J. Cell Set. 58, 211-223 (1982)
211
Printed in Great Britain © Company of Biologists Limited 1982
MEAN MACRONUCLEAR DNA CONTENTS
ARE VARIABLE IN THE CILIATE TETRAHYMENA
H.-M. SEYFERT AND G. CLEFFMANN
InstitutfQr Tierphysiologie, Justus-Liebig-Univerritdt,
Federal Republic of Germany
Wartweg 95, .D-6300 Giessen,
SUMMARY
Using cytophotometry the mean macronuclear DNA content of the ciliate Tetrahymena was
found to be variable in two cell lines examined. While environmental changes (different
culture fluids, temperature shifts) influence the mean DNA content of at least one of the two
cell lines, spontaneous changes of the means are observed in both lines. The mean DNA
contents of cultures maintained permanently in denned medium at various temperatures
suggest the existence of a lower (7-5 pg) and an upper (136 pg) limit of the mean macronuclear
DNA contents. Greatly differing means, either below or above those limits, were only occasion*
ally found in cultures shifted to different temperatures for periods of less than 3 days. Eventually, the Gj mean macronuclcar DNA contents of these cultures differed greatly from the
doubled Gl mean value (maximum: mean G, value equalling four times the Gr mean). Analysis
of the maximum and minimum G^ DNA contents found in any culture indicates no absolute
macronuclear DNA content triggering the regulation of macronuclear DNA content. There
is no stringent correlation between the average cell volume and the average macronuclear
DNA content. The data are discussed with reference to reported mechanisms of regulation
of macronuclear DNA content.
INTRODUCTION
Unlike the other eukaryotes the ciliates have two kinds of nuclei: a small, diploid
germinal micronucleus and a, usually, larger macronucleus. Although the basic
macronuclear chromatin organization may vary greatly in different ciliate species
(see Raikov, 1976, for a review), its function is always to provide the genetic material
necessary during vegetative growth.
Macronuclear 'amitotic1 division lacks the distributional precision of mitotically
dividing nuclei, thus eventually transmitting unequal amounts of DNA to each
daughter cell. Since these imbalances are compensated during successive division
cycles, apparently regulatory mechanisms of the macronuclear DNA content have
evolved.
For individual Tetrahymena cells the existence of a DNA-content increasing
(Cleffmann, 1968, 1975) and two DNA-content decreasing mechanisms (Cleffmann,
1968, 1980; Doerder & DeBault, 1978) of regulation have been established: whenever
macronuclear DNA content is low cells may quadruple their DNA content within
one division cycle, and if it is too high they may skip an entire S-phase. Additionally,
the cells extrude variable amounts of chromatin during most macronuclear divisions.
The rationalizing models of these mechanisms postulate the existence of upper and
lower threshold limits between which the macronuclear DNA content is regulated.
212
H.-M. Seyfert and G. Cleffmann
Little is known about the nature of these limits. Heat-shock experiments (Jefffrey,
Frankel, DeBault & Jenkins, 1973; Cleffmann, 1975), as well as the application of
drugs like actinomycin D (Cleffmann, 1966) and hydroxyurea (Worthington,
Salamone & Nachtway, 1976), have shown that at least the upper limit must be variable
and can be set at different DNA contents. Unfortunately, these treatments all interfere
severely with cellular metabolism, uncoupling cell growth from cell reproduction.
To avoid this, we attempted to alter the mean macronuclear DNA contents of
populations by environmental changes and asked: (a) can we identify thresholds of
macronuclear DNA content regulation in terms of absolute DNA contents; and
(b) can they be readjusted ?
MATERIALS AND METHODS
Cells and culture conditions
Two highly inbred strains of Tetrahymena thermophila (A1768II and B1868IV) were maintained as shelf cultures in 1 % Proteose Peptone (no. 3; Difco) supplemented with o-1 % yeast
extract (Difco) (1 % PPY medium) at room temperature. They were loop-transferred biweekly.
Experimental cultures (10 ml in test tubes or shaken 100 ml cultures in 500-ml Erlenmeyer
flasks) were kept either in this medium, in 2 % Proteose Peptone (2 % PP), in 1 % Proteose
Peptone (1 % PP), or in defined medium (Elliott, Brownell & Gross, 1954) supplemented with
1 mg/1 cholesterol and 0-04 % Proteose Peptone (denned medium). Unless otherwise indicated,
cells were adapted for 3 days to the respective culture fluids.
