Shifted positioning of the anticodon nucleotide residues of amberDaisuke Kiga1,2, Kensaku Sakamoto2, Saori Sato1, Ichiro Hirao1and Shigeyuki Yokoyama1,2 1 Yokoyama CytoLogic Project, ERA
Trang 1Shifted positioning of the anticodon nucleotide residues of amber
Daisuke Kiga1,2, Kensaku Sakamoto2, Saori Sato1, Ichiro Hirao1and Shigeyuki Yokoyama1,2
1 Yokoyama CytoLogic Project, ERATO, JST c/o RIKEN, Hirosawa, Wako-shi, Saitama, Japan;2Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Japan
Cytidine in the anticodon second position (position 35) and
G or U in position 36 of tRNAArgare required for
amino-acylation by arginyl-tRNA synthetase (ArgRS) from
Escheri-chia coli Nevertheless, an arginine-accepting amber
suppressor tRNA with a CUA anticodon (FTOR1D26)
exhibits suppression activity in vivo [McClain, W.H &
Foss, K (1988) Science, 241, 1804 – 1807] By an in vitro
kinetic study with mutagenized tRNAs, we showed that the
arginylation of FTOR1D26 involves C34 and U35, and that
U35 can be replaced by G without affecting the activity
Thus, the positioning of the essential nucleotides for the
arginylation is shifted to the 50 side, by one residue, in the suppressor tRNAArg We found that the shifted positioning does not depend on the tRNA sequence outside the anti-codon Furthermore, by a genetic method, we isolated a mutant ArgRS that aminoacylates FTOR1D26 more efficiently than the wild-type ArgRS The isolated mutant has mutations at two nonsurface amino-acid residues that interact with each other near the anticodon-binding site Keywords: tRNA identity; anticodon; aminoacyl-tRNA synthetase; genetic screen; kinetic analysis
The accurate recognition of a tRNA by its aminoacyl-tRNA
synthetase (aaRS) is a vital step in ensuring the fidelity of
translation An aaRS distinguishes its cognate tRNAs from
the tRNA species existing in the same cell by the recognition
of the particular nucleotide residues (identity determinants)
that are found only in the cognate tRNAs as one set In most
tRNA species, these nucleotides are located in the anticodon
moiety and at the discriminator position (position 73) [1]
The identity of tRNAArginvolves two anticodon positions:
a cytidine residue in position 35 (C35) and a G or U in
position 36 both contribute to the arginine-accepting activity,
and the effect of base substitutions is much larger for position
35 than for position 36 [2 – 4] In contrast, position 34 is not
thought to contribute to the reaction, because various base
substitutions are allowed in this position; the naturally
occurring tRNAArg species have inosine, cytidine, and a
modified uridine in this position [5], and tRNAArg
tran-scripts with A34 and C34 exhibit arginylation activity
comparable to that of the fully modified tRNAArg[4]
The irrelevance of position 34 and the ambiguous
recog-nition at position 36 are necessary, because there are several
tRNA species with different anticodon sequences for
reading the six arginine codons, which only have G as the
second letter in common Leucine and serine are also each
encoded with six codons read by several tRNA species, but
their tRNAs have identity determinants located mainly outside the anticodon [1] In addition to the anticodon residues, A20 in the D loop also contributes to the tRNAArg identity in Escherichia coli and probably in most organisms other than yeast, but its contribution to the activity is only comparable to that of G/U36 [2,4,6]
McClain & Foss [6] used amber suppressor tRNA species
to analyze the identity determinants of E coli tRNAArg First, a tRNAArg2 variant, whose anticodon was replaced by CUA, inserted more Lys than Arg in response to the amber codon, suggesting that the amber suppressor tRNAArghas structural features similar to those of tRNALys Therefore, they tried a different approach: an amber suppressor derived from tRNAPhewas engineered by the transplantation of A20 together with the three surrounding nucleotides Actually, the resultant suppressor (‘F to R’ or FTOR1) inserted Arg about 10 times more frequently than Lys or Tyr in response
to the amber codon Furthermore, only Arg was detected with FTOR1D26, which was made by the deletion of A26 from FTOR1 As these Arg-inserting amber suppressors lack both of the anticodon identity determinants, C35 and G/U36, of tRNAArg, C34 may be recognized by the C35-recognition site of ArgRS (This mode of C35-recognition is referred to hereafter as ‘shifted positioning’.) The deletion
of A26 may facilitate the shift of C34 toward the location of C35, which might enhance the Arg-accepting activity, but not the Lys- and Tyr-accepting activities [6] Alternatively, the A26 deletion may depress the Lys- and Tyr-accepting activities, but not the Arg-accepting activity, of the tRNA [6]
Recently, the crystal structure of the ArgRS:tRNA com-plex from yeast was determined, which revealed a structural basis for the anticodon recognition by ArgRS [7] An interesting finding was that C35 does not make any specific interactions with amino-acid side chains, but only with certain backbone atoms in the C-terminal domain Even with this structural information, two questions remained
Correspondence to S Yokoyama, Department of Biophysics and
Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Fax: 1 81 35841 8057,
Tel.: 1 81 35841 4413, E-mail: yokoyama@biochem.s.u-tokyo.ac.jp
Enzymes: arginyl-tRNA synthetase from Escherichia coli
(SWISS-PROT entry name ¼ SYR_ECOLI; EC 6.1.1.19).
