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Molecular and Cellular Biology, October 1999, p. 6682-6689, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Regulated Formation of Extrachromosomal Circular
DNA Molecules during Development in Xenopus laevis
Sarit
Cohen,
Sophie
Menut, and
Marcel
Méchali*
Institute of Human Genetics, CNRS, Genome
Dynamics and Development, 34396 Montpellier Cedex 5, France
Received 19 February 1999/Returned for modification 25 March
1999/Accepted 21 June 1999
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ABSTRACT |
Extrachromosomal circular DNA molecules of chromosomal origin have
been detected in many organisms and are thought to reflect genomic
plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de
novo in preblastula Xenopus embryos. This specific DNA
population is not detected in the male or female germ cells and is
dramatically reduced in later developmental stages and in adult
tissues. The activity responsible for the de novo production of
extrachromosomal circles is maternally inherited, is stored in the
unfertilized egg, and requires genomic DNA as a template. The formation
of circular molecules does not require genomic DNA replication but both
processes can occur simultaneously in the early development. The
production of extrachromosomal circular DNA does not proceed at random
since multimers of the tandemly repeated sequence satellite 1 were
over-represented in the circle population, while other sequences (such
as ribosomal DNA and JCC31 repeated sequence) were not detected. This
phenomenon reveals an unexpected plasticity of the embryonic genome
which is restricted to the early developmental stage.
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INTRODUCTION |
Plasticity of the eukaryotic genome,
characterized by rearrangements, transposition, translocation, or
amplification, is observed during evolution as well as in the
development of specific organisms. A massive occurrence of such
phenomena leads to genomic instability, which is a hallmark of
neoplastic processes in mammals (54). The production of
small extrachromosomal circular DNA molecules, also named small
polydispersed circular DNA, is one indication of genome plasticity
(18). These molecules, consisting mainly of repetitive
sequences, are found in the tissues and cells of many organisms and are
thought to emerge from the chromosomes but by a mechanism not yet
determined. Elevated levels of extrachromosomal circular DNA have been
detected in response to carcinogen treatment of human and rodent cells
(12, 14, 53), and they were proposed to play a role in gene
amplification (57). In addition, an increased amounts of
circular molecules have been observed in patients suffering from
genetic diseases which are characterized by genomic instability and
premature aging, such as Fanconi's anemia (14, 39) and Werner syndrome (30). Interestingly, it has recently been
reported that extrachromosomal circles of ribosomal DNA (rDNA)
accumulate in aged yeast cells and in mutants of Sgs 1, the yeast
homolog of the human Werner syndrome gene (50). Circular DNA
is also detected during the rearrangement of the T-cell receptor
(17, 42) and immunoglobulin class switch, which leads to
antibody diversity (36, 56). Extrachromosomal circular
molecules have been observed in Drosophila and mouse embryos
(44, 52, 59), but their specificity to embryonic stages or
their developmental significance remain obscure.
Although circular DNA has been observed in many eukaryotes for more
than two decades, the study of circular DNA has often been limited due
to the lack of convenient techniques and physiological model systems.
We have combined a well-characterized system for embryonic development,
Xenopus laevis, and a useful assay for the detection and
characterization of circular DNA, two-dimensional (2D) gel
electrophoresis (12), to investigate these phenomena during embryogenesis.
Here we describe a developmentally regulated formation of circular DNA
that occurs at a high rate during early embryogenesis of X. laevis. A specific pattern of multimeric circles consisting of the
tandemly repeated sequence satellite 1 was identified in the circular
molecules population. A cell-free system which mimics the in vivo
phenomena was used, and we found that circular DNA is formed de novo by
an activity which is stored in the egg and which is independent of DNA
replication. The possible significance of circular DNA during
development is discussed.
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MATERIALS AND METHODS |
Embryos and egg extracts.
Preparation of embryos and egg
extracts was performed as previously described (15, 25).
Injection of plasmids into fertilized eggs.
Twenty
nanoliters of a solution containing 20 pg of each plasmid (2.7, 5, and
9.4 kb) were injected into fertilized eggs as previously described
(46). Embryos were developed until the 512-cell stage
(pre-MBT embryos), and the DNA was purified.
Preparation of DNA.
