1. science
2. medicine
3. genetics

Mutation Research 434 Ž1999. 99–107
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Why do we have linear chromosomes? A matter of
Adam and Eve
Fuyuki Ishikawa ) , Taku Naito
Laboratory of Molecular and Cellular Assembly, Graduate School of Biological Information, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Accepted 30 March 1999
Keywords: Linear chromosome; Circular chromosome; Telomere; Meiosis
1. Introduction
It is usually assumed that prokaryotic cells have
circular chromosomes, whereas eukaryotic cells have
linear chromosomes. One of the consequences of
linear chromosomes is the presence of chromosomal
ends called telomeres. Simple physical ends of DNA,
such as those produced by DNA double-strand breaks
ŽDSB. by ionizing radiation, are genetically unstable,
mutagenic, and sometimes oncogenic Žreviewed in
Ref. w1x.. Telomeres are a complex composed of
telomeric DNA and a number of telomere-specific
and non-specific proteins. This large molecular assembly that forms the telomeres protects the genomic ends from end-to-end fusion or exonucleolytic
erosion Žreviewed in Ref. w2x..
Due to the end-replication problem, telomeric
DNA is shortened as the cell divides w3x. In most
eukaryotes, this shortening of telomeric DNA is
compensated by the activity of an enzyme called
telomerase that synthesizes telomeric DNA de novo
w4x. However, telomerase is strictly regulated to be
inactive in most human somatic cells, and telomere
lengths decline as an individual ages w5x. This results
)
Corresponding author. E-mail: [email protected]
in cellular senescence and cancer development due
to telomere insufficiencies Že.g., Ref. w6x, and reviewed in Ref. w7x.. Therefore, telomeres are sometimes referred to as the ‘the Achilles heel of the
chromosome’ w8x.
Why do we have linear chromosomes that lead to
senescence and cancers, instead of circular chromosomes? In this article, we review as to what extent
different chromosome configurations are conserved
among different kingdoms, and propose a hypothesis
to explain why this remarkable conservation has
evolved.
2. Chromosome configurations of prokaryotes and
eukaryotes
In this article, we operatively define chromosomes as genetic materials containing house-keeping
genes essential for the cell’s survival that replicate
synchronously with cell division to distinguish them
from extra-chromosomal genetic elements, such as
plasmids, bacteriophage and transposons w9x. The
recent invention of molecular biological tools to
analyze large DNA structures, especially pulse field
gel electrophoresis ŽPFGE., in addition to classical
0921-8777r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 1 - 8 7 7 7 Ž 9 9 . 0 0 0 1 7 - 8
100
F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
tools, such as genetic linkage studies, have greatly
enriched our knowledge about the chromosome configuration in different species, phyla and kingdoms.
Given the very large number of species on the earth,
both known and unknown, it is far from possible to
make a complete catalogue of the chromosome structures existing among the diverse range of species.
However, recent studies have identified the presence
of several exceptional cases to the general rule that
one circular chromosome is present in prokaryotic
cells and more than one linear chromosome are
present in eukaryotic cells. Nevertheless, these exceptional cases indeed further strengthen the notion
that prokaryotes and eukaryotes have maintained circular and linear chromosomes, respectively, throughout their evolution.
2.1. Prokaryotes
Generally, the small sizes of prokaryotic genomes
Žtypically 1–10 Mb. make it feasible to carry out
physical analyses, such as PFGE and total nucleotide
sequencing Žreviewed in Ref. w10x.. Genome analyses
of Archea have lagged behind that of eubacteria. For
example, the genome sequencing of only six
archebacteria had been completed, whereas 17 eubacteria had been sequenced at the time of this
review Žhttp:rrwww.tigr.orgrtdbrmdbrmdb.html..
All archebacteria examined so far contain one circular chromosome. Therefore, we will focus on several
exceptional cases in eubacteria, where this general
rule is not applied.
Borrelia, Treponema and Leptospira are members of the spirochete group ŽPhylum Spirochaetae..
They are pathogenic bacteria, and Borrelia causes
Lyme disease. Borrelia burgdorferi has been shown
to have one 910-kb linear chromosome w11–13x. This
unique feature is conserved in the other members of
the Borrelia genus w14x. In contrast, other two closely
related genera, Treponema and Leptospira, which
also belong to the Spirochaetae, contain circular
chromosomes w15–17x. This specific distribution of
linear chromosomes in the Borrelia genus suggests
that Borrelia’s linear chromosomes were evolutionarily derived from the ancestral circular chromosomes after the three genera had diverged.
