Journal of Experimental Botany, Vol. 53, No. 369, pp. 669–675, April 2002
Endoreduplication is not inhibited but induced
by aphidicolin in cultured cells of tobacco
Anne-Hélène Quélo1, John A. Bryant2 and Jean-Pierre Verbelen1,3
1
2
Department of Biology, University of Antwerp U.I.A., Universiteitsplein 1, B-2610 Wilrijk, Belgium
Department of Biological Sciences, University of Exeter, Exeter EX4 4QG, UK
Received 16 May 2001; Accepted 26 November 2001
Abstract
Introduction
Endoreduplication is a common process in plants
that allows cells to increase their DNA content. In the
tobacco cell cultures studied in this work it can be
induced by simple hormone deprivation. Mesophyll
protoplast-derived cells cultured in the presence of
NAA (auxin) and BAP (cytokinin) keep on dividing,
while elongation and concomitant DNA endoreduplication are induced and maintained in a medium
containing only NAA. If aphidicolin is given to the two
types of culture, no effect is observed on elongating,
endoreduplicating cells. However, the cells programmed for division switch to elongation and DNA
endoreduplication. Thus aphidicolin, an inhibitor of
the replicative DNA polymerases, a and d, does not
inhibit endoreduplication, and furthermore actually
induces it when the mitotic cell cycle is blocked.
DNA duplication and cell growth can only be completely blocked if ddTTP, an inhibitor of DNA
polymerase-b, is given together with aphidicolin.
This result implies that an aphidicolin-resistant
DNA polymerase, such as the repair-associated
DNA polymerase-b, can mediate DNA synthesis
during endoreduplication and can substitute for
polymerases-a and -d when the latter are inhibited.
Similar results are obtained in cultures of the BY-2
cell line by withdrawing auxins from the culture
medium. In this cell line endoreduplication is induced
only in a small proportion of the cells. A greater
proportion of the cells are blocked in the G2 phase of
the cell cycle.
DNA endoreduplication occurs widely in plants (Brodsky
and Uryvaeva, 1977; D’Amato, 1984; Nagl et al., 1985;
Galbraith et al., 1991; Larkins et al., 2001). While the
function of the mitotic cell cycle is clear, the reasons why
nuclei become polyploid in specific cells are still obscure
(D’Amato, 1984; Cavalier-Smith, 1985; Nagl et al., 1985;
Larkins et al., 2001). The mechanisms of the normal cell
division cycle have been studied more intensely than those
of endoreduplication and of the resulting polyploidy.
Cyclins and cyclin-dependent kinases (cdks) are the main
factors in the regulation of the mitotic cell cycle. Even
if they are not exactly the same in different species, a
general model for the control of the alternation between
mitosis and replication is now accepted, and a unifying
nomenclature has been proposed for plant and animal
cyclins (Renaudin et al., 1996). Changes in the expression of cdks and cyclins have also been observed
in relation to DNA endoreduplication, for example, in
Drosophila melanogaster embryos (Sauer et al., 1995), in
Arabidopsis thaliana shoot apices (Jacqmard et al., 1999)
and in cereal endosperm (Larkins et al., 2001). Nagl
suggested that changes in the expression of specific cdks
and cyclins allow a switch from a mitotic cycle to an
endoreduplication cycle (Nagl, 1995).
The enzymes that actually carry out DNA synthesis
in eukaryotes are DNA polymerases-a and -d (and
possibly -e: see below). The current model proposes that
polymerase-a, with its associated primase, initiates synthesis on both leading and lagging strands (and may also
carry out the majority of synthesis on the lagging strand)
while polymerase-d takes over from polymerase-a on the
leading strand and may also be involved in completion of
the Okazaki fragments on the lagging strand (Waga and
Key words: Aphidicolin, DNA polymerase, endoreduplication,
N. tabacum.
3
To whom correspondence should be addressed. Fax: q32 3 820 22 71. E-mail: [email protected]
ß Society for Experimental Biology 2002
670
Quélo et al.
