The Transcribed 165-bp CentO Satellite Is the Major
Functional Centromeric Element in the Wild
Rice Species Oryza punctata
Wenli Zhang, Chuandeng Yi, Weidong Bao, Bin Liu, Jiajun Cui, Hengxiu Yu, Xiaofeng Cao,
Minghong Gu, Min Liu, and Zhukuan Cheng*
State Key Laboratory of Plant Genomics and National Center for Plant Gene Research,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
China (W.Z., W.B., B.L., J.C., X.C., M.L., Z.C.); Department of Agronomy, Yangzhou University,
Yangzhou 225009, China (C.Y., H.Y., M.G.); and Graduate School of the Chinese Academy of
Sciences, Beijing 100101, China (W.Z., W.B., B.L., J.C.)
Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear what kind of sequences act
as functional centromeres and how centromere sequences are organized in Oryza punctata, a BB genome species. In this study,
we found that the CentO centromeric satellites in O. punctata share high homology with the CentO satellites in O. sativa. The
O. punctata centromeres are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons.
Immunostaining with an antibody specific to CENH3 indicates that the 165-bp CentO satellites are the major component for
functional centromeres. Moreover, both strands of CentO satellites are highly methylated and transcribed and produce small
interfering RNA, which may be important for the maintenance of centromeric heterochromatin and centromere function.
Centromeres are highly organized domains of the
eukaryotic chromosome that mediate critical mitotic
and meiotic functions, including kinetochore nucleation, spindle fiber attachment, and sister chromatin
cohesion, and thus play a critical role in chromosome
segregation and transmission. Except for the centromeres of Saccharomyces cerevisiae, which consist of only
approximately 125 bp of unique sequence (Clarke,
1990, 1998), the centromeric sequences for many other
model eukaryotes, including Saccharomyces pombe, Drosophila melanogaster, mouse, human, and Arabidopsis
(Arabidopsis thaliana), consist of long tracks of tandem
repeats, with monomer repeat units of about 160 to
approximately 180 bp (Henikoff et al., 2001; Jiang et al.,
2003). Centromeric DNA sequences are often diverged
among closely related species. For example, the approximately 171-bp a-satellite repeats are the major
centromeric sequences for human chromosomes, which
organize into long arrays from 250 kb to more than
4 Mb in different chromosomes (Wevrick and Willard,
1989). In Arabidopsis, the 180-bp centromeric satellite
repeats compose the functional centromere core region,
and each centromere contains two approximately
4 Mb of the repeats (Kumekawa et al., 2000, 2001;
Hosouchi et al., 2002). In addition to the centromeric
satellite repeats, centromeres of many eukaryotes are
* Corresponding author; e-mail [email protected]; fax
0086–10–6487–3428.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.064147.
306
enriched with centromere-specific retrotransposons
(Jiang et al., 2003).
Although centromeric DNA sequences are significantly diverged among different species, the basic
functions of centromeres are conserved among all eukaryotic species. Kinetochores differ in morphology
from species to species, but kinetochore proteins, such
as CENP-A, CENP-B, and CENP-C, which are thought
to participate in kinetochore assembly and/or maturation, are relatively conserved. CENP-A or CENH3,
a centromere-specific histone H3 variant, has been
found in all model eukaryotes (Henikoff et al., 2001;
Sullivan et al., 2001) and recently in plants (Talbert
et al., 2002; Zhong et al., 2002; Nagaki et al., 2004).
Blocks of CENH3-associated nucleosomes and regular
H3-associated nucleosomes are linearly interspersed
within functional centromeres (Blower et al., 2002).
Thus, identification of DNA sequences that interacted
with CENH3 is an effective approach to recognize
specific DNA sequences involved in centromere function.
The centromeres of rice (Oryza sativa) chromosomes
contain a 155-bp satellite repeat CentO, ranging from
approximately 60 to 2,000 kb among different centromeres. The CentO arrays are interrupted irregularly
by the centromere-specific retrotransposons (CRRs;
Cheng et al., 2002a). The two centromeres of rice chromosomes 4 and 8, which contain the shortest arrays
of the CentO satellite among the 12 centromeres, have
been completely sequenced recently (Nagaki et al.,
2004; Wu et al., 2004; Zhang et al., 2004). Moreover,
several active genes were found in the functional
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Centromeres of Oryza punctata
centromere domain of chromosome 8, within the
CENH3 binding sites (Nagaki et al., 2004).
