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Nuclear Morphology During the S Phase
Anatomia Umana Normale, Università di Bologna, 40126 Bologna, Italy
Morfologia Umana Normale, Università di Chieti, 66013 Chieti, Italy
3Istituto Citomorfologia Normale e Patologica, CNR, c/o Ist. Rizzoli Codivilla-Putti, 40136, Bologna, Italy
DNA replication; S phase; nuclear morphology; BrdU; immuno electron microscopy
In order to evaluate at the ultrastructural level the chromatin arrangement during
the S phase of the cell cycle, the detection of Bromodeoxyuridine (BrdU) by immunogold has been
performed in synchronized 3T3 fibroblasts, regenerating liver, and Friend Leukemia Cells (FLC).
After a 5-minute BrdU pulse, this label is detected in 10-nm-wide fibers, organized as lacework and
assumed to be replication units. In the early part of the S phase, DNA replication units are localized
exclusively in the dispersed chromatin domains far from the nuclear envelope. In the middle S,
replication occurs at the border between condensed and dispersed chromatin and, finally, in late S, it
mainly occurs in perinuclear heterochromatin regions. After replication, the 10-nm fibers can
condense in heterochromatin without translocation. Chromatin is highly dispersed in early S and
computer image analysis shows an increase in condensed chromatin areas ranging from 13 to 18% at
the end of the S phase with a temporal and morphological pattern of distribution characteristic for
each cell type. Scanning transmission electron microscopy demonstrates a regular and repetitive
structure of dispersed chromatin, represented by a ring-like arrangement of the 10-nm fibers;
assuming the same spatial distribution, gold particles that identify incorporated BrdU confirm this
organization. By evaluating the organization and the distribution of DNA replication units during S
phase, the results suggest that DNA replication occurs at a nucleosomal-like fiber level and that
replicating enzymes machinery moves over a fixed template. Microsc. Res. Tech. 40:418–431, 1998.
r 1998 Wiley-Liss, Inc.
The functional steps which characterize the cell cycle
are related to specific morphological modifications involving mainly the cellular dimensions, the protrusions
of the cell surface, and the different levels of chromatin
While mitosis identifies characteristic chromatin patterns, the three-dimensional arrangements of DNA
filament during G1 and S phase are still unknown,
resulting in a different and or conflicting interpretation
from several authors in the last 30 years (Huberman,
1987; Kondra and Ray, 1978; Manuelidis and Chen,
1990; Nurse, 1994).
In fact, during the interphase period, the nuclear
structure and in particular the nuclear ultrastructure
are characterized by significant variations in the proportions of condensed and dispersed chromatin. In Hyacintus orientalis, a continuous increase of chromatin condensation from G1 to metaphase has been described by
electron microscopy. In the same study it has been
demonstrated that this phenomenon results in species
and tissues specificity in plants, insects, or mammals
(Nagl, 1980).
In the macronucleus of Euplotes, Kluss (1962) and
Prescott (1962) have demonstrated a low electron opacity in the zone of DNA synthesis, and this concept was
confirmed by a study of Hay and Revel (1963) that
described a fine filamentous DNA network in the DNA
replicating areas. Similar results are also described in
regenerating rat hepatocytes where the dispersed chror 1998 WILEY-LISS, INC.
matin appears as a 12-nm thin filament (Derenzini,
1979; Derenzini et al., 1981, 1984).
High resolution electron microscopy autoradiography
demonstrated that DNA synthesis occurs in dispersed
chromatin or at the border zone between condensed
chromatin and interchromatin regions (Angelier et al.,
1976; Edemberg and Huberman, 1975; Fakan and
Bernhard, 1973; Fakan and Hancock, 1974; Kuroiwa,
1974; Williams and Ockey, 1970). In 1982 Gratzner
developed a monoclonal antibody that detects
5-bromodeoxyuridine, a synthetic analogue of thymidine that is incorporated into newly synthesized DNA.
This very simple method has a very high efficiency, low
background, is not time-consuming, and can be applied
both in flow cytometry and in light as well as electron
microscopy, thus allowing a very complete analysis of
the cell cycle (Danova et al., 1988; Moran et al., 1985;
Risio et al., 1986; Schutte et al., 1987; Sugihara et al.,
1986; Thiry, 1988; Thiry and Dombrowicz, 1988). Despite the possibility that BrdU could induce perturbation of growth and differentiation (Brown and Schildkraut, 1979; Meuth and Green, 1974), it has been
demonstrated that the results are similar to those
Contract grant sponsor: Italian CNR, PF ACRO; Contract grant sponsor:
Ministero della Ricerca Scientifica e Technologica (MURST) Regione Emilia
Romagna ISS FONDI 1%.
