Mutagenesis Advance Access originally published online on June 3, 2008
Mutagenesis 2008 23(5):407-413; doi:10.1093/mutage/gen030
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Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans
Department of Biological Sciences, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 1Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan 2Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan
Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO3-inducing cytosine deamination than wild-type strain.
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Introduction |
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DNA base moieties are continuously modified by endogenous chemical reactions such as deamination, depurination and oxidation (1
–4). Such base modifications, if unrepaired, have been suggested to play an important role in mutation, cancer and ageing (1–4). The repair of modified bases in DNA is primarily mediated by the base excision repair (BER) to prevent their deleterious consequences. DNA glycosylases hydrolyse the N-glycosylic bond between the modified base and deoxyribose, thus releasing a free base and an apurinic/apyrimidinic (AP) site in DNA (2–5). The resulting AP sites are further processed during the BER (4,6).
The loss of the amino groups in cytosine, adenine, guanine and 5-methylcytosine in DNA results in the conversion to uracil, hypoxanthine, xanthine and thymine, respectively (3,4,7,8). The number of cytosine deamination has been calculated to be 100–500 in cells/day (4). The cytosine deamination results in mutagenic U:G mispairs in DNA, which would give rise to C:G to T:A transitions upon subsequent DNA replication (7–9). Uracil also occurs in DNA through incorporation of dUMP instead of dTMP by DNA polymerases during replication (3,7,10,11). Incorporated dUMP forms U:A base pairs that are not directly mutagenic, but may be cytotoxic (12). Uracil-DNA glycosylase (Ung) acts as a major repair enzyme that protects DNA from the deleterious effects caused by uracil (3,5,7,8,13). This enzyme hydrolyses the N-glycosylic bond connecting the uracil to the DNA. Escherichia coli and Saccharomyces cerevisiae have only the family-1 Ung (4,14,15). This is one of the essential and common DNA repair pathways since ung-homologous genes and their corresponding enzymes have been identified in a variety of organisms (5,7,16,17).
It has been proposed that ageing-related dysfunction is caused by cumulative spontaneous damage in DNA (18). As the deleterious effects of DNA damage can be prevented by DNA repair, it is certain that DNA repair and ageing are interrelated. The nematode C.elegans is an excellent model system for the study of animal development and ageing. Recently, RNA interference (RNAi)-based genome-wide screen revealed that the ung-1 gene contributes to genome stability in somatic cells and prevents spontaneous mutagenesis in the germ line of C.elegans (19). Therefore, it is important to explore structure and function of Ung and examine whether or not the repair capacity for uracil in DNA directly correlates with ageing in C.elegans.
Shatilla and Ramatar (20) previously found that the extract derived from the embryos of C.elegans contained the activity to cleave the oligonucleotide substrates at the uracil position. However, cloning and purification of uracil-excising enzymes of C.elegans have not been reported to date. In this study, we cloned the ung-1 gene of C.elegans of which product (CeUng-1) shares 49% identity and 69% similarity with E.coli Ung. Purified CeUng-1 removed uracil opposite both guanine and adenine in double-stranded oligonucleotides. The activity of CeUng-1 was inhibited by Bacillus subtilis Ung inhibitor (Ugi), a specific inhibitor of the family-1 Ung (4,21). The mutation of ung-1 did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzymes. However, residual uracil excision activity could not be detected in the extract from the ung-1 mutant. The survival of the ung-1 mutant following exposure to NaHSO3, which damages DNA through deamination of cytosine to uracil, was significantly greater than wild-type strain.
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Materials and methods |
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Enzymes and oligonucleotides
Restriction enzymes and KOD plus DNA polymerase were purchased from Toyobo (Osaka, Japan). Ugi was obtained from New England Biolabs Japan (Tokyo, Japan). Thrombin was obtained from Pharmacia Biotech (Uppsala, Sweden). Oligonucleotide containing a single uracil residue (5'-CCTGGCCTGUGCAGCTGTGGG-3') was obtained from Trevigen Inc. (Gaithersburg, MD, USA). Other oligonucleotides were synthesized and purified by high-performance liquid chromatography (Takara Shuzo, Kyoto, Japan).
