From the
Division of Plant Biotechnology, Graduate school of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501,
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0101, and ¶Department of Health and Nutrition, Niigata University of Health and Welfare, Shimami-cho, Niigata-shi 950-3198, Japan
Received for publication, February 19, 2003 , and in revised form, March 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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In an attempt to obtain information for genes expressed during microsporogenesis and meiosis, we previously carried out a large scale sequencing project using a lily zygotene cDNA library (7). One of the sequenced clones, M1125, has homology with a plant-specific putative transcription factor, Scarecrow (SCR). SCR is required for asymmetric cell division in an Arabidopsis root and encodes a novel putative transcription factor (8). Recently, SCR-like genes were reported in various plant species such as maize and pea (9, 10). SCR-like gene functions are not restricted to the asymmetric cell division. Although Repressor of Ga13 (RGA) and gibberellin-insensitive (GAI) genes show a similarity to SCR in their amino acid sequences, they play important roles in the gibberellin signal transduction of Arabidopsis but not in asymmetric cell division (11, 12). In addition, PAT1 protein, which shows a similarity to SCR protein, has been shown to be involved in the phytochrome A signal transduction of Arabidopsis (13). In other species, various functions of SCR-like genes were reported; the Lateral suppressor (Ls) gene in tomato has crucial functions in the formation of lateral branches, and SLR1 of rice has been identified as an ortholog of GAI (14, 15). Pysh and co-workers (16) identified a number of Arabidopsis ESTs (expressed sequence tags) that showed a similarity to SCR amino acid sequence and designated them Scarecrow-like (SCL). They indicated that SCL genes comprised a novel gene family, referred to as the GRAS gene family. The GRAS gene products have conserved carboxyl termini, but the amino termini are structurally diverse. It has been suggested that the diversity of amino termini would be related to their functions, but no detailed studies on functional analysis have been carried out. Although the SCR product is predicted to be a transcriptional regulator on the basis of structural similarities to transcriptional regulatory proteins (8), direct evidence for the GRAS gene product as a transcriptional regulator has not been reported. It was shown that chimeric SLR1/OsGAI proteins fused to the yeast GAL4 DNA-binding domain enhanced UAS promoter activity (17, 18), but the target sequence for the GRAS protein has not been reported. Here, we describe the isolation and characterization of a novel GRAS gene from lily microsporocytes and show that the GRAS gene product is able to activate the transcription of meiosis-associated gene by transient expression assay.
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EXPERIMENTAL PROCEDURES |
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DNA SequencingDNA sequencing was performed using a Perkin-Elmer dye primer cycle system according to the manufacturer's instructions with a ABI 373 Stretch sequencer (Applied Biosystems Inc.). Homology searches were performed in the GenBankTM data base using the BLAST program (19).
RNA Isolation and Gel Blot AnalysisRNA was prepared from samples kept at -80 °C using the aurintricarboxylic acid method as described previously (20). Each lane was loaded with 10 µg of total RNA, which was then fractionated on 1% agarose-formaldehyde gels and blotted onto Hybond N+ membrane according to the manufacture's protocol (Amersham Biosciences). Hybridization of 32P-labeled DNA probes to RNA blots was performed in 50% formamide, 5% SDS, and 5x SSC. The filters were prehybridized for 2 h and hybridized overnight at 42 °C. The RNA blots were washed for 20 min in 2x SSC, 1% SDS at 42, 50, and 60 °C, briefly air-dried, and then autoradiographed with X-AR films (Fuji film).
5'-Rapid Amplification of cDNA Ends (5'-RACE)Total RNA was extracted from buds at the pachytene stage of lily PMCs by using an RNeasy Plant kit (Qiagen), and treated with RNase-free DNase (Amersham Biosciences). 5'-RACE was performed with the 5'-RACE system for Rapid Amplification of cDNA Ends kit (Invitrogen) using the following primers: 1125GSP1, 5'-GACGGTCATCTATAGC-3'; 1125A, 5'-TAGCCACTGTTTGGGCACAATGG-3'; and 1125B, 5'-GAGTTCTAAGGTCCACCACCTCAG-3'.
