Ribo- and deoxyribonucleotide triphosphate pools in synchronized populations of Tetrahymena pyriformis

12

Biochimica et Biophysica Acta, 378 (1975) 12--17 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98178 RIBO- AND DEOXYRIBONUCLEOTIDE TRIPHOSPHATE POOLS IN SYNCHRONIZED POPULATIONS OF T E T R A H Y M E N A P Y R I F O R M I S

BJORN ANDERSEN NEXO The Biological Institute of the Carlsberg Foundation, 16 Tagensvej, DK-2200 Copenhagen N (Denmark)

(Received May 24th, 1974) (Revised manuscript received September 6th, 1974)

Summary The content of ribo- and deoxyribonucleotide triphosphates has been measured in asynchronous and synchronized populations of Tetrahymena pyriformis. The concentration of ribonucleotide triphosphates does not vary much through the cell cycle. It seems to be a rule that the ATP pool is reduced during division only in cell types which also reduce macromolecular syntheses and respiration. The content of deoxyribonucleotide triphosphates varies through the cell cycle. The pools are maximal during the S-period, but measurable amounts are present in all phases.

Introduction Synchronized populations of Tetrahymena pyriform is are very convenient objects for studies of the sequence and coupling of events in the cell cycle. Several articles dealing with the control of DNA synthesis and cell division in these cells have appeared [1--4]. This article describes determinations of the eight c o m m o n ribo- and deoxyribonucleotide triphosphates in asynchronous and synchronized populations of Tetrahymena. Part of this work has been published previously [5]. Materials and Methods Cell culture and synchronization T. pyriformis GL was grown axenically in 2% proteose peptone, 0.1% liver extract and salts [6]. Cultures in 20--50 ml volumes were grown in 500 ml Fernbach flasks with constant aeration and agitation at 28°C. For synchroniza-

13 tion the temperature was raised to 34°C for 30 min separated with 160 min intervals [7]. The cells were counted electronically.

DNA synthesis DNA synthesis was followed by incorporation of [2-~4C]thymidine (Amersham) into acid precipitable materials [4].

Cell volumes Packe~t cell volumes were determined on 2.50 ml samples of culture by centrifugation for 10 min at 850 × g in cytocrit tubes, and corrected for 15% extracellular fluid.

Phosphate content o f the medium Asynchronous cultures were labelled with H3 32PO4 (1 pCi/ml) from inoculation. Samples of cells were harvested at densities from 1.5 • 104--6.5 • 10 s cells/ml. The total cellular phosphate was determined [8], and the radioactivity was counted. The phosphate concentration of the medium was then calculated from the specific activity of the cellular phosphate and the radioactivity added per ml culture. There was no sign of dilution of label during growth in the medium by gradual liberation of phosphate from organic compounds. The average phosphate concentration from several cultures, 1.56 + 0.02 mM, was used for the nucleotide determinations.

Nucleotide triphosphate determinations Cultures were labelled with 20 pCi/ml of H3 32 PO4 (Ris~b, Denmark) from inoculation at least six generations before sampling. No adverse effects of the 32p activity on the cell growth or morphology was detected. The cells were extracted in the growth medium by addition of 25 pl 6 M HCIO4 to 0.50 ml samples of culture and centrifuged. The supernatants were adjusted to pH 5 with 6 M KOH, 0.5 M EDTA. The KC104 precipitates were spun down and the supernatants stored at --20°C until they were examined chromatographically within three days. The 32p activity of the triphosphates was determined after two
Asynchronous populations of T. pyriformis grow with a generation time of 155 min in the rich medium used here. The cell number increases exponen-

14 TABLE I THE CONTENT AND CELLULAR CONCENTRATIONS OF NUCLEOTIDE ASYNCHRONOUSLY MULTIPLYING POPULATIONS OF TETRAHYMENA

T R I P H O S P H A T E S IN

Cellular v o l u m e s o f 14.1 #1/106 cells in e x p o n e n t i a l l y g r o w i n g p o p u l a t i o n s a n d 1 9 . 5 /11/106 cells in s t a t i o n a r y p o p u l a t i o n s w e r e u s e d for the c a l c u l a t i o n o f the c o n c e n t r a t i o n s . Compound

