Journal of Experimental Botany, Vol. 53, No. 370, pp. 917-925,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice
1 Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan
2 Plant Science Center, Riken, 2-1 Hirosawa, Wako 351-0198, Japan
3 Graduate School of Life Science, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan
Received 18 July 2001; Accepted 12 October 2001
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Abstract |
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 Top Abstract IntroductionTransgenic analysis for NADH...QTL analysis for contents...ConclusionReferences |
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Immunocytological studies in this laboratory have suggested that NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in developing organs of rice (Oryza sativa L. cv. Sasanishiki) is involved in the utilization of glutamine remobilized from senescing organs through the phloem. Because most of the indica cultivars contained less NADH-GOGAT in their sink organs than japonica cultivars, over-expression of NADH-GOGAT gene from japonica rice was investigated using Kasalath, an indica cultivar. Several T0 transgenic Kasalath lines over-producing NADH-GOGAT under the control of a NADH-GOGAT promoter of Sasanishiki, a japonica rice, showed an increase in grain weight (80% as a maximum), indicating that NADH-GOGAT is indeed a key step for nitrogen utilization and grain filling in rice. A genetic approach using 98 backcross-inbred lines (BC1F6) developed between Nipponbare (a japonica rice) and Kasalath were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), which is probably involved in the export of nitrogen from senescing organs and those of NADH-GOGAT. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these BC1F6. Seven chromosomal QTL regions were detected for GS1 protein content and six for NADH-GOGAT. Some of these QTLs were located in QTL regions for various biochemical and agronomic traits affected by nitrogen recycling. The relationships between the genetic variability of complex agronomic traits and traits for these two enzymes are discussed.
Key words: Glutamate synthase, nitrogen recycling, over-expression, QTL, rice.
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Introduction |
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TopAbstractIntroductionTransgenic analysis for NADH...QTL analysis for contents...ConclusionReferences |
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In japonica rice (Oryza sativa L. cv. Sasanishiki) plants, approximately 80% of total nitrogen in the ear is remobilized through the phloem from senescing organs (Mae and Ohira, 1981
). The major forms of nitrogen in the phloem sap are glutamine and asparagine (Hayashi and Chino, 1990) that is probably synthesized from glutamine (Ireland and Lea, 1999). Thus, the synthesis of glutamine in senescing organs as well as the utilization of glutamine in developing organs is the key step for nitrogen recycling in rice plants. Glutamine synthetase (GS; EC. 6.3.1.2) and glutamate synthase (GOGAT) could be involved in the process of nitrogen recycling. GS catalyses the ATP-dependent condensation of NH3 with glutamate to produce glutamine. Subsequently, GOGAT catalyses the reductive transfer of the amide group of glutamine to 2-oxoglutarate to form two glutamate molecules (Ireland and Lea, 1999). The immunocytological studies suggest that cytosolic GS (GS1) is necessary for the export of nitrogen from senescing leaves of rice plants, because the GS1 protein was detected in companion cells which are important for the phloem loading of solutes, and in vascular parenchyma cells (Sakurai et al., 1996; Obara et al., 2000). This is in contrast to the localization of chloroplastic GS (GS2) that is detected in mesophyll cells (Sakurai et al., 1996; Obara et al., 2000) and plays the major role in the photorespiratory nitrogen metabolism (Ireland and Lea, 1999). There are two GOGAT molecular species in rice plants. One is the ferredoxin (Fd)-dependent GOGAT (EC 1.4.7.1) and the other, NADH-dependent (EC 1.4.1.14). Although Fd-GOGAT is known to be involved in photorespiration (Ireland and Lea, 1999), current understanding of the physiological functions of NADH-GOGAT in plants is limited. In rice, NADH-GOGAT is active in developing organs, such as unexpanded non-green leaves and developing grains (Yamaya et al., 1992). It is hypothesized that NADH-GOGAT is probably involved in the utilization of remobilized nitrogen, because this protein is located in the specific cell types which are important for solute transport from the phloem and xylem elements (Hayakawa et al., 1994). NADH-GOGAT occurs as a single gene in rice (Goto et al., 1998). Cell-type-specific and age-dependent expression of the NADH-GOGAT gene was confirmed by promoter analysis in transgenic rice (Kojima et al., 2000). Because no mutant lacking NADH-GOGAT or GS1 has been found in the plant kingdom, the gene manipulation technology of either over-expression or reduction of these target enzymes was used to obtain more direct evidence for their physiological functions.
