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Chapter I.5
The Eukaryotic Perspective: Similarities and Distinctions
between Pro- and Eukaryotes
Conrad L. Woldringh*
Institute for Molecular Cell Biology
BioCentrum Amsterdam, University of Amsterdam
Kruislaan 316, 1098 SM Amsterdam
The Netherlands
Phone: (31) 20 525 6219
Fax: (31) 20 525 6271
E-mail: woldringh@bio.uva.nl
Roel Van Driel
E.C. Slater Instituut
BioCentrum Amsterdam, University of Amsterdam
Plantage Muidergracht 12, 1018 TV Amsterdam
The Netherlands
Phone: (31) 20 525 5150
Fax: (31) 20 525 5124
E-mail: van.driel@chem.uva.nl
*Corresponding author. Send proofs to:
C.L. Woldringh
Institute for Molecular Cell Biology
University of Amsterdam
BioCentrum Amsterdam
Kruislaan 316
1098SM Amsterdam
The Netherlands
The presence of a membrane-bound nucleus and the nucleosomal
organization of the DNA in eukaryotes represent important differences with
prokaryotic cells. Another fundamental difference lies in the mechanism of
chromosome segregation. Because of the size of prokaryotic cells, their
replicated chromosomes have only to be displaced over relatively small
distances (<2 µm). This segregation occurs along with cell growth. In contrast,
eukaryotic cells keep their replicated sister chromatids together until
segregation during mitotic anaphase. In this case chromosomes are displaced
with the help of the microtubular spindle apparatus over relatively large
distances (>10 µm).
This mitotic mechanism of segregation has such a strong appeal that it is
often considered to represent an uniform mechanism, also applicable to
prokaryotes. This has been suggested by several authors in their studies of the
bacterial cell cycle (15, 63, 65, 76). However, eubacteria as well as
archaebacteria seem to lack a cytoskeletal segregation apparatus, although they
do have cytoskeleton-like proteins (like FtsZ; 40; see for a review 53), "motor"
proteins (like MukB; 22) and condensing proteins (like Smc; 17). Moreover, the
size and complexity of the eukaryotic mitotic apparatus are such that a similar
mechanism is difficult to accommodate in a bacterial cell as illustrated in Fig. 1A.
A prerequisite for mitotic segregation is the cohesion of sister chromatids,
which is needed for bipolar attachment of kinetochores to microtubules coming
from opposite poles. The cohesion lasts until anaphase when it is abolished by
proteolysis and the chromatids are pulled apart by the action of the spindle
microtubuli. The mechanism of maintenance of cohesion is not known. It is
either the result of DNA strand entanglement or due to specific protein bridges
(19, 32). However, in metaphase chromosomes the sister chromatids are
largely disentangled and most DNA regions have separated. When considering
the separation of sister chromatids at the scale of a typical bacterial cell, the
average distance between pairs of identical genes in sister chromatids during
metaphase (1 to 2 µm) is similar to that of DNA segregation in prokaryotes
(Fig. 1B; cf. pairs of fluorescent spots as shown in Fig. 3A and B of ref. 35).
Segregation is defined here as the process that moves the replicated and
separated chromosomes apart towards opposite poles and into the prospective
daughter cells (49). Already before or during this movement, the DNA
daughter strands have to be disentangled in a process called separation.
This chapter begins by comparing the organization of DNA in bacteria and
eukaryotes. Next, the possibility that similar mechanisms are utilized by both
eukaryotes and prokaryotes to separate or disentangle sister chromatids or
daughter strands is outlined. It is suggested that transcriptional activity and
resulting RNA-protein (hnRNP) particles may play a role in DNA strand
separation in eukaryotic cells, just as coupled transcription/translation does in
prokaryotes. It is implied that genome organization is not only important for
gene expression, but also for the process of chromosome separation. First, we
will discuss how the primary separation of daughter strands in bacteria leads to
a hierarchical mode of segregation (12), which differs fundamentally from the
mitotic mode of segregation of chromosomes in the eukaryotic cell.