Reporter Gene Constructions and Transient Expression of the GFP-LlSCL Fusion Protein in Onion Epidermal CellsFor green fluorescent protein (GFP) fusions, the -glucuronidase coding region from pBI221 was replaced by the GFP coding region of plasmid pEGFP-C1 (Clontech). DNA fragments coding for the entire or partial regions of LlSCL were amplified using the following primers with KOD-plus polymerase (Toyobo): pGLlSCL-N, LlSCL-NE (5'-GGGAATTCGGTGAAAGAGCTAAAAGTAG-3'), and LlSCL-NC (5'-CCCTCGAGTGGACAGAGCAGAACAAGATC-3') (linker site is underlined); pGLlSCL-C, LlSCL-C' (5'-GGAGATCTGCAACCAGAAGTCCTCAGAATGG-3'), and 1125-C (5'-GGCTCGAGTCATCGACTATTGGGTG); pGLlSCL-CdL, LlSCL-CdL (5'-GGAGATCTGCCTACGGGCTCTATATGTCGG-3'), and 1125-C; pGLlSCL-CdV, LlSCL-CdV (5'-CCAGATCTCCACAGCCAGGTTTCCGCCC-3'), and 1125-C; pGLlSCL-CdBII, LlSCL-C', and 1125-SQ4 (5'-ATAACATTCATAGCATCCCGCCCG-3'). These PCR DNAs were digested with the appropriate enzymes and cloned into pBI221-GFP vectors. All GFP-LlSCL fusion derivatives (pGLlSCL-N, pGLlSCL-C, pGLlSCL-CdL, pGLlSCL-CdV, and pGLlSCL-CdBII) encode fusion proteins with GFP at the amino-terminal portion and with LlSCL at the carboxyl-terminal portion. Onion epidermal cells were prepared and bombarded with 1.6 µm gold particles (Bio-Rad PDS-1000/He) coated with the control plasmid DNA. Samples were kept at room temperature for 8 h, and the GFP localization was visualized using a fluorescent microscope E800 (Nikon) equipped with a color charge-coupled device camera system (CCD, Hamamatsu Photonics).
Transcriptional Activation Experiments in Plant CellsFor transcriptional activation through the GAL4 target sequence, we used the firefly luciferase gene reporter plasmid containing six copies of the GAL4 target sequence fused to the -46 region of the cauliflower mosaic virus (CaMV) 35S promoter. For the construction of the effector plasmids, the PCR-amplified LlSCL fragments were fused to the GAL4 DNA-binding domain and inserted into the pBI221 vector by replacing the -glucuronidase coding sequence: p221DBLlSCL-FL, LlSCL-5'Bam (5'-GGGGATCCATGGTGAAAGAGCTAAAAGTAGACG-3'), and 1125-C (linker site is underlined); p221DBLlSCL-N, LlSCL-5'Bam and LlSCL-N/STOP (5'-CCCTCGAGCTATGGACAGAGCAGAACAAGATC-3'); p221DBLlSCL-C, LlSCL-C'Met (5'-GGGGATCCATGGCAACCAGAAGTCCTCAGAATGG-3'), and 1125-C; p221DBLlSCL-CdL, LlSCL-CdL, and 1125-C; p221DBLlSCL-CdV, LlSCL-CdV and 1125-C; p221DBLlSCL-N(1129), LlSCL-5'Bam, and LlSCL587-STP (5'-GGCTCGAGCTAAGATGGCTGGTCAGATGGAG-3'); p221DBLlSCL-N(130202), LlSCL587-Met (5'-GGGGATCCATGTACAACAATCCTAGTCCGG-3'), and LlSCL806-STP (5'-CCCTCGAGCTATGTGGGCTCTCCCAATCCTTC-3'); p221DBLlSCL-N(203317), LlSCL806-Met (5'-GGGGATCCATGACTAACATTGAAGCTCGTG-3'), and LlSCL-N/STOP; p221DBLlSCL-N(1202), LlSCL-5'Bam, and LlSCL806-STP; p221DBLlSCL-N(130317), LlSCL587-Met, and LlSCL-N/STOP. The yeast GAL4 activation domain was amplified from pGADT7 (Clontech) with the following primers: AD5-Bg (5'-GGAGATCTGCCAATTTTAATCAAAGTGGG-3') and AD3-Xh (5'-GGCTCGAGCTACTCTTTTTTTGGGTTTGGTGGGG-3'). These PCR-amplified DNAs were digested with appropriate enzymes and cloned into the pBI221-GAL4DB vector containing a GAL4 DNA-binding domain. For the transactivation assay of LlSCL protein in plant cells, the LlSCL-FL fragment was amplified with LlSCL-5'Bam and 1125-C primers and then inserted into SacI-blunt/BamHI-digested pBI221 by replacing the
