Identification of a Giardia krr1 Homolog Gene and the Secondarily Anucleolate Condition of Giaridia lamblia
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《分子生物学进展》
* Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China; Graduate School of the Chinese Academy of Sciences, Beijing, China; and Capital University of Medical Sciences, Beijing, China
Correspondence: E-mail: wenjf@mail.kiz.ac.cn.
Abstract
Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.
Key Words: Giardia lamblia ? krr1 gene ? nucleolus ? rRNA-processing ? evolution
Introduction
In eukaryotes, ribosome biosynthesis, a process that entails rDNA transcription, pre-rRNA processing and rRNA assembly with ribosomal proteins, occurs in the specialized subnuclear compartment, the nucleolus. The pre-rRNA processing is a complex process in which a large number of proteins and small nucleolar RNAs (snoRNAs) are involved. These proteins include rRNA-modifying enzymes, endonucleases and exonucleases, RNA helicases, and components of small nucleolar ribonucleoprotein complexes (Kressler, Linder, and de La Cruz 1999). Among them, fibrillarin, nucleolin, and NOP52 have been well characterized (see http://npd.hgu.mrc.ac.uk/compartments/nucleolus.html).
The krr1 gene, which encodes KRR1p located in the nucleolus, was first identified in yeast (Gromadka et al. 1996; Gromadka and Rytka 2000). KRR1p serves as a pre-rRNA processing machinery protein that contributes to the process and synthesis of 18S and 25S rRNA (Gromadka and Rytka 2000; Sasaki, Toh, and Kikuchi 2000). A KRR1p homolog, DBE, was later identified in Drosophila and was revealed as an important protein for the processing of both 18S and 28S rRNA (Chan, Brogna, and O'Kane 2001). Homologous gene sequences were also found in expressed sequence tags from several other eukaryotes: Caenorhabditis elegans, Oryza sativa, and Homo sapiens (Gromadka et al. 1996). All these KRR1p homologs contain a putative K homology (KH) domain, a 70 to 100 amino acid module that was originally identified as a repeating sequence in heterogeneous nuclear ribonucleoprotein K (Siomi et al. 1993). It is an RNA-binding motif that is thought to make direct protein-RNA contacts (Musco et al. 1996). KH domains occur in a wide variety of proteins, and they share the only property of associating with RNA.
Giardia lamblia is one of the most widespread intestinal protozoan parasites. It has long been considered as one of the most primitive extant eukaryotes because of its initially perceived lack of mitochondria and of some other membrane-bounded organelles typical of eukaryotic cells (Gillin, Reiner, and McCaffery 1996) and its early branching position in many molecular phylogenetic trees (Roger 1999; Adam 2001). It seems that this organism has diverged before the acquisition of these cellular organelles and, thus, might provide insightful clues into the early evolution of eukaryotes. However, this opinion has been challenged by several recent studies, such as the discoveries of mitochondrial origin genes (e.g., cpn 60, mtHsp70, IscS, and IscU) (Roger et al. 1998; Tachezy, Sanchez, and Muller 2001; Arisue et al. 2002; Tovar et al. 2003) and the mitochondrial remnant organelle (mitosome) (Tovar et al. 2003). Furthermore, its basal position in phylogenetic trees was argued as a result of a long-branch attraction (LBA) artifact, which leads to their misidentification with distant prokaryotic outgroups (Philippe, Germot, and Moreira 2000).
In previous reports, it was also showed that G. lamblia lacked nucleoli, and no nucleolar skeleton structure was found in its nucleus during investigation of the nuclear matrix.This was also regarded as one of the primitive features of the organism (Li, He, and Chen 1997; Wen and Li 1998; Li 1999; Adam 2001). In practice, it is also hard to find obvious nucleoli in other diplomonads and their close relatives retortmonads, although there is suspicious denser material located against the nuclear envelope in some EM pictures (Brugerolle 1973; Brugerolle, Joyon, and Oktem 1974; Silberman et al. 2002). However, "lacking nucleoli" in Giardia has been more studied and emphasized by some authors as a primitive feature. Here, we use the word "anucleolate," which was first used by Elsdale, Fischberg, and Smith (1958) to feature the mutant of Xenopus that lacks typical nucleoli, to describe the situation of Giardia. In this brief communication, we tried to determine whether the "anucleolate" situation is indeed a primitive feature of the organism by investigating the krr1 homolog gene and its transcription in Giardia and comparing them with those of the other eukaryotes.