Identification and cloning of CeUng-1 gene
The database was searched for proteins with homology to the E.coli Ung using BLAST database. As a result, a hypothetical protein CEY56A3A.29a was detected with significant homology to the E.coli Ung. The cDNA library was used as a template for polymerase chain reaction (PCR) to amplify the ung-1 cDNA of C.elegans. The oligonucleotides 5'-GCGGATCCATGTCGAAGACTGTAAGAATTC-3' and 5'-TAGCTCGAGAATACGCAGGGTGTTCGACAG-3' were used as primers for PCR. The primers contained a BamHI or XhoI site, respectively. The amplified products were digested with BamHI and XhoI and then ligated into BamHI- and XhoI-digested pGEX-4T-1 vector (Pharmacia Biotech) to generate pGEX-CeUng-1. The sequence of the insert was checked to verify that no mutations had been introduced by the PCR.
Bacterial strains, media and plasmids
Escherichia coli BL21 (lon ompT) was used for expression of CeUng-1. Escherichia coli OP50 was used as a food for C.elegans. Bacterial cells were grown at 37°C in Luria–Bertoni (LB) medium with shaking. When necessary, ampicillin (Amp) and chloramphenicol were added to the medium at a final concentration of 100 and 30 µg/ml, respectively.
Expression and purification of CeUng-1
Escherichia coli BL21 cells bearing pGEX-CeUng-1 were grown at 37°C in LB/Amp until the optical density at 600 nm reached 
0.6. After the addition of 0.1 mM of isopropyl-β-D-galactopyranoside, the cultures were further incubated at 26°C for 12 h. The bacterial cells were collected by centrifugation and re-suspended in ice-cold phosphate-buffered saline (PBS) containing 2.7 mM KCl and 140 mM NaCl (pH 7.4). The cells were disrupted by sonication on ice with 6 x 10-sec bursts, followed by centrifugation at 26 000x g for 30 min at 4°C. The supernatant was then applied to a glutathione (GSH)–Sepharose 4B column (Pharmacia Biotech) that had been equilibrated with 50 mM Tris–HCl (pH 8.0). Glutathione-S-transferase (GST) fusion protein was eluted from the column with 10 mM of GSH in 50 mM Tris–HCl (pH 8.0). Fractions containing the GST–CeUng-1 were collected and treated with thrombin at 4°C for 24 h, followed by purification of CeUng-1 using MicroSpin GST module to remove excised GST. Purified protein in passed fraction was diluted with PBS containing 50% (v/v) glycerol and stored at –80°C before use. Purity of the protein was determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis).
Assay for enzyme activity
The 21-mer oligonucleotide containing a single uracil residue (5'-CCTGGCCTGUGCAGCTGTGGG-3') was labelled at the 5'-end with [
-32P]ATP by T4 polynucleotide kinase and then annealed to complementary strand containing guanine (dsU/G) or adenine (dsU/A) opposite the uracil. 5'-32P-labelled 21-mer oligonucleotide was also used for single-strand substrate (ssU). [-32P]ATP (>259 TBq/mmol) was obtained from ICN Biomedicals Inc. (Costa Mesa, CA, USA). The reaction mixture (10 µl) contained 60 mM Tris–HCl (pH 8.0), 1 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mg/ml of bovine serum albumin, 20 fmol of 32P-labelled oligonucleotide and purified CeUng-1 or C.elegans crude extract. DNA-nicking assay was carried out at 26°C unless otherwise stated. The oligonucleotides with resulting AP sites were cleaved at the site by treatment with 0.16 M NaOH at 95°C for 5 min. The samples were analysed by electrophoresis in 20% denaturing polyacrylamide gels in Tris–borate (pH 8.3) containing 7 M urea and 2 mM EDTA. After electrophoresis at 1300 V, the gels were dried and autoradiographed using Fuji RX film at –80°C. Enzyme activities were calculated from the quantity of the cleaved product using NIH Image software.
Caenorhabditis elegans strains and culture conditions
Caenorhabditis elegans strains Bristol N2 (wild type) and TM2862 (ung-1) were obtained from the C.elegans Genetic Center, University of Minnesota and Dr S. Mitani (Tokyo Women's Medical College) of the National Bioresource Project for the Experimental Animal ‘Nematode C.elegans’, respectively. The ung-1 mutant was isolated by treatment of C.elegans N2 with long-wavelength ultraviolet in the presence of trimethylpsoralen (22). The 293 base pairs are deleted in the ung-1 gene in the mutant strain (WormBase Gene Summary for ung-1).
