Complete Mitochondrial DNA Sequence Research Articles

The complete mitochondrial DNA sequence of the shark Mustelus manazo: evaluating rooting contradictions to living bony vertebrates.

A remarkable example of a misleading mitochondrial protein tree is presented, involving ray-finned fishes, coelacanths, lungfishes, and tetrapods, with sea lampreys as an outgroup. In previous molecular phylogenetic studies on the origin of tetrapods, ray-finned fishes have been assumed as an outgroup to the tetrapod/lungfish/coelacanth clade, an assumption supported by morphological evidence. Standard methods of molecular phylogenetics applied to the protein-encoding genes of mitochondria, however, give a bizarre tree in which lamprey groups with lungfish and, therefore, ray-finned fishes are not the outgroup to a tetrapod/lungfish/coelacanth clade. All of the dozens of published phylogenetic methods, including every possible modification to maximum likelihood known to us (such as inclusion of site heterogeneity and exclusion of potentially misleading hydrophobic amino acids), fail to place the ray-finned fishes in a biologically acceptable position. A likely cause of this failure may be the use of an inappropriate outgroup. Accordingly, we have determined the complete mitochondrial DNA sequence from the shark, Mustelus manazo, which we have used as an alternative and more proximal outgroup than the lamprey. Using sharks as the outgroup, lungfish appear to be the closest living relative of tetrapods, although the possibility of a lungfish/coelacanth clade being the sister group of tetrapods cannot be excluded.

Searching for the closest living relative(s) of tetrapods through evolutionary analyses of mitochondrial and nuclear data.

The phylogenetic relationships of the African lungfish (Protopterus dolloi) and the coelacanth (Latimeria chalumnae) with respect to tetrapods were analyzed using complete mitochondrial genome DNA sequences. A lungfish + coelancanth clade was favored by maximum parsimony (although this result is dependent on which transition:transversion weights are applied), and a lungfish + tetrapod clade was supported by neighbor-joining and maximum-likelihood analyses. These two hypotheses received the strongest statistical and bootstrap support to the exclusion of the third alternative, the coelacanth + tetrapod sister group relationship. All mitochondrial protein coding genes combined favor a lungfish + tetrapod grouping. We can confidently reject the hypothesis that the coelacanth is the closest living relative of tetrapods. When the complete mitochondrial sequence data were combined with nuclear 28S rRNA gene data, a lungfish + coelacanth clade was supported by maximum parsimony and maximum likelihood, but a lungfish + tetrapod clade was favored by neighbor-joining. The seeming conflicting results based on different data sets and phylogenetic methods were typically not statistically strongly supported based on Kishino-Hasegawa and Templeton tests, although they were often supported by strong bootstrap values. Differences in rate of evolution of the different mitochondrial genes (slowly evolving genes such as the cytochrome oxidase and tRNA genes favored a lungfish + coelacanth clade, whereas genes of relatively faster substitution rate, such as several NADH dehydrogenase genes, supported a lungfish + tetrapod grouping), as well as the rapid radiation of the lineages back in the Devonian, rather than base compositional biases among taxa seem to be directly responsible for the remaining uncertainty in accepting one of the two alternate hypotheses.

Complete mitochondrial DNA sequence of the fat dormouse, Glis glis: further evidence of rodent paraphyly.

The complete mitochondrial genome of the fat dormouse, Glis glis, has been sequenced (16,602 bp). A total of 23 complete mitochondrial mammalian genomes have been taken into account for phylogenetic reconstruction. Phylogenetic analyses were performed with parsimony, distance (stationary Markov model), and maximum-likelihood methods. In all cases, data strongly support the paraphyly of rodents, with dormouse and guinea pig in a different clade from rat and mouse, reaching bootstrap values of 95%. Rodent monophyly and the existence of Glires (Rodentia and Lagomorpha) are weakly supported, with maximum bootstrap values of 11% and 8.6%, respectively. This result agrees with the analyses of isochore patterns in the nuclear genome and the B2 and B2-like retroposons, which show a close relationship between dormice and guinea pigs rather than between dormice and rats and mice.

