Evidence of intermolecular recombination between extrachromosomal DNAs in phytoplasma: a trigger for the biological diversity of phytoplasma?b

Hisashi Nishigawa1, Kenro Oshima2, Shigeyuki Kakizawa2, Hee-young Jung1, Tsutomu Kuboyamaa,1, Shin-ichi Miyata2, Masashi Ugaki1,2 and Shigetou Namba1,2

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan1
Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan2

Author for correspondence: Shigetou Namba. Tel: +81 4 7136 3700. Fax: +81 4 7136 3701. e-mail: snamba{at}k.u-tokyo.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Recombination among bacterial extrachromosomal DNAs (EC-DNAs) plays a major evolutionary role by creating genetic diversity, and provides the potential for rapid adaptation to new environmental conditions. Previously, a 7 kbp EC-DNA, EcOYW1, with a geminivirus-like rolling-circle-replication protein (Rep) gene was isolated and characterized from an original wild-type line (OY-W) of onion yellows (OY) phytoplasma, an endocellular cell-wall-less prokaryote that inhabits the cytoplasm of both plant and insect cells. EcOYW1, found in OY-W, was not present in a mild-symptom line (OY-M) derived from OY-W. A 4 kbp EC-DNA, pOYW, was also isolated and characterized from OY-W, and its pLS1-plasmid-like rep gene was expressed. This paper describes the isolation and sequencing of an EC-DNA of 5560 nt, EcOYW2, from OY-W, and its counterpart EC-DNA of 5025 nt, EcOYM, from OY-M. EcOYW2 and EcOYM contained seven and six ORFs, respectively. They both encoded a geminivirus-like Rep and a putative single-stranded-DNA-binding protein (SSB). Southern blot analysis indicated that no more EC-DNAs with a rep gene exist in either OY-W or OY-M, which means that the complete set of EC-DNAs has been cloned from the OY-W and OY-M lines of OY phytoplasmas. Sequence analysis revealed that both EcOYW2 and EcOYM have chimeric structures of previously characterized EcOYW1 and pOYW, suggesting that they have a recombinational origin. This is the first evidence of intermolecular recombination between EC-DNAs in phytoplasma. The possible implications of these findings in increasing the biological diversity of phytoplasma are discussed.

Keywords: phytoplasma, extrachromosomal DNA, rolling circle replication, recombination

Abbreviations: EC-DNA, extrachromosomal DNA; OY, onion yellows; OY-M, mild-symptom line of onion yellows phytoplasma; OY-W, wild-type onion yellows phytoplasma

b The GenBank accession numbers for EcOYW and EcOYM are AB076262 and AB076263, respectively.

a Present address: Graduate School of Agriculture, Ibaraki University, Ami, Inashiki-gun, Ibaraki 300-0393, Japan.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phytoplasmas (formerly called mycoplasma-like organisms or MLOs) are endocellular prokaryotes without cell walls, which give rise to more than 600 diseases in at least 300 plant species (McCoy et al., 1989 ; Kirkpatrick, 1992 ). Phytoplasmas inhabit the phloem sieve element of plants and are transmitted between plants by phloem-feeding insects (Jensen, 1959 ; Hemmati, 1977 ; Tsai, 1979 ). Because of the difficulty of culturing phytoplasmas in vitro, these organisms remain one of the most poorly characterized groups of plant pathogens.

The wild-type line (OY-W) of onion yellows (OY) phytoplasma is the causal agent of onion yellows disease, which produces a wide variety of symptoms in its plant host, including virescence (excessive greening of floral tissue), yellowing, phyllody (leaf-like petals and sepals), stunting, proliferation and witches’ broom (excessive formation of shoots) (Shiomi et al., 1996 ). OY-W has been maintained in a greenhouse using a leafhopper vector, Macrosteles striifrons, and the host plant garland chrysanthemum, Chrysanthemum coronarium. In our previous work, a mild-symptom line (OY-M) was isolated from OY-W that had been maintained by insect and plant hosts (Oshima et al., 2001b ). Plants infected with OY-M still produce many lateral shoots, but show mild leaf yellowing and almost no stunting. The host range and incubation period of OY-M are not significantly different from those of OY-W.