HIERARCHICAL DNA SEGREGATION IN BACTERIA
Two characteristics of a bacterial cell such as Escherichia coli may contribute to
the difficulty in understanding its mechanism of genome segregation. First, the
occurrence of DNA synthesis throughout the whole cell cycle during rapid
growth (8). Second, the lack of a unique centromere sequence, which is
characteristic for the eukaryotic chromosome. In contrast to the multiple
replication origins per eukaryotic chromosome, bacterial chromosomes sustain
a unique origin, from which replication initiates and proceeds bidirectionally.
Although the replication forks move bidirectionally at a maximal rate (about 50
kb per min; 31), the duration of one round of replication (40 min; 8) is long
compared to the shortest generation time possible, i.e. 20 min. Despite a slow
rate of fork movement (0.5 to 5 kb per min; 31) eukaryotic cells such as
Saccharomyces cerevisiae can have an S-phase of 30 min, because replication
starts at about 400 origins (55), whereas their shortest cycle time is still about 60
min.
The bacterial cell is able to make its cycle time shorter than the duration of
the DNA replication period, by making use of multi-fork replication and
overlapping cycles (8). It should be noted that in bacteria, multi-fork replication
refers to replication forks that have initiated in previous cell cycles and that
therefore belong to different genealogical orders. In eukaryotic chromosomes,
multiple replication forks have all initiated during the same cycle and are thus
of the same genealogical order. Under conditions of rapid growth, when the
cycle time is shorter than the replication period, bacterial DNA can be
synthesized continuously throughout the cycle, while once per cycle a new
round of replication is initiated and one round is completed (see review ref. 21).
Bacterial multi-fork replication is a form of re-replication and implies the
presence of several genealogical orders of origins and of replicated daughter
strand regions. How are these multiple orders of daughter strands
disentangled and segregated?
In the case of eukaryotic chromosomes with many origins of replication (but
no re-initiations during a single cell cycle), segregation is related to the build-up
of two back-to-back oriented kinetochores on the unique centromere. These
juxtaposed structures are then attached to microtubuli of the mitotic spindle
coming from opposite poles. Subsequently, the chromatids are transported to
the poles. Inherent to this mechanism is the moving apart of the two recently
replicated sister chromatids.
In haploid cells as depicted schematically in Fig. 2A, segregation seems to
take place in the same way in prokaryotes and in eukaryotes. If a haploid cell
would skip a division it would contain two chromosomes as shown in Fig. 2B.
The mitotic segregation mechanism will, after one round of replication,
segregate each of the sister chromatids to opposite poles and to the respective
daughter cells (Fig. 2B, lower arrow). In contrast, in bacteria the replicated
daughter strands of one chromosome (sister chromatids) will remain in the
same compartment, ending up in the same daughter cell after division; they
will only become separated during the next division cycle.
In principle, a bacterial cell could segregate in a mitotic fashion. If the
bacterial chromosome contained a centromere at its terminus of replication, it
could always segregate the fully replicated chromosomes, even if re-initiation
and multi-fork replication has produced different genealogical orders of origins
and daughter strand regions. Such a mitotic segregation mechanism in E. coli
was proposed by Hiraga (21) based on the characterization of a kinesin-like
"motor protein" (MukB) and by Begg and Donachie (2) based on the
observation that nucleoids move rapidly apart after termination of replication.
However, cytological observations (77) have indicated that DNA segregation is
a continuous process and that the replicating nucleoid increases gradually in
size along the major cell axis in such a way that immediately upon termination
the two daughter nucleoids are already separated (67).
It thus seems that the origins and subsequently replicated DNA regions are
continuously being separated and segregated as each pair of replication forks
proceed from the origin to the terminus of replication. As emphasized by
Donachie et al. (12), this situation differs fundamentally from that in eukaryotes.
