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Nucleic Acids Research Pages 5045-5051  

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family
Introduction
Materials and Methods
   Isolation of cDNA for human AsnRS using the alignment-guided cross-species PCR method
   Phylogenetic analysis
Results and Discussion
   Isolation and sequence determination of human AsnRS cDNA
   Multiple sequence alignmets of 14 AsnRSs and 15 AspRSs
   Distributions of AsxRS and GlxRS orthologs in genomic sequence space
   Phylogeny of AspRS and AsnRS genes
   How old was the occurrence of AsnRS?
Acknowledgements
References

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family

Kiyotaka Shiba1,*, Hiromi Motegi1,2, Mitsutaka Yoshida2 and Tetsuo Noda1,3

1Department of Cell Biology, Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima, Tokyo 170-8455, Japan, 2Department of Physiological Chemistry, Faculty of Science, Toho University, Funahashi, Chiba 274-8510, Japan and 3CREST, Japan Science and Technology Corporation, Japan

Received September 10, 1998; Revised and Accepted October 8, 1998

DDBJ/EMBL/GenBank accession no. D84273

ABSTRACT

We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.

INTRODUCTION

Thanks to the recent progress of genome projects focused on various microorganisms, new light has been shed on the `dynamics' of genome structure. A prominent example of such dynamics is evident from analysis of the genes for aminoacyl-tRNA synthetases (ARSs) (1), a family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs and are thus essential for translation of genetic information. Although these enzymes are believed to be among the earliest proteins, the canonical orthologs (genes having strong sequence similarities to those previously identified by classical biochemical or genetic studies) for 20 ARSs are not always found within genomes whose complete sequences have been determined. For instance, the genomes of archaebacteria such as Methanococcus jannaschii (2) and Methanobacterium thermoautotrophicum (3) do not contain the canonical orthologs for asparaginyl-tRNA synthetase (AsnRS), cysteinyl-tRNA synthetase, glutaminyl-tRNA synthetase (GlnRS) or lysyl-tRNA synthetase (LysRS).

The absence of canonical orthologs must mean that either (i) ARS activity in these organisms is encoded by genes whose sequences are dissimilar to the previously identified canonical genes or (ii) the organisms lack ARS activity and form aminoacyl-tRNAs via different pathways. Ibba et al. recently found that with respect to LysRS, the former is the case for some organisms in Archaea (4). They purified a protein having LysRS activity from the archaebacterium, Methanococcus maripaludis, and discovered that the gene coding for LysRS activity is entirely unlike the canonical LysRS gene. The newly discovered non-canonical (archaeal) LysRS gene has its orthologs in the archaeal genomes of M.jannaschii (2), M.thermoautotrophicum (3), Archaeoglobus fulgidus (5) and Pyrococcus horikoshii (6).

Based on their primary and tertiary structures, the 20 canonical ARSs have been historically classified into two groups of 10 members each (classes I and II; 7,8), and LysRS has been grouped into class II. Nevertheless, the newly discovered archaeal LysRS possesses sequence motifs specific to class I enzymes (4). Thus, two entirely different LysRSs are present in genomic sequence space. Another surprising observation related to archaeal LysRS is the presence of the ortholog in the genomes of the spirochetes, Borrelia burgdorferi (9) and Treponema pallidum (10). Although these eubacteria are distant from archaebacteria according to the orthodox phylogenetic tree (based primarily on rRNA sequences), they use the archaeal rather than the canonical LysRS (11). Similarly, crossover of archaeal/eukaryotic genes for glycyl-tRNA (GlyRS), isoleucyl-tRNA (IleRS), prolyl-tRNA and methionyl-tRNA synthetases into bacterial genomes has also been observed (12-15), and transfer of the gene for valyl-tRNA synthetase (ValRS) from the mitochondrial to the eukaryotic genome has been suggested (16,17). It seems, therefore, that with respect to ARS genes, vectorial gene transfer is more widespread than previously thought (13).

