The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.     

Nucleotide Sequence and Gene Organization of the Starfish Asterina Pectinifera Mitochondrial Genome

The 16,260-bp mitochondrial DNA (mtDNA) from the starfish Asterina pectinifera has been sequenced. The genes for 13 proteins, two rRNAs and 22 tRNAs are organized in an extremely economical fashion, similar to those of other animal mtDNAs, with some of the genes overlapping each other. The gene organization is the same as that for another echinoderm, sea urchin, except for the inversion of a 4.6-kb segment that contains genes for two proteins, 13 tRNAs and the 16S rRNA. Judging from the organization of the protein coding genes, mammalian mtDNAs resemble the sea urchin mtDNA more than that of the starfish. The region around the 3' end of the 12S rRNA gene of the starfish shows a high similarity with those for vertebrates. This region encodes a possible stem and loop structure; similar potential structures occur in this region of vertebrate mtDNAs and also in nonmitochondrial small subunit rRNA. A similar stem and loop structure is also found at the 3' end of the 16S rRNA genes in A. pectinifera, in another starfish Pisaster ochraceus, in vertebrates and in Drosophila, but not in sea urchins. The full sequence data confirm the presumption that AGA/AGG, AUA and AAA codons, respectively, code for serine, isoleucine, and asparagine in the starfish mitochondria, and that AGA/AGG codons are read by tRNA(GCU)(Ser), which possesses a truncated dihydrouridine arm, that was previously suggested from a partial mtDNA sequence. The structural characteristics of tRNAs and possible mechanisms for the change in the mitochondrial genetic code are also discussed.     

Sequence Heteroplasmy of D-Loop and rRNA Coding Regions in Mitochondrial DNA from Holstein Cows of Independent Maternal Lineages

A mitochondrial DNA (mtDNA) fragment containing the D-loop, phenylalanine tRNA, valine tRNA, and 12S and 16 rRNA genes was cloned and sequenced from 36 cows of 18 maternal lineages to identify the polymorphic sites within those regions and to detect the existence of heteroplasmic mtDNA in cows. Seventeen variable sites were observed within the D-loop and rRNA coding regions of bovine mtDNA within a 2.5-kb span. The hypervariable sites in the D-loop and rRNA coding regions were identified at nucleotide positions 169, 216, and 1594. Heteroplasmic mtDNA (variable mtDNA within a tissue) existed extensively in cows and was detected within the above regions in 11 of 36 cows sequenced. The insertion, deletion, and nucleotide transversion polymorphisms were found only in homopolymer regions. Heteroplasmy was observed frequently and seemingly is persistent in cattle. Though heteroplasmy was demonstrated, most lineages and mtDNA sites showed no heteroplasmy.     

Mitochondrial DNA sequences of primates: Tempo and mode of evolution

Summary We cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of the evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.Abbreviations mtDNA mitochondrial DNA - bp base pair - URF unidentified reading frame     

The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, with a total size of 24,673 bp, was one of the smallest known mtDNAs of Pezizomycotina. It contained the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, a single intron that harbored an intronic ORF coding for a putative ribosomal protein (rps) within the large rRNA gene (rnl), and a set of 24 tRNA genes which recognized codons for all amino acids, except proline and valine. Gene order comparison with all known mtDNAs of Sordariomycetes illustrated a highly conserved genome organization for all the protein- and rRNA-coding genes, as well as three clusters of tRNA genes. By considering all mitochondrial essential protein-coding genes as one unit a phylogenetic study of these small genomes strongly supported the common evolutionary course of Sordariomycetes (100% bootstrap support) and highlighted the advantages of analyzing small genomes (mtDNA) over single genes. In addition, comparative analysis of three intergenic regions demonstrated sequence variability that can be exploited for intra- and inter-specific identification of Metarhizium. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Length Mutations in Human Mitochondrial DNA

By high-resolution, restriction mapping of mitochondrial DNAs purified from 112 human individuals, we have identified 14 length variants caused by small additions and deletions (from about 6 to 14 base pairs in length). Three of the 14 length differences are due to mutations at two locations within the D loop, whereas the remaining 11 occur at seven sites that are probably within other noncoding sequences and at junctions between coding sequences. In five of the nine regions of length polymorphism, there is a sequence of five cytosines in a row, this sequence being comparatively rare in coding DNA. Phylogenetic analysis indicates that, in most of the polymorphic regions, a given length mutation has arisen several times independently in different human lineages. The average rate at which length mutations have been arising and surviving in the human species is estimated to be many times higher for noncoding mtDNA than for noncoding nuclear DNA. The mystery of why vertebrate mtDNA is more prone than nuclear DNA to evolve by point mutation is now compounded by the discovery of a similar bias toward rapid evolution by length mutation.     