-glucuronidase coding sequence. A reporter plasmid harboring a tissue-specific promoter, pLIM10prom::221::LUC+, was constructed as described previously (21).2 The BY-2 cells, the lily leaves, and PMCs were incubated at room temperature for 12 and 24 h in the dark, respectively. Dual luciferase assay was conducted as described previously (22).
Yeast Expression Vectors, Transformation, and -Galactosidase AssayTo construct a yeast expression vector, the PCR-amplified LlSCL fragments and yeast activation domain were inserted into the pGBKT7 vector (Clontech). Constructs used in this experiments were pGB, pGBAD, pGBLlSCL-N, pGBLlSCL-N(1129), pGBLlSCL-N(130202), pGBLlSCL-N(203317), pGBLlSCL-N(1202), and pGBLlSCL-N(130317). Transformation of budding yeast Saccharomyces cerevisiae was carried out by the lithium acetate method. The strain used was AH109 (MATa, trp1901, leu23, 112, ura352, his3200, gal4
, gal80
, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1). The
-galactosidase liquid assay was performed with a luminescent
-galactosidase detection kit II according to the manufacturer's instructions (Clontech).
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RESULTS |
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LlSCL Gene Is Strongly Expressed at the Premeiotic PhaseTo characterize the expression pattern of the LlSCL gene, we conducted RNA gel blot analysis. Fig. 2 shows the results obtained by probing a blot of total RNA prepared from whole anthers collected at various stages of anther development with the radiolabeled 5'-region of the LlSCL gene. A 2.6-kb LlSCL transcript was found to be differentially regulated during the course of microsporogenesis. The maximal level of LlSCL mRNA was detected in anthers containing sporogenous cells before meiosis. During the meiosis of microsporogenesis, the transcript was detected in a slightly lower level throughout microspore development, and then it decreased to an undetectable level in mature pollen (Fig. 3). In vegetative tissues, LlSCL gene expression levels were barely detectable (data not shown).
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Nuclear Localization of LlSCL ProteinSCR and related proteins have been suggested to be involved in the transcriptional regulation (8). Thus, they are thought to have nuclear localizing activity. In fact, a previous report indicated that RGA-GFP fusion proteins are located in the nucleus of the onion epidermal cell (11). To investigate the intracellular localization of LlSCL protein, chimeric genes encoding the LlSCL protein fused to the GFP were introduced into onion epidermal cells using a particle bombardment system and expressed under control of the CaMV 35S promoter (Fig. 4). Eight hours after bombardment, green fluorescence derived from GFP fusion proteins were examined by fluorescent microscopy.