The absence of orthologs for GlnRS and AsnRS in M.jannaschii and M.thermoautotrophicum is explained by the aforementioned case (ii). Biochemical analysis has shown that in Gram-positive bacteria, archaebacteria and organelles, glutaminyl-tRNAGln is synthesized by misacylation of tRNAGln with glutamate by GluRS, followed by conversion of the glutamate moiety to glutamine by Glu-tRNAGln amidotransferase (reviewed in 18,19). Absence of the ortholog for GlnRS and identification of Glu-tRNAGln amidotransferase in organisms lacking GlnRS (20) confirmed this indirect pathway mediating glutaminyl-tRNA formation. Consistent with these observations, phylogenetic analysis of the sequences of GluRS and GlnRS suggests the comparatively late occurrence of GlnRS. A plausible evolutionary path for GlnRS, which is supported by analysis of the tertiary structure, entails the gene arising in Eucarya through duplication of a pre-existing GluRS gene and then being transferred to [gamma]-Proteobacteria by vectorial gene transfer (21-23).

Complete genome sequencing has revealed that the canonical ortholog of AsnRS is absent in some organisms, suggesting that these organisms form asparginyl-tRNA via indirect pathways (2,5,24). Indeed, it is known that the halophilic archaebacterium, Haloferax volcanii, synthesizes asparaginyl-tRNAAsn by transamidation of a mischarged aspartyl-tRNAAsn in a manner analogous to the indirect pathway for glutaminyl-tRNAGln synthesis (25). We were interested, therefore, in the occurrence and evolutionary path of AsnRS; because the sequences of AsnRS and aspartyl-tRNA synthetase (AspRS) are highly similar, they are believed to have diverged through gene duplication (26). Currently available sequences for eukaryotic ARSs (especially for higher eukaryotic ARSs) are limited. Therefore, we sought to clone human AsnRS and determine its primary structure. The identification of appended domain, which serves as a signature for eukaryotic or higher eukaryotic ARSs, in human genes would also help our evolutionary analysis of ARSs. We report here the cloning of human cytoplasmic AsnRS cDNA, distributions of AsnRS in genomic sequence space, and phylogenetic analysis of the AsnRS and AspRS (AsxRS) gene family.

Figure 1. Alignment of 14 AsnRSs (brown) and 15 AspRSs (blue). Gaps in the sequences are indicated by dots, and the numbers in black boxes represent residues that were omitted from the alignment. The numbers above the sequences refer to codon positions for human AsnRS. Elements of the secondary structure (arrows and tubes representing [beta]-strand and [alpha]-helices, respectively) of T.thermophilus AsnRS (46) are also shown above the sequences; regions corresponding to the N-terminal anticodon binding domain, the hinge domain and the C-terminal catalytic domain are depicted in blue, green and yellow, respectively, and the locations of motifs 1, 2 and 3 are indicated. Amino acids that are identical in all 29 sequences are shaded in red, and those identical in 28 sequences are shaded in pink. The amino acids shared among AsnRSs and AspRSs are shaded in light blue and light orange, respectively. The boxes identify the amino acids in the N-terminal extension of eukaryotic AsnRS or AspRS. Residues known to interact with tRNA, within or between domains, or with amino acids, ATP or Mg2+ are indicated above the sequences by t, $, a, A and M, respectively. Abbreviations, references and GenBank accession nos are as follows: Hs-N, Homo sapiens (this study; 35); Bm-N, Brugia malayi (33); Ce-N, Caenorhabditis elegans (Z71262); Sc-N, Saccharomyces cerevisiae (U10399); Bs-N, Bacillus subtilis (59); Ld-N, Lactobacillus delbrueckii (X89438); Tt-N, Thermus thermophilus (X91009); Ph-N, Pyrococcus horikoshii (6); Mg-N, Mycoplasma genitalium (60); Mp-N, Mycoplasma pneumoniae (61); Ec-N, Escherichia coli (70); Hi-N, Haemophilus influenza (71); Sy-N, Synechocystis sp. (72); Bb-N, Borrelia burgdorferi (9); Tt-D, (47); Ml-D, Mycobacterium leprae (X77655); Mt-D, Mycobacterium tuberculosis (Z77724); Ec-D, (70); Hi-D, (71); Bs-D, (59); Ph-D, (6); Py-D, Pyrococcus sp. (D45167); Mj-D, Methanococcus jannaschii (2); Mth-D, Methanobacterium thermoautotrophicum (3); Af-D, Archaeoglobus fulgidus (5); Hs-D, (J05032); Rn-D, Rattus norvegicus (J04487); Ce-D, (Z19152); Sc-D, (X03606).


