J. Cell Sci. 37, 8i-go (1977)
81
Printed in Great Britain © Company of Biologists Limited 1977
CONTINUOUS DNA REPLICATION IN THE
NUCLEUS OF THE DINOFLAGELLATE
PROROCENTRUM MICANS (EHRENBERG)
S. A. FILFILAN AND D. C. SIGEE»
Cytology Unit, Departments of Botany and Zoology,
University of Manchester, Manchester, England
SUMMARY
The uptake of tritiated thymine into cells of a heterogeneous population of Prorocentruni
micans was investigated using light-microscope and electron-microscope autoradiography.
Specificity of thymine uptake into DNA was demonstrated by the specific removal of label
from wax-embedded material using DNase and by the high degree of localization of nuclear
label to chromosomes in the electron-microscope autoradiographs.
All nuclei, including both dividing and non-dividing cells, showed a substantial uptake of
label, indicating that nuclear DNA synthesis in Prorocentrum micans is a continuous process.
The level of DNA synthesis does show considerable variation, however, with very high levels
in some interphase nuclei.
The continuous replication of nuclear DNA provides further evidence of dinoflagellate
affinity to the prokaryotes, and indicates that Prorocentrum micans is a very primitive eukaryote
cell.
INTRODUCTION
Considerable attention has been given in recent years to the fine structure of
dinoflagellates and the possibility that they may represent a condition intermediate
between the prokaryotes and eukaryotes. Dodge (1965, 1966) suggested that dinoflagellates should be placed in a separate group - the Mesokaryota, on the basis of
primitive nuclear features including an absence of nuclear spindle, membraneattached chromosomes, permanent condensation of chromosomes throughout the cell
cycle, and the absence of histochemicaUy detectable histones. Loeblich (1976), in
a review that included more recent evidence relating to dinoflagellate evolution,
supported the concept that these organisms possess both prokaryote and eukaryote
features.
The timing of DNA synthesis is a question of central interest in considering the
evolutionary affinities of the dinoflagellates. Dodge (1966) postulated a continuous
synthesis, similar to that occurring in prokaryotes, whilst Loeblich (1976) has quoted
recent evidence in support of discontinuous synthesis, similar to that of eukaryotes.
The present work was carried out to determine the nature of DNA synthesis in
Prorocentrum micans. The genus Prorocentrum is of particular interest since the
arrangement of the surface thecae places it in a primitive position within the dinoflagellates (Loeblich, 1976).
• Requests for reprints should be sent to this author.
S. A. Filfilan and D. C. Sigee
82
MATERIALS AND METHODS
Cell culture
Prorocentrum micans (Ehrenberg), obtained from the Cambridge Culture Centre (strain
LB 1136/1), was grown in Erdschreiber medium at 22 °C under fluorescent illumination
(400 ft. c. (4-3 x io 3 lux)). A high proportion of dividing cells was obtained by giving cultures
2 cycles of 32 h dark/16 h light, followed by 8 h dark/16 h light. Treatment with tritiated
thymine and counts of cell population were carried out in the final light period on 2 separate
culture samples.
Population counts
Cell population counts were carried out at intervals from samples that had been filtered on to
a squared filter membrane (Oxoid). The cell population shows a gradual increase during the
first 2 h (Fig. 1), followed by a rapid increase to about 145 % (at 4 h) of the initial level. This
means that, during the period of thymine labelling (Fig. 1), approximately half the cells were
undergoing cell division and half the cells were in interphase.
50 r
40
30
. Y////////A
20
10
1
2
3
4
5
6
7
Time, h
Fig. 1. Percentage increase in cell population during the final light period. Each value
is the mean of two counts. The period of radioactive labelling is indicated (crosshatched) with the time of fixation (arrow).
Labelling and fixation
Cells were labelled for 2 h during the final light period (Fig. 1) with tritiated thymine (Radiochemical Centre, Amersham) which was injected into the culture to give afinalconcentration of
10 fid/ml, 8p. act. 21 Ci/mM. Cells were subsequently washed in culture medium and fixed in
either glutaraldehyde or acetic ethanol. Fixation in 4 % glutaraldehyde was carried out for
2 h at 4 CC in 0-05 M sodium cacodylate buffer at pH 7-2. Cells were then washed in buffer,
postfixed in buffered 2 % osmium tetroxide for 2 h, washed in buffer, dehydrated and embedded
in Spurr resin (Spurr, 1969). Thick (4-/WT1) sections were cut on glass knives and used either
directly for light microscopy or were resectioned for electron microscopy using a resin slide
technique (Sigee, 1976). Material for enzyme studies was fixed for 30 min in acetic ethanol
at 20 °C, hydrated and washed in distilled water, then dehydrated and embedded in wax.
DNA replication in Prorocentrum
83
Enzyme treatments
Sections of acetic ethanol-fixed material were hydrated and washed in tap water for 20 min
prior to the following enzyme treatments:
DNase II, ex bovine spleen (Koch-light), 10 mg/ml in 0-2 M sodium acetate buffer, pH 5-0,
for 1 h.