Materials and Methods
We first used yeast KRR1 as a query to Blast the G. lamblia genome database (www.mbl.edu/Giardia) and found an open reading frame (ORF) ranging from position 114.922 to position 107.317 in the contig 735. Because some features of the G. lamblia genome can lead to errors in assembled contigs, we designed a pair of primers, which corresponded to the upstream and downstream regions of the ORF, respectively, to verify the ORF sequence through PCR and sequencing. The segment we sequenced is identical to the identified ORF. To examine whether the gene is actively transcribed, we performed a RT-PCR (using freshly prepared total RNA from trophozoites) and then cloned and sequenced the products (fig 1). The sequence we obtained has a poly (A) tail beginning at 37 nt downstream of the stop codon (TAA) and a polyadenylation signal AGTAAA typical to other reported Giardia genes. Thus, these data clearly demonstrated that Giardia has a krr1 gene (GenBank accession number AF541964) homolog, and it is actively transcribed.
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FIG. 1.— PCR and RT-PCR demonstrate that the krr1 gene exists in Giardia genome and is actively transcribed. RT-PCR primers: down stream primer selectively matches the poly (A) tail of mRNA; upstream primer matches the upstream of the krr1 ORF. PCR primers: upstream primer was the same to the upstream primer of the RT-PCR; downstream primer matches the tail of krr1 ORF. M indicates marker. Lane 1, PCR using gDNA of G. lamblia as templates. Lane 2, RT-PCR using total RNA of G. lamblia as templates.
Results and Discussion
Analysis of the deduced protein sequence showed 41.62% identity and 70.96% similarity to yeast KRR1p. It has a basic isoelectric point (9.54) calculated by ProtParam tool (http://us.expasy.org/tools/protparam.html), which is similar to that of yeast KRR1p (9.42). Similar to yeast KRR1p, the Giardia KRR1p contains a conserved KH motif located at 135 aa to 207 aa, which is 73 aa in length with 52% identity to the yeast KRR1p KH domain. ScanProsite program (http://au.expasy.org/tools/scanprosite/) analyses indicated the presence of two bipartial nuclear localization signals (NLS) at regions 244 to 260 (KKKNTKPYKPAKVAKRK) and 245 to 260 (KKNTKPYKPAKVAKRKR), indicating its nuclear localization.
Sequence searches using the Blast program and COGnitor showed that 22 krr1 homologs appeared in 22 eukaryotes ranging from protists, fungi, and plants to metazoa, and two krrl homologs appear in two archaebacteria (Methanococcus jannaschii and Methanopyrus kandleri) (table 1). However, no krr1 homolog was found in eubacteria. Analysis by SMART program (http://smart.embl-heidelberg.de/) revealed that all the eukaryotic KRR1p homologs have a single KH domain, whereas the archaeal homologs both possess two KH domains: one adjacent to the N-terminal, the other adjacent to the C-terminal. The latter is more similar to that of eukaryotic KRR1p homologs except without the KRR motifs located in the 1 helix (fig. 2).