(Received 2 July 2001, revised 2 October 2001, accepted
3 October 2001)
Abbreviations: aaRS, aminoacyl-tRNA synthetase; ArgRS,
arginyl-tRNA synthetase.
Trang 2First, the possibility of the ‘shifted positioning’ of C34
needed to be examined Secondly, the types of amino-acid
replacements that change the anticodon specificity of ArgRS
needed to be identified; because of the unique manner of
C35 recognition by ArgRS, it is difficult to rationally design
amino-acid replacements to change the anticodon specificity
on a structural basis
In the present study, we analyzed FTOR1D26 and its
variants, and found that position 34, but not position 35, is
crucial for the arginylation in E coli In addition, this shift
of the crucial position does not depend on the sequence
outside the anticodon Finally, by genetic selection, we
isolated a mutant ArgRS that arginylates the amber
suppressor tRNAs better than the wild-type enzyme
M A T E R I A L S A N D M E T H O D S
DNA manipulation, sequencing, and PCR amplification
Standard techniques were used for restriction endonuclease
digestion, ligation, and gel electrophoresis [8] The
nucleo-tide sequence was determined using a Sequencing Pro
Autosequencer core kit for labeled primers (Toyobo, Tokyo,
Japan) PCR was performed using the Gene Amp PCR
system 9700 (Applied Biosystems)
Preparation of ArgRS
The wild-type and mutant ArgRS species were purified by a
histidine-tag system from overproducing cells, to exclude
any contamination by the endogenous wild-type enzyme
Thus, each ArgRS preparation has 10 tandem histidine
residues at the N-terminus (the C-terminal tag inactivates
the enzyme) The tagged wild-type enzyme exhibited an
aminoacylation activity similar to the reported values [4,9]
To tag the enzymes, the argS coding sequences were
amplified by PCR with the primers, 50-GGGAATTCCATA
TGAATATTCAGGCTCTTC-30 and 50-GAGCGGATCCA
AGCTTCCATTTTCAGAATACATTTAGATGGC-30, and
were then ligated between the Nde I – Bam HI sites of
pET26b (Novagen) The E coli BLR (DE3) cells (Novagen)
were transformed with this plasmid expressing the tagged
ArgRS, and were then grown at 25 8C in Luria – Bertani
media supplemented with 6% glucose At the late log phase
of cell growth, isopropryl thio-b-D-galactoside was added
to the media to a 0.5 mM concentration, and after 1 h of
induction, the cells were harvested The enzyme was then
purified by chromatography on Ni-nitrilotriacetic acid
agarose (Qiagen), followed by FPLC using a Resource-Q
column (Amersham Pharmacia Biotech)
In vitro aminoacylation analyses
Transfer RNA variants were prepared using T7 RNA
polymerase [10] According to the standard procedure [10],
the tRNA aminoacylation assays were performed at 37 8C in
40 mL of a buffer [100 mMTris/HCl, pH 7.5, 15 mMMgCl2,
4 mM ATP, 60 mM [14C]arginine (332.1 pCi:pmol21, New
England Nuclear)] containing various concentrations of the
enzyme and the tRNA Before the reaction, the tRNA was
renatured at 65 8C for 5 min in the reaction buffer The
kinetic parameters were obtained from Lineweaver – Burk
plots The obtained data are the averages of at least two
independent assays As E coli ArgRS has a Km value of
12 mM for arginine [9], its concentration in our assay is nearly saturating
Bacterial strains and plasmids for the genetic study The E coli arginine auxotroph, which we designated as NR101, is an argE(Am) strain provided in an Interchange kit (Promega), and confers the tetracycline resistance The arginine-inserting amber suppressor tRNA, FTOR1D26, reported by McClain & Foss [6], had been expressed from the gene cloned in pUC18 (which we desig-nated as pUCsupR) under the control of the lpp promoter and the rrnC terminator This gene was cloned in the low-copy vector, pAp102, which has a ColIbP9 replication origin, and