RNase, ex bovine pancreas (Koch-light), i-o mg/ml in 02 M sodium phosphate buffer,
pH 76, for 1 h.
Preparation of autoradiographs
Sections for light-microscope autoradiography were examined, and cells with a clear nucleus
in median section were noted and photographed. The slides were then coated with Ilford G-5
emulsion, left for 7 days at 4 °C, and processed using Kodak D-19 as developer. Ultrathin
sections for electron-microscope autoradiography were coated with a monolayer of Ilford L-4
emulsion using a wire loop, left for 3-4 months at 4 °C, and developed with either Ilford D-19
or microdol-X.
RESULTS
Light-microscope autoradiography
Under the light microscope chromosomes are always apparent as discrete structures.
The phase-contrast appearance of the chromosomes varies according to the stage of
the cell cycle (Filfilan & Sigee, manuscript in preparation), ranging from dark (cells
undergoing mitosis and early interphase - Fig. 2 A) to pale (middle to late interphase,
Fig- 3A)The nuclear grain counts for 40 cells are shown in Fig. 4. All of the nuclei examined
showed a clear uptake of label. Very few silver grains occurred over the cytopiasm,
and the background count was not sufficient to make any significant contribution to
the nuclear count (Figs. 2B, 3B). In some cells, with pale chromosomes, the level of
thymine incorporation into the nucleus is exceptionally high (Figs. 3 A, B). The
majority of cells, however, including cells with both pale and dark chromosomes, have
nuclear counts in the range 20-100 grains (Figs. 2A, B).
The effect of DNase on the acetic acid-fixed material is to remove almost all
radioactivity from the cells, whilst buffer alone has no effect. Treatment with RNase
does not result in any detectable loss of radioactivity.
Electron-microscope autoradiography
Electron-microscope autoradiographs of a number of cells with varying nuclear
structure consistently show substantial levels of nuclear labelling, in agreement with
the light-microscope results. This correlation can be developed further using the
resin slide technique for resectioning 4-/im-thick sections (Sigee, 1976). The autoradiograph shown in Fig. 5 is derived from a cell with chromosomes which appeared
dense under the light microscope, while that in Fig. 6 is derived from a cell with pale
chromosomes. In these, and all other cells examined, the majority of silver grains
appear to be over or closely associated with chromosomes and are relatively infrequent
over nucleoplasm, nucleolus and nuclear envelope.
S. A. Filfilan and D. C. Sigee
2A
2B
3B
Fig. 2. A, cell with dense chromosomes (c) photographed under phase contrast prior to
coating with photographic emulsion. Peripheral chloroplast (p) vacuoles (v). B, autoradiograph of the cell in A, bright-field illumination. The nucleus contains approximately 20 silver grains. Both x 4200.
Fig. 3. A, interphase cell with pale chromosomes in a dense nuclear matrix. Legend as
in Fig. 2 A. B, autoradiograph of the cell in A with bright-field illumination. The
nucleus is heavily labelled, with an estimated 110-120 silver grains. Both x 4200.
DNA replication in Prorocentrum
85
The distribution of nuclear silver grains was considered particularly important in
supporting the specific uptake of thymine into DNA, since only chromosomes and
peripheral nucleolar chromatin contain this molecule. The nuclear grain distribution
in relation to the chromosomes is shown diagramatically in Fig. 7 A, and was critically
investigated by measuring the distance from the centre of each grain to the nearest
chromosome boundary (Fig. 8A). The results obtained are consistent with the
chromosomes being the major source of radioactivity in the nucleus, since the spread
of silver grains from the chromosome boundary approximates to the experimental
grain distribution obtained by Caro (1962) and the derived data of Salpeter, Bachman
& Salpeter (1969). If all the silver grains having centres within 0-3 /im of the boundary
are considered to be derived from the major label source (Sigee & Bell, 1971), then
over 95 % of the nuclear radioactivity can be considered chromosomal.
6 5 S 4-
d
2
3
"
2-
1
10 20 30 40 50 60 70
80 90 100
120
110
140
130
160
150
180
170
200
300
190
Nuclear grain count
Fig. 4. Light-microscope nuclear grain counts. The nuclear grain counts are shown for
40 cells, selected for the possession of a clear nucleus in median section. The results
are taken from a single autoradiographic batch, with all the slides having the same
photographic processing conditions.
The localization of radioactivity is shown by comparison with random distributions
(Figs. 7B, 8B) obtained by plotting points from random number tables on to a tracing
of the nucleus superimposed over a lined grid. Less than 70 % of the random points
occurred within 0-3 //.m of the chromosome boundary.
In all of the autoradiographs examined the silver grains appear to be dispersed over
the whole chromosome population, and are not restricted to a few profiles in section
(Figs. 5, 6). This demonstrates that chromosomal DNA replication is of general
occurrence, and that the continuous uptake of thymine cannot be accounted for in
terms of asynchrony of replication throughout the chromosome complement.