Oryza sativa is widespread and there are three types or subspecies identified for cultivated rice plants, i.e. japonica, indica, and javanica. Indica and javanica cultivars are different morphologically compared with japonica, including plant heights, sizes of leaf blade and grain, numbers of tillers, as well as their yield and biomass production (Takahashi, 1984). In general, total biomass production of indica cultivars is greater than that of japonica cultivars, whereas grain yield is less. For example, the mean value of one-spikelet weight on the main stem of Kasalath (indica) and Nipponbare (japonica) was 18.0 and 26.9 mg (n=10), respectively, when these cultivars were grown to maturity in a greenhouse at Sendai, Japan, a temperate area. When various cultivars were tested to compare the contents of protein for NADH-GOGAT in the non-green portion of the expanding leaf blade at the reproductive stage, most of the indica cultivars, including Kasalath, contained less NADH-GOGAT protein than japonica and javanica cultivars based on total leaf nitrogen (Obara et al., 2000). On the other hand, some of the indica cultivars contained GS1 protein in senescing leaves twice as high as other cultivars. If the hypothesis was true, indica cultivars could have a less efficient system in sink organs to utilize glutamine that is exported from senescing organs than japonica and javanica cultivars. In other words, over-expression of the NADH-GOGAT gene in sink organs of indica cultivars in an age- and tissue-specific manner possibly causes an increase in spikelet weight. As summarized in Table 1 there are some examples of over-expression of the GS1 gene in Lotus corniculatus (Vincent et al., 1997) and alfalfa (Ortega et al., 2001) and the GS2 gene in tobacco (Migge et al., 2000) and rice (Hoshida et al., 2000). To understand physiological functions, antisense inhibition for GS1 (Temple et al., 1998; Brugiere et al., 1999), GS2 (Migge and Becker, 2000), or NADH-GOGAT in nodules of alfalfa (Schoenbeck et al., 2000) has recently been investigated. However, over-expression of either GOGAT gene has not been reported in plants, including rice. In the first chapter of this review, recent transgenic work expressing sense RNA for NADH-GOGAT in an indica rice is described.
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Conventional approaches that involve whole plant physiology, biochemistry, and molecular physiology are limited in that they only allow the role of a single or limited number of enzymes or regulatory elements to be identified. Agronomic traits, such as the size of seeds and fruits, time for headings, and nitrogen use efficiency, could be determined by multiple gene functions. Molecular cloning of all genes is one way to understand phenotypic characteristics. This upward method is, however, not easy to cover all genes, because the individual effect of each gene on the phenotype could be relatively small. DNA markers and their linkage map on chromosomes are efficient tools and methods for mapping individual ‘quantitative trait loci’ (QTLs) (Tanksley, 1993
; Paterson, 1995), as well as the recent progress of DNA array and proteome analyses as post-genomics tools in Arabidopsis and green algae. A number of QTL analyses using interspecific crosses have been conducted to identify the loci and/or genes controlling physiological traits in various crop plants including tomato (Frary et al., 2000) and maize (Hirel et al., 2001; Wang et al., 2001). In rice, QTL analysis with DNA markers, based on a well-saturated genetic linkage map, has been employed to detect genomic regions associated with several traits exhibiting complex inheritance (Yano and Sasaki, 1997). Genes which are functioning in the regulation of time to heading have recently been identified from QTL analysis (Takahashi et al., 2001; Yano et al., 2000). As far as is known, however, there have been no trials to combine genetic traits of rice and biochemical/molecular biological characteristics in QTL analysis. Such downward analyses could provide valuable information on many genes related to the regulation of target steps for specific traits. In addition, the recent development of genome sequencing projects in a number of model plant species, including rice, will give valuable information in the near future for the identification of the precise location of key genes in QTL regions. The detection of QTLs associated with the contents of GS1 and NADH-GOGAT in rice leaves together with the agronomic traits are also described.
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Transgenic analysis for NADH-GOGAT in rice |
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TopAbstractIntroductionTransgenic analysis for NADH...QTL analysis for contents...ConclusionReferences |
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The NADH-GOGAT gene is 17.1 kb long (Goto et al., 1998
) which is probably too long to transform rice, so that construction of a chimeric gene containing the NADH-GOGAT promoter region and NADH-GOGAT cDNA was designed. Identification of cis-element(s) related to the tissue-specific expression of NADH-GOGAT gene has not yet been completed in rice (Kojima et al., 2000). There are, however, several instances that indicate the importance of the presence of the first intron and 3'-UTR in addition to the 5'-UTR as a promoter for tissue-specific gene expression in Arabidopsis thaliana (Sieburth and Meyerowitz, 1997; Larkin et al., 1993) and potato (Fu et al., 1995). Therefore, the region from –2 352 in 5'-UTR (+1 as the transcriptional start) to +1 322 in the 3rd exon of the NADH-GOGAT gene (AB001916; Goto et al., 1998) from Sasanishiki (japonica) was fused with the NADH-GOGAT cDNA (AB008845; Goto et al., 1998), from which had been deleted the overlapped region, containing the poly(A) tail (262 to 6762 in AB008845). This chimeric NADH-GOGAT gene was inserted at the XbaI site between NTPII and HTP in the uidA gene (encoding GUS) deleted Ti plasmid pBI101Hm (Kojima et al., 2000). Agrobacterium tumefaciens-mediated transformation of Kasalath (indica) was carried out as described previously (Kojima et al., 2000). Transformation of Kasalath using pBI101Hm was also adopted as a control.
