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Functional characterization of two flap endonuclease-1 homologues in rice
Gene 314 (2003) 63 – 71 www.elsevier.com/locate/gene
Functional characterization of two flap endonuclease-1 homologues in rice Seisuke Kimura a, Tomoyuki Furukawa a, Nobuyuki Kasai a, Yoko Mori a, Hiroko K. Kitamoto b, Fumio Sugawara a, Junji Hashimoto b, Kengo Sakaguchi a,* a
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan b National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan Received 13 February 2002; received in revised form 16 April 2003; accepted 12 May 2003 Received by T. Sekiya
Abstract Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed. D 2003 Elsevier B.V. All rights reserved. Keywords: OsFEN-1a; OsFEN-1b; Rice; DNA replication and repair
1. Introduction DNA replication and repair are critical for maintaining genome stability. DNA replication and repair in higher plants has been examined recently in rice, Arabidopsis, wheat and maize (Furukawa et al., 2003; Kimura et al., 2000, 2001, 2002; Hays, 2002; Ishibashi et al., 2001; Britt, 1999; Britt et al., 1993; Culligan and Hays, 1997; Landry et al., 1997; Staplenton et al., 1997). However, in general, little is known about plant DNA replication, repair and the
Abbreviations: bp, base pair; cDNA, complementary DNA; EST, expressed sequence tag; FEN-1, flap endonuclease-1; mRNA, messenger RNA; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; UV, ultraviolet; RACE, rapid amplification of cDNA ends. * Corresponding author. Tel.: +81-4-7124-1501x3409; fax: +81-47123-9767. E-mail address: [email protected] (K. Sakaguchi). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00694-2
factors involved in these processes in plants. Studies are needed to fill this knowledge gap between plants and animals or yeasts. DNA replication and repair are in part dependent on the activity of a family of structure-specific endonucleases. The RAD2 family of nucleases includes XPG (Class I), FEN-1 (Class II) and Exo1 (Class III), and the proteins exhibit a range of substrate-specific exo- and endonuclease activities. Many of these proteins feature a range of nuclease activities that contribute to DNA replication and repair. RAD2 Class I consists of XPG-like proteins that cleave at the 3V-side of the damage-containing bubble structure formed during nucleotide excision repair (Calleja et al., 2001; Evans et al., 1997; Lieber, 1997). Class II is comprised of flap endonuclease-1 (FEN-1) which remove 5V-DNA flaps produced by polymerase strand displacement and are also responsible for DNA replication and DNA repair (Matsuzaki et al., 2002; Alleva and Doetsch, 1998; Freudenreichet al., 1998; Lieber, 1997;
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Johnson et al., 1995; Reagan et al., 1995; Murray et al., 1994). Class III is made up of the Exo1-like proteins found in yeast, Drosophila, and mammals. Exo1-like proteins appear to play roles in DNA recombination, mismatch repair, and DNA replication (Lee and Wilson, 1999; Lieber, 1997). We have concentrated on investigating plant enzymes belonging to the RAD2 nuclease family in the past several years and their relationships to DNA replication and repair (Furukawa et al., 2003; Kimura et al., 2000, 2001). In the process of the study, we found that rice plants have two different species of FEN-1, termed OsFEN-1a and OsFEN1b. In this report, we describe the functional characterization of two FEN-1 homologues in rice (Oryza sativa L. cv. Nipponbare).
2. Materials and methods 2.1. Molecular cloning of OsFEN-1b The rice expressed sequence tag (EST) database was searched to identify a complementary DNA (cDNA) clone with homology to the RAD2 nuclease family. Rice EST clone C30032 (GenBank accession no. AU108651) was found to have significant homology to OsFEN-1, which we have previously isolated (Kimura et al., 2000). The fulllength cDNA was isolated using the 5V-rapid amplification of cDNA ends (RACE) method. To distinguish the two FEN-1 homologues in rice, the previously found OsFEN-1 was designated as OsFEN-1a, and the newly found cDNA was designated as OsFEN-1b. The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence database with the accession numbers AB021666 and AB088391 for OsFEN-1a and OsFEN-1b, respectively. 2.2. Mapping and analysis of genomic structure The genomic loci of OsFEN-1a and OsFEN-1b were mapped on the high-density linkage map of rice according to the method of Harushima et al. The genomic sequence of OsFEN-1a was determined using polymerase chain reaction (PCR) amplification and sequencing. The genomic sequence of OsFEN-1b was downloaded from Monsant’s and Syngenta’s genome sequence database (Goff et al., 2002). 2.3. Northern hybridization Aliquots of 20 Ag of total RNA were resolved on 1.2% formaldehyde agarose gels and transferred onto nylon membranes (Hybond-N, Amersham). After prehybridization, the filters were probed with 32P-labeled probe at 42 jC for 16 h followed by washing twice with 2SSC+0.1% SDS at room temperature for 15 min, and three times with 0.1SSC+0.1% SDS at 65 jC for 20 min.