If an E. coli cell postpones a division, the cell will contain two chromosomes and
it will become multichromosomal (or "diploid"). After replication of the
chromosomes, the next division will not take place between the recently
replicated daughter strands (sister chromatids), but between the two pairs of
chromosomes (Fig. 2B). In eukaryotic cells this situation is obtained during
meiosis I, when the replicated homologous chromosomes pair and their sister
kinetochores become associated. Because the connected kinetochores attach to
microtubuli from the same pole (49), the chromosomes separate from one
another as pairs. In prokaryotes, however, such specific pairing as during
meiosis I or a cohesion as during mitosis does not occur. Nevertheless, division
occurs normally between the pairs of chromosomes. This phenomenon, called
"hierarchical" segregation by Donachie et al. (12), explains why
multichromosomal bacteria cannot maintain heterozygosity. They thus will
always develop to homozygosity. This is because, when a mutated gene is
replicated (white star in Fig. 2B), the gene pair is not segregated over the
daughter cells, but remains in one cell because of the mechanism of
segregation. This mechanism represents a gradual and continuous movement
of the nucleoids (see below). A lateral movement of the nucleoids as in
filaments (68) and as depicted in Fig. 2B (upper arrow), suggests that cell
division will separate the pairs of chromosomes. However, this lateral
movement is not the only reason, because the same hierarchical mode of
segregation occurs when the cells assume an ovoid shape (12). The reason
probably lies in a relationship between separated nucleoids and the induction of
a division plane as suggested by Donachie et al. (12; see also 80), but this is
outside the scope of this review.
COMPARISON OF THE EUKARYOTIC NUCLEUS WITH THE
BACTERIAL CELL
Before discussing mechanisms for separation of replicated daughter strands
in prokaryotes and eukaryotes, we will first compare the organization of DNA
in both kinds of organisms. In vitro studies have shown that phase separation
(71), monomolecular collapse and intermolecular aggregation of the DNA into
a condensed state (5, 36, 51) can be induced by high concentrations of proteins
(macromolecular crowded solution) and ions. In bacterial cells high plasmid
concentrations have been observed to result in liquid-crystalline structures
(62). In growing bacteria a phase separation between nucleoid and cytoplasm is
observed by phase contrast microscopy (46) and was also predicted on the
basis of physico-chemical calculations (59).
Merely on a basis of volume, the bacterial cell can be compared with, for
instance, the nucleus of a yeast cell. Table 1 gives the DNA concentrations in
both compartments. If the DNA of E. coli would be dispersed throughout the
whole volume of the cell, the DNA concentration would be about 9 mg/ml
(Table 1). This is of the same order of magnitude as the DNA concentration in
the nucleus of a diploid yeast cell, 13 mg/ml (Table 1). The DNA concentration
in the nucleus of a mammalian cell depends on the estimate of the volume of
the nucleus and seems to be in the range of 1 to 100 mg/ml (Table 1). The
highest concentration for a human cell nucleus with a radius of 2.5 µm, would
be 100 mg/ml, which is similar to the DNA concentration within the volume of
the bacterial nucleoid. As discussed in Chapter III.2 (82), estimates of the
volume of the nucleoid visualized with a confocal scanning light microscope
operating at a lateral resolution of 130 µm, resulted in a DNA concentration of
65 mg/ml. Bohrmann et al (6) estimated the DNA concentration in the E. coli
nucleoid to be 80 - 100 mg/ml by applying low-dose ratio contrast imaging
with the scanning transmission electron microscope on resin-embedded
sections. This probably represents an upper limit, considering their use of
dehydrated and embedded cells that may have suffered shrinkage. Thus, on a
chemical basis, considering the amount of DNA in the eukaryotic nucleus and
in the bacterial cell (Fig. 3A), the DNA concentrations are similar (see Table 1).