MATERIALS AND METHODS

Isolation of cDNA for human AsnRS using the alignment-guided cross-species PCR method

The probe for a library screening was obtained using the alignment-guided cross-species PCR method (27-30). When we began the experiment, four sequences for AsnRS were available, including those from Escherichia coli (31), Saccharomyces cerevisiae (32), Brugia malayi (33) and Onchocerca volvulus (34). We chose two conserved regions that overlap with the class-defining motifs 2 and 3 (7) and synthesized the degenerated primers KY-300 (5[prime]-GCIGA RTTYT GGATG YTIGA RGTIGA-3[prime], where R = A and G, Y = C and T, and I = inosine); KY-301 (5[prime]-GCIGA RTAYG CICAY GTIGA RGCIGA-3[prime]); KY-302 (5[prime]-YTCRA AICCIA RICCR AAICC ICCRTG-3[prime]); and KY-303 (5[prime]-YTCIA RICCI ARICC RTAIC CICCRTG-3[prime]). Using cDNA prepared from human fetal fibroblasts (27) as a template, cross-species PCR was performed (35 cycles of 30 s at 94°C, 30 s at 55°C and 90 s at 72°C) using selected combinations of the degenerated primers. Two combinations, KY-301/KY-302 and KY-301/KY-303, yielded DNA fragments of equal size (0.6 kb) and with identical sequences that were very similar to the sequences of AsnRS from other organisms. The full-length cDNA was subsequently isolated from a human fetal brain cDNA library, and both of its strands were sequenced. From its similarity to S.cerevisiae cytoplasmic AsnRS, we concluded that the cloned human AsnRS cDNA encodes for a human cytoplasmic AsnRS. The nucleotide sequence presented here (2673 nt) was submitted to the DDBJ/EMBL/GenBank database in 1996 with the accession number D84273. Beaulande et al. recently reported the structure of a somewhat smaller cDNA for human cytoplasmic AsnRS isolated from a liver mRNA (1874 nt; 35). The difference in length between that sequence and ours is due to a difference in the size of the respective 3[prime] untranslated regions; the size and sequence of the open reading frames are identical in the two sequences.

Phylogenetic analysis

AsnRS and AspRS sequences were aligned using the PILEUP program from the GCG package (36) and then further optimized manually. The portion of the alignment corresponding to the catalytic domain was used for further phylogenetic analysis. For this purpose, all gaps and insertions in the alignment were manually deleted, and the remaining 297 sites were used for calculations (these data are available on request). Distance, parsimony and maximum likelihood (ML) methods were used to infer phylogenetic trees. Distances between pairs of sequences were estimated using the PROTML `-D' program with the JTT-F amino acid replacement process option (37) invoked, and a neighbor-joining tree (38) was made using the NJDIST program. Similarly, a distance matrix was calculated using the PROTDIST program with the Dayhoff matrix option invoked, and a Fitch-Margoliash tree (39) was made using the KITSCH program. The parsimony tree (40) was constructed using the PROTPARS program. Bootstrap analyses were performed with 500 random replications using the SEQBOOT and CONSENSE programs. ML analyses (41,42) were performed using the PROTML program with the JTT-F option invoked. PROTDIST, KITSCH, PROTPARS, SEQBOOT and CONSENSE were provided in the PHYLIP program package (43); PROTML and NJDIST were provided in the MOLPHY version 2.3b package (44).