Stillman, 1998). The role of polymerase-e, previously
considered to be a repair enzyme, in DNA replication is
not clear. It is essential for replication in yeast (Waga and
Stillman, 1998) and in the current model for mammalian
DNA replication may be involved in gap-filling or maturation, although it is not involved in the replication of SV40
DNA. DNA polymerase-b on the other hand is considered
to be exclusively a repair enzyme (Wang, 1996).
It is well established that the structure and properties
of the main DNA polymerase-a in plants are similar to
those of animals (Misumi and Weissbach, 1982; Bryant
et al., 1992; Garcia et al., 1997), and similarities between
the plant DNA polymerase-d and its animal counterpart
have been reported (Richard et al., 1991; Matsumoto
et al., 1994; Collins et al., 1997). However, there is as yet
no proof for the existence of a DNA polymerase-e-like
enzyme in plants. In relation to repair, there is good
evidence for the presence in plants of a DNA polymeraseb-like enzyme (Stevens et al., 1978; Chivers and Bryant,
1983; Dunham and Bryant, 1986; Luque et al., 1998)
which is similar in properties and structure to the
mammalian polymerase-b as far as has been determined.
One of the common properties of the replicative
polymerases a and d (and, in mammalian cells, of
polymerase-e) is their inhibition by aphidicolin (Syvaoja
et al., 1990; Bryant et al., 1992; reviewed by Wang, 1996).
This inhibitor has been used in many studies for the
synchronization of cell cultures by blocking entry of cells
into S-phase (e.g. tobacco BY-2 cells: Nagata et al., 1992).
In the experiments described here, this effect was tested
on cell cultures of Nicotiana tabacum L. cv. Petite Havana
where either division or endoreduplication may by
obtained by a simple hormonal control (Tao and
Verbelen, 1996). It is reported here that aphidicolin has
no inhibitory effect on endoreduplication in this culture
system, and furthermore, that it induces endoreduplication when the normal mitotic cell cycle is blocked. A
parallel is drawn between the hormonal control and the
aphidicolin effect on protoplast cultures and on BY-2
cells. These results are discussed in relation to the roles of
DNA polymerases in endoreduplication of DNA and to
regulation of the cell cycle.
Materials and methods
Cell cultures
Protoplasts were isolated from leaves of N. tabacum L. cv. Petite
Havana grown in vitro, by enzyme treatment (2% cellulase
Onozuka R10 and 0.2% macerozyme R10; Yakult Honsha Co.)
for 5–6 h at 26 8C. Protoplasts were immobilized on K3A
medium containing agarose in Petri dishes. After solidification,
1 ml of K3A, supplemented by a-naphthalene acetic acid (NAA)
and 6-benzylaminopurine (BAP), both at a concentration of
1 mg l 1, or supplemented only by NAA (1 mg l 1) was layered
on top of the agarose. Details of the protocol can be found in a
previous paper (Tao and Verbelen, 1996). To study the effect of
inhibitors, 50 mM aphidicolin or 40 mM ddTTP (dideoxythymidine triphosphate) were added at the beginning of the
culture, and cells were maintained in these conditions until the
day of observation.
BY-2 cells were cultured in an MS medium containing
30 g l 1 sucrose and 0.2 g l 1 KH2PO4 (Nagata et al., 1992).
Cells were transferred every 7 d to a fresh medium and vitamins
and 2,4-D (0.2 mg ml 1) were added to the cultures.
CLSM and flow cytometry analysis
Suspensions of nuclei were obtained by chopping plant tissue
and cells with a sharp razor blade in nuclei-stabilizing buffer
(100 mM TRIS, 50 mM NaCl, 0.1% Triton X-100, pH 7).
Propidium iodide (PI, Sigma Chemical Co), and DNase-free
RNase (Boehringer Mannheim), at final concentrations of,
respectively, 50 mg ml 1 and 25 mg ml 1, were added to the
nuclei suspension 30 min before analysis. Nuclear DNA content
was measured using FACS-scan flow cytometry. The same
staining was used for in situ measurements in individual cells.