Within the bacterial cell, the concentrations of protein and RNA are about 20
and 6 mg/ml, respectively (78; see also ref. 54). In the eukaryotic nucleus the
synthesis of mRNA is a process involving co- and post-transcriptional
processing of mRNA, including 5’-capping, 3’ cleavage, polyadenylation and
splicing. Subsequently, the processed RNA is transported as hnRNP
(heterogeneous nuclear particles), by an as yet obscure mechanism (see 61 for a
review). It seems well established that RNA synthesis in the nucleus largely
exceeds the need of cytoplasmic mRNA and rRNA, as 80 to 90% of all the
hnRNA is degraded without export to the cytoplasm (1). The synthetic
machineries that transcribe and replicate DNA and process and transport RNA,
are so extensive that it may be expected that the concentrations of RNA and
protein in the eukaryotic nucleus and in the bacterial cell are similar
Another striking similarity between the prokaryotic cell and the eukaryotic
nucleus is the occurrence of co-transcriptional processes (Fig. 3B). In
prokaryotes, ribosomes bind immediately to mRNA for the synthesis of
proteins in the so-called coupled transcription/translation process. In
eukaryotes, the pre-mRNA becomes associated during its synthesis with
proteins involved in splicing and transport of the transcripts (10, 30, 47),
forming large spliceosome structures of a size similar to the bacterial ribosome
(30).
However, as illustrated in Fig. 3B, there is a fundamental difference in the
conformation of the DNA between pro- and eukaryotes. In the bacterial
nucleoid the DNA occurs in the form of plectonemic supercoils (see ref. 82) with
relatively few bound proteins. In the eukaryotic nucleus the nucleosomal
organization involves a tenfold increased mass of proteins bound to DNA (6).
Viewed on the small scale of the double helix, the DNA (145 bp) is wrapped
around a histone octamer in two almost complete left-handed superhelical
turns, forming a thread of nucleosomes with a diameter of a 10 nm.
Subsequently, this 10 nm thread is folded as a solenoid into a 30 nm fiber (42).
Minsky et al. (48) proposed that this assembly in nucleosomes is necessary to
prevent condensation of the large amounts of DNA in the eukaryotic nucleus,
allowing accessibility to RNA polymerases throughout the chromatin.
Extremely condensed DNA molecules occur, for instance, in sperm cells, where
the DNA is compacted by basic proteins rather than structured in nucleosomes.
After fertilization these proteins are exchanged for acidic nucleoplasmin and
histones stored in the egg, resulting in a decondensation of the DNA and in the
assembly of nucleosomes (60).
In order to fit into the nucleus, the second-order level of helical folding, the
30 nm fiber, must be packed in fibers with a larger diameter. Because the
higher-order folding is very sensitive to variations in ionic conditions of nuclear
isolation buffers (3), different methods of preparation have resulted in different
concepts, e.g. that of the formation of radial loops (26, 45;) or that of further
superhelical arrangements (42). By visualizing chromosome structures both in
living cells (using optical sectioning microscopy and image deconvolution) and
in dehydrated and embedded cells (using electron microscopy), Belmont et al.
(3, 4) came to the conclusion that the 30 nm fiber must occur in threadlike
domains with a diameter of about 130 nm. Such a thread or "folded
chromonema" has been schematically depicted in the yeast nucleus of Fig. 3B.
Application of the techniques of whole-chromosome "painting" and
fluorescence in situ hybridization (FISH) support the concept that the
eukaryotic nucleus is partitioned into irregularly shaped chromosome
territories (9, 14, 32). Visualization of RNA transcription and DNA replication
has shown that these two processes occur in hundreds of different domains
scattered throughout the S-phase nucleus (25, 72, 73, 75). The chromosome
territory model predicts that in interphase interchromosomal "channels" (34, 52,
73, 83) occur between the individual chromosomes, containing RNA and
protein complexes involved in replication, transcription and RNA processing
and transport steps.
Before considering how after replication the chromatids are separated, we
will first discuss possible mechanisms used in bacteria for movement of DNA
and for nucleoid segregation.
SEPARATION OF DAUGHTER STRANDS IN BACTERIA
As previously proposed (51) and as discussed in Chapter III-2 (82), the
combined effect of supercoiling and macromolecular depletion forces lead to
DNA compaction and phase separation between nucleoid and cytoplasm (see
also ref. 71). How does the segregation mechanism displace the DNA against
this force of compaction?