Total genomic DNA was prepared by
rapidly homogenizing the eggs, embryos, or liver tissues in 5 volumes
of 30 mM EDTA-1% sodium dodecyl sulfate (SDS)-0.5% Triton
X-100-0.3 M NaCl, followed by incubation at 37°C for 16 h with
0.1 mg of proteinase K per ml. The DNA was extracted with
phenol-chloroform, precipitated with ethanol, and digested with 0.2 mg
of RNase A per ml for 2 h at 37°C. After ethanol precipitation
and resuspension in 10 mM Tris-1 mM EDTA (pH 8), the DNA samples were electrophoresed.
When cell-free systems were used, reactions were stopped by the
addition of 5 volumes of 30 mM EDTA-1% SDS-0.5% Triton X-100-0.3 M
NaCl, and total DNA was prepared as described above.
Low-molecular-weight DNA was prepared by a neutral lysis technique,
according to the method of Hirt (
23). Embryos were
homogenized
in the presence of at least 10 volumes of 10 mM Tris-HCl
(pH 7.9)-10
mM EDTA-0.6% SDS, and NaCl was added to a final
concentration
of 1 M. The homogenate was incubated 16 h at 4°C,
and subsequent
steps were performed as described earlier
(
23).
Neutral-neutral 2D gel electrophoresis.
Separation of DNA in
neutral-neutral 2D gels was performed according to the method of Brewer
and Fangman (6), as described previously (14).
Blotting and hybridization.
Southern blot analyses were done
with Hybond N+ nylon membranes (Amersham), and probes were
labelled by using the Ready-to-Go Kit (Pharmacia). Radiolabelled DNA
was detected by autoradiography (Hyperfilm MP; Amersham) and
with a PhosphorImager (Molecular Dynamics) for quantification by using
the ImageQuant 1.1 software.
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RESULTS |
Detection of extrachromosomal circular molecules in
Xenopus embryos.
In the 2D gel electrophoresis used in
this study, a population of molecules sharing the same structure but of
heterogeneous molecular mass generates a continuous arc, and thus
typical arcs of supercoiled molecules, open circles, and linear
molecules can be distinguished after hybridization with total DNA or
with specific probes (Fig. 1A).
Single-stranded DNA and mitochondrial DNA can be identified in the same
gel, and the structural identity of the DNA in each arc has been
previously determined by electron microscopy and biochemical means
(12, 14). After 2D gel analysis of a low-molecular-weight
DNA fraction from Xenopus embryos at the cleavage stage,
before mid-blastula transition (pre-MBT), we detected a continuous arc
of circles, homologous to total Xenopus DNA, as well as arcs
of double- and single-stranded linear molecules (Fig. 1B).
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FIG. 1.
Detection of extrachromosomal circular DNA in early
development by neutral-neutral 2D gel analysis. (A) Diagram of 2D gel
electrophoretic patterns of genomic DNA generated by populations of
linear and circular molecules heterogeneous in size (adapted from
previous studies (12, 14). Each arc consists of molecules
sharing the same structure but differing in mass. (B to E) A DNA sample
enriched for low-molecular-weight DNA was isolated from
Xenopus embryos at the early blastula stage (2,000-cell
stage), mixed with plasmid-derived open circle size markers (see text),
and separated on a 2D gel. The blot was first hybridized with a sperm
DNA probe to detect total genomic sequences (B) and then with a plasmid
probe to identify the open circles (C). The plasmids range from 2.7 kb
(solid arrowhead) to 11.2 kb (open arrowhead). (D) Comigration of the
nonlinear genomic DNA arc with the markers by superposition of panels B
and C. (E) Rehybridization with a Xenopus satellite 1 probe
(the insert of pE190 [31]) shows ladders of circular
and linear multimers of the satellite 1 unit (multimers of 740 to 750 bp). The sizes of the linear and circular multimers were determined by
circular (in panel C) and linear (not shown) size markers.
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The identity of the DNA which migrated with the nonlinear DNA arc was
validated by mixing the embryonic DNA with open-circle
markers
consisting of plasmid molecules of various lengths which
were relaxed
by use of DNA topoisomerase I. Upon sequential hybridization
with the
two probes (
Xenopus DNA and then plasmid DNA), comigration
of the open-circle markers with the extrachromosomal circle arc
was
observed (Fig.
1B-D). Hence, the nonlinear DNA arc is identified
as
a heterogeneous population of open circles consisting of genomic
sequences, with molecular masses of several hundreds of base pairs
up
to 12
kbp.
