MBE Advance Access originally published online on March 22, 2007
Molecular Biology and Evolution 2007 24(6):1330-1339; doi:10.1093/molbev/msm054
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Research Articles |
The Mitochondrial Genome of the Lizard Calotes versicolor and a Novel Gene Inversion in South Asian Draconine Agamids
* Graduate School of Science, Nagoya University, Nagoya, Japan
Zoology Department, Faculty of Science, Cairo University, Giza, Egypt
E-mail: yasser92us{at}yahoo.com.
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Abstract |
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A complete mitochondrial DNA (mtDNA) sequence was determined for the lizard Calotes versicolor (Reptilia; Agamidae). The 16,670-bp genome with notable shorter genes for some protein-coding and tRNA genes had the same gene content as that found in other vertebrates. However, a novel gene arrangement was found in which the proline tRNA (trnP) gene is located in the light strand instead of its typical heavy-strand position, providing the first known example of gene inversion in vertebrate mtDNAs. A segment of mtDNA encompassing the trnP gene and its flanking genes and the control region was amplified and sequenced for various agamid taxa to investigate timing and mechanism of the gene inversion. The inverted trnP gene organization was shared by all South Asian draconine agamids examined but by none of the other Asian and African agamids. Phylogenetic analyses including clock-free Bayesian analyses for divergence time estimation suggested a single occurrence of the gene inversion on a lineage leading to the draconine agamids during the Paleogene period. This gene inversion could not be explained by the tandem duplication/random loss model for mitochondrial gene rearrangements. Our available sequence data did not provide evidence for remolding of the trnP gene by an anticodon switch in a duplicated tRNA gene. Based on results of sequence comparisons and other circumstantial evidence, we hypothesize that inversion of the trnP gene was originally mediated by a homologous DNA recombination and that the de novo gene organization that does not disrupt expression of mitochondrial genes has been maintained in draconine mtDNAs for such a long period of time.
Key Words: Calotes versicolor • Agamidae • mitochondrial genome • gene inversion • homologous DNA recombination • gene rearrangement
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Introduction |
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The typical vertebrate mitochondrial genome (mtDNA) is approximately 16–18 kbp in size, containing 37 genes for 13 proteins, 2 rRNAs and 22 tRNAs, as well as a major noncoding region or control region (CR) including sites for the initiation of transcription and replication (Anderson et al. 1981). Complete or nearly complete mtDNA sequences have been reported from several hundreds of vertebrate taxa dominated by placental mammals and fishes (see, e.g., Jameson et al. 2003
). Gene organization of animal mtDNAs occasionally changes, and such gene rearrangements could provide some phylogenetic information. Gene rearrangements so far reported for vertebrate mtDNAs are most typically, but not exclusively, local rearrangements among clustering tRNA and protein genes, as well as tandem duplication of a continuous mtDNA segment (see, e.g., Moritz et al. 1987; Stanton et al. 1994; Macey et al. 1997; Mindell et al. 1998; Inoue et al. 2001; Kumazawa and Endo 2004; Mueller and Boore 2005; Kurabayashi et al. 2006; San Mauro et al. 2006). However, none of these cases accompany a gene inversion in which a gene switched its encoded strand. On the other hand, the gene inversion is common in invertebrate mitochondrial genomes (Boore 1999), suggesting some unknown difference in evolutionary constraints in gene rearrangements between vertebrate and invertebrate mtDNAs.
Agamidae, one of lizard families, is distributed in tropical and subtropical regions of Eurasia, Africa, and Australasia (Pough et al. 2004, p. 122–125). Based on molecular and morphological data, Macey et al. (2000) grouped the Agamidae into 6 subfamilies: Uromastycinae (genus Uromastyx distributed in Africa–South Asia), Leiolepidinae (Leiolepis in Southeast Asia), Amphibolurinae (Australian–New Guinean clade plus Southeast Asian Physignathus cocincinus), Hydrosaurinae (Hydrosaurus in Indonesia and Philippine), Agaminae (African–West Asian clade), and Draconinae (South or Southeast Asian clade). Monophyly of each agamid subfamily has been corroborated by some morphological (Moody 1987) and molecular (e.g., Honda et al. 2000; Macey et al. 2000; Amer and Kumazawa 2005a) studies.