During segregation, the nucleoids have been observed to move gradually
along with the elongating cell, both in normal E. coli cells and in bacterial
filaments (67, 69). This movement has been shown to be independent of DNA
replication because, upon inhibition of DNA synthesis, the nucleoids become
pulled out into small lobules dispersed throughout the filament. This dispersion
was not due to a physical fragmentation, as it was reversible; inhibition of
protein synthesis caused the small DNA regions to coalesce again into a single
region (80; see also Fig.1B, C in ref. 82). Likewise, if protein synthesis in normal
cells is inhibited either with rifampicin or with chloramphenicol, the often
lobular structure of the nucleoid is abolished and the DNA becomes confined in
smooth, sometimes spherical structures (28, 81). In the case of filaments,
already segregated nucleoids are even rapidly pushed together forming multi-
nucleoid bodies (68). Upon removal of the chloramphenicol and after recovery
of protein synthesis, the nucleoids re-segregate again (69). These observations
have led to the concept that separation of daughter strands and segregation of
nucleoids can only occur during active transcription and translation and thus
during active growth of the cell.
As a working hypothesis it has been proposed (69) that the driving force for
nucleoid movement results from the random expression of numerous genes
coding for membrane proteins. Like in eukaryotic cells, translocation of inner
membrane proteins in E. coli has been shown to require a signal recognition
particle and its receptor (39, 66). By the formation of a mRNA/ribosome/signal
recognition particle/translocon complex, these genes form DNA loops
indirectly attached to the plasma membrane as depicted in Fig. 4A (41, 57; see
for a more detailed representation of this complex Fig. 2 in ref. 81). Although
each individual loop is only formed transiently, collectively the many loops are
able to expand and move the nucleoid in the growing cell. How are loops
belonging to one daughter strand distinguished from those emanating from
the other strand? To achieve such a directionality, it is necessary that an initial
displacement occurs between the replicated origins. Once the origin regions
have separated over some distance, loops belonging to the same strand will, on
average, attach to that membrane region of the cell to where the origin has
moved. Subsequently replicated DNA regions will become pulled apart as new
loops attach to the membrane at either side of the cell (see Fig. 4A).
In sporulating B. subtilis cells it could be demonstrated that one-third of the
chromosome centered around the origin is specifically compacted in the
forespore (70), suggesting an orientation of the origin towards the cell pole.
This orientation appeared dependent on the Spo0J protein (64) which was
found to be associated with the origin and also positioned towards the cell pole
(37, 38). Similar results were obtained with a homologous protein in Caulobacter
crescentus (50). A specific positioning was also confirmed by following the
movement of the origin region in living B. subtilis cells (74) and in E. coli (16).
For visualization Green Fluorescent Protein-LacI (GFP-LacI) fusion proteins
were used that bind to an array of lacO sequences integrated near the origin.
All these observations suggest the existence of a dedicated mechanism for the
displacement of replicated DNA sequences.
Alternative to a displacement by the above mentioned specific proteins,
which are homologous to the parA and parB families of plasmid-encoded genes
(for review ref. 76), the initial displacement of the replicated origins could again
be obtained through the process of transcription. For example, in the case of
the high transcriptional activity of ribosomal RNA genes, combined with the
assembly of the ribosomal subunits, it can be envisaged that the newly
replicated daughter strands are pushed apart by the formation of a ribosome
assembly compartment as schematized in Fig. 4B (78, 80). It should be noted
that ribosomal RNA genes occur close to the origin of replication in E. coli and
in B. subtilis, but not in C. crescentus. Future studies have to elucidate the
mechanism(s) by which bacteria displace their replicated origins and give
directionality to the movement of their daughter nucleoids.
SEPARATION OF SISTER CHROMATIDS IN EUKARYOTES
In contrast to prokaryotes, eukaryotic cells have to perform two different
processes after DNA replication. The replicated sister chromatids must not only
be separated and disentangled, but they also have to be compacted, while
remaining paired for bipolar attachment to the mitotic spindle during
metaphase. Even in the yeast nucleus some mitotic condensation is necessary to
prevent the lagging ends of large chromosomes to be damaged by the process
of cytokinesis (18).
