Stable barley chromosomes without
centromeric repeats
S. Nasuda
, S. Hudakova
, I. Schubert
, A. Houben
, and T. R. Endo§
Laboratory of Plant Genetics, Graduate School of Agriculture and Core Research for Evolutional Science and Technology, Japan Science and Technology
Agency, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; and Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany
Communicated by Koichiro Tsunewaki, Fukui Prefectural University, Fukui, Japan, May 22, 2005 (received for review August 28, 2004)
The satellite sequences (AGGGAG)n and Ty3 gypsy-like retrotransposons
are known to localize at the barley centromeres. Using a
gametocidal system, which induces chromosomal mutations in
barley chromosomes added to common wheat, we obtained an
isochromosome for the short arm of barley chromosome 7H (7HS)
that lacked the barley-specific satellite sequence (AGGGAG)n. Two
telocentric derivatives of the isochromosome arose in the progeny:
7HS* with and 7HS** without the pericentromeric C-band. FISH
analysis demonstrated that both telosomes lacked not only the
barley-specific centromeric (AGGGAG)n repeats and retroelements
but also any of the known wheat centromeric tandem repeats,
including the 192-bp, 250-bp, and TaiI sequences. Although they
lacked these centromeric repeats, 7HS* and 7HS** both showed
normal mitotic and meiotic transmission. Translocation of barley
centromeric repeats to a wheat chromosome 4A did not generate
a dicentric chromosome. Indirect immunostaining revealed that all
tested centromere-specific proteins (rice CENH3, maize CENP-C, and
putative barley homologues of the yeast kinetochore proteins
CBF5 and SKP1) and histone H3 phosphorylated at serines 10 and
28 localized at the centromeric region of 7HS*. We conclude that
the barley centromeric repeats are neither sufficient nor obligatory
to assemble kinetochores, and we discuss the possible formation of
a novel centromere in a barley chromosome.
centromere chromosome aberration
Centromeric DNA sequences are not highly conserved, and
their functional importance in many higher eukaryotes is still
a matter of debate (for reviews, see refs. 1 and 2). Nevertheless,
clusters of tandem repeats interspersed with retroelements are
typical features of regular centromeres in several plant species
(2). Two repetitive sequences, CCS1 (3) and Sau3A9 (4), are
conserved within centromeres of cereal species and represent
parts of the LTR and of the integrase region of a Ty3 gypsy-like
retroelement (e.g., cereba or centromeric retrotransposon of
barley), respectively (5). Complete or truncated copies of this
retroelement family were found in centromeres of barley, maize,
sorghum, wheat, rice, rye, oats, Aegilops (5­10), and even in
dicotyledonous Beta species (11) interspersed irregularly between
species-specific tandem repeats (12­15). Preferential coprecipitation
of both centromeric sequence types with the
CENP-A-like centromeric histone H3 variant CENH3 indicates
their functional importance for centromere assembly (16), although
not all centromeric repeats were associated with CENH3
on extended chromatin fiber preparations (17).
If regular centromeres are lost, a few regions without the
typical centromeric repeats of human chromosomes (18) or
regions of Drosophila chromosomes adjacent to the centromere
(19) may assemble kinetochores and constitute a de novo-formed
centromere. Neocentromeres of plants are different because
they are observed in meiotic cells as chromatin extensions
directed to the spindle poles and are present together with a
normal centromere. Consequently, the corresponding chromosome
behaves as a dicentric at meiosis, whereas in mitosis, only
the normal centromere is functional. Plant neocentromeres, best
known from rye (20) and maize, are represented mostly by
heterochromatic terminal knobs and are composed of tandem
repeats different from those of regular centromeres (21, 22).
Kinetochore proteins such as CENP-C (23) and CENH3 are
absent from these neocentromeres (17). The neocentric activity
in maize is part of a complex meiotic drive system regulated by
several genes of an ``aberrant chromosome 10.'' Neocentromere
movement along spindle fibers differs from that of normal
centromeres (22). Neocentromeres that fully substitute for regular
centromeres have not yet been described for plants.