Complete or nearly complete mtDNA sequences have been reported from relatively fewer taxa of lizards (Kumazawa and Nishida 1999; Janke et al. 2001; Kumazawa 2004, 2007; Kumazawa and Endo 2004; Amer and Kumazawa 2005b; Macey et al. 2005, 2006; Zhou et al. 2006), among which 2 are from Agamidae: the amphibolurine Pogona vitticepes (Amer and Kumazawa 2005b) and the agamine Xenagama taylori (Macey et al. 2006). Because some novel gene rearrangements have been described in agamid mtDNAs (Macey et al. 1997, 2006; Amer and Kumazawa 2005b), agamid mtDNAs may represent one of the most frequently rearranged mitochondrial genomes among vertebrates. It thus would be intriguing to explore more gene rearrangements in diverse agamid lineages for understanding molecular evolutionary processes and mechanisms of gene rearrangements in animal mtDNAs.
Here, we report the complete mtDNA sequence of Calotes versicolor representing the 3rd agamid subfamily Draconinae. This genome has a novel gene order regarding the proline tRNA gene (trnP). Unlike mtDNAs from any other vertebrates studied so far, the C. versicolor mtDNA had a trnP gene encoded by the heavy strand. This finding prompted us to examine the occurrence of a similar gene arrangement in other South Asian agamids. By estimating reliable phylogenetic relationships and divergence times, we discuss the timing and possible mechanism of the gene inversion, which should contribute to better understanding of molecular evolutionary mechanisms operating on vertebrate mitochondrial genomes.
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Material and Methods |
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TOPAbstractIntroductionMaterial and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Sequencing and Gene Identification
Total DNA was extracted from C. versicolor muscle tissues and used as a template to amplify and sequence parts of the genes for cytochrome b (cob) and reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (nad2) with conserved primers described by Kumazawa and Endo (2004)
. These sequences were used for designing taxon-specific primers (see supplementary table S1 [Supplementary Material online] for their sequences and locations) for the long and accurate (LA)–polymerase chain reaction (PCR) amplifications of spacing regions between them. The amplified products (5 and 10 kb in length) were purified by an agarose gel electrophoresis and used as templates for nested PCRs (product length: 0.5–1 kb) that were conducted with a set of conserved reptile-oriented primers (Kumazawa and Endo 2004) and new taxon-specific primers (see supplementary table S1, Supplementary Material online). Amplified fragments by the nested PCR were directly sequenced for both strands with the amplification primers or appropriate internal primers, which were then assembled into a contiguous mtDNA sequence. For typical conditions of the LA-PCR, the nested PCR, and the sequencing reaction, see Amer and Kumazawa (2005b). The C. versicolor mtDNA sequence has been deposited in the DDBJ/EMBL/GenBank databases with an accession number AB183287. Individual genes and features in it (see supplementary table S2, Supplementary Material online) were identified in light of their counterparts from other vertebrates as described by Amer and Kumazawa (2005b).
Characterization of the trnP-Containing Region in Other Agamids
An mtDNA fragment encompassing the trnP gene and parts of the surrounding genes and the CR was successfully amplified for various agamid taxa from South or Southeast Asia (Acanthosaura capra, Acanthosaura crucigera, Calotes cf. calotes, Calotes cf. emma, Draco sp., Gonocephalus grandis, Hydrosaurus cf. weberi, Leiolepis cf. reevesi, and Physignathus cocincinus) and West Asia–Africa (Agama impalearis, and Uromastyx benti) using primers rcytb-1L and r12S-1H (Kumazawa and Endo 2004) (see supplementary table S3 [Supplementary Material online] for their sequences and figure 1 for their locations and orientations). The amplified products being approximately 2 kbp were then sequenced with the above-mentioned primers and newly designed taxon-specific internal primers (see supplementary table S3, Supplementary Material online). These sequences have been deposited in the DDBJ/EMBL/GenBank databases with accession numbers shown in table 1.
