleolar DNA synthesis [25]. Dividing cells were observed first between 270 and 300 min after refeeding while the peak division index occurred around 350 min. The division index was never greater than 19 %. The cell number had generally doubled by 430 min after refeeding. Nucleoside phosphotransferase activity increased discontinuously after starved cells were refed (fig. 3). Activity was constant for the first 75 min after refeeding and then increased 9 times from 75 to 180 min. While this increase correlated with the first incorporation of [aH]thymidine into DNA, this increase occurred well before the period of major thymidine incorporation. The addition of cycloheximide at 60 or 180 min after refeeding prevented any subsequent rise in enzyme activity (fig. 3). Enzyme activity was not inhibited by the presence of extracts of 60 min cells in equal Exp Cell Res 108 (1977)
264
Bols and Zimmerman
15( lO(__~~1 ~i~°i"" 5O
100 200 300 I
Fig. 3. Abscissa: time after refeeding starved cells (rain); ordinate: nucleoside phosphotransferase activity expressed as nmoles dTMP formed/106 cells/30 min. The rise in nucleoside phosphotransferase activity observed after starved cells are refed and the effect of cycloheximide. Activity in the absence (Q---l) and in the presence (O---O) of cycloheximide. Arrows indicate the addition of cycloheximide.
volume mixture with extracts of 140 min cells, dTMPase activity was monitored after starved cells were refed and unlike nucleoside phosphotransferase, which on the average showed an 18-fold increase in activity from the time of refeeding until cell division began, dTMPase activity increased only 3.5 times. Since the rise in nucleoside phosphotransferase activity began about the same time as the first incorporation of [aH]thymidine into DNA began, the dependence of this rise on prior or ongoing DNA synthesis was tested by blocking DNA synthesis. The addition of 50 mM hydroxyurea at the time of refeeding prevented the incorporation of [aH]thymidine into DNA for at least 280 min. Nucleoside phosphotransferase activity rose despite the absence of DNA synthesis. However, the increase in activity was delayed at least 25 min, was not as rapid, and was not as great as that observed in control cultures. Exp Cell Res 108 (1977)
Synchronization by the one heat shock per generation technique The cell cycle obtained after exposing Tetrahymena to 6 heat shocks was studied (fig. 4). A peak in division index of between 60 and 86% was observed between 80 and 85 min after the 6th heat shock and a second peak of between 38 and 56 % was observed between 190 and 200 min. Peaks of [SH]thymidine incorporation into DNA were observed to begin immediately after the division peaks. The total length of the cell Cycle was between 110 and 120 min with a S phase of approx. 70 min. The cell cycle contained no heat shock and is termed the free running cell cycle [19]. In contrast the cell cycles which contained heat shocks were 187 min in length.
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Fig. 4. Abscissa: time after the 6th heat shock (rain); ordinate: (,4) (right) % of cells showing division furrows; (left) cells/ml x 104; (B) [3H]thymidine incorporation into DNA expressed as cpm x 103/1& cells/10 rain. Characteristics of a free running cell cycle obtained by the one heat shock per generation technique of Zeuthen [19]. Cells were grown at 28°C with shaking for 157 rain at which time they received a heat shock of 34°C for 30 min. This cycle of events was repeated 6 times. From the end of the 6th heat shock cell density (&...&), the % of cells showing division furrows (O----Q), and the incorporation of [aH]thymidine into DNA (11--11) was recorded.
Nucleoside phosphotransferase activity in Tetrahymena
~6
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265
min with extracts from 80 min after the 6th heat shock resulted in activities equal to the sum of the activities of individual extracts.