RESULTS AND DISCUSSION

Isolation and sequence determination of human AsnRS cDNA

The cDNA for human AsnRS was isolated by using an alignment-guided, cross-species PCR approach as described in Materials and Methods; this approach has enabled us to also obtain cDNAs for human cytoplasmic IleRS (27), GlyRS (28), AlaRS (39) and LysRS (30). The sequence of AsnRS has a total of 2673 nt without the poly(A) track: there is a single, long open reading frame with 548 codons starting at nucleotide 33 and extending to nucleotide 1676, and a 997 nt untranslated region at the 3[prime] end. The first ATG codon and the flanking sequences (ggCATGG) are consistent with Kozak's optimal translation initiation sequence (ACCATGG; 45). The predicated molecular weight of the 548 amino acids is 62 902.

Multiple sequence alignments of 14 AsnRSs and 15 AspRSs

AsnRS, AspRS and LysRS are similar with respect to their primary, tertiary and quaternary structures and have been assigned to the class IIb subgroup of class II ARSs (26). Figure 1 shows a multiple sequence alignment of 14 AsnRSs and 15 AspRSs from various organisms including Archaea, Eucarya and Bacteria. Structural elements derived from the crystal structure of Thermus thermophilus AsnRS (46) are depicted above the sequences. Class IIb ARSs are comprised of two principle domains (47): an N-terminal anticodon binding domain (from [beta]S1 to [beta]S5) and a C-terminal catalytic domain (from H3 to H12), which are connected by a small hinge domain (H1 and H2). Across the 29 sequences that include AsnRS and AspRS, 21 amino acids are strictly conserved (shaded red in Fig. 1), and an additional six residues are conserved among 28 sequences (shaded in pink). The strictly conserved residues include three amino acids in the N-terminal domain, one in the hinge domain and 17 in the catalytic domain.

Most of the conserved residues play important roles in either substrate recognition or inter-/intra-domain communication within the AsxRS system. For example, F146, Q157 and E199 in the N-terminal domain interact with the first and second anticodon nucleotides of tRNA in the yeast AspRS-tRNAAsp complex (48). In addition, R238, located at the end of the hinge domain, interacts with the N-terminal domain, the region of motif 1 on its own subunit (AsxRS is a homodimer), and the regions of motifs 2 and 3 on the other subunit (47). Among the 18 conserved residues in the catalytic domain, nine are located in the class defining motif regions and are involved in ATP recognition, tRNA acceptor stem recognition, and inter-/intra-domain interactions (49). Of the remaining nine conserved residues, E279, S299 and Q301, located between motifs 1 and 2, and R478, located between motifs 2 and 3, are involved in the recognition of aspartate or asparagine (46,50); D463 and E471 are involved in Mg2+ binding, which stabilizes the bent conformation of ATP and is an essential first step in the aminoacylation reaction (46,51).

The alignments show that the four eukaryotic AsnRS enzymes contain an N-terminal appended domain with lengths ranging from 108 to 116 amino acids that are very rich in lysine (17-18 residues, 15-16%). Somewhat smaller N-terminal extensions are also present in four eukaryotic AspRSs: lengths range from 34 amino acids in mammalian AspRSs to 80 amino acids in yeast AspRS. The extensions of AspRSs are also rich in lysine (4-15 residues, 9-19%) (52) and one of them has been revealed to from an amphiphilic [alpha] helix by NMR measurement (53). It has been suggested that the N-terminal appended domain of AspRS plays a role in the formation of a high molecular weight multi-synthetase complex, which contains nine ARSs and is believed to be unique to metazoans (54). It seems reasonable to postulate a similar function for the appended N-terminal domains of eukaryotic AsnRSs. It should be pointed out, however, that using standard purification protocols, mammalian AsnRS has been isolated only in a free form, never as part of a multi-synthetase complex (54). It may be that AsnRS associates with a high molecular weight complex only under certain conditions, or that the appended domain has functions other than complex formation, e.g. transfer of aminoacyl-tRNA to EF1[alpha] (55) or interaction with RNA (56-58). Insertion of a domain between motifs 2 and 3 and addition of a C-terminal extension are characteristic of bacterial AspRSs (47), but are not found in AsnRS sequences from any of the three kingdoms.

