Complete mtDNA Sequences of Two Millipedes Suggest a New Model for Mitochondrial Gene Rearrangements: Duplication and Nonrandom Loss

Dennis V. Lavrov, Jeffrey L. Boore and Wesley M. Brown

Department of Biology, University of Michigan, Ann Arbor;
Département de Biochimie, Université de Montréal;
DOE Joint Genome Institute, Walnut Creek, California


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
We determined the complete mitochondrial DNA (mtDNA) sequences of the millipedes Narceus annularus and Thyropygus sp. (Arthropoda: Diplopoda) and identified, in both genomes, all 37 genes typical for metazoan mtDNA. The arrangement of these genes is identical in the two millipedes, but differs from others found in arthropod mtDNAs in the location of at least four genes or gene blocks. This novel gene arrangement is unusual for animal mtDNA in that genes with identical transcriptional polarities are clustered in the genome, and the two clusters are separated by two noncoding regions. The only exception to this pattern is the gene for cysteine tRNA, which is located in the part of the genome that otherwise contains all genes with the opposite transcriptional polarity. We suggest that a mechanism involving complete mtDNA duplication followed by the loss of genes, predetermined by their transcriptional polarity and location in the genome, could generate this gene arrangement from the one ancestral for arthropods. The proposed mechanism has important implications for phylogenetic inferences that are drawn on the basis of gene arrangement comparisons.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Animal mitochondrial DNA (mtDNA) is typically a circular molecule of 15–17 kb that encodes 37 genes: 13 for proteins (subunits 6 and 8 of the F0 ATPase [atp6 and atp8], cytochrome c oxidase subunits 1–3 [cox1cox3], apocytochrome b [cob], and NADH dehydrogenase subunits 1–6 and 4L [nad16 and nad4L]); two for ribosomal RNAs (small and large subunit rRNAs [rrnS and rrnL]); and 22 for tRNAs (Boore 1999Citation ). In addition, a single large noncoding region is typically present which, for a few animals, is known to contain sequences essential for the initiation of transcription and mtDNA replication (Shadel and Clayton 1997Citation ).

Arrangements of metazoan mitochondrial genes are relatively stable, with some having been conserved for hundreds of millions of years (e.g., most vertebrates share an identical arrangement, as do some crustaceans and insects [Boore 1999; unpublished data]). Only minor rearrangements separate the gene orders of human and the cephalochordate Branchiostoma floridae, or Drosophila yakuba and the chelicerate Limulus polyphemus, whereas numerous, but identifiable, rearrangements separate those of chordates and arthropods (Clary and Wolstenholme 1985Citation ; Boore 1999Citation ). Because of the low rearrangement rate as well as several other characteristics, mitochondrial gene arrangements promise to be a useful data set for the study of deep metazoan divergences (Boore and Brown 1998Citation ). Indeed, several problematic relationships have already been convincingly resolved using this data set, including those among classes of echinoderms (Smith et al. 1993Citation ) and of arthropods (Boore, Lavrov, and Brown 1998Citation ). However, our limited knowledge of gene rearrangement mechanisms hampers our ability to interpret this data set and is an impediment to its broader acceptance for phylogenetic studies (Curole and Kocher 1999Citation ).

The most detailed and the best supported model attributes gene rearrangements to the partial duplication of mtDNA caused by errors in replication, such as erroneous initiation or termination (Macey et al. 1997Citation ), or strand slippage and mispairing (Madsen, Ghivizzani, and Hauswirth 1993Citation ), followed by the loss of one copy of each duplicated gene (Moritz, Dowling, and Brown 1987Citation ; Boore 2000Citation ). This model is supported by the observation of mtDNAs containing duplicated regions (Moritz and Brown 1986Citation ; Moritz and Brown 1987Citation ), most of which are adjacent to or include the noncoding region; inactivating mutations are frequently found in one copy of each of the genes (Stanton et al. 1994Citation ; Arndt and Smith 1998Citation ; Kumazawa et al. 1998Citation ; Macey et al. 1998Citation ). It is commonly assumed that the loss of one of the two copies of each duplicated gene happens at random, and the model described earlier is sometimes referred to as the duplication–random loss model (Moritz, Dowling, and Brown 1987Citation ; Boore and Brown 1998Citation ; Boore 1999Citation ; Boore 2000Citation ).