Drosophila Melanogaster Mitochondrial DNA: Gene Organization and Evolutionary Considerations

The sequence of a 8351-nucleotide mitochondrial DNA (mtDNA) fragment has been obtained extending the knowledge of the Drosophila melanogaster mitochondrial genome to 90% of its coding region. The sequence encodes seven polypeptides, 12 tRNAs and the 3' end of the 16S rRNA and CO III genes. The gene organization is strictly conserved with respect to the Drosophila yakuba mitochondrial genome, and different from that found in mammals and Xenopus. The high A + T content of D. melanogaster mitochondrial DNA is reflected in a reiterative codon usage, with more than 90% of the codons ending in T or A, G + C rich codons being practically absent. The average level of homology between the D. melanogaster and D. yakuba sequences is very high (roughly 94%), although insertion and deletions have been detected in protein, tRNA and large ribosomal genes. The analysis of nucleotide changes reveals a similar frequency for transitions and transversions, and reflects a strong bias against G + C on both strands. The predominant type of transition is strand specific.     

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base

Summary A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.     

Assessment of concordance among genealogical reconstructions from various mtDNA segments in three species of Pacific salmon (genus Oncorhynchus)

Seven segments of mitochondrial DNA (mtDNA), comprising 97% of the mitochondrial genome, were amplified by polymerase chain reaction (PCR) and examined for restriction site variation using 13 restriction endonucleases in three species of Pacific salmon: pink (Oncorhynchus gorbuscha), chum (O. keta) and sockeye (O. nerka) salmon. The distribution of variability across the seven mtDNA segments differed substantially among species. Little similarity in the distribution of variable restriction sites was found even between the mitochondrial genomes of the even- and odd-year broodlines of pink salmon. Significantly different levels of nucleotide diversity were detected among three groups of genes: six NADH-dehydrogenase genes had the highest; two rRNA genes had the lowest; and a group that included genes for ATPase and cytochrome oxidase subunits, the cytochrome b gene, and the control region had intermediate levels of nucleotide diversity. Genealogies of mtDNA haplotypes were reconstructed for each species, based on the variation in all mtDNA segments. The contributions of variation within different segments to resolution of the genealogical trees were compared within each species. With the exception of sockeye salmon, restriction site data from different genome segments tended to produce rather different trees (and hence rather different genealogies). In the majority of cases, genealogical information in different segments of mitochondrial genome was additive rather than congruent. This finding has a relevance to phylogeographic studies of other organisms and emphasizes the importance of not relying on a limited segment of the mtDNA genome to derive a phylogeographic structure.     

Distribution of nucleotide substitutions in human mitochondrial DNA genes

To analyze the distribution pattern of nucleotide substitutions in human mitochondrial DNA (mtDNA), mutational spectra of the mitochondrial genes were reconstructed. The reconstruction procedure is based on the mutation distribution data for 47 monophyletic mtDNA clusters, to which 794 examined mtDNA sequences encoding for tRNAs, rRNAs, and mitochondrial proteins are attributed. One of specific features of mitochondrial mutational spectra revealed was homoplasy of the mutations (the mean mutation number per variable nucleotide site in the coding region varied from 1.09 to 1.43). It was established that in the mtDNA genes maximum mutational constraint fell onto the guanine bases, albeit the content of these bases in the mtDNA L-chains was minimal. Maximal bias towards parallel G to A transitions was observed for rRNA genes, with the protein- and tRNA-encoding genes ranking next. Despite the fact that the differences in the average G-nucleotides content and variability between the genes of two mtDNA segments located between the OriH and OriL were statistically significant, the results did not provide the conclusion that the G-nucleotide instability observed in the mtDNA L-spectra was determined by the mechanism of asynchronous mtDNA replication, along with the deamination of cytosines in the H-chain regions, which remained single-stranded during replication.     

New polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with non-syndromic hearing loss

The 12S rRNA gene was shown to be a hot spot for aminoglycoside-induced and non-syndromic hearing loss since several deafness-associated mtDNA mutations were identified in this gene. Among them, we distinguished the A1555G, the C1494T and the T1095C mutations and C-insertion or deletion at position 961. One hundred Tunisian patients with non-syndromic hearing loss and 100 hearing individuals were analysed in this study. A PCR-RFLP analysis with HaeIII restriction enzyme showed the presence of the A1555G mutation in the 12S rRNA gene in only one out of the 100 patients. In addition, PCR-RFLP and radioactive PCR revealed the presence of a new HaeIII polymorphic restriction site in the same gene of 12S rRNA site in 4 patients with non-syndromic hearing loss. UVIDOC-008-XD analyses showed the presence of this new polymorphic restriction site with a variable heteroplasmic rates at position +1517 of the human mitochondrial genome. On the other hand, direct sequencing of the entire mitochondrial 12S rRNA gene in the 100 patients and in 100 hearing individuals revealed the presence of the A750G and A1438G polymorphisms and the absence of the C1494T, T1095C and 961insC mutations in all the tested individuals. Sequencing of the whole mitochondrial genome in the 4 patients showing the new HaeIII polymorphic restriction site revealed only the presence of the A8860G transition in the MT-ATP6 gene and the A4769G polymorphism in the ND2 gene.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

Amino acid change associated with the major polymorphic Hinc II site of Oriental and Caucasian mitochondrial DNAs.