To elucidate the functional significance of centromeric repetitive
sequences, we applied a gametocidal system to induce
deletions in barley chromosomes. A gametocidal chromosome
derived from a related species, Aegilops cylindrica, when introduced
into common wheat (Triticum aestivum) in monosomic
condition, induces structural chromosome aberrations such as
deletions in the gametes not carrying the gametocidal chromosome
(24, 25). This gametocidal system can induce chromosomal
mutations in barley and rye chromosomes added to common
wheat (26, 27).
Applying the gametocidal system to the formation of aberrations
in a barley chromosome 7H added to common wheat, we
obtained an isochromosome of the short arm of 7H that was
lacking the gypsy-like retroelement cereba and the G C-rich
satellite sequence (AGGGAG)n, both representing the major
components of centromeric DNA in barley (12). Telocentric
chromosomes (or telosomes) were derived from this isochromosome
in the next generation. In this study, we used centromerespecific
DNA probes and antibodies to elucidate the molecular
organization of the centromeres of the telosomes. We also
investigated the transmission of the truncated telosomes to
understand the effect of centromeric repetitive sequences on
chromosome segregation in mitosis and meiosis, and we discuss
the possibility of neocentromere formation in barley.
Materials and Methods
Plant Material. Aberrant 7H chromosomes examined in this study
were found in a common wheat (T. aestivum cv. Chinese Spring,
2n 6x 42) line carrying a pair of barley (Hordeum vulgare cv.
Betzes) 7H chromosomes and one gametocidal chromosome 2C
of Ae. cylindrica. This line was developed for selecting 7H
aberrations (28, 29). Barley cultivars Betzes and Shinebis as well
as Chinese Spring lines carrying the whole, the short arm, or the
long arm of barley chromosome 7H (30, 31) were used as
controls.
PCR Analysis. To test in wheat background for the typical sequence
organization of barley centromeric repeats as observed
in the representative clone BAC7 (12), primers for PCR were
designed according to the LTR sequences of cereba and the
G C-rich satellite sequence (AGGGAG)n. The primers were
cerebaLTR-F (5 -GTTGATCGTGCTCCGGTGTGATCA-3 ),
Abbreviations: cereba, centromeric retrotransposon of barley; GISH, genomic in situ
hybridization.
§To whom correspondence should be addressed. E-mail: trendo@kais.kyoto-u.ac.jp.
 2005 by The National Academy of Sciences of the USA
9842­9847 PNAS July 12, 2005 vol. 102 no. 28 www.pnas.org cgi doi 10.1073 pnas.0504235102
cerebaLTR-R (5 -TGATCACACCGGAGCACGATCAAC-3 ),
(AGGGAG)3-F (5 -AGAGGGAGAGGGAGAGGGAG-3 ),
and (AGGGAG)3-RG (5 -CTCCCTCTCCCTCTCCCTCT
G-3 ). PCR was performed in 20 l containing 100 ng of
template DNA, 0.25 mM each dNTP, 4 pmol of forward and
reverse primers in 1 PCR buffer with 0.15 mM MgCl2, and 2.5
units of AccuPrime Taq DNA polymerase (Invitrogen) by using
an iCycler (Bio-Rad). The PCR conditions were as follows:
initial denaturation at 94°C for 2 min, followed by 30 cycles of
denaturion (96°C, 10 sec), annealing (67°C, 30 sec), and extension
(68°C, 1 min). The reaction was completed by additional
extension at 68°C for 7 min. Amplification products were electrophoresed
on a 1% agarose gel and were visualized by ethidium
bromide staining.
Giemsa N- and C-Banding and FISH. N- and C-banding was performed
according to Gill et al. (32). Three sequences of the two
major components of the barley centromere contained within
the barley centromere-specific clone BAC7 were used as FISH
probes to detect centromeric repeats on chromosomes: the
G C-rich satellite sequence (AGGGAG)n, the plasmid clone
pGP7 containing the RNase H and the integrase region of the
Ty3 gypsy-like retroelement cereba, and the LTRs of cereba
(12). To verify that the aberrant barley chromosomes did not
acquire centromeric or other sequences from wheat during
their formation, FISH was conducted with the 192-bp repeat
(33), the 250-bp repeat (34), and the TaiI tandem repeat (35),
which hybridize to centromeric regions of wheat chromosomes.