In this article, we describe the mitochondrial DNA of two millipedes. These share a novel gene order, which we propose has been generated from the arrangement that is primitive for arthropods by a novel mechanism: mtDNA duplication and nonrandom gene loss. According to this model, the destiny of each gene copy in the duplicated region is predetermined by its transcriptional polarity and location in the genome. We discuss the implications of this for phylogenetic inferences based on comparisons of gene arrangements.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
A specimen of Narceus annularus (order Spirobolida) was collected by Dr. Barry OConner near the Biological Station of the University of Michigan in Northern Michigan; a specimen of Thyropygus sp. (order Spirostreptida) was purchased from WARD'S Natural Science Establishment, Inc. Total DNA from body wall muscles or from eggs was prepared as described (Saghai-Maroof et al. 1984Citation ). For N. annularus, small fragments of cox3 and rrnS were amplified and sequenced, and two pairs of primers were designed based on these sequences: Narceus-cox3-F1 (5'-C3GTAGCAACAGGCTTTCATGGAC-3'), Narceus-cox3-R1 (5'-AGTCCATGAAAGCCTGTTGCTAC-3'), Narceus-rrnS-F1 (5'-CATAGTCTGAGGGACGTCAAGTC-3'), and Narceus-rrnS-R1 (5'-CCTTGACTTGACGTCCCTCAGAC-3'). For Thyropygus sp., small fragments of rrnS and cob were amplified and sequenced, and two pairs of primers were designed based on these sequences: Thyropygus-rrnS-F1 (5'-AGGACGTCAAGTCAAGGTGCAGC-3'), Thyropygus-rrnS-R1 (5'-AATCCACCTTCATGATGCACTTC-3'), Thyropygus-cob-F1 (5'-GGATTTGCAGTAGACAATGCCAC-3'), and Thyropygus-cob-R1 (5'-GGTGAATAATAACTATGGCTGCGA-3'). The whole mtDNA of each millipede was amplified in two overlapping fragments by using Perkin-Elmer's® XL PCR kit or TaKaRa LA-PCR kit, and primer pairs Narceus-rrnS-F1–Narceus-cox3-F1, Narceus-rrnS-R1–Narceus-cox3-R1, Thyropygus-rrnS-F1–Thyropygus-cob-F1, and Thyropygus-rrnS-R1–Thyropygus-cob-R1. Each PCR reaction yielded a single band when visualized with ethidium bromide staining after electrophoresis in a 1% agarose gel.

PCR reaction products were purified by three serial passages through UltrafreeTM (30,000 NMWL) columns (Millipore) and used as templates in dye-terminator cycle–sequencing reactions according to the supplier's (Perkin-Elmer®) instructions. Both strands of each amplification product were sequenced by primer walking, using an ABI 377 automated DNA sequencer. The sequences of the mtDNAs of N. annularus and Thyropygus sp. have been submitted to GenBank under the accession numbers AY055727 and AY055728, respectively.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Millipede Mitochondrial Genomes
The complete mtDNA sequences of the millipedes N. annularus and Thyropygus sp. (fig. 1 ) are 14,868 and 15,133 bp in size, respectively, and contain all 37 genes typical for animal mtDNA. These genes have an arrangement that differs from any other found in arthropod mtDNAs in the positions of at least four genes or gene clusters. In addition, the millipede gene arrangement is unusual because, with the single exception of trnC, genes that are transcribed in opposite directions are located in two different parts of the genome and separated by two noncoding regions (fig. 1 ). In contrast, in most other animal mtDNAs studied, genes are either transcribed from the same strand or arranged in several clusters with alternating transcriptional polarity, and a single large noncoding region is present (Boore 1999Citation ). Although it is possible that the millipede gene arrangement is the result of four independent rearrangements (fig. 2A ) or a large duplication followed by the random loss of genes, the unusual clustering of genes with opposite transcriptional polarity suggests that a different, nonrandom mechanism may have generated this gene order.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.—The gene maps of N. annularus and Thyropygus sp. mtDNA. Protein and rRNA genes are abbreviated as in the text; tRNA genes are identified by the one-letter code for the corresponding amino acid. Two leucine and two serine tRNA genes are differentiated by their anticodon sequence with trnL(uag) marked as L1, trnL(uaa) as L2, trnS(ucu) as S1, and trnS(uga) as S2. The direction of transcription for each gene is shown by an arrow. Filled areas depict large noncoding regions, the number attached to each indicates its size. Positive numbers at gene boundaries indicate the number of intergenic nucleotides; negative numbers indicate the number of overlapping nucleotides. Asterisks mark incomplete stop codons (T or TA) that are presumably completed by the addition of 3' A residues to the mRNA

