Department of Microbiology and Parasitology, and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
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Abstract |
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Introduction |
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Complete mt gene arrangements of over 100 animal species have been determined, and partial arrangements are known for several hundred species (Boore 1999
; see "Mitochondrial Gene Arrangement Source Guide," version 5.0, on Boore's web page). The arrangement of genes in the mt genome, particularly the arrangement of the large genes that encode proteins and rRNAs, is generally conserved in an animal phylum but varies substantially among phyla (Boore et al. 1995
; Boore and Brown 1998
). However, some exceptions have been found so far: (1) the ascidian Halocynthia roretxi (Chordata, Urochordata) has a very different gene arrangement from those of other chordates (Yokobori et al. 1999
); (2) two brachiopods, Terebratulina retusa and Laqueus rubellus, have very different gene arrangements from each other (Stechmann and Schlegel 1999
; Noguchi et al. 2000
); (3) species from four different classes of the Mollusca are very different in gene arrangement (Boore 1999
); and (4) species from the same class (Secernentea) of the Nematoda are very different in gene arrangement (Boore 1999
).
The gene content of mt genomes may also vary, since (1) a homolog of the bacterial muts gene, which is involved in mismatch repair, was found in the mt genome of the soft coral Sarcophyton glaucum (Pont-Kingdon et al. 1998
); (2) an extra trnM, but no atp8, was found in the mollusk Mytilus edulis (Hoffmann, Boore, and Brown 1992
); (3) atp8 is absent in some flatworms (Le et al. 2000; Fukunaga, unpublished but available in GenBank, accession number AB018440), nematodes (Okimoto et al. 1991, 1992
; Keddie, Higazi, and Unnasch 1998
), and the ascidian H. roretxi (Chordata, Urochordata; Yokobori et al. 1999
); and (4) some cnidarians lack all but one or two tRNA genes (Beagley, Okimoto, and Wolstenholme 1998
; Beaten, Roger, and Cavalier-Smith 1998
).
More is known about the arrangement of mt genes for the class Insecta than for any other invertebrates. The mt genomes of 12 insects have been completely sequenced. These include (1) seven flies (Diptera): Drosophila yakuba (Clary and Wolstenholme 1985
), Drosophila melanogaster (Lewis, Farr, and Kaguni 1995
), Drosophila mauritiana, Drosophila sechellia, Drosophila simulans (last three in Ballard 2000
), Ceratitis capitata (Spanos et al. 2000)
, and Cochliomyia hominivorax (Lessinger et al. 2000)
; (2) two mosquitoes (Diptera): Anopheles quadrimaculatus (Mitchell, Cockburn, and Seawright 1993
) and Anopheles gambiae (Beard, Hamm, and Collins 1993
); (3) the domestic silkworm (Lepidoptera) Bombyx mori (Lee et al., unpublished but available in GenBank, accession number AF149768); (4) the honeybee (Hymenoptera) Apis mellifera (Crozier and Crozier 1993
); and (5) the locust (Orthoptera) Locusta migratoria (Flook, Rowell, and Gellissen 1995
). Moreover, partial gene arrangements of over 300 species of insects are known. Full or partial mt gene arrangements are thus available from 10 orders of insects, but the vast majority of the species studied are from three orders: the Diptera, the Lepidoptera, and the Hymenoptera, which are all Endopterygota (flies, fleas, beetles, moths, wasps, etc.).
All the insects studied so far have the same arrangement of protein-coding genes, rRNA genes, and most tRNA genes. Only the positions of a few tRNA genes vary, particularly the positions of those near the control region and in the tRNA gene cluster trnA-trnR-trnN-trnS1-trnE-trnF (genes underlined are encoded by the minority strand). The most common arrangement of mt genes of insects studied, which is present in D. yakuba and many other species, is inferred to be ancestral for insects (Boore, Lavrov, and Brown 1998
). In contrast to the Endopterygota, little is known about the arrangements of genes in many other lineages of insects, such as the Apterygota, the Palaeoptera, the hemipteroid assemblage, etc. The hemipteroid assemblage (bugs, thrips, psocopterans, and lice) is particularly interesting because it is thought to be the sister group of the Endopterygota (Kristensen 1991
). Oncopeltus fasciatus, a milkweed bug (Hemiptera), is the only species so far from the hemipteroid assemblage for which we have any information on the arrangement of mt genes. This species has the arrangement of trnL2-?-cox2-?-trnK, which is the same as the ancestral arrangement of insects (Liu and Beckenbach 1992
; "?" indicates that the amplifying primers in this study annealed in the two tRNA genes, but the sequence data reported are not sufficient to verify that there are no other tRNA genes between these tRNA genes and cox2).
