Journal of Experimental Botany, Vol. 54, No. 380,
Plant Reproductive Biology Special Issue, pp. 11±23, January 2003
DOI: 10.1093/jxb/erg042
Meiosis, recombination and chromosomes: a review of
gene isolation and ¯uorescent in situ hybridization data in
plants
Trude Schwarzacher1
1
Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
Received 1 July 2002; Accepted 17 September 2002
Abstract
Evidence is now increasing that many functions and
processes of meiotic genes are similar in yeast and
higher eukaryotes. However, there are signi®cant
differences and, most notably, yeast has considerably higher recombination frequencies than higher
eukaryotes, different cross-over interference and
possibly more than one pathway for recombination,
one late and one early. Other signi®cant events are
the timing of double-strand breaks (induced by
Spo11) that could be either cause or consequence of
homologous chromosome synapsis and SC formation
depending on the organisms, yeast plants and mammals
versus Drosophila melanogaster and Caenorhabditis
elegans. Many plant homologues and heterologues to
meiotic genes of yeast and other organisms have now
been isolated, in particular in Arabidopsis thaliana,
showing that overall recombination genes are very
conserved while synaptonemal complex and cohesion
proteins are not. In addition to the importance of unravelling the meiotic processes by gene discovery, this
review discusses the signi®cance of chromatin packaging, genome organization, and distribution of speci®c repeated DNA sequences for homologous
chromosome cognition and pairing, and the distribution of recombination events along the chromosomes.
Key words: Centromere, chiasmata, cross-overs, genome
organization, homologous chromosome pairing, repeated
DNA sequences, synaptonemal complex.
Introduction
Meiosis, the unique and essential part of the life cycle of all
sexually reproducing organisms, is the process by which a
1
Fax: +44 (0)116 252 3330. E-mail: [email protected]
ã Society for Experimental Biology 2003
diploid cell of the sporophyte gives rise to haploid cells
which develop further to the gametophyte and gametes (for
review see John, 1990). It involves two divisions that are
linked together without any further DNA replication.
While the second division resembles mitosis and segregates sister chromatids, the ®rst division is unique
involving the pairing of homologous chromosomes and
their subsequent segregation (see Armstrong and Jones,
2003). This process is accompanied by the normally
random disjunction of parental chromosomes and recombination of chromosomes and genes so the haploid cells
have different and new combinations from those of the
organism's parents giving rise to much of nature's
diversity. Between different organisms, there are many
remarkably conserved features of meiosis while at the same
time showing striking differences; however, all involve the
following most critical events of meiosis: (1) initiation; (2)
cognition and pairing; (3) synapsis and the synaptonemal
complex; (4) recombination; (5) cohesion and segregation;
and (6) tetrad formation and gamete development.
This review discusses some aspects of these important
events and shows how gene discovery, in combination with
cell biological observations, can lead to a comprehensive
picture of meiosis.
Meiotic genes and gene isolation
Meiotic gene discovery has been led by studies in budding
yeast, Saccharomyces cerevisiae, over the past decades,
but with the use of insertional mutagenesis and the
availability of whole genome DNA sequences, knock-out
mutants and differential screening, many homologues to
yeast genes have now been identi®ed in other organisms
including Schizosaccharomyces pombe, Caenorhabditis
elegans, Drosophila melanogaster, Arabidopsis thaliana,
mouse, and man. About 200 genes speci®c to meiosis and
12 Schwarzacher
gamete formation had been identi®ed by classical methods
by 2000 (Primig et al., 2000). Probably as many as 1500
genes show altered gene expression as analysed by
microarrays (Chu et al., 1998; Primig et al., 2000;
Andrews et al., 2000; Reinke et al., 2000) estimating the
total of core genes speci®c for meiosis at 300 and those
speci®c for sporulation/gametogenesis at 600.
Microarray analyses study the expression of many
thousands of genes simultaneously and semi-quantitatively, giving information not only about genes that are
induced during certain processes, developmental stages or
disease, but also those that are repressed. However, in
many cases only a global picture results and only genes
with signi®cant expression changes can be identi®ed.
Microarrays with all known yeast ESTs have been used to
compare expression pro®les between different yeast
strains, growth conditions and times during sporulation
(Chu et al., 1998; Primig et al., 2000): a large category of
genes identi®ed in the meiotically induced yeast cells were
stress and metabolism-related genes because of the
response to growth medium changes. Nevertheless, distinct temporal patterns of induction could be identi®ed, and
the transcript pro®les correlated with the distribution of
de®ned meiotic promoter elements and with the time of
known gene functions.
Microarray hybridization generates huge amounts of
data about expression pro®les, and with its comparative
and high throughput and exploitation capabilities this
approach has revolutionized genomic analysis, but it rarely
assigns function to individual, hitherto unknown, ESTs or
genes, and ®nding these functions will be a massively
dif®cult task. Rabitsch et al. (2001) have recently deleted
301 unknown ORFs in yeast that were preferentially
expressed in meiotic cells according to published microarray gene expression data (Chu et al., 1998; Primig et al.,
2000): of these, 33 genes showed a meiotic phenotype
(three no replication, eight chromosome mis-segregation,
four formation of abnormal asci, 15 no spore and ascus
formation), 15 genes were essential for vegetative growth,
while 253 genes showed no apparent phenotype illustrating
the problem at hand.