Extrachromosomal circular molecules homologous to total DNA were not
detected in DNA isolated from unfertilized eggs, indicating
the absence
of maternal stocks of such molecules (data not shown;
see also Fig.
7C).
As repetitive DNA may be involved in genomic plasticity
(
55), we tested for the presence of the
Xenopus
satellite 1 DNA
sequence, a tandemly repeated element of 750 bp
representing 1
to 2% of the
Xenopus genome (
1,
31,
37) in the extrachromosomal
DNA population. We detected
extrachromosomal satellite 1 DNA in
two series of spots defining two
arcs (Fig.
1E) when intact DNA
was analyzed. Using circular and linear
size markers, we determined
that these ladders consist of circular and
linear multimers of
the 750-bp unit of satellite 1. The arc of circular
DNA was sensitive
to restriction enzymes which cut inside the satellite
1 unit (e.g.,
HindIII,
HinfI, and
BglII; also data not shown) but was resistant
to those that
cut outside the unit (e.g.,
AlwNI,
EcoRI, and
XhoI)
(data not shown; see also Fig.
8C). We conclude that
multimers
of satellite 1 DNA are present in the extrachromosomal
circular
DNA. Transcription followed by reverse transcription, a
mechanism
involved in the formation of some classes of transposons, is
unlikely
to be responsible for the production of the extrachromosomal
satellite
1 DNA. Satellite 1 DNA is transcribed only after the
midblastula
stage (
35), and its transcript, which is 180 bp
long (
1),
would not give multimers of 750 bp and would not
be sensitive
to
BglII, whose site is external to the
transcription
unit.
The broad size range of multimers in the satellite 1 extrachromosomal
population suggests that their formation involves intramolecular
homologous recombination, an activity previously detected in activated
Xenopus eggs, and has been suggested to serve for embryonic
functions
(
32).
We did not detect supercoiled DNA in our DNA preparations from embryos,
although the same purification method successfully
recovered from
developing embryos supercoiled plasmids which were
injected into
fertilized eggs (Fig.
2). The DNA content
of the
injected embryos was analyzed after eight cell divisions
(512-cell
stage). Supercoiled and relaxed forms of all injected
plasmids
were observed, while linear sequences were not detected (Fig.
2A). This result shows that supercoiled DNA can persist in the
embryos
and that it can be successfully purified with genomic
DNA. It further
indicates that the injected molecules were not
subjected to nuclease
activity or other mechanical breakage. Rehybridization
of the same blot
with satellite 1 probe revealed linear DNA and
open circles (Fig.
2C)
that comigrate with the open circle form
of the injected plasmids (Fig.
2D).
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FIG. 2.
Supercoiled plasmids can be recovered from embryos while
endogenous circles are relaxed. Supercoiled plasmids (2.7, 5, and 9.4 kbp) were injected into fertilized eggs before the first cellular
division. Total DNA was purified from the embryos at the pre-MBT stage
(512 cells/embryo) and separated on a 2D gel. (A) Hybridization with
plasmid probe revealed the supercoiled and the relaxed forms of each
plasmid but no linear DNA. (B) Scheme indicating the identity of the
plasmid spots found in panel A and the positions of the linear and
circular DNA. (C) Rehybridization with satellite 1 probe revealed the
position of the genomic circular DNA which contained only open circles.
(D) Superposition of panels A and C.
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We cannot eliminate the possibility that supercoiled molecules appear
only in a transient manner in the cells and are immediately
metabolized
into other forms (e.g., linearization or integration)
and therefore are
not accumulated as standard extrachromosomal
episomes.
It should be noted that throughout this study, normal development of
the embryos was verified prior to DNA extraction and,
thus, circular
DNA is unlikely to result from necrosis. As an
additional control, DNA
was purified from abnormally developing
embryos at the pre-MBT stage
and was found to contain a reduced
amount of extrachromosomal circular
satellite 1 DNA molecules
compared to the normally developing
counterparts (data not shown).
Apoptosis is also unlikely to be
responsible for our observations
since (i) apoptosis is not detected in
Xenopus embryos before
the gastrula stage (
22,
49,
51) and (ii) apoptosis would
generate DNA fragments which are
multimers of 180 bp and not sequence
specific.