The genus Oryza includes two cultivated species, O.
sativa and O. glaberrima, and 21 wild species containing AA, BB, CC, BBCC, CCDD, EE, FF, GG, and HHJJ
genomes (Ge et al., 1999). Results of PCR combined
with southern-blot hybridization using rice centromeric DNA sequences confirm that several wild rice
species, such as those with the AA, CC, BBCC, CCDD,
and EE genomes, contain the CentO satellite repeats
and CRR-related sequences (Hass et al., 2003). However, the centromeric DNA composition of O. punctata,
which contains the BB genome, is still unclear. In this
study, we sequenced one bacterial artificial chromosome (BAC) clone derived from the centromeric region
of O. punctata. We demonstrate that the 165-bp CentO
repeat is the major functional centromeric element in
O. punctata by immunostaining using an antibody
against rice CENH3 followed by chromosome fluorescence in situ hybridization (FISH). We also provide
evidence that the centromeric repetitive DNA in O.
punctata is transcriptionally active.
RESULTS
Isolation of the Centromere-Specific Tandem Repeat
in O. punctata
The centromere is a discrete domain on each chromosome with a complex substructure and usually a
prominent heterochromatic region. Recent evidence
suggests that satellite DNA regions around centromeres may be important for sister chromatid cohesion.
To isolate the centromere-specific satellite DNA in O.
punctata, we screened an O. punctata BAC library using
sheared genomic DNA from O. punctata as a probe. A
total of 20 positive clones showing strong hybridization signals were selected and labeled as FISH probes
to hybridize to O. punctata pachytene chromosomes.
Eighteen of them hybridize to the centromeric regions
of all chromosomes (Fig. 1A). One BAC clone, 02M23,
showed consistent strong FISH signals to all centromeres and was selected for subcloning. We further
probed 02M23 to the meiotic metaphase I chromosomes of O. punctata and found the signals were consistently detected at the most poleward positions on
the bivalent chromosomes (Fig. 1B), suggesting that
02M23-related DNA sequences are located at chromosomal regions associated with the kinetochore
complex.
We randomly sequenced 10 subclones derived from
02M23 and found most of the sequences consist
of CentO-like tandemly repeated DNA with a consensus length of 165 bp (A and B in Fig. 2). We used the
CentO consensus sequence to generate PCR primers
and subsequently created a plasmid library of CentOderived PCR product. To further confirm that the
165-bp CentO is the dominant centromere satellite in
O. punctata, we sequenced several of the CentO plasmid clones (C, D, E, and F in Fig. 2) and found most of
the plasmids contained only a 165-bp monomer. A few
of the plasmids contained 155-bp monomers as well as
incomplete CentO monomers. Moreover, when only
a single primer of the CentO satellite (either forward
or reverse primer) was used in PCR reactions, ladder
bands were produced (data not shown). This suggests
that some monomers may be connected in inverted
orientation among the genome.
According to the sequence length and structure, the
CentO monomers from O. punctata can be classified
into three groups: 155 bp, 165 bp, and incomplete
CentO subfamilies. The 165-bp subfamily has a
10-nucleotide insertion (TTT ATA GGC A) compared
with the 155-bp monomer (Fig. 2). An initial search in
GenBank using several monomers of CentO satellite
from O. punctata revealed high sequence identity with
155- and 164-bp satellite repeats of pRCS2 (G in Fig. 2)
isolated from O. sativa. Detail alignment of the CentO
sequences from O. punctata and those from O. sativa
revealed small conserved regions (such as GGT GCG
A) as well as hypervariable regions with both single
base deletion and substitution (indicated by gray
rectangle below the sequence in Fig. 2). However,
no single base insertion was found in our limited sequence set. Moreover, we could not identify any monomer variants specific to either species, indicating
the CentO monomers from O. punctata and from O.
sativa have limited divergence.
Quantification of the CentO Satellite in
Different Centromeres
The 5S ribosomal DNA (rDNA) is organized in
tandem arrays with homologous units, forming clusters on one rice chromosome (Kamisugi et al., 1994),
and the sizes of 5S rDNA repeat blocks are significantly different between the indica and japonica rice
genome, which suggested that 5S rDNA repetitive sequences underwent dramatic changes in amount between the two rice subspecies (Ohmido et al., 2000). To
determine the locus of 5S rDNA in O. punctata, the 5S
rDNA and CentO satellite DNA were labeled with
biotin-11-dUTP or digoxigenin-16-dUTP, respectively,
and probed together to the pachytene chromosomes
of O. punctata. The 5S rDNA probe localized to a single
location close to the centromere of chromosome 11.
The single 5S rDNA locus allowed us to precisely
measure the length of this 5S locus using fiber-FISH
(Fig. 1E). Ten fiber-FISH signals were measured with
an average length of 136.01 6 4.13 mm, which corresponds to 436.59 6 13.28 kb according to a 3.21 kb/mm
conversion rate (Cheng et al., 2002b).
The CentO satellite signals were generally strong
and highly specific to the centromere of each pachytene chromosome. We used FISH signal intensities to
estimate the length of the CentO array on chromosome 11. Five slides with pachytene chromosomes of
O. punctata were probed with digoxigenin-16-dUTPlabeled CentO and biotin-11-dUTP-labeled a0025K19,
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Zhang et al.