2.4. In situ hybridization Riboprobes for in situ hybridization were labeled with digoxigenin-11-rUTP using a DIG RNA Labeling Kit (Roche) according to the manufacturer’s protocol. The riboprobes were subjected to mild alkaline hydrolysis by heating at 60 jC in carbonate buffer and used at a concentration of 2 Ag/ml. Plant tissues from 10-day-old rice seedlings were fixed for 16 h at 4 jC with a mixture of 4% (W/ V) paraformaldehyde and 0.25% (V/V) glutaraldehyde in 50 mM sodium phosphate buffer (pH 7.2). The fixed tissues were dehydrated in a series of xylene and ethanol and embedded in paraffin (HISTPREP 568, Wako). Embedded tissues were sectioned at a thickness of 10 Am, and placed on microscope slides precoated with APS (3-aminopropyltriethoxysilane). Sections were deparaffinized with xylene and rehydrated through a graded ethanol series. They were subsequently pretreated with 10 mg/ml of proteinase K in 100 mM Tris – HCl, pH 7.5, and 50 mM EDTA at 37 jC for 30 min, dehydrated in a graded ethanol series, and dried under vacuum for 2 h. Hybridization and detection of hybridized riboprobes were performed as described by Kimura et al. (2000). 2.5. Sequence and phylogenetic analyses and molecular modeling Sequence and phylogenetic analyses were performed using GENETYX-MAC ver. 10 (Software Development). Modeled 3D structures for OsFEN-1a and OsFEN-1b proteins were generated automatically with the Swiss Model program http://www.expasy.ch/swissmed/SWISS-MODEL. html) (Guex et al., 1999; Guex and Peitsch, 1997) using the X-ray structure of Pyrococcus furiosus FEN-1 as a template (Hosfield et al., 1998). The structures were visualized using Insight II (Molecular Simulations). The secondary classifications were performed using the Kabsch Sander calculation.
3. Results and discussion 3.1. Identification and molecular cloning of OsFEN-1b, another homologue of FEN-1 from rice The rice EST database was searched to identify a plant RAD2 nuclease family member protein. Rice EST clone C30032 (GenBank accession no. AU108651) was found to have significant homology to FEN-1 (one of the members of the RAD2 nuclease family), and full-length cDNA was isolated as described in Materials and methods. The length of the cDNA was 1674 base pair (bp), and the open reading frame of the cDNA encoded a predicted product of 412 amino acid residues with a molecular mass of 45.7 kDa. Previously, we also isolated and characterized a rice homologue of FEN-1 which was designated as OsFEN-1
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(Kimura et al., 2000). As shown in Figs. 1 and 2, the cDNA was significantly similar to OsFEN-1, indicating that rice has two different species of FEN-1 homologue. Therefore, we designated the newly found OsFEN-1 cDNA as OsFEN-1b, and the previously found OsFEN-1 cDNA as OsFEN-1a to distinguish the two FEN-1 homologues. As described in the later part of this report, OsFEN-1b is not a pseudogene because the messenger RNA (mRNA) was detected by Northern hybridization analysis. The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence database with the accession numbers, AB021666 and AB088391 for OsFEN-1a and OsFEN1b, respectively. 3.2. Mapping and genomic structures of OsFEN-1a and OsFEN-1b The chromosomal locations of cDNAs of OsFEN-1a and OsFEN-1b on the linkage map of rice were determined by restriction fragment length polymorphism (RFLP) mapping (Harushima et al., 1998). The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and chromosome 3,
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respectively (Fig. 1A). These results clearly indicated that rice has two genes for FEN-1. The genomic sequences of OsFEN-1a and OsFEN-1b were analyzed as described in Materials and methods. Both genes have 17 exons and 16 introns (Fig. 1B). These exon patterns are very similar to each other, implying that OsFEN-1b was generated by genome duplication. 3.3. Comparison of amino acid sequences of OsFEN-1a and OsFEN-1b Fig. 2 shows a comparison of the deduced amino acid sequences of OsFEN-1a and OsFEN-1b. OsFEN-1b protein has 60.0% sequence identity with OsFEN-1a protein, and 45.5% identity with human FEN-1 (Nolan et al., 1996). Functional analysis of point mutations in the human FEN-1 active site revealed that Asp-34, Asp-86 and Glu-160 are important for substrate binding and cleavage, Glu-158, Asp179 and Asp-233 are essential for substrate binding, and Asp-181 is involved in substrate cleavage (Shen et al., 1997). All of these amino acid residues were conserved in OsFEN-1a and OsFEN-1b proteins (indicated by asterisks in Fig. 2).
Fig. 1. Mapping and genomic structure of the OsFEN-1a and OsFEN-1b. (A) Maps of OsFEN-1a and OsFEN-1b loci. (B) Genomic organizations of the OsFEN-1a and OsFEN-1b genes. Boxed areas are exons, lines are introns and 5Vand 3Vupstream regions. The open boxes correspond to the coding region and hatched boxes correspond to the untranslated region.
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Fig. 2. Alignment of the amino acid sequences of OsFEN-1a and OsFEN-1b. Common residues are boxed, and gaps added for sequence alignment are indicated with dashes. The two highly conserved regions, N domain and I domain, are shown. Asterisks indicate the amino acids essential for substrate binding or nuclease activity.
Much of the amino acid sequence conservation among the RAD2 nuclease family is concentrated within the three domains termed the N (N-terminal), I (Internal) and C (Cterminal) domains. (Lieber, 1997). As shown in Fig. 3, the N and I domains were highly conserved in the OsFEN-1b sequence, but the C domain was not conserved. The N and I domains of OsFEN-1b protein showed 74.3% and 79.7% sequence identity with those of OsFEN-1a, respectively (Fig. 3B). The C-terminal region of OsFEN-1b was larger than OsFEN-1a and human FEN-1 (Fig. 3B). 3.4. Modeled 3D structures for OsFEN-1a and OsFEN-1b The crystal 3D structures of P. furiosus FEN-1 and Methanococcus jannaschii have been reported (Hosfield et al., 1998; Hwang et al., 1998). This enables us to predict the 3D structures of OsFEN-1a and OsFEN-1b using the Swiss Model service provided on the ExPASy server (Guex et al., 1999; Guex and Peitsch, 1997). As shown in Fig. 4, comparison of the modelings suggests substantial conservation among the 3D structures of P. furiosus FEN-1, OsFEN-1a and OsFEN-1b proteins. According to this model, the 3D structures of the region from the N to the I domain are quite similar to each other. On the other hand, the 3D structure of the C domains are thought to differ among them. As described previously (Kimura et al., 2000), the OsFEN-1a protein has the activities of flap endonuclease and 5Vto 3Vexonuclease. Since the 3D structures are quite similar, OsFEN-1b protein is also thought to have the activities of flap endonuclease and 5V to 3Vexonuclease. 3.5. Phylogenetic analysis The RAD2 nuclease family includes XPG (Class I), FEN-1 (Class II) and EXO1 (Class III). Recently, we
Fig. 3. Comparison of conserved domain structures. (A) Schematics depict the protein structure and highly conserved domains (N, I, and C domain). (B) Pairwise comparison of the conserved domains of human FEN-1, OsFEN-1a, and OsFEN-1b.