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The fluorescent signals derived from the control plasmid vector expressing GFP alone were observed in both the cytoplasm and the nucleus of onion epidermal cells (Fig. 5A). Likewise, the GFP signals from cells transfected with pGLlSCL-CdV and pGLlSCL-CdL constructs were preferentially observed in the cytoplasm (Fig. 5, D and E, respectively). The GFP-LlSCL-N fusion proteins strongly aggregated in cytoplasm (Fig. 5G). Although GFP signals of the pGLlSCL-CdBI construct were observed in both the nucleus and cytoplasm, the signals in the nucleus were stronger than those in cytoplasm (Fig. 5C). On the contrary, the signals from the pGLlSCL-C were located exclusively in the nucleus (Fig. 5B). Interestingly, the signals from the construct pGLlSCL-CdBII were observed in the cytoplasm, even though the pGLlSCL-CdBII construct expressed a fusion protein including the BRI region (Fig. 5F). These results indicate that both BRI (amino acids 351359) and BRII (amino acids 697704) are important for the nuclear localization of LlSCL protein. It is necessary to confirm the requirement of basic regions in the context of full-length protein; however, we could not carry out a mutational analysis using full-length protein because of our inability to detect fluorescent signals from samples transfected with pGLlSCL-FL.
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The LlSCL Protein Contains a Transcriptional Activation DomainTo evaluate the function of the LlSCL protein as a transcription factor, we performed transcriptional activation experiments by transient assay in tobacco BY-2 cells. The reporter gene was the firefly (Photinus pyralis) luciferase gene preceded by a promoter containing six repeats of the GAL4 target sequence (UAS) fused to the CaMV 35S promoter TATA-box region. The effector genes were designed to express fusions of various parts of the LlSCL protein with the DNA-binding domain (DB) of yeast GAL4. The activation domain (AD) of yeast GAL4 protein was used as a positive control. The plasmid constructs used and the results are shown in Fig. 6. The full-size LlSCL protein fused to GAL4-DB, DB-FL, was able to raise the activation level the UAS promoter 2.5-fold higher than the activation level of GAL4-DB alone. However, three truncated LlSCL proteins, LlSCL-C, LlSCL-CdL, and LlSCL-CdV, fused to GAL4-DB did not activate the UAS promoter. On the other hand, the amino-terminal portion of the LlSCL protein fused to GAL4-DB strongly activated the UAS promoter. The transcriptional activation levels of GAL4-DB-LlSCL fusion protein derivatives containing the amino-terminal region were equivalent to the level of the GAL4-AD. These results suggest that the amino terminus of the LlSCL protein possesses the capability of strong transcriptional activation.
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The amino terminus of LlSCL protein has two acidic regions. The domain responsible for transcriptional activation often belongs to the sequence that is rich in acidic residues (25, 26). Therefore, we dissected the amino-terminal region of LlSCL protein to investigate the activities of transcriptional activation with respect to these acidic regions. As shown in Fig. 7, DB-N(1129), which includes the first acidic domain (amino acids 4110), showed strong transcriptional activation equivalent to that of DB-N(1317). The neutral domain fused to GAL4-DB, DB-N(130202), had up to 3-fold higher activity than GAL4-DB alone. On the other hand, DB-N(203317) and DB-N(130317) showed no transcriptional activity, even though both constructs included the second acidic domain. Interestingly, the highest activity was obtained by the expression of DB-N(1202) harboring the first acidic domain and the neutral domain. These results suggest that the region containing the first acidic domain and the neutral domain but not the second acidic domain of LlSCL protein is responsible for the transcriptional activation.