Figure 1. FISH analysis of the BAC clone related
to centromeres in O. punctata. A, Chromosomes
in a pachytene cell of O. punctata probed by
02M23 (green signals). B, The metaphase I chromosomes of O. punctata probed by 02M23
(green signals), showing the signals are located
in stretched regions of the bivalent. C, The
pachytene chromosomes of O. punctata probed
by digoxigenin-16-dUTP-labeled CentO (red signals) and biotin-11-dUTP-labeled a0025K19 (green
signal, arrow), a marker specific to the short arm
of chromosome 11. D, The pachytene chromosomes of O. punctata were probed by digoxigenin-16-dUTP-labeled 5S rDNA (red signal) and
biotin-11-dUTP-labeled a0025K19 (green signal,
arrow). E, The extended DNA fibers probed by
biotin-11-dUTP-labeled 5S rDNA. Bars in A to D,
5 mm. Bar in E, 10 mm.
a marker specific to the short arm of chromosome
11 (Fig. 1C). Another set of five slides was probed with
digoxigenin-16-dUTP-labeled 5S ribosomal RNA
(rRNA) gene and biotin-11-dUTP-labeled a0025K19
using the same hybridization and signal developing
conditions (Fig. 1D). Fifty FISH signals of CentO from
chromosome 11 and the 5S rDNA locus were respectively measured for intensity using IPLab Spectrum
software. By comparing the signal intensities of both
CentO and 5S rRNA gene of chromosome 11, the
amount of CentO from chromosome 11 was estimated
to be 1.50 6 0.73 Mb.
The 12 O. punctata pachytene chromosome pairs
could be unambiguously identified based on pachytene morphology as well as chromosome-specific BAC
markers. By comparison of the FISH signal intensities
of CentO from different centromeres with those from
chromosome 11, the amount of CentO in every centromere was estimated using IPLab Spectrum software
(Table I). Among the 12 O. punctata centromeres,
chromosome 2 has the highest amount of CentO,
approximately 1.67 Mb, while chromosome 10 has the
smallest amount at 1.23 Mb.
Distribution and Organization of the CentO Satellite
and the CRR in the O. punctata Genome
Previous studies demonstrated that the centromeres
of grass species contain a Ty3/gypsy class of retrotransposon family (Miller et al., 1998; Presting et al.,
1998; Langdon et al., 2000). In O. sativa, CRR elements
preferentially inserted into CentO satellite arrays
(Cheng et al., 2002a). This centromere preferential insertion was verified by sequence analysis of the complete centromeres of both chromosome 8 (Nagaki et al.,
2004; Wu et al., 2004) and chromosome 4 (Zhang et al.,
2004). To reveal the distribution of CRR elements in
O. punctata, six subclones containing CRR elements, including pRCS1, pRCH1, pRCH2, pRCH3, pRCE1, and
pRCE2 (Dong et al., 1998), were labeled together and
probed to the pachytene chromosomes of O. punctata.
We also labeled CentO probe in a different color to
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Centromeres of Oryza punctata
Figure 2. Sequence comparison between different CentO variant monomers derived from O. punctata (A–F) and O. sativa (G),
respectively. From top to bottom: A and B are CentO monomers derived from 02M23 subclones; C, D, E, and F are CentO
monomers generated from CentO PCR segment clones; G is pRCS2 plasmid clone. Sequence conservation between different
CentO monomers is highlighted by dark shading. The gray rectangle represents hypervariable sequences among different CentO
monomers. Asterisks represent digital numbers 10, 30, 50, etc.
track the centromeres of each pachytene chromosome.
In general, the CRR signals on each chromosome are
mainly located in a region covering both sides of the
CentO signal (Fig. 3, A–F). Some dispersed weak
signals could also be detected uniformly on the entire
length of the chromosomes. The densities and intensities of CRR signals on different pachytene chromosomes are not as strong as those derived from the
CentO probe. The amount of CRR is variable among
different centromeres. Chromosome 3 and 12 showed
strongest signals, while chromosome 2 and 9 showed
weakest signals. The CRR signals on the centromere of
chromosome 5 were brighter and more compact than
those on the other centromeres. All these data indicated that the CRR elements are enriched in the centomeres, but they are not centromere specific.
We also labeled CRR and CentO in different colors
and probed together to extended DNA fibers prepared
from O. punctata. The CentO signals were bright and
contiguous; however, the CRR signals are weak and
consist of two to three continuous dots. Most CentO
signals are very long and are sometimes interrupted
by a few dots of CRR signal (Fig. 3G). The majority of
CRR signals are not inserted into the CentO signals but
are normally located on both sides of the CentO tracks
or are not associated with CentO. The fiber-FISH data
agree with the pachytene chromosome FISH data,
showing that CRR preferentially inserted into CentO
and its outside regions. We also found a few long tracks
of CRR signals, which are more bright and contiguous
than those of the other regions. According to the
pachytene chromosome FISH data, they might be generated from the centromere region of chromosome 5
(Fig. 3H).