Distributions of AsxRS and GlxRS orthologs in genomic sequence space

Table 1 summarizes the distributions of orthologs for GluRS, GlnRS, AspRS and AsnRS in various organisms whose genomes have been sequenced completely (the orthologs for Homo sapiens represent cDNAs). The distribution of GlnRS is restricted to Eucarya and [gamma]-Proteobacteria, reflecting its late occurrence in Eucarya and subsequent vectorial transfer to a subgroup of Proteobacteria (see Introduction). Unlike GlnRS, which is absent in Gram-positive bacteria, the AsnRS ortholog is present inmany Gram-positive bacteria including Bacillus subtilis (59), Mycoplasma genitalium (60) and Mycoplasma pneumoniae (61). Similar to GlnRS, the ortholog for AsnRS is absent in archaebacterial genomes with the exception of the hyper-thermophilic archaebacterium, P.horikoshii OT3 (6). In addition, the deeply branching Aquifex aeolicus (62), the Gram-positive Mycobacterium tuberculosis (63), and the [epsis]-subgroup of Proteobacteria Helicobacter pylori (24) all lack the ortholog for AsnRS. The data gathered so far do not permit us to draw any conclusion about the similarity between the evolutionary paths of AsnRS and GlnRS.

Phylogeny of AspRS and AsnRS genes

Phylogenetic trees were inferred from the sequence alignments of the catalytic domains (from 238R to 548P of human AsnRS; Fig. 1) using two distance methods (neighbor-joining and Fitch-Margoliash) and a parsimony method. Because preliminary analyses have revealed that the inclusion of the T.thermophilus AspRS in the calculations resulted in the poor resolution within the clade of prokaryotic AspRS, we excluded the sequence from subsequent analyses. All three protocols produced similar trees with high bootstrap values and were made up of five major sequence clusters (Fig. 2A): bacterial AspRS (B-Asp), eukaryotic AspRS (E-Asp), archaeal AspRS (A-Asp), bacterial AsnRS (B-Asn) and eukaryotic AsnRS (E-Asn). In both the neighbor-joining and parsimony trees, P.horikoshii AsnRS was within the clade of Bacteria, but it was within the clade of T.thermophilus, B.subtilis and Lactobacillus delbrueckii in the Fitch-Margoliash tree (shown by a dotted line in Fig. 2A). Thus, the evolutionary position of P.horikoshii AsnRS, which is the only archaebacterial AsnRS known, was not well resolved using this data set. Other branches were consistent among the three protocols, although there was a minor difference between the parsimony and distance trees with respect to the position of B.subtilis AspRS (data not shown).


Figure 2. (A) Phylogeny based on the alignment for the catalytic domains of 28 AsxRS sequences (Fig. 1). Similar trees were obtained by three independent methods (see text) with some exceptions, including P.horikoshii AsnRS (see text). The solid line represents the tree inferred from the NJ method. The dotted line represents an alternative branching of P.horikoshii AsnRS inferred from the Fitch-Margoliash method. The numbers indicate the percent bootstrap values obtained from Fitch-Margoliash calculation. Scale bar calibrates the number of amino acid substitutions per site. (B) Three simplified topologies for the occurrence of AsnRS. A, Archaea; B, Bacteria; E, Eucarya; Asp, AspRS; Asn, AsnRS.