Additionally, genomic in situ hybridization (GISH) was conducted
with genomic DNA of common wheat as a probe and
with barley genomic DNA in 300-fold excess for blocking. To
identify the arms of the 7H chromosome, C-banding, FISH
with the barley-specific subtelomeric repeat HvT01 (36), and
GISH with total barley genomic DNA were combined. The
DNA probes were labeled with digoxigenin-11-dUTP or with
biotin-11-dUTP according to the manufacturer's instructions
(Roche Applied Science, Indianapolis) and detected with
either fluorescein-conjugated anti-digoxigenin antibody
(Roche Applied Science) or streptavidin-Cy3 (Amersham
Pharmacia Biosciences). Chromosomes were counterstained
with DAPI or propidium iodide. FISH, GISH, and sequential
C-banding FISH GISH were performed as described in refs.
37 and 38. Chromosome images were recorded with a fluorescence
microscope equipped with a charge-coupled device
camera (SenSys, Photometrics, Tucson, AZ).
Indirect Immunostaining. Polyclonal rabbit antibodies against histone
H3 phosphorylated at serine-10 (Upstate Biotechnology,
Lake Placid, NY) and a rat monoclonal antibody against H3
phosphorylated at serine-28 (39) were diluted 1:400 in PBS 3%
BSA. After a 12-h incubation of slides containing squashed root
tip meristems in primary antibody solution at 4°C and washing
for 15 min in PBS, the slides were incubated in rhodamineconjugated
anti-rabbit IgG and FITC-conjugated anti-rat IgG,
both from Dianova (Hamburg, Germany), in PBS 3% BSA for
1 h at 37°C. After final washes in PBS, the preparations were
mounted in antifade containing DAPI as a counterstain (40).
For immunostaining of kinetochore proteins, suspensions of
nuclei and chromosomes were prepared as described in refs. 41
and 42 from synchronized root tip meristems of seedlings of the
wheat barley telosomic addition line containing the telocentric
chromosome without the centromeric repeats (7HS*, see Results).
Suspended nuclei and chromosomes were spun down onto
microscopic slides (at 18 g for 5 min) by using a Cytospin3
(Shandon, Pittsburgh) cytological centrifuge and stored in glycerol
at 4°C until use.
Polyclonal rabbit antisera raised against synthetic peptides
derived from sequences encoding putative barley homologues of
the yeast kinetochore proteins CBF5 and SKP1 (43) and rabbit
antisera against the centromeric CENH3 (the histone H3 variant
corresponding to the human CENP-A) protein of rice (44) and
against maize ZmCENP-C (23) were used as primary antibodies.
Slides with isolated nuclei chromosomes were washed three
times for 20 min each in PBS, postfixed in 4% formaldehyde
(vol vol) in PBS for 10 min, blocked (3% BSA 10% horse
serum, in PBS) for 1 h at 37°C, and then incubated for 1 h at room
temperature in primary antiserum in AK buffer (1% BSA 10%
horse serum 0.1% Tween 20, in PBS). After three 5-min washes
in PBS, the slides were incubated for 1 h at room temperature
in secondary antiserum (Alexa Fluor 488-conjugated goat antirabbit
IgG, Molecular Probes, or FITC-conjugated goat antirabbit
IgG, Sigma) in AK buffer and washed in PBS. DNA was
counterstained with DAPI in Vectashield (Vector Laboratories).
For subsequent FISH, coverslips were removed by using
liquid nitrogen. After washing in PBS and postfixation in 4%
paraformaldehyde 3.4% sucrose for 10 min at room temperature,
FISH with the rhodamine-labeled subtelomeric repeat
HvT01 was carried out as described in ref. 37.
Results
Aberrant Derivatives of Chromosome 7H Without Centromeric Satellite
Sequences and cereba Retroelements Are Stably Transmitted. The
primary constrictions of wild-type barley chromosomes appeared
after N-banding as thin stretched chromatin threads that
were oriented to the opposite spindle poles and thus formed a
diamond shape (Fig. 1A). This chromatin structure colocalizes
with the cereba sequence pGP7 (Fig. 1A) and the (AGGGAG)n
repeat located between the dark pericentromeric C bands of the
short arm (7HS) and the long arm (7HL) of chromosome 7H
(Fig. 1B) and at the centric ends of the telocentrics 7HS (Fig. 1C)
and 7HL (Fig. 1D). These data indicate that the 7H centromeric
region contains many of these repeats.