a copy number of 1.7 per cell [11], to generate pApsupR The pAp102 vector was a gift from K Mizobuchi (Department of Applied Physics and Chemistry, University
of Electro-Communications, Tokyo, Japan)
The E coli argS gene, with its native promoter and SD sequence, was inserted between the Bam HI – HindIII sites of the pACYC184 plasmid to generate pACargS
Construction of a mutantargS library and genetic selection
In order to prepare a mutant ArgRS library, part of the argS sequence (0.5 kb), from the Eco RV site (amino-acid residue 318) to the C-terminal end, was amplified by error-prone PCR with the primers, 50-CACCACTGATATCGCCTG TGCG-30 and 50-GAGCAAGCTTCCATTTTCAGAATAC ATTTAGATGGC-30, where the HindIII site is underlined [12] After digestion with these restriction enzymes, the PCR product was cloned in pACargS, in place of its counterpart in the wild-type argS sequence, to generate the pACargSmuplasmids
In order to isolate mutant ArgRS species that amino-acylate FTOR1D26, strain NR101 was transformed with pApsupR together with pACargSmu, and was then grown for
36 h at 37 8C on an M9 plate supplemented with proline, methionine, and thiamine (1 mM each), but not with argi-nine The colonies that formed on the plate were isolated, and after the pACargSmuplasmids were extracted, they were tested again for the potential to suppress the arginine auxotroph The Eco RV – HindIII fragments of the recovered argS genes from the plasmids thus selected were cloned again in pACargS, in place of the counterpart in the wild-type argS, and were then examined again for their suppressing potential Finally, the Eco RV – HindIII frag-ments thus selected were subjected to sequence determination
R E S U L T S A N D D I S C U S S I O N
ArgRS recognition of tRNAArgspecies with a CUA anticodon
In E coli and yeast, C35 and G or U in position 36 are crucial for the arginylation [2,4,13] In contrast, position
34 is not thought to contribute to the reaction [4,5] With this in mind, we investigated the manner by which ArgRS recognizes FTOR1D26 A wild-type tRNAArg, tRNAArg [14], and FTOR1D26 variants with various
Trang 3anticodon sequences were prepared by in vitro transcription
with T7 RNA polymerase, and were subjected to in vitro
kinetic analyses The wild-type tRNAArg2 exhibits an
arginine-accepting activity similar to those reported so far
[4,9], while FTOR1D26 exhibits a 6 105-fold lower
activity than the wild-type (Table 1) The replacement of
C34 by either A or U in FTOR1D26 further reduced the
arginine-accepting activity This result is in sharp contrast
to the wild-type tRNAArgwith C35, in which position 34
is not relevant, but position 35 is strictly required to be C
for the arginine-accepting activity [4,5] For position 35 of
FTOR1D26, the U35A substitution also reduces the activity,
but U35G does not affect it, which is reminiscent of the
effects of the base substitutions in position 36 of the
wild-type tRNAArg species As the specificities of ArgRS for
positions 34 and 35 of FTOR1D26 are exactly the same as
those for positions 35 and 36, respectively, of the wild-type
tRNAArg, this indicates that C34 and U35 of FTOR1D26 are
recognized by the binding pockets for C35 and G/U36,
respectively, of the enzyme, as previously proposed [6]
We then examined the effect of the deletion of A26 The
arginine-accepting activity of FTOR1 is higher than that of
FTOR1D26 in vitro (Table 1) However, FTOR1 inserts Lys
and Tyr, in addition to Arg, but FTOR1D26 predominantly
inserts Arg, in response to the amber codon [6] As previously
