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Nucleic Acids Research, 2002, Vol. 30, No. 7 1585-1592
© 2002 Oxford University Press

A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.)

Seisuke Kimura, Yukinobu Uchiyama, Nobuyuki Kasai, Satoshi Namekawa, Ai Saotome, Tadamasa Ueda1, Tsuyu Ando2, Toyotaka Ishibashi, Masahiko Oshige, Tomoyuki Furukawa, Taichi Yamamoto, Junji Hashimoto1,3 and Kengo Sakaguchi*

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, 1National Institute of Agrobiological Sciences, Tsukuba , Ibaraki 305-0854, Japan, 2Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan and 3Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Ochanomizu, Chiyodaku, Tokyo101-0062, Japan

Received December 13, 2001; Accepted January 30, 2002.

DDBJ/EMBL/GenBank accession no. AB047689.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase {gamma} and {theta}. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Cell proliferation requires maintenance of genomic integrity, which requires coordination among cell cycle-regulated DNA replication, repair and recombination to avoid mutation and genomic instability. Analysis of proteins that are required for these processes provides insight into the mechanism of this coordination. Our research interest lies especially with the roles of multiple DNA polymerase systems in DNA replication, repair and recombination, and subsequently in cell proliferation and development (1–6).

The normal development of higher plants is regulated by the initiation of cell proliferation of the meristematic tissue, and organ formation is accomplished in a small-specialized region, the meristem. Since the plant tissues and organs are quite simple, we can investigate the distribution of each DNA polymerase in the differentiated tissues, and subsequently can infer their functional roles. Higher plants are also an interesting system in which to study the mechanism of coordination between DNA replication and repair. The meristematic tissue and the organs on the ground must proliferate under more severe UV bombardment than that to which animals or yeast are exposed (7–14). Unlike mammals, in which the germ cells are set aside early and are completely shielded from radiation, plant cells enter meiosis only after significant vegetative growth. Mutations occurring in the shoot apical meristem may be passed on to the gametophytes. This raises the question of how the coordination between replication and repair is achieved. Finally, higher plants have another subcellular self-proliferating organelle, the chloroplast, which is not present in animals. We have analyzed DNA polymerase and related factors in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (6,7,9–14).

To date, at least 12 classes of DNA polymerase have been identified in animals, {alpha}, ß, {gamma}, {delta}, {varepsilon}, {zeta}, {eta}, {theta}, {iota}, {kappa}, {lambda} and µ (15–24). However, information concerning plant DNA polymerases is still very limited (6,25–31). To date, only two DNA polymerases from higher plants have been isolated, the catalytic subunit of DNA polymerase {alpha} (31) and catalytic and small subunits of DNA polymerase {delta} (unpublished data).

We screened for DNA polymerases in rice and found a novel DNA polymerase homologous to Escherichia coli DNA polymerase I. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Interestingly, the expression level of the DNA polymerase was suggested to be closely correlated with cell proliferation. We report here the molecular cloning and characterization of a novel DNA polymerase, designated as OsPolI-like, which is possibly localized in the plastid, and describe its pattern of expression in the higher plant, rice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Molecular cloning of O.sativa PolI-like DNA polymerase, OsPolI-like
The rice EST database was searched using the BLAST program to identify cDNA clones with homology to E.coli DNA polymerase I protein. Rice EST clone C0843 (GenBank accession no. D15567) was found to have significant homology. Screening of a rice cDNA library with the insert DNA of the EST clone resulted in isolation of a 3.8 kb clone designated OsPolI-like. The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession no. AB047689. The genomic sequence of the OsPolI-like gene was determined by PCR amplification and sequencing. The genomic locus of the cDNA of OsPolI-like was mapped on the high-density linkage map of rice according to Harushima et al. (32).

Production of polyclonal antibody
The truncated sequence (residues from 432 to 791) of the OsPolI-like coding region was cloned into the pET21a expression vector and transformed into E.coli for protein induction. Extracts prepared from the cells induced for 3 h were shown to contain a six histidine C-terminal-tagged OsPol-like fusion peptide. The OsPolI-like peptide was purified by His-bind resin column chromatography and SDS–PAGE. The purified peptide was used for immunization of rabbits.