Table 1 KRR1p Homologs Obtained by Searching GenBank
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FIG. 2.— KH domain alignment of STAR proteins with KRR1p homologs. In comparison with STAR protein KH domains, all KRR1p KH domains have a big deletion between the ?2 and ?3 sheets. Different from those of archaebacteria, all eukaryote KRR1p have a unique highly conserved KR(K)R motif (frame), which is located in the 1 helix and carries strong positive charges. The aligned KH domains in the SMART database were used to guide our alignment. The secondary structure is determined according to the crystal structure of 1KHM (PDB entry). ? indicates beta sheet; indicates alpha helix. The accession numbers of the five STAR proteins are as follows: Sam68, NP_006550 [GenBank] ; GRP33, P13230 [GenBank] ; Quaking-1, AAC52491 [GenBank] 1; GLD-1, NP_492143 [GenBank] ; How, AAB51251 [GenBank] 1. The accession numbers of KRR1p homologs and the abbreviations are in table 1.
Proteins with KH domains usually have more than one such domain (between two and 14) (Musco et al. 1996). The only known exception is the STAR subfamily proteins, which have only one KH domain (Vernet and Artzt 1997). We compared the STAR subfamily proteins with our eukaryotic KRR1p homologs and found that sequence similarity between them was limited to the KH domain. Figure 2 shows KH domain alignment of five STAR proteins with our KRR1p homologs. In comparison with the KH domains in STAR protein, the KH domain in KRR1p has a big deletion between the ?2 and ?3 sheets. Moreover, all eukaryotic KRR1p homologs have a unique highly conserved KR(K)R motif, which is located in the 1 helix and carries strong positive charges, whereas STAR proteins do not have such a motif. The unique single-KH domain of all these KRR1p homologs suggests a novel subfamily in the KH domain protein family.
Because KRR1p physically and functionally interacts with a protein Kri1p to form a complex, which is required for 40S ribosome biogenesis in the nucleolus in yeast (Sasaki, Toh, and Kikuchi 2000), we also used Krilp as a query to Blast genomic data available in GenBank. We identified Kri1p homologs in both G. lamblia and other eukaryotic lineages but not in eubacteria or archaebacteria (data not shown).
Basing on the above analyses, we suggest that the anucleolate eukaryote, G. lamblia, has a homolog of yeast KRR1p, which is present in diverse eukaryotes, not in prokaryotes.
Fibrillarin, another important protein involved in pre-rRNA processing and ribosome assembly, is conserved in both eukaryotes and archaebacteria (Tollervey et al. 1991, 1993). It had also been identified in G. lamblia previously (Narcisi, Glover, and Fechheimer 1998). The identified Giardia fibrillarin has a GAR domain (glycine and arginine–rich N-terminal domain), which is found only in eukaryotic fibrillarins and clusters with its eukaryotic homologs in phylogenetic trees (Narcisi, Glover, and Fechheimer 1998; Hickey, Macario, and Conway de Macario 2000).
We next used 207 S. cerevisiae nucleolus-localized proteins (http://mips.gsf.de/) as queries to Blast the G. lamblia genomic database, and 100 potential homologs (E value <1e-05 as cutoff) were identified. Most of them are similar to those involved in pre-rRNA processing and ribosomal assembly (see table 1 in Supplementary Material online).
Conclusion
Overall, our data imply that although G. lamblia lacks nucleolar structure, the mechanism of its pre-rRNA processing and ribosomal assembly should be similar to those of the other eukaryotes and dissimilar to those of prokaryotes. Therefore, we suggest that like the initially perceived "lack of mitochondria," the "lack of nucleolus" is not a primitive feature of G. lamblia but probably arose secondarily. Indeed, in addition to the discovery of mitosome (Tovar et al. 2003), accumulating evidence, such as the discoveries of intron and Golgi in Giardia (Lujan et al. 1995; Dacks and Doolittle 2001; Nixon et al. 2002), has also proved some previously so-called "primitive features" to be shaky. Our present work also implies that the anucleolate feature of other diplomonads and their close relatives retortamonads might arise secondarily. To confirm this, it is necessary to find nucleolar homologs in them.
Acknowledgements
In this work, the database of Giardia lamblia Genome Project (http://jbpc.mbl.edu/Giardia-HTML/), Marine Biological Laboratory at Woods Hole was used. We thank authors of the database. This work was supported by grants (30070362, 30170135, 30021004) from the National Natural Science Foundation of China to J.F.W and a grant (KSCX2-SW-101C) from the Chinese Academy of Sciences to J.F.W.