Because of the termination of many converging replication forks, and
because of the various orders of helical folding, DNA replication results in a
complex plectonemic intertwining of the daughter strands. Duplantier et al. (13)
propose that the disentanglement of the sister chromatids occurs in two steps.
First, a separation that generates two catenated daughter chromosomes.
Remarkably, this partial separation of the chromatids occurs also when the
mitotic spindle has been disrupted through treatment with colchicine (49).
Second, a pulling at the start of anaphase by the spindle, which is coupled with
and gives direction to the strand-passing reaction of topoisomerase II. In this
second step, the chromatids are pulled and moved over relatively large
distances.
It has been suggested that the driving force for an initial disentanglement of
the DNA strands could be given by the process of DNA condensation as such
(44) and that as compaction proceeds the dissolution of the sister chromatids
becomes more efficient (24). The condensation could be obtained with the help
of force generating systems, possibly involving SMC- (structural maintenance
of chromosome) proteins that act as chromatin "motors" (32) and
topoisomerase II (23). In addition, the generation of superhelical tension by
wrapping the DNA around multi-subunit protein complexes (condensin; 29),
has been suggested as a mechanism to compact the chromatin fiber. It is
interesting that SMC-proteins have a nucleotide binding motif and extended
coiled-coil domains that bear structural similarity to proteins occurring in the
cytoplasm of Salmonella enterica (TlpA; 33), E. coli (MukB; 56) and Mycoplasma
hyorhinis (P115; 58).
For the disentanglement of chromatin fibers the strand-passing activity of
topoisomerase II is essential. However, this enzyme can only be functional if it
is given a direction for strand-passing (see for a theoretical analysis ref. 27).
What mechanism can give force and direction to the activity of these enzymes
if not the traction force of the spindle? How can mere condensation of
entangled fibers lead to a dissolution of the sister chromatids? Here, an analogy
with the transcription/translation-mediated separation of daughter strands in
bacteria could suggest a possible mechanism.
The continuing process of transcription during G2 or even during S phase
and a similar phenomenon of phase separation between nucleosomal DNA and
hnRNP particles as observed in the bacterial cell (cf. Fig. 3B) and as suggested
by Cremer et al. (9), could provide such a driving force. The formation of a
domain of transcriptional activity on one replicated daughter strand and the
fusion of this domain with other transcriptional domains at the border of a
chromosome territory could result in a pulling that gives directionality to the
strand-passing activity of topoisomerases and separate local entanglements
between the daughter strands (Fig. 5A). At a later stage, the condensation of
the daughter strand could again exert a pulling force, with the hnRNP particles
locally separating the chromatids and allowing them to slide past each other
(Fig. 5B). An indication for a structural role of continuous transcriptional
activity in nuclear organization was obtained from experiments with
transcriptional inhibitors which induced a dispersion of chromosome territories
(20). The occurrence in mammalian nuclei of separate replication and
transcription sites grouped into distinct clusters has been described by Wansink
et al. (73) and Wei et al. (75). The dynamic interdigitation of these transcription
and replication domains may reflect the transition from one functional state to
the other and serve to separate and disentangle the replicated DNA strands.
CONCLUSION
In a bacterial cell chromosomal DNA occurs in a compact nucleoid region
separated from the cytoplasmic phase. During segregation, the geometrical
centers of the duplicated nucleoids have to move apart in the elongating cell
only over a distance equal to the length increase of the cell during its growth
cycle. Thus, the doubling in cell length already suffices to move the compact
nucleoids apart, assuming that they move the maximum distance. In the
eukaryotic cell, however, the nucleus does not grow along a linear axis as in a
(rod-shaped) bacterium. During S phase it grows isometrically rather than
linearly. Moreover, replicated chromosomes have to be transported within the
cell over distances that largely exceed the diameter of the nucleus. Even in the
small yeast cell, chromosomes are transported over more than 6 µm (7),
whereas this is less than 2 µm in a bacterium (Fig. 1B).