GTP ATP CTP UTP dGTP dATP dCTP dTTP

E x p o n e n t i a l l y m u l t i p l y i n g cells ( ~ 2 × 10 5 cells/ml)

S t a t i o n a r y cells ( ~ 10 6 c e l l s / m l )

Content ( p m o l e / l O 6 cells)

Concn (NM)

Content (pmole/lO6cells)

Concn (NM)

3 230 18 3 0 0 1490 2970 40 73 37 117

229 1300 105 211 2.9 5.1 2.6 8.3

1390 6170 890 1340 28 21 7 22

72 340 46 69 1.4 1.1 0.4 1.1

tially t o a d e n s i t y a r o u n d 7 . 1 0 s cells/ml, a n d divisions s t o p a r o u n d 10 6 cells/ml. T h e a m o u n t o f N T P in s u c h a culture f o l l o w s general cell g r o w t h t o a d e n s i t y b e t w e e n 2 • 10 s a n d 3 • 10 s cells/ml, t h e n g r o w t h b e c o m e s u n b a l a n c e d . T a b l e I s u m m a r i z e s t h e results o f several e x p e r i m e n t s w i t h a s y n c h r o n o u s l y mul(•) 80 o~

O X'

60

40

A

A

w

--~ 20 LJ

S

(o) °1o 100

~0 I t I

50

(~) _~ vl

[A}

600

2 4OO

x v

E r~ U

40

> -- 20

2OO 10

0

60 T i m e ( min )

120

180

I. -60

I

I

I

a

0

60

120

[~ 180

T i m e (rain)

Fig. 1. Cell divisions a n d D N A s y n t h e s i s in a s y n c h r o n i z e d Tetrahymena p o p u l a t i o n . T h e u p p e r half of t h e figure s h o w s t h e cell n u m b e r (o) a n d t h e division i n d e x (o). T h e l o w e r half s h o w s t h e i n c o r p o r a t i o n o f [2 - 1 4 C ] t h y m i d i n e i n t o t r i c h l o r o a c e t i c a c i d p r e c i p i t a b l e m a t e r i a l w i t h c o n t i n u o u s labelling (A) a n d w i t h 9 rain pulses (~). T h e r e c t a n g l e s o n the abscissa s h o w t h e 6 t h a n d t h e 7 t h s h o c k used f o r s y n c h r o n i z a t i o n . Fig. 2. Cell v o l u m e s i n s y n c h r o n i z e d Tetrahymena p o p u l a t i o n s . T h e v o l u m e s h a v e b e e n n o r m a l i z e d to a p o p u l a t i o n c o n s i s t i n g of 1 0 6 cells a t 0 rain. T h e r e c t a n g l e s o n t h e abscissa s h o w t h e 6 t h a n d t h e 7 t h h e a t s h o c k u s e d f o r s y n c h r o n i z a t i o n . T h e a r r o w s i n d i c a t e cell divisions.

15

o

800

8 600

I1\

q

d

•

6

4OO

~oo 8

?'.

4 1

i

I

I

I

I

Jl

u

-~ 200 0

J

I

II

I

~

t80

60

[

I

L

~

0

I

I

i

60 T i m e (min)

120

-5

',, ,,."