Sequence content of circular DNA is not random.
Hybridization
of the same blot to different probes revealed that satellite 1 is
over-represented in the population of circular DNA relative to its
proportion in the genome, since the ratio of circular to linear
satellite 1 sequences is at least threefold higher than the ratio of
circular to linear total genomic DNA (compare Fig. 1B and E, 3B and C,
6A and C, and 7A and B).
We have also identified 5S rDNA sequences in the extrachromosomal DNA
molecules, at a level similar to satellite 1 DNA (Fig.
3A and C). The
5S rDNA genes are organized in tandem repeats close
to the telomeres of
most of the chromosomes. Their size (ca. 690
bp) and frequency are
similar to those of satellite 1. However,
the repeating units are very
heterogeneous in size (
10), and
even adjacent repeats can be
heterogeneous (
9). The circular
form of 5S rDNA sequences
are observed in a continuous arc (Fig.
3A), a finding in agreement with this
size heterogeneity. The
circles containing 5S rDNA were resistant to
cleavage with
EcoRI,
which does not have a site in the rDNA
sequence, and sensitive
to cleavage with
DraI, which has two
sites (data not shown).
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FIG. 3.
5S rDNA in circular DNA from early embryos. A DNA sample
enriched for low-molecular-weight DNA was isolated from
Xenopus embryos at the early blastula stage and separated on
a 2D gel. The blot was hybridized with 5S rDNA probe (pXbs115/77)
(3) (A), with total Xenopus genomic DNA (B), and
with satellite 1 (C).
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With total cellular DNA extracted from pre-MBT embryos, circular DNA
migrated with the same pattern as with low-molecular-weight
DNA,
namely, extrachromosomal circles separated from the bulk
of chromosomal
DNA (Fig.
2C,
4A and C, and 5A).
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FIG. 4.
Sequence content of circular DNA. (A and C) Total
genomic DNA from early embryos was separated on 2D gel and hybridized
with satellite 1. One blot was then hybridized with a 5-kb coding
sequence of the rDNA gene cluster (probe B [25]) (B),
and the second was hybridized with a 500-bp dispersed repetitive
sequence (JCC31 [11]) (D). To compare the relative
amounts of circular DNA of each probe and to avoid the effect of
different copy numbers, the exposure time was adapted to obtain similar
intensity of the linear DNA arc. The position of maternally amplified
circular rDNA is marked by an arrowhead.
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High-molecular-weight extrachromosomal rDNA is maternally amplified
during early oogenesis by a rolling circle mechanism and
is accumulated
in the egg (
7). Electron microscopy studies
have shown that
most of the molecules are linear, and only a small
fraction (2 to 5%)
of the amplified DNA consisted of circular
molecules representing
multimers of the rDNA unit (
24,
48),
which varies between 11 and 15 kbp (
47,
58). When analyzed
on 2D gel
electrophoresis, the vast majority of rDNA present in
the egg migrated
with the arc of linear DNA, but a low amount
of circular rDNA
corresponding to one unit could be identified
(data not shown and Fig.
4B). Molecules larger than one unit are
beyond the resolution limits of
the technique. The population
of circular extrachromosomal rDNA
molecules decreased in embryos,
in agreement with the degradation of
the amplified rDNA population
during early development (
8).
An arc of small circular molecules
homologous to the rDNA gene cluster
but smaller than the size
of one unit was never detected either in eggs
or embryos (Fig.
4B), implying that an abundant sequence is not prone
to form extrachromosomal
circles by stochastic events of breakage and
ligation.
In addition, a repetitive sequence of 500 bp (pJCC31) which is
dispersed in the
Xenopus genome (
11) is not
detected in the
population of circular DNA (Fig.
4D), further
demonstrating that
the formation of extrachromosomal circles does not
occur at
random.
Quantification of the gels of Fig.
4A and C and Fig.
5A and gels from additional experiments
revealed that ca. 1% of the cellular
satellite 1 DNA sequences is
present as circular molecules during
early development. As it was not
possible to quantify the linear
satellite 1 multimers comigrating with
linear chromosomal DNA,
this is probably an underestimation of the
extrachromosomal satellite
1. Interestingly, in HeLa cells a similar
fraction (1%) of the
genomic alphoid satellite
Sau3A
element is represented as extrachromosomal
multimers of the basic
170-bp unit (
27,
28).