Measuring Inconsistency in Phylogenetic Trees

Suppose that we seek a treeTgiving the phylogenetic relationships among the species in a setS. A common method selects for such a tree a maximum parsimony tree using the genome of the species inS. Suppose thatKis a proper subset ofS. ThenTinduces a treeUwhich gives the same relationships among the species inKbut omits the species ofSwhich are not inK. Unfortunately, whenTis a maximum parsimony tree for the species inS, thenUneed not be a maximum parsimony tree for the species inK. This phenomenon exhibits an inconsistency in the criterion of maximum parsimony—maximum parsimony trees for different groups of species may be “inconsistent”. It implies that the addition of a new species scan change relationships already “established” for prior species if the trees are obtained by the criterion of maximum parsimony. The phenomenon occurs both in artificial examples and with real data.An alternative method for generating phylogenetic trees seeks to minimize such inconsistencies. For each groupJconsisting of four of the species, we find a treeT(J) describing the relationship only among the four species inJ, for example by the use of maximum parsimony on those four species alone. In favorable cases one may combine all the treesT(J) into a single treeTthat is consistent with all the treesT(J). If such a treeTexists, then it is unique, and there is a computationally efficient algorithm for finding the treeT. In unfavorable bases such a treeTdoes not exist, but there may still be a tree containing only “mild” inconsistencies with the treesT(J). A numerical measure is given for the inconsistencyI(T) of a treeTin terms of the treelengths of the various trees with setJof leaves in comparison with the treeT. We may then seek a “minimally inconsistent treeT” that minimizes the inconsistencyI(T). We describe procedures which find a treeTwith low inconsistencyI(T). Examples are provided using both artificial strings and data from the complete mitochondrial DNA sequences for 16 species. In particular, minimally inconsistent trees are identified for the 16 species. The definition permits a proof that the trees are in fact minimally inconsistent. The criterion can be applied in both a relative and an absolute sense.

Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus L.: conservation of major features among arthropod classes.

Numerous complete mitochondrial DNA sequences have been determined for species within two arthropod groups, insects and crustaceans, but there are none for a third, the chelicerates. Most mitochondrial gene arrangements reported for crustaceans and insect species are identical or nearly identical to that of Drosophila yakuba. Sequences across 36 of the gene boundaries in the mitochondrial DNA (mtDNA) of a representative chelicerate. Limulus polyphemus L., also reveal an arrangement like that of Drosophila yakuba. Only the position of the tRNA(LEU)(UUR) gene differs; in Limulus it is between the genes for tRNA(LEU)(CUN) and ND1. This positioning is also found in onychophorans, mollusks, and annelids, but not in insects and crustaceans, and indicates that tRNA(LEU)(CUN)-tRNA(LEU)(UUR)-ND1 was the ancestral gene arrangement for these groups, as suggested earlier. There are no differences in the relative arrangements of protein-coding and ribosomal RNA genes between Limulus and Drosophila, and none have been observed within arthropods. The high degree of similarity of mitochondrial gene arrangements within arthropods is striking, since some taxa last shared a common ancestor before the Cambrian, and contrasts with the extensive mtDNA rearrangements occasionally observed within some other metazoan phyla (e.g., mollusks and nematodes).

The complete mitochondrial DNA sequence of the bichir (Polypterus ornatipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order.

The evolutionary position of bichirs is disputed, and they have been variously aligned with ray-finned fish (Actinopterygii) or lobe-finned fish (Sarcopterygii), which also include tetrapods. Alternatively, they have been placed into their own group, the Brachiopterygii. The phylogenetic position of bichirs as possibly the most primitive living bony fish (Osteichthyes) made knowledge about their mitochondrial genome of considerable evolutionary interest. We determined the complete nucleotide sequence (16,624 bp) of the mitochondrial genome of a bichir, Polypterus ornalipinnis. Its genome contains 13 proteincoding genes, 22 tRNAs, two rRNAs and one major noncoding region. The genome's structure and organization show that this is the most basal vertebrate that conforms to the consensus vertebrate mtDNA gene order. Bichir mitochondrial protein-coding and ribosomal RNA genes have greater sequence similarity to ray-finned fish than to either lamprey or lungfish. Phylogenetic analyses suggest the bichir's placement as the most basal living member of the ray-finned fish and rule out its classification as a lobefinned fish. Hence, its lobe-fins are probably not a shared-derived trait with those of lobe-finned fish (Sarcopterygii).

