J. Cell Sci. 37, 117-124 (1979)
Printed in Great Briain © Company of Biologists Limited 1979
117
INCREASE IN MACROMOLECULAR AMOUNTS
DURING THE CELL CYCLE OF
TETRAHYMENA:
A CONTRIBUTION TO CELL CYCLE CONTROL*
Gt)NTER CLEFFMANN, WOLF-OTTO REUTER AND HANSMARTIN SEYFERT
Institut fOr Tierphysiologie, Universitat Giessen, Germany
SUMMARY
Increases in RNA, protein and cell size were determined cytophotometrically during the cell
division cycle of Tetrahymena. For these parameters different patterns were found. RNA accumulates slowly during Gi period and faster during macronuclear S. This agrees with the
changing uridine incorporation rate which is at least partly related to the varying macronuclear
DNA amount. Increases in protein content and cell size occur mainly during Gx and G2. This
pattern was confirmed by determining the RNA : protein ratio in individual cells. It is minimal
at the end of the G, period. These findings and evidence from the literature suggest that initiation of DNA replication is under negative control by the relative RNA content of the
cell.
INTRODUCTION
In an exponentially multiplying cell system the cell division cycle of individual cells
is to be considered as a set of regularly repeating events which occur in an ordered
sequence. The significance of each of these events for the completion of the cycle can
be studied by inhibiting single events or by experimental alteration of external conditions. By several different approaches causal relationships between parts of the cell
cycle have been described. It was found e.g. that in Tetrahymena replication of macronuclear DNA can be separated in different ways from the cell division cycle, demonstrating that the completion of the division cycle is not directly dependent on the
replication of DNA and vice versa (Hjelm & Zeuthen, 1967; Cleffmann, 1968; Frankel,
Jenkins & DeBault, 1976; Zeuthen, 1978). Nevertheless, in the vast majority of cell
cycles the coupling of replication and division is maintained and replication occurs
invariantly at the same stage of the cell cycle. Therefore, some connexion between
these 2 events must exist. The nature of this coupling mechanism is not known. It may
consist of a more complex situation of the cell in which cell constituents reach specific
ratios. To find such situations we describe in the present study the development of the
main macromolecular components during the cell cycle in Tetrahymena. The results
are based on cytophotometric and autoradiographic determinations in single cells. This
allows one to relate different parameters for individual cells. The findings suggest that
Correspondence to: Dr Gfinter Cleffmann, Institut filr Tierphysiologie, Justus-LiebigUniversitat, Wartweg 95, 6300 Giessen, Germany.
• Dedicated to Professor Peter Karlson on the occasion of his sixtieth birthday.
n8
C. Cleffmann, W.-O. Renter and H.-M. Seyfert
the initiation of DNA synthesis is under negative control of relative RNA and/or
DNA content in the cell.
MATERIAL AND METHODS
Cells. Cultures of Tetrahymena sp. strain HSM # were grown in synthetic medium at
29 °C. By regular transfer the cells were kept in exponential growth phase: the time for
doubling in cell number ( = average generation time) was 210 min and constant within the
range of 500 to 30000 cells per ml. The multiplication rate was determined by measuring
cell densities in culture samples (Coulter Counter) or by averaging individual generation
times of cell samples in capillaries. Cells for experiments described below were taken from
cultures at about 1000 cells per ml. They were collected at late division, transferred to
capillaries, and prepared for autoradiography or cytophotometry at the indicated times. They
were usually dried onto gelatine-coated slides and fixed in ethanol acetic acid (3:1, 20 min).
Cell size was measured as the area of dried and stained cells taken by a planimeter on photographs. Since the data of Morrison & Thompkins (1973) as well as our own demonstrate that
determinations of projected area, Coulter volume, and volume of fixed cells in suspension
measured microscopically are in good correlation in samples of different developmental
stages and exhibit the same pattern of development, area has been taken as the measure for
cell size.
RNA content was determined cytophotometrically at 600 nm after staining with gallocyanine according to Kiefer, Kiefer & Sandritter (1966). For cytophotometry a Zeiss microscope photometer SMP 05 equipped with a scanning stage and a o-5-/tm diaphragm was
used throughout this study. Since gallocyanine stains all nucleic acids parallel samples were
taken from Gl and Gt cells treated with RNase and stained with gallocyanine. The average
absorbence of these cells was subtracted from the readings from the experimental cells.