A wheat­barley Robertsonian translocation involving 7HS
and an unidentified wheat chromosome arm with a centromere
containing the barley-specific centromeric (AGGGAG)n repeats
(Fig. 1E) but no detectable wheat-specific centromeric 192-bp
repetitive sequence (33) was found. From this translocation
chromosome, a 7HS isochromosome was derived that no longer
contained (AGGGAG)n sequences detectable by FISH within its
primary constriction (Fig. 1F) but at least a part of the pericentromeric
C-band was retained (Fig. 1G). Thus, the (AGGGAG)n
repeats were demonstrated to be located proximally to the
pericentromeric C-band of 7HS and were absent at a cytological
level from the 7HS isochromosome. Minor (AGGGAG)n signals
were detected on wheat chromosomes 3B and 5B when images
were exposed longer than usually required for detecting the
corresponding signals on normal barley chromosome 7H (Fig. 1
F and R).
Of 26 plants examined in the selfed progeny of the wheat line
with the 7HS isochromosome, 5 plants contained the same
isochromosome as a monosomic addition and another 5 had
single telocentric chromosomes derived from this isochromosome.
We analyzed the 7HS telosomes by sequential C-banding,
FISH, and GISH, and identified two aberrant derivatives of 7HS:
one with the pericentromeric C-band (7HS*, Figs. 1H and 2) and
the other without the pericentromeric C-band (7HS**, Figs. 1I
and 2).
Both types of truncated telosomes were stably transmitted to
subsequent generations at rates similar to that of a regular
telosome 7HS. In the monosomic addition, 7HS was transmitted
to 24.1% (7 of 29 plants) of the selfed progeny, whereas 7HS*
and 7HS** were transmitted to 30.2% (119 of 394 plants; 10
plants were disomic for 7HS*) and to 25.8% (17 of 66 plants; two
plants were disomic for 7HS**) of the selfed progeny, respectively.
We established a stable disomic 7HS* addition line of
Chinese Spring wheat that showed the regular bivalent formation
Nasuda et al. PNAS July 12, 2005 vol. 102 no. 28 9843
GENETICS
of 7HS* during meiotic metaphase I (Fig. 1J), normal fertility,
and regular transmission of 7HS* to the subsequent generations.
We confirmed the absence of the centromeric repeats from
the centromere of 7HS* in hybrid plants that carry one 7HS and
one 7HS*. As expected, no (AGGGAG)n-specific signal was
detected on 7HS*, whereas a strong signal occurred at the centric
Fig. 1. Characterization of barley centromeres in regular and altered chromosomal positions. (A) Partial mitotic metaphase cell of barley cv. Shinebis after
N-banding (Left) and FISH using the pGP7 probe (green signal) counterstained with propidium iodide (Right). (B­D) Chromosomes 7H (B), 7HS (C), and 7HL (D)
after C-banding (Left) and FISH using the (AGGGAG)n probe (Right). (E) Robertsonian translocation between 7HS and a wheat chromosome segment after
C-banding (Left), FISH using the (AGGGAG)n probe (green, Center), and FISH GISH (Right) using the HvT01 (green) and genomic barley DNA (red, Right). (F) The
7HS isochromosome after FISH with (AGGGAG)n and GISH with genomic barley DNA (red, Inset). (G) The 7HS isochromosome after C-banding (Left) and
subsequent FISH GISH with HvT01 (green) and barley genomic DNA (red, Right). (H) 7HS* after C-banding (Left) and subsequent FISH GISH with HvT01 (green)
and barley genomic DNA (red, Right). (I) 7HS** after C-banding (Left) and subsequent FISH GISH with HvT01 (green) and barley genomic DNA (red, Right). (J)
The 7HS* bivalent at meiotic metaphase I after FISH with HvT01 (green) and GISH (red). (K) Mitotic chromosomes 7HS* and normal 7HS in the same cell after
FISH with HvT01 (green) and (AGGGAG)n (red). (L) 7HS* and normal 7HS after FISH with LTR sequences of cereba (green), 7HS* after subsequent FISH with HvT01
(green) and GISH (red) (Inset Left), and 7HS after subsequent FISH with (AGGGAG)n (red, Right). (M) 7HS** after FISH with LTR (green) and after subsequent