Recombination
Many genes have now been identi®ed that are important
for meiotic recombination and establishing their function,
interaction and timing enables the building of a comprehensive picture of the meiotic recombination pathway (for
a review see Lichten, 2001; Keeney, 2001; Villeneuve and
Hillers, 2001). In general, recombination genes are highly
conserved and direct homologues are found in diverse
organisms, although some variation of function is possible;
Table 1 lists plant homologues of some of the most
important genes and gives a brief summary of their
function. More detailed descriptions of these plant genes
and comparisons of their function can be found in recent
review articles by Bhatt et al. (2001) and Anderson and
Stack (2002).
There is now no doubt that recombination is initiated by
the enzyme Spo11 which generates double-strand breaks
(DSBs) in yeast and possibly in most if not all organisms
(see Lichten, 2001; Keeney, 2001). This is evident from
the isolation of SPO11 homologues in most organisms that
have genome projects or are commonly used to study
meiosis: for example, A. thaliana (Table 1), mouse, man,
D. melanogaster, and C. elegans. Borde et al. (2000) have
shown elegantly that DNA replication is needed for DSBs
and suggest that this causal link functions as a safety check
to ensure that breakage is not induced before sister
chromatids are available for repair, in case corresponding
sequences on homologous chromosomes cannot be found.
Further, DNA replication in pre-meiotic S-phase is
important for Rec-8 dependent processes and the establishment of meiotic cohesin complexes (Watanabe et al.,
2001).
The Spo11 protein has homology to Top6A, the
catalytic subunit of an archaebacterial type 2 topoisomerase (Keeney, 2001; Villeneuve and Hillers, 2001) and is
linked covalently to the 5¢ ends of the double-stand breaks
forming a Spo11p-DNA intermediate. Spo11p is then
removed and 3¢OH single strand tails are formed by the
Mre11±Rad50±XrS2 protein complex (Lichten, 2001).
The action of Spo11 is possibly controlled region-byregion and tied to the establishment of the higher-order
chromosome structure needed for DSBs (Borde et al.,
2000; Baudat and Keeney, 2001). In A. thaliana two
further Spo11 genes are found, AtSPO11-2 and -3 (Table 1)
that interact with a subunit B of archaebacterial-type
topoisomerase 6 found in plants, but not in other
eukaryotes (Hartung and Puchta, 2001).
Despite the high sequence homology between Spo11
genes from different species, two distinct patterns of
function are emerging (for review see Lichten, 2001;
Villeneuve and Hillers, 2001). DSBs are found in mutants
without SC formation in yeast, and spo11 mutants do not
have SCs, indicating that DSBs are not only initiated
before SC formation but are required for homology search
and SC formation. This is also the case in mouse and
Arabidopsis. By contrast, in D. melanogaster and
C. elegans pairing centres initiate synapsis and doublestrand breaks follow, indicating that DSBs are not needed
for SC formation in these species. These differences raise
the issue as to whether ¯ies and nematode worms have
evolved recombination mechanisms that differ substantially from those of yeast, mouse and man.
The recombination pathway in yeast continues as the
generated single strand ends invade homologous sequences and the missing strands are resynthesized
(Keeney, 2001). Homologues and orthologues of the
yeast genes DMC1, RAD51, RAD 54, and MND1, and of
Table 1. Plant homologues and heterologues of meiotic recombination, cohesion and synaptonemal complex genes
Yeast or
mammalian gene
Plant homologue or
orthologue
Species
Description
Function
Reference
SPO11
AtSPO11-1
A. thaliana
Forms double-strand breaks
Hartung and Puchta, 2000
AtSPO11-2 and -3
A. thaliana
Corresponds to Spo-11
gene of others
New in plants
Interact with a subunit B of archaebacterial
topoisomerase 6 not found in other eukaryotes
Hartung and Puchta, 2001
Grelon et al., 2001
MRE11
XRS2
RAD50
AtMRE11
±
AtRAD50
A. thaliana
A. thaliana
Single copy
Not identi®ed in plants yet
Single copy
MRE11, XRS2 and RAD50 form a complex that
processes DSBs to yield 3¢overhanging ends and
possibly alters chromatin structure for DSBs
Hartung and Puchta, 1999
Gallego et al., 2001
DMC1
AtDMC1
A. thaliana
Two gene family in plants
LIM15
OsDMC1
RiLIM15A and B
Lillium longi¯orum
Oryza sativa
Oryza sativa
Meiotic homologue to Rad 51
Conversion of DSBs with DNA
tails into hybrid joint molecules.
Associated with early recombination
nodules
HvDMC1
Zea mays
Hordeum vulgare
Klimyuk et al., 1997
Couteau et al., 1999
Kobayashi et al., 1993
Ding et al., 2001
Shimazu et al., 2001
Kathiresan et al., 2002
Franklin et al., 1999
Klimyuk et al., 2000
A. thaliana MND1
A. thaliana
A. thaliana
O. sativa
Related to yeast sequence
Two subunits isolated
(three subunits have been
found in human,
¯y and fungi)
Involved in strand invasion
DNA-single stand binding protein.
Responsible for DNA replication,
repair and recombination. Component
of early recombination nodules
Gerton and DeRisi, 2002
Ishibashi et al., 2001
Co-localizes with RPA. Activates a cell
cycle checkpoint/DNA repair pathway
after mitotic and meiotic DNA damage
Component of EN and LN
Garcia et al., 2000
MND1
RPA
ATM
RECQ HELICASES
Six homologues,
some meiotic
Active in DNA replication and
recombination at later stages of DSBs
repair, possibly associated with branch
migration at the Holliday junction.