Synchronization by differential labelling and density centrifugation
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Nucleoside phosphotransferase activity was observed at all stages of this free running cell cycle and increased in a continuous manner through this period (fig. 5). However, activity increased only 1.4 times over this cell cycle. In contrast during a cell cycle which contained a heat shock nucleoside phosphotransferase activity increased 2 times, dTMPase activity fluctuated through the cell cycle in a manner very similar to that observed for nucleoside phosphotransferase. The increase in nucleoside phosphotransferase activity from 5 to 80 min after the 6th heat shock was blocked by the addition of cycloheximide (10 /zg/ ml). The mixing (1:1) of extracts from 5
A cell cycle obtained by the selection of dividing and newly divided cells from late logarithmic phase of the growth curve was studied (fig. 6). A peak in the division index of between 28 and 38 % was observed between 180 and 206 min after selection. The peak incorporation of [SH]thymidine into DNA was observed between 60 and 135 min after selection. Nucleoside phosphotransferase activity was observed at all stages of this cell cycle (fig. 7). The activity increased in a con-
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24 Fig. 6. Abscissa: time atter the selection of cells (min); ordinate: (A) (right) % of cells showing division furrows; (left) % increase in cell no.; (B) [SH]thymidine incorporation into DNA expressed as cpmx liP/ 105 cells/10 rain. Characteristics of a cell cycle obtained by the selection of dividing cells from a logarithmic population. Cells were grown at 28°Cwith shaking to a density of 381)11tl cells/ml. Dividing cells were then selected by differential density labelling and gradient centrifugation. Drop cultures of the selected cells were set up and at various times after the selection procedure the % increase in cell number (&" • "A) and the % of cells showing division furrows noted (O--O). The bulk of the selected cells was used to study [~H]thymidine incorporation into DNA (B---II). Exp Cell Res 108 (1977)
266
Bols and Zimmerman
tinuous manner through the cell cycle. However, the increase in activity was only 1.5 times so that when the cells divided the activity, expressed on a per cell basis, dropped. However, when cells were selected from an early logarithmic population, enzyme activity increased 2.6 times over one cell cycle time. Unfortunately, the synchrony obtained by selecting cells from an early logarithmic population was very poor and precise pattern of increase could not be determined.
Effect of culture conditions on nucleoside phosphotransferase activity Because the level of nucleoside phosphotransferase activity varied with the different synchronization procedures, shaking, a variable of the culture conditions which changed with the different synchronization procedures, was tested for a possible effect on the level of activity. A culture that was shaken gently during growth gave much more activity on a per cell basis than did a culture that was not shaken (table 1). Previously, Levy [26] has shown that the level of certain enzymes depends on whether a culture is kept static or not.
DISCUSSION The patterns of nucleoside phosphotransferase activity observed in this study appeared to reflect the patterns of nucleoside phosphotransferase synthesis. The increases in activity were blocked by cycloheximide while mixed extract experiments ruled out the possibility that the patterns were due to fluctuations in activators or inhibitors. The patterns of dTMPase activity were similar to those of nucleoside phosphotransferase which ruled out the possibility that the nucleoside phosphoExp Cell Res 108 (1977)
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Fig. 7. Abscissa: time after the selection of cells (rain); ordinate: (A) (right) % of cells showing division furrows; (left) % increase in cell no.; (B) nucleoside phosphotransferase activity expressed as nmoles of dTMP formed/10 e cells/30 min. Nucleoside phosphotransferase activity through a cell cycle obtained by the selection of dividing cells from a logarithmic population. Cells were grown at 28°C with shaking to a density of 36000 cells/ml at which time a population was selected by differential density labeling and gradient centrifugation. The % increase in cell number ( A . . . A ) and the % of cells showing division furrows (O----O) was followed in drop cultures. The bulk of the selected cells was incubated in fresh PPL at 32000 cells/ml at 28°C with shaking. This culture was used to measure nucleoside phosphotransferase activity (11----II) through the cell cycle.