The genome size of phytoplasmas ranges from 530 to 1350 kb, representing the smallest genome in the class Mollicutes and of all known living organisms (Whitley & Finch, 1990 ; Neimark & Kirkpatrick, 1993 ; Carle & Neimark, 1995 ; Gibb et al., 1995 ; Zreik et al., 1995 ; Marcone et al., 1999 ). Moreover, this variability in genome size is strikingly large (Pyle et al., 1990 ; Ladefoged & Christiansen, 1992 ; Marcone et al., 1999 ). It has been observed that dynamic variation in the genome size occurs after maintenance of a phytoplasma isolate for a certain period of time (Oshima et al., 2001b ). However, little is known about the dynamics of extrachromosomal DNA (EC-DNA) species of phytoplasmas.

Genes encoded in EC-DNAs, such as plasmids, are known to play important roles in the pathogenicity and virulence of many plant-pathogenic bacteria (Panopoulos & Peet, 1985 ; Vivian et al., 2001 ). The isolation and characterization of EC-DNAs in some phytoplasmas have been described (Denes & Sinha, 1991 ; Kuske et al., 1991 ; Schneider et al., 1992 ; Goodwin et al., 1994 ; Nakashima & Hayashi, 1997 ; Kuboyama et al., 1998 ; Oshima et al., 2001a ; H. Nishigawa and others, unpublished). The western aster yellows phytoplasma contains at least four different species of EC-DNA (Kuske & Kirkpatrick, 1990 ). However, isolation of the complete set of EC-DNAs from one phytoplasma isolate has not yet been reported. In our previous work, we isolated and characterized two different classes of EC-DNAs from OY phytoplasma. One class of EC-DNA, which is 7 kbp in length and has a rep gene similar to that of the geminiviruses, has been shown to exist only in the OY-W line (EcOYW1) and not in the OY-M line (Nishigawa et al., 2001 ). Another class of EC-DNA, which is 4 kbp in size and has a rep gene similar to that of pLS1-like plasmids, has been isolated from the OY-W (pOYW) (Kuboyama et al., 1998 ; Oshima et al., 2001a ) and OY-M (pOYM) (H. Nishigawa and others, unpublished) lines. The rep gene of pOYW was found to encode a unique protein, pOY plasmid Rep. The N-terminal region of pOY plasmid Rep is similar to the Rep of the pLS1 family of plasmids, but, unlike the Rep of other plasmids, it has a C-terminal extension unexpectedly similar to the helicase domain of Rep of eukaryotic viruses, especially the circoviruses (single-stranded-DNA viruses of vertebrates). It was suggested that an ancestral phytoplasma plasmid, pOYW, may have acquired a helicase domain from host phytoplasmal DNA, entered the surrounding eukaryotic cytoplasm, and subsequently evolved into an ancestral eukaryotic single-stranded-DNA virus (Oshima et al., 2001a ).

This paper reports the isolation and characterization of another class of EC-DNA species, EcOYW2 and EcOYM, which are approximately 5 kbp in size, from OY-W and OY-M, respectively. Southern blot analysis indicated that no more EC-DNAs exist in either OY-W or OY-M, which means that the complete set of EC-DNAs has been cloned from OY phytoplasma. Interestingly, both EcOYW2 and EcOYM have chimeric structures of two other classes of EC-DNAs: EcOYW1 and pOYW. We postulate that the new class of EC-DNAs originated from intermolecular recombination between different classes of EC-DNAs. To our knowledge, such an event has never been reported for phytoplasmal EC-DNAs. The roles of recombination in the evolution of phytoplasmal EC-DNAs and in increasing the biological diversity of phytoplasma are discussed.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Extraction of phytoplasmal DNA from infected plants.
OY-W (Shiomi et al., 1996 ) and its mild-symptom variant line OY-M, isolated from OY-W during maintenance in a greenhouse by an insect vector and a plant host (Oshima et al., 2001b ), were used. Healthy garland chrysanthemum plants were caged with infected leafhopper vectors for 5–7 days to transmit the phytoplasmas to the plants. After removal of the insects, the plants were grown in the greenhouse (at 20–30 °C) for about 2 months. DNA was extracted from plants with well-developed symptoms. Since phytoplasmas are localized in the phloem, the vascular bundle tissue was excised aseptically from the stems, and the midribs were collected from the leaves, as described by Nishigawa et al. (2001) . Extraction of EC-DNA from phytoplasma-infected plants has been described previously (Lee & Davis, 1983 ; Denes & Sinha, 1991 ).