The Mitochondrial DNA of Allomyces macrogynus: The Complete Genomic Sequence from an Ancestral Fungus

We have determined the complete nucleotide sequence of the circular mitochondrial DNA (mtDNA) of the chytridiomycete fungus, Allomyces macrogynus(57,473 bp; A + T content 60.5%). The identified genes that are typical for most fungal mitochondria include those for the large ( rnl) and small subunit ( rns) ribosomal RNAs, a complete set of 25 tRNAs, three ATPase subunits ( atp6, atp8and atp9), apocytochrome b(cob), three subunits of the cytochrome oxidase complex ( cox1, cox2and cox3), and seven subunits of the NADH dehydrogenase complex ( nad1, nad2, nad3, nad4, nad4L, nad5and nad6). A total of 28 introns of both groups are found, some of which contain open reading frames (ORFs) coding for potential endonucleases (group I) or reverse-transcriptases (group II). All mitochondrial genes are transcribed from the same DNA strand, as is the case in many other eufungi. Particular features of the A. macrogynus mtDNA include: (1) the first documented case of a fungal mitochondrial ribosomal protein gene ( rps3) that is clearly identified by similarity with bacterial homologues; (2) four unique ORFs; (3) the presence of an insert in the atp6gene that may have been acquired by interspecific transfer; (4) more than 67 short, highly structured and conserved DNA elements inserted in intergenic spacers, introns, and variable regions of the rnland rnsgenes: these elements are unusually G + C rich; (5) rRNA structures that resemble more closely those of eubacteria than their counterparts in other fungal mitochondria. The high degree of conservation of the A. macrogynusmitochondrial rRNA secondary structures, the existence of a mitochondrial rps3gene (common to protist but unique in fungal mtDNAs), and phylogenetic relationships inferred from highly conserved protein genes, demonstrate consistently the ancestral character of this fungal mitochondrial genome.

Chloroplast and Mitochondrial Genomes from a Liverwort,Marchantia polymorpha—Gene Organization and Molecular Evolution—

The complete nucleotide sequence of chloroplast DNA (121,025 basepairs, bp) from a liverwort, Marchantia polymorpha, has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. We also identified the complete nucleotide sequence of the liverwort mitochondrial DNA and deduced 96 possible genes in the sequence of 186,608 bp. The complete nucleotide sequence from chloroplast DNA comprises twenty introns (19 group II and 1 group I) in 18 different genes. One of the chloroplast group II introns separated the ribosomal protein gene in the trans-position. The mitochondrial genome also has thirty-two introns (25 group II and 7 group I) in the coding regions of 17 genes. From the evolutionary point of view, we describe the origin of organellar introns which gave the evidence for their vertical and horizontal transfers and their intragenic propagation, and RNA editing which was apparently lacking in the liverwort chloroplast and mitochondrial genomes.

Complete Sequence of the Mitochondrial DNA of the Rhodophyte Chondrus crispus(Gigartinales). Gene Content and Genome Organization

The complete nucleotide sequence of the circular mitochondrial (mt) DNA from the red alga Chondrus crispuswas determined (25, 836 nucleotides, A+T content 72.1%). Fifty one genes were identified. They include genes encoding three subunits of the cytochrome oxidase ( cox1 to 3), apocytochrome b ( cob), seven subunits of the NADH dehydrogenase complex ( nad1to 6, nad4L), two ATPase subunits ( atp6and atp9), three ribosomal RNAs ( rrn5, srnand Irn), 23 tRNAs and four ribosomal proteins ( rps3, rps11, rps12and rpl16). Two subunits of the succinate dehydrogenase complex ( sdhBand sdhC), usually found on nuclear genomes, are also located on the mtDNA of C. crispus.One group IIb intron is inserted in the tRNA IIegene. Six potentially functional open reading frames were identified, four of them having counterparts among green plant mtDNAs. The use of a modified genetic code and the absence of RNA editing, previously reported for the cox3gene, appears as a general characteristic of this molecule. Mitochondrial genes are encoded on both DNA strands, in two opposite major transcriptional directions, suggesting the existence of two main transcriptional units. Two long and stable stem-loops were identified in intergenic regions, which are believed to be involved with transcription and replication. The main structural features of this genome are compared with the overall organization of mtDNAs and are discussed in view of the evolution of mitochondria.