Streptomyces is another genus that has been shown
to possess linear chromosomes w18x. It should be
noted that Streptomyces, a member of Phylum Actinomycete, is phylogenically distant from Borrelia.
The closely related genus, Mycobacterium, which is
within the same phylum, contains a conventional
single circular chromosome w19x, again suggesting
the relatively recent origin of the linear chromosome
in Streptomyces.
Finally, Agrobacterium tumefaciens, a member of
Proteobacteria has one 2.1-Mb linear and three 3Mb, 450-kb and 200-kb circular replicons. It is known
that at least the two 1-Mb and 3-Mb replicons contain metabolically essential genes, which classifies
them as chromosomes w20x.
The conventional DNA replication mechanism
does not replicate the very ends of linear DNA,
because all DNA polymerases need primers for initiating synthesis. The diverse range of linear genomes
solves this end-replication problem by different
strategies. Adenovirus initiates the replication of its
linear genome using a protein primer Žterminal protein, TP. w21x. TP forms a covalent bond with the
5X-OH of dNMP, and the DNA polymerase starts
synthesis using this base as the first nucleotide to be
incorporated. As a result, adenovirus DNA has a
covalently associated TP at its 5X-ends. On the other
hand, vaccinia virus has a hairpin structure at both
ends of its linear genome w22x. One strand is continuous to the other strand, and DNA synthesis continues
onto the next strand after completing one strand. A
palindromic sequence is left after nicking the hairpin
DNA to resolve the two daughter duplex DNAs. The
terminal structures of the linear genomic DNAs of
Borrelia and Streptomyces have also been reported.
Streptomyces has 5X-end associated proteins, suggesting that the telomeres are replicated by TP primers in
this bacterium w23,24x. In contrast, Borrelia has hairpin structures with 26-bp inverted repeats at both
telomeres, suggesting that its telomeres are replicated
in a way similar to vaccinia virus w25x.
These studies indicate that at least three prokaryotic genera possess linear chromosomes, instead of
circular ones. However, the appearance of linear
chromosomes in prokaryotes seems rather sporadic.
First, these three genera are distantly related to each
other. Second, closely related genera belonging to
the same phylum contain conventional circular chromosomes. Finally, the solutions for the end-replication problems differ between Borrelia and Strep-
F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
tomyces. Accordingly, it is suggested that prokaryote
linear chromosomes have not been inherited directly
from one ancient prokaryote that had linear chromosomes. Instead, they most likely have developed
recently from the circular chromosomes of an ancestor species. Therefore, it may be concluded that the
prokaryote genomes have been maintained phylogenically in circular forms.
2.2. Eukaryotes
Eukaryotes contain larger genomes than prokaryotes Žtypically larger than 10 Mb.. Accordingly, in
many cases, the chromosomes can be visualized by
microscopy to analyze the gross structures. However,
large chromosome sizes are a disadvantage in another respect, since it is usually difficult to construct
a physical map of the genome. As will be reviewed
here, many reports have been published showing the
presence of ‘circular chromosomes’. However, the
evidence for covalently linked circular chromosomes
is not available in most cases. To avoid possible
confusion, circular chromosomes judged solely on
morphological criteria will be called ‘ring chromosomes’ in this review.
Circular chromosomes have been reported both in
budding yeast w26,27x and fission yeast w28,29x. They
were isolated spontaneously or artificially, and have
been shown to be circular by either a genetic or a
physical approach. In each case, only one circular
chromosome was identified, and it was unstable
mitotically and meiotically.
As there are many opportunities to examine karyotypes in a variety of medical settings, many cases of
ring chromosomes have been reported to be associated with a variety of clinical manifestations. In most
cases, the ring chromosomes have been found in
somatic cells, either normal or cancerous, and either
constitutively or in mosaicism. However, few notable cases in which one ring chromosome had apparently been inherited from one of the parents who
also had the same ring chromosomes have been
reported Že.g., Ref. w30x.. In these cases, the parents
generally showed mosaic ring chromosomes, suggesting that an individual who has a ring chromosome constitutively is infertile. There has been no
report describing the inheritance of more than one
ring chromosomes. These results have suggested that
101
in some rare cases, a single ring chromosome may be
normally segregated in meiosis, and fertilized.
In summary, circular or ring chromosomes have
been found sporadically in eukaryotes. With some
rare exceptions, they are not usually inherited. However, there have been no reports describing eukaryotic cells having more than one circular or ring
chromosome that have been meiotically transmitted.