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A quick PCR survey was also conducted for the presence/absence of the trnP gene inversion in more agamid taxa available in our laboratory. Primers rGlu-1L and rPro-1L (see supplementary table S3, Supplementary Material online) were used for this assay. PCR products being approximately 1.2 kbp would be obtained if the inversion exists in the corresponding mtDNA (see fig. 1A).
Phylogenetic Analyses
Nucleotide sequences of an mtDNA segment between nad1 and cytochrome oxidase subunit I (cox1) genes for various agamid taxa were collected from the database (see supplementary table S4 [Supplementary Material online] for accession numbers) and used for phylogenetic analyses after unalignable and gap-containing sites were deleted (1,490 bp in total). The aligned nucleotide sequences can be obtained from S.A.M.A. upon request. The phylogenetic analyses were done primarily by maximum likelihood (ML) method with PAUP* 4.0b10 (Swofford 2003) by heuristic searches with the NNI branch swapping and 10 random taxon additions. The general reversible model (GTR + I + G) and parameters optimized by Modeltest 3.0 (Posada and Crandall 1998) were used. Bootstrapping replicates were set to 500.
Assuming the topological relationships thus obtained, divergence times at nodes were estimated using the multidistribute package (Thorne et al. 1998) after optimizing parameters for the model F84 and the gamma distribution for 8 categories by PAML (Yang 1997). We then used estbranches/multidivtime programs to estimate divergence times by Bayesian Markov Chain Monte Carlo method without assuming the molecular clock. Some key parameters used for this were the following: numsamps, 10,000; sampfreq, 100; burnin, 100,000; rttm and rttmsd, 1.0 (see Kumazawa 2007); rtrate and rtratesd, 0.6; brownmean and brownsd, 0.4; and minab, 1.0. Two iguanid taxa were used as an outgroup. In the multidivtime analyses, we used 2 kinds of constraints for the nodal divergence times. Assuming that Laudakia microlepis of Zagros vicariantly diverged from northern Laudakia caucasia 9 MYA due to mountain uplifts caused by the Indian and Arabian collision (Macey et al. 1998), we constrained this divergence to be 8–10 MYA. We also constrained the divergence between Agamidae and Chamaeleonidae to be 123–76 MYA. This second constraint is based on independent molecular time estimates for this divergence using complete mtDNA sequences including other reptiles, mammals, birds, and amphibians (Kumazawa 2007).
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Results and Discussion |
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TOPAbstractIntroductionMaterial and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Genomic Features of C. versicolor mtDNA
The complete mtDNA sequence of C. versicolor determined as described in Material and Methods was 16,670-bp long and had the usual set of 37 genes and a major noncoding region (fig. 1A). Genes for 2 rRNAs, 13 proteins, and 20 out of 22 tRNAs were encoded in the same relative position and orientation as found in other vertebrate mtDNAs reported so far. One of the other tRNA genes specifying glutamine was present at the 5' end of the isoleucine tRNA gene rather than at the 3' end of it in the typical vertebrate gene organization. This is a gene arrangement that occurs widely in Agamidae and Chamaeleonidae (Macey et al. 1997
, 2000).
The remaining tRNA gene specifying proline conserved its relative position in the mtDNA gene organization but was encoded by the heavy strand, rather than by the light strand as found for other vertebrates. Thus, a gene inversion for trnP likely occurred on an agamid lineage leading to C. versicolor by perfectly conserving organizations for 36 other genes and the major noncoding region (fig. 1A). To the best of our knowledge, this is the first example of gene inversion in vertebrate mtDNAs after >600 complete mtDNA sequences were reported from various groups of vertebrates (Jameson et al. 2003).