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FIG. 5.
Circular DNA is produced during the early stages of
development. (A and B) Total genomic DNA hybridized with a satellite 1 probe: A, 590 ng of early blastula embryos (pre-MBT, 1,000-cell stage);
B, 1.4 µg of late-neurula-stage embryos (stage 23, 90,000 cells/embryo). The quantity of DNA was estimated from the number and
the stage of the embryos and was verified directly on the gel by
hybridization with a genomic DNA probe and comparison to a genomic
standard calibrated spectrophotometrically at 260 nm and loaded on the
same gel (not shown). (C to F) Low-molecular-weight (LMW) DNA was
isolated from early blastula embryos (pre-MBT [C and E]) and from
stage 13 embryos (neurula [D and F]), and similar amounts of DNA were
compared on the same 2D gel. The blots were hybridized with a satellite
1 probe (C and D) and with a sperm DNA probe (total genomic probe [E
to F]). The pre-MBT panels (C and E) represent low-molecular-weight
DNA from 32 embryos of the 1,000- to 2,000-cell stage (i.e., maximum of
60,000 cells) and the neurula panels (D and F) represent
low-molecular-weight DNA from five embryos at the 80,000-cell stage
(i.e., 400,000 cells).
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The production of extrachromosomal DNA circles is developmentally
regulated.
To determine whether the accumulation of circular DNA
was stage specific, we compared on the same blot samples of DNA taken from pre-MBT embryos and from late-neurula-stage embryos. While circular DNA of satellite 1 could be easily identified in the early
embryos (Fig. 5A), we could not detect circular molecules in neurula
embryos when using 2.4-fold more DNA (Fig. 5B). To further increase the
sensitivity of the assay, the analysis was repeated with the
low-molecular-weight fraction of embryonic DNA. In Fig. 5C and D,
similar amounts of low-molecular-weight DNA purified from pre-MBT and
neurula embryos were loaded on the same gel. Hybridization to satellite
1 probe shows a reduced amount (seven- to eightfold) of circular
molecules in the DNA sample extracted from neurula embryos. As the
neurula DNA was extracted from 6-fold-more cells, our data indicate a
50-fold decrease in the amount of extrachromosomal circles per cell at
the neurula stage compared to pre-MBT stage. In addition, the linear
multimers of satellite 1 are no longer detected in the
low-molecular-weight neurula DNA, even with shorter exposures (data not
shown). Hybridization of the low-molecular-weight DNA samples with a
total genomic DNA probe further indicates that the decrease in the
amount of circular DNA is not restricted to satellite 1 DNA (Fig. 5E
and F).
The earliest developmental stage that could be tested by the 2D gel
analysis was that of the embryos of the 32- to 64-cell
stage. Circular
DNA can be detected at this stage, and its proportion
relative to total
genomic satellite 1 content was similar to that
observed in 1,000- to
2,000-cell-stage
embryos.
Circular molecules were not detected in adult liver tissue (Fig.
6) or in
Xenopus somatic A6
cells in culture (data not shown).
We conclude that the formation of
extrachromosomal circular DNA
is a developmentally regulated
phenomenon, one specific to early
development.
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FIG. 6.
Circular DNA is not detected in adult liver tissue.
Low-molecular-weight (LMW) DNA was isolated from early blastula embryos
(pre-MBT [A and C]) and from adult liver (B and D) and compared on
the same 2D gel. Hybridizations with a sperm DNA probe (total genomic
probe [A and B]) and with a satellite 1 probe (C and D) show that, in
adult liver cells, circular DNA is not detectable, even when loading an
amount of low-molecular-weight DNA which was three times greater than
that of the early embryo.
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Extrachromosomal circles are generated de novo by an activity
present in the egg.
To further investigate the mechanism that
produces extrachromosomal circles, we used cell-free systems derived
from Xenopus eggs (2, 34). After incubation of
sperm nuclei in egg extract, extrachromosomal circles were detected
both by the total genomic probe (Fig. 7A)
and the satellite 1 probe (Fig. 7B). In some cases, a small amount of
supercoiled molecules was also detected, which might result from a
less-efficient processing mechanism of the circular DNA in vitro.
Hence, the in vitro system reproduced the phenomena observed in vivo.
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FIG. 7.
De novo formation of circular DNA in a cell-free system.