Here, we report the complete mt genome of the wallaby louse, Heterodoxus macropus (Phthiraptera, Amblycera, Boopiidae). Heterodoxus macropus infests agile wallabies, Macropus agilis, throughout almost all of the geographic range of this marsupial in Australia (unpublished data). Relative to the ancestral gene arrangement of insects, numerous rearrangements of protein-coding and tRNA genes have occurred in the mt genome of this louse. This is the first species from the hemipteroid assemblage for which the entire mt genome has been sequenced, and this paper represents the first report of rearrangements of protein-coding genes in the mt genome of an insect. The number of gene rearrangements required to account for the arrangement of genes in this species is unprecedented for an arthropod.
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Materials and Methods |
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The protein-coding and rRNA genes were identified by BLAST searches (Altschul et al. 1990
) of GenBank. The tRNA genes were found with the program tRNAscan-SE (Lowe and Eddy 1997
) and by eye from the anticodons and inferred secondary structures of tRNAs.
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Results and Discussion |
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For the BLAST searches, the closest matches to the putative genes atp6, cox1cox3, cob, nad2, rrnL, nad1 and nad6 of H. macropus were the corresponding genes of other insects. The closest matches to the putative genes atp8, nad3nad5, nad4L, and rrnS of H. macropus were the corresponding genes of noninsect arthropods or nonarthropods. We identified trnS1 by eye and other tRNA genes by the program tRNAscan-SE search. Thus, all 37 genes were identified putatively. The tRNA genes were 6369 bp long. The putative sizes of the protein-coding and rRNA genes are as follows: atp6, 657 bp; atp8, 162 bp; cox1, 1,539 bp; cox2, 660 bp; cox3, 792 bp; cob, 1,101 bp; nad1, 894 bp; nad2, 897 bp; nad3, 327 bp; nad4, 1,347 bp; nad4L, 273 bp; nad5, 1,674 bp; nad6, 588 bp; rrnL, 1,170 bp; rrnS, 776 bp. The total size of the 37 genes is 14,299 bp. The majority strand encodes 19 genesseven protein-coding genes and 12 tRNA genes, 7,263 bp in total; the minority strand encodes 18 genessix protein-coding genes, two rRNA genes, and 10 tRNA genes, 7,036 bp in total.
Six pairs of adjacent genes apparently overlap in the mt genome of H. macropus: (1) 4 bp between cox1 and trnC, (2) 6 bp between trnL2 and nad4, (3) 2 bp between nad4L and nad2, (4) 4 bp between trnN and trnA, (5) 4 bp between rrnS and atp6, and (6) 1 bp between atp6 and atp8. For the first four pairs of genes, the overlapping genes are encoded by different strands; for the last two pairs, the overlapping genes are encoded by the same strand.
Nucleotide Composition and Codon Usage
The nucleotide composition of the majority strand of the mt genome of H. macropus is as follows: A, 5,680, 38.7%; T, 5,950, 40.6%; C, 1,539, 10.5% G, 1,501, 10.2%. The A+T-richness of the mt genome of H. macropus is reflected further in the codon usage. There is a strong bias against C+G (A, 44.6%; T, 46.6%; C, 4.2%; G, 5.1%) at the third codon positions of the protein-coding genes (table 2
), as observed in the mt genomes of other insects (Crease 1999
). Six A+T-rich codons (encoding amino acids Asn, Ile, Lys, Met, Phe, and Tyr) appear 1,558 times (42.8%), whereas four C+G-rich codons (encoding amino acids Ala, Arg, Gly, and Pro) are present 398 times (10.9%) (table 3
). The overall ratio of C+G-rich codons to A+T-rich codons is 0.26. All codons are present in the protein-coding genes of this genome except for CGC and CCG. Four different codons apparently initiate translation of protein-coding genes: ATT for nad3 and nad4, ATC for cob and nad6, ATG for cox1 and cox3, and ATA for the other seven genes. Nine protein-coding genes have complete termination codons: TAG for cox3 and TAA for cox2, cob, nad1nad3, nad4L, nad5, and nad6. The other four genes, cox1, nad4, atp6, and atp8, could have the complete termination codon TAA if we accept gene overlaps 1, 2, 5, and 6 described above.
