Biochimica et Biophysica Acta 1574 (2001) 137^144
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Pseudouridylation of U35 in the anticodon of Arabidopsis thaliana
pre-tRNATyr depends on length rather than structure of an intron
Joanna Pien¤kowska a , Daniel MichaIowski b , WIodzimierz J. Krzyzçosiak b ,
Zo¢a Szweykowska-Kulin¤ska a; *
a
Department of Gene Expression, Adam Mickiewicz University, Miedzychodzka 5, 60-371 Poznan¤, Poland
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan¤, Poland
L
b
Received 24 August 2001 ; received in revised form 29 October 2001; accepted 7 November 2001
Abstract
In order to establish the structure and sequence requirements for pseudouridine (835 ) biosynthesis in Arabidopsis thaliana tRNATyr five
mutants of nuclear pre-tRNATyr have been prepared and analyzed: vI-tRNATyr transcript depleted of an intron, and 5UI, 7UI, 9UI and
12UI transcripts containing tracts of five, seven, nine and 12 U residues, respectively, instead of the wild type tRNATyr intron. The in vitro
transcripts were incubated in a lupin seed extract containing 835 synthase activity, and those containing an artificial intron composed of 12
or nine U residues turned out to be good substrates for 835 synthase. The transcript with an intron composed of seven uridine residues was
pseudouridylated up to 40%, whereas the remaining two were not pseudouridylated at all. The secondary structures of all transcripts were
determined using enzymatic and chemical probes: S1 , V1 , T1 , A, P1 and Pb2þ . All mutant pre-tRNAs show similar structural features: their
anticodon arm contains a five base pair stem and a large loop which consists of five natural tRNATyr AC loop nucleotides to which five,
seven, nine and 12 U residues are added. As the structure of the wild type pre-tRNATyr is different we propose that the role of its intron in
the process of U35 pseudouridylation is simply to expand the anticodon region to the required critical length. ß 2001 Elsevier Science B.V.
All rights reserved.
Keywords : Pseudodouridine ; pre-tRNATyr transcript ; Intron-dependent modi¢cation ; RNA structure
1. Introduction
Pseudouridine synthases are a family of enzymes that
catalyze the synthesis of pseudouridine, the most abundant
modi¢ed nucleoside present in various RNA molecules:
tRNAs, rRNAs, snRNAs and snoRNAs [1]. The enzymes
are known to display a variable degree of speci¢city for
their RNA substrates [2] and ¢rst details of their interactions with RNA molecules have been recently revealed
[3,4]. It has been shown that pseudouridine synthase I
from Escherichia coli that modi¢es positions 38, 39 and/
or 40 in tRNAs is functional only as a dimer containing
two positively charged, RNA-binding clefts responsible for
interactions with two tRNA molecules. Each of the RNAbinding clefts contains a critical aspartic acid residue,
which is known to be responsible for catalytic activity of
* Corresponding author. Fax: +48-61-829-2730.
E-mail address : [email protected]
(Z. Szweykowska-Kulin¤ska).
pseudouridine synthases [3,4]. One interesting member of
the pseudouridine synthase family that has drawn considerable attention is eukaryotic tRNA 835 synthase.
Pseudouridylation of U35 located in the central position
of the anticodon loop of all known eukaryotic cytoplasmic
tRNATyr , which is catalyzed by 835 synthase, is strictly
dependent on the presence of an intron [5^8]. Moreover,
the 835 synthase activity also depends on the nucleotide
sequence that surrounds U35 [9]. The consensus sequence
U33 N34 U35 A36 Pu37 has been shown to be required by both
plant and vertebrate enzymes. Insertion or deletion of one
base pair in the anticodon stem of pre-tRNATyr does not
in£uence its substrate activity [9]. On the other hand, an
intronic insertion of dinucleotide, which extends the double-stranded stem of the Arabidopsis thaliana tRNATyr primary transcript by two additional base pairs and results in
hiding U35 in the helix structure, makes the transcript inactive towards 835 synthase. A comparison of intervening
sequences in tRNATyr gene families of Nicotiana rustica
and Xenopus laevis shows signi¢cant di¡erences in both
their length and sequence [10,6]. This suggests that 835
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 3 5 5 - 4
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synthases are highly tolerant to the diverse intron structures. The shortest natural intervening sequences of 11 bp
were identi¢ed in the tRNATyr genes from Nicotiana, Arabidopsis, Polytrichum, Rumhora and Tetrahymena [11,12].