These results suggest that ring chromosomes face
some difficulty in sexual reproduction. Since mitochondrial DNAs are circular in most eukaryotes,
chromosome circularity itself is obviously not incompatible with inheritance. Circular chromosomes
may be incompatible with a process specific to sexual reproduction, such as meiosis.
This section has shown that two chromosome
configurations, circular and linear, are remarkably
conserved in prokaryotes and eukaryotes, respectively. In an evolutionary sense, linear chromosomes
require extra energy to maintain intact telomeres,
which is not a requirement for circular chromosomes. This reasoning suggests that there must be
some advantages to eukaryotes that have been acquired in a trade-off for this extra burden. One of the
most direct experiments to test this hypothesis would
be to construct a eukaryotic cell that maintains its
genome in a circular form, and to see what biological
functions this cell has lost. However, until recently,
there has been no report that describes the existence
of eukaryotic cells that maintain completely circular
genomes.
3. ATM family genes and telomeres
Telomeres are comprised of many components to
accomplish its functions. Proteins involved in telomere maintenance are now being studied in some
detail, especially in simple eukaryotic cells such as
yeast Žreviewed in Ref. w31x.. One group of interesting proteins thus identified is the ATM family. The
Saccharomyces cereÕisiae TEL1 gene was originally
identified by screening for mutants with short telomere phenotypes w32x. When this gene was cloned, it
turned out to have a significant level of homology
with the human ATM gene, whose mutations cause
the hereditary disease, ataxia telangiectasia ŽA-T.
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F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
w33,34x. Interestingly, telomeres in A-T cells also
showed excessive shortening w35,36x, suggesting that
the ATM family genes are involved in telomere
maintenance in all cells from yeast to human. There
is at least more than one member of the ATM family
genes present in one species w37x. The budding yeast
Saccharomyces has TEL1 and MEC1 genes, and
human has ATM, ATR and the relatively distant
member DNA-PK. All these proteins have a PI3
Žphosphatidylinositol 3.-kinase-like domain at the
C-termini, yet they have protein kinase activity. Fission yeast, Schizosaccharomyces pombe, also has
two ATM family genes, named rad3q and tel1q
w38,39x. The mutant defective for rad3q or tel1q
shows moderate to minimal telomere shortenings
w39,40x. However, when both ATM family genes
were mutated, there was an additive effect, and the
fission yeast chromosomes essentially lost all telomere sequences w39x. Thus, the ATM family genes are
redundant but essential for stable telomere maintenance. The rad3 tel1 double mutant cells grow very
slowly with a low viability, and showed aberrantly
irregular colony shapes, as expected for cells suffering from extensive telomere shortening. However,
derivative cells that showed apparently normal colony
shapes appeared spontaneously among these double
mutants at a relatively high frequency. Surprisingly,
these derivative cells contain three self-circularized
chromosomes Žfission yeast contains three linear
chromosomes. w39x. This case is the first report
describing the existence of eukaryotic cells that
maintain the genome exclusively in circular forms.
plete circular genomes in yeast should be highly
unstable and lead to cell death. Indeed, the fission
yeast rad3 tel1 mutant with the three circular chromosomes showed anaphase bridges and some degree
of aneuploidy ŽNaito and Ishikawa, unpublished..
However, the fact that this mutant grew well mitotically as a mass suggests that the SCE of dicentric
circular chromosomes may have a relatively small
effect. Indeed, SCE happens in prokaryotic cells to
produce circular dimers. In Escherichia coli, these
circular dimers are known to be resolved by both the
recA-independent resolvases, XerC and XerD, that
act on a specific locus called dif located at the
replication terminus, and the recA-dependent recombination pathway Žreviewed in Refs. w43,44x.. In
higher eukaryotes, genomic DNA is organized into
multiple loops by tight association of matrix-associated region ŽMAR. on DNA with nuclear scaffolds.
In a topological sense, each loop can be assumed
microscopically to be a closed circle. Therefore,
closed circular oligomers may also be formed by
SCE in linear eukaryotic genomes, making the hypothesis that the circular dimer formation prohibits
circular chromosomes in eukaryotes unlikely. Eukaryotes may have mechanisms similar to XerCD
and recA-dependent recombination in E. coli to
resolve these microscopic oligomers. Indeed,
RAD51-deficient chicken cells, the eukaryote recAhomologue, are shown to be arrested in G2rM phase
and to accumulate chromosome breaks, suggesting
the possible involvement of the Rad51 protein in
resolving SCE-intermediates w45x.