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Fig. 4. Modeled 3D structure of OsFEN-1a and OsFEN-1b. (Left) The X-ray structure of P. furiosus FEN-1 was used as a template for modeling. (Center) Modeled 3D structure of OsFEN-1a protein (amino acids 12-346). (Right) Modeled 3D structure of OsFEN-1b protein (amino acids 18-336).
isolated and characterized a new RAD2 nuclease family member, OsSEND-1, that is thought to belong to Class IV (Furukawa et al., 2003). To determine the phylogenetic relationship between OsFEN-1b and other RAD2 nuclease family members, phylogenetic analysis was performed (Fig. 5). The phylogenetic tree was drawn based on the amino acid sequence of the N domain of various RAD2 nuclease family members by the UPGMA method. As shown in Fig. 5, OsFEN-1a and OsFEN-1b were closely related to each other. OsFEN-1a and OsFEN-1b belong to Class II. OsFEN-1a was closer to Arabidopsis FEN-1. Moreover, the Arabidopsis genome had only one FEN-1 gene. Therefore, OsFEN-1a must be a prevalent FEN-1 homologue in plants, and OsFEN-1b might be a rice-specific enzyme.
that OsFEN-1b expressed in matured leaves very weakly, while OsFEN-1a did not express at all (Fig. 6B). OsFEN1b may function in the mature leaves without proliferating cells.
3.6. Expression of OsFEN-1a and OsFEN-1b in different organs of the rice plant To determine the expression patterns of OsFEN-1a and OsFEN-1b in different organs, Northern hybridization analysis was performed. As shown in Fig. 6A, specific probes for OsFEN-1a and OsFEN-1b were used for the expression pattern analysis. Total RNA samples isolated from various organs of 50day-old rice plants were blotted and probed with 32Plabeled OsFEN-1a- and OsFEN-1b-specific cDNA probe (Fig. 6B). Transcripts of each of OsFEN-1a and OsFEN-1b were detected in the shoot apical meristem, young leaves, panicles and roots, and very weakly in flag leaves. Cell proliferation in these meristematic tissues is very active, and thus the level of DNA replication in the meristem must be high. Transcription of OsFEN-1a and OsFEN-1b might be related to the level of DNA replication. It is interesting
Fig. 5. Phylogenetic analysis. A phylogenetic tree was constructed by the UPGMA method, based on the amino acid sequences of the N domain of a RAD2 nuclease family member. Horizontal distances are proportional to evolutional divergence expressed as substitutions per site.
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Fig. 6. Determination of expression pattern of OsFEN-1a and OsFEN-1b by Northern hybridization analysis. (A) Schematic representation of the specific probes for OsFEN-1a and OsFEN-1b. (B) Expression of OsFEN-1a and OsFEN-1b in different organs. Each lane contained 20 Ag of total RNA isolated from the shoot apical meristem (lane 1), mature leaves (lane 2), young leaves (lane 3), flag leaves (lane 4), panicles (lane 5) or roots (lane 6). The blot was probed with 32P-labeled OsFEN-1a (upper panel) or OsFEN-1b (middle panel). Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (lower panel). (C) Effects of sucrose starvation on the level of OsFEN-1a and OsFEN-1b expression. Rice cells were cultured in suspension for 6 (lanes 1 and 2) or 10 days (lane 3) with (lane 1) or without (lanes 2 and 3) sucrose, or cultured for 6 days without sucrose, then sucrose was added to the medium and culture was continued for a further 4 days (lane 4). Aliquots of 20 Ag of total RNA isolated from the cultured cells were separated on a 1.2% agarose gel containing formaldehyde and then blotted. The blot was probed with 32P-labeled OsFEN-1a cDNA (upper panel) or OsFEN-1b (middle panel). Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (lower panel).
3.7. Expression levels of OsFEN-1a and OsFEN-1b are correlated with cell proliferation The effects of sucrose starvation on the expression levels of OsFEN-1a and OsFEN-1b were tested. OsFEN-
1a and OsFEN-1b were actively transcribed in rice cells in suspension culture. Fig. 6C shows the expression of OsFEN-1a and OsFEN-1b in the rice cells cultured with or without sucrose. OsFEN-1a and OsFEN-1b were actively transcribed in rice cells in suspension culture (lane 1
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3.8. Spatial expression patterns of OsFEN-1a and OsFEN-1b
Fig. 7. Spatial expression pattern of OsFEN-1a and OsFEN-1b. In situ hybridization analysis was performed using longitudinal sections from the shoot apex region of 10-day-old rice seedlings. The plant tissues were probed with (A) OsFEN-1a or (B) OsFEN-1b antisense riboprobe labeled with digoxigenin-UTP.
in Fig. 6C). When cell proliferation was temporarily halted for 6 or 10 days by removal of sucrose from the growth medium, the levels of OsFEN-1a and OsFEN-1b expression were significantly reduced (lanes 2 and 3 in Fig. 6C). When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at a high level (lane 4 in Fig. 6C). These results suggested that OsFEN-1a and OsFEN-1b expression might be required for cell proliferation.
