 1998 Oxford University Press
`
`Nucleic Acids Research, 1998, Vol. 26, No. 6
`
`1481–1486
`
`A liquid chromatography/electrospray mass
`spectrometric study on the post-transcriptional
`modification of tRNA
`Hisaaki Taniguchi* and Nobuhiro Hayashi
`
`Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University,
`Toyoake, Aichi 470-1192, Japan
`
`Received October 31, 1997; Revised and Accepted February 3, 1998
`
`ABSTRACT
`Liquid chromatography/electrospray mass spectrometry
`is one of the rapidly developing techniques with which
`mass of large hydrophilic polymers such as proteins
`and nucleic acids can be determined precisely. The
`technique was applied to studies on the modifications
`of tRNAs. Various tRNA species purified from
`Escherichia coli were directly injected into a capillary
`reversed-phase column and
`the desalted and
`concentrated tRNAs were analyzed on-line with an
`electrospray mass spectrometer. In some cases, small
`but significant differences were noted between the
`theoretical and observed molecular masses, suggesting
`that there exist still unknown modifications. Under
`high
`resolution measurements, multiple peaks
`corresponding to species modified to a varying extent
`were resolved. To study the structures in detail, the
`isolated tRNA species were digested with ribonuclease
`T1, and the resulting mixture of fragments were
`analyzed by the same liquid chromatography/mass
`spectrometry. In this way, most of the fragments were
`easily identified solely from their masses, and the
`positions where the expected and real structures differ
`were revealed. The results obtained showed the
`presence of micro-heterogeneity among tRNAs and
`demonstrated at the same time the power of the
`hyphenated technique for the structural analysis on
`nucleic acids.
`
`INTRODUCTION
`
`Recent development in mass spectrometry has made it possible
`to ionize large hydrophilic polymers such as proteins and
`determine their molecular mass with high precision and resolution
`(1–3). The precision obtained with a conventional quadrupole
`mass spectrometer coupled with electrospray ionization is often
`better than ±0.01%, so that even a slight deviation from the
`theoretical mass based on the gene sequence can be easily
`detected. We have previously shown that the application of the
`method to studies of protein phosphorylation and those of protein
`
`acylation is very successful (4–6). Peaks of proteins phosphorylated
`to different degrees were resolved, and the presence of in vivo
`phosphorylated species could be demonstrated (7,8). Capillary
`high performance liquid chromatography connected on-line to
`the electrospray mass spectrometer (LC/MS) was further used to
`identify various in vivo phosphorylation sites (6,8,9).
`However, the application of the mass spectrometry to another
`important biopolymer, nucleic acids, is still in its infancy (for
`review see reference 10). This is mainly due to the lack of suitable
`solvents (for the electrospray ionization) or matrices (for
`matrix-assisted laser desorption ionization) which can be used to
`ionize large polynucleotides efficiently. Furthermore, the negatively-
`charged phosphate backbones have high affinities for non-volatile
`cations such as Na+ and K+. This necessitates either the extensive
`removal of the ions (11) or the use of elaborate machinery such
`as a Fourier transform mass spectrometer (12). Recent studies
`from several laboratories overcame these problems by choosing
`suitable solvents for the ionization (13,14). The addition of
`organic bases was also found to be efficient in suppressing the
`Na+ and K+ adduct ions (15). Furthermore, the direct on-line
`LC/MS analysis of oligonucleotides has been reported (16).
`In the present report, we describe the application of the LC/MS to
`structural studies of tRNA. Choice of suitable solvents made it
`possible to observe only molecular peaks without any adduct peaks.
`Mixtures of polynucleotide fragments produced by endonuclease
`digestion could be separated by the column chromatography, and
`their masses were immediately determined with the mass
`spectrometer. Time consuming and often troublesome off-line
`fractionation and sample preparations used in the previous studies
`(11,13) became unnecessary. The results thus obtained revealed
`micro-heterogeneity of tRNA and demonstrated the usefulness of
`the LC/MS analysis to elucidate the structures of nucleic acids
`including the various post-transcriptional modifications.