proposed [6], the two possible reasons why the deletion of
A26 makes the tRNA much more specific to arginine are (a)
the arginine-accepting activity, but not the lysine- and
tyrosine-accepting activities, of the tRNA was increased,
and (b) the recognitions of the tRNA by LysRS and TyrRS
were more impaired than that by ArgRS The present results
clearly indicate that the latter reason is the case for the
A26 deletion of FTOR1 The C34A and C34U substitutions
of FTOR1 both reduce the arginine-accepting activity, as
in the case of FTOR1D26 (Table 1) Therefore, the shifted
positioning occurred in both the presence and absence of
A26 in FTOR1
We next assayed the amber suppressor tRNA derived
from tRNAArg2 , whose nucleotide sequence is significantly
different from that of FTOR1D26 This amber suppressor tRNA, tRNAArg2 (CUA), shows a higher arginine-accepting activity in vitro than those of FTOR1D26 and FTOR1 (Table 1) In contrast, tRNAArg2 (CUA) inserts more lysine than arginine in vivo in response to the amber codon, while the other two tRNAs mainly insert arginine [6] The nucleo-tide sequence of tRNALys is more similar to that of tRNAArg2 (CUA) than to those of FTOR1D26 and FTOR1, which are derived from tRNAPhe Again, the substitution of C34 in tRNAArg2 (CUA) decreased the arginine acceptance (Table 1), indicating that neither the irregular conformation
of FTOR1D26 nor the tRNA sequence outside the anticodon
is important for the shifted positioning In summary, the change in the tRNA framework from tRNAArg2 (CUA) to FTOR1, as well as the A26 deletion of FTOR1, increased the arginine specificity by decreasing the recognition by LysRS and TyrRS much more than that by ArgRS Genetic selection of mutant ArgRS molecules
To explore the accommodation of the suppressor tRNA by ArgRS, mutant enzymes facilitating the amber suppression
by the tRNA were isolated by genetic methods In the complex structure of ArgRS:tRNA, C35 is recognized only
by the main-chain atoms of the enzyme [7], and this finding raised the question of what types of amino-acid replace-ments change the anticodon specificity of the enzyme For the mutant isolation, we constructed a genetic system
to detect the increased activity of a mutant enzyme for FTOR1D26 When expressed from a high-copy plasmid (pUCsupR), FTOR1D26 by itself exhibits a suppression activity When expressed from a single copy plasmid (pApsupR), FTOR1D26 does not complement the poor growth of the E coli argE(Am) mutant on minimal medium (data not shown): this cannot be rescued by the over-production of ArgRS from the plasmid pACargS (a multi-copy plasmid carrying argS ), which allowed us to isolate mutant argS genes that promote the amber suppression by FTOR1D26
Table 1 The kinetic parameters for the aminoacylation of the tRNAArgvariants by the wild-type ArgRS and the argS1 mutant enzyme ND, not determined because of the very low activation of the tRNA by the enzyme variant “Framework” indicates a moiety of tRNA outside the anticodon.
Framework Anticodon
k cat
(s 21 )
K m
(m M )
k cat /K m
( M21:s 21 )
k cat
(s 21 )
K m
(m M )
k cat /K m
( M21:s 21 ) tRNAArg2
(wild-type) ACG 17 1.0 1.7 107 2.8 0.44 6.4 106 FTOR1D26 CUA 9.3 1024 34 27 3.1 1023 32 97
FTOR1D26 CGA 1.1 1023 38 29 3.7 1023 32 1.2 102
FTOR1 CUA 2.8 1023 14 2.0 102 6.4 1023 12 5.3 102
tRNAArg2 CUA 3.1 1022 4.5 6.9 10 3 8.4 1022 5.6 1.5 10 4
Trang 4Fig 1 Amino-acid replacements found in the ArgRS mutants The amino-acid sequences from residue 318 to the C-terminus (residue 577) of the wild-type E coli ArgRS and its mutants are shown The secondary structures are assigned according to the crystal structure of yeast ArgRS determined Cavarelli et al [20].