Estimation of DNA contents
Macronuclear DNA contents were measured cytofluorometrically after Feulgen staining,
essentially as previously described (Seyfert & Preparata, 1979). Usually, 50 cells at a defined
stage in the cell cycle were measured. Values for G, phase were obtained from cells that were
visibly in preparation for macronuclear division as judged from the elongated shape of the
macronucleus, and from the fact that the micronuclei had already divided at that stage. Values
for Gx phase were derived from separated sister macronuclei. Inclusion of bull sperm standards
allowed, in most experiments, conversion of relative DNA contents into pg of DNA, using
3-3 pg/bull sperm as the reference value (Leuchtenberger, Leuchtenberger, Vanderly &
Vanderly, 1952).
Cell counting and sizing
Cells were counted and their sizes measured with a Coulter Counter model ZB connected
to a Coulter Channelyzer as previously described (Seyfert & Preparata, 1979). The sizemeasuring system was calibrated with Tetrahymena populations of different mean cell volumes,
which had been measured microscopically, using the equation:
VtM = i7,a* x b,
where a — halved short axis, b = halved long axis.
Thus, the 'electronic cell volumes' could be converted into /*m*.
RESULTS
Nutritional effects upon macronuclear DNA content
In the first set of experiments we assessed the effect of different culture media upon
the average Gt DNA content of cells maintained constantly at 29 °C. All samples
Variable macronuclear DNA contents
213
were drawn from cultures in the early phase of logarithmic growth (Table 1). Reproducibly, the mean DNA content was highest in 1 % PPY medium and lowest in
defined medium. These differences are statistically significant at the 5% level
(Student's <-test), whereas all other combinations of differences reveal a lower level
of statistical significance. Comparison of the means indicates a DNA content 22%
lower in defined medium than in 1 % PPY. While this reduction in DNA content is
paralleled by a reduction in cell size (mean cell volume in 1% PPY = 18000 /mi 3 ,
compared to 8000/im* in defined medium) the apparently much larger cells kept in
2 % PP had a DNA content roughly 10% lower than the cells maintained in 1 % PPY.
Table 1: Gx macronuclear DNA content of T. thermophila BIV cells maintained
at 29 °C in various culture fluids
Expt
Culture fluid
X* (±S.E.)
n
1a
b
Def. mediumj
7 6 4 ± 2-55
883 ± 2-68
9 8 2 ± 368
8 9 4 ± 3-12
14-11 ± 0-24
1677 ± o-49
17-77 ± 0-28
40
42
40
40
c
d
1 % PP
1 % PPY
2%PP
Def. medium
b
C
i%PP
i%PPY
168
60
124
c.v.
DMf
2I-I
8
19-7
16
18
237
221
22-5
22'6
17-8
3°
—
—
—
Ratio
X&/Xc = 078
Xa/Xh = 087
ATb/Xc = 0-90
—
Xa/A'c = 0 7 9
Xa/Xb = 084
XbfXc = 094
• Mean Gx macronuclear DNA contents in arbitrary units (expt i, extinction units at
550 ran; expt 2, fluorescences units), S.E., standard error of the mean.
f Most frequently occupied size channel in the Coulter Channelyzer representing an
approximate measure of the average cell volume in fim3 x io" 8 . c.v., coefficient of variation.
X Def. medium, defined medium as described in Materials and Methods.
The observed variation in the individual Gx DNA contents was similar in all samples,
and the mean coefficient of variation of 21-4% agrees well with previous reports
(Seyfert, 1979; Doerder, 1979).
Effects of temperature on macronuclear DNA content
Previous experiments (Seyfert & Cleffmann, 1979) indicated significantly higher
macronuclear DNA contents, if T. thermophila BIV cells were grown at 38 °C
instead of 29 °C in all culture fluids tested (1 % PPY, 1 % PP, defined medium). The
greatest temperature effect was observed in defined medium, in which we found
almost twice the DNA content of the 29 °C controls, at 19 °C as well as at 38 °C
(Table 2). This temperature-mediated readjustment of macronuclear DNA limits
was examined in greater detail in order: (a) to obtain confirmation of the results
and (b) to sample cultures during the process of changing the mean macronuclear
DNA content of the population. Because of reported strain-dependent differences in
the timing of downward regulation of macronuclear DNA content after macronuclear
reorganization (Doerder & Debault, 1978), we used two different strains (BIV and
All) of T. thermophila and ran the following experiment. Cells were adapted as
10-ml test-tube cultures for 3 days to 29 °C in defined medium and then inoculated
into 100 ml shaken batch cultures at 19, 29 and 38 °C to cell densities of 1000 cells/ml.