*Correspondence to: Giovanni Mazzotti, MD, Ist. Anatomia Umana Normale, v.
Irnerio 48, 40126, Bologna, Italia.
Received 21 November 1995; accepted in revised form 12 January 1996
obtained with tritiated thymidine (Lin and Allison,
1993; Mazzotti et al., 1990).
So multiple DNA replicon domains spatially and
temporally organized during the S phase of the mammalian cells (Nakamura et al., 1986) have been demonstrated and computer analysis (Van Dierendonck et al.,
1989) or the confocal microscopy of immunostained
cells (Neri et al., 1992) show evidence within the S
phase of different staining patterns. Electron microscopy studies on synchronous and asynchronous cells
allowed a better definition of these events both in vitro
and in vivo (Mazzotti et al., 1990; Rizzoli et al., 1992;
Vitale et al., 1991).
After incorporation of H3 thymidine, using biotinylated dUTP into permeabilized cells or antibodies against
5-bromodeoxyuridine to detect in vivo or in vitro DNA
replication, it has been demonstrated that DNA synthesis occurs at fixed sites in close association with the
nuclear matrix (Carrı̀ et al., 1986; McCready et al.,
1980; Nakayasu and Berezney, 1989; Neri et al., 1992;
Smith and Berezney, 1982).
In the columnar cell on the crypt base of mouse
duodenum El-Alfy and Leblond (1987, 1988, 1989) and
El-Alfy et al. (1994), comparing Feulgen-stained semithin Epon sections and autoradiography, have demonstrated that during the transition from G1 to S phase,
there is an increase of distinguishable dots and filaments.
Despite these results many questions are still unresolved. In particular, we need to know if in a cycling cell
it is possible to discriminate G1 and S phase exclusively
from the morphological point of view.
The first requirement to answer this question is the
identification of a correct experimental model in which
it should be possible to detect the whole cell cycle
starting from a given unquestionable point. Nowadays
the system employed to evaluate the S phase is via the
detection of labelled precursors of DNA synthesis. In
our experience, we have utilized 5-bromo-2-deoxyuridine in synchronized cells both in vitro and in vivo.
Synchronization has been induced ‘‘in vitro’’ on 3T3
fibroblasts by the starvation method, and ‘‘in vivo’’
utilizing rat regenerating liver after surgical removal of
3 of the organ. All the samples have been evaluated by
transmission electron microscopy and monitored with a
cytofluorimeter to exactly evaluate the DNA content of
the cell population and the percentage of synchronization, in order to obtain a tight correlation between the
different moments (early, middle, and late) of the S
phase and nuclear morphology. Moreover, by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM), we have analyzed the
dimension and the organization of chromatin fibers in
replicating DNA.
Cell Culture
Murine Friend erythroleukemia cells (FLC-745), with
a cell cycle of 10–12 hours, were grown in RPMI 1640
medium supplemented with 10% foetal calf serum in a
5% CO2 atmosphere. Swiss 3T3 cells were grown
in Dulbecco’s modified minimum essential medium
(D-MEM), supplemented with 10% newborn calf serum
(NCS). Cells were initially seeded onto flask at a
density of 3 3 103/cm2 and usually became confluent
after 4 days of culture. The cell synchronization in low
serum medium according to Nakayasu and Berezney
(1989) was performed incubating them for 72 hours in
D-MEM containing 0.5% NCS. In this way, the cells
were arrested at G0. Successively, the cells were reseeded at a density of 7 3 103/cm2 onto glass slides in
D-MEM containing 10% NCS.
Regenerating Rat Liver
Sprague Dawley male rats of an average body weight
of 180–200 g were used. Two animals were used as
controls (Vitale et al., 1991). Partial hepatectomy (about
⁄4 of the liver was left) was performed according to
Grisham (1962) and Higgins and Anderson (1931) in
order to obtain different times of regeneration before
the sacrifice of the animal at 18, 22, 26, 30, and 34
hours. Three rats for each experimental point were
sacrificed and the regenerating removed livers processed for electron microscopy.
Bromodeoxyuridine Incorporation
Exponentially growing FLC were exposed, according
to Gratzner (1982), to 5-bromo-2-deoxyuridine (BrdU)
at a final concentration of 50 µM, for periods of time
ranging from 5 minutes to 12 hours at 37°C.
Synchronized Swiss 3T3 fibroblasts at 1-hour intervals from the cell release were exposed to 20 µM BrdU
for either 5 or 30 minutes. Control cells, which were
cultured for 96 hours in low serum medium, were
incubated for 5 or 30 minutes in the presence of BrdU.