 


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 2.—Comparison of gene arrangements in mtDNA of L. polyphemus and two millipedes (A) and the mechanism proposed for the generation of the latter gene arrangement (B). The rearrangement of genes or gene blocks in A is shown by arrows. The proposed position for transcription initiation and termination in B are marked by TI and TT. Genes are not to scale; protein and rRNA genes are indicated by bigger boxes, tRNA genes by smaller boxes, and noncoding regions are dotted. Genes are transcribed from left to right except when underlined; underlining indicates the opposite transcriptional polarity. The copies of the genes that became pseudogenes in a hypothetical intermediate arrangement are indicated by filled boxes, the unexpected pattern of retention or loss for two copies of trnC (marked by ovals) is discussed in the text. Genes are abbreviated as in fig. 1

 
Gene Rearrangement Mechanism
The proposed mechanism is presented in figure 2B . We assume that the original mitochondrial genome had a gene arrangement identical to that in L. polyphemus, which appears to be ancestral for arthropods (unpublished data), and that both the mtDNA strands were transcribed in their entirety either from the same bidirectional promoter or from two unidirectional promoters, both located in the same noncoding region. Such arrangements have been found in vertebrate mtDNAs, in which most studies on mitochondrial transcription mechanisms have been conducted (Tracy and Stern 1995Citation ), and are consistent with the generally observed scarcity of intergenic sequences in arthropod mitochondrial genomes that otherwise could serve as transcriptional promoters (nevertheless, see Clary, Wahleithner, and Wolstenholme 1983Citation ). We further assume that there are unidirectional transcription termination signals located in the same noncoding region of the mtDNA. The presence of such signals has been inferred based on RNase mapping experiments in rats (Sbisa et al. 1990Citation ); however, no mechanistic details are known for this or for other animal species.

We envision the first step leading to the millipede gene arrangement to have been a tandem duplication of the entire mtDNA, resulting in a dimeric molecule with the two monomers covalently linked head to tail. Such complex molecules are usually associated with cellular abnormalities and tissue cultures (Clayton and Vinograd 1967Citation ; Wolstenholme et al. 1973Citation ), but have also been observed in the normal tissues of rat (Wolstenholme et al. 1973Citation ), several species of Drosophila (Shah and Langley 1977Citation ), and the isopod Armadillidium vulgare (Raimond et al. 1999Citation ). In addition to duplicated structural genes, such a dimeric molecule would contain duplicated transcriptional regulators located in the two copies of the noncoding regions. If the functionality of transcriptional promoters in one of the noncoding regions was lost or severely impaired (e.g., by a deletion encompassing the two unidirectional promoters or by a point mutation in a single bidirectional promoter), whereas the accompanying sets of transcriptional termination signals were retained, then the sets of genes under the control of the disabled promoter(s) would immediately become pseudogenes, and the sequences containing them would be free to mutate and to disappear from the genome (fig. 2B ). As a result, the loss of genes would be predetermined by their transcriptional polarity. All genes having one polarity would be lost from one genome copy, and all genes having the opposite polarity would be lost from the other.