4. Sister chromatid exchanges (SCEs) and circular dimer formation
5. Meiosis and telomeres
Previously, it had been postulated that eukaryotic
cells do not have circular chromosomes because if an
odd number of crossing-over events occurs between
the two sister chromatids by SCE, this would result
in the formation of dicentric circular chromosomes.
Thus, formed circular dimer chromosomes would be
eventually broken during mitosis by the two spindles
pulling them apart Že.g., see discussions in Refs.
w41,42x.. Since yeasts undergo efficient homologous
recombination and SCEs, we would expect that com-
Telomeres perform a number of important functions in different biological situations. Recently, the
role of the telomeres in meiosis has come into light
Žreviewed in Ref. w46x.. The first hint came from the
cytological observation that telomeres are closely
clustered with each other at a specific stage of
meiosis. There have been ample observations that
telomeres and centromeres are positioned asymmetrically in nuclei Žreviewed in Ref. w47x.. In mitotic
interphase, the centromeres have a tendency to cluster around the centriole, presumably reflecting the
F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
association between these two structures during the
last anaphase. This centromere clustering is named
the Rabl w48x orientation after the German scientist
who first described it. However, during meiotic
prophase, this relative distribution of centromeres
and telomeres is reversed: In the leptotenerzygotene
stage, telomeres, instead of centromeres, are clustered at the inner surface of the nuclear envelope.
The centromeres are distributed randomly in the
nucleus at this stage. This polarized chromosomal
distribution is called the bouquet arrangement w49x,
and is found during meiosis in many species Žreviewed in Ref. w50x.. The functional significance of
this peculiar conformation has been recently revealed. In fission yeast, two haploid cells of opposite
mating types conjugate to produce diploid cells
Žkaryogamy., and enter meiosis Žzygotic meiosis..
Immediately after karyogamy, the fused nucleus
forms an elongated shape, called a horse-tail w51x.
Using time-elapsed image recording, the fused
horse-tail nuclei were found to undergo a dynamic
to-and-fro oscillating movement w52x. Moreover, by a
combination of telomere-specific fluorescence in situ
hybridization ŽFISH. and immunostaining of the
spindle pole body ŽSPB, a centriole-counterpart in
yeast., it was shown that six telomeres of the three
fission yeast chromosomes are closely associated
with SPB, and they lead the front edge of this
horse-tail movement. In fission yeast, the bouquet
arrangement is established by an association between
the telomeres and SPB. In this way, the microtubule
enucleated from the SPB promotes the dynamic nuclear movement by pulling the telomeres and dragging the chromosomes behind as a mass Žreviewed in
Ref. w53x..
Recently, three additional genetic studies have
further indicated the importance of telomeres in the
meiotic process. Telomere DNA consists of wellconserved G-rich simple tandem repeats. Telomere
DNA-specific binding proteins are known to exist in
several species. These include TRF1 and TRF2 in
human w54x, Rap1 in budding yeast w55x, and taz1p in
fission yeast w56x. Fission yeast mutants defective for
taz1q failed to form the telomere clustering at the
horse-tail stage of pre-meiosis, and showed reduced
spore viability w57,58x. Taz1p is presumably involved
in the SPB-telomere association w53x. The rad3 tel1
fission mutant with the three circular chromosomes
103
was examined for spore viability after azygotic
meiosis w39x. In this case, diploid cells derived from
two haploid cells harboring circular chromosomes
produced no viable spores, a phenotype more profound than that of the taz1 mutant. These studies
clearly indicated that telomeres are essential for a
productive meiotic process.
6. How telomeres are essential for meiosis
Several scenarios can be proposed to explain why
functional telomeres are essential for meiosis. Meiosis consists of two successive cell divisions, called
meiosis I and meiosis II. Meiosis II is similar to
mitotic cell division, but meiosis I is unique. Meiosis
is a process that produces four haploid cells from
one diploid cell. Meiosis I is responsible for this
reduction of ploidy by segregating the two homologous chromosomes to the two daughter cells. To
accomplish this reductional segregation, each pair of
homologous chromosomes needs to be paired before
the onset of meiosis I Žreviewed in Ref. w59x.. Homologous chromosome pairing has another important
role in inducing homologous recombination between
the two homologues. This homologous recombination shuffles the two alleles originally derived from
different individuals Žfather and mother., and ensures
that the haploid cells contain chimeric genetic information. Moreover, the recombination and segregation are interdependent, since the covalent associations formed by the recombinational Holiday junction between the two homologues Žchiasmata. are
thought to be essential for stable homologue pairing,
and ensuring proper segregation. Therefore, homologue-pairing is at the heart of the mechanism of
meiosis.