2D gel analysis of DNA purified from 2 × 105
demembranated sperm nuclei after incubation in 200 µl of egg extract
for 2 h at 23°C as described previously (15) (A and
B), from 200 µl of low-speed activated egg extract which was
incubated without addition of sperm nuclei (C), and from 2 × 105 demembranated sperm nuclei (D). For panels A, C, and D,
blots were hybridized with a sperm DNA probe, and the arc of open
circles is indicated in panel A by an open arrowhead. The vast majority
of the DNA in the egg extract is circular and linear mitochondrial DNA
(4 ng per egg [21], ca. 800 ng in this sample). It is
slightly labelled due to nonspecific hybridization with the sperm DNA
probe (solid arrowheads in panel C). Rehybridization of blots from
panels C and D with the satellite 1 probe did not reveal circular DNA
(data not shown), while rehybridization of the blot from panel A with
satellite 1 probe revealed ladders of circular multimers in an arc of
open circles (solid arrow) and a faint arc of supercoiled circles (open
arrow), as well as a massive linear-DNA arc (B). The structural
identity of the arc DNA and the size of the multimers was determined by
their comigration with supercoiled and relaxed plasmid markers, which
were mixed with the DNA sample and visualized after hybridization with
a plasmid probe (data not shown).
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Egg extracts incubated in vitro without additional DNA template did not
produce circular DNA homologous to total genomic sequences
(Fig.
7C) or
to satellite 1. We conclude that the circles are
not produced from a
preexisting free cytoplasmic egg
DNA.
Total DNA purified from sperm nuclei did not contain detectable
extrachromosomal circles after hybridization with total sperm
DNA probe
or with satellite 1 probe (Fig.
7D and data not shown).
This result
confirms that the formation of extrachromosomal circles
occurs de novo
due to an activity already present in the egg and
that the template for
this activity is nuclear genomic
DNA.
Circular DNA is produced by egg extract which reproduces the early
cell cycles in vitro.
Demembranated sperm nuclei are replicated in
Xenopus activated egg extracts that mimic the early cell
cycles, including genomic DNA replication (2, 41). DNA
isolated from sperm nuclei that were incubated in egg extract was
digested with EcoRI to produce a specific rDNA fragment and
then subjected to 2D gel electrophoresis. The satellite 1 repeated
sequence does not contain an EcoRI site and therefore
remains intact in these conditions.
DNA replication in early embryos and in egg extracts is characterized
by three types of replication intermediates while testing
a single DNA
fragment (
26). These are bubbles for initiations
in the
fragment, Y shape for forks that pass through, and H shapes
for
termination events. After hybridization with an rDNA probe,
all of the
expected chromosomal rDNA replication intermediates
were identified,
including replication bubbles (Fig.
8A).
Rehybridization
of the same gel with the satellite 1 probe revealed
both the linear
and circular extrachromosomal multimers (Fig.
8B and C,
respectively).
These data show that the egg extracts competent for DNA
replication
can also form circular extrachromosomal DNA molecules. The
presence
of intact replication intermediates further rules out the
possibility
that circle production refers to abnormal processes of
defective
extracts. The amount of circles detected was proportional to
the
concentration of sperm nuclei in the extract (data not shown).
This
observation suggests that the activity that produces circles
is present
in excess in the egg. The formation of extrachromosomal
DNA in extracts
that reproduce the normal cell cycle shows that
the extracts are
competent for both processes but does not necessarily
indicate that
circle formation requires DNA replication.
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FIG. 8.
Formation of circular DNA in egg extracts competent for
DNA replication. DNA purified after in vitro incubation of sperm nuclei
in egg extract was cleaved with EcoRI and then analyzed by
2D electrophoresis. (A) Hybridization with a 5-kb coding sequence of
the rDNA gene cluster (probe B [25]) reveals the
replication intermediates of this fragment in a typical triple-arc
pattern, representing initiation (- -), termination (>-<), and
elongation (>-) events as expected in this system (26).
Rehybridization with the satellite 1 probe reveals linear multimers of
satellite 1 unit after a short exposure (B) and circular multimers with
a longer exposure (C), similar to the patterns observed with early
embryo DNA (e.g., Fig. 1E and 2C).
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The formation of extrachromosomal circular DNA does not require
chromosomal replication.