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Correspondence: E-mail: wenjf@mail.kiz.ac.cn.
Abstract
Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.
Key Words: Giardia lamblia ? krr1 gene ? nucleolus ? rRNA-processing ? evolution
Introduction
In eukaryotes, ribosome biosynthesis, a process that entails rDNA transcription, pre-rRNA processing and rRNA assembly with ribosomal proteins, occurs in the specialized subnuclear compartment, the nucleolus. The pre-rRNA processing is a complex process in which a large number of proteins and small nucleolar RNAs (snoRNAs) are involved. These proteins include rRNA-modifying enzymes, endonucleases and exonucleases, RNA helicases, and components of small nucleolar ribonucleoprotein complexes (Kressler, Linder, and de La Cruz 1999). Among them, fibrillarin, nucleolin, and NOP52 have been well characterized (see http://npd.hgu.mrc.ac.uk/compartments/nucleolus.html).
The krr1 gene, which encodes KRR1p located in the nucleolus, was first identified in yeast (Gromadka et al. 1996; Gromadka and Rytka 2000). KRR1p serves as a pre-rRNA processing machinery protein that contributes to the process and synthesis of 18S and 25S rRNA (Gromadka and Rytka 2000; Sasaki, Toh, and Kikuchi 2000). A KRR1p homolog, DBE, was later identified in Drosophila and was revealed as an important protein for the processing of both 18S and 28S rRNA (Chan, Brogna, and O'Kane 2001). Homologous gene sequences were also found in expressed sequence tags from several other eukaryotes: Caenorhabditis elegans, Oryza sativa, and Homo sapiens (Gromadka et al. 1996). All these KRR1p homologs contain a putative K homology (KH) domain, a 70 to 100 amino acid module that was originally identified as a repeating sequence in heterogeneous nuclear ribonucleoprotein K (Siomi et al. 1993). It is an RNA-binding motif that is thought to make direct protein-RNA contacts (Musco et al. 1996). KH domains occur in a wide variety of proteins, and they share the only property of associating with RNA.
Giardia lamblia is one of the most widespread intestinal protozoan parasites. It has long been considered as one of the most primitive extant eukaryotes because of its initially perceived lack of mitochondria and of some other membrane-bounded organelles typical of eukaryotic cells (Gillin, Reiner, and McCaffery 1996) and its early branching position in many molecular phylogenetic trees (Roger 1999; Adam 2001). It seems that this organism has diverged before the acquisition of these cellular organelles and, thus, might provide insightful clues into the early evolution of eukaryotes. However, this opinion has been challenged by several recent studies, such as the discoveries of mitochondrial origin genes (e.g., cpn 60, mtHsp70, IscS, and IscU) (Roger et al. 1998; Tachezy, Sanchez, and Muller 2001; Arisue et al. 2002; Tovar et al. 2003) and the mitochondrial remnant organelle (mitosome) (Tovar et al. 2003). Furthermore, its basal position in phylogenetic trees was argued as a result of a long-branch attraction (LBA) artifact, which leads to their misidentification with distant prokaryotic outgroups (Philippe, Germot, and Moreira 2000).
In previous reports, it was also showed that G. lamblia lacked nucleoli, and no nucleolar skeleton structure was found in its nucleus during investigation of the nuclear matrix.This was also regarded as one of the primitive features of the organism (Li, He, and Chen 1997; Wen and Li 1998; Li 1999; Adam 2001). In practice, it is also hard to find obvious nucleoli in other diplomonads and their close relatives retortmonads, although there is suspicious denser material located against the nuclear envelope in some EM pictures (Brugerolle 1973; Brugerolle, Joyon, and Oktem 1974; Silberman et al. 2002). However, "lacking nucleoli" in Giardia has been more studied and emphasized by some authors as a primitive feature. Here, we use the word "anucleolate," which was first used by Elsdale, Fischberg, and Smith (1958) to feature the mutant of Xenopus that lacks typical nucleoli, to describe the situation of Giardia. In this brief communication, we tried to determine whether the "anucleolate" situation is indeed a primitive feature of the organism by investigating the krr1 homolog gene and its transcription in Giardia and comparing them with those of the other eukaryotes.
