The Mitochondrial Genomes of Two Nematodes, Caenorhabditis Elegans and Ascaris Suum

The nucleotide sequences of the mitochondrial DNA (mtDNA) molecules of two nematodes, Caenorhabditis elegans [13,794 nucleotide pairs (ntp)], and Ascaris suum (14,284 ntp) are presented and compared. Each molecule contains the genes for two ribosomal RNAs (s-rRNA and l-rRNA), 22 transfer RNAs (tRNAs) and 12 proteins, all of which are transcribed in the same direction. The protein genes are the same as 12 of the 13 protein genes found in other metazoan mtDNAs: Cyt b, cytochrome b; COI-III, cytochrome c oxidase subunits I-III; ATPase6, Fo ATPase subunit 6; ND1-6 and 4L, NADH dehydrogenase subunits 1-6 and 4L: a gene for ATPase subunit 8, common to other metazoan mtDNAs, has not been identified in nematode mtDNAs. The C. elegans and A. suum mtDNA molecules both include an apparently noncoding sequence that contains runs of AT dinucleotides, and direct and inverted repeats (the AT region: 466 and 886 ntp, respectively). A second, apparently noncoding sequence in the C. elegans and A. suum mtDNA molecules (109 and 117 ntp, respectively) includes a single, hairpin-forming structure. There are only 38 and 89 other intergenic nucleotides in the C. elegans and A. suum mtDNAs, and no introns. Gene arrangements are identical in the C. elegans and A. suum mtDNA molecules except that the AT regions have different relative locations. However, the arrangement of genes in the two nematode mtDNAs differs extensively from gene arrangements in all other sequenced metazoan mtDNAs. Unusual features regarding nematode mitochondrial tRNA genes and mitochondrial protein gene initiation codons, previously described by us, are reviewed. In the C. elegans and A. suum mt-genetic codes, AGA and AGG specify serine, TGA specifies tryptophan and ATA specifies methionine. From considerations of amino acid and nucleotide sequence similarities it appears likely that the C. elegans and A. suum ancestral lines diverged close to the time of divergence of the cow and human ancestral lines, about 80 million years ago.     

Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.     

Genetic characterization of the mitochondrial DNA from Lepeophtheirus salmonis (Crustacea; Copepoda). A new gene organization revealed

The mitochondrial DNA (mtDNA) from the salmon louse, Lepeophtheirus salmonis, is 15445 bp. It includes the genes coding for cytochrome B (Cyt B), ATPase subunit 6 and 8 (A6 and A8), NADH dehydrogenase subunits 1-6 and 4L (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), cytochrome c oxidase subunits I-III (COI, COII and COIII), two rRNA genes (12S rRNA and 16S rRNA) and 22 tRNAs. Two copies of tRNA-Lys are present in the mtDNA of L. salmonis, while tRNA-Cys was not identified. Both DNA strands contain coding regions in the salmon louse, in contrast to the other copepod characterized Tigriopus japonicus, but only a few genes overlap. In vertebrates, ND4 and ND4L are transcribed as one bicistronic mRNA, and are therefore localized together. The same organization is also found in crustaceans, with the exceptions of T. japonicus, Neocalanus cristatus and L. salmonis that deviate from this pattern. Another exception of the L. salmonis mtDNA is that A6 and A8 do not overlap, but are separated by several genes. The protein-coding genes have a bias towards AT-rich codons. The mitochondrial gene order in L. salmonis differs significantly from the copepods T. japonicus, Eucalanus bungii, N. cristatus and the other 13 crustaceans previously characterized. Furthermore, the mitochondrial rRNA genes are encoded on opposite strands in L. salmonis. This has not been found in any other arthropods, but has been reported in two starfish species. In a phylogenetic analysis, using an alignment of mitochondrial protein sequences, L. salmonis groups together with T. japonicus, being distant relatives to the other crustaceans.     