The major noncoding region between the trnP and trnF genes (1,504 bp in size) harbored 6 direct repeats of a 66-bp unit and an extremely AT-rich region in the right part close to trnF. Intensive search for possible cloverleaf structures in the left part of the major noncoding region failed to find any functional tRNA genes in it. This major noncoding region containing motifs of the conserved sequence blocks I and III (see supplementary table S2, Supplementary Material online) may be regarded as the CR for replication and transcription (Clayton 1992). The base composition of the C. versicolor mtDNA is skewed similarly as in other vertebrate mtDNAs with more A-T base pairs than G-C base pairs and much more A and C contents in the light strand than in the heavy strand (Asakawa et al. 1991). The overall base composition of the light-strand sequences is A, 33.8%; T, 26.0%; C, 27.0%; and G, 13.3%.
Size Reduction of Some Structural Genes
When protein genes were aligned to their counterparts from other vertebrates, C. versicolor genes for cob, nad2, and nad6 were somewhat (10–15 bp) shorter than those from most other vertebrates (data not shown). In addition, C. versicolor small rRNA gene (rrnS) was approximately 100-bp smaller than those from most other vertebrates, which is due to considerable truncation in 4 distinct regions of its secondary structure (data not shown). Alignment of individual gene sequences suggested that the size shortening of these structural genes is a phenomenon not specific to C. versicolor but applicable for wider taxonomic groups of agamids (data not shown).
Twenty-two tRNA genes encoded in the C. versicolor mtDNA, having identical anticodon triplet sequences to those for other vertebrates, can be folded into the cloverleaf secondary structures. However, truncation of the T
C arm (T arm), previously described for snake mitochondrial tRNAs (Kumazawa et al. 1996), was also notable for some of C. versicolor mitochondrial tRNA genes. The C. versicolor trnT gene had only 3 bp in the T stem. The C. versicolor trnM, trnC, and trnP genes also had a truncated T stem (e.g., 4 bp in trnP as contrasted to 5 bp for most mitochondrial tRNAs; see fig. 2) or an extremely shorter number of bases in the T loop (only 1 or 2 bases if the regular 5 bp in the T stem are allowed; data not shown). This tendency of shortening the T arm size is also notable for the corresponding tRNA genes of other agamids (Macey et al. 2000; data not shown). Taken together, it is likely that the agamid mtDNAs have evolved under an evolutionary pressure to shorten their gene sizes.
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Timing of the trnP Gene Inversion
In order to examine phylogenetic distribution of the inverted state of trnP, we amplified and sequenced an mtDNA region enclosed by the cob and rrnS genes for diverse groups of agamids using procedures described in Material and Methods. Table 1 lists agamid taxa for which sequence information of this region is available, and they cover all 6 subfamilies of Agamidae defined by Macey et al. (2000)
. The results indicated that all 7 draconine taxa sequenced have the inverted trnP, whereas all nondraconine agamids do not (table 1). In addition, the PCR assay (see Material and Methods) supported that 3 more draconines (Bronchocela cristatella, Japalura splendida, and Pseudocalotes sp.) have the inverted trnP (data not shown). We confirmed that the inverted trnP genes of all the 7 draconines can assume a functional secondary structure (fig. 2), arguing against the possibility that they are pseudogenes. These tRNA genes retain not only universally conserved bases (e.g., T8, T33, and R37) but also those conserved within trnP genes from other vertebrates encoded by the opposite strand (see bold nucleotides in fig. 2).
Among the nondraconine taxa examined, those from Uromastycinae, Leiolepidinae, Amphibolurinae, and Hydrosaurinae showed the typical organization of genes in this region (i.e., cob–trnT–trnP–CR–trnF–rrnS with underlined genes encoded by the light strand), whereas 2 representative species from Agaminae showed different gene arrangements. In Xenagama taylori (Macey et al. 2006), the trnP gene appears to have been translocated to the 3' end of CR in the vicinity of trnF (see fig. 1B). Agama impalearis showed another different arrangement in which trnF was translocated into the genes for trnT and trnP (fig. 1C). These arrangements found in Agaminae do not involve inversion of genes from their typical coding orientation.
Figure 3 shows an ML tree for the agamid phylogeny constructed using partial mtDNA sequences between nad1 and cox1 genes. Topological relationships supported by the ML analyses with >50% bootstrap values (see fig. 3) were also reconstructed by Bayesian analyses with appreciably high (>90%) posterior probabilities (data not shown). This phylogeny is consistent with trees with larger taxonomic samplings constructed by Macey et al. (2000) using the same gene region. The trees (fig. 3; Macey et al. 2000) supported basal divergence of Leiolepidinae and Uromastycinae, followed by divergences of amphibolurines and hydrosaurines.