The Confocal microscope was a Bio-Rad MCR 600 mounted
on a Zeiss Axiophot; a 20 3 objective (NA 0.4) was used with
1.25 mW laser power at 514 nm wavelength. The total fluorescence of nuclei, proportional to their DNA content, was
calculated by multiplying the mean pixel density by the number
of relevant pixels and is expressed in fluorescence units (fu).
In vitro-grown tobacco leaves were used as a standard for 2C
nuclei (for details see Valente et al., 1998).
Results
Cells derived from tobacco mesophyll protoplasts divide
extensively in the presence of NAA and BAP while they
only elongate in NAA. After 15 d of culture, divided cells
had an average diameter of 38 mm and their nuclei had
diameters between 6 mm and 12 mm. Elongated cells had
an average length of 272 mm and the nuclear diameter
reached 40 mm (Fig. 1a, b). Microfluorimetry of the nuclei
by CLSM illustrates the relative differences in DNA
content. This method was used because of the relatively
small number of cells available and the difficulties
encountered in preparing isolated nuclei from elongated
cells. As the large nuclei were fragmented during isolation, no significant results could be obtained by flow
cytometry. The tobacco leaf nuclei used as a standard
exhibit a main peak at 15 000 fu (fluorescence units) that
corresponds to a 2C DNA content (Fig. 2A). Nuclei from
dividing cells after 20 d of culture exhibit 2C and 4C
DNA levels (Fig. 2B) and the intermediate levels are
indicative of a large proportion of the cells being in
the S-phase of the cell cycle. In elongated cells of the
same age, the DNA content reaches 8C and 16C levels
(60 000 and 120 000 fu; Fig. 2C). This clearly shows
that the elongated cells had undergone extensive endoreduplication in the medium containing NAA but
lacking BAP.
The continuous presence of aphidicolin (50 mM) in the
cultures gave surprizing results. In the NAA-only medium
cell elongation was slightly affected (the average cell size
Aphidicolin effect on endoreduplication
671
Fig. 1. Protoplasts derived cells after 15 d of culture in different conditions. Nuclei are stained with PI (propidium iodide). Pictures on the left side
show cells cultured in a medium supplemented with auxin N, and cytokinin B, and pictures on the right side show cells cultured in the presence of N
only. (a, b) Control, (c, d) with aphidicolin (50 mM), (e, f ) with dTTP (40 mM), (g, h) with aphidicolin and dTTP. The scale bar represents 200 mm.
of 214.1 mm was less than the control), but their nuclei
enlarged in the same way as in the control (Fig. 1d).
Microfluorimetry shows that 4C–8C–16C nuclei also predominate in the aphidicolin-treated cells (Fig. 2E). These
unexpected results suggest that aphidicolin does not block
DNA endoreduplication and calls into question the role
of the replicative polymerases in this process.
When aphidicolin was added to the medium that
supports cell division (i.e. containing NAA and BAP) cell
division was inhibited as expected, but very unexpectedly
the cells switched to DNA endoreduplication and cell
elongation. Indeed, cell size, nuclear size and DNA content were similar to those observed in the cells endoreduplicating under cytokinin deprivation (Figs 1c, 2D).
Thus aphidicolin, a specific inhibitor of the polymerase-a
and -d, not only fails to block endoreduplication, but it
can actually induce it.
The failure of aphidicolin to block endoreduplication
suggests that a DNA polymerase other than polymerasesa -d (and -e) can mediate this process. Therefore, 40 mM
ddTTP (2,3-dideoxythymidine triphosphate), an inhibitor
of the repair enzyme DNA polymerase-b was added to
the cultures. In the absence of aphidicolin no effect
was observed on dividing cells, nor on elongated cells
(Fig. 1e–f ), suggesting that polymerase-b did not participate in either normal DNA replication or endoreduplication. However, if ddTTP was added to cells together
with aphidicolin then cells did not elongate any more
672
Quélo et al.