`
`MATERIALS AND METHODS
`
`Materials
`tRNAPhe purified from brewers’ yeast was purchased from
`Sigma. Crude Escherichia coli tRNA mixture was obtained from
`E.coli A 19 cells at the late log phase, and tRNALys was purified
`
`*To whom correspondence should be addressed. Tel: +81 562 93 9381; Fax: +81 562 93 8832; Email: htanigut@fujita-hu.ac.jp
`
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`from the mixture as described (17,18). tRNAGlu was purified
`from the same source using conventional chromatography as
`described previously (19). A mixture of two Met-specific tRNAs,
`tRNAfMet and tRNAMet, was purified from E.coli (20). The in
`vitro transcript of E.coli tRNALys was made with T7 RNA
`polymerase (19,21). Digestion with ribonuclease T1 (Sankyo,
`Tokyo, Japan) was carried out as described previously (19).
`
`LC/MS analysis
`
`A capillary high performance liquid chromatography column
`(0.3 × 15 cm) packed with a polymer-based reversed-phase material
`(PerSeptive Biosystems, R2/H perfusion chromatography material)
`was connected on-line to the electrospray interface of a quadrupole
`mass spectrometer (PE Sciex API-III) as described previously
`(6,8). The mass spectrometer was operated in negative mode with
`the ion source voltage set at –3500 V. The column was eluted with
`a linear gradient of water-acetonitrile containing 0.3 mM
`tributylamine-acetate (pH 5.5). The mass values of neutral
`molecules are used throughout the manuscript.
`
`RESULTS
`LC/MS analysis of yeast tRNAPhe
`
`We have already reported that the addition of organic amine such
`as tripropylamine or tributylamine as a counter ion during the
`reversed-phase chromatography not only affected the chromato-
`graphic behaviour but also the efficiency of the ionization of
`oligonucleotides (22). With increasing chain length of the
`substituent group of the amine, both DNA and RNA bind more
`strongly to the reversed-phase column and the retention time
`increases accordingly. At the same time, the ionization efficiency
`increases with a concomitant decrease in the Na+ and K+ adduct
`peaks. Triethylamine, which has been used in the previous study,
`was found to be less effective both in the adduct suppression and
`ionization efficiency (22). In the presence of 0.3 mM tributylamine,
`almost only molecular peaks were observed even with a large
`polynucleotide such as tRNAPhe (Fig. 1a). The molecular mass of
`the major species was calculated to be 24 953.4 ± 3.5 Da from the
`multiply-charged ions. This is very close to the theoretical mass
`based on the expected structure including various post-
`transcriptional modifications (24 951.3 Da). This clearly indicates
`the lack of any adduct ions that should shift the apparent mass
`peaks. Due to the effective desalting and concentration by the
`on-line capillary reversed-phase column chromatography, the
`rigorous removal of salts by pretreatment (11,13) was unnecessary.
`As can be seen in the spectrum (Fig. 1a), two series of the peaks
`belonging to minor species were also observed. The presence of
`two minor components with molecular masses smaller than that
`of the major component was clearly seen in the mass spectrum
`obtained by deconvolution (Fig. 1b). One component with a
`molecular mass of ~ 24 623 Da may be a truncated form of the
`tRNAPhe. The mass difference between the major peak and this
`minor peak (~ 330 Da) suggests that it is truncated at the 3′-end,
`since the omission of one ‘A’ would cause a mass decrease of
`329.2 Da. On the other hand, the identity of the middle peak is not
`clear. While the patterns of the distribution of the multiply-
`charged ions were similar between the major component and the
`truncated form, the ions belonging to the middle component
`showed a quite different distribution pattern (Fig. 1a). Since the
`distribution of the multiply-charged ions reflects the accessibility
`
`Figure 1. Electrospray mass spectra of yeast tRNAPhe obtained by LC/MS.
`Yeast tRNAPhe (300 pmol) was directly injected into the LC/MS apparatus as
`described under ‘Experimental Procedures.’ With the electrospray ionization,
`a series of multiply-charged ions are observed (a). Peaks belonging to the
`contaminating component are labeled with asterisks. The original spectrum was
`deconvoluted to give a reconstructed spectrum (b). The best estimates for the
`mass values can be obtained by averaging the experimental values obtained for
`multiple peaks in the original spectra such as shown in (a). The mass values may
`differ slightly from those shown in the deconvoluted spectra such as shown in (b).