Alternatively, 13 hours after the block release cells
were exposed to BrdU for 30 minutes. One hour before
sacrifice Sprague Dawley rats were injected intraperitoneally with 50 mg/kg BrdU. Rat liver nuclei were
prepared from regenerating liver by the method of
Widnell and Tata (1964), before December 31, 1993.
Briefly, after sacrifice, livers were quickly excised,
minced with a scalpel, and manual homogenized with
20 strokes in a Dounce pestle type A with a tissue/buffer
TM ratio of 1:3 (buffer TM 5 0.25 M sucrose; 0.05 M
Tris, pH 7.4; 4.5 mM MgCl2). After filtration through
four layers of cheesecloth, the homogenate was centrifuged at 770g for 10 minutes to yield a crude nuclear
pellet. The pellet was resuspended in 2.2 M sucrose TM
buffer and centrifuged at 40,000g for 90 minutes. This
purified nuclear pellet was washed two times and
stored in the same buffer at 0°C.
Electron Microscopy
Murine Friend erythroleukemia cells and nuclear
matrices were fixed, after centrifugation at 900 rpm for
5 min, with 1% glutaraldehyde in 0.1 M phosphate
buffer, pH 7.4, for 1 hour at room temperature, rinsed
three times in 0.15 M phosphate buffer, dehydrated in
increasing concentrations of ethanol, and embedded in
Epon and London Resin White. Moreover FLC was also
embedded in Unicryly (Scala et al., 1992).
Swiss mouse 3T3 adherent cells, after two washes in
PBS, were fixed as described. The cells were then rinsed
three times in 0.15 M phosphate buffer, detached by
scraping, and centrifuged at 800 rpm for 10 minutes
and embedded in Epon.
Regenerating rat livers, 2 3 2 3 1 mm pieces, were
processed as described before and embedded in Epon
Silver-gold ultrathin sections of all specimens were
mounted on nickel grids. Semithin sections (0.1 µm) of
Unicryl-embedded FLC cells were deresinated with
several washes in acetone and critical point dried.
Immunocytochemical Labelling
A postembedding immunogold technique for all
samples was used as reported by De Mey et al. (1981),
involving the following steps: etching of the sections
with 10% H2O2 for 10 minutes; incubation with normal
goat serum for 30 minutes at room temperature, incubation for different periods of time (2–14 hours for FLC
cells and nuclear matrices; 14 hours for 3T3 cells and
regenerating livers) at 4°C with an anti-BrdU monoclonal antibody (Becton Dickinson, San Jose, CA) 1 µg/ml
in 0.05 M Tris HCl, pH 7.6, containing 0.1% bovine
serum (BSA) and incubation with goat anti-mouse IgG
conjugated with 15-nm colloidal gold particles (Janssen, Beerse, Belgium) diluted 1:10 in 0.02 M Tris HCl,
pH 8.2, containing 0.1% BSA for 1 hour at room
temperature. The grids were stained with uranyl acetate and lead citrate and observed employing a Philips
(Mahwah, NJ) CM 10 and a Zeiss (Thornwood, NY) EM
109 transmission electron microscope (TEM).
After the immunocytochemical labelling, the deresinated sections were coated with a thin platinum-gold
layer with a Balzers (Balzers, FL) MED 010 evaporating system and observed with a Philips CM 12 scanning
transmission electron microscope (STEM).
Computer Image Analysis
About 100 TEM pictures with good contrast of each
cell type were scanned by a Hewlett Packard (Corvallis,
OR) Scanjet and analysed by a Microsoft Image-pro
Plus 1.3 PC program in order to obtain, after a repetitive binarization of each image, an automatic recognition of the nuclear envelope and the border between
nucleus and nucleolus. With this system it was possible
to determinate nuclear and nucleolar diameter and,
consequently, the nuclear and nucleolar total area. The
exclusion of the nucleolar area permits the estimation
of the percentage of the condensed chromatin, computable as dark fields on the digitally acquired image, in
comparison to the total nuclear area.
The controls consisted, for FLC and 3T3 synchronized fibroblasts, of cells not exposed to BrdU or not
incubated with the primary antibody or incubated with
a non-immune mouse serum, instead of the primary
For the regenerating rat livers, the following controls
were performed: samples obtained from non-hepatectomized rats injected with BrdU or from hepatectomized
rats not injected with BrdU. Sections of regenerating
liver exposed to BrdU (26 hours after the hepatectomy)
were incubated without the primary antibody or with a
non-specific immune serum followed by the secondary
gold-conjugated antibody.