Protein content was determined cytophotometrically at 410 nm after staining with dinitrofluorbenzene (DNFB) according to Kimball et al. (1971).
RNA synthesis was determined by grain counting in autoradiographs after 15-min labelling
with [*H]uridine at a final concentration of 2 /tCi 3H-U per ml. The technical details have
been described earlier. For discussion of the method see section Results.
Protein synthesis was determined as incorporation rate of PHJleucine. Samples of 20-30
cells were dried on slides after a 10-min pulse and radioactivity determined after solubilizing
the cells.
RNA to protein ratio of individual cells was determined by successive staining and measuring
both parameters. Since absorption spectra of DNFB and gallocyanine are sufficiently different
that DNFB contributes at 600 nm less than 1 % to gallocyanine absorption, protein content
was measured first, and after recording the location of the cells RNA content was determined
as described above.
The relation of RNA synthesis to macronuclear DNA amount was investigated by first
performing autoradiography for [*H]uridine incorporation and staining the same cells with
Feulgen as described earlier (Cleffmann, 1968). Hydrolysis at 55 °C removes the autoradiographic emulsion together with the silver grains so that cytophotometry of Feulgen-positive
material at 550 nm can be applied (Kimball et al. 1971).
RESULTS
The timing of the macronuclear DNA replication phase (5-phase) of Tetrahymena
has been determined in a number of previous papers (e.g. Stone & Cameron, 1964).
Under the culture conditions applied in this study DNA is replicated between 55 and
• This strain was formerly designated as Tetrahymena pyriformis HSM. Its assignment to
one of the phenosets of the Tetrahymena group (Nanney & McCoy, 1976) is under investigation.
Increase of macromolecules in cell cycle
119
135 min after cell division, which amounts to 26-65% of the cell cycle. Initiation and
termination of replication may vary between cells by ± 15 min. All available data show
that in Tetrahymena the amount of DNA in the macronucleus is doubled during Sphase. Furthermore it was found that DNA is regularly lost during division (Cleffmann,
1968). On the average 3 % of the DNA of a Ga macronucleus is extruded from the
dividing macronucleus. Therefore, the sum of the DNA in the daughter nuclei is less
than the DNA in the macronucleus of the mother.
20
40
60
% of cell cycle
80
100
Fig. 1. Increase of KNA(A), protein(#) and cell size(B) during the cell cycle of Tetrahymena. RNA: Absorption at 600 run in scanning microphotometry after gallocyanine
staining. Means of 18-30 cells. Corrected for RNase-insensitive material. Standard
errors of the mean (s.E.) are covered by the symbols. Protein: Absorption at 410 nm
after DNFB staining. Means of 20-30 cells ± s.E. Cell size: Projected area in /im 1 .
Average of 3 experiments. In each experiment 10-25 cells per sample were measured
( ± S . E . ) . Bar line indicates S-phase.
Cytophotometric determination of RNA in individual cells of known cell cycle age
show that the rate of increase is not constant throughout the cycle (Fig. 1). During
Gx phase RNA increase is very low or even absent. During S-phase RNA increase
accelerates. During G2 the RNA increase on the average remains at the same high rate
as at the end of 5-phase. Repetitions of the experiment reveal essentially the same
pattern in particular with respect to the low rate of RNA accumulation during Gx and
the increasing rate duiing S. The increase of RNA during a complete cycle does not
result in a doubling of the content at the beginning of the cycle.
120
G. Ckffmann, W.-O. Renter and H.-M. Seyfert
The increased accumulation of RNA during S and Gt may depend on the number of
templates which increases during S. Since the macronuclear DNA amount is variable
in Tetrahymena it is possible to test whether the amount of RNA produced per cell is
related to the macronuclear DNA content. Therefore, G1 cells were incubated in
•
60 -
•
•
C
o
B
5
f 40
u
c
8UIC
•
*•
:"
••
•
•
•
•
5 20 —>
z
I
1
1
1
i
20
40
60
80
100
Macronuclear DNA
Fig. 2. Correlation between incorporation of [*H]uridine into acid-insoluble material
and macronuclear DNA content. Abscissa: photometer readings after Feulgen staining in scanningmicrophotometry at 550 nm. Ordinate:silvergrainspercell.Regression
line y = 0-25*+ 28-3. r = +0-34 (p = o-oi).