Worms were fed on 9 cm diameter enriched nematode growth medium (NGM) agar plates with E.coli cells at 22°C. The NGM plates contained 0.3% NaCl, 0.25% polypeptone, 0.002% cholesterol, 1 mM MgSO4, 1 mM CaCl2 and 0.17% agar (pH 6.0).
Preparation of C.elegans crude extract
Mixed stage of worms were collected by centrifugation at 2000x g for 15 sec at room temperature and washed with sterile water three times. After being leaved for 1 h to digest E.coli remaining in worms’ intestine, the worms were collected by centrifugation and re-suspended into buffer A [50 mM Tris–HCl (pH 7.5), 1 mM DTT, 5 mM KCl and 1.5 mM MgCl2]. The extract was prepared by homogenization and subsequent sonication on ice, followed by centrifugation at 13 000x g for 30 min at 4°C. The supernatant thus prepared was divided into portions and stored at –80°C before use. The BCA Protein Assay Kit was obtained from Pierce (Rockford, IL, USA) and used to quantify proteins.
Assay for lifespan
Lifespan was determined as described by Ishii et al. (23). L1 larvae were allowed to hatch by overnight incubation in S buffer [0.05 M phosphate buffer (pH 7.2) containing 0.1 M NaCl] and transferred to NGM plates to develop to the L4 stage. A hundred of the L4 larvae were transferred to NGM plates supplemented with 40 µM of 5-fluoro-2'-deoxyuridine to suppress the production of their progenies. The worms were examined daily, and dead worms, which did not move after we touched their heads with a platinum wire, were removed to count.
Assay for survival of C.elegans after treatment with NaHSO3
The eggs were treated with various concentrations of NaHSO3 at 16°C for 12 h and then placed on NGM plates, followed by incubation at 16°C for 24 h. The number of dead embryos was scored to estimate survival.
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Identification of C.elegans homologue of the E.coli Ung
BLAST searching of the Wormbase database retrieved a sequence Y56A3A.29a, highly homologous to the E.coli ung gene product (WormBase Gene Summary for ung-1). The database showed a high degree of identity (49%) and similarity (69%) with E.coli Ung. The ung-1 gene of C.elegans locates on chromosome III: 13.69 ± 0.087 cM and has three exons, encoding a 282-amino acid protein (32 KDa) (WormBase Gene Summary for ung-1). The GenBank accession number for the C.elegans Ung-1 cDNA sequence is CAB60520 [GenBank] . Reverse BLAST search of the protein sequence database retrieved many of the eukaryotic Ung with high accuracy.
Figure 1 shows comparative primary sequence homology of Ung of various species, analysed by CLUSTALW. The homology between human Ung, Mus musculus Ung, E.coli Ung and C.elegans Ung-1 (CeUng-1) spans the whole sequence. The structural element analysis indicated that a protein potentially belonging to the Ung family has eight conserved DNA-contacting elements in its sequence: 
1–8 and β1–β4 (5,24,25). These domains were highly conserved among the known Ung sequences including CeUng-1 in the database (Figure 1).
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CeUng-1 contained certain conserved residues that have counterparts in E.coli and human Ung. The CeUng-1 polypeptide had highly conserved active sites A and B (4
,24,26–29). The active site A was present between residues 116–137 in CeUng-1, 58–77 in E.coli Ung and 138–159 in human Ung (5,26–28). The active site B was also highly conserved between residues 247 and 253 in CeUng-1 (Figure 1). The position of a conserved Apn182 was involved in catalysis and conserved for distinguishing cytosine and uracil, which plays a major role in establishing the high degree of base specificity displayed by Ung enzyme (5,26–29). Aromatic residues, Tyr125, Phe136 and His247, are involved in the stacking interaction with uracil (5,27). The sequence Gly121–Gln122 (5,26–29) was conserved together with Leu251 in CeUng-1 (Figure 1).
Cleavage of uracil-containing oligonucleotide by purified CeUng-1
We purified the CeUng-1 protein from the GST–CeUng-1-over-expressing E.coli cells. The GST fusion protein was subsequently cleaved by thrombin to obtain CeUng-1. The CeUng-1 thus obtained showed apparent homogeneity band (Figure 2). The activity of purified CeUng-1 was measured at various temperatures to know the optimal temperature. The 9-mer product was generated by cleavage of 21-mer U:A-containng oligonucleotides by CeUng-1, indicating that the enzyme cleaved the oligonucleotide at the site of the uracil (Figure 3). CeUng-1 showed high level of activity 26°C. The same profile was observed with dsU/G and ssU (data not shown). Hence, we assayed enzymatic activities of CeUng-1 at 26°C during this study.