FISH GISH with HvT01 (green) and genomic barley DNA (red, Inset). (N) 7HS* after FISH with the 192-bp centromeric repeat of wheat (green) and after subsequent
FISH GISH with HvT01 (green) and genomic barley DNA (red, Inset). (O) 7HS* after FISH with 250-bp centromeric repeat of wheat (green) and after
subsequent FISH GISH with HvT01 (green) and genomic barley DNA (red, Inset). (P) 7HS* after FISH with TaiI (green) and after subsequent FISH GISH with HvT01
(green) and genomic barley DNA (red, Inset). (Q) 7HS* (counterstained red) after GISH with wheat labeled genomic DNA (green) and an excess of unlabeled barley
DNA. (R) C-banding (Center) and FISH (Left and Right) with (AGGGAG)n (red) on chromosomes of the 4A line; note that much longer exposure was required
to detect the minor signals on chromosomes 3B and 5B (Left) than to detect those of 4A (Right). (S) Partial metaphase of wheat including chromosome 7HS*
after immunostaining with antibodies against histone H3 phosphorylated at serines 10 (red, Lower) and 28 (green, Upper). (T) Isolated telosomes 7HS* after
immunostaining with antibodies (green) against rice CENH3 (Upper Left), ZmCENP-C (Upper Second), CBF5 (Upper Third), and SKP1 (Upper Right). Red signals
of subsequent FISH with HvT01 are shown in Lower, demonstrating that the telosomes were 7HS*. (Scale bars, 5 m. The magnification is the same for A­P, except
for Insets.)
9844 www.pnas.org cgi doi 10.1073 pnas.0504235102 Nasuda et al.
end of 7HS (Figs. 1K and 2). After FISH with the LTR probe of
the centromere-specific Ty3 gypsy-like retroelement cereba, no
signals were found in the centromeric regions of 7HS* (Figs. 1L
and 2) and 7HS** (Figs. 1M and 2), whereas an intense
hybridization signal was present on 7HS that was comparable
with that observed on wheat chromosomes (Figs. 1L and 2).
FISH on the same 7HS, re-probed with (AGGGAG)n repeats,
revealed that these sequences were present in a region extending
beyond the region containing the centromeric cereba sequences
(Fig. 1L Right). No FISH signals of the three wheat centromeric
repeats, 192-bp, 250-bp, and TaiI, were detected on 7HS* (Fig.
1 N­P), and no GISH signals of wheat genomic DNA were found
on 7HS* (Fig. 1Q).
Translocation of the Barley Centromeric (AGGGAG)n Repeat onto a
Wheat Chromosome Does Not Generate a Dicentric Chromosome. In
the siblings of the Robertsonian wheat­barley translocation line
from which plants with the 7HS isochromosome were derived,
we found a wheat chromosome 4A carrying many (AGGGAG)n
repeats at the tip of the long arm (4A in Fig. 1R, Center and
Right). The 4A chromosome was regularly transmitted through
male and female gametes. Disomic 4A plants behaved normally
throughout their life cycle. Thus, no dicentric was formed
by translocation of this barley centromere repeat onto a wheat
chromosome.
Truncated 7HS Telosomes Lack the Barley-Specific Sequence Organization
of Centromeric Repeats. To verify the in situ hybridization
results that the barley centromeric (AGGGAG)n repeats and
retroelements had been lost in the truncated 7HS telosomes, we
investigated the Chinese Spring lines having 7H, 7HS, 7HS*,
7HS**, and 4A , together with Betzes barley and Chinese
Spring wheat, by PCR using four primer-pair combinations:
cerebaLTR-F (AGGGAG)3-F, cerebaLTR-F (AGGGAG)3RG,
cerebaLTR-R (AGGGAG)3-F, and cerebaLTR-R
(AGGGAG)3-RG. The former two primer combinations yielded
no distinct PCR product (data not shown). With the latter two
primer combinations, two fragments of similar size ( 800 bp)
were amplified in Betzes but not in Chinese Spring (Fig. 3). The
primer pair cerebaLTR-R (AGGGAG)3-F amplified the barleyspecific
fragments from the 7H and 7HS additions and from the
4A line but not from the 7HS* and 7HS** additions (Fig. 3).