214
H.-M. Seyfert and G. Cleffmann
Table 2. Macronuclear DNA content ofT. thermophila BIV cells, adapted for 8 days to
defined medium and the indicated temperatures
Temperature
(°C)
X{±s.v.)
19
29
38
n
c.v.
Source
24-01 ± 071
124
331
I3'i9 ± °'39
46
2O-i
24-38 ± 080
49
229
Symbols as in Table 1.
Random sample
Gt population
Gx population
Table 3. DNA content ofT. thermophila cells adapted for various periods to different
temperatures*
Expt1: BIV cells
Starting
culture
(3 days
at 29 °C)
Xf
After 24 h at
29 °C
I2-S-I
±083
n
c.v.§
15
256
X
29 °C
14-79
±0-74
n
23
C.v.
>
A
/
19 °C
38 °C
After
48 h at
38 °C
489
±023
319
±036
8-22
490
±055
23
22-6
35
675
±090
6
3017
20
A
29 °C
44-85
Expt 2: All cells
After
After 24 h at
A
48 h at
19 °C
19°C
38°C
1
11 06
1323
83
±071
±075
±o-54
24
12
17
2606
31 -2t
2010
23-85
After 6 days at
t
19 °C
38 °C
607
±035
870
±0-41
±065
8
1620
17
1965
28-1
9-00
15
After 5 days at
A
r
29 °C
290
±0-7611
48
1804
19 °C
12-44
±0-56
16
38 °C
3029
± I-2I ||
179
1695
18
Expt 3: ComparisonAll versus BIV cells, all 10 days adapted
All at
BIV cells at
A
X
29 °G
6-6i
±050
n
c.v.
12
19 °C
779
±024
20
1387
A
38 °C
6-57
±°45
29 °C
7-29
±032
19 °C
756
±046
7
22
10
180
19-19
16-70
2598
• Each experiment represents one Feulgen-staining series.
t X, Gi mean macronuclear DNA content ( ± s.E.) in pg of DNA.
|| Gt mean macronuclear DNA content ( ± S.E.) in pg of DNA.
§ C.v., coefficient of variation in %.
38 °C
1458
±0-98
8
1907
Variable macronuclear DNA contents
215
Generation times and cell volumes were recorded throughout culture growth. After
24 h samples were removed for determination of cytophotometric DNA content.
Furthermore, from these briefly temperature-treated cultures we inoculated test-tube
cultures. They were kept at the respective temperatures in permanent logarithmic
growth by daily transfers. After at least 5 days of adaptation they were sampled for
determination of DNA content. Data from these experiments are shown in Table 3,
together with results of a direct comparison between both strains, which had been
adapted for 10 days to the different temperatures in test-tube cultures.
Considering first the temperature effects upon macronuclear DNA contents of
BIV cells adapted for long periods (Table 3, Expt 1), it is clear that the cells kept at
19 °C and at 38 °C had higher DNA contents than the 29 °C controls (19 °C, + 43 %;
38 °C, +49%), as was expected from previous results (Table 2). Repetition of the
experiment (Table 3, Expt 3) again revealed higher DNA content at 38 °C than at
29 °C ( + 84%), but at 19 °C the cells had almost the same DNA contents as the
29 °C controls.
All cells do not show this temperature sensitivity: the cells of this strain adapted
for long periods (Table 3: Expts 2,3) have similar DNA contents at all the temperatures
tested.
This strain-dependent difference in the temperature sensitivity of macronuclear
DNA contents was also observed in cultures temperature-treated for short periods
(Table 3; Expts 1, 2; 24 h cultures). While the All cells had similar DNA contents
at 19 °C and 29 °C, and a reduced DNA content at 38 °C, the BIV cells showed
great variations (Table 3, Expt i, 24 h cultures). Upon shift to 19 °C the mean
macronuclear DNA content dropped from 12-51 pg to 319 pg within 24 b. The
generation time of 401 min, measured in this exponentially growing culture, was
normal for this growth condition, indicating healthy cells. The average cell volume
dropped from 17000 to ioooo/mi 3 . The cells shifted to 38 °C had a mean DNA
content of 8-22 pg. After 48 h of culture growth this particular culture was sampled
again for determination of DNA content, since the average cell volume had increased
from 13000 to 18000/tm3 (Fig. 1). The DNA content was reduced to 4-90 pg,
indicating no close relationship between cell volume and macronuclear DNA content.