3T3 Fibroblasts
Immunocytochemical detection of incorporated BrdU
can be performed in TEM without previous acid hydrolysis treatment or proteolytic digestion, as required for
light microscopy or flow cytometry, since the antigen is
directly exposed on the sectioned surface of the embedding media; in these conditions it is possible to preserve
nuclear morphology at the ultrastructural level (Mazzotti et al., 1990). To prevent the possibility of modifications of chromatin organization related to the detachment of the cells after trypsinization and to the
consequent modifications of flattened to a spherical
shape, 3T3 fibroblasts have been fixed directly in situ
onto the culture flask and scraped out. Moreover, some
of the samples have been grown on a slide and directly
embedded and sectioned as monolayers.
Flow cytometer analysis demonstrates that when
using the serum deprivation method in G0 phase arrested 3T3 fibroblasts, DNA synthesis takes place 9
hours after the release of the block (Rizzoli et al., 1992).
At the 9th hour about 10% of the cells are synthesizing DNA, while at the 13th hour the percentage of cells
increases to 70% and exceeds 80% from the 15th to the
20th hour, decreasing to about 50% at the 24th hour.
Moreover, the evaluation of the DNA content by
propidium iodide (PI) counterstain shows that most of
the cells have a 2n DNA content from 9 to 13 hours,
suggesting that they could be in early S phase (Rizzoli
et al., 1992). With the progression of the DNA synthesis, synchronized cells that have incorporated BrdU
show a 3n DNA content from the 15th to the 18th hour,
and 4n DNA content from the 21st to 24th hour and
they have been interpreted as being, respectively, in the
mid and late S phase of the cell cycle (Rizzoli et al.,
1992). Immunocytochemical detection of incorporated
BrdU in newly synthesized DNA is also detectable 9
hours after the block release and for a pulse of 15–30
minutes demonstrates specific labelling patterns for
each of the three periods. In early S, colloidal gold
particles are localized exclusively in the interchromatin
regions of the dispersed chromatin. The heterochromatin localized at the periphery along the nuclear envelope is not labelled (Fig. 1). Mainly at 9–10 hours but
also in the other periods, for short times of incubation
with the precursor (1–58) the labelling appears organized in isolated clusters (Fig. 2). At higher magnifications, the gold particles are associated to a regular
netting structure of 10–15-nm-wide chromatin fibers
organized in a ring-like structure of 100–150 nm in
diameter (Fig. 3). From 15 to 18 hours in about 80% of
the nuclei, the gold particles are localized at the border
between the dispersed and the condensed chromatin
associated with the nuclear envelope or the nucleolus
(Fig. 4). While in early S phase the inner nuclear
regions are characterized by dispersed chromatin, the
middle-late S phase is characterized by regularly distributed masses of condensed chromatin about 0.3–0.5 µm
in diameter. For the times of incubation with BrdU up
to 308, gold particles decorate the periphery of this
condensed chromatin, while for longer time periods
(more than 608) gold labelling is evident on chromatin
clumps, demonstrating the composition of newly synthesized DNA. Twenty-one to 22 hours after the block
release, flow cytometry analysis demonstrates that
about 80% of the cells contains 4n DNA, so that this
period can be considered to be the late S phase of the
cells. This stage is characterized by the localization of
the gold particles exclusively on condensed chromatin
along the nuclear envelope or nucleolus. Dispersed
Fig. 1. 3T3 fibroblasts. At the early S phase BrdU is detectable in
interchromatin regions (arrows). No gold markers are evident at the
perinuclear heterochromatin. Bar 5 400 nm.
Fig. 2. 3T3 fibroblasts after short pulse (10 min) of BrdU. At the
early S phase gold particles are detectable as isolated clusters (p).
Bar 5 400 nm.
Fig. 3. At higher magnification of the same case as Figure 2 gold
particles (W) appear localized in a regular net structure of 10–15-nm
chromatin fibers organized in a ring-like structure of 100–150 nm in
diameter (arrows). Bar 5 50 nm.
Fig. 4. Middle S phase in 3T3 cell. BrdU gold markers appear at
this stage at the border between dispersed and condensed chromatin
at the nuclear envelope and near the nucleolus (arrows). Bar 5 250 nm.
Fig. 5. A late step in the S phase of 3T3 fibroblasts. The absence of
colloidal gold particles in the dispersed chromatin is evident as well as
the arrangement of heterochromatin in regularly distributed clumps
(p). Bar 5 250 nm.
Fig. 6. Late S phase after short BrdU pulse, high magnification.
Gold markers (●) are arranged in cluster near the heterochromatin
masses (p) at the nuclear border (arrows). Bar 5 50 nm.
chromatin and the inner chromatin masses do not show
any gold particle decoration at this stage (Fig. 5). After
58 of incubation with BrdU also at this stage gold
particles organized in isolated cluster are detectable.
Higher magnifications demonstrate that in these regions too the antibodies coupled to the gold particles
detect BrdU that has been incorporated into a 10-nm
chromatin fiber (Fig. 6).