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Macey et al. (1997
, 2000) found that a tRNA gene rearrangement from trnI–trnQ–trnM to trnQ–trnI–trnM occurred in an ancestral lineage of Acrodonta (Agamidae + Chamaeleonidae). Amer and Kumazawa (2005b) found that duplication and insertion of CR into the nad5–nad6 boundary occurred in the amphibolurine lineage after the divergence of Physignathus cocincinus. The present study shows that the inverted state of trnP is shared by all examined draconine taxa, strongly suggesting a single occurrence of the gene inversion in the draconine ancestral lineage (fig. 3). Divergence time estimation further suggested that this inversion event took place approximately 37–55 MYA during the Paleogene period (fig. 3). Because genus Draco represents an earliest shoot-off among the draconines (Macey et al. 2000; fig. 3), it seems difficult to further narrow down the timing of the gene inversion by the current approach. Within the Agaminae, we conducted the PCR assay with some more taxa (e.g., Laudakia nupta, Laudakia stellio, and Pseudotrapelus sinaitus) and did not find evidence for the trnP inversion (data not shown). This further corroborates our conclusion for the single occurrence of the trnP gene inversion within Draconinae. In order to elucidate the timing for occurrence of 2 different gene translocations within Agaminae (figs. 1B and C), relevant mtDNA regions will be sequenced from more diverse agamine taxa and reported elsewhere.
Possible Mechanism of Gene Inversion
Although gene rearrangements have been found in mtDNAs of various vertebrate groups (Macey et al. 1997; Mindell et al. 1998; Inoue et al. 2001; Kumazawa and Endo 2004; Mueller and Boore 2005; Kurabayashi et al. 2006; San Mauro et al. 2006; references therein), these rearrangements did not accompany a switch of the sense strand for any genes. Because polymorphic duplications and/or deletions of an mtDNA segment are present in diverse groups of vertebrates (see, e.g., Moritz et al. 1987; Holt et al. 1988; Stanton et al. 1994; Gach and Brown 1997; Kajander et al. 2000), molecular mechanisms for these rearrangements have been ascribed to the tandem duplication/random loss model (Moritz et al. 1987; Boore 2000; San Mauro et al. 2006). However, this model does not account for gene rearrangements involving a switch of an encoded strand, such as the one found for trnP of draconine agamids.
Figure 4 depicts 2 possible candidates of molecular mechanisms that might account for the trnP inversion in draconines. The 1st model invokes the tRNA gene remolding proposed to account for some tRNA gene rearrangements in invertebrate mtDNAs (Cantatore et al. 1987; Higgs et al. 2003; Rawlings et al. 2003; Lavrov and Lang 2005). There is a strong evidence for the gene remolding in these cases based on sequence similarities of the corresponding tRNA genes. This mechanism can potentially mediate the strand switch of a tRNA gene if a template tRNA gene for duplication was encoded by an opposite strand in relation to a gene that would be replaced and deleted. The template tRNA gene for the model of figure 4A is an adjacent trnT gene which is encoded by the heavy strand, whereas a trnP gene, which will later be replaced and deleted, is encoded by the light strand.
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We attempted to evaluate this model using available tRNA gene sequences for draconine and nondraconine agamids (figs. 5 and 6). If the model of figure 4A were the case, one would expect to observe a higher sequence similarity between trnT and trnP genes in draconines than in nondraconines. Alternatively, if the other mechanisms such as the homologous DNA recombination (fig. 4B) were the case, one would not observe the significant increase of the trnT–trnP similarity in draconines. Figure 6 shows that the trnT–trnP similarities are not significantly higher (P > 0.05 by Student's t-test) in draconines than in nondraconines. Figure 6 also shows that the trnT–trnP similarities in draconines are not significantly higher (P > 0.05 by Student's t-test) than an accidental level of sequence similarity between other neighboring tRNA genes (i.e., trnI vs. trnM, trnA vs. trnN, and trnC vs. trnY) using the same taxon sampling. These results do not provide supportive evidence for the tRNA remolding model of figure 4A. The same conclusion was reached when trnT and trnP sequences at the immediate common ancestor of draconines were inferred (fig. 5) and compared directly (fig. 6).