`
`of the solvent and hence the three-dimensional structure of the
`sample molecules, this suggests that the third component is a
`contaminating tRNA species having a structure quite different
`from those of the other two species.
`
`LC/MS analysis of E.coli tRNALys
`
`Several tRNA species purified from E.coli were then subjected to
`the same LC/MS analysis. One example is shown in Figure 2a.
`Deconvolution of a typical electrospray mass spectrum showing
`multiply-charged ions (inset) gave a mass spectrum of tRNALys
`with a sharp single peak. A small peak of a minor species well
`resolved from the major peak was also observed. However, the
`molecular mass calculated for the main peak (24 796 Da) is
`significantly larger than the theoretical one (24 781 Da) (23–26).
`Since the reliability and the precision is an important issue, we
`then measured an in vitro transcript of E.coli tRNALys under the
`same conditions. The in vitro transcript should have the same
`structure as the previous sample except for the post-transcriptional
`modifications. As shown in Figure 2b, one major species together
`with at least two minor species with higher molecular masses
`were observed both in the original mass spectrum (inset) and in the
`deconvoluted mass spectrum. The mass of the major species was
`calculated from the multiply-charged ions to be 24 444.2 ± 3.3 Da,
`which is within experimental error of the theoretical mass
`(24 441.6 Da). The minor species contain probably one or two
`more additional bases. This demonstrated the accuracy of the
`mass determination of the present method and suggested that the
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`Figure 2. Mass spectra of E.coli tRNALys. tRNALys purified from E.coli A19 cells
`by successive column chromatography was directly injected into the LC/MS
`apparatus (a). The original electrospray mass spectrum with multiply-charged ion
`series (inset) was deconvoluted to give a mass spectrum. An in vitro transcript of
`tRNALys was made with T7 RNA polymerase (21) and analyzed by the LC/MS
`(b). A part of the original mass spectrum was expanded to show the presence of
`three species (inset). Peaks belonging to each species are labeled as a, b and c.
`
`difference between the expected and real structure of tRNALys
`purified from E.coli was not due to the artifacts associated with the
`measurement itself but to the presence of unknown modifications.
`Furthermore, it should be noted that the accuracy and the
`resolution obtained in the present study are good enough to
`sequence the polynucleotides, although C and U nucleotides in
`RNA cannot be distinguished due to their small mass difference.
`The difference (330 Da) between the major peak (24 444 Da) and
`the contaminating peak (24 774 Da) is very close to the mass
`difference expected with the addition of an A (329.2 Da). For
`comparison, the addition of a G, C or U would give a mass
`difference of 345.2, 305.2 or 306.2 Da, respectively.
`
`LC/MS analysis of E.coli tRNAMet
`Another example is tRNAMet. Since the preparation used
`contained tRNAs for both formyl Met (initiator) and Met
`(elongator), this is an interesting case to demonstrate the power
`of the method to measure complex mixtures. At least three peaks
`were resolved as shown in Figure 3a. The major peak had a
`molecular mass of 24 910 Da, while the minor peak of higher
`mass was 25 132 Da. These two peaks should correspond to the
`initiator tRNAfMet (theoretical mass, 24 926.1 Da) and the
`elongator tRNAMet (theoretical mass, 25 144.3 Da), although the
`observed masses were slightly but significantly lower than the
`theoretical ones. The peak areas of the two peaks in the
`deconvoluted spectra correspond well to the amounts of the two
`
`Figure 3. Mass spectra of E.coli tRNAMet and tRNAGlu. A mixture of two
`Met-specific tRNAs, tRNAfMet and tRNAMet, was purified from E.coli (20)
`and analyzed by the same LC/MS technique (a). tRNAGlu purified from E.coli
`A19 cells by ion exchange column chromatography was directly analyzed by
`the LC/MS (b). The preparation used has previously shown to contain
`Glu by sequencing (19). Two settings of the resolution of the mass
`tRNA2
`spectrometer were used. Under normal low resolution conditions, a shoulder on
`the high mass side was observed (dotted line). More than four peaks were
`resolved under high resolution conditions (solid line). In the latter experiment,
`the mass resolution of the quadrupole mass analyzer was increased and the mass
`step of the data acquisition was decreased from 0.5 to 0.1 a.m.u. The major
`species is larger than the species corresponding to the know structure (24 528.9 Da)
`by 13 Da (inset).