The primer pair cerebaLTR-R (AGGGAG)3-RG amplified
both fragments from the 7H addition, only one of the fragments
from the 7HS line, and no fragment from the 7HS* and 7HS**
additions and from the 4A line (Fig. 3). These results suggested
that (i) 7H and 7HS both contained the cereba LTR and
(AGGGAG)n sequences in adjacent position and in different
orientations, (ii) 4A contained both sequences adjacently in
one orientation, and (iii) 7HS* and 7HS** had lost the centromeric
region where both sequences were in close vicinity.
Derivatives of Chromosome 7H Without Centromeric Repeats Possess
Centromere-Specific Proteins. During mitosis, histone H3 phosphorylation
levels at serine positions 10 and 28 are high in plant
pericentromeric regions (40, 45, 46). We explored whether H3
undergoes the same posttranslational modification at the centromeric
end of the telosome 7HS* that normal pericentromeres
do. The histone H3 phosphorylation revealed no difference
between regular wheat chromosomes and the aberrant telosome
7HS* (Fig. 1S). Our observation agrees with previous findings
that only regions around functional centromeres contain H3
phosphorylated at serine-10 from prophase until telophase (40).
Immunostaining with antibodies against rice CENH3, maize
ZmCENP-C, and CBF5 and SKP1, barley homologues of yeast
kinetochore proteins, revealed the presence of all these centromere-specific
proteins at the centric end of isolated 7HS*
telosomes (Fig. 1T Upper). The opposite terminus of the same
telosomes showed signals after FISH with the barley subtelomeric
repeat HvT01 (Fig. 1T Lower).
Discussion
Are Centromeric Repeats Necessary for Assembling Functional Kinetochores?
It is generally assumed that the centromere is located in
the primary constriction of monocentric chromosomes where
chromatin is weakly stained after chromosome banding. The
primary constriction of regular barley chromosomes contains
extended repetitive sequences as shown by FISH and, as reFig.
2. C-bands (solid bars) and the presence ( ) or absence ( ) of FISH
signals in chromosomes 7H, 7HS, 7HS*, and 7HS**.
Fig. 3. PCR amplification of the regions between cereba LTR and adjacent
(AGGGAG)n sequences. Primer combinations used were cerebaLTR-R
(AGGGAG)3-F (Left) and cerebaLTR-R (AGGGAG)3-RG (Right). The barleyspecific
amplification products are indicated with arrows. Opposite orientations
of cereba LTR and (AGGGAG)n repeats inferred from the different
amplification products are shown below the gel.
Nasuda et al. PNAS July 12, 2005 vol. 102 no. 28 9845
GENETICS
ported by Singh and Tsuchiya (47), forms a diamond-shaped
structure after N-banding (Fig. 1A). The kinetochore, where
specific proteins accumulate and mediate attachment to spindle
fibers during nuclear divisions, is positioned at the centromere,
hence at the primary constriction.
The retroelement cereba and (AGGGAG)n sequences represent
all centromeric repeats reported so far for barley (12). The
(AGGGAG)n sequence in 7HS extends from the centric end
beyond the cereba LTR sequences (Fig. 1L). As summarized in
Fig. 2, both repetitive sequences are absent from 7HS* and
7HS**, and 7HS** also lacks the centromere-adjacent C-band
(Fig. 1I). PCR analyses using the primer pairs designed to
amplify the region between the cereba LTR and (AGGGAG)n
revealed neither for 7HS* nor for 7HS** the centromere-specific
organization of both sequences that has been described for the
BAC7 insert. This finding indicates that 7HS* and 7HS** have
lost most of these repeats. GISH with barley genomic DNA and
unlabeled excess BAC7 DNA yielded only very weak signals at
the barley centromeres, indicating the absence of considerable
amounts of repeats other than cereba and (AGGGAG)n (12).