Unexpectedly, both strains revealed temperature-independent fluctuations of the
mean macronuclear Gx DNA contents throughout the experiments. For BIV cells this is
exemplified in Expt 1 (Table 3). In the cultures maintained permanently at 29 °C, the Gx
DNA content decreased from 12-51 pg in the starting culture to 4-89 pg in the shaken
100-ml batch culture (29 °C) within 24 h of vigorous culture growth (generation
time = 196-7 min). The average cell volume remained unchanged. After 6 days
at 29 °C the mean DNA content was 6-07 pg, still about only half the DNA content
of the starting culture. All cells are also capable of readjusting their mean macronuclear DNA content, since a comparison of the average G1 DNA contents of the
cells adapted for long periods, from Expt 2 (X = 14-0 pg) and Expt 3 (X = 7-0 pg),
reveals a precisely twofold difference in the mean macronuclear G1 DNA contents.
Summarizing the results concerning the temperature effects upon the mean macronuclear DNA content, it is noted: (1) in BIV cells adapted for long periods the DNA
216
H.-M. Seyfert and G. Cleffmann
contents at 38 °C were always found to be significantly higher than at 29 °C; at 19 °G
it was twice found higher and once found similar to the 29 °C control. The mean
macronuclear DNA contents of this cell line are greatly affected by temperature
shifts to either 19 °C or 38 °C. These aberrations of the mean macronuclear DNA
contents caused by temperature shifts are erased by prolonged maintenance at the
different temperatures. (2) The All cells behave differently, since their mean G1
DNA contents were alike at all temperatures tested, and are unaffected by temperature
shifts. (3) Temperature-independent fluctuations of mean macronuclear DNA
contents occur in both lines.
15-
_
10-
5-
~T~
10
20
Mean cell volume I f i m 1 X 10~3)
Fig. i. Relationship between mean cell volume and macronuclear DNA content of
T. thermophila BIV ( # ) and All cells (x ). From short-time temperature-treated
cultures sampled for DNA content determinations (Table 3) cell volumes were
recorded and plotted against the average macronuclear DNA content. Circled values
belong to the same culture sampled twice, 24 h apart (cf. Table 3, Expts 1, 2).
The range of the observed mean Gx DNA contents of the All cells (6-97 to i4-79 pg)
was not quite as large as that of the BIV cells (319 to 1458 pg). This variability of
the mean G1 DNA contents was also observed when the effects of starvation upon
macronuclear DNA contents were assessed. For this purpose, we grew BIV and All
cells at 29 °C in 1 % PPY medium as 100-ml batch cultures, and starved (10 mM-Tris,
pH 7-4) for 3 days samples that had been removed during: (a) exponential culture
growth, and (b) during stationary phase. Afterwards DNA contents were measured
from 200 randomly selected specimens and compared with G1 and Gt cells obtained
from the early logarithmic cultures, as well as with samples from the stationary
Variable macronuclear DNA contents
217
cultures. The distributions (not shown) of most samples were compatible with the
assumption that the DNA contents remained unchanged during stationary phase or
during starvation. However, the All sample removed from the starved stationaryphase batch culture had twice the DNA content of the stationary culture, from
which it had been removed. The BIV cells remained unchanged.
To examine a possible relationship between the macronuclear DNA content and
the cell volume, we recorded the cell volume distributions throughout the culture
growth in the experiments shown in Table 3. The plot of the mean cell volumes
versus the mean Gx DNA contents (Fig. 1) shows no correlation.