Overexpression of DNA polymerase domain of OsPolI-like
DNA polymerase domain of OsPolI-like protein was overexpressed and purified as follows. The truncated sequence (residues 444–959) of OsPolI-like coding region was cloned into the pET21a expression vector (Novagen). Protein expression was performed by transforming the construct into BL21 (DE3) (Novagen) and growing these bacteria in 500 ml of LB medium containing 50 µg/ml of ampicillin. Cells were grown to an OD of 0.8 and isopropyl-ß-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM. Cells were harvested after 3 h by centrifugation at 3000 g for 10 min. Cell pellets were resuspended in 4 ml of ice-cold binding buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 5 mM imidazole, 0.1% NP-40) and sonicated with 20 bursts of 10 s each. Cell lysates were centrifuged at 39 000 g for 20 min and the soluble protein fraction was collected as the crude extract. The crude extract was loaded onto 10 ml of His-Bind resin (Novagen). The column was washed with 100 ml of binding buffer and then washed with 100 ml of wash buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 60 mM imidazole, 0.1% NP-40). The bound protein was eluted with 30 ml of elution buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 1 M imidazole). The eluted protein was dialyzed against buffer A (50 mM Tris–HCl pH 7.5, 1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, 0.1% NP-40). The dialysate was loaded onto a Mono Q HR5/5 column (Amersham Biosciences) equilibrated with buffer A. After washing, the fraction was collected with 20 ml of a linear gradient of 0–0.5 M NaCl in buffer A. The eluted OsPolI-like DNA polymerase domain was dialyzed against buffer A and used in the subsequent experiments. DNA polymerase activity was measured as described previously (6).

Isolation of plastid
The ground tissue of 10-day-old rice seedlings was briefly homogenized with a blender in buffer G (50 mM HEPES–KOH pH 7.5, 0.33 M sorbitol, 2 mM EDTA, 1 mM MgCl2, 1 mM MnCl2 and 5 mM Na-ascorbate). The ruptured cells were filtered through two layers of nylon membrane, then the crude plastid fraction was collected by centrifugation at 5000 g. The pellet was gently resuspended in buffer G and layered onto 30% Percoll solution (30% percoll, 50 mM HEPES–KOH pH 8.0 and 0.33 M sorbitol). The intact plastid fraction was isolated by centrifugation at 700 g for 15 min, then the pellet was resuspended in buffer W (50 mM HEPES–KOH pH 8.0, 0.33 M sorbitol). The plastid fraction was again centrifuged at 700 g for 3 min. The plastid pellet was used for western blotting analysis.

Other methods
In situ hybridization analysis was performed as described previously (7,33). A modeled 3D structure for OsPolI-like was generated automatically with the program Swiss Model (http://www.expasy.ch/swissmod/SWISS-MODEL.html) (34–36) and visualized using Insight II (Molecular Simulations Inc.). Phylogenetic analysis was performed based on the amino acid sequence by the UPGMA method using GENETYX-MAC var. 10 (Software Development Co. Ltd.). DNA polymerase domains of various family A DNA polymerases were aligned and used to produce the tree.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Identification and molecular cloning of O.sativa PolI-like DNA polymerase (OsPolI-like), a novel DNA polymerase from a higher plant
To identify plant DNA polymerase with homology to E.coli DNA polymerase I, the rice EST database was searched using the BLAST program. Rice EST clone C0843 (GenBank accession no. D15567) was found to have significant homology and was used for cDNA library screening. Screening of a rice cDNA library with the fragment resulted in the isolation of a 3765 bp clone which had significant homology with E.coli DNA polymerase I and was designated as OsPolI-like (O.sativa Pol I-like DNA polymerase). The open reading frame of OsPolI-like encoded a predicted product of 977 amino acid residues with a molecular mass of 100 kDa. As described later, western blotting analysis revealed one major band of 100 kDa, suggesting that the molecular mass of OsPolI-like was ~100 kDa. The nucleotide sequence data reported in this paper appear in the DDBJ nucleotide sequence database with the accession number AB047689.