I 1-0
o
a
8
20
40
60
80
100
%of cell cycle
Fig. 3. Rate of protein synthesis at different times of the cycle. Ordinate: Radioactivity
normalized to the first value. Mean of 3 experiments ± s.E. Bar line indicates 5-phase.
[3H]uridine. After autoradiography the amount of label incorporated in acid-insoluble
material was determined by counting grains per cell. Thereafter cells were stained
with Feulgen and the amount of DNA measured cytophotometrically. Uridine incorporation alone is not an unequivocal measure for transcription rate. Several parameters like rate of uptake, pool size, turnover, selfabsorption may influence the results.
Increase of macromolecules in cell cycle
121
In the present experiment, however, cells of the same physiological state are compared.
Therefore, the intensity of labelling is taken to indicate RNA-synthetic rate.
Fig. 2 shows that uridine labelling and DNA amount are positively correlated. The
correlation between the parameters is rather weak due to the scatter of data. Therefore,
only qualified conclusions can be drawn. Two repetitions of the same experiment,
however, produced essentially the same results. This suggests that one of the factors
determining the rate of RNA synthesis is the amount of DNA. This holds true not
only for the changing DNA content during progress of the cell division cycle as stated
above but also for DNA content varying between individual cells.
40
60
% of cell cycle
Fig. 4. Ratio of RNA to protein during the cell cycle. RNA and protein have been
determined by microphotometry in the same cells. The ratio was set to 1 at the beginning of the cycle. 30—50 cells per sample. Means ± S.E. Bar line indicates 5-phase.
The rates of protein increase differ from the rates of RNA increase (Fig. 1). Protein
accumulation is lowest during 5-phase and higher in Gx and particularly G2. This pattern is due to changing rate in protein synthesis which decreases by about one third
during early 5-phase (Fig. 3). The same pattern as for protein increase is found for
the increase in cell size (Fig. 1). In this case the retardation of increase during iS-phase
is even more pronounced. Cell size and protein content develop at similar rates during
the cell cycle. This is also demonstrated by a close correlation (r = + 0-85) between
cell size and cellular protein for individual cells (n = 52). Again protein content is not
completely doubled during the cell cycle. Whereas the factor by which RNA accumulates is i-6, it is 1-9 for cell size and 1-7 for protein.
The increase of RNA and the increase of protein (or size) during the cell cycle are
inverse with respect to their relative rate. This leads to changing ratios of the 2 components. These ratios were measured directly in individual cells during the cell cycle.
The RNA : protein ratio at the beginning of the cell cycle was set to 1. In Fig. 4 it is
shown that the ratio decreases during the Gj-phase and reaches a minimum during
early 5. It increases during 5-phase and remains approximately constant during G2 at
a level which is characteristic for the beginning of the cycle.
122
G. Cleffmam, W.-O. Renter and H.-M. Seyfert
DISCUSSION
There are some reports on the growth pattern of Tetrahymena within the cell cycle.
Determination of cell volume (Cameron & Prescott, 1961) and 3H-amino acid incorporation into protein (Prescott, i960) revealed a linearity of growth except for the short
period prior to division. Using the cartesian diver for a survey of growth of single cells
in dry mass it was found that individual cells may exhibit different patterns such as
linear or exponential increase (Lovlie, 1963). The pattern of cell growth varies even
more if one considers different cell systems. Every cell type, therefore, has to be analysed separately, distinguishing between different parameters of cell growth. The data
presented here for Tetrahymena show that protein and RNA increase during the cell
cycle at different rates. This is particularly obvious during Gv Whereas the RNA content of the cell remains almost constant, protein and cell size increase considerably.
During S, on the other hand, the increase in RNA is pronounced and the accumulation
of protein slows down. The pattern leads to a relative decrease of RNA as referred
to protein content and cell size. This could also be demonstrated directly in single
cells. The absolute rates of increase of these 2 compounds are of such magnitude that
they sum up to an almost linear increase in macromolecular dry weight throughout
the cell cycle (Reuter, 1974).
The rate of increase of cellular RNA is subject to several limiting factors. The
present study suggests that one of them is connected to macronuclear DNA amount.