The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria

The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3' downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.

The complete mitochondrial DNA sequence of the horse, Equus caballus: extensive heteroplasmy of the control region.

The sequence of the mitochondrial (mt) DNA of the horse (Equus caballus) was determined. The length of the sequence presented is 16,660 bp. This figure, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif GTGCACCT in the control region of different molecules. Boundaries of the 13 peptide-coding genes were determined by the presence of start and stop codons, and by analogy with other eutherian mtDNAs. Three genes (COIII, NADH3 and NADH4) were not terminated by a stop codon. Comparison among the peptide-coding genes of the horse and eight other mammals suggests that the boundaries of some mt genes should be redefined. The number of repeats in the control region was determined by sequencing 77 different clones (20 direct plus 57 PCR clones). The number of repeats ranged from 2 to 29. There was a pronounced overrepresentation of clones with many repeats (22-27). Very few clones had a repeat number that was close to the mean number of repeats.

Complete Sequence of the Mitochondrial DNA of the Chlorophyte Alga Prototheca wickerhamii: Gene Content and Genome Organization

The complete nucleotide sequence of the circular mitochondrial (mt) DNA of the chlorophyte alga Prototheca wickerhamii has been determined (55,328 base-pairs, A+T content 74·2%). The genes identified encode three subunits of the cytochome oxidase, apocytochrome b, nine subunits of the NADH dehydrogenase complex ( nad1 to 7, nad4L and nad9), three ATPase subunits ( atp6, atp9, atp1 (also referred to as atpA )), three ribosomal RNAs (5 S ( rrn5), small subunit ( srn) and large subunit ( lrn) RNA), 26 tRNAs, and 13 ribosome proteins. A total of five group I introns reside in lrn and cox1, two of which include intronic open reading frames (ORFs). Five free-standing ORFs longer than 60 codons are present. Three of these ORFs are counterparts to genes encoding proteins of unknown function in plant mitochondria ( orf25 and orfB of angiosperms and orf244 of liverwort), whereas two of them are unique. Mitochondrial genes are encoded on both DNA strands in a way that suggest the existence of two transcription units, each including approximately one half of the mitochondrial genome. The two intergenic regions in which transcription is believed to initiate and terminate are about ten times longer than other intergenic regions (1118 and 1993 nt versus 100 to 150 nt). A total of 29 recurring sequence motifs (30 to 200 nt long) have been found in intergenic regions. Nine different types of motifs are present, most of them arranged as tandem repeats. These motifs may be implicated in transcription, e.g. as signals for initiation and/or processing. Phylogenetic analysis on the basis of the cox1 gene strongly suggested that P. wickerhamii and plant mitochondrial genomes are monophyletic. The finding of plant-specific mitochondrial genes such as orf25, orf244, orfB and rrn5 in P. wickerhamii mitochondria corroborates this idea.

A method for calibrating molecular clocks and its application to animal mitochondrial DNA.

A generalized least-squares procedure is introduced for the calibration of molecular clocks and applied to the complete mitochondrial DNA sequences of 13 animal species. The proposed technique accounts for both nonindependence and heteroscedasticity of molecular-distance data, problems that have not been taken into to account in such analyses in the past. When sequence-identity data are transformed to account for multiple substitutions/site, the molecular divergence scales linearly with time, but with substantially more variation in the substitution rate than expected under a Poisson model. Significant levels of divergence are predicted at zero divergence time for most loci, suggesting high levels of site-specific heterozygosity among mtDNA molecules establishing in sister taxa. For nearly all loci, the baseline heterozygosity is lower and the substitution rate is higher in mammals relative to other animals. There is considerable variation in the evolutionary rate among loci but no compelling evidence that the average rate of mtDNA evolution is elevated with respect to that of nuclear DNA. Using the observed patterns of interspecific divergence, empirical estimates are derived for the mean coalescence times of organelles colonizing sister taxa.