Fig. 2. DNA content measurements made on 20-d-old protoplast-derived cells using CLSM, and expressed in fluorescence units (fu) measured at
514 nm. The values of 15 000, 30 000, 60 000, and 120 000 fu correspond to a DNA content of 2C, 4C, 8C, and 16C, respectively. For each sample the
fluorescence of 300 nuclei was measured. (A) The standard: isolated nuclei from in vitro tobacco leaves have 2C nuclei. (B) Divided cells in
NAAqBAP. (C) Elongated cells in NAA. (D) Elongated cells in NAAqBAPqAphidicolin. (E) Elongated cells in NAAqAphidicolin.
(the average size was 122 mm compared with 214 mm with
aphidicolin only) (Fig. 1g–h), and the endoreduplication
that previously took place in the presence of aphidicolin
did not occur (data not shown). The simplest conclusion from these data is that, normally, endoreduplication
is mediated by polymerase-a and -d and thus ddTTP,
an inhibitor of polymerase-b, has no effect. However,
when aphidicolin has blocked polymerases-a and -d, then
endoreduplication can be mediated by polymerase-b.
When all three of these polymerases are blocked
by adding both aphidicolin and ddTTP, endoreduplication does not occur. A further conclusion is that
cells programmed for cell division enter a polymeraseb-mediated endoreduplication programme when DNA
replication is blocked by aphidicolin.
Both the hormonal change and the aphidicolin induction of endoreduplication were also investigated in BY-2
cells. This cell line is only auxin (2,4-D)-dependent, therefore it was transferred to a fresh culture medium without
2,4-D. After 6 d of culture, cells showed extensive enlargement with an average size of 176.8 mm compared to
62.0 mm for the dividing cells (Fig. 3A, B). The measurement of DNA content by flow cytometry was straightforward for dividing BY-2 cells, but the preparation of
Aphidicolin effect on endoreduplication
673
Fig. 3. 6-d-old BY-2 cell cultures. Cells were observed with bright field fluorescence microscopy after PI staining (left side) and their DNA content was
measured by flow cytometry (right side). The dot plots represent cytograms of the area fluorescence versus width. Only dots within the regions
displayed are included in the histogram analysis (fluorescence area versus events). (A) Cells cultured in presence of 2,4-D. (B) Cells cultured without
2,4-D (bar: 100 mm).
isolated nuclei from the elongated cells was more difficult,
which is reflected by the noise in the dot plot (Fig. 3B).
It would be expected that cells transferred from the
stationary phase to a medium without 2,4-D would
remain in G1 phase with a 2C DNA content (Fig. 3A).
However, they did not: 34% of the cells went through
one round of replication (4C) and 7% went through two
rounds (8C) (Fig. 3B). Although the percentage of endoreduplicating cells was small (7%) compared with the
protoplast culture, the effect of hormonal deprivation was
similar in the two culture systems.
The authors did not succeed in obtaining flow cytometry data for aphidicolin-treated cells. For this reason
the DNA content was measured by microfluorimetry.
Aphidicolin is commonly used in BY-2 cells for cell cycle
synchronization. The cells are blocked in G1 for a maximum of 24 h, and after an intensive wash a high percentage of the cells enter the S-phase synchronously. In the
experiments described here the effect of aphidicolin was
studied over a longer period of time: 24 h after their
transfer to fresh medium with aphidicolin (50 mM) the
cells were still blocked in the G1 phase (2C DNA content
corresponding to 15 000 fu) as expected (Fig. 4B). After
48 h the G1 peak had disappeared and the DNA content values were spread between 10 000 and 30 000 fu
(Fig. 4C). At 72 h a 4C peak (30 000 fu) appeared
corresponding to cells in the G2 phase (Fig. 4D), and
this situation remained until after 6 d of culture (Fig. 4E).