Circular chromosomes potentially undergo more
than one pathway during meiosis, and in all cases,
they have very small probabilities of proper segregation ŽFig. 1.. In normal meiotic prophase, linear
chromosomes gather together by telomere-clustering
ŽFig. 1A.. Telomere associations of homologous
chromosomes may help the homologue pairing by
aligning the two chromosomes that are now tethered
at both ends. After successful pairing, homologous
recombination occurs between the two homologues,
104
F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
Fig. 1. Meiosis I of linear chromosomes ŽA. and possible pathways in meiosis I of circular chromosomes ŽB.. For details, see the text.
F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
and this covalent association further contributes to
the stable chromosome pairing. In anaphase I, the
Holiday junctions are resolved and the two homologues are segregated to different daughter cells. In
contrast, circular chromosomes may undergo several
different pathways ŽFig. 1B.. As circular chromosomes lack functional telomeres, two homologues
cannot be positioned in proximity. In this case, no
homologue pairing and recombination occur, and the
homologues are randomly segregated to daughter
cells Žpathway 1.. Two homologues may be positioned closely by chance, and somehow may pair and
undergo recombination Žpathway 2.. However, if an
odd number of crossing-over events occurs between
two homologues, this results in the formation of
dicentric circular chromosomes Žpathway 3.. If resolvase fails to resolve this form into monomers, the
dicentric circle enters anaphase. When the spindles
of different origins attach to each of the two kinetochores, the chromosome will be pulled apart and tear
Žpathway 4.. When a common spindle attaches to
both of the two kinetochores, the chromosome is
segregated to only one cell, with the other cell
receiving no homologue. In either case, daughter
cells will lose a significant amount of genetic information. When an even number of crossing-over
events occurs Žpathway 7., or the dicentric circles are
resolved into monomeric circles Žpathway 6., the two
homologues may be segregated properly to the two
daughter cells. However, even in this case, it is not
known if spindles correctly attach to the kinetochores of chromosomes that have not been associated with telomeres during the meiotic prophase.
Overall, the chance that one particular circular chromosome is segregated properly in meiosis I is very
small. All eukaryotes contain more than one and
usually many chromosomes. The chance, that one
daughter cell will have all chromosomes properly
segregated in circular forms, is the multiple of these
small probabilities for each circular chromosome,
and should be negligible. In conclusion, there is
essentially no chance that all circular chromosomes
are properly segregated during meiosis. This essential role of telomeres in accomplishing reductional
chromosome segregation in meiosis must be the
major reason that linear chromosomes are strikingly
conserved in eukaryotes, which are characterized by
the presence of sexual reproduction in most cases.
105
7. Conclusion
We have stated that linear chromosomes are essential for productive meiosis. Meiosis Ža mechanism
to generate haploid cells. is a prerequisite for shuffling the genetic information between individuals. It
has been proposed that the production of genetically
diverse offspring is advantageous in an ever-changing or saturated environment w60x. Indeed, the number of absolutely asexual eukaryotic organisms is
very small Žreviewed in Refs. w61,62x., and the conservation of the potential of sexual reproduction
seems to be as strong as the conservation of linear
chromosomes in eukaryotes. Recent studies have
indicated that chromosome linearity is important for
meiosis, and we would like to propose that the
correlation between sex and linear chromosomes is
based on a mechanistic reason, and not on a superficial parallelism.
Once upon a time, two groups of living creatures
emerged from a common ancestor. One group decided to maintain genomes in circular forms, because
this form is more economical without the need to
maintain telomeres. However, the progeny of this
group Ž Bacteria. is not able to exchange genomic
information by meiosis and fertilization, and thus,
needs to grow faster and keep the genome size as
small as possible. The other group decided to maintain the genomes in linear forms. Although this
strategy requires extra energy to maintain telomeres,
these organisms have enjoyed the dynamic flow of
genomic information by sexual reproduction. This
process has allowed this group Ž Eukaryota. the
chance to produce a variety of offspring. Accordingly, eukaryotes have complicated systems, and
grow less rapidly than prokaryotes.
Acknowledgements
We thank E.A. Kamei ŽGunma University. and H.
Niki ŽKumamoto University. for critical reading of
and comments on the manuscript. The excellent secretarial works of F. Nishizaki, K. Saito and K.
Yokoyama are acknowledged. This work was supported by a grant-in-aid from the Organization for
Pharmaceutical Safety and Research, Japan.
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F. Ishikawa, T. Naitor Mutation Research 434 (1999) 99–107
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