Associated with the SC and co-localizes
with Rad51/Dmc1 and Rpa loci
Hartung et al., 2000
MSH4, MSH5
MSH2, MSH3,
MSH6, MSH7
A. thaliana
MutS-related mismatch
repair family
Active in later steps of recombination and
promotes the formation of cross-overs and
are required for cross-over interference
Culligan and Hays, 1997
Culligan and Hays, 2000
MLH1
AtMLH1
A. thaliana
MutL homologue, single
copy
Interacts with Mlh3 to promote cross-over.
Part of LNs, foci correspond in number to
cross-overs
Jean et al., 1999
REC8/RAD21
DIF1/SYN1
A. thaliana
Small gene family of
cohesins
Shows low sequence homology
to yeast genes
Required for chromosome condensation and
sister chromatid cohesion at mitosis;
chromosome condensation and pairing
at meiosis
Bhatt et al., 1999
Bai et al., 1999
Dong et al., 2001
HOP1
ASY1 and 2
A. thaliana
Similarities at the amino
acid level
Asy1 is associated with chromatin in close
proximity to the axial/lateral element of the SC
Caryl et al., 2000
Armstrong et al., 2002
Meiosis, recombination and chromosomes
A. thaliana
13
14 Schwarzacher
the mammalian genes RPA and ATM, involved in these
processes have been identi®ed in plants (Table 1) with high
conservation in possible gene structure and function.
Dmc1, the meiotic homologue of Rad51, is important in
early recombination events in eukaryotes by converting
double-strand breaks into hybrid joint molecules. Dmc1
and Rad51 are homologous to RecA, the major protein that
catalyses homologous pairing and DNA strand exchange in
prokaryotes (Bishop, 1994). It has been isolated as cDNA
and genomic clones from many plants (Table 1) and is a
large gene, 8±11 kb with 12±15 exons in A. thaliana and
H. vulgare (Klimyuk et al., 2000; Klimyuk and Jones,
1997) and shows not only meiotic, but also mitotic
expression in several plants, possibly with differential
splicing (Shimazu et al., 2001).
Rad51 proteins were found to form discrete nuclear foci
from early zygotene to pachytene, to co-localize with
lateral element proteins in yeast, mouse, rat, human, and
lily meiosis (Barlow et al., 1997; Bishop, 1994; Terasawa
et al., 1995) and are components of early recombination
Meiosis, recombination and chromosomes
nodules (Anderson et al., 1997; Tarsounas et al., 1999).
Rad51 has also been proposed to have a role in the
homology search phase of chromosome pairing (Franklin
et al., 1999). Using antibodies against Rad51, in early
meiotic prophase nuclei from rye (Fig. 1a) distinct foci are
seen that are localized near the DAPI positive subtelomeric
heterochromatin indicating that the initiation of recombination and DNA sequence homology testing is concentrated near the ends of rye chromosomes.
Once DSBs have been repaired, double Holliday
junctions form and can be resolved as either reciprocal
recombination (cross-over) or gene conversion events
(non-cross-over). It is now speculated that yeast has two
pathways of recombination (Gilbertson and Stahl, 1996;
Borts et al., 2000; Allers and Lichten, 2001; Hunter and
Kleckner, 2001; Villeneuve and Hillers, 2001): cross-overs
are formed by resolution of Holliday junction intermediates, dependent on Dmc1, while non-cross-overs are
resolved by a second early mechanism of synthesisdependent strand annealing. MSh4/5 mediated cross-over
resolution generates interference when Type II intermediates are programmed in an SC-dependent manner.
In summary, many genes responsible for recombination
have been identi®ed and their interaction and timing are
now well known. Most genes are highly conserved and
direct homologues have been found in different organisms
increasing the likelihood of evolutionarily conserved
recombination machinery. However, some variation of
function and multiple pathways seem possible.
Synaptonemal complex
The SC is a tripartite proteinaceous structure that forms
between homologous chromosomes as they synapse during
15
zygotene (for reviews see von Wettstein et al., 1984;
Gillies, 1985; Moens and Pearlman, 1988). Before pairing
at leptotene, each single chromosome develops a proteinaceous axial core, or axial element, along its entire
length to which the two sister chromatids are attached in a
series of loops. When homologous chromosomes synapse
these axial elements come together to become the lateral
elements of the SC. Perpendicular, thin transverse ®laments traverse the gap between the lateral elements. The
width of the gap varies slightly between different species,
usually being somewhere between 100±300 nm. Lying
parallel between the lateral elements is the central element.
This, together with the lateral elements, makes up the
tripartite structure of the synaptonemal complex.
Early nodules (ENs), darkly stained ellipsoid bodies
along the central element (Fig. 1b) are found at many axial
element convergence sites where homologous chromosomes initiate synapsis (Albini and Jones, 1987; Anderson
and Stack, 2002). ENs are thought to be involved in
recognition and alignment of homologous chromosomes
and in early events of recombination as they are the sites of
recombination-related and homology searching proteins,
such as Rad51-p/Dmc1-p, Rap-p and Atm-p (Anderson
et al., 1997; Tarsounas et al., 1999; Anderson and Stack,
2002; Table 1). Most ENs are lost from the SCs by midpachytene and only those that are involved in reciprocal
recombination events remain. They are now called late
nodules (LNs) and have been shown to correspond in
number and locations to cross-over events.
The synaptonemal complex is found in most eukaryotic
organisms and shows remarkable morphological and
structural conservation (Moens and Pearlman, 1988).