To study the expression patterns further, the spatial expression patterns of OsFEN-1a and OsFEN-1b in the shoot apex region were investigated by in situ hybridization analysis using digoxigenin-labeled antisense RNA of OsFEN-1a or OsFEN-1b as a probe. In situ hybridization was performed on paraffin sections of the plant tissues from 10-day-old rice seedlings, which were fixed for 16 h at 4 jC as described in Materials and methods. No hybridization signals were detected when digoxigeninlabeled sense probe was used (data not shown). In the shoot apex region, the antisense probe for OsFEN-1a (Fig. 7A) and OsFEN-1b (Fig. 7B) showed strong hybridization signals in the shoot apical meristem and young leaves. On the other hand, both OsFEN-1a and OsFEN-1b were not expressed in the matured leaves where cell proliferation does not occur (Fig. 7A,B). These spatial expression patterns mostly coincided with the results of Northern hybridization analysis, except the data for OsFEN-1b in the matured leaves (Fig. 6B). The expression level of OsFEN-1b in the matured leaves was very low, and subsequently, hard to detect by the in situ hybridization analysis. These results confirmed that OsFEN-1a and OsFEN-1b were mainly expressed in actively proliferating tissues. 3.9. Functional complementation assay The budding yeast Saccharomyces cerevisiae has a homologue of FEN-1 (RAD27), and RAD27 null mutant (Drad27) fail to grow at a temperature of 37 jC (Reagan et al., 1995). To demonstrate that the OsFEN-1a and OsFEN-1b cDNA encode a functional FEN-1, we investigated the ability of these cDNA to complement the Drad27 mutation and restore growth at the restrictive temperature to a yeast strain carrying this mutation. For this, OsFEN-
Fig. 8. Complementation assays of Sacharomyces cerevisiae rad27 mutant by OsFEN-1a and OsFEN-1b. Plates show growth of (i) BY4741a (wild) transformed with the plasmid pYES2/NTA. (ii) Drad27 mutant transformed with the plasmid pYES/NTA. (iii) Drad27 mutant transformed with the plasmid pYES/NTA into which OsFEN-1a cDNA has been cloned. (iv) Drad27 mutant transformed with the plasmid pYES/NTA into which OsFEN-1b cDNA has been cloned.
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Table 1 Conserved PCNA binding motif of FEN-1
1a and OsFEN-1b cDNA were cloned into the yeast expression vector pYES2/NTA (Invitorogen) such that the expressions of the cDNA were under the control of GAL1 promoter. These constructs were used to transform the Drad27 strain, and the transformants were assayed for growth at 37 jC. As shown in Fig. 8, the OsFEN-1a cDNA was able to partially complement the growth at 37 jC, but OsFEN-1b cDNA transformant fails to grow. A region of nine conserved amino acid residues (residues Gln-337 through Lys345) in the C terminus of human FEN-1 was shown to be responsible for the interaction with proliferating cell nuclear antigen (PCNA) (Frank et al., 2001). As shown in Table 1, PCNA binding motif of FEN-1 nucleases was not conserved in OsFEN-1b protein. These results suggested that PCNA binding motif might be important for the complementation of Drad27 strain. FEN-1 is a multifunctional structure-specific endonuclease that is important for DNA replication and repair. Its main function in DNA replication is proposed to be the removal of the displaced RNA – DNA primers during lagging strand DNA synthesis. FEN-1 has also been shown to be involved in DNA repair. Vertebrate cells lacking FEN-1 are viable but hypersensitive to methylating agents and H2O2 (Matsuzaki et al., 2002). In yeast (S. cerevisiae and Schizosaccharomyces pombe), genetic studies showed that deleting FEN-1 homologues resulted in marked sensitivity to alkylating reagents, ultraviolet (UV) sensitivity and chromosome instability (Johnson et al., 1995; Reagan et al., 1995; Murray et al., 1994). The FEN-1 gene is also required for prevention of trinucleotide repeat (TNR) expansion (Freudenreichet al., 1998). These data suggested that this protein plays a role in genomic integrity. In this report, we described the functional characterization of two FEN-1 homologues in rice (O. sativa L. cv. Nipponbare). Both FEN-1 homologues are mainly expressed in proliferating tissues, suggesting their function in DNA replication. Functional complementation assay revealed that OsFEN-1a cDNA could complement the Drad27, but OsFEN-1b cDNA could not. This result may suggest that the functions or roles of OsFEN-1b are different from those of OsFEN-1a. Detailed investigations of their phenotypes in which each of the OsFEN-1a and OsFEN-1b have been knocked down are required for further clarification of the roles. Our long-range goal is to determine the mechanism of plant DNA replication and repair. Further studies on the biological function of each of OsFEN-1a and OsFEN-1b may allow us to understand the mechanism.
Acknowledgements This work was supported in part by a grant from Futaba Electronics Memorial Foundation (Japan) and by the grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project IP5006, MP2105 and EF-S2). We thank the Rice Genome Research Program (RGP) of Japan for providing the rice EST clone C0843. We thank the companies of Monsant and Syngenta for the download of the rice genome sequence. We also thank T. Yamamoto, T. Ishibashi and Y. Uchiyama of Tokyo University of Science for their technical advice and helpful discussion.