Twenty-two lines of the NADH-GOGAT transformant (T0) and 11 lines of the control transformant (TC) were obtained at a frequency of 3.1% and 3.6%, respectively. The occurrence of the chimeric gene (1–6 copies for the NADH-GOGAT chimeric gene and 3–4 copies of hpt for the control) in each line was confirmed by DNA gel blot analysis (data not shown). These regenerated lines as well as the wild-type Kasalath were grown hydroponically (Hayakawa et al., 1994) in an isolated greenhouse until maturity. Molecular biological and biochemical analyses were performed as described previously (Hayakawa et al., 1994; Kojima et al., 2000; Obara et al., 2000). As shown in Fig. 1, there were large variation in the NADH-GOGAT protein contents, when unexpanded non-green leaf blades of three productive tillers from each line and from wild-type plants were analysed with immunoblotting. The NADH-GOGAT protein content increased in several lines. Line 34 contained the protein approximately 1.8-fold higher than the control line or wild-type plant. On the other hand, some lines severely reduced the content of NADH-GOGAT protein, which is probably caused by so-called ‘co-suppression’ or ‘homology-dependent gene silencing’ (Iyer et al., 2000; Matzke and Matzke, 1995). This phenomenon is sometimes seen when a gene or cDNA from a plant is introduced into the same plants in a sense-orientation (Voinnet and Baulcombe, 1997). There was also some variation in the content of Fd-GOGAT protein and that of soluble protein in those lines. The changes in the contents of Fd-GOGAT protein and soluble protein were apparently independent of that of NADH-GOGAT protein. The variation seen in the soluble protein content could not be caused by the efficiency of protein extraction from different lines, since the values of standard error were small.
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Three lines were chosen from either the over-expressed or co-suppressed T0-transformants, and NADH-GOGAT activity in unexpanded non-green leaf blades of three productive tillers from each line and agronomic characteristics at a particular stage of harvest were investigated (Fig. 2
). These lines are associated with the higher content of NADH-GOGAT protein seen in Fig. 1
and also showed higher NADH-GOGAT activity. Plant height and the number of spikelets on the main stem were identical among these transformants and wild-type plants. It should be noted, however, that the mean value of one spikelet weight of the over-expressed lines was higher than that of the wild-type or control transformants, although the rate of increase in the NADH-GOGAT protein in unexpanded non-green leaf blades was not correlated with that in the one-spikelet weight among these three lines. This correlation should be evaluated in future by estimating the NADH-GOGAT protein contents and its activity in spikelets using progenies of the T0-transformants. Co-suppressed lines showed a much lower one-spikelet weight. Although these results were obtained from the T0 generation having multiple trans-genes, other results with a homozygous T3-progeny from transgenic Sasanishiki (japonica) expressing an antisense RNA for NADH-GOGAT under the control of cauliflower mosaic virus 35S promoter also showed a severe reduction in the one-spikelet weight (K. Ishiyama et al., unpublished results). In this case, total nitrogen percentage (about 1.4%) in the dry weight of spikelets was identical in the antisense progeny and the wild type, so that a reduction of NADH-GOGAT caused a reduction in the entire process of grain filling. Isolation of a progeny of the over-expressed transgenic Kasalath having homozygous and fewer sense genes is now in progress.