A detailed analysis of the A. thaliana pre-tRNATyr intron
has shown that its truncation from 12 nucleotides (nt) to
7 nt still allowed partial, up to 40%, pseudouridylation of
U35 , whereas the 5 nt intron did not promote any pseudouridine formation [13]. However, the question of
whether the e¡ects were due to the reduced length of the
intron sequence itself or to some changes in the intron
structure caused by the deletion, has not been answered.
In this study, we show that the A. thaliana pre-tRNATyr
mutants containing unstructured tracts of 12, nine and
seven U residues retain signi¢cant pseudouridylation activity. We conclude from these results that the activity of
U35 synthase depends on length rather than the speci¢c
structure of an intron.
which the 12 bp long intron was deleted, and pATY2T75UI, pATY2T77UI, pATY2T79UI and pATYT712UI,
in which the wild type intron was replaced by an oligo(dT/
A) tract composed of ¢ve, seven, nine and 12 T:A pairs,
respectively.
2.4. Preparation of yellow lupin S-23 extract
Yellow lupin extract was prepared according to Stange
and Beier [15] with some modi¢cations as described in [8].
2.5. In vitro transcription of tRNATyr gene variants with
T7 RNA polymerase
Plasmids carrying the tRNATyr gene or its variants were
linearized by digestion with BstNI isoschizomer MvaI and
transcribed using T7 RNA polymerase as described in [8].
2.6. In vitro assay of pseudouridine formation and
quanti¢cation of pseudouridine in the 32 P-labeled
RNA samples
2. Materials and methods
2.1. Bacterial strains and plasmids
E. coli TG1 and CJ 236 strains were the hosts for propagation of plasmid DNA and M13mp19 phages. The recombinant plasmid used in this study was pATY2T7 carrying the A. thaliana tRNATyr gene under control of a T7
RNA polymerase promoter and containing the BstNI restriction site used to linearize plasmid and terminate in
vitro transcription [13].
2.2. Enzymes and reagents
RNase T2 was obtained from Calbiochem and Sigma,
nucleases S1 , V1 , P1 and ribonuclease T1 were from Amersham/Pharmacia Biotech and ribonuclease A was from
Reanal (Budapest, Hungary). Lead acetate was purchased
from Sigma. [Q-32 P]ATP (spec. act. 185 TBq/mmol),
[K-32 P]ATP (spec. act. 29.6 TBq/mmol), [K-35 S]ATP
(spec. act. s 37 TBq/mmol), and the T7 DNA polymerase
sequencing kit were from Amersham/Pharmacia Biotech.
The Muta-gene site-directed mutagenesis kit was from
Promega. The ‘stains all’ dye was from Serva. Restriction
enzymes, Taq DNA polymerase and all other enzymes
used were from Boehringer Mannheim. Yellow lupin seeds
cv. Ventus were obtained from the Plant Breeding Station
in Wierzonka, Poland.
2.3. Construction of A. thaliana tRNATyr gene mutants
Plasmid pATY2T7 harboring the A. thaliana tRNATyr
gene containing the 12 bp intron was mutagenized using
the Kunkel method [14]. Introduced mutations were con¢rmed by DNA sequencing. Five plasmids containing mutants of the tRNATyr gene were prepared : pATY2T7vI, in
Pseudouridine formation in an in vitro assay in yellow
lupin extract and the e⁄ciency of pseudouridine formation
were carried out as described in [8,13,16].