DISCUSSION
Thymine, rather than thymidine, was used in this experiment, since a previous
attempt to label cells with the nucleoside only achieved a low level of incorporation
(Sigee, unpublished observations), and there is some evidence that thymidine is not
taken up by other dinoflagellates (Allen et al. 1975). Radioactive thymine has been
86
S. A, Filfilan and D. C. Sigee
DNA replication in Prorocentrum
Fig. 7. Localization of nuclear silver grains. Tracings taken from a single nucleus to
show oudines of nuclear membrane and chromosomes, with distributions of autoradiographic silver grains (A) and random points (B).
Fig. 5. Electron-microscope autoradiograph showing details of nucleus. The ultrathin
section has been resectioned from a cell with dense chromosomes (light microscopy).
Silver grains occur largely over or close to the chromosomes (c) with very few over the
nucleoli. The surrounding cytoplasm includes chloroplasts (p), vacuoles (i>) and
mitochondria (m). x 10000.
Fig. 6. Electron-microscope autoradiograph showing part of nucleus and surrounding
cytoplasm. The ultrathin section has been resectioned from a cell with pale chromosomes (light microscopy). The chromosomes are heavily labelled throughout, x 9600.
S. A. Filfilan and D. C. Sigee
- 40
- 30
ilver grains
A
1
/7
IL
r'' ' 1
0-4
0-6
Interna grains
- 10
/ /
%
0-2
\ •'—I
0-2.
r-r-H 7 7 1
1
0-4
n
0-6
II
1
0-8
i
1-0
1 ' T
1
External grains
- 30
B
- 20
- 10
I
0-6
0-4
0-2
0-2
0-4
0-6
0-8
1
10
Distance from boundary of nearest chromosome, /im
Fig. 8. Localization of nuclear silver grains
A, autoradiograph. The distribution of nuclear silver grains in relation to the
boundaries of chromosomes. The results are taken from 3 separate autoradiographs
of the same nucleus.
B, random distribution. The distributions of random points have been plotted in
numbers equal to the silver grains over a tracing of the 3 nuclei.
used as a specific precursor in several previous instances (Werner, 1971; Numan &
Wilson, 1975). The specificity of labelling in Prorocentrum is shown both by the localization to the chromosomes, which are known to contain DNA but not RNA or basic
proteins (Dodge, 1964a, b), and also by the specific action of DNase in removing the
label from the cells.
Within the population of Prorocentrum cells used in this study, all the cells show
nuclear uptake of label irrespective of the stage of cell cycle. This result has been
confirmed by repeat experiments, and must be interpreted as continuous DNA
synthesis throughout a large part of the cell cycle. This synthesis is variable, however,
reaching a high level during interphase.
The continuous DNA synthesis found in Prorocentrum has not been found in any
other dinoflagellate so far investigated. Studies on the uptake of thymidine in an
endozoic dinoflagellate of Anthopleura (Franker, 1971) and 32P incorporation into
alkali-insoluble material of Cryptothecodinium cohnii (Franker, Sakhrani, Pritchard &
Lamden, 1974) both reveal a discrete phase of DNA synthesis. A similar conclusion
was reached for Cachonina niei (Loeblich, 1976) on the basis of fluorimetric analysis,
though it must be noted that the precise timing of 5-phase differs for each of these
3 species.
DNA replication in Prorocentrum
89
Prorocentrum resembles these dinoflagellates, and other eukaryotes, in having the
highest levels of nuclear DNA synthesis during interphase, but is very unusual in the
synthesis being continuous over most, if not all, of the cell cycle. The implications of
continuous DNA synthesis in Prorocentrum are a matter for speculation, but may relate
to 2 unusual and quite separate characteristics of dinoflagellate chromosomes polyteny and prokaryote affinities.
Several authors have proposed that chromosomes in dinoflagellates are polytenic
(Grasse & Dragesco, 1927; Haapala & Soyer, 1973). In other polytene cells, such as
those of dipteran salivary glands or pulvilli, the synthesis of nuclear DNA occurs as
a discrete but prolonged S-phase (Pavan, 1963; Roberts, Whitten & Gilbert, 1974).
This comparison goes some way to account for the observations on Prorocentrum, but
was not considered likely in view of the continuation of replication during cell division.
It would also not explain why other dinoflagellates do not have a similar DNA
replication pattern.
The prokaryote affinities of dinoflagellate chromosomes are now widely appreciated,
and have been summarized by Soyer & Haapala (1974). The replication of nuclear
DNA in Prorocentrum clearly shows some similarity to prokaryotes, where replication
can occur continuously throughout the cell cycle, depending on the generation time
and the duration of replication (Holland, 1970). This similarity extends also to mitochondria and plastids, where autoradiographic studies have generally revealed continuous but variable DNA synthesis (Sigee, 1972).
It seems most likely that the pattern of DNA synthesis in Prorocentrum relates to its
primitive prokaryote characteristics rather than to any secondarily derived feature,
such as chromosome polyteny. This implies a uniquely primitive position for the
genus within the dinoflagellates, and is supported by the conclusions reached by
Loeblich (1976), who described Prorocentrum as a 'living fossil' on the basis of its
surface morphology.
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(Received 12 February 1977)