`
`species in the preparation (20), suggesting that the relative
`quantification of the various tRNA species by the present method
`is possible. The origin of the third component of 24 765 Da is not
`clear; the deletion of a single nucleotide would not account for the
`differences from the two other peaks.
`
`LC/MS analysis of E.coli tRNAGlu
`
`The next tRNA species subjected to the mass analysis was
`tRNAGlu (Fig. 3b, dotted line). Only one single peak without any
`minor contaminants was observed, indicating an apparent
`homogeneity, corresponding to a single band observed in gel
`electrophoresis (data not shown). However, the molecular mass
`calculated (24 540 Da) was again significantly larger than the
`theoretical one (24 528.9 Da). Furthermore, a shoulder at the high
`mass side was noted, and the peak gave a broader impression than
`those obtained with other tRNA species (for example see Fig. 2).
`To resolve the underlying components, the same sample was
`subjected to the LC/MS under high resolution measurement
`conditions, where the mass resolution of the quadrupole mass
`analyzer was increased (Fig. 3b, solid line). The shoulder on the
`higher mass side was now resolved into at least two peaks with a
`separation of ~ 14 Da (Fig. 3b, inset), while the major peak is also
`split into at least two peaks. The mass of the shoulder (24 527 Da)
`corresponds very well to the theoretical mass of the known
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`Figure 4. A total ion chromatogram of tRNAGlu RNase T1 digest and the
`assignments obtained by the LC/MS analysis. tRNAGlu purified from E.coli
`was digested with RNase T1 was directly injected into the LC/MS apparatus as
`described under Materials and Methods. The intensity of the total ion current
`from the detector was plotted against the retention time (a). Most of the
`fragments were identified solely from the masses. The identified fragments are
`Glu (b).
`indicated by arrows under the sequence of tRNA2
`
`structure (24 528.9 Da). The mass difference between the major
`peak and the shoulder was ~ 13 Da. Note that the mass difference
`caused by methylation, a common base modification, is 14 Da,
`suggesting that the major species observed contains one more
`methylated base that has been left unnoticed. In addition, the
`tRNAGlu preparation seems to contain a number of species
`having molecular masses of small differences. Other possibilities
`such as the presence of contaminated tRNA species can be ruled
`out, since the LC/MS analysis of the RNase T1 digests excluded
`the presence of other structurally-unrelated tRNA species in the
`preparation used as will be described below.
`
`LC/MS analysis of E.coli tRNAGlu RNase T1 digests
`
`To elucidate the cause of the micro-heterogeneity observed, the
`same LC/MS analysis was conducted with RNase digests of
`tRNAGlu. In this case, the purified tRNAGlu was first digested
`with RNase T1, which cleaves RNA specifically at the 3′ side of
`G, and the resulting mixture of polynucleotides was directly
`injected into the reversed-phase column. As shown in the total ion
`chromatogram (Fig. 4a), the polynucleotide fragments were
`separated during the reversed-phase chromatography, and the eluted
`peaks were analyzed by mass spectrometry. From the known
`sequence, theoretical masses of fragments can be easily calculated,
`and they were compared with those obtained experimentally. Most
`
`of the fragments were unequivocally identified solely from their
`masses (Table 1). The fragments thus identified cover most of the
`whole structure except for short segments containing successive
`Gs (Fig. 4b). Some deviations from the known structure were, as
`expected, observed. One fragment that was tentatively assigned
`as fragment G2 showed the most notable heterogeneity (Fig. 5a).
`The peak having a mass close to the theoretical one (2808.8 Da)
`was a minor component, and three peaks of higher masses were
`observed. Another fragment was tentatively assigned as G3, but
`the mass obtained was larger than the theoretical one by 6 Da.
`These results suggest that the micro-heterogeneity and the
`discrepancy between the observed and theoretical masses of
`tRNAGlu originate mainly from the regions close to the 5′-end.
`The hitherto unnoticed modifications near the 5′-end may be
`functionally very important, since the part of the molecule, i.e., the
`acceptor stem, is involved in the recognition of tRNA species by
`specific amino-acyl tRNA synthase (27,28).