The gene order in the two millipede mtDNAs corresponds to the hypothetical gene order that would be derived from the primitive arthropod arrangement by the proposed mechanism, but with two discrepancies present. The first, already mentioned, is the location of trnC in the part of the genome that otherwise contains all genes with the opposite transcriptional polarity. The second is the location of trnT, which is different from the one expected after the proposed rearrangement. Neither discrepancy necessarily invalidates the proposed mechanism. The product of trnC carries the least-frequent amino acid in mitochondrial proteins. There are only 31 cysteine residues encoded in N. annularus mitochondrial protein genes and 30 in those of Thyropygus sp., less than 1% of the total number of amino acids in both cases. Thus, a weak residual level of transcription could potentially produce a sufficient supply of this gene product. The unexpected location of trnT can be explained by an independent translocation event, such as that depicted in figure 2B. Such translocations are relatively common in animal mitochondrial genomes (Boore 1999Citation ), and the translocation of trnT could have happened either before or after the duplication and loss events postulated in the model. Further sampling of this group of animals may provide data with which to test this hypothesis.

Support for the Proposed Model
The model proposed here has greater explanatory power for the arrangement found in two millipede mitochondrial genomes than the duplication–random loss model. Unlike the latter model, it predicts the pattern of the gene loss and, therefore, the resulting gene order based on the genes' transcriptional polarities and their positions in the genome. It also explains the presence and predicts the locations of the two noncoding regions that are found in genomes that otherwise have very few noncoding nucleotides (fig. 1 ).

Indirect support for the proposed model also comes from the study of the mitochondrial genome of the red alga Chondrus crispus (Leblanc et al. 1995Citation ). This protist has a relatively small and gene-rich mitochondrial genome, with an overall genome organization surprisingly similar to that found in millipedes. As in millipedes, the C. crispus mitochondrial genes that are transcribed in opposite directions are located in two different parts of the genome and separated by two noncoding regions. Also as in millipedes, a single tRNA gene (trnH) defies this rule and is located in the wrong part of the genome. In addition, the mitochondrial genomes of the millipedes and C. crispus share the presence of two large stem-loop structures in the noncoding regions. In C. crispus and N. annularus, one stem-loop structure is found in each of the two noncoding regions, whereas in Thyropygus sp. both structures are present in the same noncoding region (fig. 3 ). The study of mitochondrial transcription by Northern hybridization in C. crispus revealed the presence of two large transcriptional units, with transcription initiating in one noncoding region (corresponding to the noncoding region adjacent to trnI and rrnS in millipedes), and terminating in the other noncoding region in one direction and near trnH in the other (Richard et al. 1998Citation ). Interestingly, although it has been suggested that the stem-loop structures found in C. crispus mtDNA may be involved in transcription initiation and termination (Leblanc et al. 1995Citation ; Richard et al. 1998Citation ), those in millipede mtDNA resemble the stem-loop structure at the origin of light-strand replication in vertebrates (Wong and Clayton 1985Citation ). If these are functional analogues, then a novel replication mechanism may exist which utilizes both these structures, perhaps for the separate initiation of replication on each strand of millipede mtDNA.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.—Potential stem-loop structures in the noncoding regions of millipede mtDNA. Structural genes adjacent to noncoding regions are shown by arrows; their transcriptional polarities are indicated by the arrows' directions. Runs of thymidines or adenosines in the loop regions are in boldface. The boldface T at the base of the second stem-loop structure in Thyropygus sp. mtDNA is inferred to be part of trnQ. The 5' end of N. annularus rrnS directly adjacent to the stem-loop structure has been inferred based only on sequence comparisons. The gene names are as described in the text