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Lieber, M.R., 1997. The FEN-1 family of structure specific nucleases in eukaryotic DNA replication, recombination and repair. BioEssays 19, 233 – 240. Matsuzaki, Y., Adachi, N., Koyama, H., 2002. Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2. Nucleic Acids Res. 30, 3273 – 3277. Murray, J.M., Tavassoli, M., Al-Harithy, R., Sheldrick, K.S., Lehmann, A.R., Carr, A.M., Watts, F.Z., 1994. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Mol. Cell Biol. 14, 4878 – 4888. Nolan, J.P., Shen, B., Park, M.S., Sklar, L.A., 1996. Kinetic analysis of human flap endonuclease-1 by flow cytometry. Biochemistry 35, 11668 – 11676. Reagan, M.S., Pittenger, C., Siede, W., Friedberg, E.C., 1995. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J. Bacteriol. 177, 364 – 371. Shen, B., Nolan, J.P., Sklar, L.A., Park, M.S., 1997. Functional analysis of point mutations in human flap endonuclease-1 active site. Nucleic Acids Res. 25, 2228 – 3332. Staplenton, A.E., Thornber, C.S., Walbot, V., 1997. UV-B component of sunlight causes measurable damage in field grown maize (Zea mays L.): development and cellular heterogeneity of damage and repair. Plant Cell Environ. 20, 279 – 290.
Functional characterization of two flap endonuclease-1 homologues in rice Seisuke Kimura a, Tomoyuki Furukawa a, Nobuyuki Kasai a, Yoko Mori a, Hiroko K. Kitamoto b, Fumio Sugawara a, Junji Hashimoto b, Kengo Sakaguchi a,* a
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan b National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan Received 13 February 2002; received in revised form 16 April 2003; accepted 12 May 2003 Received by T. Sekiya
Abstract Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed. D 2003 Elsevier B.V. All rights reserved. Keywords: OsFEN-1a; OsFEN-1b; Rice; DNA replication and repair
1. Introduction DNA replication and repair are critical for maintaining genome stability. DNA replication and repair in higher plants has been examined recently in rice, Arabidopsis, wheat and maize (Furukawa et al., 2003; Kimura et al., 2000, 2001, 2002; Hays, 2002; Ishibashi et al., 2001; Britt, 1999; Britt et al., 1993; Culligan and Hays, 1997; Landry et al., 1997; Staplenton et al., 1997). However, in general, little is known about plant DNA replication, repair and the
Abbreviations: bp, base pair; cDNA, complementary DNA; EST, expressed sequence tag; FEN-1, flap endonuclease-1; mRNA, messenger RNA; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; UV, ultraviolet; RACE, rapid amplification of cDNA ends. * Corresponding author. Tel.: +81-4-7124-1501x3409; fax: +81-47123-9767. E-mail address: [email protected] (K. Sakaguchi). 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00694-2
factors involved in these processes in plants. Studies are needed to fill this knowledge gap between plants and animals or yeasts. DNA replication and repair are in part dependent on the activity of a family of structure-specific endonucleases. The RAD2 family of nucleases includes XPG (Class I), FEN-1 (Class II) and Exo1 (Class III), and the proteins exhibit a range of substrate-specific exo- and endonuclease activities. Many of these proteins feature a range of nuclease activities that contribute to DNA replication and repair. RAD2 Class I consists of XPG-like proteins that cleave at the 3V-side of the damage-containing bubble structure formed during nucleotide excision repair (Calleja et al., 2001; Evans et al., 1997; Lieber, 1997). Class II is comprised of flap endonuclease-1 (FEN-1) which remove 5V-DNA flaps produced by polymerase strand displacement and are also responsible for DNA replication and DNA repair (Matsuzaki et al., 2002; Alleva and Doetsch, 1998; Freudenreichet al., 1998; Lieber, 1997;
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Johnson et al., 1995; Reagan et al., 1995; Murray et al., 1994). Class III is made up of the Exo1-like proteins found in yeast, Drosophila, and mammals. Exo1-like proteins appear to play roles in DNA recombination, mismatch repair, and DNA replication (Lee and Wilson, 1999; Lieber, 1997). We have concentrated on investigating plant enzymes belonging to the RAD2 nuclease family in the past several years and their relationships to DNA replication and repair (Furukawa et al., 2003; Kimura et al., 2000, 2001). In the process of the study, we found that rice plants have two different species of FEN-1, termed OsFEN-1a and OsFEN1b. In this report, we describe the functional characterization of two FEN-1 homologues in rice (Oryza sativa L. cv. Nipponbare).
2. Materials and methods 2.1. Molecular cloning of OsFEN-1b The rice expressed sequence tag (EST) database was searched to identify a complementary DNA (cDNA) clone with homology to the RAD2 nuclease family. Rice EST clone C30032 (GenBank accession no. AU108651) was found to have significant homology to OsFEN-1, which we have previously isolated (Kimura et al., 2000). The fulllength cDNA was isolated using the 5V-rapid amplification of cDNA ends (RACE) method. To distinguish the two FEN-1 homologues in rice, the previously found OsFEN-1 was designated as OsFEN-1a, and the newly found cDNA was designated as OsFEN-1b. The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence database with the accession numbers AB021666 and AB088391 for OsFEN-1a and OsFEN-1b, respectively. 2.2. Mapping and analysis of genomic structure The genomic loci of OsFEN-1a and OsFEN-1b were mapped on the high-density linkage map of rice according to the method of Harushima et al. The genomic sequence of OsFEN-1a was determined using polymerase chain reaction (PCR) amplification and sequencing. The genomic sequence of OsFEN-1b was downloaded from Monsant’s and Syngenta’s genome sequence database (Goff et al., 2002). 2.3. Northern hybridization Aliquots of 20 Ag of total RNA were resolved on 1.2% formaldehyde agarose gels and transferred onto nylon membranes (Hybond-N, Amersham). After prehybridization, the filters were probed with 32P-labeled probe at 42 jC for 16 h followed by washing twice with 2SSC+0.1% SDS at room temperature for 15 min, and three times with 0.1SSC+0.1% SDS at 65 jC for 20 min.