A modeled 3D structure for OsPolI-like protein
Escherichia coli DNA polymerase I is comprised of a 5'–3' exonuclease domain in the N-terminal region, a 3'–5' exonuclease domain in the internal region, and a DNA polymerase domain in the C-terminal region (37–39). The DNA polymerase domain of OsPolI-like shares 123 of the 426 amino acid residues with E.coli Pol I (28.8%), the 5'–3' exonuclease domain shares 65 of the 360 amino acid residues (18.1%) and 3'–5' exonuclease domain shares 35 of the 230 amino acid residues (15.2%). OsPolI-like protein showed a high degree of conservation in the C-terminal DNA polymerase domain. This degree of sequence identity is sufficient for 3D structure prediction, modeled on the X-ray structure of the Klenow fragment of E.coli Pol I using the Swiss Model service of the Expasy server (34–36). Modeling suggested substantial conservation of structure between Klenow fragment and the last 664 amino acids of OsPolI-like protein. According to this model, the C-terminal region of OsPolI-like would be organized in four different subdomains corresponding to the thumb, palm, finger and 3'–5' exo defined in the Klenow fragment. Except the 3'–5' exonuclease domain, the structures of these two molecules are very similar to each other (Fig. 1). We propose that the C-terminal domain of OsPolI-like protein is homologous to E.coli DNA polymerase I.



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  		Figure 1. A modeled 3D structure for OsPolI-like protein. (Left) X-ray structure of the Klenow fragment of E.coli Pol I (amino acids 329–927). This structure was used as a template for modeling. (Right) Modeled 3D structure for OsPolI-like protein (amino acids 296–959).

 
Map of OsPolI-like locus and exon patterns
The chromosomal location of cDNA of OsPolI-like on the linkage map of rice was determined by restriction fragment length polymorphism (RFLP) mapping (32). The OsPolI-like gene was mapped to chromosome 8 (Fig. 2A) and was shown to contain 12 exons and 11 introns (Fig. 2B). Southern blotting analysis revealed that theOsPolI-like gene exists as a single copy per genome (data not shown). These results suggested that the OsPolI-like gene is present in a single copy in the genome of rice, O.sativa L.



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  		Figure 2. Map of the OsPolI-like locus and exon patterns. (A) Map of chromosome 8 of rice (O.sativa L.) showing the locus of OsPolI-like. CEN, centromeric region. (B) Genomic organization of OsPolI-like gene. Boxed areas are exons, lines are introns and 5' and 3' upstream regions. The open boxes correspond to coding regions and hatched boxes correspond to the untranslated regions.

 
Phylogenetic analysis
DNA polymerase {gamma} (pol {gamma}) and {theta} (pol {theta}), members of DNA polymerase family A, are known to have sequence homology to E.coli Pol I (18,40,41). To determine the phylogenetic relationship between OsPolI-like and other family A DNA polymerases, the phylogenetic tree was drawn based on alignment by the UPGMA method (Fig. 3). As shown in Figure 4, OsPolI-like was not closely related to pol {gamma} or pol {theta}, but was closely related to DNA polymerase I, suggesting that OsPolI-like was different from pol {gamma} and {theta}. We also identified homologs of OsPolI-like and pol {theta} from the genomic sequence of Arabidopsis thaliana (Tables 1 and 2), indicating that both polymerases are different from each other (Fig. 4 and Tables 1 and 2). Interestingly, no nucleotide sequence similar to OsPolI-like was found in the genomic sequences of Drosophila melanogaster or Caenorhabditis elegans. Therefore, OsPolI-like was considered to be a novel DNA polymerase present in higher plants that belongs to DNA polymerase family A. In the family A DNA polymerase, OsPolI-like was most closely related to DNA polymerase I from a cyanobacterium, Synechocytis, implying that OsPolI-like belongs to a chloroplast or plastid DNA polymerase.



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  		Figure 3. Phylogenetic analysis. A phylogenetic tree was constructed by the UPGMA method, based on the amino acid sequences of DNA polymerase domain of OsPolI-like and the other family A DNA polymerases. Horizontal distances are proportional to evolutionary divergence expressed as substitutions per site.