Regenerating Rat Liver Nuclei
Liver regeneration that occurs after the removal of 3⁄4
of the organ represents a very interesting example of in
vivo non-malignant proliferation. By 16–18 hours after
surgical hepatectomy proliferative activity may be detected in the parenchymal cells surrounding the portal
areas. Flow cytometry evaluation of DNA synthesis
progression can be obtained observing BrdU incorporation and PI presence on isolated nuclei (Vitale et al.,
Eighteen hours after hepatectomy, the parenchymal
cells assume a steatosis-like state with diffuse lipid
droplets and a large amount of dispersed glycogen
granules in the cytoplasm. The chromatin mainly appears to have a dispersed organization, while the
condensed one, close to the nuclear envelope, is represented by a very thin layer regularly interrupted in
correspondence with the nuclear pore complexes. At
this early stage, BrdU detection in newly synthesized
DNA shows a labelling pattern regularly distributed on
the dispersed chromatin that appears to be represented
mainly by a delicate 10–15-nm fiber network where
groups of not gold labelled RNP particles are constantly
present (Fig. 7). The progression throughout the S
phase is characterized by the same staining patterns
described in 3T3 fibroblasts with a gold labelling at the
boundary of condensed and dispersed chromatin from
23 to 26 hours and on the peripheral condensed chromatin from 26 to 30 hours (Fig. 8). Although in vivo results
are more difficult to evaluate as to time and amount of
uptake of the precursor than the in vitro models,
sometimes, also in regenerating liver nuclei both in the
early and in the late stage, isolated cluster of gold
particles can be detected both in dispersed and condensed chromatin domains. The condensed chromatin
masses of about 0.3–0.5 µm described in the fibroblasts
are also present but, in this model, they appear in a
more precocious stage, at the end of the early S phase of
the cell cycle. Also these masses, at a longer (60 min)
time of incubation with BrdU appear intensively decorated by gold particles, demonstrating the presence of
newly synthesized DNA (Fig. 9). Nevertheless this last
aspect is more clearly detectable in the second half of
the S phase.
Friend Leukemia Cells (FLC)
FLC have been analyzed without synchronization. In
a cycling cell population after the administration of
BrdU for 58, flow cytometry demonstrates that more
than 50% of cells are BrdU positive (Mazzotti et al.,
1990). In these labelled cells, electron microscopy detection of the precursor demonstrates gold particles localized in the interchromatic domains in 30% of cells, at
the border between inter and heterochromatin in 50%
of cells, and on the condensed chromatin in 20% of cells.
RNP aggregates of about 0.3 µm are present among the
fibers while only some randomly distributed heterochromatin masses can be detected in a single section (Figs.
10 and 11).
In all the examined stages and in particular for
shorter times (i.e., 5–10 minutes) of incubation with
BrdU, it is possible to detect isolated clusters gold
particles about 150–200 nm wide both in dispersed and
condensed chromatin. In these nuclei, the inner chromatin appears decondensed in all the three observed
labelling patterns and there is no morphological evidence of any interchromatin fibers condensation (Figs.
12 and 13). By comparing the labelling patterns and
chromatin arrangement distribution, we could assume
that in FLC no premature chromosome condensation
occurs during the S phase.
Computer image analysis performed over about 100
different sections for each cell type shows that the
degree of chromatin condensation from early to late S
phase increases in all the examined models ranging
from about 13% in FLC and 3T3 to 18.2% in hepatocytes (Fig. 14). Despite the different progression with
the presence of a peak in middle S for regenerating
liver, the final amount of heterochromatin is quite
similar in the three different cell types.
Finally, to overcome the limitation of the thin section
for TEM analysis, in order to identify a three-dimensional organization of chromatin fibers, we utilized
STEM analysis. To preserve the spatial architecture of
the nucleus, the cells have been embedded in removable
resin. After deresination, the cellular structure appears
to be recognizable, even also at the higher magnifications (Fig. 15). The method does not preserve the
organization of condensed chromatin but, in the dispersed one, it is possible to clearly detect a regular
three-dimensional network where fibers of different
diameter but mainly of 12–15 nm (including the metal
coating of about 3 nm) form meshes of 150–200 nm
(Fig. 16).
Immunostaining with anti-BrdU in STEM analysis
demonstrates that gold particles can assume a ring-like
distribution (Fig. 17), limiting areas of 150–200 nm
corresponding to the dimensions of the meshes detectable by TEM in dispersed chromatin of the same
samples. Moreover, this distribution is similar to the
cluster organization of gold particles observed in TEM
in all three analyzed cell types.