Southern hybridization.
Southern blot analysis was performed with an AlkPhos alkaline phosphatase labelling kit (Amersham Pharmacia) and by chemiluminescent detection with CDP-Star (Amersham Pharmacia) according to the manufacturer’s instructions. DNA fragments containing ORF1 (rep) from EcOYW1 were amplified by primers Rep-N and Rep-C (Nishigawa et al., 2001 ) and used as a probe. A DNA fragment containing the rep gene from pOYW was amplified by primers 105Rep1 (5'-TATTT ATCAA AATGA TAAAG AGGCT C-3') and 105Rep2 (5'-CAACG ACGTT TTAAT TGAGT AATAC-3') and also used as a probe (Oshima et al., 2001a ). Total DNA, extracted from either OY-W- or OY-M-infected plants, was digested with EcoRI. For hybridizations, this digested DNA was electrophoresed on 0·7% agarose gels, transferred to nylon membranes (Roche Diagnostics) and detected by chemiluminescence on X-ray films (Fuji Medical X-ray Film RX-U; Fuji Photo Film).

Cloning of EC-DNA.
EC-DNA species were extracted, by gel purification from OY-W and OY-M lines, and digested with EcoRI. The DNA was ligated into pUC18 cloning vectors and introduced into a host Escherichia coli strain, JM109. The ends of the cloned fragments were amplified by PCR using primers EW1 (5'-CATAA TTAGG TTCTA ATGAT TCAGC-3') and EW2 (5'-TATTC TCATT ACTCC TTTAC TGCC-3') for OY-W, and EM1 (5'-CAATT CTTTA ATATC AGATT CAAAG AT-3') and EM2 (5'-TGCGT ATATG TAGGG TCATT TTTAT-3') for OY-M. Amplification of these fragments was performed in a thermal cycler (Perkin Elmer 9700) for 30 cycles, under the following conditions: denaturation for 15 s at 94 °C, and annealing and extension for 3 min at 60 °C.

Sequence analysis.
The EC-DNA species were sequenced by the dideoxynucleotide chain termination method using a PRISM 377 DNA Sequencer (Applied Biosystems). Sequence analysis was performed with DNASIS version 3.7 (Hitachi Software Engineering). Amino acid sequence similarity analysis by BLAST-P and PSI-BLAST (Altschul et al., 1990 ) was performed with the server of the National Center for Biotechnology Information, USA.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of a new class of EC-DNA
We previously showed that an EC-DNA, EcOYW1, resides in the OY-W line and not in the OY-M line (Nishigawa et al., 2001 ). To see whether any other EC-DNAs with a similar replication mechanism reside in either the OY-W or OY-M line, Southern hybridization was performed using the rep gene (ORF1) of EcOYW1 as a probe. The target DNA was EcoRI-treated total DNA from phytoplasma-enriched plant extracts. Two bands, 7 and 5·5 kbp in length, were detected from the OY-W line, and one 5 kbp band was detected from the OY-M line (Fig. 1). The 7 kbp band detected in the OY-W line was EcOYW1 (Nishigawa et al., 2001 ), while the 5·5 kbp band from the OY-W line and the 5 kbp band from the OY-M line were thought to be newly detected EC-DNA species. The bands were not detected in tissue from a healthy plant, indicating that the detected bands were derived from OY phytoplasma. The two new species of EC-DNA were successfully cloned. We designated these EC-DNAs, derived from the OY-W and OY-M lines, as EcOYW2 and EcOYM, respectively.