Because of this universality and because some organisms
and many mutants lacking SC formation do not have
Fig. 1. (a) Immunocytochenistry of the Rad51 antibody from tomato to meiotic nuclei from a rye anther. Distinct foci are detected close to the
DAPI positive subtelomeric heterochromatin that clusters at zygotene. Magni®cation: 10003. (b) Electron micrograph of a thin transverse section
of a wheat meiocyte nucleus at zygotene. Darkly stained chromatin is arranged at either side of the synaptonemal complex. The central element is
visible running in the middle of the SC and shows an ellipsoidal early recombination nodule (arrow). Magni®cation: 100 0003. (c) Karyotype of
rye (2n=14) metaphase chromosomes after DAPI staining (cyan, top row) and in situ hybridization with subtelomeric repeated DNA clones
(overlay of all signals underneath), The metaphase was ®rst probed with pSc200 (labelled with biotin and detected with Cy3, red) and pSc250
(labelled with digoxigenin and detected with FITC, green), and then reprobed after photography with pSc119.2 (labelled with biotin detected with
Cy3, displayed in blue). For description of clones see Vershinin et al. (1995). Magni®cation 20003. (d, e) Localization of the major 180 bp
centromeric repetitive sequence to metaphase and interphase chromosomes of A. thaliana stained with DAPI (blue). In situ hybridization with the
cloned sequence, Atcon, shows approximately equal strength of hybridization (red) to all ten centromeres (d) while in situ primer extension
(PRINS) using short oligonucleotides to variants of AtCon shows different signal strengths indicating that that the variants differ in abundance (e).
For details of Atcon and PRINS results see Heslop-Harrison et al. (1999). Magni®cation: 50003. (f, g, h) Chromosomal organization of tandem
satellite repeats (green) and simple sequence repeats (SSRs, red) in rye. SSRs are clustered at intercalary positions giving diagnostic distribution
patterns (Cuadrado and Schwarzacher, 1998) and are generally not found in the subtelomeric heterochromatin that contains the long tandemly
repeated DNA families (f). In situ hybridization to surface spread synaptonemal complex preparations shows the contrasting organization of
satellite repeats in tight clusters (g) and SSRs in extended rows of signal (h, compare signal indicated by arrows). The SC was stained with silver
and is depicted in white. Magni®cation: 20003 (f) and 10003 (g, h). (i) In situ hybridization with two subtelomeric repeat families (red and
green, overlay yellow) to metaphase I chromosomes of rye stained with DAPI (blue) showing chiasmata close to the telomeres. Magni®cation:
25003. (j, left) Model of rye chromosome 1R showing the location of the major genomic components: tandem satellite repeats: red pSc200,
yellow pSc250, blue pSc119.2, and green 45s rDNA; pink SSRs, white centromere, cyan telomere, and brown retroelements. (j, right) In situ
hybridization with two subtelomeric repeat families (red and green, overlay yellow) to an interphase of rye stained showing clustering of
telomeres at one end of the nucleus that shows less DAPI staining indicative of the more decondensed chromatin in this region of the nucleus.
Magni®cation: 20003. (k) Genomic in situ hybridization to detect chromosome 1RS (red) at pachytene of a wheat±rye line. The distance between
the subtelomeric heterochromatin visible as a large knot and 45s rDNA site (white) is larger than at metaphase (compare with the model of j).
Magni®cation: 10003.
16 Schwarzacher
recombination, the SC has long been viewed as being
central and essential for initiating and mediating recombination. However, recombination has now been shown to be
initiated well before the formation of the SCs (see above)
and there are a few cases where reciprocal recombination
occurs without the presence of an SC, for example,
S. pombe and Aspergillus nidulans. Accordingly, the
importance of the SC has changed to re¯ect its role in
the maturation of cross-over events into chiasmata, crossover interference and chromatin cohesion, all events that
happen later in meiotic prophase (Moens, 1994; Heyting,
1996; Zickler and Kleckner, 1999). Despite the conserved
nature of the synaptonemal complex, only few components
and genes have been isolated: in yeast, Zip1 and Red1, are
associated with the central element and transversal
®laments, and Hop1, is associated with the lateral elements
(Zickler and Kleckner, 1999) corresponding to the mammalian proteins Scp1, Scp2 and Scp3 (Heyting, 1996). In
plants, Asy1 and 2 show some similarities to Hop1, but
might have different speci®cities (Caryl et al., 2000;
Armstrong et al., 2002; Table 1). Generally, SC proteins
show remarkably low conservation and sequence homology; anti-bodies against SC components of related species
have shown limited and variable cross-reaction, and no
cross-reactions have been reported between vertebrates,
invertebrates, plants, and fungi (Moens, 1993). Similarly,
cohesin proteins, responsible for the cohesion between
sister chromatids and essential for the bipolar attachment
of bivalents to the spindle that promotes proper disjunction
(Nasmyth, 2001), have generally shown low levels of
conservation.
Homologous chromosome pairing
Fluorescent in situ hybridization allows direct observation
of the behaviour of individual chromosomes during
interphase (Lichter et al., 1988; Schwarzacher et al.,
1989, 1992a); and the technology has recently been
applied to studies of early meiotic prophase from yeast
to plants and mammals, starting to assemble a comprehensive although contrasting picture of chromosome
behaviour immediately before and at meiotic prophase.
Comparing different models, it is becoming clear that they
are not necessarily contradictory, but that different organisms exploit different mechanisms and that pairing is a
multi-step and often multi-path process. A consequence is
that compensation occurs when one route is blocked,
making mutant approaches to gene discovery dif®cult.