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The Amino Terminus of LlSCL Protein Functions as a Transcriptional Activator in Yeast CellsTo investigate whether the LlSCL protein activates transcription through a plant-specific mechanism, we also examined the activity of transcriptional activation of LlSCL protein in yeast cells. We constructed various plasmids expressing the GAL4 DNA-binding domain fusions of different regions of LlSCL protein in yeast. These plasmids were introduced into yeast cells carrying the lacZ reporter gene under the control of the GAL1 promoter. (Fig. 8). The results of the -galactosidase assay indicated that the properties of transcription activation of LlSCL protein in yeast cells were the same as those in plant cells. The region covering the first acidic domain and the neutral domain of the amino terminus of LlSCL protein caused transcriptional activation in the yeast as well as in the plant cell. Interestingly, the transcriptional activation levels of the GAL4-DB fusion proteins containing the LlSCL acidic domain were higher than that of GAL4-DB-AD fusion in yeast cells. These results suggest that plant-specific factors are not required for the strong activity of transcriptional activation of LlSCL acidic domain.
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LlSCL Protein Is a Transcriptional Activator of the Meiosis-associated Gene in PMCsTo study whether the LlSCL gene functions as a transcriptional activator at microsporogenesis, we investigated the effect of the full-length LlSCL protein expression on the transcriptional activity of the meiosis-associated promoter that directs microsporogenesis-specific gene expression. We exploited a meiosis-associated gene, LIM10, which encodes a small molecular weight heat shock protein specifically induced at the meiotic prophase (zygotene) in lily PMCs. To measure the transcriptional activation of the meiosis-associated promoter, the firefly luciferase coding region was placed downstream of the LIM10 promoter sequence.3 The full-length LlSCL cDNA preceded by the CaMV 35S promoter was used as the effector gene. The activity of the CaMV 35S promoter was higher than the meiosis-associated promoter in tobacco BY-2 cells and lily leaves, whereas the higher activity of the meiosis-associated promoter was detected in lily PMCs (Fig. 9). When the LlSCL protein was co-expressed together with reporter genes, the activity of CaMV 35S promoter was slightly decreased in all plant cells tested. Similarly, the activity of the meiosis-associated promoter was slightly decreased by co-expression with LlSCL in tobacco BY-2 cells and lily leaves. In PMCs, the activity of the meiosis-associated promoter was enhanced by LlSCL co-expression, whereas the activity of CaMV 35S promoter was down-regulated (Fig. 9). These results suggest that the LlSCL protein plays a role in the transcriptional activation of the meiosis-associated (LIM10) promoter during meiosis in conjunction with PMC-specific factor(s).
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DISCUSSION |
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Amino Terminus of LlSCL Protein Has a Strong Transcriptional ActivityAlthough the GRAS gene products share highly conserved motifs located within the carboxyl terminus, the amino termini were variable (16). The variable structures of the amino termini of GRAS gene products would be related to their functions. In fact, the DELLA domain, which is positioned in the amino termini of the RGA, GAI, RGL1, and SLR1 proteins, is required for the specific functions of those proteins in the gibberellin response (11, 18, 28). On the contrary, the SCR protein, which has a function distinct from other GRAS proteins, does not contain any DELLA domains in the amino terminus. The LlSCL protein does not contain any DELLA domains and shows no similarity to the amino terminus of SCR protein. Therefore, it is speculated that the LlSCL gene would have specific functions distinct from the gibberellin response and from asymmetric cell division in the root.
Except for the DELLA domain, the molecular functions of the amino terminus of GRAS gene products have not been reported. In this study, however, we have demonstrated that the amino terminus of LlSCL protein (amino acids 1317) is able to direct transcriptional activation equivalent to the level of the activation domain of yeast GAL4 protein. It is well known that hydrophobic residues interspersed between the acidic residues are associated with the transcriptional activation (26). This is consistent with the fact that the amino terminus of LlSCL (amino acids 1317) is highly acidic with a net negative charge of 32. The amino terminus of LlSCL protein can be further divided into three domains: first acidic domain, neutral domain, and second acidic domain. Figs. 7 and 8 clearly indicate that both the first acidic and neutral domains, but not the second acidic domain, show strong activity. The transcriptional activation of both domains was observed not only in plant cells but also in yeast cells. This indicates that the mechanism of transcriptional activation by the amino terminus of LlSCL protein is evolutionarily conserved.
