Department of Microbiology and Parasitology and Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
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Abstract |
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Key Words: Thrips imaginis hemipteroid assemblage mtDNA rRNA gene transcription duplicate control regions
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Introduction |
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Animal mt genomes are generally circular, about 16 kb long and have 37 genes (rrnL and rrnS for large and small rRNA subunits, atp6 and atp8 for ATP synthase subunits 6 and 8, cox1-3 for cytochrome oxidase subunits 1 to 3, cob for cytochrome b, nad1-6 and nad4L for NADH dehydrogenase subunits 1 to 6 and 4L, one tRNA gene for each amino acid [except for leucine and serine, which have two genes]: trnL1 [anticodon sequence nag], trnL2 [yaa], trnS1 [nct] and trnS2 [nga] [Wolstenholme 1992]). With few exceptions, animal mt genomes also have a large noncoding region, the control region, which, based on studies of flies, humans, bovines, mice, frogs, and chickens, contains elements that control the transcription and/or replication of mt genomes (Goddard and Wolstenholme 1980; Tracy and Stern 1995; Taanman 1999). The arrangements of genes are usually the same or very similar within a phylum but differ substantially among phyla of animals (Boore and Brown 1998).
Much is known about the arrangement of genes in the mt genomes of hexapods; only vertebrates have been studied more. Entire sequences of mt genome are available for 17 hexapods from eight orders; parts of the mt genomes of many other hexapods have also been sequenced. All the hexapods studied, except those from three of the four orders of hemipteroid insects, have an identical arrangement of protein-coding genes and rRNA genes and similar arrangements of tRNA genes. The gene arrangement of the fruit fly Drosophila yakuba (Clary and Wolstenholme 1985) is inferred to be ancestral for hexapods. This inference is based on the following evidence: (1) Eleven of the 17 hexapods that have been sequenced for the entire mt genome have this arrangement, and five of the other six hexapods differ from D. yakuba by the position of only one to eight tRNA genes (these hexapods and many other animals are listed on Boore's web page http://www.jgi.doe.gov/programs/comparative/). (2) Two crustaceans have this arrangement (Crease 1999; Wilson, Ballment, and Benzie 2000). (3) Two chelicerates differ from D. yakuba by only the position of trnL2 (Staton, Daehler, and Brown 1997; Black and Roehrdanz 1998).
In contrast to other hexapods, all species studied from three of the four hemipteroid orders, Phthiraptera (lice), Psocoptera (psocids, book lice and bark lice), and Thysanoptera (thrips) have rearrangements of protein-coding genes and tRNA genes relative to the putative ancestral arrangement (Shao, Campbell, and Barker 2001; Shao et al. 2001). Moreover, the gene arrangements differ among these orders (Shao et al. 2001). However, all species from the fourth hemipteroid order, the Hemiptera (bugs, cicadas, aphids, and kin), have the putative ancestral gene arrangement of hexapods (Dotson and Beard 2001; Shao et al. 2001).
The abundance of gene rearrangements in the Phthiraptera, Psocoptera, and Thysanoptera, and the ancestral gene arrangement in the Hemiptera, make the hemipteroid assemblage an ideal group for studies of the evolution of animal mt genomes. Entire mtDNA sequences are desirable for such studies. However, only two hemipteroid insects, Heterodoxus macropus (Phthiraptera) (Shao, Campbell, and Barker 2001) and Triatoma dimidiata (Hemiptera) (Dotson and Beard 2001) have been sequenced for entire mt genome. For the other two hemipteroid orders, Thysanoptera and Psocoptera, only partial mtDNA sequences are available (Shao et al. 2001).
Here, we present the entire nucleotide sequence of the mt genome of the plague thrips, Thrips imaginis (Thysanoptera). This genome is highly rearranged and has many unusual features. T. imaginis is the first known species of arthropod which has inverted and distantly separated rRNA genes. Further, T. imaginis is the first known species of animal which has both duplicate control regions and rearrangements of most mt genes. We discuss the possible mechanisms of rRNA gene transcription, evolution of duplicate control regions, and gene rearrangements in T. imaginis. We evaluate the phylogenetic value of two novel gene boundaries shared by T. imaginis (Thysanoptera) and a lepidopsocid species (Psocoptera). Finally, we discuss the potential of the hemipteroid assemblage as a model system for studies of the evolution of animal mt genomes and outline some fundamental questions, which may be addressed with this system.