Therefore, it is unlikely that cereba LTR and (AGGGAG)n
sequences were eliminated preferentially to leave only other
unknown centromeric repeats in 7HS* and 7HS**. The absence
of any wheat chromatin (see Fig. 1 N­Q) does not support the
idea that the truncated 7HS telosomes gained wheat-specific
centromeric sequences during their formation from the 7HS
isochromosome. The stable mitotic and meiotic transmission of
7HS* and 7HS** suggests that both telosomes retain functional
centromeres and that presently known centromeric repeats in
such amounts detectable by FISH are not essential for the
normal centromere function. The conserved epitopes with high
similarity to the yeast kinetochore proteins SKP1 and CBF5, to
ZmCENP-C, and to rice CENH3, as well as the signals for
histone H3 phosphorylated at serines 10 and 28, were all
detected at one terminus of chromosome 7HS*, confirming the
presence of a functional centromere. The 4A chromosome
with a cluster of the (AGGGAG)n sequences, together with the
cereba LTR, at the terminus of the long arm showed regular
mitotic and meiotic transmission, suggesting that these barley
centromeric repeats are not sufficient to organize a functional
centromere on the wheat chromosome.
Where Is the Position of the Kinetochore? The kinetochore of 7HS
might be located on the distal side of the pericentromeric C-band
(see Fig. 1H), not in the primary constriction, because 7HS**
had a normal centromere function despite the loss of the
pericentromeric C-band. If this is the case, the normal 7H
chromosome should have at least two functional centromeres,
one on 7HS and the other on 7HL or in the primary constriction.
Alternatively, if the kinetochore of 7HS is organized at the
centromeric repeats within the primary constriction, as generally
assumed, 7HS* and 7HS** should have centromeres that might
have been established close to the position of original centromeric
sequences in a similar way as Drosophila neocentromeres
were formed (19). Three arguments support the view that the
telosomes 7HS* and 7HS** contain fully functional centromeres
different from the neocentromere-type of maize that also lacks
repeats of regular centromeres: (i) Centromeric satellite and
retroelement sequences of maize associate preferentially with
the centromeric isoform CENH3 of histone H3 (17), therefore
it seems reasonable to assume that the same basic step of
kinetochore assembly takes place also at the corresponding
repeats of barley. Then, these regions represent the position of
regular barley centromeres and are deleted from the truncated
telosomes. (ii) Functional human neocentromeres and the centric
ends of 7HS* but not the transient meiotically neocentric
knobs of maize (48) are associated with all tested kinetochoric
proteins. (iii) The centric end of 7HS* is enriched in histone H3
phosphorylated at serine-10, which is typical for fully functional
barley centromeres but not for inactive ones such as found on a
semidicentric chromosome (40).
About 60 positions within the human chromosome complement
have been reported to develop centromeric activity without
containing detectable amounts of centromeric repeats (18). This
results in an average of 1.3 potential neocentromeres per chromosome.
However, frequent acentric chromosome fragments,
which appear during the first nuclear division after genotoxin
exposure, form micronuclei and eventually disappear rather than
establishing a neocentromere spontaneously. According to the
hypothesis of Mellone and Allshire (49), stretching centromeric
regions by attachment to spindle fibers during nuclear divisions
is required to exchange nucleosomal histone H3 by CENP-A
CENH3. If this happened somewhere on an acentric fragment,
full kinetochores might become established and perpetuated
during subsequent nuclear divisions. The observation of Drosophila
neocentromeres at chromosomal positions close to that
of the original centromeres could be due to remnants of the
original centromere kinetochore, which allow attachment of a
few spindle fibers to generate tension during nuclear division.
The consequence might be an evolutionary ``migration'' of the
centromeric position in case of (partial) deletion of the original
centromere. On the one hand, the presence of CENH3 as well
as of other kinetochore proteins and of pericentromere-specific
histone modifications at the centric end of chromosome 7HS*
suggests that a fully functional neocentromere might have originated
after deletion of the original centromere at stretched
chromatin-containing sequences other than centromere-specific
repeats. On the other hand, the centromeric repeats translocated
to the terminus of the wheat chromosome 4A do not generate
an additional centromere at this chromosomal position, possibly
because the translocated regions did not become exposed to
stretching forces exerted by spindle microtubules.
We thank Paul Talbert (Fred Hutchinson Cancer Research Center,
Seattle, WA) for providing anti-rice CENH3 antibodies and Kelly Dawe
(University of Georgia, Athens, GA) for anti-maize CENP-C antibodies,
Rigomar Rieger and Richard Pickering for critical reading of the
manuscript, and Joachim Bruder for technical assistance. This work was
supported by grants from the Deutsche Forschungsgemeinschaft (to I.S.
and A.H.). This paper is contribution no. 585 from the Laboratory of
Plant Genetics, Graduate School of Agriculture, Kyoto University.
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