Silverstone et al. (11) proposed a model for gibberellin signal transduction through O-GlcNAc modification at serine/threonine-rich region(s) of GAI/RGA proteins. Because the LlSCL protein also contains serine stretches within the first acidic and neutral domains, it may be possible that the serine stretches could be O-GlcNAc modified by unknown factors in tobacco BY-2 cells and yeast cells, modulating the levels of transcriptional activation.
Compared with the high activity of transcriptional activation of the amino terminus of LlSCL protein, the full-length LlSCL protein exhibited lower activity (Fig. 6). The carboxyl terminus region of the LlSCL includes two parts, a leucine heptad repeat and an LXXLL motif, both of which mediate protein-protein interaction. Thus, it is possible that the LlSCL protein interacts with factors involved in the regulation of transcription via these protein domains that modulate the level of transcriptional activation.
Richards et al. (29) proposed that GRAS gene products were related to the STAT family of proteins based on structural similarities between GRAS and STAT family proteins. Because STATs are activated by the receptor kinase (30), the LlSCL protein could also be phosphorylated by an unknown protein kinase for a suitable function. Because the O-GlcNAc modification and serine/threonine phosphorylation are broadly observed in eukaryotic cells (31), it will be interesting to analyze the properties of mutated LlSCL proteins with amino acid substitutions within the serine stretch region.
LlSCL Protein May Play a Role in Transcriptional Regulation during MicrosporogenesisBy the experiments of transient expression of the full-length LlSCL protein in plant cells, we found that the LlSCL protein may activate the expression of the meiosis-associated gene in lily PMCs (Fig. 9). To our knowledge, this is the first report indicating that the GRAS gene product is directly involved in regulation of a specific gene expression. Together with the result of mRNA accumulation pattern, we report here for the first time that a GRAS gene is involved in the specific gene expression during microsporogenesis. The results obtained in this study indicated that the LlSCL protein requires a specific factor(s) for specific activation in PMCs, because the LlSCL protein did not elevate the activities of LIM10 promoter in BY-2 and leaves. Thus, the LlSCL protein functions as a co-activator for activating a gene expression with the specific factor(s). To elucidate the mechanisms involved in LlSCL-mediated gene expression, it is necessary to isolate the specific factor(s) that activate LIM10 gene expression in concert with the LlSCL protein.
Further studies will be needed to define the biological functions and biochemical properties of LlSCL protein in the microsporogenesis and anther development in lily. However, because of our inability to conduct a molecular genetics approach, studies using a lily system are rather limited. The GRAS gene, which shows a high similarity to LlSCL gene, would have a similar function in the anthers from various species. Recently, we identified a gene encoding the LlSCL homolog from Arabidopsis and rice.4 The availability of LlSCL homologs from these species would allow further study of the function and biological implication of the LlSCL gene.
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FOOTNOTES |
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* This work was supported by Grant-in-aid 14360205 for Scientific Research (B) from the Japanese Society for the Promotion of Science, by Research for the Future Program JSPS-RFTF 00L01604, and by Grant-in-aid 12052217 for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed: Division of Plant Biotechnology, Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan. Tel.: 81-45-339-4413; Fax: 81-45-339-4413; E-mail: hiratsk{at}ynu.ac.jp.
1 The abbreviations used are: PMC, pollen mother cell; UAS, upstream activating sequence; 5'-RACE, 5'-rapid amplification of cDNA ends; GFP, green fluorescent protein; CaMV, cauliflower mosaic virus; NLS, nuclear localization signal; BR, basic region; DB, DNA-binding domain; AD, activation domain; STAT, signal transducers and activators of transcription.
2 H. Takase, M. Minami, Y. Hotta, and K. Hiratsuka, unpublished.
3 M. Minami, H. Takase, and K. Hiratsuka, manuscript in preparation.
4 K. Morohashi, H. Takase, and K. Hiratsuka, unpublished data.
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REFERENCES |
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