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Materials and Methods |
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Analysis of Nucleotide Sequence
Nucleotide sequences were aligned with SequencherTM (Gene Codes Corporation). We identified tRNA genes with tRNAscan-SE (Lowe and Eddy 1997) or by eye. BLAST searches (Altschul et al. 1997) identified rRNA and protein-coding genes. Hydrophilicity profile comparisons (Hopp and Woods 1981) (MacVectorTM) were also used to identify or confirm protein-coding genes.
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Results |
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Protein-Coding Genes
BLAST searches failed to identify atp8, nad4L, and nad6; these three genes were identified by comparisons of the hydrophilicity profiles of their putative proteins with those of D. yakuba and Homo sapiens (Anderson et al. 1981; Clary and Wolstenholme 1985) (fig. 2). Among the 3,661 codons of the 13 protein-coding genes, 1,607 (44%) are A+T-rich codons (those with A or T at the first and second codon positions, but termination codons and those coding for leucines were excluded), whereas only 448 (12%) are G+C-rich codons (those with G or C at the first and second codon positions (Foster, Jermiin, and Hickey 1997) (table 1). Further, 3,186 (87%) codons have A or T at the third codon position, whereas only 475 (13%) codons have G or C at the third codon position (table 2). ATA and ATT apparently initiate the translations of eight and four genes, respectively. Comparisons of hydrophilicity profiles and amino acid sequences of nad4 between T. imaginis and D. yakuba suggest that ATAA probably initiates the translation of nad4 in T. imaginis, as is the case for cox1 of D. melanogaster (de Bruijn 1983), D. yakuba (Clary and Wolstenholme 1985), and Daphnia pulex (Crease 1999). Four genes apparently have incomplete termination codons: TA for nad4 and nad5 and T for atp8 and nad2. TAG apparently terminates the translation of nad4L, and TAA terminates the translations of the other eight genes.
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Noncoding Regions
There are apparently 1,136 noncoding nucleotides in the mt genome of T. imaginis: 236 bp in 16 intergenic regions and 900 bp in two large noncoding regions. The two large noncoding regions are 440 bp and 460 bp long, and they have 437 bp in common (fig. 4). We propose that these two regions are the control regions since these regions have four features in common with the control regions of other insects (Zhang and Hewitt 1997): (1) tandem repeats, (2) a T-stretch, (3) an A+T-rich (84%) segment, and (4) a stem-loop (figs. 4 and 5).
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Discussion |
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Remarkably little is known about the transcription of rRNA genes in arthropods. However, in all arthropods studied, except T. imaginis, the two rRNA genes are arranged in a similar way as in H. sapiens: the two rRNA genes are close to one another and rrnS is adjacent to the control region. Therefore, it is likely that the arthropods studied, other than T. imaginis, have the same or similar mechanism of rRNA gene transcription as H. sapiens. In T. imaginis, however, the two rRNA genes have inverted and are distant from one another. Moreover, both rRNA genes are distant from the putative control regions. This raises a question: How are rRNA genes transcribed in T. imaginis? It appears that the most efficient transcription of rRNA genes occurs when the promoter and termination element are adjacent to the rRNA genes, as in H. sapiens. If this is the case for T. imaginis, then there may be two sets of promoter and termination elements for rRNA gene transcription in T. imaginis. Further, the promoters would more likely be in the genes upstream of rRNA genes than in the control regions. Arrangements of rRNA genes similar to that of T. imaginis also occur in some mollusks and nematodes (see Boore's web page). Biochemical studies, such as mapping experiments (Montoya et al. 1982) and mutagenesis experiments (Shadel and Clayton 1993), are needed to elucidate the mechanism of rRNA gene transcription in these organisms.
Duplicate Control Regions and Gene Rearrangements
Duplicate control regions have been found in ticks (Black and Roehrdanz 1998; Campbell and Barker 1998, 1999), sea cucumbers (Arndt and Smith 1998), a fish (Lee et al. 2001), parrots (Eberhard, Wright, and Bermingham 2001), and snakes (Kumazawa et al. 1996, 1998). T. imaginis is the first known species of hexapod which has duplicate control regions and, further, the first species of animal which has both duplicate control regions and rearrangements of the majority of mt genes.