The mitochondrial DNAs (mtDNAs) from 116 Oriental and Caucasian blood samples were analyzed for their Hinc II restriction endonuclease cleavage patterns using Southern analysis and 32P human mtDNA probes. Seven distinct patterns were found, all of which could be interrelated by single nucleotide changes. The predominant pattern (mtHinc II-2) was found in 97% of the Caucasians and 73% of the Orientals. This mtDNA morph had one more Hinc II site than did the second most common morph (mtHinc II-1), which was found only in 20% of the Orientals. Three additional patterns were in a single Oriental sample, a fourth in a single Caucasian sample, and a fifth in one member of each population. The polymorphic site that differentiated mtHinc II-1 and mtHinc II-2 was cloned and sequenced. A single nucleotide change was found that created an Hinc II site and changed the amino acid sequence of the URF5 gene. Comparison of these sequences with those of other primates [15] revealed that the Asian mtHinc II-1 and mtHinc II-4 mtDNAs were identical in this region with those of chimpanzees and orangutans. These results suggest that the Asian mtHinc II-1 mtDNA may have been ancestral to other human mtDNAs.     

Complete DNA Sequence of the Mitochondrial Genome of the Black Chiton, Katharina Tunicata

The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for tRNAs. The gene arrangement resembles those of arthropods much more than that of another mollusc, the bivalve Mytilus edulis. Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however, at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is unpaired in tRNA(ser(AGN)), which is typical of metazoan mtDNAs, and also in tRNA(ser(UCN)), a condition found previously only in nematode mtDNAs. There are two additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA(glu) and CO3 contains a 72-bp tract of alternating A and T.     

The Rates of Molecular Evolution in Rodent and Primate Mitochondrial DNA

A higher rate of molecular evolution in rodents than in primates at synonymous sites and, to a lesser extent, at amino acid replacement sites has been reported previously for most nuclear genes examined. Thus in these genes the average ratio of amino acid replacement to synonymous substitution rates in rodents is lower than in primates, an observation at odds with the neutral model of molecular evolution. Under Ohta's mildly deleterious model of molecular evolution, these observations are seen as the consequence of the combined effects of a shorter generation time (driving a higher mutation rate) and a larger effective population size (resulting in more effective selection against mildly deleterious mutations) in rodents. The present study reports the results of a maximum-likelihood analysis of the ratio of amino acid replacements to synonymous substitutions for genes encoded in mitochondrial DNA (mtDNA) in these two lineages. A similar pattern is observed: in rodents this ratio is significantly lower than in primates, again consistent only with the mildly deleterious model. Interestingly the lineage-specific difference is much more pronounced in mtDNA-encoded than in nuclear-encoded proteins, an observation which is shown to run counter to expectation under Ohta's model. Finally, accepting certain fossil divergence dates, the lineage-specific difference in amino acid replacement-to-synonymous substitution ratio in mtDNA can be partitioned and is found to be entirely the consequence of a higher mutation rate in rodents. This conclusion is consistent with a replication-dependent model of mutation in mtDNA. Received: 24 September 1999 / Accepted: 18 September 2000     

The design of synthetic genes

Computer programs are described that aid in the design of synthetic genes coding for proteins that are targets of a research program in site directed mutagenesis. These programs "reverse-translate" protein sequences into general nucleic acid sequences (those where codons have not yet been selected), map restriction sites into general DNA sequences, identify points in the synthetic gene where unique restriction sites can be introduced, and assist in the design of genes coding for hybrids and evolutionary intermediates between homologous proteins. Application of these programs therefore facilitates the use of modular mutagenesis to create variants of proteins, and the implementation of evolutionary guidance as a strategy for selecting mutants.     

Distribution of nucleotide substitutions in human mitochondrial DNA genes

To analyze the distribution pattern of nucleotide substitutions in human mitochondrial DNA (mtDNA), mutational spectra of the mitochondrial genes were reconstructed. The reconstruction procedure is based on the mutation distribution data for 47 monophyletic mtDNA clusters, to which 794 examined mtDNA sequences encoding for tRNAs, rRNAs, and mitochondrial proteins are attributed. One of specific features of mitochondrial mutational spectra revealed was homoplasy of the mutations (the mean mutation number per variable nucleotide site in the coding region varied from 1.09 to 1.43). It was established that in the mtDNA genes maximum mutational constraint fell onto the guanine bases, albeit the content of these bases in the mtDNA L-chains was minimal. Maximal bias towards parallel G to A transitions was observed for rRNA genes, with the protein-and tRNA-encoding genes ranking next. Despite the fact that the differences in the average G-nucleotides content and variability between the genes of two mtDNA segments located between the OriH and OriL were statistically significant, the results did not provide the conclusion that the G-nucleotide instability observed in the mtDNA L-spectra was determined by the mechanism of asynchronous mtDNA replication, along with the deamination of cytosines in the H-chain regions, which remained single-stranded during replication.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 93–99.Original Russian Text Copyright © 2005 by Malyarchuk.