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA : A primitive form of plant mitochondrial genome

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H +-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 ± 10.9%, 37.0 ± 13.2% and 26.4 ± 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes ( rrn18 and rrn26), a transfer RNA gene ( trnS) and a pseudogene (ψ nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.

Mitochondrial genome in animal cells

In the past decade, the development of new DNA, RNA, and protein technologies has greatly incremented the knowledge about the organization and expression of mitochondrial DNA. The complete base sequence of mitochondrial DNA of several animals is known and many data are rapidly accumulating on the mitochondrial genomes of other systems. Here we discuss the results so far obtained that disclosed unexpected features of mitochondrial genetics. Furthermore, mitochondrial DNA has become established as a powerful tool for evolutionary studies in animals. Evidences are presented demonstrating that the evolution of mitochondrial DNA has proceeded in different ways in the various taxonomic groups. Data on heteroplasmic animals, which demonstrate the rapid evolution of mitochondrial DNA, are also presented.

41] Analysis of human mitochondrial RNA

Publisher Summary This chapter focuses on the human mitochondrial RNA. The powerful molecular techniques have allowed the dissection of the mammalian, in particular human, mitochondrial genome with a resolution that has approached and often surpassed that of the finest genetic analysis. Two fundamental approaches are followed in these investigations. First, with the availability of the cloning technology and of rapid sequencing methods, it has been possible to determine the complete sequence of mitochondrial DNA (mtDNA) from several mammalian species and the partial sequence of mtDNA from other species. Second, the transcription of mtDNA in HeLa cells is analyzed in depth, and the structural, metabolic, and mapping properties of the transcription products are described. The availability of a method of HeLa cell growth giving a high yield of cells and the development of in vivo labeling procedures producing mitochondrial nucleic acids of high specific activity have alleviated the difficulties associated with the presence of the mitochondrial RNA species in extremely small amounts. Sensitive techniques are used for sequencing, mapping, and studying the metabolism of mitochondrial RNAs.

Complete sequence of bovine mitochondrial DNA conserved features of the mammalian mitochondrial genome

We present here the complete 16,338 nucleotide DNA sequence of the bovine mitochondrial genome. This sequence is homologous to that of the human mitochondrial genome (Anderson et al., 1981) and the genes are organized in virtually identical fashion. The bovine mitochondrial protein genes are 63 to 79% homologous to their human counterparts, and most of the nucleotide differences occur in the third positions of codons. The minimum rate of base substitution that accounts for the nucleotide differences in the codon third positions is very high: at least 6 × 10 −9 changes per position per year. The bovine and human mitochondrial transfer RNA genes exhibit more interspecies variation than do their cytoplasmic counterparts, with the “TΨC” loop being the most variable part of the molecule. The bovine 12 S and 16 S ribosomal RNA genes, when compared with those from human mitochondrial DNA, show conserved features that are consistent with proposed secondary structure models for the ribosomal RNAs. Unlike the pattern of moderate-to-high homology between the bovine and human mitochondrial DNAs found over most of the genome, the DNA sequence in the bovine D-loop region is only slightly homologous to the corresponding region in the human mitochondrial genome. This region is also quite variable in length, and accounts for the bulk of the size difference between the human and bovine mitochondrial DNAs.

Different pattern of codon recognition by mammalian mitochondrial tRNAs.

Analysis of an almost complete mammalian mitochondrial DNA sequence has identified 23 possible tRNA genes and we speculate here that these are sufficient to translate all the codons of the mitochondrial genetic code. This number is much smaller than the minimum of 31 required by the wobble hypothesis. For each of the eight genetic code boxes with four codons for one amino acid we find a single specific tRNA gene with T in the first (wobble) position of the anticodon. We suggest that these tRNAs with U in the wobble position can recognize all four codons in these genetic code boxes either by a "two out of three" base interaction or by U.N wobble.