2.7. RNA structure probing
In vitro transcripts were phosphorylated at their 5P-ends
with [Q-32 P]ATP and polynucleotide kinase in standard
conditions. Labeled RNAs were puri¢ed in 10% polyacrylamide denaturing gel, identi¢ed by autoradiography and
recovered as described above. The unlabeled carrier RNA
was added to the labeled RNA solution for a ¢nal concentration of 8 WM. RNA was denatured at 55‡C in a
bu¡er containing: 10 mM Tris^HCl, pH 7.2, 10 mM
MgCl2 , 40 mM NaCl, and allowed to renature at 25‡C
for 20 min. All structure probing reactions were carried
out at 25‡C. The reaction times and probe concentrations
are speci¢ed in the legend to Fig. 4. Reactions were
stopped by mixing the reaction mixtures with an equal
volume of loading bu¡er, 8 M urea/20 mM EDTA/dyes,
and the samples were kept frozen before gel loading.
2.8. Analysis of products of RNA structure probing
reactions
Products of RNA enzymatic digestions and metal ion
induced cleavages were analyzed by gel electrophoresis in
15% polyacrylamide 7 M urea gel. RNA fragments generated by speci¢c probes were run along RNA ladders, i.e.
the products of RNA limited hydrolysis with formamide
and ribonuclease T1 . The formamide ladder was generated
by incubating the labeled RNA solution with a 5 times
larger volume of formamide/0.5 mM MgCl2 at 100‡C for
8 min. Partial digestion of the denatured RNA with ribo-
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139
Fig. 1. The cloverleaf structure of intron-containing A. thaliana wild type pre-tRNATyr and the structures of the anticodon arms of its oligo(U) mutants
and of tRNATyr without intron. The secondary structures shown in a^f are in agreement with structure probing and structure prediction data obtained
in this work. Intron sequences are shown in bold and numbered independently. (a) Wild type of pre-tRNATyr containing natural intron. The numbers
19, 35 and 55 mark selected positions in the mature tRNA molecule. (b^e) Fragments of pre-tRNATyr mutants representing their anticodon arm, containing instead of the wild type intron 12, nine, seven and ¢ve uridine residues, respectively. Arrows point to the existing or putative splice sites.
(f) Fragment of pre-tRNATyr showing the anticodon arm without intron. In wt pre-tRNATyr and all its mutants the numbering of nucleotides corresponding to the mature tRNATyr is the same. Separate numbering has been introduced for intron nucleotides (U1i etc.).
nuclease T1 was performed in the presence of 50 U/ml
enzyme, 10 mM sodium citrate, pH 4.5; 0.5 mM EDTA,
3.5 M urea at 55‡C for 13 min. The gels were autoradiographed at 380‡C with an intensifying screen.
3. Results
The goal of our research was to determine the minimum intron length in A. thaliana pre-tRNATyr which still
promotes conversion of U35 to 835 by plant 835 synthase.
Four pre-tRNATyr mutants were prepared in which the
intron of the wild type (wt) sequence was replaced by U
tracts with lengths of 12, 9, 7 and 5 nt, designated 12U,
9U, 7U and 5U, respectively. In addition, a vI mutant
completely deprived of the intron sequence was prepared.
The A. thaliana tRNATyr wt gene and its ¢ve mutants
(shown in Fig. 1) were transcribed by T7 RNA polymerase in the presence of [K-32 P]ATP. Gel puri¢ed labeled
transcripts were incubated in yellow lupin S-23 cell-free
extract under conditions which inhibited splicing, i.e.
without Triton X-100 and ATP. Then, all pre-tRNAs
were again puri¢ed, subjected to RNase T2 hydrolysis,
and analyzed for pseudouridine (835 ) formation. Nucleosides were resolved in a two-dimensional chromatographic system that separates common nucleotides from
their modi¢ed counterparts as shown in Fig. 2. It turned
out that the wild type pre-tRNATyr and pre-tRNATyr
Fig. 2. Analysis of pseudouridine 35 content in A. thaliana pre-tRNATyr and in its selected mutant by two-dimensional thin layer chromatography. PretRNAs were incubated in yellow lupin seed S-23 extract. Precursor tRNAsTyr were synthesized and treated as described in Section 2. (a) Wild type pretRNATyr ; (b) Pre-tRNATyr mutant containing intron composed of ¢ve U residues.