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QTL analysis for contents of GS1 and NADH-GOGAT protein in rice |
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TopAbstractIntroductionTransgenic analysis for NADH...QTL analysis for contents...ConclusionReferences |
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In rice, there are two cytosolic GS isozymes encoded by distinct genes, i.e. GS1 (mainly expressed in leaves) and GSr (mainly expressed in roots) (Sakamoto et al., 1989
). By contrast, NADH-GOGAT occurs as a single gene (Goto et al., 1998). Because nitrogen recycling largely occurs in leaves and spikelets, the focus was on GS1 and NADH-GOGAT in the QTL analyses. Plant phenotype, including the content of target enzyme protein as well as agronomic and physiological traits, is determined by a number of gene activities in which some genes contribute positively and other genes work negatively (Goldman et al., 1993). There is a large variation in the content of GS1 protein in senescing leaf blades and that of NADH-GOGAT protein in the unexpanded young leaf blades among rice cultivars (Obara et al., 2000). As the first step towards identifying some of the many genes related to the regulation of nitrogen recycling, the locus of GS1 was mapped and the QTLs associated with GS1 protein content in the senescing leaf blade and NADH-GOGAT protein content in the developing leaf blade were detected. A genetic approach using 98 backcross-inbred lines (BIL: BC1F6) developed between Nipponbare (japonica) and Kasalath (indica) was employed to detect putative QTLs associated with the contents of GS1 and NADH-GOGAT in leaves. Although a given QTL region would still contain many genes, some of them could be tightly related to the physiological and biochemical traits. The GS1 protein content in senescing leaf blades of the BILs showed a continuous distribution with one peak ranging from 2.55 to 16.18 µg g-1 FW, and demonstrated transgressive segregation in both directions. Also, the NADH-GOGAT protein content in developing leaf blades of the BILs was continuously distributed with one peak ranging from 1.27 to 4.48 µg g-1 FW, the mean value of the BILs being within the range of the parents. Thus, the contents of GS1 and NADH-GOGAT proteins in rice plants are considered to be quantitative traits. Details of analytical methods and results have been recently published (Obara et al., 2001).
Seven chromosomal regions were detected having QTLs associated with GS1 protein content in the senescing leaf blade and six with NADH-GOGAT protein content in the developing leaf blade. Three putative QTLs for GS1 protein content including the major QTL (linked to the marker S2260: positive allele from Kasalath) on chromosome 11 were detected in the vicinity of QTL regions for physiological traits (Fig. 3). The GS1 gene was mapped between C560 and C1408 on chromosome 2 (Fig. 3). The genetic distance was 10.8 cM from C560 and 0.8 cM from C1408, where the GS1 gene was detected to overlap with a putative QTL for one-spikelet weight. Alleles from Nipponbare and Kasalath that were identified with a higher GS1 protein content were also associated with a higher rate of leaf discoloration (rate of leaf senescence) in the case of QTLs linked with the markers C950 (positive allele from Nipponbare) and R728 (positive allele from Kasalath) on chromosome 11. Therefore, the alleles that increase the GS1 protein content apparently promote the rate of leaf senescence. These results suggest that variation of the GS1 protein content in this population might change the rate of export of glutamine from senescing organs to developing organs and then lead to changes in these physiological traits. This possibility should be confirmed in future experiments by identifying genes.
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Similarly, two putative QTLs for NADH-GOGAT protein content on chromosome 2 were detected in the vicinity of QTLs for physiological traits (Fig. 3
). The major QTL linked to the marker R712 (positive allele from Nipponbare) was located close to the QTL regions for soluble protein content in developing leaves, spikelet number per panicle on the main stem, and rate of leaf discoloration. In this case, the alleles from Nipponbare contributed to increases in NADH-GOGAT protein content and spikelet number whilst also promoting the rate of discoloration. One QTL for spikelet number on the main stem linked to marker G227 (positive allele from Kasalath) was located close to the marker R712. Therefore, the alleles from Nipponbare in this region negatively contributed to an increase in spikelet number on the main stem. Another QTL for NADH-GOGAT protein content linked to the marker C1221 (positive allele from Kasalath) on chromosome 2 was located in QTL regions for spikelet number on the main stem. In this case, the alleles from Kasalath contributed to increases in NADH-GOGAT protein content and spikelet number on the main stem. One putative QTL for one-spikelet weight linked to marker R3393 (positive allele from Nipponbare) was located close to the marker C1221 on chromosome 2. This suggests that alleles from Kasalath in this QTL region negatively contributed to an increase in one-spikelet weight. It should be noted that a major QTL linked to the marker R728 (positive allele from Kasalath) on chromosome 11 was located in the QTL region for one-spikelet weight. A QTL for spikelet number on chromosome 1 linked to the marker R886 (positive allele from Nipponbare) indicates that the allele from Kasalath in this region negatively contributes spikelet number on the main stem.