Limits of mean macronuclear DNA contents
The plot of G1 mean values from cultures adapted for long periods (Fig. 2A)
strongly suggests a non-random distribution. Rather, the values are scattered around
a low {X = 753 ± 0-37 pg) and a high (X = 13-58 ± 064 pg) mean. These means
correspond favourably with the calculated 32 C and 64 C levels of macronuclear DNA
contents, which are 7-36 pg and 1472 pg of DNA, using 092 pg as the 4C micronuclear
I
i •
10
10
•
•
\
•
i
10
15
10
15
•
20
20
•
•
•
r
^
30
30
DNA(pg)
Fig. 2. Distribution of mean Gx or G, macronuclear DNA contents of populations
(in pg of DNA, data from Table 3). The Gj means of cells adapted for long periods
(A) are clustered around low and high means (arrows), whereas the values of shorttime temperature-treated cultures are scattered over a wide range (B), the largest
mean representing 4-5 times more DNA than the smallest. The G, means of stable
cultures (c) are grouped around 15 and 28 pg (means indicated by arrowheads; value in
parenthesis is not incorporated into calculation of means). The distribution of G,
means of short-time temperature-treated cultures (D) is different from the corresponding Gt distribution, since the ratio of the largest to the smallest value is only
2-25. Asterisks emphasize that the abscissa is halved in c and D compared to A and B.
The encircled values are ones for which the respective G, or G, mean is missing.
8
CEL58
218
H. -M. Seyfert and G. Cleffmann
DNA content (Seyfert, 1979). Likewise, the corresponding G2 means are clustered
around values of 14-2 ± 0-78 pg and 27-9 + 1-14 pg (Fig. 2c). These values were
obtained by averaging all values smaller than 18 pg and larger than 23 pg, respectively.
Considering the distribution of G1 values from the short-time temperature-treated
cells (Fig. 2 B) the much wider spread is as obvious as the lack of clustering. The
latter is also true for their G2 means. Unlike their Gx correlates, however, the distribution spans only the range also observed in the stable cultures. Specifically, the G2
means of the short-time temperature-treated cultures with the lowest DNA contents
are much closer to the lower mean limits of the stable cultures, than the respective
Gt means.
While these lower and upper limits of mean macronuclear DNA content found in
stable cultures may have some significance for the regulation of the mean DNA
contents of populations, individual cells may acquire different DNA contents. The
20-
15-
o
o
o
o
a
10-
5-
10
I
15
Mean DNA (pg)
Fig. 3. Correlation between extreme DNA contents of individual cells with the
population mean DNA content. The maximum (O) and the minimum ( + ) G, DNA
contents of each sample (ordinate, in pg of DNA) are plotted against the respective
population mean DNA content (abscissa, in pg of DNA). In this plot the halved,
measured Gt values were combined with the G, values.
Variable macronuclear DNA contents
219
largest measured G1 DNA content exceeds the 64 C level by 60%. The smallest and
largest Gj DNA contents measured in each sample are positively correlated with the
population means (Fig. 3). Only the largest Gx macronuclei in the sample with the
smallest mean DNA content are unexpectedly high. This is also expressed by the
high coefficient of variation of the Gx mean DNA content in this sample (*%Gi
macromolecular DNA content: 675). Furthermore, a comparison of the coefficients
of variation of the Gx macronuclear DNA contents from perturbed cultures with
those of cultures adapted for long periods shows, that this parameter is variable
(perturbed cultures: Xa% = 346%, n — 7; long-time adapted cultures: Xg% =
19-9%, n = 11). Thus, the extent of macronuclear DNA content variability is not
fixed. The average variability of Gx and G2 populations was almost the same (mean
*%<?i = 25-5%; mean 5% G2 = 22-5%).
100 —
50-
®
f
•
®
+10 -
-10
-
•
(
1
1
•
1
1 '
-1
10
1
1
1
15
Mean G, DNA content (pg)
Fig. 4. Interdependence of the differences between the halved mean Gt macronuclear
DNA contents and the corresponding Gj macronuclear DNA content as a function of
the population mean DNA content. The measured G, mean macronuclear DNA contents were halved and the percentage difference from the measured Gx mean (ordinate)
was plotted against the Gt mean DNA content (abscissa). Values for which the halved
G, mean DNA content was significantly different from the respective Gx mean at the
5% level in the t-test are circled. Apostrophes denote values from perturbed cultures.
Arrows indicate the low and high mean DNA contents of cultures adapted for long
periods as calculated for Fig. 2A.