Determination of the Key Functional Elements of
O. punctata Centromere by CENH3 Immunostaining
The centromere-specific location of repetitive DNA
elements often leads to the suggestion that these
repeats are required for centromere function. These
repetitive arrays have been postulated to form essential higher-order structures, bind key centromeric
proteins, or serve as targets for critical DNA modification. Thus, identifying the DNA sequences that
interacted with CENH3 is an effective approach to
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Zhang et al.
Table I. Distribution of the CentO satellite among different
centromeres in O. punctata
Distribution values are based on 10 measurements for each data
point.
Chromosome No.
Relative Intensity of
FISH Signala
Centromeric Satellite
Repeat Content
Mb
1
2
3
4
5
6
7
8
9
10
11
12
0.94
1.11
0.98
0.95
0.97
0.99
1.02
0.96
1.05
0.82
6
6
6
6
6
6
6
6
6
6
1
0.92 6
0.38
0.40
0.53
0.46
0.28
0.23
0.58
0.47
0.31
0.49
0.55
1.41
1.67
1.47
1.43
1.45
1.48
1.52
1.44
1.57
1.23
1.50
1.38
6
6
6
6
6
6
6
6
6
6
6
6
0.58
0.61
0.80
0.69
0.42
0.34
0.87
0.71
0.46
0.74
0.73b
0.83
a
The intensity of the FISH signal in centromere 11 is calibrated as 1,
and the intensity of the signals in the other centromeres are converted
b
into relative values.
The physical size of the CentO array in
centromere 11 is measured by comparing the intensities of CentO
signal and 5S signal on chromosome 11. The sizes of the CentO loci in
other centromeres are calculated based on the values of relative
fluorescence intensity.
recognize specific DNA sequences involved in centromere function.
We stained rice pachytene chromosomes with the
rice anti-CENH3. After recording the immunostaining
signals, the pachytene chromosomes were sequentially probed with CentO and CRR probes, respectively (Fig. 4). In each pachytene spread of O. punctata,
we observed 12 immunostaining signals with similar
size and intensity. For most of the centromeres, the
regions of anti-CENH3 signals were smaller than those
of CentO signals. But there are also one or two centromeres with less CentO content, the antibody to CENH3
signals are almost overlapped with CentO signals. The
signals generated from CRR are mainly located on
both sides of the CENH3 signals. Only a few of the
small dots from CRR signals are located in the CENH3
signals. The results suggested that the 165-bp CentO
satellite repeats are the major functional elements of
different centromeres in O. punctata.
sine of its recognition site allowing detection of CpG
and CpNpG methylation, whereas MspII is only inhibited by methylation of the 5# cytosine allowing
detection of CpNpG methylation. We found that MspII
can cleave the genomic DNA of O. punctata and produce ladder bands, while HpaII cannot digest the O.
punctata genomic DNA, indicating that the internal
cytosines in the CCGG sites of CentO are extensively
methylated in O. punctata (Fig. 5A).
To test whether both strands of CentO repeats are
transcribed, we conducted strand-specific RT-PCR and
northern analysis. For RT-PCR analysis, specific primer pairs were designed to generate top and bottom
strand-specific cDNA (see ‘‘Materials and Methods’’).
The cDNAs were used as template to do PCR amplification. Ladder banding patterns with sizes ranging
from 100 bp to 1 kb were produced from these reactions. Band patterns were different based on the
template used (Fig. 5B). The top strand transcripts of
CentO generated bigger and more abundant bands
compared with those from bottom strand transcripts.
Northern analysis showed that the CentO top and
bottom single-strand riboprobes hybridize to the total
RNA of O. punctata. In this case, no obvious difference
for the band patterns could be detected between the
two strands (Fig. 5C). Both RT-PCR and northern
analysis indicated that the two strands of CentO are
extensively transcribed.
The simultaneous presence of forward and reverse
transcripts may activate the RNA interference (RNAi)
pathway and produce small interfering RNA (siRNA).
To verify whether the transcribed CentO repeats were
processed into siRNA, we conducted small RNA
northern hybridization using strand-specific riboprobes of CentO. We could detect two types of siRNA,
21 to 22 nucleotides and 24 to 25 nucleotides in size,
with both top and bottom probes, respectively (Fig. 5,
D and E). The result suggested that transcripts of both
strands of CentO could generate two classes of siRNA.