2.4. In situ hybridization Riboprobes for in situ hybridization were labeled with digoxigenin-11-rUTP using a DIG RNA Labeling Kit (Roche) according to the manufacturer’s protocol. The riboprobes were subjected to mild alkaline hydrolysis by heating at 60 jC in carbonate buffer and used at a concentration of 2 Ag/ml. Plant tissues from 10-day-old rice seedlings were fixed for 16 h at 4 jC with a mixture of 4% (W/ V) paraformaldehyde and 0.25% (V/V) glutaraldehyde in 50 mM sodium phosphate buffer (pH 7.2). The fixed tissues were dehydrated in a series of xylene and ethanol and embedded in paraffin (HISTPREP 568, Wako). Embedded tissues were sectioned at a thickness of 10 Am, and placed on microscope slides precoated with APS (3-aminopropyltriethoxysilane). Sections were deparaffinized with xylene and rehydrated through a graded ethanol series. They were subsequently pretreated with 10 mg/ml of proteinase K in 100 mM Tris – HCl, pH 7.5, and 50 mM EDTA at 37 jC for 30 min, dehydrated in a graded ethanol series, and dried under vacuum for 2 h. Hybridization and detection of hybridized riboprobes were performed as described by Kimura et al. (2000). 2.5. Sequence and phylogenetic analyses and molecular modeling Sequence and phylogenetic analyses were performed using GENETYX-MAC ver. 10 (Software Development). Modeled 3D structures for OsFEN-1a and OsFEN-1b proteins were generated automatically with the Swiss Model program http://www.expasy.ch/swissmed/SWISS-MODEL. html) (Guex et al., 1999; Guex and Peitsch, 1997) using the X-ray structure of Pyrococcus furiosus FEN-1 as a template (Hosfield et al., 1998). The structures were visualized using Insight II (Molecular Simulations). The secondary classifications were performed using the Kabsch Sander calculation.
3. Results and discussion 3.1. Identification and molecular cloning of OsFEN-1b, another homologue of FEN-1 from rice The rice EST database was searched to identify a plant RAD2 nuclease family member protein. Rice EST clone C30032 (GenBank accession no. AU108651) was found to have significant homology to FEN-1 (one of the members of the RAD2 nuclease family), and full-length cDNA was isolated as described in Materials and methods. The length of the cDNA was 1674 base pair (bp), and the open reading frame of the cDNA encoded a predicted product of 412 amino acid residues with a molecular mass of 45.7 kDa. Previously, we also isolated and characterized a rice homologue of FEN-1 which was designated as OsFEN-1
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(Kimura et al., 2000). As shown in Figs. 1 and 2, the cDNA was significantly similar to OsFEN-1, indicating that rice has two different species of FEN-1 homologue. Therefore, we designated the newly found OsFEN-1 cDNA as OsFEN-1b, and the previously found OsFEN-1 cDNA as OsFEN-1a to distinguish the two FEN-1 homologues. As described in the later part of this report, OsFEN-1b is not a pseudogene because the messenger RNA (mRNA) was detected by Northern hybridization analysis. The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence database with the accession numbers, AB021666 and AB088391 for OsFEN-1a and OsFEN1b, respectively. 3.2. Mapping and genomic structures of OsFEN-1a and OsFEN-1b The chromosomal locations of cDNAs of OsFEN-1a and OsFEN-1b on the linkage map of rice were determined by restriction fragment length polymorphism (RFLP) mapping (Harushima et al., 1998). The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and chromosome 3,
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respectively (Fig. 1A). These results clearly indicated that rice has two genes for FEN-1. The genomic sequences of OsFEN-1a and OsFEN-1b were analyzed as described in Materials and methods. Both genes have 17 exons and 16 introns (Fig. 1B). These exon patterns are very similar to each other, implying that OsFEN-1b was generated by genome duplication. 3.3. Comparison of amino acid sequences of OsFEN-1a and OsFEN-1b Fig. 2 shows a comparison of the deduced amino acid sequences of OsFEN-1a and OsFEN-1b. OsFEN-1b protein has 60.0% sequence identity with OsFEN-1a protein, and 45.5% identity with human FEN-1 (Nolan et al., 1996). Functional analysis of point mutations in the human FEN-1 active site revealed that Asp-34, Asp-86 and Glu-160 are important for substrate binding and cleavage, Glu-158, Asp179 and Asp-233 are essential for substrate binding, and Asp-181 is involved in substrate cleavage (Shen et al., 1997). All of these amino acid residues were conserved in OsFEN-1a and OsFEN-1b proteins (indicated by asterisks in Fig. 2).
Fig. 1. Mapping and genomic structure of the OsFEN-1a and OsFEN-1b. (A) Maps of OsFEN-1a and OsFEN-1b loci. (B) Genomic organizations of the OsFEN-1a and OsFEN-1b genes. Boxed areas are exons, lines are introns and 5Vand 3Vupstream regions. The open boxes correspond to the coding region and hatched boxes correspond to the untranslated region.
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Fig. 2. Alignment of the amino acid sequences of OsFEN-1a and OsFEN-1b. Common residues are boxed, and gaps added for sequence alignment are indicated with dashes. The two highly conserved regions, N domain and I domain, are shown. Asterisks indicate the amino acids essential for substrate binding or nuclease activity.