In eukaryotes partitioning of the chromosomes over the daughter cells
occurs in two steps. First the separation of replicated daughter strands,
involving both disentanglement or decatenation. Second, the transport or
movement of separated chromatids to opposite poles by the cytoskeletal
spindle apparatus in the abrupt process of mitotic segregation. In bacteria,
segregation occurs in one step in which physical strand separation occurs
continuously and gradually along with replication and cell growth. The
hierarchical mode of segregation (12) implies that soon after termination of a
round of replication, the daughter nucleoids are also structurally separated
while they can have started new rounds of replication (cf. Fig. 2B).
In the bacterial cell the process of strand separation is mediated by
transcription in combination with supercoiling and phase separation between
nucleoid and cytoplasm. Together with cell elongation, transcriptional activities
represent an expansion force which is sufficient to fully disentangle the
replicated chromosomes and to position them in the prospective daughter cells.
In the eukaryotic cell a similar phase separation between chromatin and hnRNP
particles, may help in the process of chromatin condensation, and may cause an
initial disentanglement of the sister chromatids and their partial separation over
a small distance perpendicular to the long axis of the chromatids (Fig. 5A). The
distance of this separation is comparable to that in the bacterium (Fig. 1B).
However, because eukaryotic cells are large relative to bacterial cells they
require additional mitotic transport mechanisms to fully dissolve the
chromatids and to bring them to the daughter cells.
While many microbiologists compare DNA segregation in bacteria with the
second step of eukaryotic segregation, i.e. mitosis, it should rather be
compared with the disentanglement or separation of sister chromatids
occurring during the first step. Like in bacteria, this separation could already
start soon after replication initiation (Fig. 5A) and continue throughout G2
phase until prophase when it is sustained and enhanced by chromosome
condensation (Fig. 5B). This suggests that a transcription-mediated process
could be the driving force behind chromosome separation in both prokaryotes
(Fig. 3B and 4B) and eukaryotes (Fig. 5). In both organisms therefore, the
genomic organization may help in chromosome segregation, the expression of
genes fulfilling a role in the fundamental process of daughter strand separation.
Acknowledgments
We thank N. Nanninga and J.Veuskens for critical reading of the manuscript
and H. Oud, A. Houtsmuller, J.-L. Sikorav and A.C. Fijnvandraat for helpful
discussions.
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Table 1. Comparison of the bacterial cell with the eukaryotic nucleus:
concentration of macromolecules
E. coli S. cerevisiae human cell
cell volume (µm3) 1.061) 552) 1200 (R=6.5 µm)
3)
nucleoid or nuclear volume
(µm3)
0.144) 2.5 (R=0,75 µm)
2)
65.5 (R=2.5 µm)
5)
524 (R=5 µm)3)
genome size (kbp) 4. x10313.5 x 103
(diploid)
6 x 106
(diploid)
amount of DNA per cell (pg) 0.01 0.032 6.7
DNA concentration (mg/ml)
in nucleoid 654) - -
in cell 9 - -
in nucleus - 13 1 - 1006)
in metaphase chromosome - - 1205)
1) Calculated from average cell length (2.5 µm) and diameter (0.5 µm),
assuming the shape of the cell to be a right cylinder with hemispherical polar
caps.
2) See ref. 79.
3) See refs. 1 and 43.
4) See ref. 82.
5) Calculated for the haploid human chromosome 1 in metaphase (300 MB)
using dimensions given by Manuelidis and Chen (43).
6) See ref. 6.
LEGENDS
Fig. 1. (A) Comparison of the size of the spindle apparatus in a HeLa cell and
the size of an E. coli cell grown in glucose minimal medium. (B) The
displacement of DNA in a metaphase chromosome between the sister
chromatids is comparable to that of the segregated nucleoids in E. coli. Spots
indicate tubuline or, in E. coli, the tubuline-like FtsZ proteins, which form a
ring structure prior to division (40, 53).