We also found that the amount of the two classes of
siRNA in different tissues was different. For example,
the amount of the longer siRNAs (25 nucleotides) is
almost the same as that of shorter siRNA (22 nucleotides) in callus, while the longer siRNA is more
abundant than the shorter siRNA in spikelets.
DISCUSSION
CentO Is Highly Methylated and Actively Transcribed,
Producing Top and Bottom RNA in O. punctata
The Centromeric Elements and Their Relationship
with Genome Diversity
In general, methylation is preferentially targeted to
repeated sequences such as centromere-associated repeats, rRNA encoding repeats, and transposable elements (Bender, 2004). To investigate the methylation of
the CentO repeat in O. punctata, we applied southernblot analysis using HpaII and MspII digestion. Both
HpaII and MspII recognize the CCGG sequence; however, HpaII is inhibited by methylation of either cyto-
Exploiting the genome diversity within Oryza species is one of the most important post-sequencing research subjects. Plant centromeres have DNA elements
that are shared across species, yet they diverge rapidly
through large- and small-scale changes (Hall et al.,
2004). The association between repetitive DNA and centromere structure and function is a long-standing and
intriguing question. Although the primary centromeric
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Centromeres of Oryza punctata
Figure 3. Distribution and organization of the CentO satellite and the CRR
in O. punctata chromosomes. A, FISH
mapping of the CentO satellite (red)
and the CRR elements (green) on meiotic pachytene chromosomes. B, Both
signals of CentO satellite (red) and the
CRR elements (green) digitally separated from A. C to F, Images of individual rice centromeres of pachytene
chromosomes hybridizated with the
CentO satellite (red) and the CRR
probe (green). The composite images
are separated digitally into images of
FISH signals derived from the CentO
satellite (D), images of FISH signals
derived from the CRR probe (E), and
merged images of FISH signals from
both probes (F). G and H, Fiber-FISH
with CRR (green) and CentO (red) as
probes. G, Representative fiber-FISH
signals of most CentO regions are
inserted with a few CRR elements. H,
The biggest insertion of CRR in the
O. punctata genome. Bars in A and B,
5 mm. Bars in G and H, 10 mm.
DNA sequences vary among diverse species, the
molecular architectures of these sequences are generally the same: blocks of tandem repeated DNA bracketed by clusters of various classes of retrotransposons
(Henikoff et al., 2001; Jiang et al., 2003). The presence
of satellite DNA and other repetitive DNA in the
centromeres is thought to favor the assembly of
the kinetochore, thereby ensuring the efficient and
timely meiotic and mitotic segregation of chromosomes (Tyler-Smith et al., 1993). A complete understanding of centromere function requires identifying
centromeric DNA components and determining their
organization in vivo. In this study, we show that
CentO and CRR sequences are highly intermingled
throughout all centromeres analyzed in O. punctata.
The centromeric satellite arrays in CentO also display
a high degree of sequence homogeneity, and it is characterized by a chromosome-specific higher-order organization that is similar to those of human (Willard
and Waye, 1987), Drosophila (Sun et al., 1997), maize
(Zea mays; Ananiev et al., 1998), and Arabidopsis
(Martinez-Zapater et al., 1986).
In most cases, centromeric satellites are present in
vast quantities, but the sizes of reported functional
centromeres in a variety of species are almost similar.
The centromere of a Drosophila minichromosome is
contained within a 420-kb region of centromeric repetitive DNA (Murphy and Karpen, 1995; Sun et al.,
1997), and a centromere misdivision study in maize
suggested that 500 kb of centromeric repeats is the
minimum for fully functional B centromeres (Kaszas
and Birchler, 1998). Our results show that centromeres
of all individual chromosomes in O. punctata contain
similar amounts of the CentO satellite sequences,
ranging from 1.23 to 1.67 Mb (Table I). In O. sativa,
several centromeres contain only a limited amount of
the CentO satellites, especially chromosome 8, which
has only approximately 60 kb CentO. For these centromeres, the CentO satellites act only as part of the
functional centromere, and the sequences flanking the
CentO array are recruited as part of the centromere
(Nagaki et al., 2004). In this study, the CENH3 antibody localizes similarly to all O. punctata centromeres,
and these signals are fully covered by CentO FISH
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Zhang et al.
Figure 4. Association of CENH3 with CentO and
CRR in O. punctata. A, The pachytene chromosomes of O. punctata are stained with antiCENH3. B, The same cell is probed with CentO.
C, The same cell is sequentially probed with CRR.
D, Merged signals of CENH3 (red) and CentO
(green). E, Merged signals of CENH3 (red) and
CRR (green). F, Merged signals of CENH3 (blue),
CentO (red), and CRR (green). Bar, 5 mm.
signals. This indicates that the CentO cores of all centromeres may be large enough for formation of fullsized kinetochores in O. punctata.