Much of the amino acid sequence conservation among the RAD2 nuclease family is concentrated within the three domains termed the N (N-terminal), I (Internal) and C (Cterminal) domains. (Lieber, 1997). As shown in Fig. 3, the N and I domains were highly conserved in the OsFEN-1b sequence, but the C domain was not conserved. The N and I domains of OsFEN-1b protein showed 74.3% and 79.7% sequence identity with those of OsFEN-1a, respectively (Fig. 3B). The C-terminal region of OsFEN-1b was larger than OsFEN-1a and human FEN-1 (Fig. 3B). 3.4. Modeled 3D structures for OsFEN-1a and OsFEN-1b The crystal 3D structures of P. furiosus FEN-1 and Methanococcus jannaschii have been reported (Hosfield et al., 1998; Hwang et al., 1998). This enables us to predict the 3D structures of OsFEN-1a and OsFEN-1b using the Swiss Model service provided on the ExPASy server (Guex et al., 1999; Guex and Peitsch, 1997). As shown in Fig. 4, comparison of the modelings suggests substantial conservation among the 3D structures of P. furiosus FEN-1, OsFEN-1a and OsFEN-1b proteins. According to this model, the 3D structures of the region from the N to the I domain are quite similar to each other. On the other hand, the 3D structure of the C domains are thought to differ among them. As described previously (Kimura et al., 2000), the OsFEN-1a protein has the activities of flap endonuclease and 5Vto 3Vexonuclease. Since the 3D structures are quite similar, OsFEN-1b protein is also thought to have the activities of flap endonuclease and 5V to 3Vexonuclease. 3.5. Phylogenetic analysis The RAD2 nuclease family includes XPG (Class I), FEN-1 (Class II) and EXO1 (Class III). Recently, we
Fig. 3. Comparison of conserved domain structures. (A) Schematics depict the protein structure and highly conserved domains (N, I, and C domain). (B) Pairwise comparison of the conserved domains of human FEN-1, OsFEN-1a, and OsFEN-1b.
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Fig. 4. Modeled 3D structure of OsFEN-1a and OsFEN-1b. (Left) The X-ray structure of P. furiosus FEN-1 was used as a template for modeling. (Center) Modeled 3D structure of OsFEN-1a protein (amino acids 12-346). (Right) Modeled 3D structure of OsFEN-1b protein (amino acids 18-336).
isolated and characterized a new RAD2 nuclease family member, OsSEND-1, that is thought to belong to Class IV (Furukawa et al., 2003). To determine the phylogenetic relationship between OsFEN-1b and other RAD2 nuclease family members, phylogenetic analysis was performed (Fig. 5). The phylogenetic tree was drawn based on the amino acid sequence of the N domain of various RAD2 nuclease family members by the UPGMA method. As shown in Fig. 5, OsFEN-1a and OsFEN-1b were closely related to each other. OsFEN-1a and OsFEN-1b belong to Class II. OsFEN-1a was closer to Arabidopsis FEN-1. Moreover, the Arabidopsis genome had only one FEN-1 gene. Therefore, OsFEN-1a must be a prevalent FEN-1 homologue in plants, and OsFEN-1b might be a rice-specific enzyme.
that OsFEN-1b expressed in matured leaves very weakly, while OsFEN-1a did not express at all (Fig. 6B). OsFEN1b may function in the mature leaves without proliferating cells.
3.6. Expression of OsFEN-1a and OsFEN-1b in different organs of the rice plant To determine the expression patterns of OsFEN-1a and OsFEN-1b in different organs, Northern hybridization analysis was performed. As shown in Fig. 6A, specific probes for OsFEN-1a and OsFEN-1b were used for the expression pattern analysis. Total RNA samples isolated from various organs of 50day-old rice plants were blotted and probed with 32Plabeled OsFEN-1a- and OsFEN-1b-specific cDNA probe (Fig. 6B). Transcripts of each of OsFEN-1a and OsFEN-1b were detected in the shoot apical meristem, young leaves, panicles and roots, and very weakly in flag leaves. Cell proliferation in these meristematic tissues is very active, and thus the level of DNA replication in the meristem must be high. Transcription of OsFEN-1a and OsFEN-1b might be related to the level of DNA replication. It is interesting
Fig. 5. Phylogenetic analysis. A phylogenetic tree was constructed by the UPGMA method, based on the amino acid sequences of the N domain of a RAD2 nuclease family member. Horizontal distances are proportional to evolutional divergence expressed as substitutions per site.
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Fig. 6. Determination of expression pattern of OsFEN-1a and OsFEN-1b by Northern hybridization analysis. (A) Schematic representation of the specific probes for OsFEN-1a and OsFEN-1b. (B) Expression of OsFEN-1a and OsFEN-1b in different organs. Each lane contained 20 Ag of total RNA isolated from the shoot apical meristem (lane 1), mature leaves (lane 2), young leaves (lane 3), flag leaves (lane 4), panicles (lane 5) or roots (lane 6). The blot was probed with 32P-labeled OsFEN-1a (upper panel) or OsFEN-1b (middle panel). Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (lower panel). (C) Effects of sucrose starvation on the level of OsFEN-1a and OsFEN-1b expression. Rice cells were cultured in suspension for 6 (lanes 1 and 2) or 10 days (lane 3) with (lane 1) or without (lanes 2 and 3) sucrose, or cultured for 6 days without sucrose, then sucrose was added to the medium and culture was continued for a further 4 days (lane 4). Aliquots of 20 Ag of total RNA isolated from the cultured cells were separated on a 1.2% agarose gel containing formaldehyde and then blotted. The blot was probed with 32P-labeled OsFEN-1a cDNA (upper panel) or OsFEN-1b (middle panel). Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (lower panel).
3.7. Expression levels of OsFEN-1a and OsFEN-1b are correlated with cell proliferation The effects of sucrose starvation on the expression levels of OsFEN-1a and OsFEN-1b were tested. OsFEN-
1a and OsFEN-1b were actively transcribed in rice cells in suspension culture. Fig. 6C shows the expression of OsFEN-1a and OsFEN-1b in the rice cells cultured with or without sucrose. OsFEN-1a and OsFEN-1b were actively transcribed in rice cells in suspension culture (lane 1
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3.8. Spatial expression patterns of OsFEN-1a and OsFEN-1b
Fig. 7. Spatial expression pattern of OsFEN-1a and OsFEN-1b. In situ hybridization analysis was performed using longitudinal sections from the shoot apex region of 10-day-old rice seedlings. The plant tissues were probed with (A) OsFEN-1a or (B) OsFEN-1b antisense riboprobe labeled with digoxigenin-UTP.
in Fig. 6C). When cell proliferation was temporarily halted for 6 or 10 days by removal of sucrose from the growth medium, the levels of OsFEN-1a and OsFEN-1b expression were significantly reduced (lanes 2 and 3 in Fig. 6C). When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at a high level (lane 4 in Fig. 6C). These results suggested that OsFEN-1a and OsFEN-1b expression might be required for cell proliferation.