Trang 5On average, a few base substitutions were introduced in
random positions in the C-terminal region of ArgRS by
error-prone PCR; this region ranges from residue 318, a
position between the HIGH and KMSKS sequences, to the
C-terminus Of the 107 argE(am) cells with pApsupR that
were transformed by these randomly mutagenized argS
genes, 102 cells formed colonies on the minimal plates
without arginine after an incubation at 37 8C for 36 h After
excluding the revertant cells, 26 different mutant argS genes
were isolated with the amino-acid replacements listed in
Fig 1, which will be discussed later
Recognition of the suppressor tRNA species by a
mutant ArgRS
To characterize the mutant ArgRSs, we tried to purify the
mutant enzymes The histidine tag was added to the
N-terminus of the enzyme to exclude the contamination by
the endogenous wild-type ArgRS As all but one of the
mutant enzymes with the histidine tag were found in the insoluble fraction of the cell lysates, we hereafter focused on the only soluble mutant enzyme, argS1, with two amino-acid replacements, Met to Val in position 460 and Tyr to Asp
in position 524 In order to investigate the effects of these replacements individually, the argS mutants with either replacement were constructed by site-directed mutagenesis, and were subjected to the same test used for the mutant selection; neither of the single mutants could form colonies
on the plate for the selection Both replacements were thus shown to be necessary for the ability of argS1 to suppress argE(Am) when FTOR1D26 is expressed simultaneously (data not shown)
As shown in Table 1, the argS1 mutant enzyme arginylates FTOR1D26 3.6-fold more efficiently, and the wild-type tRNAArg2.7-fold less efficiently, than the wild-type ArgRS Thus, the tRNA specificity of argS1 changes by 9.7-fold, compared with that of the wild-type ArgRS The activities of argS1 for the other two tRNAs with the CUA anticodon, FTOR1 and tRNAArg2 (CUA), were also higher than that of the wild-type enzyme
The effects of the anticodon base substitutions in FTOR1D26 were examined for argS1, and the shifted positioning of the anticodon residues was also observed for this mutant enzyme (Table 1); the substitutions of C34U, C34A, and U35A all reduce the arginine-accepting activity, whereas U35G has no effect on the activity In addition, the substitutions of C34, in both FTOR1 and tRNAArg2 (CUA), all reduce the arginine-accepting activity by the mutant enzyme
Between the wild-type and mutant enzymes, the differ-ence in the aminoacylation activity of the suppressor tRNA species is mainly derived from the difference in the kcat values, not in the Kmvalues It reminds us that the substi-tution of the identity determinants in the anticodon of tRNAArgmainly decreases the kcatvalues [2,4] The muta-tions therefore seem to facilitate the unusual signal
Fig 2 Mapping of the yeast ArgRS residues that correspond to the two amino-acid replacements in the argS1 mutant of E coli ArgRS
on the yeast ArgRS:tRNA complex structure (A) The crystal structure of the yeast ArgRS:tRNA complex [7] used as a working model of the E coli system In the argS1 mutant of E coli ArgRS, Met460 and Tyr524 are replaced by Val and Asp, respectively The
E coli Met460 and Tyr524 correspond to Leu489 and Phe555, respectively, of yeast ArgRS On the ribbon model of yeast ArgRS, the positions of Leu489 and Phe555 are indicated with the side chains (ball-and-stick) in green and magenta, respectively The tRNA main chain is shown by a wire model, mainly colored yellow, while a part of the anticodon stem (positions 39 – 41), which interact with the H18 and H22 helices bearing Leu489 and Phe555, respectively, are shown in red The three nucleotide residues of the anticodon are indicated by orange sticks (B) The detailed structure around Leu489 and Phe555 in the yeast ArgRS:tRNA complex The H18 helix and the Leu489 side chain are shown in green, and the H22 helix and the Phe555 side chain are shown in magenta The side chains of Tyr488 on the H18 helix and those of His559, Ser562, and Ser563 on the H22 helix, shown by balls and sticks, form a protein surface that contacts the anticodon-stem residues in positions 39 – 41, shown by sticks The E coli ArgRS residues that correspond to the yeast ArgRS residues are indicated in parentheses All the figures were prepared using the programs
[21] and 3 [22].