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Although current taxonomic sampling includes more closely related taxa in the draconine group than in the nondraconine one (fig. 3), this should not affect the above-mentioned conclusion because these comparisons were not made for orthologous genes. This conclusion also does not appear to be misled by the differential base composition between mtDNA strands (Asakawa et al. 1991
). Indeed, trnP genes encoded by the heavy strand in draconines possess a somewhat different base composition from that for trnP genes encoded by the light strand in nondraconines (see Supplementary fig. S1, Supplementary Material online), suggesting that the inverted trnP genes of extant draconines have already been adapted to the general trend of the strand-specific base composition after 37–55 Myr of the inversion event. However, in the tRNA remolding model of figure 4A, this factor, if anything, would operate to increase the trnT–trnP similarities for draconines as compared with those for nondraconines, which was not actually detected by the sequence comparisons (fig. 6). Thus, our conclusion of no tRNA gene remolding is likely not an artifact by the base composition bias.
In the tRNA gene remolding model (fig. 4A), anticodon sequences of trnT and trnP genes can be converted by a single-base substitution (TGT to TGG) but a number of other base substitutions, especially in the acceptor and T stem regions, must be assumed to convert from trnT to trnP in draconines (fig. 5). By contrast, trnP gene sequences of draconine and nondraconine agamids, which are encoded by a different strand from each other, are clearly more similar to each other than they are to trnT genes (figs. 5 and 6). Because identity elements for each tRNA are usually confined to relatively small numbers of nucleotide positions (Giegé et al. 1998), this pattern of sequence similarities cannot be explained by rapid sequence convergence after the tRNA gene remolding. We also considered the possibility that a template tRNA gene for the tRNA remolding was supplied by a tRNA gene other than the neighboring trnT gene (e.g., an antisense sequence of the trnP gene itself). However, none of other tRNA gene and its antisense sequence was found to have a prominently high sequence similarity with the trnP gene in Calotes versicolor (data not shown).
An alternative model shown in figure 4B takes advantage of accidentally formed inverted repeat sequences (IRS) to invert the trnP gene by homologous DNA recombination. The role of IRS in causing a gene inversion by the homologous DNA recombination is well known (see, e.g., Watson et al. 2004, p. 294–295). For example, circular chloroplast genomes contain long IRS, with which intervening unique sequences between them are frequently inverted and the chloroplast genomes exist as equimolar mixtures of 2 isomers differing from each other in the relative orientation of the intervening sequences (Stein et al. 1986; Hiratsuka et al. 1989).
Additional support for the model of figure 4B comes from the following arguments.
- The inversion of trnP gene in draconines took place by conserving its relative position in the mtDNA gene organization (fig. 1A), favoring the local DNA recombination (fig. 4B) rather than other mechanisms, such as retrotransposition.
- As described above, nucleotide sequences of the inverted and noninverted trnP genes are quite similar and likely homologous, supporting the direct gene inversion (fig. 4B) rather than a homoplasious origin of the trnP function (fig. 4A). However, inspection of the mtDNA sequences of extant draconines did not show a more direct evidence of the DNA recombination (e.g., a remnant of IRS) presumably because of substantial sequence changes for 37–55 Myr (data not shown).
- There is now growing evidence for the occurrence of homologous DNA recombination in vertebrate mtDNAs (Thyagarajan et al. 1996; Hoarau et al. 2002; Kraytsberg et al. 2004; Tsaousis et al. 2005).