Antibodies against synthetic peptides reveal that the unidentified reading frame A6L, overlapping the ATPase 6 gene, is expressed in human mitochondria

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to detect human mitochondrial gene products. In particular, antibodies directed against either the NH2-terminal decapeptide or the COOH-terminal undecapeptide of cytochrome c oxidase subunit II (COII) were both very effective in immunoprecipitating the previously identified COII polypeptide from an SDS lysate of mitochondria from HeLa cells. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the unidentified reading frame A6L, which overlaps the ATPase 6 gene, immunoprecipitated specifically a component (#25) of the HeLa cell mitochondrial translation products; antibodies directed against the NH2-terminal octapeptide also precipitated protein 25, although less efficiently. The size of protein 25, as estimated from its electrophoretic mobility, is compatible with its being the unidentified reading frame A6L product. Furthermore, a fingerprinting analysis of this protein after trypsin digestion has given results consistent with this identification.     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

A Novel Mitochondrial Genome Organization for the Blue Mussel, Mytilus Edulis

The sequence of 13.9 kilobases (kb) of the 17.1-kb mitochondrial genome of Mytilus edulis has been determined, and the arrangement of all genes has been deduced. Mytilus mitochondrial DNA (mtDNA) contains 37 genes, all of which are transcribed from the same DNA strand. The gene content of Mytilus is typically metazoan in that it includes genes for large and small ribosomal RNAs, for a complete set of transfer RNAs and for 12 proteins. The protein genes encode the cytochrome b apoenzyme, cytochrome c oxidase (CO) subunits I-III, NADH dehydrogenase (ND) subunits 1-6 and 4L, and ATP synthetase (ATPase) subunit 6. No gene for ATPase subunit 8 could be found. The reading frames for the ND1, COI, and COIII genes contain long extensions relative to those genes in other metazoan mtDNAs. There are 23 tRNA genes, one more than previously found in any metazoan mtDNA. The additional tRNA appears to specify methionine, making Mytilus mtDNA unique in having two tRNA(Met) genes. Five lengthy unassigned intergenic sequences are present, four of which vary in length from 79 to 119 nucleotides and the largest of which is 1.2 kb. The base compositions of these are unremarkable and do not differ significantly from that of the remainder of the mtDNA. The arrangement of genes in Mytilus mtDNA is remarkably unlike that found in any other known metazoan mtDNA.     

Sources of incongruence among mammalian mitochondrial sequences: COII, COIII, and ND6 genes are main contributors

To investigate the origins of incongruence among mammalian mitochondrial protein-coding genes, we compiled a matrix that included 13 protein-coding-genes for 41 mammals from 14 different orders. This matrix was examined for congruence using different partitioning strategies. The incongruence length difference test showed significant incongruence among the 13 gene partitions used simultaneously, and the result was not affected by third codon or transversion weighting. In the pair-wise comparisons, significant incongruence was detected between NADH:ubiquinone oxidoreductase subunit 6 gene (ND6), cytochrome oxidase subunit II (COII), or cytochrome oxidase subunit III (COIII) gene partitioned individually against the rest of the genes. Omission of any of the 14 mammalian orders alone or in combinations from the matrix did not result in a statistically significant improvement of congruence, suggesting that taxonomic sampling will not improve congruence among the data sets. However, omission of the ND6, COII, and COIII significantly improved congruence in our data matrix. Possible origins of unusual phylogenetic properties of the three genes are discussed.     

Understanding molecular biology of codon usage in mitochondrial complex IV genes of electron transport system: Relevance to mitochondrial diseases