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Fig. 1. Southern blot analysis detected EC-DNA with a virus-like rep gene. ORF1 (rep) of EcOYW1 was used as a probe. Lanes: EcoRI-treated total DNA extracted from a healthy plant (lane H), from an OY-W-infected plant (lane W) and from an OY-M-infected plant (lane M).

 
To determine whether the new EC-DNAs were originally circular in the cell and whether the cloned fragments were full-length, we sequenced the cloned DNAs and designed a set of primers to amplify the junction of the ends of the cloned fragments. Using these primers, PCR amplified a 2·5 kbp fragment from both the OY-W line and the OY-M line (data not shown), which means that the original EC-DNAs were circular. Cloning and sequencing of the PCR-amplified fragments and comparison of the sequences with those of the originally cloned fragments revealed that both of the originally cloned fragments were full-length.

Sequence analysis
Sequence analysis of the cloned full-length EC-DNAs revealed that EcOYW2 was 5560 bp and EcOYM 5025 bp in length. The physical map of these EC-DNAs and the locations of putative ORFs encoding more than 100 amino acids are shown (Fig. 2). Seven ORFs were found in EcOYW2, and six were found in EcOYM. ORFs 1, 2, 3, 4 and 5 of EcOYW2 and EcOYM were similar to each other, suggesting that EcOYW2 and EcOYM share the same ancestry.



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Fig. 2. Map of the EC-DNAs and plasmids of the OY-W and OY-M lines. A counterpart of EcOYW1 does not exist in OY-M. EcOYW2 is 5560 bp in length and has seven ORFs, while EcOYM is 5025 bp in length and has six ORFs. The ORFs and their orientations are indicated by arrows. ORFs sharing similarity are marked in the same colour. Two ORFs, encoding Rep and the single-stranded-DNA-binding protein gene (ssb), are contained in each EC-DNA. The EcoRI site was used for cloning. Small arrows indicate EW1, EW2, EM1 and EM2, the primers for PCR of the cloned fragment used to determine the sequence around the EcoRI site. Similar regions are indicated by the same character. EcOYW2 has some parts similar to EcOYW1 and pOYW. EcOYM also has some parts similar to EcOYW1, EcOYW2 and pOY plasmids.

 
Putative proteins encoded in ORF5 of both EC-DNAs were similar to the Rep of geminiviruses, single-stranded-DNA viruses belonging to the family Geminiviridae. A motif called the P-loop, GXXXXGKT/S, conserved in all geminivirus Rep proteins, was found in the C-termini of both ORF5-encoded proteins (Fig. 3). Other motifs of Rep were also conserved in the N-termini of both ORF5-encoded proteins, as shown in the alignment in Fig. 3. This substantial similarity suggests that EcOYW2 and EcOYM replicate via a geminivirus-like rolling-circle replication mechanism. ORFs 4 of EcOYW2 and EcOYM were similar to the single-stranded-DNA-binding protein (ssb) genes. None of the other ORFs showed significant similarity to any reported genes.



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Fig. 3. Comparison of the deduced amino acid sequence of ORF5 (rep) with other rep genes from geminiviruses. Putative common domains (motif 1, 2 and 3, helix 1 and helix 2, and the P-loop) are indicated. Residues in black boxes are identical residues; dots indicate gaps. BGMV, bean golden mosaic virus (Begomovirus; GenBank accession no. P05175); BCTV, beet curly top virus (Curtovirus; GenBank accession no. U56975).

 
When we compared the deduced proteins encoded by the EcOYW2 and EcOYM ORFs with those of EcOYW1 and pOYW, two previously reported classes of EC-DNA of OY phytoplasma (Nishigawa et al., 2001 ; Oshima et al., 2001a ), many similarities were revealed (Table 1). The rep genes of EcOYW2 and EcOYM (ORF5) were similar to the EcOYW1 rep gene (ORF1), and ORFs 1, 2, 3 and 4 (ssb) of EcOYW2 and EcOYM were similar to ORFs 1, 2, 3 and 4 (ssb) of pOYW and pOYM, respectively. In addition, ORF8 of EcOYM, which has no counterpart in EcOYW2, was found to be similar to ORF7 of EcOYW1.