Kumazawa et al. (1998) proposed two models for the evolution of duplicate control regions: (1) gene conversion and (2) tandem duplication and deletion. Southern hybridization experiments on snakes supported the tandem duplication and deletion model but did not exclude the possibility of gene conversion (Kumazawa et al. 1998). Tandem duplication and deletion is also the most plausible model for gene translocations in animal mt genomes (Boore 2000). In fact, except for the fish, all the animals which have duplicate control regions have also had translocations of genes. The duplicate control regions and gene translocations of the above animals, except T. imaginis, can be explained by a single tandem duplication followed by deletions of one copy of the duplicate genes, but retention of both the control regions.
Tandem duplication and deletion is also a plausible model for the translocations of genes and the evolution of duplicate control regions in T. imaginis, although the possibility of gene conversion cannot be excluded. Tandem duplication and deletion may have occurred a number of times in the lineage leading to T. imaginis since the number of genes translocated in T. imaginis (24 genes) is much higher than in ticks (two tRNA genes plus a block of 10 genes), sea cucumbers (six tRNA genes), parrots (a block of three genes), and snakes (one tRNA gene). We noticed that in T. imaginis, all the 18 genes between control regions #1 and #2 have translocated and/or inverted, whereas only six of the 20 genes outside this region have translocated and/or inverted (fig. 6). Further, the extra gene, trnS3, and the two pseudo-tRNA genes are either in this section or immediately adjacent to one of the control regions. These observations suggest that duplicate control regions may facilitate the tandem duplication of the genes between them.
Intramitochondrial recombination (Dowton and Austin 1999; Dowton and Campbell 2001) may account for the gene inversions in the mt genome of T. imaginis. All the eight genes which have inverted in T. imaginis are on the minority-strand of the mt genome of the putative ancestral hexapod (fig. 6). Five of these genes, nad1, trnL1, rrnL, trn,V and rrnS, probably inverted together since theses genes are in one block in the ancestral arrangement.
Convergence of Two Novel Gene Boundaries
The interordinal phylogeny of the hemipteroid assemblage is still an unresolved trichotomy (Kristensen 1991) (fig. 7a). One of the aims of our study was to see if mt gene rearrangements could shed light on this trichotomy. It is generally accepted that the hemipteroid assemblage, Phthiraptera, Psocodea (Phthiraptera + Psocoptera), Thysanoptera, and Hemiptera are monophyletic (Boudreaux 1979, pp. 139143; Kristensen 1991; Wheeler et al. 2001), whereas the Psocoptera may be paraphyletic (Lyal 1985).
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Further, we noticed that T. imaginis also shares cox3-trnR and another novel gene boundary, cob-trnY, with the hermit crab, Pagurus longicarpus (Crustacea) (Hickerson and Cunningham 2000). It is almost certain that cox3-trnR and cob-trnY have evolved independently in T. imaginis and P. longicarpus. Based on the above evidence, we propose that the two novel gene boundaries shared by T. imaginis and the lepidopsocid species evolved by convergence and thus are not informative for the resolution of interordinal phylogeny of the hemipteroid assemblage.
Hemipteroid Assemblage As a Model System
The hemipteroid assemblage is a good model system for studies of the evolution of animal mt genomes for two reasons. First, various gene rearrangements have occurred in the hemipteroid assemblage: translocations and inversions of tRNA genes, rRNA genes and protein-coding genes, evolution of duplicate control regions, and evolution of extra tRNA gene and pseudogenes. Second, there is much variation in the rate of gene rearrangement among the four orders of hemipteroid insects. The number of genes rearranged ranges from zero in T. dimidiata (Hemiptera) (Dotson and Beard 2001) to eight in the lepidopsocid species (Psocoptera) (Shao et al. 2001) to 24 in T. imaginis (Thysanoptera) and to 31 in H. macropus (Phthiraptera) (Shao, Campbell, and Barker 2001).
Questions which may be addressed with the hemipteroid assemblage include (1) Why does the rate of gene rearrangement vary so much among closely related lineages? (2) What are the mechanisms of gene rearrangement? (3) How are the rRNA genes which are distant from one another and from the control region transcribed? (4) How do the mt genomes which have duplicate control regions replicate? (5) Is the rate of gene rearrangement correlated with the rate of nucleotide substitution? (6) What types of gene arrangements are more reliable as phylogenetic markers?
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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E-mail: r.shao{at}mailbox.uq.edu.au.
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