In budding yeast, it has been observed that some
homologues are paired, possibly via multiple interstitial
interactions involving unstable side-by-side joints between
intact DNA duplexes. These interactions occur at multiple
sites along each chromosome pair in premeiotic and
probably also in vegetative cells, implying that homologues align prior to synapsis (Loidl et al., 1994; Roeder,
1997; Zickler and Kleckner, 1998). Work on spo11
mutants in yeast now has shown that double-strand breaks
are required for homology search and SC formation (Allers
and Lichten, 2001; and see above), but whether DSBs
actually promote homology search and the exact timing are
still debated.
In man, Scherthan et al. (1996) postulated movements of
centromeres and then telomeres to the nuclear envelope
and subsequent bouquet formation as conserved motifs of
the pairing process. At the onset of meiotic prophase, at
leptotene, the compact and separate chromosome territories developed into long thin threads. Subsequently,
telomeres moved towards the clustered site and produced
numerous encounters among the now elongated chromosomes that are suggested to contribute to homology testing
at exposed pairing sites. Dawe et al. (1994) and Bass et al.
(1997) demonstrated that the homologous chromosomes of
maize, similar to mouse and man, are apart when entering
meiosis, but undergo a dramatic structural reorganization
prior to synapsis at zygotene. Telomeres are localized
peripherally, and cluster de novo before the initiation of
pairing.
In the large cereal genomes, such as wheat (17 000
Mbp), homologous chromosomes associate during the
interphase before leptotene. Total genomic DNA from rye,
used as a probe for in situ hybridization identi®ed the rye
chromosome arm in a wheat±rye translocation line
(T5AS´5RL) at meiotic prophase and the preceding
interphase (Schwarzacher, 1997): three stages of pairing
were identi®ed. First, cognition occurs during the interphase before leptotene bringing the homologous chromosome domains into close proximity, apparently starting at
the centromeres. Secondly, the chromosomes are organized into the meiotic chromosome `threads', as recognized
in conventionally stained preparations, and the homologous sequences align during leptotene and early
zygotene. The third step of pairing, is the synapsis of the
homologous chromosomes and the formation of the
tripartite SC during zygotene after further alignment of
homologous sequences. In many cases, the telomeric
region was among the ®rst to synapse, while the middle of
the rye chromosome arm synapsed last, often with several
small interstitial non-paired sites, con®rming earlier studies using SC surface spreading in rye, wheat and other
plants (Albini and Jones, 1987; Gillies, 1985; Jenkins and
White, 1990).
Aragon-Alcaide et al. (1997) and Mikhailova et al.
(1998) have also demonstrated that homologous chromosomes associate before leptotene and that centromeres are
clustered at that stage. In lines with deletions of the
homologous paring gene Ph1, centromere morphology is
altered and centromeres fail to associate indicating that
Ph1 in¯uences chromatin organization at the interphase
before meiosis (Martinez-Perez et al., 1999; Mikhailova
et al., 1998; and see also below).
Meiosis, recombination and chromosomes
Homology recognition
While pairing models are now well established, homologous recognition and testing mechanisms are less clear.
For yeast, random search models have been considered
(Loidl, 1998) and have some attraction. However, the
applicability of any single-cell yeast model to most
multicellular animals and plants is unknown. In particular,
direct scaling-up of homology search processes that would
work with the 14 Mbp genome of yeast to even the 100
Mbp genomes of A. thaliana or C. elegans, let alone the
3000 Mbp of humans or 17 000 Mbp of wheat, may not be
possible.
Premeiotic and mitotic association of homologues is
proposed in budding (see above) and ®ssion yeast (Kohli,
1994), and such associations seem to be a universal
condition in somatic Diptera cells (Hiraoka et al., 1993).
Three-dimensional reconstructions of human cells (Leitch
et al., 1994) and cereal plant species (Heslop-Harrison
et al., 1988) were not able to ®nd evidence for homologous
association of chromosomes, even at the last mitotic
division before meiosis (Bennett, 1984; Schwarzacher
et al., 1992b). Chromosome painting in human spermatogonia (meiotic stem cells) revealed compact, largely
mutually exclusive chromosome territories that did not
show homologue association (Armstrong et al., 1994;
Cheng and Gartler, 1994; Scherthan et al., 1996).
Chromosome morphology
While homologous synapsis at zygotene is dependent on
direct DNA homology check mechanisms, it is likely that
chromosome recognition and initial pairing cannot be
achieved by DNA±DNA interactions alone (Sybenga,
1999). Chromosome morphology, speci®c sequence distribution, proteins bound to DNA, and the resulting
chromatin condensation patterns are likely to be involved
directly or indirectly in chromosome homology recognition. For example, repeated sequences could in¯uence
recognition through their secondary folding structures,
protein binding sites, and the condensation patterns that
give a chromosome a characteristic shape. Speci®c coiling
patterns with apparent denser and weaker zones, presumably re¯ecting more or less condensed chromatin were
observed in the homologous chromosome domains of
wheat. When the homologous domains associate, these
condensation patterns were also aligned (Schwarzacher,
1997) and the specialized prezygotene chromosome
morphology observed in maize may facilitate homology
recognition (Dawe et al., 1994). Karpen et al. (1996) have
analysed pairing of achiasmatic chromosomes in
D. melanogaster and proposed that DNA and protein
structures inherent to heterochromatin could produce a self
complementary chromosome `landscape' that ensures
partner recognition and alignment by `best ®t' mechan-
17
isms. Cook (1997) argued that each chromosome in a
haploid set has a unique array of transcription units strung
along its length and that, therefore, chromatin ®bres will be
folded into a unique array of loops and that only
homologues share similar arrays.