We also found that the contents of both CentO and
CRR in O. punctata showed significant differences with
those in O. sativa. In O. sativa (variety Nipponbare),
most centromeres contain ,1 Mb of CentO, and the
total amount of CentO satellite is approximately 7 Mb,
accounting for 1.6% of the rice genome (Cheng et al.,
2002a). In O. punctata, most centromeres contain .1 Mb
of CentO, and the total amount of CentO is approximately 17.55 Mb, accounting for 3.3% of the BB
genome. The CRR also showed a much wider distribution in O. punctata than in O. sativa. As the genome
size of O. punctata is about 535 Mb, compared with 438
Mb for O. sativa (variety Nipponbare; Uozu et al.,
1997), the centromeric regions in O. punctata contain
significantly more repetitive DNA than those in O.
sativa. As most of the basal splits within Oryza were
diverged approximately 9 million years ago (Guo and
Figure 5. Analysis of CentO methylation and transcription level in O. punctata. A, Southern-blot
analysis of the 165-bp CentO. Equivalent amounts
of genomic DNA digested with HpaII (left) and MspI
(right) and probed with CentO. B, Results of RT-PCR.
Using total RNA as reverse transcriptional templates,
top (lane 1) and bottom strand CentO transcripts
(lane 2) were synthesized with either CentO oligo as
the reverse transcriptional primer, then the CentO
amplification was conducted with cDNA sample as
template in the presence of CentO primer pairs. C,
RNA bolt hybridization of total O. punctata RNA
using single-stranded probes derived from either
strand of CentO. Each lane was loaded with 30 mg
total RNA and hybridized with either single stranded
riboprobe paired with bottom (lane 1) or top (lane 2)
strands of CentO, respectively. D and E, Total small
RNA was extracted from callus (right lane) and
spikelet (left lane) tissues, respectively. Each lane
was loaded with 20 mg total RNA, and two classes of
siRNA (22 and 25 nucleotides) derived from bidirectional transcription of centromeric satellite CentO
were detected by hybridization with hydrolyzed top
(D) and bottom (E) RNA transcribed from each strand
of CentO. nt, Nucleotides.
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Centromeres of Oryza punctata
Ge, 2005), it showed that the copy number of centromeric repeats had been changed a lot among different
rice species.
The centromere-specific satellites are highly divergent in Oryza species containing different genomes.
Results of PCR combined with southern analysis using rice centromeric sequences confirm that wild rice
species, such as the AA, CC, BBCC, CCDD, and EE
genomes, contain CentO tandem repeats and other
centromeric transposable elements (Hass et al., 2003).
In this study, we found that the BB genome of O.
punctata also has CentO similar satellites. According to
the sequence length and structure, the CentO monomers from O. punctata could be classified into three
groups: 155 bp, 165 bp, and incomplete CentO. The
165-bp CentO has a 10-nucleotide insertion (TTT ATA
GGC A) compared with the 155-bp monomer. The 165-bp
monomers are the dominant element in O. punctata
revealed by both PCR and southern analysis. CentO is
not the major centromeric element in the CC genome
and is only present in a few centromere regions (our
unpublished data). Among the six diploid genomes
(AA, BB, CC, EE, FF, and GG) of the Oryza genus, BB
and EE genome species contain the CentO satellite in
the centromeres, while the CC, FF, and GG genome
species do not contain CentO, and it is likely that the
centromeres in these species contain different satellite
DNA families. It seems that the BB and EE genomes
have a closer evolutionary relationship with the AA
genome compared with that of the CC, FF, and GG
genomes.
ates CENH3 localization, providing strong evidence
that transcription is involved in establishing the centromere state (Chen et al., 2003). Mutations in the
RNAi pathway release pericentromeric repeats from
transcriptional repression and disturb normal centromere function (Volpe et al., 2002, 2003; Hall et al.,
2003). Topp et al. (2004) demonstrated that centromeric
RNA originating from the transcription of centromere
repeats remains bound to the centromere or kinetochore complex. All these data suggest that centromere
transcription may contribute to both the deposition
and stabilization of kinetochore chromatin structure.
In this study, we found that CentO in O. punctata is
highly methylated, and both strands of CentO satellites are actively transcribed and produce siRNA in
O. punctata. Although no direct evidence about the
relationship between the two processes has been
obtained yet, the siRNA transcribed from the CentO
satellites may target to the enzymes related to chromatins so as to methylate histone H3 and keep the
centromere in a heterochromatic status, just like the
same process in S. pombe (Grewal and Klar, 1997;
Guarente, 2000). The heterochromatic structure is
important to inhibit the recombination between homologous repeats, stabilize the repetitive centromeric
sequences, and keep the centromere as an entire functional centromere.