`
`Table 1. Assignment of E.coli tRNAGlu RNase T1 digest
`
`G2
`
`G3
`
`G6
`
`G8
`
`G6–8
`
`G5–11 or G6–12
`
`G9
`
`G13
`
`G17
`
`G18
`
`G18–22
`
`G20–23
`
`Mass of M (Da)
`Theoretical
`
`2807.7
`
`1610.0
`
`1608.0
`
`1937.2
`
`3872.4
`
`8405.1
`
`3210.0
`
`1962.2
`
`1294.8
`
`3183.9
`
`5199.1
`
`2547.7
`
`Observed
`2808.8/2819.6a
`1616.3a
`
`1607.9
`
`1937.4
`
`3871.1
`
`8412.9
`
`3210.9
`
`1961.2
`
`1295.3
`
`3182.7
`
`5198.5
`2541.6a
`
`aFragment where the expected and observed masses differ significantly.
`
`Post-transcriptional modifications in the anticodon loop of
`E.coli tRNAMet
`
`Other tRNA species were also subjected to the same LC/MS
`analysis. One interesting observation was the heterogeneity of a
`fragment containing the anticodon loop of tRNAMet. Although
`the sample used was a mixture of at least three species (Fig. 3a),
`most of the fragments produced by the RNase T1 digestion were
`assigned to either to tRNAfMet or to tRNAMet (data not shown).
`Since no other fragments were observed in significant amounts,
`the third component of 24 765 Da (Fig. 3a) is probably closely
`related to one of the two Met specific tRNAs. Among various
`fragments thus assigned, one fragment corresponding to the
`anticodon loop of tRNAMet showed heterogeneity (Fig. 5b). In
`addition to the major species of 5273.7 Da, which is in good
`agreement with the theoretical mass (5276.2 Da), a minor peak of
`5231.4 Da was observed. Since the mass difference between the
`two peaks, 42.3 Da, is very close to that associated with acetylation
`(42.04 Da), we conclude that the two peaks arise from species
`modified differently at the first base of the anticodon. It is less likely
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`in the eluent that helps the ionization process. Second, the
`formation of the Na+ or K+ adduct peaks is well suppressed with
`a concomitant increase of molecular peaks. The substitution of
`the tributylamine with less hydrophobic organic amines such as
`triethylamine, which has been used in the previous study (16),
`produced mass spectra with many adduct ion peaks too complex
`to be analysed satisfactorily.
`
`Table 2. Theoretical and observed molecular masses of various tRNA species
`
`tRNA
`
`E.coli tRNALys
`E.coli tRNALys
`
`(in vitro transcript)
`
`E.coli tRNAGlu
`E.coli tRNAfMet
`E.coli tRNAMet
`yeast tRNAPhe
`
`aMean $ S.D.
`
`Massa (Da)
`Theoretical
`
`24 781.0
`
`24 441.6
`
`24 528.9
`
`24 926.1
`
`25 144.3
`
`24 951.3
`
`Observed
`
`24 796.4 $ 1.9
`
`+15.4 $ 1.9
`
`24 444.2 $ 3.3
`
`+2.6 $ 3.3
`
`24 540.6 $ 4.7
`
`–11 $ 4.7
`
`24 910.3 $ 4.8
`
`–15.8 $ 4.8
`
`25 131.7 $ 7.1
`
`+12.6 $ 7.1
`
`24 953.4 $ 3.5
`
`+2.1 $ 3.5
`
`The results of the mass measurements indicate that the structures
`of some of the tRNA species purified from E.coli differ from the
`expected structure (Table 2). Here, the precision of the measurement
`is clearly not the issue, as the in vitro transcript of tRNALys as well
`as yeast tRNAPhe showed masses very close to the theoretical
`values. Furthermore, the mass spectrum of tRNAGlu obtained at high
`resolution indicates that the tRNA contains not a single species but
`a group of species with small mass differences. Mass differences
`between some of the species are ~ 14–15 Da, suggesting that they are
`due to methylation (causing a mass difference of 14 Da), a common
`post-transcriptional modification of bases. Therefore, the tRNAGlu
`species corresponding to the expected structure is probably the
`minor species, and the majority contains various unknown
`modifications to different degrees. The observed mass of
`tRNALys and that of tRNAfMet are either larger or smaller than the
`theoretical mass values by ~ 14 Da. This suggests again that there
`is an unknown methylation site, or the known methylation is only
`partial. Although the extent of several modifications has been
`reported to be only partial and the presence of micro-heterogeneity
`has been documented, the extent of partial modifications could
`not be well determined experimentally. The mass spectrometry
`can ‘visualise’ such micro-heterogeneity. The LC/MS analysis on
`the ribonuclease digests described in the present report should
`help the determination and the identification of the unknown
`modifications. McCloskey and co-workers have, in fact, shown
`the usefulness of the electrospray mass spectrometry in the
`elucidation of modified bases in RNase digests (13,29). The
`present on-line LC/MS, however, has an advantage over the
`previous off-line experiments; a complex mixture of polynucleotides
`can be analyzed without any tedious and often troublesome
`fractionation and/or pretreatment. It doesn’t matter whether the
`mixture contains various intact tRNA species or fragments
`produced by enzymatic or chemical cleavage.
