Department of Biology, University of Michigan, Ann Arbor;
Département de Biochimie, Université de Montréal;
DOE Joint Genome Institute, Walnut Creek, California
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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of years (e.g., most vertebrates share an identical arrangement, as do some crustaceans and insects [Boore 1999; unpublished data]).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 1985
; Boore 1999
). 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 1998
). Indeed, several problematic relationships have already been convincingly resolved using this data set, including those among classes of echinoderms (Smith et al. 1993
) and of arthropods (Boore, Lavrov, and Brown 1998
). 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 1999
).
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. 1997
), or strand slippage and mispairing (Madsen, Ghivizzani, and Hauswirth 1993
), followed by the loss of one copy of each duplicated gene (Moritz, Dowling, and Brown 1987
; Boore 2000
). This model is supported by the observation of mtDNAs containing duplicated regions (Moritz and Brown 1986
; Moritz and Brown 1987
), 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. 1994
; Arndt and Smith 1998
; Kumazawa et al. 1998
; Macey et al. 1998
). 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 duplicationrandom loss model (Moritz, Dowling, and Brown 1987
; Boore and Brown 1998
; Boore 1999
; Boore 2000
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR reaction products were purified by three serial passages through UltrafreeTM (30,000 NMWL) columns (Millipore) and used as templates in dye-terminator cyclesequencing 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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 1967
; Wolstenholme et al. 1973
), but have also been observed in the normal tissues of rat (Wolstenholme et al. 1973
), several species of Drosophila (Shah and Langley 1977
), and the isopod Armadillidium vulgare (Raimond et al. 1999
). 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 1999
), 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 duplicationrandom 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. 1995
). 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. 1998
). 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. 1995
; Richard et al. 1998
), those in millipede mtDNA resemble the stem-loop structure at the origin of light-strand replication in vertebrates (Wong and Clayton 1985
). 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.
|
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 1999
).
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 1983
) and is likely to occur in many other animal mtDNAs (Valverde, Marco, and Garesse 1994
). 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 1981
), 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. 1998
). 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. 1990
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Keywords: mitochondrial DNA
gene rearrangement
genome duplication
phylogenetic inference
diplopoda
Narceus annularus
Thyropygus sp
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
.
![]() |
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
Boore J. L., 2000 The duplication/random loss model for gene rearrangement exemplified by mitochondrial genomes of deuterostome animals Pp. 133147 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
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
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
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
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
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
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
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]