Methylation and Transcription of the
Centromeric Elements
Oryza punctata (accession no. 103896), a diploid wild Oryza species, was
used for cytological studies. Young panicles were collected and fixed in 3:1
(100% ethanol:glacial acetic acid) Cannoy’s solution. Microsporocytes at the
pachytene stage were squashed in acetocarmine solution and slides were
stored at 220°C until use. After soaking in liquid nitrogen and removing the
coverslips, the slides were dehydrated through an ethanol series (70%, 90%,
and 100%) prior to being applied in FISH. The FISH procedure was conducted
according to Jiang et al. (1995). Image capture and measurement were performed as described by Zhang et al. (2005). Genomic DNA fiber-FISH preparation and two-color fiber-FISH signal detection procedures were as described
previously (Jackson et al., 1998).
The plasmid clone, pTa794, with 410-bp insertion containing about three
monomers of the coding sequences for the 5S rRNA genes of wheat (Triticum
aestivum; Gerlach and Dyer, 1980) was used to quantify the size of 5S rDNA in
O. puncata. Another plasmid clone, pRCS2, with 639-bp insertion containing
four different monomers of CentO was also used as FISH probe to quantify the
CentO content of each centromere (Dong et al., 1998).
Centromere regions may require methylation to
maintain their heterochrmatin formation. Patients
with immunodeficiency, centromere instability, and
facial abnormalities carry a mutation in the de novo
methyltransferase Dnmt3b gene and undergo chromosome breaks in the heterochromatin adjacent to certain centromeres (Hansen et al., 1999; Xu et al., 1999).
The methylation status of the centromere region may
also be critical for the assembly of binding proteins
(CENPs). For example, human cells treated with
5-aza-2#-deoxycytidine experience a redistribution of
CENP-B protein (Mitchell et al., 1996).
The accumulated evidence suggests that RNAi facilitates the targeting of chromatin-modifying complexes to specific regions of the genome (Grewal and
Moazed, 2003). RNAi was first found in Caenorhabditis
elegans as a mechanism of gene silencing. The related
processes were identified in Neurospora as well as in
plants. Recently, the evidence has also been obtained
that RNAi in these processes correspond to heterochromatic centromere repeat sequences. Volpe et al.
(2002) demonstrated that RNAi is required to establish
a heterochromatic state in the pericentromeric regions
flanking the (CENH3-binding) centromere core of S.
pombe. Ams2, a transcription factor of S. pombe, medi-
MATERIALS AND METHODS
Materials and Cytology
BAC Library and Subclone Construction and
Sequencing Analysis
Megabase-sized plant genomic DNA embedded in low-temperature agarose plugs was extracted from 4- to 5-week-old greenhouse-grown O. punctata
seedlings according to Peterson et al. (2000). The BAC vector pBeloBAC11 was
used to construct the HindIII library of O. punctata using published protocols
(Woo et al., 1994). Partial HindIII digestion of high Mr DNA, size selection, and
ligation were conducted as described by Peterson et al. (2000). Positive recombinant colonies were stored individually in 384-well microtiters. The BAC
library was screened with 32P-labeled genomic DNA of O. punctata, and positive BACs were further used as FISH probes.
DNA from BAC 02M23 was extracted, digested with EcoRI and fractionated by electrophoresis in 0.8% agarose gels. DNA fragments with sizes
ranging from 2 to 4 kb were extracted with the Qiaquick gel extraction kit
Plant Physiol. Vol. 139, 2005
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Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Zhang et al.
(Qiagen) and cloned into T-easy vectors (Promega). Using genomic DNA as
template, ladder bands were produced by PCR using CentO primers [foward
primer, 5#-CAA AA (A/C) TCA TGT TT (TG) GGT G (ATGC)-3#; reverse
primer, 5#-GGA C (A/C) T A (T/A) (A/T) G (G/T) A GTG (G/T) AT (AGTC)3#]. The PCR products were cloned into the T-easy vector for use in sequencing
and FISH probes. Sequence alignments were edited manually and displayed
using DNASTAR software.
Total RNA Extraction and RT-PCR Analysis
Total RNA was extracted from leave tissues of O. punctata using RNeasy
plant mini kits (Qiagen). RNA samples were treated with 20 units of RNasefree RQ1 DNase in the presence of 20 units of RNasin at 37°C for 2 h. After
extraction twice with phenol, the RNA was precipitated with ethanol and then
dissolved in RNase-free double-distilled water.