 


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  		Figure 4. DNA polymerase activity of OsPolI-like. (A) SDS–PAGE analysis of purified protein. The concentration of the SDS–polyacrylamide gel was 10%, and the gel was stained with CBB. (B) Neutralization assay performed with a polyclonal antibody against OsPolI-like. DNA polymerase activity of the protein was measured with poly(dA)/oligo(dT)12–18 as template.

 

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  		Table 1. Characterization of OsPolI-like DNA polymerase domain: template/primer specificity
 

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  		Table 2. Characterization of OsPolI-like DNA polymerase domain: effect of DNA polymerase inhibitor
 
DNA polymerase activity of OsPolI-like protein
To determine whether OsPolI-like protein has DNA polymerase activity, the DNA polymerase domain (residues 444–959) was overexpressed in E.coli and purified as described in Materials and Methods. The DNA polymerase domain (60 kDa) of OsPolI-like was purified to near homogeneity as shown by SDS–PAGE analysis (Fig. 4A). The purified protein showed the DNA polymerase activity with poly(dA)/oligo(dT)12–18 as template (Fig. 4B). To confirm that the activity was associated with OsPolI-like protein, a neutralization assay was performed. As described in Materials and Methods, a polyclonal antibody against a partial peptide region (residue 432–791) of OsPolI-like was raised in rabbit. As shown in Figure 4B, the antibody against OsPolI-like significantly neutralized the DNA polymerase activity. The activity was not inhibited by pre-immune serum (data not shown). These results indicated that OsPolI-like protein has DNA polymerase activity.

The biochemical properties of the OsPolI-like DNA polymerase domain are shown in Tables 1 and 2. The protein showed a preference for poly(dA)/oligo(dT)12–18 as template. Monovalent cations (KCl) showed an inhibitory effect on the activity. The activity was strongly inhibited by dTTP, which is known to be an inhibitor on mammalian DNA polymerase ß and {gamma}, but was not insensitive to aphidicolin and NEM; both are inhibitors of DNA polymrase {alpha}, {delta} and {varepsilon}.

Subcellular localization of OsPolI-like
We next investigated the subcellular localization of OsPolI-like by comparison between the plastid fraction and total cell extract fraction from 10-day-old rice seedlings. The plastid fraction was isolated by Percoll density gradient centrifugation as described in Materials and Methods. Figure 5 shows the results of western blotting analysis of the fractions probed with antibodies against OsPolI-like, DS9 protein and OsPCNA protein. DS9 is a homolog of bacterial FtsH protein and is localized in chloroplasts (42). OsPCNA is an important enzyme involved in DNA replication and repair, and is localized in the nucleus (9). The OsPolI-like DNA polymerase is present in the plastid fraction similarly to DS9 protein (Fig. 5), suggesting that it is a plastid DNA polymerase.



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  		Figure 5. Subcellular localization of OsPolI-like. Western blotting analysis of total extract and plastid fractions of 10-day-old rice seedlings probed with anti-OsPolI-like antibody (lanes 1 and 2), anti-DS9 antibody (lanes 3 and 4) or anti-OsPCNA antibody (lanes 5 and 6). T and P represent ‘total extract’ and ‘plastid fraction’, respectively. Arrowheads indicate the positions of the bands.

 
The expression level of OsPolI-like is correlated with cell proliferation
To determine the expression pattern of OsPolI-like in various organs, northern hybridization analysis was performed. Total RNA samples isolated from various organs of 50-day-old rice plants (Fig. 6) were blotted and probed with 32P-labeled OsPolI-like cDNA. A 3.8-kb transcript was detected in roots, root tips and young leaves, but not in the mature leaves (Fig. 6). The level of expression in the root tips was higher than that in the whole roots (lane 3 and 4 in Fig. 6), suggesting that OsPolI-like is expressed in root apex regions that contain root apical meristem. The young leaves have meristem to increase the leaf width, whereas mature leaves have no proliferating cells. Transcription of OsPolI-like might be related to the level of cell proliferation.