During S this amount is doubled and RNA accumulation increases markedly at this
time. An increase in RNA synthesis in connexion with the S-phase has also been
found in previous studies (Cleffmann, 1965; Jauker, Seyfert & Sgonina, 1975; Keiding
& Andersen, 1978). One of these reports (Jauker et al. 1975) demonstrates that increase
in RNA-synthetic rate is always coupled to S-phase under different growth conditions.
This gene dose effect on transcription rate does not only occur during the cell cycle
of the average cell but also in individual cells since in cells with lower macronuclear
DNA content less RNA is produced. This is not surprising but has not yet been shown
on the level of individual cells. It is evident from Fig. 2 that the correlation between
DNA amount and uridine incorporation is not proportional in the sense that a doubling in DNA does not result in a doubling in incorporation. From this and from the
finding that incorporation rate may be higher during S (Jauker et al. 1975) than during
Ga it is evident that the amount of DNA i9 not the only factor determining the
transcriptional activity and the gain in cellular RNA.
There is no definite information on the regulation of the particular pattern of protein
accumulation in Tetrahymena. It is demonstrated here that the specific pattern of protein increase is a result of changing rate of protein synthesis.
Earlier findings (Jauker, 1977), this study, and recent unpublished results from this
laboratory demonstrate that RNA increase as well as protein and volume increase do
not result in a complete doubling within the cell cycle of exponentially growing Tetrahymena cultures. The mode of regulation of this unbalanced growth has been found
and discussed with respect to RNA by Jauker (1977).
Several lines of evidence point out that the reduction of RNA/protein ratio during
Increase of macromolecules in cell cycle
123
Gx and at the beginning of S is not only a temporal coincidence but that a low RNA
concentration is causally related to initiation of DNA replication: Decrease in macronuclear DNA as it occurs during successive cell generations and which is accompanied
by reduction in transcription induces extra replication periods as soon as the DNA
amount has reached a lower threshold (Cleffmann, 1968). Experimental inhibition
of DNA synthesis by hydroxyurea releases replication of DNA immediately after
division excluding a Gx period (Worthington, Salamone & Nachtwey, 1976.) It was
further shown by Seyfert (1977) that low macronuclear DNA content is connected
to an early beginning of replication. These findings show that the lower the number of
templates the sooner replication is initiated. This relationship is not confined to Tetrahymena but has also been found for tissue culture cells (Cress & Gerner, 1977). The
amount of DNA in fact acts through transcriptional activity as was shown by Jauker
(1975), who experimentally reduced transciiptions at the end of a cell cycle and found
a significantly advanced replication phase.
All these findings suggest that replication is initiated when RNA in relation to protein reaches a lower threshold. This point will be attained if at increasing protein the
net increase of RNA is low. Concentration of RNA or a certain fraction of it falls
below the level that prevents initiation of DNA synthesis. If only a fraction of RNA is
responsible for this mechanism it is likely to be mRNA because of its turnover rate.
The inhibiting effect may be established via nucleotide pools. When RNA synthesis
in relation to the cell size and overall nucleotide metabolism is low, pool size may
increase to a level that is inducing DNA replication.
The negative control of DNA replication by low RNA to protein ratios fulfills all
requirements for the model of regulation by an unstable inhibitor as described by
Fantes et al. (1975). The amount of overall RNA and therefore most likely also of
certain RNA fractions remains constant throughout Gv Its concentration decreases
reaching a lower threshold at the beginning of S. The difference between the present
case and their model is, that the threshold reaction is not the initiation of mitosis but
that of replication. Replication in turn causes a rise in concentration so that the cycle
starts again at this point. Since it is known that Gt is the most variable part of the
cell cycle, the timing of the beginning of S also determines the length of the cell cycle.
REFERENCES
I. L. & PRESCOTT, D. M. (1976). Relation between cell growth and cell division.
Expl Cell Res. 23, 354-360.
CLEFFMANN, G. (1965). Die Schwellen derHemmung der NucleinsSuresynthese und derTeilung
durch Actinomycin bei Tetrahymena pyriformis. Z. Zellforsch. mikrosk. Anat. 67, 343-3 30.
CLEFFMANN, G. (1968). Regulierung der DNS-Menge im Makronukleus von Tetrahymena.