We named the strand of CentO that sequences were homologous to the
165-bp CentO monomer sequence as top strand (Cheng et al., 2002a); thus,
another strand of CentO paired with top strand of CentO was designated as
bottom strand. Top- and bottom-specific primers were designed according to
the conserved sequences of CentO. First-strand cDNA was prepared for the
respective analysis of top and bottom CentO transcripts. Using total RNA as
template and either CentO oligo as the primer, reverse transcriptions of firststrand cDNA were performed with SuperScript II reverse transcriptase
(200 units; Invitrogen). The top and bottom reverse transcriptase reactions
were each incubated at 42°C for 1 h. CentO cDNA amplification was performed on 50 ng of the cDNA samples using the CentO primer pairs. PCR
conditions were as follows: 4 min at 94°C, 35 cycles of 40 s at 94°C, 1 min at
56°C and 90 s at 72°C, and 10 min at 72°C. The RT-PCR products were further
cloned into T-easy vector for sequence analysis.
Immunodetection of CENH3
Fresh young panicles were fixed in 4% (w/v) paraformaldehyde for 5 min
at room temperature. Anthers in the proper stage were squashed on a slide
with phosphate-buffered saline (PBS) solution and covered with a coverslip.
After soaking in liquid nitrogen and removing the coverslip, the slide was
dehydrated through an ethanol series (70%, 90%, and 100%) prior to being
used in immunostaining. Slides were then incubated in a humid chamber at
37°C for 0.5 h in the rabbit antibody to CENH3 primary sera (Biosource
International) diluted 1:5000 in TNB buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M
NaCl, and 0.5% blocking reagent). After three rounds of washing in PBS,
Texas-red conjugated goat anti-rabbit antibody was added to the slides. The
chromosomes were counterstained with 4#,6-diamidino-phenylindole in an
antifade solution (Vector Laboratories). After recording of the immunostaining signal, the same slides were washed in PBS buffer, dehydrated in the
ethanol series, and probed with digoxigenin-16-dUTP-labeled CentO and
CRR sequentially using the same FISH procedure described above.
ACKNOWLEDGMENTS
We thank Steven Henikoff for providing the anti-OsCENH3 peptide
antibody against rice CENH3 and Jiming Jiang and Robert M. Stupar for
critical reading of the manuscript. This work was supported by grants from
the Ministry of Sciences and Technology of China (2002AA225011 and
2005CB120805), the Chinese Academy of Sciences, and the National Natural
Science Foundation of China (30325008, 3017056, 30325015, and 30428019).
Received April 14, 2005; revised June 22, 2005; accepted June 22, 2005;
published August 19, 2005.
DNA and RNA Gel-Blot Analysis
Total genomic DNA was isolated from the leave tissues of O. punctata.
Genomic DNA was digested with 20 units of restriction enzyme in the
recommended buffer (New England Biolabs), such as methylation-sensitive
and methylation-insensitive isoschizomers (HpaII and MspII). The digested
DNA was fractionated by electrophoresis in 0.8% agarose gel overnight. After
depurinating in 0.25 N HCl, DNA fragments were transferred to Hybond-N1
membranes (Amersham).
Northern analyses were also performed after removing DNA using RNasefree RQ1 Dnase. Aproximately 30 mg of total RNA per lane was electrophoresed on 1% agarose-formaldehyde gels and capillary blotted onto
Hybond-N1 membranes (Amersham).
DNA probes used in southern analysis were labeled with a-32P-dCTP
using the primer-a-gene labeling system (Promega). Two riboprobes for the
CentO top and bottom strands were labeled by annealing the respective
CentO primers to total RNA and labeling with a-32P-CTP using the riboprobe
in vitro transcription systems (Promega).
Southern or northern hybridization conditions were the same. The membranes were incubated in standard prehybridization solution at 65°C for 6 h
and then hybridized with 32P-labeled CentO probe at 65°C for 12 h. Following
hybridization and sequential washing, the radioactive membranes were then
exposed to x-ray film.
Detection of siRNA
RNA preparations enriched for small-sized RNA were obtained as described by Park et al. (2002). Detection of small RNA was performed by
following the protocol of Hutvagner et al. (2000) with a few modifications.
Briefly, 30 mg total testing RNA was loaded in each lane of a 15% denaturing
polyacryamide gel containing 8 M urea, electrophoresed, and electroblotted to
Hybond N1 membrane (Amersham) using 0.5 3 Tris-borate/EDTA buffer.
The riboprobes were transcribed by RNA polymerase (Promega) using the
CentO linearized fragment containing T7 promoter obtained by PCR as
a template and labeled with 32P-UTP to produce a top or bottom RNA probe
for hybridization. After synthesis, the labeled RNA was partially hydrolyzed
by NaHCO3/Na2CO3 (80 mM/120 mM) incubated at 60°C for 1 h. Hybridization was performed in 125 mM sodium phosphate buffer (pH 7.2) containing
50% deionized formamide, 7% SDS, and 250 mM sodium chloride. After
overnight hybridization, the membrane was washed twice in 2 3 SSC at 42°C
for 15 min each. Exposure to x-ray film overnight was sufficient to detect
signals from small RNA.
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