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Noncoding Regions and the Control of Gene Transcription and DNA Replication
There are 392 bp unassigned to genes in the mt genome of H. macropus. The largest noncoding region is 73 bp long. It is between atp8 and trnQ and has 93% A+T. The second-largest noncoding region is 47 bp long. It is between cox2 and nad3 and has 92% A+T. Both of these regions seem to be able to form stem-loop structures (fig. 3
). Besides these two regions, there are 272 bp unassigned to genes, scattered in short runs (132 bp) between neighbor genes. In contrast to H. macropus, most animal species studied so far have a single large noncoding region in their mt genomes, which is thought to control gene transcription and DNA replication (Clayton 1982, 1991
). Thus, it is called the control region, or the "A+T-rich region," in insects. This region varies from about 350 bp in some butterflies (Lepidoptera) (Taylor et al. 1993
) to 13,000 bp in some weevils (Coleoptera) (Boyce, Zwick, and Aquadro 1989
). In insects, conserved stem-loop structures have been found in the control regions of fruit flies (Diptera) and locusts (Orthoptera) (Zhang and Hewitt 1997
). In mammals, initiation of DNA replication of the minority strand (commonly called the light strand in vertebrates) occurs in a small (
30-bp) noncoding region which has the potential to form a stem-loop structure (Shadel and Clayton 1997
). Macey et al. (1997)
proposed that nucleotide mutations to the stem-loop region in vertebrates cause errors during the replication of the minority strand, and these errors might lead to rearrangements of mt genes. Lavrov, Boore, and Brown (2000)
suggested that the stem-loop structures in the noncoding regions of the horseshoe crab Limulus polyphemus and the prostriate tick Ixodes hexagonus might correspond to the origin of DNA replication. They also suggested that the change of position of this origin might have caused gene rearrangements in the metastriate ticks Rhipicephalus sanguineus and Boophilus microplus (see also Campbell and Barker 1998, 1999
).
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Gene Rearrangements
Compared with the ancestral gene arrangement of insects, numerous gene rearrangements have apparently occurred in the mt genome of H. macropus (fig. 1
). All 22 of the tRNA genes have moved and/or inverted relative to their ancestral positions. Of the 13 protein-coding genes and two rRNA genes, only cox1, cox3, nad4, nad4L, rrnL, and rrnS are in the same positions relative to each other in H. macropus and the ancestral insect. If we assume that these six genes are in their ancestral positions, then the other nine protein-coding genes (atp6, atp8, cox2, cob, nad1nad3, nad5, and nad6) have moved. Moreover, four of these genes (atp6, atp8, nad1, and nad3) have inverted.
With a few notable exceptions, the gene arrangement in the mt genome is very conserved within closely related groups of animals. Before our study, the class Insecta typified this conservatism, since rearrangements of the protein-coding and rRNA genes had not been found in insects despite the fact that the Insecta were the most studied group of invertebrates.
The number of gene rearrangements implied by the gene arrangement in the mt genome of H. macropus indicates that the rate of change in the arrangement of mt genes may vary substantially among closely related lineages. Arthropods evolved more than 600 MYA (Bergström 1979
). The inferred ancestral arrangement of mt genes of arthropods is the same as that of insects except for the position of one tRNA gene (Staton, Daehler, and Brown 1997
; Boore, Lavrov, and Brown 1998
). Thus, in the lineage leading to D. yakuba, there has been only one change in the arrangement of mt genes in more than 600 Myr. However, in the lineage leading to H. macropus, most genes have been rearranged over the course of about 300 Myr since it diverged from the lineage that leads to D. yakuba (Kukalová-Peck 1991
).
Interestingly, the milkweed bug, O. fasciatus (Hemiptera), has the arrangement trnL2-?-cox2-?-trnK (Liu and Beckenbach 1992
), which is the same as the ancestral arrangement of insects. Thus, the gene rearrangements in the mt genome of H. macropus might have occurred after the lineage leading to lice (Phthiraptera) and the lineage leading to bugs (Hemiptera) diverged about 280 MYA (Kukalová-Peck 1991
). We are studying a variety of hemipteroid insects to determine the phylogenetic extent of the various gene rearrangements. We are also studying other species of lice to see when the gene rearrangements in the mt genome of H. macropus evolved and whether any intermediate states can be found. Doubtless, informative rearrangements would improve our understanding of the phylogeny and evolution of lice and other hemipteroid insects.
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Acknowledgements |
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Footnotes |
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1 Keywords: Heterodoxus macropus,
insect
hemipteroid assemblage
Paraneoptera
mtDNA
gene order
2 Address for correspondence and reprints: Renfu Shao, Department of Microbiology and Parasitology, University of Queensland, Brisbane, Queensland 4072, Australia. r.shao{at}mailbox.uq.edu.au
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