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Table 1. Similarities among ORFs of the EC-DNAs, and their putative functions

 
As summarized in Fig. 2, the EC-DNAs of OY phytoplasma share similarities not only in the ORFs, but also in the non-coding regions around them. This again supports the hypothesis of a recombinational origin for EcOYW2 and EcOYM. The fact that there are no inversions among the similar regions also agrees with the hypothesis that these EC-DNAs replicate via a rolling-circle-replication mechanism.

Southern blot analysis
To confirm that the OY phytoplasma has no more EC-DNA species, Southern blot analysis was performed. When a virus-type rep gene was used as a probe (Fig. 1), no DNA bands were detected other than EcOYW1, EcOYW2 and EcOYM. Therefore, we concluded that there were no other virus-like EC-DNAs present in OY-W or OY-M. When a plasmid-like rep gene of pOYW was used as a probe (Fig. 4), two bands from OY-W and one band from OY-M were detected. We previously reported that pOYW was composed of heterogeneous molecules; a newly cloned full-length clone of pOYW (Oshima et al., 2001a ) contains two EcoRI sites, while a newly cloned almost full-length clone of pOYW (Kuboyama et al., 1998 ) did not possess one of the EcoRI sites found in the full-length clone. We presume that the 2·2 kbp band represents the pOYW molecule with two EcoRI sites, and that the 3·9 kbp band represents that with only one EcoRI site. The 3·9 kbp band from OY-M should be pOYM. Therefore, we conclude that there are no other plasmid-type EC-DNAs present in OY-W or OY-M.



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Fig. 4. Southern blot analysis used to confirm whether other EC-DNAs with plasmid-like rep exist in OY phytoplasma. No signals were present in lane H. Lanes H, W and M are as described in the legend of Fig. 1. ORF5 (rep) of pOYW was used as a probe.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This work, and our previous studies (Kuboyama et al., 1998 ; Nishigawa et al., 2001 ; Oshima et al., 2001a ; H. Nishigawa and others, unpublished), describes the isolation and characterization of all the EC-DNA species from the OY-W and OY-M lines derived from OY phytoplasma. We believe this is the first report on the isolation of the complete set of EC-DNAs from a phytoplasma isolate. The EC-DNAs of OY phytoplasma were classified into two categories, depending on their replication proteins: those having a rep gene similar to those of geminiviruses (EcOY-DNAs); and those having a rep gene similar to those of pLS1-family plasmids (pOY plasmids). EcOYW1 and EcOYW2, from OY-W, have been identified as EC-DNA with a virus-like rep, while pOYW, from OY-W, has a plasmid-like rep.

Most interestingly, sequence analysis strongly suggests that EcOYW2 and EcOYM might be the products of DNA recombination between EcOYW1 and pOYW. We know of no reports of intermolecular recombination among EC-DNAs in phytoplasmas. The rolling-circle-replication plasmids of Bacillus subtilis are notoriously unstable (Ehrlich et al., 1986 ; Ehrlich, 1989 ), probably because their replication generates intermediates that are susceptible to rearrangement, and they show high levels of recombination (Niaudet et al., 1984 ; Jannière & Ehrlich, 1987 ). Since both the geminivirus-like rep gene and the pLS1-plasmid-like rep gene mediate rolling-circle replication, it may be possible that the EC-DNAs of phytoplasmas can also undergo recombination (Saunders & Stanley, 1999 ).