'" ,'T

"",.-"

O&

100 2 L

-60

I

I

L

0

60 Time (min)

i

120

[

Ji

180

Fig. 3. T h e cellulvx c o n c e n t r a t i o n s of r N T P in a s y n c h r o n i z e d Tetrahymena p o p u l a t i o n . Cell n u m b e r a t 0 man: 71 0 0 0 cells/m1. A T P , o; G T P , A, U T P , e; CTP, 4. T h e r e c t a n g l e s at t h e abscissa s h o w t h e t i m e of t h e 6 t h and t h e 7 t h h e a t s h o c k used f o r s y n c h r o n i z a t i o n . T h e a r r o w s i n d i c a t e t h e t i m e of cell divisions, and t h e h o r i z o n t a l lines s y m b o l i z e t h e S p e r i o d s . Fig. 4. T h e cellular c o n c e n t r a t i o n s of d N T P in a s y n c h r o n i z e d Tetrahymena p o p u l a t i o n , d T T P , e; d A T P , o; d G T P , ~; d C T P , 4. F o r o t h e r s y m b o l s , see Fig. 3.

tiplying populations. The concentrations of all nucleotides decrease gradually, as the cells enter the stationary phase. The cell cycle of the synchronized Tetrahymena populations is represented in Fig. 1. The cells divide 100 min after a heat shock (upper half) and enter the S period almost immediately (lower half). The next shock comes late in the S period. The increase in cell volume (Fig. 2), RNA and protein are rather smooth except for plateaus during the heat shocks. The cellular concentrations of rNTP in a synchronized population are shown in Fig. 3. The concentrations do n o t vary much during the cell cycle, i.e. the accumulation of rNTP follows general cell growth. The only larger exception is a decrease in UTP during the heat treatment. The curves show the lowest rNTP values obtained. In other experiments they were up to 50% larger, but the ratios between the nucleotide concentrations were almost constant. The concentrations of dNTP in the same population are shown in Fig. 4. All concentrations have a m a x i m u m during the S period, but measurable concentrations are present in all cell phases. At the onset of S, dTTP is high and dATP drops. The experiments were repeated with cultures kept continuously at 28°C after the sixth heat shock (results not shown). The earlier observed decrease in UTP during the heat shocks (Fig. 3) and the minima of all dNTP just after the heat shocks (Fig. 4) were absent, and thus probably a direct effect of the temperature changes. Apart from this the results were similar to those shown.

16 Discussion The turnover of the nucleotide pool measured can be roughly estimated. From the cellular content of macromolecules [11] and the composition of these [12] one can calculate that the dNTP pools in exponentially growing Tetrahymena suffice for only 1--2 min of DNA synthesis. CTP, UTP and GTP suffice for 2--4 min of RNA synthesis. The half-life of the terminal phosphate group in ATP may be estimated from the oxygen uptake of the cells [13], if one assumes a P/O ratio of 3. An upper limit to the half-life of the terminal phosphate in GTP may be estimated from the protein content of the cells [11], if one assumes that all proteins are stable and that 2 GTPs are used per peptide linkage formed. In both ATP and GTP the half-life of the terminal phosphate is estimated to 1--2 s. The constant ribonucleotide concentrations in synchronized cultures here described are at variance with earlier reports of several-fold changes in the ATP and GTP content prior to and during cell division in Tetrahymena [14,15]. In view of the fast turnover of the NTP it is natural to suggest that harvesting the live cells by centrifugation, as used in the early reports mentioned, modified the pools. It seems that cells when dividing either strongly reduce their macromolecular syntheses, respiration and ATP pool (Physarurn polycephalum, mammalian cells) or t h e y maintain all these parameters fairly constant (Escherichia coli, Schizosaccharomyces pombe, eggs from Strongylocentrotus purpuratus) (refs 16--21 and Nex¢, B.A., unpublished). In contrast to the earlier reports the present results make Tetrahymena conform to this: it belongs to the second group. Quantitative determinations of the dNTP pools in Tetrahymena have not been published before. Our estimates (2--8 pM cellular concentrations, 3--10 pmole/pg DNA) are in agreement with those found in a number of other cells [22--24]. The observed variations in the dNTP pools in Tetrahymena are also in keeping with results with other cell types. It is a general observation that the dNTP pools increase with DNA synthetic activity both in growth stimulated [25,26] and in synchronized cells [22--24]. The presence of distinct amounts of all four dNTP in Tetrahymena cells with low DNA synthetic activity (G2 and stationary cells} suggests that the DNA synthesis in these cells is not quenched by lack of precursors. This conclusion is supported by autoradiographic experiments indicating that low molecular weight thymidine compounds survive in the nucleus between two consecutive S periods [27]. Acknowledgements The author wishes to t h a n k Professor Erik Zeuthen, Dr Leif Rasmussen and Dr Helge Andersen for their encouragement and advice in the course of the work and for help during the preparation of the manuscript. He thanks Dr Per Nygaard for teaching him the chromatographic technique, and the entire staff of the Biological Institute for their helpfulness. The author was supported by a Carlsberg Foundation Scholarship during the work.