At that time the control cells in division had already
entered the stationary phase and the majority of them
had a 2C DNA content (Fig. 3A). Cells were obviously
blocked by aphidicolin during the first 24 h of culture, but
after a period of 48 h they overrode the aphidicolin block
and underwent at least one round of replication. They did
not divide at any time and they reached a size comparable
to the size of cells cultured without 2,4-D.
Discussion
Current ideas on the role of cytokinin in the regulation of
the plant cell cycle involve regulation of entry into the
S-phase and into mitosis (Soni et al., 1995; Frank and
Schmülling, 1999; Laureys et al., 1998). In contrast to the
expectations (Frank and Schmülling, 1999) omission of
cytokinin from the medium for protoplast culture not
only prevented normal cell division in mesophyll protoplast-derived cell cultures, but it also led to cell elongation
and DNA endoreduplication (Valente et al., 1998, and
results presented here).
This suggests that different controls (cdks, cyclins) act
on the entry into S-phase during endoreduplication and
during normal replication. Lines of support come from
674
Quélo et al.
Fig. 4. DNA content of aphidicolin treated BY-2 cells measured
by CLSM. 15 000 and 30 000 fu correspond to a DNA content of 2C
(G1 phase) and 4C (G2 phase), respectively. The fluorescence of 100
nuclei was measured for each sample. (A) Day of transfer in a fresh
medium with aphidicolin (50 mM). (B) 24 h after transfer. (C) 48 h after
transfer. (D) 72 h after transfer. (E) day 6 (144 h after transfer).
the findings that in Drosophila melanogaster (Sauer et al.,
1995), Arabidopsis thaliana (Jacqmard et al., 1999) and
Zea mays (Larkins et al., 2001) there are indeed differences in the populations of cyclins and cdks between
replicating and endoreduplicating cells.
It is well known that aphidicolin inhibits the replicative
DNA polymerases of plants (Bryant et al., 1992), and
the synchronization of cultures of tobacco cells by
aphidicolin relies on this. However, the data presented
here show that DNA endoreduplication can occur in the
presence of aphidicolin. The most obvious explanation
of these data is that DNA endoreduplication may be
mediated by another polymerase, such as DNA polymerase-b. Indeed, only the presence of both ddTTP and
aphidicolin in the culture medium blocks the entry of
the cells in endoreduplication. There are precedents for
this. For example, in rat giant trophoblast cells, DNA
endoreduplication is not inhibited by aphidicolin, but is
inhibited by ddTTP, an inhibitor of DNA polymerase-b
(Siegel and Kalf, 1982). However, in the results presented
here, ddTTP does not inhibit endoreduplication in the
absence of aphidicolin, which suggests that polymerases-a
and -d are certainly able to mediate endoreduplication
and presumably normally do so.
Most surprisingly the inhibition of the replicative
polymerases in cells maintained in the auxin plus cytokinin medium also leads to entry into the elongationu
endoreduplication programme, as if the cells had been
maintained under cytokinin deprivation.
BY-2 cells are only auxin dependent as they produce
endogenously the cytokinins necessary for their development. When 2,4-D was omitted from the culture medium,
cells did not go into division but enlarged considerably.
Their DNA was replicated at least once, and 7% of the
cells went through two cycles of replication. The auxin
2,4-D is thus necessary for the entry of BY-2 cells into
mitosis, but not for their progression through S-phase.
Also in BY-2 cells, aphidicolin inhibited M phase, but
failed to inhibit the replication process. Replication could
not occur during the first 24 h of the aphidicolin block
presumably because of the inhibition of the polymerases
a and d. But then BY-2 cells managed to override this
block, entered S-phase and reached a 4C DNA content.
The mitotic process was, however, blocked. Exceptionally, a cell went through a second round of DNA
replication. This suggests that BY-2 cells can also use
another enzyme to replicate their DNA, such as polymerase b, and that aphidicolin has an effect on the G2uM
checkpoint and does not allow cells to enter mitosis.