If AsnRS, like GlnRS, had occurred in Eucarya after their separation from Archaea, and was then transferred to Bacteria, eukaryotic AspRS (E-Asp) and AsnRS (B-Asn and E-Asn) should form a sister group (Fig. 2B, middle). All three inferred trees indicate, however, that eubacterial AspRS (B-Asp), but not eukaryotic AspRS (E-Asp), form sister groups with AsnRS (B-Asn and E-Asn; Fig. 2B left).

That AsnRS and eubacterial AspRS are neighbors was confirmed using the ML method (41,42). Because the number of lineages that can be simultaneously analyzed using the ML method is limited, we divided the sequences into seven groups according to the tree shown in Figure 2A: (i) archaebacterial AspRS; (ii) eukaryotic AspRS; (iii) bacterial AspRS; (iv) eukaryotic AsnRS; (v) P.horikoshii AsnRS; (vi) T.thermophilus, L.delbrueckii and B.subtilis AsnRS; and (vii) other bacterial AsnRS. Within each group, the topology was constrained to match that shown in Figure 2A. The 945 possible topologies derived from the seven lineages were exhaustively searched, and the 10 trees exhibiting the greatest log-likelihood are summarized in Table 2. The topology of the ML tree (tree-1) is identical to that of the NJ tree shown in Figure 2A.

Tree-2 was not significantly different from the ML tree on the basis of the criterion of -1 [delta]Li/SE unit (Table 2; 64), and both trees possess topologies that place AsnRS (B-Asn and E-Asn) and eubacterial AspRS (B-Asp) in the same neighborhood (Fig. 2B, `early model'). Nevertheless, the two trees differ in the position of P.horikoshii AsnRS: in the ML tree, P.horikoshii AsnRS is within the clade of Bacteria, whereas in tree-2, it is on the earliest branch of any AsnRS. Bootstrap pseudoprobability (64) assessing the frequency with which a given tree topology might occur in 10 000 replicates predicted that out of 945 topologies, trees 1 and 2 would occur 72.3% of the time. The remaining trees were well discriminated from the ML tree ([delta]Li/SE values less than -1), and their bootstrap probabilities were very low. Thus, ML analysis strongly supports the notion that AsnRS (B-Asn and E-Asn) and eubacterial AspRS (B-Asp) are neighbors. In contrast, GlnRS and eubacterial GluRS do not appear to be neighbors (21,22). Based on these data, we conclude that AsxRS and GlxRS must have evolved separately along independent paths.

Table 1. Distributions of orthologs for GluRS, GlnRS, AspRS and AsnRS in various genomes
Domain Species orthologsa
GluRS GlnRS AspRS AsnRS
Archaea Methanococcus jannaschii (2) [bull] × [bull] ×
Methanobacterium thermoautotrophicum (3) [bull] × [bull] ×
Archaeoglobus fulgidus (5) [bull] × [bull] ×
Pyrococcus horikoshii (6) [bull] × [bull] [bull]
Eucarya Homo sapiensb [bull] [bull] [bull] [bull]
Saccharomyces cerevisiae (32) [bull] [bull] [bull] [bull]
Bacteria Aquifex aeolicus (62) [bull] × [bull] ×
Synechocystis sp. (72) [bull] × [bull] [bull]
Bacillus subtilis (59) [bull] × [bull] [bull]
Mycoplasma genitalium (60) [bull] × [bull] [bull]
Mycoplasma pneumoniae (61) [bull] × [bull] [bull]
Mycobacterium tuberculosis (63) [bull] × [bull] ×
Borrelia burgdorferi (9) [bull] × [bull] [bull]
Helicobacter pylori (24) [bull] × [bull] ×
Haemophilus influenzae (71) [bull] [bull] [bull] [bull]
Escherichia coli (31) [bull] [bull] [bull] [bull]
a[bull], genomes containing the ortholog; ×, genomes that do not contain the ortholog.
bThe data for human cytoplasmic ARSs are based on cDNAs (DDBJ/EMBL/GenBank accession nos X07466, X76013, J05032, D84273).