The last variable parameter to note is the relationship between the mean G2 DNA
contents and the respective G1 mean within a given population. In truly asynchronous
populations the halved G2 mean should be larger than the Gx mean of the same
population by the average amount of chromatin extruded during macronuclear
division, i.e. by a small percentage (Cleffmann, 1980). This was not always found
(Fig. 4). Indeed, we observed only insignificant (f-test, 5% level) differences of the
8-2
220
H.-M. Seyfert and G. Cleffmann
halved G2 mean DNA content from the respective Gt mean, if the latter were close
to the limits mentioned above. In 9 out of 18 samples the G2 DNA contents were
significantly different (in 8 larger, in 1 smaller) than expected, the frequency being
higher in perturbed cultures (5 out of 7) than in cultures adapted for long periods
(4 out of 11). The extent of this difference is more pronounced in perturbed cultures
(n = 7, X = +35 - 6%) than in cultures adapted for long periods (n = 11, X =
DISCUSSION
Different mean macronuclear DNA contents
Using cytophotometry we have demonstrated in this study that the mean macronuclear DNA contents of T. thermophila may vary greatly (eventually more than
twofold) within single staining series (Table 3). Careful specimen handling ensured
that this was not artifactual but inherent in the samples.
Superficially, these differences may be thought to be caused by maintaining cells:
(1) in different culture media at the same temperature (Table 1); or (2) at different
temperatures in the same culture fluid. Consideration of the effects of different culture
fluids, especially the good reproducibility of the extent of differences between cells
kept in various culture fluids from both experiments shown in Table 1, suggests
precisely controlled limits, and hence thresholds of mean macronuclear DNA contents
under given culture conditions. This is also suggested from a survey of the literature
(for references, see Gorovsky, 1980; Seyfert & Preparata, 1979; Doerder, 1979;
Doerder & DeBault, 1978). However, the occurrence of temperature-independent
fluctuations of the mean macronuclear DNA contents of cells maintained in the same
culture fluid (Table 3: Expt 1; All cells in Expt 2 versus Expt 3) limits the significance
of the observed differences, even though they were found repeatedly. This seems to
indicate that both cell lines studied may readjust their macronuclear DNA contents,
at least in defined medium. The BIV cells show, in addition to these spontaneous
fluctuations, a pronounced temperature effect upon the mean macronuclear DNA
content: BIV cells adapted for long periods had, in three out of three experiments, a
significantly smaller mean Gt DNA content at 29 °C than at 38 °C. Hence, temperature treatment of these cells may be used as a tool to induce a readjustment of the
mean macronuclear DNA contents.
This readjustment of Tetrahymena DNA content thresholds has been reported to
occur only early during the vegetative life cycle, reducing the macronuclear DNA
contents of young cells after 50-100 fissions by 50% (Doerder & DeBault, 1978).
The reported strain-specific differences concerning the timing of this process are
matched by our results on the different temperature sensitivity of macronuclear
DNA contents in both strains.
Levels of macronuclear DNA content limits
The values of macronuclear DNA content limits in terms of absolute DNA content
need to be considered at two levels. First, it may be asked where these limits are;
Variable macronuclear DNA contents
221
and second, how alterations of the latter influence the regulation thresholds of
individual cells. The distribution of the Gx mean values found in stable cultures
(Fig. 2 A) clearly suggests, that under stable conditions, and at least in defined
medium, there exist two distinct levels of DNA contents between which T. thermo
phila macronuclear DNA contents may vary. The lower level is 7-7 pg and the upper
13-6 pg. Though the values are too sparse for a conclusive statistical analysis of their
scatter around these limits, the percentage standard errors of either mean are similar
(s.E.%, low mean = 4 9 % ; S.E.%, upper mean = 47%). Mean macronuclear
DNA contents deviating greatly from either limit were found only in cultures
perturbed by short-time temperature shifts, or by transition from test-tube to
shaken-culture conditions (Table 3). The significance of their locations close to
the 32C and 64 C levels of macronuclear DNA content is not easily understood
considering the still unresolved macronuclear chromatin organization in this
organism (for reviews, see Gorovsky, 1980; Seyfert, 1979). But of course it is
intriguing to take this as an indication that the basic micronuclear chromatin organization (which is chromosomal) has a fundamental impact on the macronuclear chromatin
organization (for which a chromosomal organization has been challenged) of the
vegetative cell, despite the reorganization events occurring during development
(see Gorovsky, 1980). The 32 C and 64 C levels of macronuclear DNA contents were
postulated to mark threshold levels for the regulation macronuclear DNA content
(Doerder, 1979). To our knowledge, the data presented here provide the first subtantial experimental evidence of their relevance to the adjustment of macronuclear
DNA contents throughout vegetative growth. The position of these limits in terms
of absolute DNA contents may vary in different culture fluids (Table 1), explaining
the higher mean DNA contents reported by other workers (Doerder, 1979; Doerder &
DeBault, 1978, 1975; Woodward, Kaneshiro & Gorovsky, 1972; Gibson & Martin,
1971), none of whom used defined medium. The frequency with which the lower
and upper limits are reached is temperature-sensitive in the BIV cells, with high
temperature favouring the high limit.