To determine whether DNA replication was
involved in the production of the extrachromosomal circular DNA, newly
replicated DNA was radiolabelled by adding [
-32P]dATP
and sperm nuclei simultaneously to the egg extracts. 2D gel analysis
revealed a distinct arc of labelled extrachromosomal circles (Fig.
9A). No labelled arcs were detected in
the absence of sperm nuclei (data not shown). However, the labelled
extrachromosomal DNA circles could have arisen from replicated DNA in
this experiment. To test whether DNA replication could be uncoupled
from the production of extrachromosomal circular molecules, DNA
replication was blocked by aphidicolin, which was added at the
beginning of the reaction. Extrachromosomal circles were not detected
by direct labelling (Fig. 9B) but were revealed by hybridization with a
total genomic DNA probe (Fig. 9C). These data demonstrate that the
formation of extrachromosomal circles can be uncoupled from genomic DNA replication since it is resistant to aphidicolin.
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FIG. 9.
The production of circular DNA does not require DNA
replication. To label the newly replicated DNA, 105 sperm
nuclei were incubated in 100 µl of activated egg extract as described
above in the presence of 5 µl of [-32P]dCTP (100 µCi). Aphidicolin (50 µg/ml) was added in panels B and C (aphi).
Samples were taken after 95 min of incubation, and the DNA was
separated on 2D gels and transferred to a nylon membrane. An arc of
circular DNA (solid arrowhead) was detected among the labelled newly
replicated DNA (A), while labelled circular molecules were not detected
in the presence of aphidicolin (B). The weak labelling of the large
amount of mitochondrial DNA present in the egg (21) in the
presence of aphidicolin (panel B, open arrowheads) may be due to its
replication by DNA polymerase- (16), which is aphidicolin
resistant. (C) Hybridization of the blot from panel B with a total
Xenopus genomic DNA probe revealed circular molecules (solid
arrowhead).
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Circular DNA was already detected at 30 min of incubation of sperm
nuclei in the egg extract. At that time, DNA synthesis
had not yet
started, as determined by incorporation of [
-
32P]dATP,
which was added to a sample of the tested reaction mixture
at time zero
(data not shown). This result is in accordance with
the appearance of
circular DNA in the presence of aphidicolin.
The formation of circles
before the onset of DNA synthesis or
in the presence of aphidicolin may
suggest an excision by recombination.
We cannot yet determine whether
the circular molecules can be
further replicated as extrachromosomal
DNA in a DNA polymerase
-dependent manner or are excised from newly
replicated chromosomal
molecules, generating the labelled circular
DNA.
| |
DISCUSSION |
In this study we report a phenomenon of genomic plasticity
identified by the formation of extrachromosomal circles from a chromosomal origin during early embryonic development in X. laevis. We show that this activity is developmentally regulated
and is particularly pronounced in the early embryos.
Several features strongly argue against an artificial or a random
production of extrachromosomal circles. (i) The circular DNA population
was detected after several different procedures for DNA isolation which
involve phenol detergent and high salt extraction. Random breakage, if
it occurs during extraction, is unlikely to be followed by ligation
under these conditions. (ii) Nuclease activity in the embryos or during
DNA preparation is unlikely since supercoiled plasmids that were
injected into fertilized eggs were successfully recovered from embryos
after eight cell divisions and using our standard extraction procedures
(Fig. 2). Furthermore, replication intermediates remained intact (Fig.
6A), as well as most of mitochondrial DNA (17 kbp, circular), which is
very abundant in the eggs and in early embryos (Fig. 7C and 9B). (iii)
Circles are not likely be formed artificially by reannealing of
single-stranded DNA during DNA purification since we did not observe a
correlation between the presence of single-stranded DNA and the
formation of extrachromosomal circles. In addition, pretreatment of the
input DNA used in vitro with nuclease S1 does not affect circle
formation (13). Still, we cannot exclude the possibility
that the mechanism by which circular DNA is formed includes
single-stranded intermediates as in the single-strand annealing
mechanism (32, 33, 45). (iv) Apoptosis or necrosis cannot be
responsible for circle formation since apoptosis is not detected in
early embryos (22, 49, 51) and normally developing embryos
were used in this study. When abnormal embryos were deliberately
selected, their DNA contained a reduced amount of circular DNA. (v) The
formation of circular DNA can be reproduced in egg extracts which mimic
the cell cycle in vitro. (vi) The under-representation of the
repetitive pJCC31 sequence in the circular molecule population compared
to a random genomic sequence rules out the possibility of accidental
breakage-ligation in vivo. The maternally amplified rDNA did not form
circles smaller than the size of its repeating unit, thus further
supporting the conclusion that random breakage-ligation of abundant
sequences is not likely. (vii) Furthermore, the over-representation of
satellite 1 sequence and the formation of sharply defined circular
multimers of satellite 1 suggest the presence of a controlled mechanism
which may involve intramolecular homologous recombination.