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  		Figure 6. (A) Expression of OsPolI-like in different organs. Each lane contained 20 µg of total RNA isolated from mature leaves (lane 1), young leaves (lane 2), roots (lane 3) or root tips (lane 4) from 50-day-old rice plants. The blot was probed with 32P-labeled OsPolI-like cDNA (top). Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (bottom). Numbers on the left indicate the approximate length of the mRNA. (B) Effects of sucrose starvation on the level of OsPolI-like expression. Rice cells were cultured in suspension for 6 days (lanes 1 and 2) or 10 days (lane 3) with sucrose (lane 1) or without sucrose (lanes 2 and 3), 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 µg 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 OsPolI-like cDNA. Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (bottom). Numbers on the left indicate the approximate length of the mRNA.

 
OsPolI-like was actively transcribed in rice cells in suspension culture (lane 1 in Fig. 6). When cell proliferation was temporarily halted for 6 or 10 days by removal of sucrose from the growth medium, the level of OsPolI-like expression was significantly reduced (lanes 2 and 3 in Fig. 6). When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsPolI-like was again expressed at high levels (lane 4 in Fig. 6). These results indicated that OsPolI-like expression is correlated with cell proliferation.

Figure 7 shows the results of western blotting analysis of OsPolI-like protein in the tissues of 50-day-old rice plants on the ground. As shown in Figure 7A, the rice plants were separated into three segments: A, upper leaves (mostly mature leaves); B, lower leaves; and C, containing shoot apical meristem. The extracts from each segment were blotted, and probed with the polyclonal antibody (Fig. 7B). The protein was present at high levels in segment C containing the shoot apex (Fig. 7B). On the other hand, the protein was not detected in segment A composed of mature leaves (Fig. 7B). The cultured cells showed relatively high levels of the protein (Fig. 7B). These results suggested that OsPolI-like protein was distributed mostly in the meristematic tissues, but not in non-proliferating tissues.



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  		Figure 7. Immunoblotting analysis. (A) The plant organs used for immunoblotting analysis are illustrated. Segment C contained the shoot apex region. (B) Each lane contained the same amount of protein extracted from cultured rice cells (lane 1), segment C (lane 2), B (lane 3) or A (lane 4).

 
Spatial expression pattern of OsPolI-like
The results of northern and western blotting analyses clearly indicated that the level of OsPolI-like expression was correlated with cell proliferation. To study the expression pattern further, in situ hybridization using digoxigenin-labeled antisense OsPolI-like RNA as a probe was performed. When digoxigenin-labeled sense OsPolI-like RNA was used as a probe, no hybridization signals were detected (data not shown). In the shoot apex, the antisense probe for OsPolI-like showed strong hybridization signals in the shoot apical meristem, leaf primordium and marginal meristem of young leaves (Fig. 88A). In the root apex, hybridization signals were observed in root apical meristem (Fig. 88B). On the other hand, OsPolI-like was not expressed in the mature leaves or root caps, tissues in which cell proliferation does not occur (Fig. 88A–C). These results confirmed that OsPolI-like was mainly expressed in actively proliferating tissue. These spatial expression patterns coincided well with the results of northern and western hybridization analyses shown in Figures 6 and 7. With growth from the leaf primordia to young and then mature leaves, OsPolI-like antisense probe gradually showed weaker hybridization signals. Our observations indicated that OsPolI-like is expressed in tissue rich in proliferating cells, and that its expression may be necessary for cell growth. OsPolI-like might be required for plastid DNA replication in plant meristem because plastid proliferation might be accompanied by cell proliferation.



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  		Figure 8. Spatial expression pattern of OsPolI-like by in situ hybridization. (A) Longitudinal section from the shoot apex region of 10-day-old rice seedling. (B) Longitudinal section from the root apex region of 10-day-old rice seedling. (C) Cross-section of mature leaf of 30-day-old rice plant. The plant tissues were sectioned and probed with OsPolI-like antisense riboprobe labeled with digoxigenin-UTP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
OsPolI-like, a novel plant DNA polymerase
In this study, we isolated and characterized a novel DNA polymerase, designated as OsPolI-like, from the higher plant, rice (O.sativa L. cv. Nipponbare). OsPolI-like showed a high degree of conservation in the C-terminal DNA polymerase domain, implying that OsPolI-like has DNA polymerase activity. Sequence comparison among the family A DNA polymerases indicated that OsPolI-like was obviously different from DNA polymerase {gamma} and {theta}, other eukaryotic DNA polymerases known to have some homology with E.coli pol I protein. The results of subcellular fractionation and western blotting analysis suggested that OsPolI-like protein is localized in the plastids, premature chloroplasts. Therefore, this represents the first report of molecular cloning and extensive characterization of a novel DNA polymerase for plant plastid DNA synthesis, a novel eukaryotic DNA polymerase and the third cloned plant DNA polymerase after Pol {alpha} (31) and plant Pol {delta} (unpublished data).

