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First published online August 19, 2005; 10.1104/pp.105.064147

Plant Physiology 139:306-315 (2005)
© 2005 American Society of Plant Biologists

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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

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.)


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
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, 1990Go, 1998Go), 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., 2001Go; Jiang et al., 2003Go). Centromeric DNA sequences are often diverged among closely related species. For example, the approximately 171-bp {alpha}-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, 1989Go). 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., 2000Go, 2001Go; Hosouchi et al., 2002Go). In addition to the centromeric satellite repeats, centromeres of many eukaryotes are enriched with centromere-specific retrotransposons (Jiang et al., 2003Go).

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., 2001Go; Sullivan et al., 2001Go) and recently in plants (Talbert et al., 2002Go; Zhong et al., 2002Go; Nagaki et al., 2004Go). Blocks of CENH3-associated nucleosomes and regular H3-associated nucleosomes are linearly interspersed within functional centromeres (Blower et al., 2002Go). 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., 2002aGo). 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., 2004Go; Wu et al., 2004Go; Zhang et al., 2004Go). Moreover, several active genes were found in the functional centromere domain of chromosome 8, within the CENH3 binding sites (Nagaki et al., 2004Go).

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., 1999Go). 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., 2003Go). 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
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

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.



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  		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 µm. Bar in E, 10 µm.

 
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 CentO-derived 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.



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  		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.

 
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., 1994Go), 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., 2000Go). 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 ± 4.13 µm, which corresponds to 436.59 ± 13.28 kb according to a 3.21 kb/µm conversion rate (Cheng et al., 2002bGo).

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-dUTP-labeled CentO and biotin-11-dUTP-labeled a0025K19, 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 ± 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.


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  		Table I. Distribution of the CentO satellite among different centromeres in O. punctata

Distribution values are based on 10 measurements for each data point.

 

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., 1998Go; Presting et al., 1998Go; Langdon et al., 2000Go). In O. sativa, CRR elements preferentially inserted into CentO satellite arrays (Cheng et al., 2002aGo). This centromere preferential insertion was verified by sequence analysis of the complete centromeres of both chromosome 8 (Nagaki et al., 2004Go; Wu et al., 2004Go) and chromosome 4 (Zhang et al., 2004Go). 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., 1998Go), were labeled together and probed to the pachytene chromosomes of O. punctata. We also labeled CentO probe in a different color to 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.



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  		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 µm. Bars in G and H, 10 µm.

 
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 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.



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  		Figure 4. Association of CENH3 with CentO and CRR in O. punctata. A, The pachytene chromosomes of O. punctata are stained with anti-CENH3. 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 µm.

 

CentO Is Highly Methylated and Actively Transcribed, Producing Top and Bottom RNA in O. punctata

In general, methylation is preferentially targeted to repeated sequences such as centromere-associated repeats, rRNA encoding repeats, and transposable elements (Bender, 2004Go). To investigate the methylation of the CentO repeat in O. punctata, we applied southern-blot analysis using HpaII and MspII digestion. Both HpaII and MspII recognize the CCGG sequence; however, HpaII is inhibited by methylation of either cytosine 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).



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  		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 µg 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 µg 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.

 
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
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 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 

The Centromeric Elements and Their Relationship with Genome Diversity

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., 2004Go). The association between repetitive DNA and centromere structure and function is a long-standing and intriguing question. Although the primary centromeric 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., 2001Go; Jiang et al., 2003Go). 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., 1993Go). 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, 1987Go), Drosophila (Sun et al., 1997Go), maize (Zea mays; Ananiev et al., 1998Go), and Arabidopsis (Martinez-Zapater et al., 1986Go).

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, 1995Go; Sun et al., 1997Go), 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, 1998Go). 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., 2004Go). In this study, the CENH3 antibody localizes similarly to all O. punctata centromeres, and these signals are fully covered by CentO FISH signals. This indicates that the CentO cores of all centromeres may be large enough for formation of full-sized 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., 2002aGo). 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., 1997Go), 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 Ge, 2005Go), 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., 2003Go). 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.


Methylation and Transcription of the Centromeric Elements

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., 1999Go; Xu et al., 1999Go). 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., 1996Go).

The accumulated evidence suggests that RNAi facilitates the targeting of chromatin-modifying complexes to specific regions of the genome (Grewal and Moazed, 2003Go). 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)Go 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, mediates CENH3 localization, providing strong evidence that transcription is involved in establishing the centromere state (Chen et al., 2003Go). Mutations in the RNAi pathway release pericentromeric repeats from transcriptional repression and disturb normal centromere function (Volpe et al., 2002Go, 2003Go; Hall et al., 2003Go). Topp et al. (2004)Go 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, 1997Go; Guarente, 2000Go). 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.


   MATERIALS AND METHODS
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 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Materials and Cytology

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 –20°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)Go. Image capture and measurement were performed as described by Zhang et al. (2005)Go. Genomic DNA fiber-FISH preparation and two-color fiber-FISH signal detection procedures were as described previously (Jackson et al., 1998Go).

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, 1980Go) 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., 1998Go).


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)Go. The BAC vector pBeloBAC11 was used to construct the HindIII library of O. punctata using published protocols (Woo et al., 1994Go). Partial HindIII digestion of high Mr DNA, size selection, and ligation were conducted as described by Peterson et al. (2000)Go. 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 (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 RNase-free 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., 2002aGo); 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 first-strand 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.


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-N+ membranes (Amersham).

Northern analyses were also performed after removing DNA using RNase-free RQ1 Dnase. Aproximately 30 µg of total RNA per lane was electrophoresed on 1% agarose-formaldehyde gels and capillary blotted onto Hybond-N+ membranes (Amersham).

DNA probes used in southern analysis were labeled with {alpha}-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 {alpha}-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)Go. Detection of small RNA was performed by following the protocol of Hutvagner et al. (2000)Go with a few modifications. Briefly, 30 µg total testing RNA was loaded in each lane of a 15% denaturing polyacryamide gel containing 8 M urea, electrophoresed, and electroblotted to Hybond N+ membrane (Amersham) using 0.5x 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 2x SSC at 42°C for 15 min each. Exposure to x-ray film overnight was sufficient to detect signals from small RNA.


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; returned for revision June 22, 2005; accepted June 22, 2005.


   FOOTNOTES
 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.064147.

* Corresponding author; e-mail zkcheng{at}genetics.ac.cn; fax 0086–10–6487–3428.


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