To study the expression patterns further, the spatial expression patterns of OsFEN-1a and OsFEN-1b in the shoot apex region were investigated by in situ hybridization analysis using digoxigenin-labeled antisense RNA of OsFEN-1a or OsFEN-1b as a probe. In situ hybridization was performed on paraffin sections of the plant tissues from 10-day-old rice seedlings, which were fixed for 16 h at 4 jC as described in Materials and methods. No hybridization signals were detected when digoxigeninlabeled sense probe was used (data not shown). In the shoot apex region, the antisense probe for OsFEN-1a (Fig. 7A) and OsFEN-1b (Fig. 7B) showed strong hybridization signals in the shoot apical meristem and young leaves. On the other hand, both OsFEN-1a and OsFEN-1b were not expressed in the matured leaves where cell proliferation does not occur (Fig. 7A,B). These spatial expression patterns mostly coincided with the results of Northern hybridization analysis, except the data for OsFEN-1b in the matured leaves (Fig. 6B). The expression level of OsFEN-1b in the matured leaves was very low, and subsequently, hard to detect by the in situ hybridization analysis. These results confirmed that OsFEN-1a and OsFEN-1b were mainly expressed in actively proliferating tissues. 3.9. Functional complementation assay The budding yeast Saccharomyces cerevisiae has a homologue of FEN-1 (RAD27), and RAD27 null mutant (Drad27) fail to grow at a temperature of 37 jC (Reagan et al., 1995). To demonstrate that the OsFEN-1a and OsFEN-1b cDNA encode a functional FEN-1, we investigated the ability of these cDNA to complement the Drad27 mutation and restore growth at the restrictive temperature to a yeast strain carrying this mutation. For this, OsFEN-
Fig. 8. Complementation assays of Sacharomyces cerevisiae rad27 mutant by OsFEN-1a and OsFEN-1b. Plates show growth of (i) BY4741a (wild) transformed with the plasmid pYES2/NTA. (ii) Drad27 mutant transformed with the plasmid pYES/NTA. (iii) Drad27 mutant transformed with the plasmid pYES/NTA into which OsFEN-1a cDNA has been cloned. (iv) Drad27 mutant transformed with the plasmid pYES/NTA into which OsFEN-1b cDNA has been cloned.
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Table 1 Conserved PCNA binding motif of FEN-1
1a and OsFEN-1b cDNA were cloned into the yeast expression vector pYES2/NTA (Invitorogen) such that the expressions of the cDNA were under the control of GAL1 promoter. These constructs were used to transform the Drad27 strain, and the transformants were assayed for growth at 37 jC. As shown in Fig. 8, the OsFEN-1a cDNA was able to partially complement the growth at 37 jC, but OsFEN-1b cDNA transformant fails to grow. A region of nine conserved amino acid residues (residues Gln-337 through Lys345) in the C terminus of human FEN-1 was shown to be responsible for the interaction with proliferating cell nuclear antigen (PCNA) (Frank et al., 2001). As shown in Table 1, PCNA binding motif of FEN-1 nucleases was not conserved in OsFEN-1b protein. These results suggested that PCNA binding motif might be important for the complementation of Drad27 strain. FEN-1 is a multifunctional structure-specific endonuclease that is important for DNA replication and repair. Its main function in DNA replication is proposed to be the removal of the displaced RNA – DNA primers during lagging strand DNA synthesis. FEN-1 has also been shown to be involved in DNA repair. Vertebrate cells lacking FEN-1 are viable but hypersensitive to methylating agents and H2O2 (Matsuzaki et al., 2002). In yeast (S. cerevisiae and Schizosaccharomyces pombe), genetic studies showed that deleting FEN-1 homologues resulted in marked sensitivity to alkylating reagents, ultraviolet (UV) sensitivity and chromosome instability (Johnson et al., 1995; Reagan et al., 1995; Murray et al., 1994). The FEN-1 gene is also required for prevention of trinucleotide repeat (TNR) expansion (Freudenreichet al., 1998). These data suggested that this protein plays a role in genomic integrity. In this report, we described the functional characterization of two FEN-1 homologues in rice (O. sativa L. cv. Nipponbare). Both FEN-1 homologues are mainly expressed in proliferating tissues, suggesting their function in DNA replication. Functional complementation assay revealed that OsFEN-1a cDNA could complement the Drad27, but OsFEN-1b cDNA could not. This result may suggest that the functions or roles of OsFEN-1b are different from those of OsFEN-1a. Detailed investigations of their phenotypes in which each of the OsFEN-1a and OsFEN-1b have been knocked down are required for further clarification of the roles. Our long-range goal is to determine the mechanism of plant DNA replication and repair. Further studies on the biological function of each of OsFEN-1a and OsFEN-1b may allow us to understand the mechanism.
Acknowledgements This work was supported in part by a grant from Futaba Electronics Memorial Foundation (Japan) and by the grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project IP5006, MP2105 and EF-S2). We thank the Rice Genome Research Program (RGP) of Japan for providing the rice EST clone C0843. We thank the companies of Monsant and Syngenta for the download of the rice genome sequence. We also thank T. Yamamoto, T. Ishibashi and Y. Uchiyama of Tokyo University of Science for their technical advice and helpful discussion.
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