Expl Cell Res. 50, 193-207.
CRESS, A. E. & GERNER, E. W. (1977). Hydroxyurea treatment affects the G1 phase in next
generation CHO Cells. Expl Cell Res. n o , 347-353.
FANTES, P. A., GRANT, W. D., PRITCHARD, R. H., SUDBERRY, P. E. & WHEALS, A. E. (1975).
The regulation of cell size and the control of mitosis. J. theor. Biol. 50, 213-244.
FRANKEL, J., JENKINS, L. M. & DEBAULT, L. E. (1976). Causal relation among cell cycle
processes in Tetrahymena pyriformis. J. Cell Biol. 71, 242-260.
HJELM, K. K. & ZEUTHEN, E. (1967). Synchronous DNA sythesis following heat-synchronized cell division in Tetrahymena. Expl Cell Res. 48, 231-232.
CAMERON,
124
G. Cleffmann, W.-O. Renter and H.-M. Seyfert
F. (1975). A feedback control of cell cycle parameters in Tetrahymena. J. Cell Biol.
67, 901-904.
JAUKER, F. (1977). Analyse eines Zelkoachstums-Programms am Beispiel der quantitativen Enttoicklung ribosomaler RNS-Gehalte bet Tetrahymena pyriformis. Habilitationsschrift,
Fachbereich 15-Biologie der Justus-Liebig-Universitat Giessen.
JAUKER, F., SEYFERT, H. M. & SGONINA, J. (1975). Temperature dependent changes of cell
growth parameters in Tetrahymena. Exp Cell Res. 96, 439—442.
KEIDING, J. & ANDERSON, H. A. (1978). Regulation of ribosomal RNA synthesis in Tetrahymena
pyriformis. J. Cell Sci. 31, 13-23.
KIEFER, G., KIEFER, R. & SAKDRITTER, W. (1966). Cytophotometric determination of nucleic
acids in UV-light and after gallocyanine chromalum staining. Expl Cell Res. 45, 247-249.
KIMBALL, R. F., PERDUE, S. W., CHU, E. H. Y. & ORTIZ, J. R. (1971). Microphotometric and
autoradiographic studies on the cell cycle and cell size during growth and decline of Chinese
hamster cell cultures. Expl Cell Res. 66, 17-32.
LOVLIE, A. (1963). Growth in mass and respiration rate during the cell cycle of Tetrahymena
pyriformis. C. r. Trav. Lab. Carlesberg 33, 377-413.
MORRISON, G. A. & TOMKINS, A. L. (1973). Determination of mean cell size of Tetrahymena in
growing cultures. .7. gen. Microbiol. 77, 383-392.
NANNEY, D. L. & MCCOY, J. W. (1976). Characterisation of the species of the Tetrahymena
pyriformis complex. Trans. Am. microsc. Soc. 59, 664-682.
PRESCOTT, D. M. (i960). Relation between cell growth and cell division. IV. The synthesis of
DNA, RNA, and protein from division to division in Tetrahymena. Expl Cell Res. 19, 228238.
REUTER, W. O. (1974). Interferenzoptische Massenbestimmungen an logaritltmisch wachsenden
Zellen von Tetrahymena pyriformis HSM. Ein Beitrag zur quantitativen Erfassimg des
Zellicachstums. Diplomarbeit, Fachbereich 15-Biologie der Justus-Liebig-Universitat
Giessen.
SEYFERT, H. -M. (1977). A short Gi period is correlated with low macronuclear DNA contents
in Tetrahymena. Expl Cell Res. 108, 456-459.
STONE, G. E. & CAMERON, I. L. (1964). Methods for using Tetrahymena in studies of the
normal cell cycle. In Methods in Cell Physiology, vol. 1 (ed. D. M. Prescott), pp. 127-141.
New York: Academic Press.
WORTHINGTON, D. H., SALAMONE, M. & NACHTWEY, D. S. (1976). Nucleocytopla8mic ratio
requirements for the initiation of DNA replication and fission in Tetraltymena. Cell Tiss.
Kinet. 9, 131-145.
ZEUTHEN, E. (1978). Induced reversal of order of cell division and DNA replication in Tetrahymena. Expl Cell Res. 116, 39-46.
(Received 19 September 1978)
JAUKER,