Function of OsPolI-like
We analyzed the expression pattern of OsPolI-like by northern and western blotting and in situ hybridization analyses. Our results showed that OsPolI-like was expressed in the meristematic tissue such as shoot apical meristem and root apical meristem. OsPolI-like was not expressed in the mature leaves or the root caps. Therefore, we considered OsPolI-like expressed only in meristematic tissues. The chloroplasts in the mature leaves do not proliferate, suggesting that plastids, premature chloroplasts, proliferate in the meristem and may mature to chloroplasts with leaf maturation. In the cell, the proliferation of the plastids may be link with nuclear proliferation. It is possible that plastid DNA replication may require not only OsPolI-like but also other protein factors involved in nuclear DNA replication.

Higher plants are exposed to UV for much longer periods than animals or yeasts (7–10). They cannot avoid or escape from the effects of UV exposure. The observation that the OsPolI-like transcript was not detected in mature leaves despite continual exposure to high levels of UV is of interest. These observations raise the question of whether OsPolI-like is dispensable for repairing UV-damaged DNA in the chloroplasts, which are severely bombarded by sunlight. Studies in OsPolI-like-deficient mutant plants are required for further clarification of the role of OsPolI-like.

Plant DNA polymerases
To date, at least 12 classes of eukaryotic DNA polymerases have been identified in eukaryotes (15–24). However, little is known about DNA polymerases in higher plants in comparison with those in animals and yeasts (26,28,29,31). In plants, only two DNA polymerases, Pol {alpha} and Pol {delta}, have been cloned (31 and our unpublished data). There have been reports of wheat DNA polymerases designated as A, B, CI and CII (25,27,29,30,43), but which of the 12 mammalian polymerases correspond exactly to DNA polymerases A, B, CI and CII from wheat embryos is not yet known.

Table 3 summarizes plant DNA polymerases along with other eukaryotic DNA polymerases. Our search for convincing homologs of DNA polymerase in A.thaliana in the GenBank database showed that the plant must have at least nine species of DNA polymerase, {alpha}, {delta}, {varepsilon}, {zeta}, {eta}, {theta}, {kappa}, {lambda} and OsPolI-like (Table 3). It would be interesting to determine whether the biological functions of plant DNA polymerases are conserved as observed in mammalian cells. As described in the Introduction, our research interest lies with the roles of multiple DNA polymerase systems in relation to DNA synthesis and development.


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  		Table 3. Summary of plant DNA polymerases
 
Higher plants are interesting systems in which to study the mechanism of coordination between DNA synthesis and development and to investigate the relationship between DNA replication at nuclear division and at proliferation of subcellular organelles. Therefore, studies of plant DNA polymerases may shed light on their roles from a different viewpoint.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at NAR Online.


   ACKNOWLEDGEMENTS
 
We thank the Rice Genome Research Program (RGP) of Japan for providing the rice EST clone C0843. We also thank Dr S. Seo (National Institute of Agrobiological Sciences) for providing the polyclonal antibody against DS9. This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project MA-2201).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 471 24 1501; Fax: +81 471 23 9767; Email: kengo{at}rs.noda.sut.ac.jp Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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  6. Seto,H., Hatanaka,M., Kimura,S., Oshige,M., Tsuya,Y., Mizushina,Y., Sawado,T., Aoyagi,N., Matsumoto,T., Hashimoto,J. and Sakaguchi,K. (1998) Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence. Biochem. J., 332, 557–563.

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