Trang 6transduction from the C35- and G/U36-binding pockets
upon the binding of some nucleotides in the noncognate
CUA anticodon to the catalytic domain of the enzyme
Structural elements for the change in the tRNA specificity
ofargS1
The argS1 mutant enzyme involves two mutations, M460V
and Y524D, as described above In the sequence alignment
of the ArgRSs from E coli and yeast, the E coli Met460
corresponds to the yeast Leu489, which is located on an a
helix, H18 (Fig 2), in the reported structure of the yeast
ArgRS:tRNA complex [7] The other argS1 mutation,
Y524D, of the E coli ArgRS occurs in the position that
corresponds to position 555 in the yeast ArgRS This
position is located on another a helix, H22 (Fig 2) These
two helices form part of the a helix bundle that constitutes
the anticodon-binding site In the helix bundle of the yeast
ArgRS, these two amino-acid residues are below the protein
surface, and interact directly with each other (Fig 2) The
mutations in these positions should induce some structural
change on the surface of the helices, which contact the
backbones of the nucleotide residues in positions 39 – 41
within the anticodon stem of the tRNA (Fig 2)
Upon binding of the anticodon of the wild-type tRNAArg
to the site on the yeast ArgRS, the anticodon loop undergoes
a large conformational change [7]; upon the putative binding
of C34 and U35 of the suppressor tRNA species to the
C35- and G/U36-binding sites, respectively, of the enzyme
(‘shifted positioning’), a somewhat different conformational
change of the anticodon loop is likely to be required This
alternative anticodon-loop conformational change of the
suppressor tRNA is probably much less favorable for
amino-acylation than that of the wild-type tRNA In the argS1
mutant enzyme, the unusual structure due to the mutations
of the anticodon-stem-docking site may facilitate the
alter-native anticodon-loop conformational change of the
sup-pressor tRNA, but it may hamper the anticodon-loop
conformational change of the wild-type tRNA
It has been reported that mutations in positions other than
the anticodon-interacting amino-acid residues increase the
aminoacylation of a nonsense suppressor tRNA; for E coli
glutaminyl-tRNA synthetase, the mutations are outside the
anticodon-binding site, and are located near the core region
of tRNA in the enzyme:tRNA complex [15] It was argued
that these mutations affect the process of transmitting the
signal from the anticodon binding domain to the active site,
and make the enzyme bind C35 in the pocket for U35, which
is conserved in all of the glutamine tRNAs On the other
hand, on the surface of the putative anticodon-recognition
helix of E coli methionyl-tRNA synthetase, there are two
acidic residues that reportedly serve as negative
discrimi-nants against noncognate tRNA anticodons through a direct
electrostatic repulsion; the replacement of one of these
residues does not affect the activity of the cognate tRNA, but
rather increases the activity of the noncognate ones [16]
The mutations in the 26 ArgRS mutants isolated in this
work exhibit a tendency to converge at certain positions
(Fig 1), although their contributions to the mutant
pheno-type have not yet been confirmed, and the mutant enzymes
have not been characterized Structural and genetic studies
have suggested that the H16, H18, and H22 helices and
the V loop of yeast ArgRS are probably involved in the
recognition of the anticodon moiety [7,17] The conver-gence of the above mutations in these domains suggests that their effects are associated with the anticodon recognition by the E coli enzyme
Misrecognition of an apparently irrelevant anticodon sequence has been also reported for yeast ArgRS [13,18,19], which misarginylates tRNAAsptranscripts with an efficiency similar to to that of the arginylation of the cognate tRNAArg species This misrecognition is due to the existence, in the anticodon region, of an alternative set of nucleotides con-tributing to the arginylation In addition, for the yeast ArgRS, the replacement of Y491H (position 462 in the E coli numbering) probably affects its anticodon specificity, causing a misarginylation of the noncognate tRNAAspwith
a GUC anticodon sequence [17] Notably, Tyr491 is also located on the H18 helix, and is only two residues down-stream of Leu489, the counterpart of Met460 found in the
E coli argS1 mutant
A C K N O W L E D G E M E N T S
We thank Dr J Cavarelli (Biologie et Genomique Structurales, Institut
de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ ULP) and coworkers, for allowing us to use the published coordinates of the ArgRS:tRNA complex before public release We also thank Dr
K Mizobuchi for providing us the pAp102 vector This work was supported in part by the Japan Society for the Promotion of Science under the Research for the Future program (JSPS-RFTF 96I00101).
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