The two tobacco cell cultures used in this research have
different requirements, they are cultured in different conditions and under different hormonal controls. However,
they show similarities in their reaction to hormonal
deprivation and to aphidicolin. In both cases there is
a change in the regulation of the cell cycle that does not
allow cells to go into mitosis, but allows DNA replication
that eventually drives them into endoreduplication. The
hormone deprivation acts directly on the G2uM transition
(Soni et al., 1995; Frank and Schmülling, 1999; Laureys
et al., 1998), but the explanation of the dual aphidicolin
Aphidicolin effect on endoreduplication
effect is not obvious. First the normal process of
replication is blocked, but the blockage induces cells to
find an alternative pathway to replicate their DNA. Then
after the DNA has been replicated, mitosis is blocked,
possibly because the cells fail a DNA ‘quality’ checkpoint as suggested by work on fucoid zygotes (Corellou
et al., 2000).
Further experiments on the process of replication
and on the control of the cell cycle will help in the
understanding of the mechanisms of this complex but
interesting switch from division to endoreduplication.
Acknowledgements
We would like to thanks Professor D Van Bockstaele and
Dr M Van der Planken for their help and advice for the analysis
by flow cytometry. This work was supported by a grant from the
Flemish Community of Belgium.
References
Brodsky WY, Uryvaeva IV. 1977. Cell polyploidy: its relation to
tissue growth and function. International Review of Cytology
50, 275–332.
Bryant JA, Fitchett PN, Hughes SG, Sibson DR. 1992. DNA
polymerase-a in pea is part of a large mutiprotein complex.
Journal of Experimental Botany 43, 31–40.
Cavalier-Smith T. 1985. Cell volume and the evolution of
eukaryotic genome size. In: Cavalier-Smith T, ed. The evolution of genome size. New York, USA and Chichester, UK:
John Wiley and Sons, 105–184.
Chivers HJ, Bryant JA. 1983. Molecular weights of the major
DNA polymerases in a higher plant, Pisum sativum L. (pea).
Biochemical and Biophysical Research Communications 110,
632–639.
Collins JTB, Heinhorst S, Cannon GC. 1997. Characterization of
DNA polymerases and a replication accessory protein from
the Glycine max cell line SB-1 and detection of a delta-like
DNA polymerase gene in soybean cDNA. Plant Physiology
114, Supplement 3, 156.
Corellou F, Bisgrove SR, Kropf DL, Meijer L, Kloareg B,
Bouget F-Y. 2000. A SuM DNA replication checkpoint
prevents nuclear and cytoplasmic events of cell division
including centrosomal axis alignment and inhibits activation
of cyclin-dependent kinase-like proteins in fucoid zygotes.
Development 127, 1651–1660.
D’Amato F. 1984. Role of polyploidy in reproductive organs and
tissues. In: Johri BM, ed. Embryology of angiosperms. New
York, USA: Springer-Verlag, 523–566.
Dunham VL, Bryant JA. 1986. DNA polymerase activities in
healthy and cauliflower mosaic virus-infected turnip (Brassica
rapa) plants. Annals of Botany 57, 81–89.
Frank M, Schmülling T. 1999. Research news: cytokinin cycles
cells. Trends in Plant Science 4, 243–244.
Galbraith DW, Harkins KR, Knapp S. 1991. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiology 96, 985–989.
Garcia E, Orjuela D, Canacho Y, Zumiga JJ, Plasencia J,
Vasquez Ramos JM. 1997. Comparison among DNA polymerases 1, 2 and 3 from maize embryo axes: a DNA primase
co-purifies with DNA polymerase 2. Plant Molecular Biology
33, 445–455.
Jacqmard A, De Veylder L, Segers G, de Almeida Engler J,
Bernier G, Van Montagu M, Inzé D. 1999. Expression of
675
CKS1At in Arabidopsis thaliana indicates a role for the
protein in both the mitotic and the endoreduplication cycle.
Planta 207, 496–504.