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The substrate oligonucleotide (20 fmol), dsU/G, dsU/A or ssU, was incubated with purified CeUng-1 at 100 fmol for 0, 0.5, 1, 3, 10 and 60 min. The results are shown in Figure 4A and B. The amount of cleavage product increased with reaction time (until
10 min). Next, the double-stranded oligonucleotide containing U:G or U:A was incubated with the CeUng-1 at 0, 0.1, 1, 5, 10, 50 and 100 fmol for 5 min. The cleavage product increased with the amount of the CeUng-1 (Figure 4C). CeUng-1 was active on dsU/G and dsU/A. It removed uracil most efficiently from U:G mismatches. CeUng-1 had less efficient activity for ssU than double-stranded oligonucleotides.
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Ugi, a uracil glycosylase inhibitor of B.subtilis bacteriophage PBS1, is a small protein, which specifically inhibits E.coli Ung, as well as the family-1 Ung from other species (21
,30). The oligonucleotide, dsU/A, dsU/G or ssU, was incubated with excess amount of purified CeUng-1 (10 ng) in the presence of Ugi. The results are shown in Figure 5. DNA cleavage activity of CeUng-1 was completely inhibited. Thus, we confirmed earlier results by Shatilla and Ramotar (20) with the homogeneously purified Ung-1 protein.
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Physiological effects of the ung-1 mutation
To investigate physiological roles of CeUng-1, we selectively interfered with the activity of the CeUng-1 by deletion mutation of the ung-1 gene [WormBase Gene Summary for ung-1 (22
)]. The ung-1 mutant was viable and fertile and laid eggs at normal rate. Larval development was unaffected, as both wild-type and ung-1 mutant worms began producing progeny on the same day. We further compared lifespan for ung-1 mutant worms with that for wild-type N2 strain. Similar lifespan profiles were observed in the two strains (Figure 6). These results indicated that the mutation of the ung-1 gene did not couple with viability, development and lifespan of C.elegans.
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Uracil excision activity in extract prepared from ung-1 mutant of C.elegans
To examine whether or not C.elegans contains other uracil excision enzymes than CeUng-1, the substrates dsU/G, dsU/A and ssU were incubated with the extract (0.4 µg protein) derived from wild-type N2 and ung-1 mutant TM2862 strains of C.elegans. The results are shown in Figure 7. The uracil excision activity was almost completely lost in extract derived from TM2862 worms (lanes 4 and 10). There was no Ugi-resistant uracil-excising activity in the extract derived from N2 and TM2862 strains (lanes 3, 5, 9 and 11). It was the case when ssU was incubated with excess amount of crude extract (8 µg protein) (data not shown).
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Sensitivity of wild-type and ung-1 mutant strains to lethality by NaHSO3
We examined the sensitivity of wild-type N2 and CeUng-1-deficient mutant TM2862 to lethal effect of NaHSO3 (up to 30 mM). NaHSO3 damages DNA primarily through deamination of cytosine to uracil (31
). The results are shown in Figure 8. The survival of the ung-1 mutant was much greater than wild-type strain when the reagent was added at >20 mM.
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Discussion |
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TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
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In this study, we first cloned and characterized the C.elegans homologue of E.coli Ung. The CeUng-1 protein shares 49% identity and 69% similarity in amino acid sequence with E.coli Ung. The ung gene of C.elegans encodes a 282-amino acid protein with a calculated molecular weight of 32 KDa. The purified CeUng-1 catalysed the removal of uracil from both U:G and U:A base pairs in double-stranded DNA (Figure 4). Escherichia coli Ung and S.cerevisiae Ung also remove uracil from U:G and U:A base pairs in DNA (4
,14,15). Furthermore, the activity of CeUng-1 was inhibited by Ugi (Figure 5). The peptide inhibitor Ugi potently inhibits both prokaryotic and eukaryotic Ungs belonging to the family 1 Ung (4,21,30). These results demonstrated that CeUng-1 is a member of the family-1 Ung family (4,5,17). The argument was supported by the following fact; the expression of CeUng-1 was capable of complementing the increased spontaneous rifampicin-resistant mutations in E.coli ung mutant (data not shown). The results also indicated that the removal of deaminated cytosine from DNA is relevant in vivo. Therefore, Ung enzyme is the major enzyme for the repair of uracil in C.elegans as in E.coli, yeast and humans (4,5).