Despite the fact that the interphase represents the
larger parts of the cell cycle, very little is known about
the organization of chromatin fibers during this period,
especially concerning the DNA replication process. In
eucaryotic systems different models and hypotheses
have been proposed, but all authors agree about the
possibility that DNA synthesis involves initiation processes at multiple sites. However, the amount of those
sites has been differently estimated: Edemberg and
Huberman (1975) and Hand (1978), by autoradiographic data, suggested that there are about 100,000
initiation sites in a mammalian nucleus, spaced by
tracts ranging from 50,000 to 300,000 bp. Recently, in
rat fibroblasts incubated with BrdU, Nakamura et al.
(1986) observed about 150 foci containing about 20
replication units each. Similar results have been obtained with biotinylated dUTP by Nakayasu and Be-
Fig. 7. Early S phase morphology in regenerating rat liver. A
regularly distributed pattern of gold particles is detectable (arrows) in
the dispersed chromatin. RNP particles without gold labelling are
present (p). Bar 5 700 nm.
Fig. 8. Late S phase nuclear morphology in regenerating liver. The
markers appear at this stage on the condensed perinuclear chromatin
masses (arrows). Chromatin clumps are also evident in the nuclear
area (p). Bar 5 1 µm.
Fig. 9. In the same condition as in Figure 8 but after a longer
period of incubation with BrdU, chromatin clumps are also highly
labelled by gold particles (arrows). Bar 5 500 nm.
Fig. 10. In FLC cells BrdU appears distributed in the dispersed
chromatin (arrows) in a arrangement similar to that of early S phase;
not labelled RNP aggregates (p) are detectable. Bar 5 600 nm.
Fig. 11. A ‘‘late S phase’’ pattern of distribution (arrows) is evident
in this FLC image. RNP (p) masses are also detectable without gold
particles. Bar 5 700 nm.
Fig. 12. FLC cells. At high magnification, an ‘‘early S’’ distribution
of gold marker for BrdU. No chromatin clumps are evident in the
interchromatin regions. Bar 5 400 nm.
Fig. 13. No interchromatin clumps are evident, in FLC cells, in a
‘‘late S’’ view. Bar 5 400 nm.
Fig. 14. Condensed chromatin area/nuclear surface ratio. Results are expressed as mean percent of condensed chromatin nuclear
rezney (1989), Kill et al. (1991), and Tomilin et al.
Independently from the number of the activated
units, all these models accept the hypothesis that the
initiation sites are linked to physical sites, as nuclear
skeleton, nuclear cage, or nuclear matrix (Smith and
Berezney, 1982; Smith et al., 1984; Vogelstein et al.,
1980). Even if other authors suggest that nuclear
skeleton is not the native site of DNA replication in
eukaryotic cells (Djondjurov et al., 1986; KrzyzowskaGruca et al., 1983), or that its conformation reflects
exclusively the original status of condensation of DNA
fibers (Martelli et al., 1991), the main questions about
DNA synthesis still concern (1) the level of DNA
filament organization that allows the DNA duplication
and (2) the localization of the first replication sites.
In our experiments, the TEM results demonstrate
that, after few minutes of incorporation of BrdU, both
in dispersed chromatin and at the border of the condensed one, the chromatin structure appears represented by fibers 10–12 nm in diameter that surround
areas about 150–200 nm in diameter. Deresinated
samples observed by STEM analysis confirm the threedimensional organization of these 12–15-nm fibers and
the presence of gold particles in a ring-like regular
distribution. These structures appear the smallest detectable with this method and could be referred to
single DNA replication units (Rizzoli et al., 1992).
Both by STEM and TEM, these fibers appear as
nucleosomic fibers about 10 nm in diameter, which
correspond to the first order of DNA condensation
(Cocco et al., 1986; De Jong et al., 1990; Maraldi et al.,
1979). Recently, by high resolution in situ hybridisation
on metaphase chromosomes, we have demonstrated
that this kind of fiber represents the lowest order of
DNA organization that permits transient DNA denaturation (Rizzi et al., 1995; Rizzoli et al., 1994). Moreover
Horowitz et al. (1994) and Woodcock and Horowitz
(1995), by electron tomography analysis of chromatin
fibers, have proposed a new concept of chromatin fibers
architecture represented by open structures of a continuously variable zigzag nucleosomal ribbon. This hypothesis is alternative to the supercoiled solenoid model
actually strongly conflicting (Cook, 1995) but it offers a
more open substrate to the transcriptional regulatory
machinery. This more dynamic process is in agreement
with the demonstration of the possibility of the transition between the two forms by changing ionic strength
in vitro and in vivo also in reversible mode (Giannasca
et al., 1993; Labhart et al., 1981; Lepault et al., 1980;
Thoma et al., 1979). All these findings also suggest that
DNA replication could occur at nucleosomal-like fiber
The localization of the initiation sites after DNA
precursor administration has been described in the
interchromatin domains (Belmont et al., 1989; Brown
and Schildkraut, 1979; Carrı̀ et al., 1986; Fakan, 1978;
Kay et al., 1971; Mazzotti et al., 1990; Rizzoli et al.,
1992), at the boundary of condensed and dispersed
chromatin (Babu and Verma, 1987), or close to the
nuclear envelope with peripheral localization of both
initiation and termination sites (Nicolini et al., 1986).