Repeated DNA sequences
Repeated sequences play an important part in several
aspects of meiosis, both structurally and functionally. They
might be responsible or at least aid the ®rst recognition of
homologous chromosomes when they search for pairing
partners and synapse at early meiosis. This could be
achieved at the DNA level itself (Roeder, 1997) through
chromosome speci®c sequence motifs or chromosome
speci®c patterns of several motifs.
Telomeres
Knowledge of the importance of telomeres for synapsis
comes from studies that, in the absence of homology in the
distal regions of chromosome arms, very long homologous
segments may remain unrecognized in meiosis
(Lukaszewski, 1997). Bouquet formation heavily depends
on telomere movement and attachment to the nuclear
envelope followed by de novo clustering of telomeres
before the initiation of pairing (Scherthan et al., 1996;
Dawe et al., 1994; Bass et al., 1997).
Subtelomeric repeats with chromosome-speci®c distribution would be essential for recognition of homologous
chromosome ends. The sequence at the telomere is highly
conserved, and consists of a short repeat, similar to
(TTTAGGG)n in most plants and (TTAGGG)n in mammals, tandem arrays many hundreds of units long at the
physical ends of chromosomes of most eukaryotes (for
review see Fuchs et al., 1995; Zakian, 1995). In plants, the
enzyme adding the telomeric sequence, telomerase, is
apparently active in all tissues. The length of arrays of
telomeric repeats is species-speci®c, ranging from 2±5 kb
in Arabidopsis thaliana (Richards and Ausubel, 1988),
through 12±15 kb in cereals (Schwarzacher and HeslopHarrison, 1991), up to 60±160 kb in tobacco (Fajkus et al.,
1995). The number of copies of the repeat differs between
chromosome arms of the karyotype (Schwarzacher and
Heslop-Harrison, 1991) and varying from cell-to-cell and
tissue-to-tissue (Kilian et al., 1995). It will be interesting to
know about the activity of telomerase and telomere length
changes through meiocyte mother cell development in
plants.
Adjacent to the simple telomere repeats most organisms
have more complex, often chromosome-speci®c, proximal
sequences (Richards et al., 1993) and various interacting
proteins (Biessmann and Mason, 1994). Analysis of these
sequences on rye chromosomes shows that they are able to
evolve in copy number rapidly (Alkhimova et al., 1999),
18 Schwarzacher
and may be part of a complex chromosome end structure
(Vershinin et al., 1995; Fig. 1c).
Centromeres
Centromeres at premeiotic interphase through to pachytene
and anaphase I have a more diffuse structure in hexaploid
wheat, exhibiting high homoeologous pairing compared to
low homeologous pairing wheat (Aragon-Alcaide et al.,
1997; Mikhailova et al., 1998). Suzuki et al. (1997) found
a speci®c antiserum that did not stain centromeres during
mitotic division in somatic cells of lily, but stained
centromeres during the meiotic division (male and female)
and postulate that the meiosis-speci®c centromere protein
is associated with conversion of a mitotic to a meiotic
chromosome, that meiosis is regulated by modi®cation of
the structure of chromosomes and particular centromeres,
and that a meiosis-speci®c centromere protein is required
for the meiosis-speci®c behaviour of the centromere. In
wheat, it has been noted that the association of homologous
domains apparently starts predominantly at the centromeres (Schwarzacher, 1997; Aragon-Alcaide et al., 1997;
Mikhailova et al., 1998). Hence cognition of homology
could be mediated by proteins or sequences associated
with the wheat centromeres. However, there is no evidence
that centromeres have a speci®c role in chromosome
pairing in A. thaliana (Armstrong et al., 2001).
Eukaryotic centromeres and kinetochores are responsible for sister chromatid cohesion, the correct alignment
of chromosomes on the metaphase plate, the attraction of
microtubules and organization of the spindle assembly, all
leading to the proper segregation of chromosome during
anaphase of division (for review see Earnshaw, 1994;
Allshire, 1997). The functional centromere of budding
yeast is attributed to a 125 bp sequence carrying three
characteristic `centromere DNA elements', CDEI, CDEII
and CDEIII (Clarke, 1990). However, the budding yeast
and even ®ssion yeast, with more complex and longer
centromeric sequences, do not seem a particularly good
model for understanding the centromeres in the plant or
animal kingdoms. Despite their highly conserved function
and detailed knowledge of the proteins involved, the DNA
sequences at the centromeres of higher eukaryotes remain
poorly understood and no universal features have been
found.
In many species, the centromere is associated with
blocks of heterochromatin containing highly repetitive
DNA sequences which include tandem satellite repeats and
retroelement-like components, representing a considerable
fraction of the genomic DNA. In humans, many, but not
all, authors regard the tandemly repeated a-satellite
sequences (0.3% of the human genome) as playing a key
role in centromere function and chromosome segregation
(Tyler-Smith et al., 1998). At each human centromere, the
tandemly arranged 170 bp units of the a-satellites form
large arrays of megabase pair length showing a chromo-
some-speci®c subpattern of sequence variants and characteristic multimers of slightly variant units (Willard, 1985).