The mitochondrial cytochrome oxidase (CO) genes are involved in complex IV of the electron transport system, and dysfunction of CO genes leads to several diseases. However, no work has been reported on the codon usage pattern of these genes. We used bioinformatic methods to analyze the compositional properties and the codon usage pattern of the COI, COII, and COIII genes in fishes, birds, and mammals to understand the similarities and dissimilarities of codon usage in these genes, which gave an insight into the molecular biology of these genes. The effective number of codons (ENC) value of genes was high in different species of fishes, birds and mammals, which indicates that the codon bias of CO genes was low and the ENC values were significantly different among fishes, birds, and mammals, as revealed from the t test. The overall guanine and cytosine (GC) content in fishes, birds, and mammals was lower than 50% in all genes, indicating that the genes were AT-rich and significantly different among fishes, birds, and mammals. The TCA codon was overrepresented in fishes, birds, and mammals for the COI gene, in birds and mammals for the COII gene, but it was not overrepresented in others. Only three codons, namely CTA, CGA, and AAA, were overrepresented in all three groups for the COI, COII, and COIII genes, repectively. From the neutrality plot in fishes, birds, and mammals, it was observed that the slopes of the regression lines (regression coefficients) in the COI, COII, and COIII genes were <0.5, suggesting that natural selection played a major role, whereas mutation pressure played a minor role.     

Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema

Animal mitochondrial DNA genomes are generally single circular molecules, 14-20 kb in size, containing a number of functional RNAs and 13 protein-coding genes. Among these, the COI, COII and COIII genes encode three subunits of cytochrome c oxidase. We have isolated and characterized these three mitochondrial genes from the mesozoan Dicyema, a primitive multicellular animal. Surprisingly, the COI, COII and COIII genes are encoded on three small, separate circular DNA molecules (minicircles) of length 1700, 1599 and 1697 bp, respectively. We estimated the copy number of each minicircle at 100 to 1000 per cell, and have shown a mitochondrial localization of the minicircles by in situ hybridization. Furthermore, we could not detect a putative "maxicircle" DNA molecule containing any combination of the COI, COII and COIII genes using either PCR or genomic Southern hybridization. Thus, our results show a novel mitochondrial genome organization in the mesozoan animal Dicyema.     

Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans

Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa₃). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits of the Paracoccus enzyme show remarkable homology to the corresponding subunits of the mitochondrial cytochrome oxidase. Possible protein products of the open reading frames have not yet been identified.     

Three patterns of mitochondrial DNA nucleotide divergence in the meadow vole,Microtus pennsylvanicus

Summary The DNA sequence was determined for the cytochrome c oxidase II (COII), tRNA^Lys, and ATPase 8 genes from the mitochondrial genome of the meadow vole, Microtus pennsylvanicus. When compared to other rodents, three different patterns of evolutionary divergence were found. Nucleotide variation in tRNA^Lys is concentrated in the TC loop. Nucleotide variation in the COII gene in three genera of rodents (Microtus, Mus, Rattus) consists predominantly of transitions in the third base positions of codons. The predicted amino acid sequence in highly conserved (>92% similarity). Analysis of the ATPase 8 gene among four genera (Microtus, Cricetulus, Mus, Rattus) revealed more detectable transversions than transitions, many fixed first and second position mutations, and considerable amino acid divergence. The rate of nucleotide substitution at nonsynonymous sites in the ATPase 8 gene is 10 times the rate in the COII gene. In contrast, the estimated absolute mutation rate as determined by analysis of nucleotide substitutions at fourfold degenerate sites probably is the same for the two genes. The primary sequences of the ATPase 8 and COII peptides are constrained differently, but each peptide is conserved in terms of predicted secondary-level configuration.     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

A novel mitochondrial gene order in the crinoid echinoderm Florometra serratissima

The complete nucleotide sequence of the mitochondrial genome of the crinoid Florometra serratissima has been determined. It is a circular DNA molecule, 16,005 bp in length, containing the genes for 13 proteins, small and large ribosomal RNAs, and 22 transfer RNAs (tRNAs). Three regions of unassigned sequence (UAS) greater than 73 bp have been located. The largest, UAS I, is 432 bp long and exhibits sequence similarity to the putative mitochondrial control regions seen in other animals. UAS II (77 bp) and UAS III (73 bp) are located between the 5' ends of coding sequences and may play roles as bidirectional promoters. Analyses of nucleotide composition revealed that the major peptide-encoding strand is high in T and low in C. This bias is reflected in a specific pattern of codon usage. Molecular phylogenetic analyses based on cytochrome c oxidase (COI, COII, and COIII) amino acid and nucleotide sequences did not resolve all the relationships between echinoderm classes. The overall animal mitochondrial gene content has been maintained in the crinoid, but there is extensive rearrangement with respect to both the echinoid and the asteroid mtDNA gene maps. Florometra serratissima has a novel genome organization in a segment containing most of the tRNA genes, large and small rRNA genes, and the NADH dehydrogenase subunit 1 and 2 genes. Potential pathways and mechanisms for gene rearrangements between mitochondrial gene maps of echinoderm classes and vertebrates are discussed as indicators of early deuterostome phylogeny.     