Materials and Methods
We first used yeast KRR1 as a query to Blast the G. lamblia genome database (www.mbl.edu/Giardia) and found an open reading frame (ORF) ranging from position 114.922 to position 107.317 in the contig 735. Because some features of the G. lamblia genome can lead to errors in assembled contigs, we designed a pair of primers, which corresponded to the upstream and downstream regions of the ORF, respectively, to verify the ORF sequence through PCR and sequencing. The segment we sequenced is identical to the identified ORF. To examine whether the gene is actively transcribed, we performed a RT-PCR (using freshly prepared total RNA from trophozoites) and then cloned and sequenced the products (fig 1). The sequence we obtained has a poly (A) tail beginning at 37 nt downstream of the stop codon (TAA) and a polyadenylation signal AGTAAA typical to other reported Giardia genes. Thus, these data clearly demonstrated that Giardia has a krr1 gene (GenBank accession number AF541964) homolog, and it is actively transcribed.
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FIG. 1.— PCR and RT-PCR demonstrate that the krr1 gene exists in Giardia genome and is actively transcribed. RT-PCR primers: down stream primer selectively matches the poly (A) tail of mRNA; upstream primer matches the upstream of the krr1 ORF. PCR primers: upstream primer was the same to the upstream primer of the RT-PCR; downstream primer matches the tail of krr1 ORF. M indicates marker. Lane 1, PCR using gDNA of G. lamblia as templates. Lane 2, RT-PCR using total RNA of G. lamblia as templates.
Results and Discussion
Analysis of the deduced protein sequence showed 41.62% identity and 70.96% similarity to yeast KRR1p. It has a basic isoelectric point (9.54) calculated by ProtParam tool (http://us.expasy.org/tools/protparam.html), which is similar to that of yeast KRR1p (9.42). Similar to yeast KRR1p, the Giardia KRR1p contains a conserved KH motif located at 135 aa to 207 aa, which is 73 aa in length with 52% identity to the yeast KRR1p KH domain. ScanProsite program (http://au.expasy.org/tools/scanprosite/) analyses indicated the presence of two bipartial nuclear localization signals (NLS) at regions 244 to 260 (KKKNTKPYKPAKVAKRK) and 245 to 260 (KKNTKPYKPAKVAKRKR), indicating its nuclear localization.
Sequence searches using the Blast program and COGnitor showed that 22 krr1 homologs appeared in 22 eukaryotes ranging from protists, fungi, and plants to metazoa, and two krrl homologs appear in two archaebacteria (Methanococcus jannaschii and Methanopyrus kandleri) (table 1). However, no krr1 homolog was found in eubacteria. Analysis by SMART program (http://smart.embl-heidelberg.de/) revealed that all the eukaryotic KRR1p homologs have a single KH domain, whereas the archaeal homologs both possess two KH domains: one adjacent to the N-terminal, the other adjacent to the C-terminal. The latter is more similar to that of eukaryotic KRR1p homologs except without the KRR motifs located in the 1 helix (fig. 2).
Table 1 KRR1p Homologs Obtained by Searching GenBank
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FIG. 2.— KH domain alignment of STAR proteins with KRR1p homologs. In comparison with STAR protein KH domains, all KRR1p KH domains have a big deletion between the ?2 and ?3 sheets. Different from those of archaebacteria, all eukaryote KRR1p have a unique highly conserved KR(K)R motif (frame), which is located in the 1 helix and carries strong positive charges. The aligned KH domains in the SMART database were used to guide our alignment. The secondary structure is determined according to the crystal structure of 1KHM (PDB entry). ? indicates beta sheet; indicates alpha helix. The accession numbers of the five STAR proteins are as follows: Sam68, NP_006550 [GenBank] ; GRP33, P13230 [GenBank] ; Quaking-1, AAC52491 [GenBank] 1; GLD-1, NP_492143 [GenBank] ; How, AAB51251 [GenBank] 1. The accession numbers of KRR1p homologs and the abbreviations are in table 1.