Circular DNA in mammalian cells is correlated with genomic instability
and aging (18) and was proposed to appear as an early intermediate of gene amplification during malignant processes (57). We found that they are also produced in early
Xenopus embryos. Hence, the formation of circular DNA may be
a marker of genome plasticity important for early development which is downregulated throughout the normal life of the organism.
Although the biological significance of the production of
extrachromosomal circular DNA is difficult to address, we showed here
that this phenomenon exists in normal Xenopus embryos, is developmentally regulated, affects the cellular genome, and does not
proceed at random. A genetic rearrangement of some discrete sequences
may be one explanation for this phenomenon during early development.
Genetic rearrangements in developmental processes have been observed in
unicellular organisms and specifically in the early development of some
nematodes and hagfish (43). For example, a dramatic
exception to the rule of DNA constancy is the developmentally
controlled chromatin diminution first reported by T. Boveri in
Parascaris, where specific chromosome fragmentation and
telomere addition occurs in the presomatic blastomeres during the first
divisions of the embryo (4, 40). In the presumptive somatic
cells, 85% of the genomic DNA, including most repetitive DNA
sequences, is eliminated (38). In hagfish (cyclostomata, vertebrata), highly repetitive DNA elements are restricted to the germ
line and are deleted during early embryonic development (19,
29). However, significant changes in the amount of genomic satellite 1 sequences were not detected upon comparison of sperm DNA
and somatic tissues (spleen and erythrocytes) of the same individual
(13). Therefore, a massive elimination of satellite 1 elements in somatic tissues, as described in parascaris and in hagfish,
is unlikely in X. laevis. An additional support to the idea
that a loss of a large set of sequences during development is not
likely in X. laevis arises from the experiments involving nuclear transplantation of somatic nuclei into enucleated eggs which
resulted in normal fertile animals (20).
In higher vertebrates, large global programmed rearrangements of the
genome have not yet been observed, but localized gene rearrangements
have been demonstrated during the differentiation of the immune system
cells (5). Small extrachromosomal circles are produced by
intramolecular DNA deletions during the rearrangement of the T-cell
receptor and 
genes in mouse thymocytes (17, 42) and
during immunoglobulin class switch recombination (36, 56).
The presence of large amounts of specific repetitive sequences in the
extrachromosomal population might represent by-products of cellular
differentiation pathways or, alternatively, might indicate an active
role for these sequences in developmental processes such as remodeling
of the organization of the genome during embryogenesis. Extrachromosomal circles may also reflect elimination of sequences necessary for germ line functions, including fertilization and chromosome pairing, but unnecessary for further development. In any
case, the production of extrachromosomal circles during
Xenopus early embryogenesis is a novel example of genomic
plasticity of a vertebrate genome during development. It occurs at a
stage when there is no transcription in the embryo but when
determination of the cell lineage is already taking place
(16).
We believe that the physiological experimental system presented here
will facilitate further study of genomic plasticity during early
vertebrate embryogenesis and help with our understanding of the
evolution and function of repetitive sequences in the genome.
| |
ACKNOWLEDGMENTS |
We thank P. Brooks for a critical reading of the manuscript.
This study was supported by grants from the CNRS, the Association pour
la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, and
the Fondation pour la Recherche Médicale. S.C. was supported by
postdoctoral fellowships from the European Molecular Biology
Organization and the Fondation pour la Recherche Médicale.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Human Genetics, CNRS, Genome Dynamics and Development, 141, Rue de la Cardonille, 34396 Montpellier Cedex 5, France. Phone: 33-(0)
4-99-61-99-17. Fax: 33-(0) 4-99-61-99-20. E-mail:
mechali{at}igh.cnrs.fr.
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Abstract
Introduction