However, the importance of nuclear envelope on replication process has been clearly demonstrated (Mills et al.,
1989; Newport, 1987; Sheean et al., 1988).
Swiss 3T3 fibroblasts synchronized by starvation
method and surgically induced liver regeneration represent, respectively, in vitro and in vivo, the models for
the study of S phase progression avoiding the use of
chemicals or drugs that could interfere with enzymes or
molecules involved in DNA replication. In both these
cell lines, the incorporation of BrdUrd in early S phase
occurs on dispersed chromatin in the inner regions of
the nucleus. In middle S phase DNA replication units
are activated at the border between condensed and
dispersed chromatin tight to the nuclear envelope and
to the nucleolus and in late S exclusively on perinuclear
condensed chromatin.
Unsynchronized cycling HeLa cells show the same
distribution of the staining patterns, thus confirming
that DNA replication units localization is specific for
each step of the S phase, starting from the inner and
moving to the peripheral chromatin domains. Moreover, in synchronized 3T3 fibroblasts we have demonstrated that no translocation of the newly synthesized
DNA occurs during S phase. In fact, cells that have
incorporated BrdU for 308, 13 hours after metabolic
block release, and were cultured until the 23rd hour
showed a labelling in the interchromatin domains as in
cells collected at 13 hours, immediately after the administration of BrdU (Rizzoli et al., 1992), while the cells
that in the same conditions received BrdUrd at the 23rd
hour showed the labelling exclusively at the periphery
of the nucleus. This observation suggest that chromosomes maintain fixed sites in the nucleus during S
phase and that they can replicate different tracts at
different times.
In 1993 Hozak et al. described replication factories
fixed in the inner part of the nucleoskeleton and in cells
Fig. 15. Deresinated FLC at STEM analysis. Nuclear morphology
with condensed areas (p) in opposition to decondensed ones (arrows).
Bar 5 200 nm.
Fig. 16. Higher magnification of the same samples as in Figure 14.
Evident is the three-dimensional architecture of the decondensed
chromatin, arranged as a network with meshes of 150–200 nm (p).
Bar 5 100 nm.
Fig. 17. Backscattered STEM image of the samples in Figure 14
allows detection of the gold particle markers distribution in a ring-like
array of about 200 nm. Bar 5 45 nm.
incubated with biotinylated dUTP, and suggested that
the template moves through these structures. In 1994
Hozak et al. demonstrated that some replication occurs
outside these factories at discrete sites of the nucleus on
the diffuse skeleton; it becomes significant by mid S
phase and later becomes concentrated beneath the
lamina. These last results are more in agreement with
our data according to the hypothesis that the replication machinery moves with respect to the template.
The demonstration of several replication sites activated at different times in the nuclear domains is
consistent with the possibility that the DNA synthesis
is a continuous process during all the S phase. Van
Dierendonk et al., in 1989, evaluating BrdU incorporation, divided S phase in five staining patterns, suggesting that spatial and temporal organization of DNA
synthesis seems to be characterized by at least three
successive stages of replications. BrdU incubated in
situ nuclear matrices also revealed five different staining patterns (Neri et al., 1992).
By electron microscopy, the same precursor allows
the identification of three or five specific aspects (Mazzotti et al., 1990; Rizzoli et al., 1992). El-Alfy and
colleagues (1988, 1994), by comparing H3 thymidine
autoradiography and Feulgen reaction, identified four
stages characterized by dots and filaments having
different sizes. Nevertheless, all these aspects were
obtained after short pulses with the precursor, generally below 308. For longer times of incubation, we can
observe a continuous growing and diffusion of the
labelling around the initiation sites that form structures looking like granules clustered into ring-like,
horseshoe-like, and rope-shaped arrays (Nakamura et
al., 1986). By considering also that transitional patterns have been observed, Neri et al. (1992) suggested
the hypothesis of a dynamic nature of DNA synthesis
that is continuous through the S phase.
The last question concerns the condensation of the
newly synthesized DNA to the final metaphasic chromosomal state. In all the cell models analyzed, the inner
chromatin is completely decondensed in early S phase.