Similar chromosome-speci®c variants have been identi®ed
in the centromeric minor satellite of the mouse (Kipling
et al., 1994). In Arabidopsis, a major 180 bp satellite
sequence, some 3% of the genome, is located at the
centromeres of all ®ve chromosome pairs (Maluszynska
and Heslop-Harrison, 1991; Murata et al., 1994; Fig. 1d),
although several other repetitive DNA sequences, including retroelements and degenerate telomeric motifs, have
also been located in this region (Brandes et al., 1997;
Fransz et al., 2000). Retrotransposon-like elements have
also been found in cereal and beet centromeric repeats and
many centromeric repeats include regions with homology
to the 17 bp mammalian CENP-B binding motifs, although
not always recognized in the source reference (AragonAlcaide et al., 1996; Gindullis et al., 2001; Murata, 2002).
Detailed sequence analysis of the units of a 180 bp long
tandem repeat motif in Arabidopsis (Heslop-Harrison et al.,
1999) identi®ed not only conserved regions including the
CENP-B and CEDII binding site motifs, but several
regions with alternate base pair changes. Using speci®c
primers for these regions, PCR or primed in situ
hybridization ampli®ed sequences on some chromosomes
more than others, indicating that chromosome-speci®c
variants are present (Heslop-Harrison et al., 1999; Fig.1e).
Such speci®c variants at centromeres or other pairing sites
would be a requisite for recognition of chromosomes using
repetitive sequence motifs. Different proteins associated
with DNA, including histone H3 and CENP-A or others
could be involved.
Simple sequence repeats
Microsatellites or simple sequence repeats (SSRs), runs of
repetitions of single nucleotides or motifs up to about 5 bp
long, are ubiquitous elements of eukaryotic genomes
(Tautz and Renz, 1984). However, the genomic organization of different microsatellite sequences varies widely,
with implications for ampli®cation and dispersion mechanisms, and hence evolution, and their utility for mapping
(SchloÈtterer, 2000). Schmidt and Heslop-Harrison (1996)
in beet and Gortner et al. (1998) in chickpea, used different
SSRs as probes for in situ hybridization and found, apart
from the dispersed overall signal, motif-speci®c patterns of
distribution with site-speci®c enrichment or depletion of
some motifs at centromeric or intercalary positions. In
wheat and rye, several SSR motifs cluster in similar sites in
the two species, although conventional staining systems
give very different chromosome bands, suggesting that the
pattern was established before their evolutionary separation (Cuadrado and Schwarzacher, 1998; Cuadrado et al.,
2000). In Fig. 1f, in situ hybridization with the motif AAC
to chromosomes of rye is shown. With many intercalary
sites, AAC cluster distribution is diagnostic, but notably
Meiosis, recombination and chromosomes
different and rarely overlaps with the long tandemly
repeated sequences of the subtelomeric heterochromatin.
During meiotic prophase, each single chromosome
along its entire length develops the synaptonemal complex
(see above), to which the two sister chromatids are
attached in a series of loops. The average size of the
chromatin loops attached to the SC are species-speci®c
(Moens and Pearlman, 1988; Zickler and Kleckner, 1999)
and it has been postulated that one mechanism for the
regulation of the loop size is the existence of specialized
DNA sequences that associate with the meiotic chromosome core. DNA sequences isolated from puri®ed,
DNAse-treated rat SCs did not contain sequences that
are unique to chromosome cores, but proved to be notably
different from random genomic fragments and contained
an excess of simple sequence repeats (mostly the
dinucleotide motif GT or, on the complementary DNA
strand, CA) and retroelement-related repetitive sequences
(LINE and SINE elements, long and short interspersed
sequences; Pearlman et al., 1992). Fluorescent in situ
hybridization to mouse SCs has shown that unique
sequences, the mouse minor satellite DNA sequence and
some other tandemly repeated sequences are mainly found
in the chromatin loops, while the signal from the telomere
sequence does not come from the loops (Moens and
Pearlman, 1993). Similarly, in humans, telomeric
sequences were seen tightly associated with the SCs
while centromeric alpha-satellites and classical satellites
were all found to form loops that are associated with the
SC only at their base (Barlow and Hulten, 1996).
In rye, two tandemly repeated sequences, the 18S±25S
rDNA and a 120 bp repeat from rye heterochromatin are
closely associated with the bivalent axes, corresponding to
the SC, and also located in the surrounding chromatin
loops (Albini and Schwarzacher, 1992). The relative
lengths of the bivalent axes covered with signal from the
120 bp repeat appears to be less than expected from
somatic metaphases (compare Fig. 1f and g), supporting
the speculation that heterochromatin is under-represented
in the SC length (Stack, 1984), although perhaps only with
respect to somatic metaphase. In contrast to the tight
packing and close association of these classes of long
tandem repeats, studies using simple sequence repeats as
probes for ¯uorescent in situ hybridization showed that
very little signal is associated with the SC, but is mainly
found in the chromatin loops (Fig. 1h).
Sites of recombination and cross-over
In organisms with conventional meiosis, at least one crossover is needed per bivalent to guarantee proper alignment
of bivalents on the equatorial plate of the ®rst meiotic
division and subsequent proper disjunction of homologous
chromosomes. Interference increases the distance between
two or more cross-overs on the same chromosome and is
19
possibly promoted by SCs (see also above), as interference
was found to be absent in fungi and yeast mutants without
SCs (Moens, 1994). To some extent chiasma frequency
and distribution is determined genetically, as differences
between male and female (Lagercrantz and Lydiate, 1995)
and between strains (Sall et al., 1993) have been found in
the same species. Chromatin, chromosome and genome
structure, perhaps under genetic control, play a part in
determining how many and where chiasma happen. When
comparing recombination frequency in different organism,
Anderson and Stack (2002) have shown that it correlates
better with total pachytene SC lengths and gene number
than with total genome size, except for yeast that has a
remarkable higher recombination frequency: an average of
100 cross-overs per 10 Mbp and 29 mm of SC (2n=32),
compared to 10 cross-overs per 180 Mb and 145 mm SC of
Arabidposis (2n=10) and 40 cross-overs per 17 330 Mbp
and 1475 mm SC of wheat (2n=42) are reported in their
table.