 
Implications for Phylogenetic Studies
Most studies comparing mitochondrial gene arrangements for phylogenetic inference have not considered the mechanisms for gene rearrangements, but have made the tacit assumption that gene rearrangements are random events in which there is an extremely small probability of convergence. Some studies have also used combinatorics to estimate the significance of shared gene boundaries, further assuming that each rearrangement has an equal chance of occurring (Noguchi et al. 2000Citation ). It is clear, however, that if a rearrangement happens by the duplication-loss mechanism, the resulting gene order will not be completely random because (1) the transcriptional polarity of the genes would not change, (2) a gene would only be able to relocate within the boundaries of the duplicated region, and (3) not all rearrangements that preserve the transcriptional polarity of the genes in this region would be obtainable in a single duplication-loss event (e.g., duplication of ABC to ABCABC cannot lead to CBA by gene loss alone). In addition, the presence of duplication hot spots, as revealed by comparisons of mtDNAs from different species (Boore and Brown 1998Citation ; Boore 1999Citation ) and by analysis of different mtDNA molecules from the same organism (Moritz and Brown 1986Citation ; Moore, Gudikote, and Van Tuyle 1998Citation ), contributes further to the nonrandomness in mitochondrial gene rearrangements. Some of these considerations have led to our earlier suggestions for down-weighting the sharing of certain types of changes (such as exchange of nearest-neighbor tRNA genes and movements of genes flanking the control region) in phylogenetic analysis (Boore and Brown 1998Citation ).

The present study adds a new dimension to the problem by showing that the destiny of genes in a duplicated region may be determined by biological constraints, rather than by chance. If a duplication includes a noncoding region containing transcriptional control sequences, a condition that appears to be common among metazoan mtDNA duplications, an inactivating mutation in one of those sequences could facilitate the nearly simultaneous and nonrandom loss of an entire subset of the duplicated genes. If, as proposed here, the entire mtDNA duplicates with the monomers linked head to tail, it would lead to gene arrangements similar to those found in millipede and red alga mtDNAs. If, instead, duplication resulted in a dimeric mtDNA molecule in which two monomers were linked head to head so that two copies of each gene had the opposite transcriptional polarities, an inactivating mutation in one set of transcriptional control sequences would result in an arrangement in which all genes were transcribed in the same direction. This latter variation of the model may explain the relatively frequent and apparently independent emergence of such arrangements during metazoan mtDNA evolution: mtDNAs in which all the genes are transcribed from the same strand have been found for some species of nematodes, mollusks, annelids, brachiopods, and flatworms (Boore 1999Citation ).

Some additional constraints on mitochondrial genome organization could also cause nonrandom gene loss after a duplication. A transcriptional attenuator is located downstream from the two ribosomal genes in human mtDNA (Montoya, Gaines, and Attardi 1983Citation ) and is likely to occur in many other animal mtDNAs (Valverde, Marco, and Garesse 1994Citation ). This may constrain the position of the rRNA genes relative to the origin of transcription, to other genes, and to each other. If and when the secondary structures of tRNA sequences serve as the processing signals for mitochondrial primary transcripts, as has been proposed (Ojala, Montoya, and Attardi 1981Citation ), the possible rearrangements would be restricted to those that have at least one tRNA gene between each pair of neighboring protein or rRNA genes (or both). The conservation of some mitochondrial gene arrangements across diverse phylogenetic groups may also be the result of the physical interaction among their gene products, some of which may require cotranslational folding (Dandekar et al. 1998Citation ). Possible candidates for this include the pairs nad4L-nad4 and atp8-atp6 which, at least in some animals, are translated from the same messenger RNA. Thus, selection for a certain gene order may operate even in cases in which a complete mtDNA is transcribed as a single operon. An additional constraint on rearrangement may occur if subsets of genes are transcribed from different transcriptional units, as has been reported for the sea urchin Paracentrotus lividus (Cantatore et al. 1990Citation ).

Mitochondrial gene rearrangements are rare events in animal evolution and, therefore, appear to be well suited for the study of ancient relationships. However, as we accumulate more and more gene arrangement data and try to use them for global phylogenetic studies, we must also consider the possible mechanisms that underlie the rearrangements, both to improve our inference of evolutionary relationships and to be able to evaluate the robustness of our conclusions. The model we present provides a means to explain a specific gene rearrangement that has been observed and is an important step in this direction.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
This work was supported by the NSF dissertation improvement grant (DEB 9972712) to W.M.B. and D.V.L., the NSF grant (DEB 9807100) to W.M.B. and J.L.B., and the University of Michigan predoctoral fellowship to D.V.L. Thanks to John Moran, B. Franz Lang Kevin Helfenbein, and two anonymous reviewers for helpful comments on the manuscript.