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Fig. 3. Kinetics of pseudouridine 35 formation in A. thaliana pretRNATyr mutants. Two pmoles of appropriate 32 P-labeled pre-tRNATyr
transcripts were incubated with cell-free lupin extract at 37‡C for 10, 20,
40, 60 and 90 min and analyzed for the relative amount of pseudouridine residue present at position 35. wt, wild type pre-tRNATyr ; mutants
12UI, 9UI, 5UI and pre-tRNATyr without intron (vI).
having the tract of nine uridyl residues instead of the
natural intron were fully pseudouridylated at position
U35 (Fig. 2a and 3), and pre-tRNATyr with the 12U
long intron modi¢ed to about 80% (Fig. 3). The pretRNATyr with the 7U long intron was modi¢ed to a lower
extent (40%) (Fig. 3), whereas mutants having either a 5U
intron or no intron were not pseudouridylated at all (Fig.
2b and 3). The kinetic data showing formation of 835 in
A. thaliana pre-tRNATyr and in its variants are displayed
in Fig. 3.
Trying to ¢nd a satisfactory explanation for the observed di¡erences in modi¢cation e⁄ciencies of di¡erent
substrates, we analyzed solution structures of all in vitro
transcripts used in this study. The natural and model pretRNAs labeled at their 5P-end were subjected to limited
hydrolysis with well characterized structure probes: lead
ions [17,18], nucleases S1 , V1 , P1 and ribonucleases T1 and
A [19,20]. All structure probing reactions were conducted
under similar conditions and important conclusions were
drawn from comparisons of the results obtained for di¡erent transcripts with the same probes [21]. The experimental approach was supplemented by RNA secondary structure prediction by free energy minimization [22].
As concerns the global structure of the investigated
transcripts, the most profound di¡erence in hydrolysis
patterns obtained for the wt pre-tRNATyr (having a 12
nt long intron) and for its variant with the 12U intron is
reversed susceptibility of the D loop and AC loop regions
to chemical cleavages and enzymatic digestions (Fig. 4).
In particular, the cuts generated by nucleases S1 , P1 , T1 in
the wt transcript are more prominent in the D loop re-
Fig. 4. Chemical and enzymatic probing of the 5P-end-labeled wild type pre-tRNATyr (a) and its 12U mutant (b). The following probe concentrations
and reaction times were used: lanes : A, B, C, nuclease S1 5000, 2500, 1250 U/ml, respectively, in the presence of 1 mM ZnCl2 , for 20 min; D, E, F,
lead ions 1.25, 0.875, 0.5 mM, for 20 min; G, H, I, nuclease P1 12.5 Wg/ml for 5, 10 and 20 min, respectively; J, K, L, nuclease V1 7.5 U/ml for 5, 10
and 20 min; M, N, O, ribonuclease T1 125 U/ml for 5, 10 and 20 min; P, R, S, ribonuclease A 0.001, 0.0005, 0.0001 Wg/ml for 10 min; Ci, incubation
control; L, formamide ladder ; T, T1 ribonuclease ladder. Positions of selected guanine residues are marked. Nucleotide numbering in this ¢gure and
Fig. 5 is the same as described in the legend to Fig. 1.
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141
Fig. 5. Chemical and enzymatic probing of pre-tRNATyr mutants: 9U (a), 7U (b), 5U (c) and intronless transcript vI (d). Lane designations are the
same as shown in the legend to Fig. 4, except for vI where the P1 and V1 lanes are exchanged.
gion, whereas in the 12U transcript they are much stronger in the AC region. This di¡erence holds also true for
other transcripts with oligo(U) containing introns (Fig.
5). Careful inspection of the D loop cleavages shows
that both those in the wt transcript and those observed
in 12U and other model transcripts (Figs. 4 and 5) occur
at the same sites and involve all internucleotide bonds
between G15 -U16 and G19 -U20 (for nucleases S1 and P1 )
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and at G15 -U16 , G18 -G19 and G19 -U20 (for ribonuclease
T1 ). The observed di¡erence between the wt transcript
and transcripts containing oligo(U) introns means either
a signi¢cant protection of the D loop region against nucleases, or decreased £exibility of this region in all oligo(U) containing transcripts in comparison to the wt pretRNATyr .