In general, chiasmata are more frequent in euchromatin
that contains actively transcribed genes, while they are
reduced in heterochromatin containing repeated sequences. DSBs in yeast are preferentially located in promotor
regions of genes and correlate with DNase sensitivity (Wu
and Lichten, 1995) and their non-random distribution
de®ne large, 39±105 kb, chromosomal domains that are
either hot or cold spots (Baudat and Nicholas, 1997).
Overall, recombination hot-spots correspond to regions
with high CG contents and a certain transcriptional pro®le,
while cold spots are found near telomeres and centromeres.
(Klein et al., 1996; Gerton et al., 2000). In Arabidopsis,
Fransz et al. (2000) described a hot spot of recombination
that correlated with a region of low chromatin condensation while the core centromeric region has suppressed
recombination (Haupt et al., 2001). In maize, recombination hot-spots in a 140 kb multigenic regions were located
in gene as well as non-gene regions, but the retrotransposons present in the region were recombinationally
inert (Yao et al., 2002).
The physical distances between genes and markers
along chromosomes correlate poorly with genetic map
distances, particularly in the large cereal genomes (HeslopHarrison, 1991; Lukaszewski and Curtis, 1993;
Schwarzacher, 1996), but also in Arabidopsis (Lin et al.,
1999; Mayer et al., 1999) and humans (Dunham et al.,
1999). In wheat and rye, many genes and RFLP markers
are clustered near the ends of some chromosome arms,
while genetically they are far apart, indicating that genetic
recombination frequency is high near the telomeres, but
rare towards centromeres. In situ hybridization of cloned
probes to meiotic metaphase I preparations in rye (Fig. 1i)
shows that chiasmata are very close to the subtelomeric
heterochromatin. The chiasmata which are visualized to
occur in different segments of the rye chromosome 1R,
distally or proximal of the rDNA site (Schwarzacher,
20 Schwarzacher
1996) show close correlation with genetic map distances
described by Devos and Gale (1993). In tomato, chiasmata
were found almost exclusively in more distal, rather
subterminal chromosome segments in Lycopersicum
esculentum3L. peruvianum back crosses (Parokonny
et al., 1997) and Festuca3Lolium hybrids (King et al.,
1998). In early meiotic prophase nuclei from rye, Rad51
foci are seen that are localized near the telomeres (Fig. 1a)
indicating that already the initiation of recombination is
concentrated near the ends of rye chromosomes.
In the physical model of a rye chromosome (Fig. 1j left),
blocks of simple sequence repeats are located near the
centromere, long tandemly repeated sequences make up
the subtelomeric heterochromatin. Dispersed repetitive
sequences, related to retroelements are distributed over
most of the chromosome arms (Moore et al., 1991), and
genes are enriched distally close to the subtelomeric
repetitive sequences. During interphase, the subtelomeric
repetitive sequences do not decondense and form tight
clusters, called chromocentres (Fig. 1j, right). The generich distal chromosome regions are very loose, indicated
by the weak DAPI staining between the chromocentres and
in contrast to the relatively more condensed, more
intensely DAPI-stained, centromeric regions. Similarly,
at pachytene, the distal chromosome segment is more
decondensed. (Fig. 1k).
Conclusions
While great advances have been made in unravelling the
mechanism of recombination by isolating the genes
involved and analysing their functions and interactions,
important questions in meiosis remain unanswered by
looking at meiotic genes alone. In particular, the nature and
mechanism of homologous chromosome recognition and
pairing and the distribution of recombination sites are
in¯uenced by local and regional chromatin conformation.
Further in¯uences on meiotic processes stem from the
enormous differences in genome sizes of different organisms that vary greatly between yeast with 14 Mbp per
haploid genome, C. elegans with 100 Mbp, mammals with
3000 Mbp and plants ranging between 150 Mbp for
Arabidopsis to well over 25 000 Mbp for lily or pine.
Genome size has considerable consequences for chromosome and genome organization, the packing of DNA into
chromatin and the distribution of genes and repeated DNA
sequences that, in turn, in¯uence pairing mechanism and
recombination site choice.
The tools are now available to explore meiotic gene
discovery and to combine the molecular sequence data
with high throughput expression analysis and studies of the
organization and behaviour of chromatin and chromosomes to build a comprehensive structural, temporal and
functional model of meiosis. Its application to plant
research will be of fundamental and applied signi®cance,
allowing the understanding and exploitation of the variation nature has provided by inventing recombination.
Acknowledgements
I would like to thank Pat Heslop-Harrison and Josef Loidl for fruitful
discussions and Shaobin Wu and Tanja Garkoucha for technical
assistance. The following are acknowledged for collaboration:
Angelines Cuadrado University Alcala, Madrid, Spain (Fig. 1f±h);
Umesh Lavania, Lucknow, India (Fig. 1c); Chris Gillies, University
Sidney, Australia (Fig. 1i); Lorrie Anderson, University Fort Collins,
Colorado, USA (Fig. 1a, and sharing her in-press Current Genomics
Article). Figures 1c and f±i are reproduced from Schwarzacher and
Heslop-Harrison (2000) with permission from the publisher. This
work was partially supported by BBSRC and CREST of JST (Japan
Science and Technology Corporation).
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