`Compared to the other ionization methods such as fast atom
`bombardment (FAB) ionization and matrix-assisted laser desorption
`ionization (MALDI), the electrospray ionization seems to be less
`affected by the composition of or presence of other components
`
`Figure 5. Mass spectra of RNase T1 fragments showing heterogeneity.
`tRNAGlu was digested with RNase T1 and analyzed by the LC/MS. A part of
`the scans containing the heterogeneous fragment G2 was shown (a). A
`deconvoluted spectrum showing the heterogeneity of a fragment containing the
`anticodon loop of tRNAMet was observed during the LC/MS analysis of the
`RNase T1 digest of tRNAfMet-tRNAMet mixture (b). The position of an
`N4-acetyl C in the anticodon of tRNAMet is indicated by an arrowhead.
`
`that the deacylation occurred during the mild treatments
`employed in the present study. Since the base modification of the
`anticodon plays important roles in the codon–anticodon recognition
`(wobbling) and the identity of tRNAs (recognition by specific
`amino-acyl tRNA synthase), the present method should give a
`means to analyze the structure–function relationship.
`
`DISCUSSION
`
`The LC/MS analysis that has been previously used for studies of the
`post-translational modifications of proteins is now successfully
`applied to the structural studies of tRNA. The use of the on-line
`reversed-phase column chromatography allows a direct analysis
`of samples containing various salts and additives without any
`pretreatment. Previously, extensive desalting has been necessary
`to obtain electrospray mass spectra of reasonable quality, even
`with small oligo DNAs (11). Under these conditions, the
`intensities of the Na+ or K+ adduct ion peaks increase with
`increasing chain length, and large RNA molecules pose more
`problems than DNA molecules of similar lengths. The present
`LC/MS analysis, on the other hand, allowed us to observe only
`molecular ion peaks free from adduct peaks, and can be used to
`analyze even larger RNA and DNA up to ~ 200 bases (>60 kDa)
`under the same measuring conditions. The advantage of the addition
`of the organic amine with long substituents is twofold. First, the
`binding of hydrophilic polynucleotides to the reversed-phase
`column becomes stronger with increasing chain length of the
`substituents. This results not only in better chromatographic
`separation but also in the increase of organic solvent concentration
`
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`in the samples. Therefore, peak relative intensities observed in the
`deconvoluted spectra give at least rough, in some instances
`quantitative, estimates of the relative amounts of the species
`present in the samples. A notable example in the present study is
`the heterogeneity of the fragment containing the anticodon loop
`of tRNAMet. The presence of a modified base in the anticodon
`may regulate the activities of the tRNA, and it would be of interest
`to analyse the dynamic regulation of the modification. The present
`method should allow a rapid, relative quantification of the
`modifications under various cellular conditions such as heat shock.
`
`ACKNOWLEDGMENTS
`
`This work was supported in part by Grants-in-Aid from the Fujita
`Health University, a Grant-in-Aid for Developmental Scientific
`Research (07558219) and Grants-in-Aid for Scientific Research
`on Priority Areas (08283220 and 09272222) from the Ministry of
`Education, Science, Sports and Culture, Japan.
`
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