More relevant to the questions asked in this study are
structural di¡erences that occur in the anticodon region
of the investigated transcripts. Comparison of the wt pretRNATyr and the 12U transcript shows that cleavages in
this region are slightly stronger in the latter molecule. The
cleavage patterns are dissimilar both in terms of their
locations and relative intensities. In the wt transcript a
minor RNase T1 cut occurs at the G34 -U35 bond and a
stronger RNase A cut takes place at the U35 -A36 bond.
Weak digestions by nucleases S1 and P1 and a strong cut
by T1 RNase occur at the G37 -U1i bond. Starting with
this internucleotide bond, a whole series of cleavages generated by the single strand speci¢c nucleases occur in the
3P direction to end up at the intron 3P-terminus. All guanines and pyrimidines from this region are recognized by
ribonucleases T1 and A, but not all internucleotide bonds
in this region are digested by nucleases S1 and P1 . The
observed pattern of cleavages is basically consistent with
the structure of wt pre-tRNATyr shown in Fig. 1a. The
weak T1 cut at the G34 -U35 bond which occurs at the
border of the stem and loop portion indicates that base
pair G34 :C7i in the stem is relaxed to some extent. A
much stronger T1 cut at the G6i -C7i bond marks the 3Pend of the terminal loop. The weaker T1 digestion at the
G9i -A10i bond shows loosening of the C32 :G9i base pair in
the immediate vicinity of the four nucleotide bulge. The
bulge itself is well recognized by the single strand speci¢c
nucleases. Interestingly, the weak lead-induced cleavages
that begin at the C32 -U33 bond and also occur at the sites
digested by nucleases in the anticodon region may suggest
the relaxed structure of the entire region. On the other
hand, in the 12U transcript the C32 -U33 bond is strongly
cleaved by lead ions, the U33 -G34 bond is cleaved by lead
ions, P1 nuclease and RNase A, and strong S1 and P1 cuts
begin at the G34 -U35 bond. The RNase A cut marks the
3P-end of the U tract, lead ions recognize the 3P part of
this tract, whereas nucleases S1 and P1 tend to cut its 5Pend and central portion, respectively. The above data
taken together are consistent with the large 19 nt terminal
loop present in the anticodon arm of the 12U transcript
(Fig. 1b). Very weak lead cleavages in the central part of
this loop might suggest that this region is structured.
However, the very strong S1 , P1 , T1 , and A cuts that
occur in this portion speak against any strong secondary
structure in that region. According to the structure probing data shown in Fig. 5, terminal loops begin and end at
the same positions as in the 12U, but encompass 16, 14,
12 and 7 nt in the 9U, 7U, 5U and vI transcripts, respectively (Fig. 1c^f).
4. Discussion
As shown by the in vitro and in vivo expression studies
of tRNATyr genes, the presence of an intron is absolutely
required for 835 synthesis [5^8]. In spite of signi¢cant
progress in our understanding of 835 biosynthesis in pretRNATyr , the in£uence of intron architecture on this process remains elusive. It has been shown earlier that the
activity of X. laevis 835 synthase is independent of the
sequence and the size of the introns tested, which varied
in length from 20 nt to 113 nt [6]. Studies with plant pretRNATyr demonstrated that the same is true for plant 835
synthase [10]. Our results described in this study revealed
that the shortest arti¢cial intron that promotes e⁄cient
U35 pseudouridylation is 9 nt long. The reactivity of the
U residue being isomerized to pseudouridine is signi¢cantly lower in the pre-tRNATyr mutant containing the
7U intron, but the transcript containing the 5U intron,
as well as the intronless transcript, are completely inactive.
The reason for their inactivity could be either ine⁄cient
binding of the model substrates to the enzyme active site
or hindered access of the enzyme catalytic groups to the
substrate isomerization site.