    Footnotes
 
Pekka Pamilo, Reviewing Editor

Keywords: mitochondrial DNA gene rearrangement genome duplication phylogenetic inference diplopoda Narceus annularus Thyropygus sp Back

Address for correspondence and reprints: Dennis V. Lavrov, Département de Biochimie, Université de Montréal, C.P. 6128, succursale Centre-ville, Montréal, Québec. H3C 3J7, Canada. dlavrov{at}bch.umontreal.ca . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 

    Arndt A., M. J. Smith, 1998 Mitochondrial gene rearrangement in the sea cucumber genus Cucumaria Mol. Biol. Evol 15:1009-1016[Abstract]

    Boore J. L., 1999 Animal mitochondrial genomes Nucleic Acids Res 27:1767-1780[Abstract/Free Full Text]

    Boore J. L., 2000 The duplication/random loss model for gene rearrangement exemplified by mitochondrial genomes of deuterostome animals Pp. 133–147 in D. Sankoff and J. H. Nadeau, eds. Comparative genomics. Kluwer Academic Publishers, Dordrecht

    Boore J. L., W. M. Brown, 1998 Big trees from little genomes: mitochondrial gene order as a phylogenetic tool Curr. Opin. Genet. Dev 8:668-674[ISI][Medline]

    Boore J. L., D. V. Lavrov, W. M. Brown, 1998 Gene translocation links insects and crustaceans Nature 392:667-668[ISI][Medline]

    Cantatore P., M. Roberti, P. L. Polosa, A. Mustich, M. N. Gadaleta, 1990 Mapping and characterization of Paracentrotus lividus mitochondrial transcripts: multiple and overlapping transcription units Curr. Genet 17:235-245[ISI][Medline]

    Clary D. O., J. A. Wahleithner, D. R. Wolstenholme, 1983 Transfer RNA genes in Drosophila mitochondrial DNA: related 5' flanking sequences and comparisons to mammalian mitochondrial tRNA genes Nucleic Acids Res 11:2411-2425[Abstract]

    Clary D. O., D. R. Wolstenholme, 1985 The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code J. Mol. Evol 22:252-271[ISI][Medline]

    Clayton D. A., J. Vinograd, 1967 Circular dimer and catenate forms of mitochondrial DNA in human leukaemic leucocytes Nature 216:652-656[ISI]

    Curole J. P., T. D. Kocher, 1999 Mitogenomids: digging deeper with complete mitochondrial genomes Trends Ecol. Evol 14:394-398[ISI][Medline]

    Dandekar T., B. Snel, M. Huynen, P. Bork, 1998 Conservation of gene order: a fingerprint of proteins that physically interact Trends Biochem. Sci 23:324-328[ISI][Medline]

    Kumazawa Y., H. Ota, M. Nishida, T. Ozawa, 1998 The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control regions Genetics 150:313-329[Abstract/Free Full Text]

    Lavrov D. V., J. L. Boore, W. M. Brown, 2000 The complete mitochondrial DNA sequence of the horseshoe crab Limulus polyphemus Mol. Biol. Evol 17:813-824[Abstract/Free Full Text]

    Leblanc C., C. Boyen, O. Richard, G. Bonnard, J. M. Grienenberger, B. Kloareg, 1995 Complete sequence of the mitochondrial DNA of the rhodophyte Chondrus crispus (Gigartinales). Gene content and genome organization J. Mol. Biol 250:484-495[ISI][Medline]

    Macey J. R., A. Larson, N. B. Ananjeva, Z. Fang, T. J. Papenfuss, R. J. Macey, 1997 Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome Mol. Biol. Evol 14:91-104[Abstract]

    Macey J. R., J. A. Schulte II,, A. Larson, T. J. Papenfuss, 1998 Tandem duplication via light-strand synthesis may provide a precursor for mitochondrial genomic rearrangement Mol. Biol. Evol 15:71-75[Abstract]