In fact, in 3T3 fibroblasts, regular masses of condensed
chromatin appear in middle-late S phase, in rat liver, in
early-middle S phase, while in FLC the correlation of
the labelling patterns demonstrates that heterochromatin increase mainly in middle S. From middle to late S
with short pulses of BrdU we observed the labelling at
the border between condensed and dispersed chromatin; after 1 hour administration or more the gold
particles also cover the condensed chromatin; this is
particularly evident in synchronized fibroblasts in the
inner masses of condensed chromatin. This suggests
that DNA replication occurs in dispersed chromatin
and, later on, the newly synthesized DNA is assembled
in a more condensed form.
In regenerating liver, different hepatocyte populations have been provided to perform DNA synthesis at
different times 18 hours after hepatectomy. In parenchymal cells the inner chromatin masses are regularly
covered by gold particles if administration of BrdU for
more than 1 hour occurs during middle-late S, while
some of them appear without labelling for BrdU pulse
during the first half of the S phase. This observation
could explain the peak obtained in regenerating rat
liver nuclei by image analysis at middle S, suggesting
that all heterochromatin domains are not necessarily
represented from condensed DNA fibers. In fact, chromatin condensation may be the result of poly (ADPribose)
polymerase activity that transfers the adenosine diphosphate ribose moiety of nicotinamide adenin dinucleotide (NAD) to the nuclear proteins and in particular to
H1 histone (Saldit-Geogieff et al., 1980). Both the
terminal region of the homopolymer can link to chromosomal proteins, inducing a chromatin condensation
that cannot be differentiated by electron microscopy
from chromosome fiber condensation (Berger et al.,
1979; Farina et al., 1979; Nagl, 1980). This enzyme is
activated in proliferating tissues (Menegazzi et al.,
1988, 1991); in mouse liver poly (ADP) ribose polymerase transcripts can be found 8 hours after partial
hepatectomy and increase up to sixfold within 1–2 days.
Both the amount of mRNA and enzymatic activity
return to their basal values with the decline of the DNA
synthesis (Menegazzi et al., 1990).
Moreover, molecules like histones are synthesized all
along the S phase (Epner et al., 1981; Kondra and Ray,
1978); while Raska et al. (1990) and Hassan et al.
(1994) have demonstrated that transcription and replication occur in complementary regions of the nucleus;
Hozak et al. (1994) demonstrated that in structures
named nuclear factories, transcription and replication
occur simultaneously and can be discriminate exclusively by immunolocalization of the precursors.
Recently, in mouse duodenal crypt cell, El Alfy et al.
(1995) have been able to divide the cell cycle into 11
stages and have demonstrated that nucleofilaments are
assembled in compacted structures that increase in size
from the beginning to the end of the S phase. The
condensation of newly synthesized DNA could prevent
the possibility that the same tract of the genome is
duplicated more times during S phase (Babu and
Verma, 1987; Harland, 1981). Moreover, it has been
demonstrated that the DNA accessibility to various
fluorochromes, drugs, or DNase treatment changes
during the cell cycle and in particular during S phase
(Evenson et al., 1986; Fujiwara, 1967; Pederson and
Robbins, 1972; Zamai et al., 1993).
For all this evidence we can confirm that S phase is
characterized by modifications of the chromatin arrangement, which is very dispersed in early S phase and
more condensed in the second part of the S phase.
At the moment there are still two classical morphological and histochemical techniques: the detection of precursor of DNA synthesis and the evaluation of the DNA
content. This information can be obtained simultaneously with Feulgen reaction combined with H3 thymidine autoradiography (El-Alfy et al., 1994) or by considering that Feulgen hydrolysis denaturation of DNA in
fixed cells allows measurement of DNA content, tritiated thymidine, and BrdU incorporation in the same
cell (Lin and Allison, 1993).
One of the next goals of morphological science in this
field is represented by the specific localization of genes
in the interphasic nucleus for the evaluation of the
exact time of expression and duplication, the position
within the nucleus, and the evaluation of gene translo-
cation in the different conditions (Yokota et al., 1995).
In fact, it has been demonstrated that the translocation
of specific oncogenes can completely change the proliferating cell rate in B cells lymphomas (Dalla-Favera et
al., 1983). The localization of molecular probes combined with morphological and histochemical data will
provide new insights for basic and diagnostic purposes
in order to evaluate, at the single cell level, the growth
kinetic of heterogeneous cell populations in solid tumours, or to predict the effects of gene transplantation
in non-proliferating cells as in nervous tissues or in
precursors of cycling elements, as stem cells of the bone
The authors thank Mr. Aurelio Valmori for photographic assistance. This work was supported by grants
from the Italian CNR, PF ACRO, and by 40 and 60%
grants from the Ministero della Ricerca Scientifica e
Tecnologica (MURST), Regione Emilia Romagna, ISS
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