A survey of nucleotide sequences of tyrosine tRNA
genes deposited in GenBank has shown that the shortest
natural introns are 11 bp long. However, it has to be kept
in mind that besides undergoing base modi¢cation in
vivo, the pre-tRNAs studied also undergo splicing. The
splicing reaction that occurs after pseudouridylation has
its own requirements for the minimum length and structure of an intron present in the anticodon stem. It has
been shown earlier that a distorted secondary structure in
plant pre-tRNATyr and pre-tRNAMet in the extended
anticodon stem results in a severe splicing de¢ciency in
plant cell extracts [23,24]. Plant splicing endonucleases
require the presence of a double-stranded structure between the 5P and 3P splice sites, and the minimal length
of an intron in plant tRNAMet genes is the 10 bp found in
Polytrichum and Ginkgo [24]. Thus, also with respect to
splicing, it is necessary for tRNATyr genes to contain at
least 10 bp long introns. Accordingly, the A. thaliana pretRNAsTyr having their natural intron shortened to 7 or
5 nt were not spliced in wheat germ extract. However, at
least for transcripts with a 7 nt long intron, the structural
requirements of plant splicing endonuclease for the expanded anticodon stem were partially ful¢lled [13]. We
have concluded from these experiments that both pretRNA maturation events, i.e. splicing and pseudouridylation, require the presence of an intron of a minimum of
10^11 nucleotides in length in order to be e⁄cient. Interestingly, as shown in this study, the process of U35 pseudouridylation in plant pre-tRNATyr , unlike splicing, does
not require any speci¢c secondary structure of the expanded anticodon arm. Our study has demonstrated
that the anticodon arms that contain oligo(U) instead
of natural intron have rather simple structures. They
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are all composed of ¢ve base pairs that form a hairpin
stem that is closed by a large terminal loop accommodating tracts of ¢ve, seven, nine or 12 U residues. The only
structural di¡erence between the pre-tRNAs that are
pseudouridylated and those that are not is the size of
the terminal loop. Thus, for e⁄cient pseudouridylation
of U35 not the speci¢c structure of the expanded anticodon stem is required, but only the appropriate length of
the intron, which is critical for pre-tRNATyr substrate
activity.
Recently, structural requirements for tRNA recognition
and pseudouridine formation by E. coli pseudouridine synthase I (8S I) were established. The enzyme is responsible
for pseudouridylation of positions 38, 39 and/or 40 in
tRNAs. Its active form is a homodimer that contains
two positively charged RNA-binding clefts along its surface. 8S I recognizes the speci¢c shape and charge of the
entire tRNA molecule, not only its anticodon stem. These
results are consistent with biochemical data showing that
8S I neither binds nor catalyzes 8 formation in RNA
minisubstrates corresponding to the anticodon stem loop
of tRNA [3]. It is therefore likely that plant 835 synthase,
similar to E. coli 8S I, requires a critical mass and charge
of the pre-tRNATyr molecule containing an intron longer
than 7 nt for e⁄cient substrate recognition, binding and
catalysis. Earlier we have shown that the A. thaliana pretRNATyr derived minisubstrate corresponding to the expanded anticodon arm was not pseudouridylated in the
wheat germ extract, but was slightly pseudouridylated, to
about 10%, in heterologous yeast S-100 extract [13]. In any
e¡ort to explain the latter fact, it cannot be ruled out that
the conversion of U35 to 835 in the minisubstrate was
catalyzed by another Pus1 pseudouridine synthase. This
enzyme is known to act on tRNA minisubstrates in vitro
and recognizes pre-tRNATyr as a substrate for U35 modi¢cation [16,25]. In light of the accumulated data, it seems
reasonable to divide the pseudouridine synthases into two
categories with respect to their substrate requirements.
Members of the ¢rst group recognize only a part of the
RNA molecule, leaving the rest exposed to solvent. Members of the second group recognize the shape and charge
of the whole RNA substrate. The Tru B enzyme from
E. coli [26] may be an example of the former group, whereas the 8S I from E. coli [3] may be a representative of the
latter. Based on the results of this study, we postulate that
plant 835 synthase belongs to the second category of pseudouridylating enzymes.
Progress in isolating genes for di¡erent pseudouridine
synthases and characterizing their products is rapid (e.g.
[27^30]). However, no 835 synthase gene or its product
has been isolated and described. Even in Saccharomyces
cerevisiae, no putative ORF coding for the 835 synthase
has been identi¢ed thus far. Search for this gene and its
product is therefore highly encouraged in order to improve
our knowledge of 835 biosynthesis.
143
Acknowledgements
This work was supported by the Polish Committee for
Scienti¢c Research project No. 6 P04A00312.
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