Recombination of EC-DNA plays a major evolutionary role by creating genetic diversity, and provides the potential for rapid adaptation to new environmental conditions (Riley, 1993 ). A cluster of genes encoding bactericidal colicins and related proteins of E. coli resides on plasmids, and its diversity results from plasmid mobilization and subsequent recombination within and between the clusters (Riley, 1993 ). In addition, a type I restriction/modification system in Lactococcus lactis was reported to acquire novel specificities via the recombination of two plasmids, each having different hsdS genes encoding a specificity-determining subunit. The recombination of two plasmids at the hsdS loci generates a large plasmid with two chimeric hsdS genes with novel specificities (O’Sullivan et al., 2000 ). Recombination also plays a major evolutionary role by creating genetic diversity within prokaryotic and eukaryotic virus populations (Domingo & Holland, 1997 ; Saunders & Stanley, 1999 ). Interspecific recombination has been well documented in many virus families (Roossinck, 1997 ), for example, between RNA viruses (White et al., 1995 ), between DNA viruses (Zhou et al., 1997 ), and between RNA and DNA viruses (Morse et al., 1992 ). Indeed, some instances of recombination between different viral rep genes have been reported (Gibbs & Weiller, 1999 ; Saunders & Stanley, 1999 ).

The mechanism of EC-DNA recombination in phytoplasmas is not clear. It should be noted, however, that the non-coding region between ORF4 (ssb) and ORF5 (rep) of EcOYW2 has similarity to sequences upstream from ORF1 (rep) of EcOYW1 and to sequences downstream from ssb of pOYW (Fig. 5), indicating that this common region between EcOYW1 and pOYW may be the recombination site for generating EcOYW2. No other regions showed as clear junctions as this region. It should also be noted that the homologous, non-coding region ‘b’ shown in Fig. 2 is located differently between EcOYW2 and EcOYM. This could suggest that these chimeric EC-DNAs are not a result of a simple single recombination event of EcOYW1 (or related EC DNAs) and pOYW (or related ones), but further deletions, insertions or recombination events should have taken place.



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Fig. 5. An alignment of a partial DNA sequence of EcOYW2, EcOYW1 and pOYW is shown. EcOYW2 is similar to the region of EcOYW1 upstream of ORF1 (rep) and the region of pOYW downstream of ssb. A, The ends of ORF6 (EcOYW1), ORF4 (EcOYW2) and ORF4 (pOYW). B, The initiation site of rep (EcOYW1 and EcPYW2). C, A stem–loop structure, the putative nick site for replication.

 
Taking the above discussions into consideration, it may be that the recombination between EC-DNAs is one of the mechanisms responsible for increasing biological diversity in phytoplasma populations. Further analysis of the relationship between EC-DNA structure and the biological characteristics of phytoplasmas will clarify the significance of the rearrangement events. Interestingly, we observed no recombinant EC-DNA encoding a pOY plasmid Rep. This could be because of the stability or compatibility of the replicons. Although we do not yet know the mechanism and principle behind this, these observations will encourage further investigations into possible links between evolution and recombination.

Another speculation is that in the OY-W line there may have been a considerable number of variant EC-DNAs consisting of EcOYW1, pOYW and recombinant EcOYW2, each of which may have been heterogeneous itself. The phytoplasmal cells of the OY-W line may contain several different combinations of the three replicons. In the serial maintenance of the OY-W line, some may have lost EcOYW1, resulting in an OY-M line that may have contained only ancestral sets of EcOYM and pOYM.

Comparison of the gene organization of EC-DNAs of OY phytoplasma (Fig. 2) revealed that the EC-DNAs differ in OY-W and OY-M. Since genes encoded in EC-DNAs have been reported to play important roles in the pathogenicity and virulence of plant-pathogenic bacteria (Panopoulos & Peet, 1985 ; Vivian et al., 2001 ), it is possible that the difference in the genes of these EC-DNAs is responsible for the difference in the pathogenicity of the OY lines. This is the first evidence of intermolecular recombination between extrachromosomal DNAs in phytoplasma.


   ACKNOWLEDGEMENTS
 
We thank Mr Shigeru Hatano for his excellent technical assistance.This work was supported partly by grants-in-aid from the Ministry of Education, Science and Culture of Japan (no. 09460155), and by the programme for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of the Ministry of Agriculture, Forestry and Fisheries of Japan.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 22 October 2001; revised 15 January 2002; accepted 28 January 2002.