    Madsen C. S., S. C. Ghivizzani, W. W. Hauswirth, 1993 In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria Proc. Natl. Acad. Sci. USA 90:7671-7675[Abstract/Free Full Text]

    Montoya J., G. L. Gaines, G. Attardi, 1983 The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units Cell 34:151-159[ISI][Medline]

    Moore C. A., J. Gudikote, G. C. Van Tuyle, 1998 Mitochondrial DNA rearrangements, including partial duplications, occur in young and old rat tissues Mutat. Res.—Fundam. Mol. Mech. Mutagen 421:205-217

    Moritz C., W. M. Brown, 1986 Tandem duplications of D-loop and ribosomal RNA sequences in lizard mitochondrial DNA Science 233:1425-1427[ISI][Medline]

    Moritz C., W. M. Brown, 1987 Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content among lizards Proc. Natl. Acad. Sci. USA 84:7183-7187[Abstract]

    Moritz C., T. E. Dowling, W. M. Brown, 1987 Evolution of animal mitochondrial DNA: relevance for population biology and systematics Annu. Rev. Ecol. Syst 18:269-292[ISI]

    Noguchi Y., K. Endo, F. Tajima, R. Ueshima, 2000 The mitochondrial genome of the brachiopod Laqueus rubellus Genetics 155:245-259[Abstract/Free Full Text]

    Ojala D., J. Montoya, G. Attardi, 1981 tRNA punctuation model of RNA processing in human mitochondria Nature 290:470-474[ISI][Medline]

    Raimond R., I. Marcade, D. Bouchon, T. Rigaud, J. P. Bossy, C. Souty-Grosset, 1999 Organization of the large mitochondrial genome in the isopod Armadillidium vulgare Genetics 151:203-210[Abstract/Free Full Text]

    Richard O., G. Bonnard, J. M. Grienenberger, B. Kloareg, C. Boyen, 1998 Transcription initiation and RNA processing in the mitochondria of the red alga Chondrus crispus: convergence in the evolution of transcription mechanisms in mitochondria J. Mol. Biol 283:549-557[ISI][Medline]

    Saghai-Maroof M. A., K. M. Soliman, R. A. Jorgensen, R. W. Allard, 1984 Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics Proc. Natl. Acad. Sci. USA 81:8014-8018[Abstract]

    Sbisa E., M. Nardelli, F. Tanzariello, A. Tullo, C. Saccone, 1990 The complete and symmetric transcription of the main non-coding region of rat mitochondrial genome: in vivo mapping of heavy and light transcripts Curr. Genet 17:247-253[ISI][Medline]

    Shadel G. S., D. A. Clayton, 1997 Mitochondrial DNA maintenance in vertebrates Annu. Rev. Biochem 66:409-435[ISI][Medline]

    Shah D. M., C. H. Langley, 1977 Complex mitochondrial DNA in Drosophila Nucleic Acids Res 4:2949-2960[Abstract]

    Smith J. M., A. Arndt, S. Gorski, E. Fajber, M. J. Smith, 1993 The phylogeny of echinoderm classes based on mitochondrial gene arrangements J. Mol. Evol 36:545-554[ISI][Medline]

    Stanton D. J., L. L. Daehler, C. C. Moritz, W. M. Brown, 1994 Sequences with the potential to form stem-and-loop structures are associated with coding-region duplications in animal mitochondrial DNA Genetics 137:233-241[Abstract/Free Full Text]

    Tracy R. L., D. B. Stern, 1995 Mitochondrial transcription initiation: promoter structures and RNA polymerases Curr. Genet 28:205-216[ISI][Medline]

    Valverde J. R., R. Marco, R. Garesse, 1994 A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria Proc. Natl. Acad. Sci. USA 91:5368-5371[Abstract]

    Wolstenholme D. R., J. D. McLaren, K. Koike, E. L. Jacobson, 1973 Catenated oligomeric circular DNA molecules from mitochondria of malignant and normal mouse and rat tissues J. Cell Biol 56:247-255[Free Full Text]

    Wong T. W., D. A. Clayton, 1985 In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis Cell 42:951-958[ISI][Medline]

Accepted for publication September 21, 2001.