Proteins with KH domains usually have more than one such domain (between two and 14) (Musco et al. 1996). The only known exception is the STAR subfamily proteins, which have only one KH domain (Vernet and Artzt 1997). We compared the STAR subfamily proteins with our eukaryotic KRR1p homologs and found that sequence similarity between them was limited to the KH domain. Figure 2 shows KH domain alignment of five STAR proteins with our KRR1p homologs. In comparison with the KH domains in STAR protein, the KH domain in KRR1p has a big deletion between the ?2 and ?3 sheets. Moreover, all eukaryotic KRR1p homologs have a unique highly conserved KR(K)R motif, which is located in the 1 helix and carries strong positive charges, whereas STAR proteins do not have such a motif. The unique single-KH domain of all these KRR1p homologs suggests a novel subfamily in the KH domain protein family.
Because KRR1p physically and functionally interacts with a protein Kri1p to form a complex, which is required for 40S ribosome biogenesis in the nucleolus in yeast (Sasaki, Toh, and Kikuchi 2000), we also used Krilp as a query to Blast genomic data available in GenBank. We identified Kri1p homologs in both G. lamblia and other eukaryotic lineages but not in eubacteria or archaebacteria (data not shown).
Basing on the above analyses, we suggest that the anucleolate eukaryote, G. lamblia, has a homolog of yeast KRR1p, which is present in diverse eukaryotes, not in prokaryotes.
Fibrillarin, another important protein involved in pre-rRNA processing and ribosome assembly, is conserved in both eukaryotes and archaebacteria (Tollervey et al. 1991, 1993). It had also been identified in G. lamblia previously (Narcisi, Glover, and Fechheimer 1998). The identified Giardia fibrillarin has a GAR domain (glycine and arginine–rich N-terminal domain), which is found only in eukaryotic fibrillarins and clusters with its eukaryotic homologs in phylogenetic trees (Narcisi, Glover, and Fechheimer 1998; Hickey, Macario, and Conway de Macario 2000).
We next used 207 S. cerevisiae nucleolus-localized proteins (http://mips.gsf.de/) as queries to Blast the G. lamblia genomic database, and 100 potential homologs (E value <1e-05 as cutoff) were identified. Most of them are similar to those involved in pre-rRNA processing and ribosomal assembly (see table 1 in Supplementary Material online).
Conclusion
Overall, our data imply that although G. lamblia lacks nucleolar structure, the mechanism of its pre-rRNA processing and ribosomal assembly should be similar to those of the other eukaryotes and dissimilar to those of prokaryotes. Therefore, we suggest that like the initially perceived "lack of mitochondria," the "lack of nucleolus" is not a primitive feature of G. lamblia but probably arose secondarily. Indeed, in addition to the discovery of mitosome (Tovar et al. 2003), accumulating evidence, such as the discoveries of intron and Golgi in Giardia (Lujan et al. 1995; Dacks and Doolittle 2001; Nixon et al. 2002), has also proved some previously so-called "primitive features" to be shaky. Our present work also implies that the anucleolate feature of other diplomonads and their close relatives retortamonads might arise secondarily. To confirm this, it is necessary to find nucleolar homologs in them.
Acknowledgements
In this work, the database of Giardia lamblia Genome Project (http://jbpc.mbl.edu/Giardia-HTML/), Marine Biological Laboratory at Woods Hole was used. We thank authors of the database. This work was supported by grants (30070362, 30170135, 30021004) from the National Natural Science Foundation of China to J.F.W and a grant (KSCX2-SW-101C) from the Chinese Academy of Sciences to J.F.W.
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