As described previously (Obara et al., 2001), one-spikelet weight in Kasalath was 33% lower than that in Nipponbare. Over-expression of NADH-GOGAT in transgenic Kasalath showed an increase in panicle weight and in spikelet weight, but had no effect on the spikelet number on the main stem, as mentioned in the previous chapter. On the other hand, the existence of QTL regions for NADH-GOGAT content linked to the markers C1221/R3393 and R712/G227 indicates that alleles from Kasalath in these regions negatively contribute to one-spikelet weight and promote the increase in spikelet number, respectively. Although this seems inconsistent with what was observed in the over-expressed transgenic Kasalath, it is suggested that other effective QTLs, such as a QTL linked to the marker R728 (positive allele from Kasalath) on chromosome 11, could overcome the QTLs linked to R712, resulting in an increase in one-spikelet weight. Similarly, a QTL linked to the marker R886 (positive allele from Nipponbare) on chromosome 1 may overcome the QTLs linked to G227 and C499 (positive alleles from Kasalath) on chromosome 2, resulting in a small difference in spikelet number on the main stem. Furthermore, putative QTLs for NADH-GOGAT protein content on chromosome 1 were located in the QTL region for soluble protein content in developing leaves. At these QTLs, the alleles from Kasalath were identified with an increase of NADH-GOGAT protein content and soluble protein content in developing leaves. These findings suggested that the variation in NADH-GOGAT protein content in this population is related to changes in the rate of regeneration of glutamic acid from glutamine transported via the phloem and xylem in developing organs leading to changes in protein content. A QTL region for NADH-GOGAT protein content was detected at the position mapped for the NADH-GOGAT structural gene on chromosome 1. A QTL region for soluble protein content in developing leaves was also detected in this region. Although fine mapping is required to identify individual genes, QTL analysis could be a useful post-genomic tool to study the gene functions for regulation of nitrogen recycling in rice.
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Conclusion |
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TopAbstractIntroductionTransgenic analysis for NADH...QTL analysis for contents...ConclusionReferences |
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Nitrogen recycling and grain-filling in rice are complex traits that depend on many factors such as rate of senescence to provide nitrogen source, photosynthesis and respiration to provide carbon skeleton and energy for amino acid biosynthesis and transport, re-utilization of precursors in sink organs for storage of protein and starch, and so on. Morphological traits, such as size of leaves and spikelets, the number of spikelets and panicles, thickness of leaves and stem, development of root system, etc. all affect the productivity of rice. The findings of the over-expression of NADH-GOGAT in sink organs of Kasalath as well as the co-suppression or the antisense inhibition in Sasanishiki (Ishiyama et al., 1999
) strongly supported the hypothesis that NADH-GOGAT in spikelets at the early stage of ripening is important to re-utilize glutamine. Not only that, but also the grain-filling process is co-regulated by the status of the re-utilization in which the precise mechanisms are largely unknown. This is a good example of the limitation of upward analysis by manipulating a limited number of target genes.
QTL analysis provides multiple independent loci expected to have regulatory functions on the gene products, although fine mapping is needed to identify these genes. QTLs for GS1 protein and rate of leaf senescence detected on chromosome 11 could be a good candidate gene that can, at least partially, explain variations in nitrogen export from senescing leaf blades. Two QTLs for NADH-GOGAT on chromosome 2 could be related to grain yield. From QTL analysis using 99 recombinant inbred lines of maize, leaf nitrate accumulation and reactions catalysed by nitrate reductase and GS are recently suggested to be co-regulated and represent key elements controlling nitrogen use efficiency (Hirel et al., 2001). Prioul et al. showed that QTLs for the activities of sucrose-phosphate-synthase and acid-soluble invertase were detected in the regions where each structural gene was mapped in maize (Prioul et al., 1999). Furthermore, these QTLs were located close to QTLs for the contents of products and substrates of each enzyme. In these experiments, two out of seven QTLs for GS1 protein content and three out of six QTLs for NADH-GOGAT protein content were detected in different regions from other biological and physiological traits (Fig. 3). To identify the candidate genes from the QTL analysis more precisely, it is necessary to investigate lines that are near isogenic in the QTL region. Elucidation of the genetic nature regulating GS1 and NADH-GOGAT protein contents as a whole could aid in the efficient breeding of high-performance rice cultivars. A combination of genetic manipulation and QTL analysis should be useful for the elucidation of the nitrogen recycling mechanism.
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Acknowledgments |
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We thank Miss Aiko Shudo for technical assistance and Dr Masahiro Yano, National Institute of Agrobiological Resources, Tsukuba, Japan, for providing seeds of the backcross inbred lines. We also thank to two referees for helpful comments and critical reading of the manuscript. This work was supported in part by a program of CREST of JST (Japan Science and Technology) and in part by Grant-in-Aid for Scientific Research (Nos 10556075 and 12460029) from the Ministry of Education, Science, Sport, Technology, and Culture of Japan.
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Notes |
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4 To whom correspondence should be addressed. Fax: +81227178787. E-mail: tyamaya{at}biochem.tohoku.ac.jp1.
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