For individual cells no absolute DNA content could be found triggering regulation.
The observed minimum and maximum DNA contents are positively correlated with
the population means over the entire range (Fig. 3). This excludes the possible
existence of fixed minimum or maximum absolute values for DNA contents, which
must not be surpassed. The smallest measured G1 macronucleus of a newly divided
cell had only i - 56pg of DNA and the largest 23-81 pg, thus making it extremely
unlikely that the absolute DNA content of a macronucleus itself may serve as a
regulation trigger.
Mechanisms for readjusting mean macronuclear DNA content
Of the three possible regulating mechanisms for macronuclear DNA content
(extra 5-phase, skipping of 5-phases and chromatin extrusion during division), the
latter was shown to be an active mechanism in the downward regulation of DNA
content in cells previously division-blocked (Worthington et al., 1976). Since, however, the appearance and frequency of chromatin extrusion bodies were similar in
222
H.-M. Seyfert and G. Cleffmann
our cultures adapted for long periods compared to the short-time temperaturetreated cultures, it is unlikely that altered chromatin extrusion is the major regulating
mechanism responsible for the gross changes of macronuclear DNA contents reported
here. The readjustment can be explained instead by the performance of two 5-phases
and the skipping of S-phases within one division cycle. As a consequence of the
action of the former mechanisms the 38 °C cells (Table 2) had twice the DNA
content of the 29 °C controls, with the same coefficient of variation for the macronuclear DNA contents in both populations. This suggests that previously all cells
underwent one extra 5-phase (cf. Doerder, 1979). If, on the other hand, only a
fraction of the population performed an extra 5-phase, the coefficient of variation for
the macronuclear DNA content would increase, and the increase in the mean DNA
content would be less than twofold (Table 3: Expt 1, 38 °C cells after 6 days). Further,
if cultures are partly synchronized for this event, great positive differences between the
halved G2 mean DNA content and the corresponding Gx mean will result. Hence, we
interpret the large differences eventually found between both values (Fig. 4) as the expression of a continuing regulation. The higher frequency of such large deviations found
in perturbed cultures (5 out of 7) than in long-time temperature-adapted cultures
(4 out of 11) agrees with this rationalization. Positive deviations result from the partly
synchronous performance of extra 5-phases within the culture, whereas negative
deviations are signs of synchronous skipping of 5-phases. The latter was found in
only two samples (Fig. 4). This low frequency indicates either that the event itself
is rare or that it happens much more quickly than upward regulation. The latter
alternative is favoured, since we found evidence for downward regulation in some
cultures during a 24 h period (Table 3: Expt 1), but at the sampling time all cultures
showed signs of upward regulation in this experiment.
Questionable cell-size control over macronuclear DNA content
Data collected from long-time adapted cells (Table 1) suggest a correlation between
both parameters only in the lower size-range. The largest cells kept in 2% PP medium
had a DNA content about 10% lower than the cells kept in 1% PPY medium
(30000 versus 18000/on3 average cell volume). Values shown in Fig. 1 were collected
from temperature-shocked cells in which the DNA contents had not yet stabilized
in some cases. Thus, they may be of only limited significance. Nevertheless, combination of data from Fig. 1 with those from Table 1 suggests no stringent relationship
between the absolute DNA contents of Tetrahymena populations. This is so, despite:
(1) evidence of size-mediated regulation of macronuclear DNA content in Paramecium tetraurelia (Berger, 1978); (2) size-mediated control over replication and cell
division in yeast (Fantes & Nurse, 1977; Nurse & Thuriaux, 1977); and (3) suggested
possible control over replication in T. pyriformis GL mediated by size or protein
content (Worthington et al. 1976) and HSM (Cleffmann, Reuter & Seyfert, 1979).
While some size-linked regulation mechanisms may operate during the cell cycle,
control by absolute cell size over the absolute macronuclear DNA content is not
obvious from our data.
Variable macronuclear DNA contents
223
We appreciate the skilled assistance of Mrs C. Schwarz. The work was supported by grant3
from the Sonderforschungsbereich 103, Marburg, F.R.G.
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(Received 1 September 1981 - Revised 15 April 1982)