transferase patterns were due to changes in dTMPase activity. Different patterns of nucleoside phosphotransferase synthesis through the cell cycle were observed with different synchronization techniques. The enzyme was synthesized continuously in cells synchronized by the physical induction technique of heat shocks [19], which resulted in the most synchronous population. The selection technique of differential density centrifugation [20] also gave a continuous pattern. On the other hand, a discontinuous pattern was observed in cells synchronized by the chemical induction technique of starvation-refeeding [18]. The discontinuous
Nucleoside phosphotransferase activity in Tetrahymena
267
Table 1. The effect of culture conditions on nucleoside phosphotransferase activity in log phase cells
synthesis as thymidine kinase synthesis does. However, thymidine kinase synthesis continues also when DNA replication has been blocked with hydroxyurea [5]. Instead nmoles dTMP formed of following the pattern of DNA replication, Culture condition per l0 s cells/30 min nucleoside phosphotransferase synthesis Static 64.4+ 10.4 (n---5) was similar to the pattern of RNA syntheShaken 470.6+72.9 (n---4) sis, which takes place continuously through Cells were grown to a density of between 54000 and the course of the cell cycle [19, 30]. If the 76000 cells/ml when they were collected, lysed, and nucleoside phosphotransferase of Tetraassayed for nucleoside phosphotransferase activity as described in the Methods and Materials. hymena is similar to the nucleoside phosphotransferase of other organisms, this enzyme would be expected to phosphorylate pattern observed with this technique ap- a wide range of nucleosides [14], which peared to be a consequence of the starva- would be utilized for both RNA and DNA tion treatment. Tetrahymena that had been synthesis. Thus the pattern of continuous starved for 24 h had very few polysomes synthesis might reflect the enzyme's inand polysomes only reappeared 40 min after volvement in RNA metabolism. refeeding while incorporation of [aH]uriThe results of this study show that the dine into RNA did not begin until between periodic synthesis of the thymidine phos60 and 90 min after refeeding [27]. Other phorylating enzyme is not an event characworkers have observed that the pattern of teristic of all cell cycles. However, whether synthetic events during the cell cycle are this reflects different regulatory mechaninfluenced by the methods used to obtain a isms, patterns of enzyme synthesis, or nusynchronous culture [2, 28, 29]. cleoside metabolism during the TetrahyThe pattern of nucleoside phosphotrans- mena cell cycle is unclear. Other enzymes ferase synthesis through the Tetrahymena have been reported to be synthesized pericell cycle differs greatly from the pattern odically during the Tetrahymena cell cycle observed for the other thymidine phosphor- [31] and thus both continuous and disconylating enzyme, thymidine kinase, which is tinuous patterns of enzyme synthesis probfound in other organisms. Nucleoside phos- ably exist in this organism as they do in photransferase is synthesized continuously. other organisms. As in other cells, the deIn contrast, thymidine kinase is synthesized oxyribonucleotide pools vary through the discontinuously through many animal cell Tetrahymena cell cycle [32]. cycles and this synthesis begins about the The failure of nucleoside phosphotranstime DNA replication starts [4-12]. In the ferase to double through all cell cycles sugcase in Tetrahymena where nucleoside gests that Tetrahymena are able to synthephosphotransferase synthesis was discon- size some molecules in excess in certain cell tinuous (after starved cells were refed), the cycles and that they are able to go through synthesis began well before macronuclear subsequent cell cycles without synthesizing DNA synthesis and continued when DNA these molecules or synthesizing them at replication was blocked with hydroxyurea. reduced levels. The absence of a doubling Thus nucleoside phosphotransferase syn- in nucleoside phosphotransferase activity thesis does not appear as coupled to DNA through the free running cell cycle obtained Exp Cell Res 108 (1977)
268
Bols and Zimmerman
by the heat shock procedure appears to be a consequence of the synchronization technique. The free running cell cycle is shorter than a normal cell cycle which indicates that cells build up excesses of certain compounds during the cell cycles which contain heat shocks. However, cells selectively synchronized from a late logarithmic population also failed to double their nucleoside phosphotransferase activity over one cell cycle time. This does not appear to be a result of the manipulations required to select these cells as cells selected in the same manner from an early logarithmic population did show a doubling in nucleoside phosphotransferase activity. More importantly, in a study of non-synchronized cells the results indicated that nucleoside phosphotransferase activity increased less rapidly than cell number during late logarithmic phase. Previous workers have presented evidence that the composition of Tetrahymena changes during logarithmic phase [33, 34]. Thus during logarithmic growth no two cell cycles might be the same although certain synthetic events would have to be reproduced faithfully from one cell cycle to the next. This investigation w a s supported by the National Research Council of Canada. N. C.B. was a recipient of a NRC Postgraduate Scholarship.
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