Larkins BA, Dilkes BP, Dante RA, Coelho CM, Woo Y-M,
Lui Y. 2001. Investigating the hows and whys of endoreduplication. Journal of Experimental Botany 52, 183–192.
Laureys F, Dewitte W, Witters E, Van Montagu M, Inzé D,
Van Onckelen H. 1998. Zeatin is indispensable for the G2-M
transition in tobacco BY-2 cells. FEBS Letters 426, 29–32.
Luque AE, Benedetto JP, Castroviejo M. 1998. Wheat DNA
polymerase C1: a homologue of rat DNA polymerase-b. Plant
Molecular Biology 38, 647–654.
Matsumoto T, Hata S, Suzuka I, Hashimoto J. 1994. Expression
of functional proliferating cell nuclear antigen from rice
(Oryza sativa) in Escherichia coli: activity in association with
human DNA polymerase-d. European Journal of Biochemistry
223, 179–187.
Misumi M, Weissbach A. 1982. The isolation and characterization of DNA polymerase a from spinach. Journal of Biological
Chemistry 257, 2323–2329.
Nagata T, Nemoto Y, Hasezawa S. 1992. Tobacco BY-2 cell line
as the HeLa cell in the cell biology of higher plants.
International Review of Cytology 132, 1–30.
Nagl W. 1995. Cdc2-kinases, cyclins and the switch from
proliferation to polyploidization. Protoplasma 188, 143–150.
Nagl W, Pohl J, Radler A. 1985. The DNA endoreduplication
cycles. In: Bryant JA, Francis D, eds. The cell division cycle
in plants. Cambridge, UK: Cambridge University Press,
217–232.
Renaudin J-P, Doonan JH, Freeman D et al. 1996. Plant cyclins:
a unified nomenclature for plant A-, B- and D-type cyclins
based on sequence organization. Plant Molecular Biology
32, 1003–1018.
Richard MC, Litvak S, Castroviejo M. 1991. DNA polymerase-B
from wheat embryos: a plant delta-like DNA polymerase.
Archives of Biochemistry and Biophysics 287, 141–150.
Sauer K, Knoblich JA, Richardson H, Lehner CH. 1995. Distinct
modes of cyclins Eucdc2c kinase regulation and S-phase
control in mitotic and endoreduplication cycles of Drosophila
embryogenesis. Genes and Development 9, 1327–1339.
Siegel RL, Kalf GF. 1982. DNA polymerase b involvement
in DNA endoreduplication in rat giant trophoblast cells.
Journal of Biological Chemistry 257, 1785–1790.
Soni R, Carmichael JP, Shah ZH, Murray JAH. 1995. A family
of cyclin D homologs from plants differentially controlled
by growth regulators and containing the conserved retinoblastoma protein interaction motif. The Plant Cell 7, 85–103.
Stevens C, Bryant JA, Wyvill PC. 1978. Chromatin-bound DNA
polymerase from higher plants: a DNA polymerase-b-like
enzyme. Planta 143, 127–132.
Syvaoja J, Suomensaari S, Nishida C, Goldsmith JS, Chui GSJ,
Jain S, Linn S. 1990. DNA polymerases a, d and e: Three
distinct enzymes from HeLa cells. Proceedings of the National
Academy of Sciences, USA 87, 6664–6668.
Tao W, Verbelen J-P. 1996. Switching on and off cell division
and cell expansion in cultured mesophyll protoplasts of
tobacco. Plant Science 116, 107–115.
Valente P, Tao W, Verbelen J-P. 1998. Auxins and cytokinins
control DNA endoreduplication and deduplication in single
cells of tobacco. Plant Science 134, 207–215.
Waga S, Stillman B. 1998. The DNA replication fork in
eukaryotic cells. Annual Review of Biochemistry 67, 721–752.
Wang TS-F. 1996. Cellular DNA polymerases. In:
DePamphilis ML, ed. DNA replication in eukaryotic cells.
Cold Spring Harbor, NY, USA: Cold Spring Harbor Press,
461–493.