Molecular Evolution at the Cytochrome Oxidase Subunit 2 Gene Among Divergent Populations of the Intertidal Copepod, Tigriopus californicus

The cytochrome c oxidase subunit 2 gene (COII) encodes a highly conserved protein that is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX) crucial to the production of ATP during cellular respiration. Despite its integral role in electron transport, we have observed extensive intraspecific nucleotide and amino acid variation among 26 full-length COII sequences sampled from seven populations of the marine copepod, Tigriopus californicus. Although intrapopulation divergence was virtually nonexistent, interpopulation divergence at the COII locus was nearly 20% at the nucleotide level, including 38 nonsynonymous substitutions. Given the high degree of interaction between the cytochrome c oxidase subunit 2 protein (COX2) and the nuclear-encoded subunits of COX and cytochrome c (CYC), we hypothesized that some codons in the COII gene are likely to be under positive selection in order to compensate for amino acid substitutions in other subunits. Estimates of the ratio of nonsynonymous to synonymous substitution (ω), obtained using a series of maximum likelihood models of codon substitution, indicated that the majority of codons in T. californicus COII are under strong purifying selection (ω << 1), while approximately 4% of the sites in this gene appear to evolve under relaxed selective constraint (ω = 1). A branch-site maximum likelihood model identified three sites that may have experienced positive selection within the central California sequence clade in our COII phylogeny; these results are consistent with previous studies showing functional and fitness consequences among interpopulation hybrids between central and northern California populations. [Reviewing Editor: Dr. Willie Swanson]     

Novel features of metazoan mtDNA revealed from sequence analysis of three mitochondrial DNA segments of the land snail Albinaria turrita (Gastropoda: Clausiliidae)

The mitochondrial DNA (mtDNA) size of the terrestrial gastropod Albinaria turrita was determined by restriction enzyme mapping and found to be approximately 14.5 kb. Its partial gene content and organization were examined by sequencing three cloned segments representing about one-fourth of the mtDNA molecule. Complete sequences of cytochrome c oxidase subunit II (COII), and ATPase subunit 8 (ATPase8), as well as partial sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 6 (ND6), and the large ribosomal RNA (IrRNA) genes were determined. Nine putative tRNA genes were also identified by their ability to conform to typical mitochondrial tRNA secondary structures. An 82-nt sequence resembles a noncoding region of the bivalve Mytilus edulis, even though it might contain a tenth tRNA gene with an unusual 5-nt overlap with another tRNA gene. The genetic code of Albinaria turrita appears to be the same as that of Drosophila and Mytilus edulis. The structures of COI and COII are conservative, but those of ATPase8 and ND6 are diversified. The sequenced portion of thelrRNA gene (1,079 nt) is characterized by conspicuous deletions in the 5 and 3 ends; this gene represents the smallest coelomate IrRNA gene so far known. Sequence comparisons of the identified genes indicate that there is greater difference between Albinaria and Mytilus than between Albinaria and Drosophila. An evolutionary analysis, based on COII sequences, suggests a possible nonmonophyletic origin of molluskan mtDNA. This is supported also by the absence of the ATPase8 gene in the mtDNA of Mytilus and nematodes, while this gene is present in the mtDNA of Albinaria and Cepaea nemoralis and in all other known coelomate metazoan mtDNAs.