Rarity of Gene Inversion in Vertebrate mtDNAs
Why are gene inversions so rare in vertebrate mtDNAs although they appear to be more frequent in invertebrate mtDNAs (Boore 1999; references therein)? As far as invertebrate chordates are concerned, there exist multiple examples of gene inversions. Gene orders of cephalochordate mtDNAs retain a certain level of similarity to vertebrate counterparts but a lineage of cephalochordates did allow an inversion of 4 neighboring genes (Nohara et al. 2005). Ascidian mtDNAs appear to have allowed numerous gene inversions that led to a quite extreme gene order in which all genes are now encoded by 1 of 2 mtDNA strands (Yokobori et al. 1999).
Here, we discuss 2 main possibilities to account for the rarity of gene inversion in vertebrate mtDNAs although we do not necessarily rule out other potential causes. First, inversional mutations may be less frequent due to a lower recombinational activity in vertebrate mtDNAs. However, to our knowledge, there is no study in which mitochondrial recombinational activities were compared between vertebrates and invertebrates. Description of frequent recombinations in selectively neutral CR sequences of vertebrate mtDNAs (Hoarau et al. 2002; Kraytsberg et al. 2004) may not support this possibility.
Second, inverted gene organizations may be more frequently deleterious and thus less frequently tolerated in vertebrate mtDNAs. The typical gene organization of vertebrate mtDNAs may be at an optimum state in some respects. In vertebrate systems, large polycistronic RNA molecules transcribed from major promoters inside the CR are processed into individual RNA components (mRNAs, rRNAs, and tRNAs) by enzymatic activities that target tRNA sequences between mRNA and rRNA sequences (Anderson et al. 1981; Ojala et al. 1981). On the other hand, gene expressions from some invertebrate mtDNAs may proceed in a somewhat different way using multiple promoters (Elliott and Jacobs 1989). In addition, the tendency of clustering tRNA genes (see, e.g., Jacobs et al. 1989) does imply relaxation of their processing constraints in some invertebrate mtDNAs.
As a positive evidence of such processing constraints consistently operating in the evolution of vertebrate mitochondrial tRNA genes, a transposed methionine tRNA gene in the parrot fish mtDNA left its pseudogene in its original location as a processing signal for an adjacent protein gene (Mabuchi et al. 2004). If tRNA genes with such constraints were inverted, they would no longer play the original processing role. The trnP gene resides in a position where it does not serve as a processing signal for any adjacent protein or rRNA gene (see fig. 1A). Only a limited number of tRNA genes that are apparently free or relaxed from such constraints (i.e., trnQ, trnA, trnN, trnC, 2 trnS isoacceptor genes, and trnP, all of which are encoded by the light strand in the typical gene organization) may be allowed to be inverted in vertebrates.
In addition to the processing constraints operating on tRNA genes, the relative location and orientation of individual structural genes in vertebrate mtDNAs may be important to ensure controlled gene expressions in response to, for example, exogenous regulatory factors such as temperature and adenosine triphosphate concentration, as well as the requirement of synthesizing increased amounts of rRNAs relative to mRNAs (reviewed in Attardi 1993). The less frequent gene inversion may therefore reflect the strength of these regulatory constraints in vertebrate mtDNAs. Unfortunately, biochemical experiments that implied such regulatory constraints have been carried out mostly using mammalian systems (Clayton 1992; Attardi 1993) and very little is known on invertebrate systems. Elucidation of molecular mechanisms for regulatory gene expressions in invertebrate mitochondria should therefore be encouraged to examine this hypothesis in future.
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Supplementary Material |
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TOPAbstractIntroductionMaterial and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary tables S1, S2, S3, and S4 and figure S1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterial and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank Mr K. Yagi (Remix Peponi Co., Nagoya, Japan) for providing animal samples, Drs T. Nishikawa (Nagoya University Museum) and S. Kawada (National Science Museum, Tokyo) for help in depositing our specimens to museums, and Ms C. Aoki for experimental assistance. We also thank Dr T. P. Satoh (University of Tokyo) for critically reading an earlier version of manuscript and providing useful comments. This work was supported by grants from the Daiko Foundation (the Visiting Fellowship for Foreign Scholars number 10093) and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (numbers 14902202 and 17570076).
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Footnotes |
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Yoko Satta, Associate Editor
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