How old was the occurrence of AsnRS?

Although a rooted tree can be inferred from an analysis of a set of paralogous ARSs that are believed to have duplicated prior to separation of the three kingdoms, e.g. the tree inferred from the sequences of IleRS and ValRS (16,17), the tree inferred from sequences of class IIb ARSs has to be an unrooted one. This is because we cannot presume that AsnRS (or LysRS) occurred before separation of the three kingdoms; instead, the occurrence of these enzymes is deduced from the analysis, per se. We can discuss the occurrence of AsnRS in the context of the topology described above, however, if we assume that AspRS initially diverged from an ancestral gene yielding bacterial AspRS (B-Asp) and the common ancestor of both archaeal (A-Asp) and eukaryotic AspRS (E-Asp), from which they subsequently diverged. This assumption is acceptable because many rooted trees inferred from the sequences of translation apparatuses have shown the same evolutionary scenario (16,17,65,66).

Given this assumption, the topology obtained in the present study tells us that the occurrence of AsnRS must not have preceded the separation of Eucarya and Archaea, which contrasts with the late occurrence (after separation of Eucarya and Archaea) of GlnRS. Thus, AsnRS must have occurred earlier than GlnRS. Whereas the bacterial GlnRSs found in Proteobacteria are regarded as resulting from vectorial transfer of eukaryotic GlnRS (21), the scarcity of AsnRS orthologs in bacterial genomes must be explained differently. One plausible scenario is that during the course of bacterial evolution, some organisms may have altered their system for decoding asparagine from one using AsnRS to one that is AsnRS independent and uses amidotransferase instead (67).

The current data do not distinguish between the possibility that Archaea possessed AsnRS in the past and then lost it at a later stage in the evolution of the domain-in that case, P.horikoshii AsnRS may represent a remnant of archaeal AsnRS-and the possibility that Archaea never possessed orthodox AsnRS, and P.horikoshii AsnRS is the product of vectorial transfer from a eubacterial genome. In that regard, further information about AsnRS from archaeal genomes should deepen our understanding of the evolution of AsxRS family, and the nature and the evolution of the ancestral genome (68,69)

Table 2. Phylogenetic relationships among AspRS and AsnRS family by the ML analysisa
Tree Topologyb [delta]Lic ± SE [delta]Li/SEd Pie
1 early model (-12641.1)f 0.0 0.556
2 early model -2.9 ± 4.7 -0.61 0.167
3 third model -3.6 ± 3.1 -1.16 0.023
4 late model -3.6 ± 3.1 -1.16 0.048
5 early model -4.6 ± 3.9 -1.18 0.028
6 late model -5.6 ± 5.5 -1.02 0.031
7 third model -5.7 ± 5.5 -1.04 0.011
8 third model -7.7 ± 4.8 -1.60 0.001
9 late model -7.7 ± 4.8 -1.60 0.003
10 early model -10.3 ± 7.5 -1.37 0.044
aOf the 945 topologies, only top 10 are shown.
bSimplified topologies are shown in Figure 2B.
cThe difference of log-likelihood of a given tree from that of the ML tree.
d[delta]Li/SE less than -1 indicates that the tree is significantly different from that of the ML tree (64).
eBootstrap psuedoprobability estimated by 10 000 replications (64).
fLog-likelihood of the ML tree.

ACKNOWLEDGEMENTS

We thank Dr Tetsuo Hashimoto and Prof. Paul Schimmel for providing helpful comments on the manuscript. This work was supported in part by a grant from the Ministry of Education, Science and Culture and by Cubist Pharmaceuticals, Inc. (Cambridge, MA).

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*To whom correspondence should be addressed. Tel: +81 3 3918 0111; Fax: +81 3 5394 3903; Email: kshiba@jfcr.or.jp

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