17

References 1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Zeuthen, E. (1970) Exp. Cell Res. 6 1 , 3 1 1 - - 3 2 5 Jeffery, W.R. (1972) J. Cell Biol. 5 3 , 6 2 4 - - 6 3 4 Andersen, H.A. (1972) Exp. Cell Res. 74, 610--613 Andersen, H.A. (1972) Exp. Cell Res. 75, 89--94 Nex~b, B.A. and Zeuthen, E. (1973) Sixth Meeting of the European Study Group for Cell Proliferation, Moscow. Abstracts p. 5 Plesner, P,, Rasmussen, L. and Zeuthen, E. (1964) in S y n c h r o n y in Cell Division and Growth (Zeuthen, E., ed.), pp. 543--563. Interscience, New York Zeuthen, E. (1971) Exp. Cell Res. 68, 49--60 Leloir, L,F. and Cardini, C.E. (1957) in Methods in E n z y m o l o g y (Colowick, S.P. and Kaplan, N.O., eds), Vol. 3, pp. 840--850, Academic Press, New York Neuhard, J., Ran der ath, E. and Randerath, K. (1965) Anal. Biochem. 1 3 , 2 1 1 - - 2 2 2 Neuhard, J. (1968) J. Bacteriol. 96, 1519--1527 Leick, V. (1967) C. R. Trav. Lab. Carlsberg 36, 113--126 Hill, D.L. (1972) The Biochemistry and Physiology of Tetrahymena, p. 138, Academic Press, New York Hamburger, K. and Zeuthen, E. (1957) Exp. Cell Res. 13, 443--453 Plesner. P. (1964) C. R. Trav. Lab. Carlsberg 34, 1--76 Scherbaum, O.H.. Chou, S., Seraydarian, K.H. and Byfield, J.E. (1962) Can. J. Microhiol. 8, 753--760 Chin, B. and Bemstein, I.A. (1968) J. Bacteriol. 96, 330--337 Sachsenmayer, W., Immich, H., Grunst, J., Schols, R. a nd Biicher, Th. (1969) Eur. J. Biochem. 8, 557--561 Gerschenson, L.E., Strasser, F.F. and Rounds, D.E. (1965) Life Sci. 4 , 9 2 7 - - 9 3 5 Huzyk, L. and Clark, D.J. (1971) J. BacterioL 108, 74--81 Epel, D. (1963) J. Cell Biol. 1 7 , 3 1 5 - - 3 1 9 Mitchison, J.M. (1971) The Biology of the Cell Cycle, Cambridge University Press, London Bray, G. and Brent, T.P. (1972) Biochim. Biophys. Acta 2 6 9 , 1 8 4 - - 1 9 1 Skoog, K.L., NordenskjSld, B.A. and Bjursell, K.G. (1973) Eur. J. Biochem. 3 3 , 4 2 8 - - 4 3 2 Waiters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 3 1 9 , 3 3 6 - - 3 4 7 NordenskjSld, B.A., Skoog, L., Brown, N.C. and Reichard, P. (1970) J. Biol. Chem. 245, 5360--5368 Tyrsted, G., Munch-Petersen, B. and Cloos, L. (1973) Exp. Cell Res. 7 7 , 4 1 5 - - 4 2 7 Stone, G.E., Miller, O.L. and Prescott, D.M. (1965) J. Cell Biol. 2 5 , 1 7 1 - - 1 7 7