The mutation of ung-1 gene did not result in severe growth phenotypes and development in C.elegans. Similar characteristics of Ung-1-deficient worms were observed by Dengg et al. (32). In addition, we showed that lifespan was also unaffected in C.elegans ung-1 mutant worms (Figure 6). Hence, it was suggested that other DNA glycosylases may serve a relatively efficient backup for CeUng-1 in the repair of uracil in DNA in C.elegans. Recent studies revealed that there are at least four additional families of uracil-excising enzymes according to their differences in substrate recognition and amino acid sequence. They are the MUG/TDG family (family-2), the SMUG family (family-3), the thermostable Ung family (family-4) and the Ung-B family (family-5) (4,5,33–36). However, it is not likely that C.elegans has any uracil-removing enzymes belonging to such other families because the C.elegans database did not reveal any homologues of other family Ung enzymes including Mug, SMUG1, TDG and GBD4. SMUG1 is also not inhibited by Ugi (34). Furthermore, SMUG1 shares extremely limited amino acid sequence homology with the Ung protein (34,36). These findings suggest that such enzymes do not exist and serve as a backup for Ung in C.elegans. In fact, there were not any residual activities in the extract derived from C.elegans ung-1 mutant. In addition, there were no cleavage activities for U:G mismatch-containing DNA that were not inhibited by Ugi (Figure 5). CeUng-1 showed low activity for ssU (Figure 4) compared with E.coli Ung (4,5,7). So, C.elegans might have SMUG-like enzyme that efficiently excises uracil from single-stranded DNA.
Pothof et al. (19) recently reported that RNAi-based genome-wide screen identified 60 genes that contribute to genome stability in somatic cells and prevent spontaneous mutagenesis in the germ line of C.elegans. The ung-1 gene is included as such a mutator gene (19). It is necessary to clarify the spectra of spontaneous mutation in C.elegans ung-1 mutant, in particular to examine whether the frequency of C:G to T:A transitions is increased in the mutant worms.
Other repair systems such as nucleotide excision repair or mismatch repair may operate on the deaminated cytosine in C.elegans. Methanococcus jannaschii uracil-DNA glycosylase (MjUDG) efficiently removes uracil from both single- and double-stranded DNA (37). MjUDG also catalyses the excision of 8-oxoguanine from DNA. It is of interest to examine whether or not C.elegans possesses such novel type of DNA glycosylase as MjUDG because C.elegans has no homologues of E.coli MutM and mammalian Ogg1 that remove 8-oxoguanine from DNA.
Saccharomyces cerevisiae ung-1 mutants defective for Ung activity have an increased sensitivity to NaHSO3 (31). Interestingly, the survival of the ung mutant of C.elegans following exposure to NaHSO3 was much higher than the wild-type strain (Figure 8). This suggested that persistent BER intermediates such as AP sites generated by the action of Ung and/or single-stranded DNA formed during repair of misincorporated uracil, if not efficiently processed, might be a lethal damage for C.elegans. To examine whether developmental defects and apoptosis can be induced in NaHSO3-treated C.elegans are under investigation in our laboratory.
Ung enzymes are highly conserved in evolution and the active site is almost completely conserved (4,5,38). The cloning of genes or cDNA for Ung from bacteria to humans has demonstrated a striking similarity between these enzymes, ranging from 40.3% (yeast) to 90% (mouse) amino acid identity relative to human Ung (38). Unlike many other Ung enzymes, the optimal temperature of CeUng-1 was 26°C (Figure 4), which might reflect the optimal temperature for C.elegans. This property may be required during evolution.
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Funding |
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Global Center of Excellence Program ‘Formation of a Strategic Base for Biodiversity and Evolutionary Research; from Genoome to Ecosystem’ (A-06) of the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan; Central Research Institute of Electric Power Industry (Tokyo) and Takeda Science Foundation (Osaka) to Q.-M.Z.
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Acknowledgments |
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The authors express their gratitude Dr. S. Mitani, Tokyo Women's Medical College for kindly supplying C. elegans ung-1 mutant. Conflict of interest statement: None declared.
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Notes |
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* To whom correspondence should be addressed. Tel: +81 75 753 4097; Fax: +81 75 753 4087; Email: qmzhang{at}kingyo.zool.kyoto-u.ac.jp
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Received on October 31, 2007; revised on April 1, 2008; accepted on April 3, 2008.
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