Fig. 2. The fundamental difference in the mode of segregation between
prokaryotes and eukaryotes becomes evident in the case of
multichromosomal cells. (A) In a schematic haploid cell the chromosome,
depicted as a black rod, is semi-conservatively replicated generating two
daughter chromosomes, depicted as a black and a dashed rod. In the
prokaryotic cell, these chromosomes are segregated into the daughter cells
as indicated. Likewise, in the eukaryotic cell, they are segregated as sister
chromatids pulled by spindle microtubuli to opposite poles. (B) In a
multichromosomal or diploid cell, replication of the two chromosomes
results in two pairs of chromosomes (chromatids), depicted schematically as
dark- and grey-coloured rods and dashed rods. In the prokaryotic cell each
pair of daughter chromosomes ends up in a prospective daughter cell (like
during eukaryotic meiosis I); these daughter chromosomes become only
segregated in the next cycle. This has been called "hierarchical segregation"
by Donachie et al. (12). In the eukaryotic cell each pair of daughter
chromosomes or sister chromatids becomes segregated into different
daughter cells, because the cohesion between chromatids and because of the
attachment of microtubuli from opposite poles to the kinetochores. If a
mutation occurs in one of the chromosomes as indicated by the white star,
the cell becomes heterozygous. As proposed by Donachie et al. (12),
hierarchical segregation does not maintain this heterozygosity, whereas
mitotic segregation does.
Fig. 3. (A) Comparison of a diploid yeast cell, its nucleus, and the diameter of a
HeLa-cell nucleus with an E. coli cell. See Table 1 for a calculation of the DNA
concentrations in these compartments. (B) 200 nm wide regions of a yeast
nucleus and an E. coli cell. The chromatin in the yeast nucleus (left panel) is
depicted as afolded 130 nm-wide thread (cf. ref. 4) formed by folding of the
30 nm-fiber (see text). Nucleosomes are depicted as 10 nm circles. The large
circles represent hnRNP particles with a diameter of 20 nm (30), involved in
co-transcriptional processing of pre-mRNA. The DNA in the E. coli nucleoid
is drawn as plectonemic supercoils with a diameter of 20 nm (see ref. 82).
Ribosomes with a diameter of about 30 nm are involved in co-transcriptional
translation.
Fig. 4. (A) Working model of transcription-mediated segregation assuming (i) a
dedicated mechanism for the initial displacement of the two replicated
origins (oriC and oriC'indicated by the round and square symbols) and (ii)
the expansion of the nucleoid by the transient attachment of DNA loops
through co-transcriptional and co-translational translocation of membrane
proteins, forming two membrane growth zones that cause cell elongation.
Black- and gray-coloured loops to the membrane represent DNA from the
two replicated daughter strands. See Fig. 2 in ref. 81 for a more detailed
representation of this attachment complex. (B) Initial displacement of the
origins could occur by the formation of a hypothetical ribosome assembly
compartment formed at ribosomal RNA genes near the unique origin of
replication. Through this co-transcriptional assembly the origins are pushed
apart (open arrows). The movement of this initial displacement is
subsequently taken over and enhanced by DNA loops pulled to the
membrane during the co-transcriptional and co-translational translocation of
membrane proteins ("transertion"; 57). This segregation mechanism is also
suitable for multifork replication. When, after reinitiation, the origins of each
pair are moved apart along the cell's length axis, the subsequently replicated
daughter strands will again generate loops that attach to the membrane,
now forming two pairs of growth zones.
Fig. 5. (A) Recently replicated sister chromatids, drawn as black- and gray-
coloured 10 nm-fibers, are locally disentangled by topoisomerases and a
pulling and pushing force exerted by transcriptional microcompartments.
These are formed around one daughter strand by activities of co-
transcriptional splicing and transport of pre-mRNA. Compare with the initial
separation of replicated oriC in a bacterium (Fig. 4B). The force is directed
perpendicularly to the long axis of the chromatids (open arrows). (B)
Condensation of the DNA in two separated (euchromatin) regions exerts a
pulling force of the chromatids (small arrows) and gives directionality for
further disentanglement by topoisomerases. Transcription may separate the
strands locally, allowing them to slide past each other to the regions of
condensation. The cohesion between sister chromatids is here drawn as the
result of residual entanglements, but could also be achieved by specific
binding proteins (cf. ref. 32).
