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.     

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.     

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.     

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.     

Identification of Japanese <Emphasis Type="Italic">Lymantria</Emphasis> species (Lepidoptera: Lymantriidae) based on DNA sequences

We examined the utility of the cytochrome c oxidase subunit I (COI) and II (COII) genes of mitochondrial DNA (mtDNA) as a tool to identify nine Japanese Lymantria species, including four Asian gypsy moth species [Lymantria dispar japonica (Motschulsky), Lymantria umbrosa (Butler), Lymantria albescens Hori and Umeno, and Lymantria postalba Inoue]. In phylogenetic trees constructed for the COI and COII genes using the maximum likelihood methods, we could identify seven out of the nine Lymantria species (L. albescens and L. postalba could not be identified). These results suggest that the DNA sequences of the COI and COII genes are useful for identifying Japanese Lymantria species.     

Identification and transcription of transfer RNA genes in dinoflagellate plastid minicircles

The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene (psbD, psbE, petD) and one that appears to be an "empty" circle. Using the tRNA prediction programs ARAGORN and tRNAscan-SE, tRNA-Met was found in the petD circle immediately downstream of the end of petD, while tRNA-Trp and tRNA-Pro were detected in the psbE and petD circles as well as in several chimeric circles of H. triquetra and the psbA minicircles of Heterocapsa pygmaea. RT-PCR showed that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA.     

Mitochondrial DNA Restriction Map and Cytochrome c Oxidase Subunits I and II Sequence Divergence of Corn Stalk Borer Sesamia nonagrioides (Lepidoptera: Noctuidae)

Corn stalk borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) is among the most important insect pests of corn in the Mediterranean basin. The mitochondrial DNA of this insect was purified and a restriction map was constructed. The size of the mtDNA genome is 16.3 kb. Genetic analysis of four corn stalk borer populations, collected from Greece (three populations) and Spain (one population), was undertaken using DNA sequences of the mtDNA cytochrome oxidase (CO) I and II genes. Sequencing of a 2079 bp region of these genes revealed 25 polymorphic sites among the populations. Five molecular RFLP markers, located in the mtDNA COI and COII genes, were surveyed, and two different haplotypes were detected. Phylogenetic analysis based on COI/COII nucleotide sequences revealed genetic differentiation between samples, and the results are discussed in relation to the geographic distribution of the corn stalk borer in two Mediterranean countries.     

Mitochondrial DNA restriction map and cytochrome c oxidase subunits I and II sequence divergence of corn stalk borer Sesamia nonagrioides (Lepidoptera: Noctuidae)

Corn stalk borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) is among the most important insect pests of corn in the Mediterranean basin. The mitochondrial DNA of this insect was purified and a restriction map was constructed. The size of the mtDNA genome is 16.3 kb. Genetic analysis of four corn stalk borer populations, collected from Greece (three populations) and Spain (one population), was undertaken using DNA sequences of the mtDNA cytochrome oxidase (CO) I and II genes. Sequencing of a 2079 bp region of these genes revealed 25 polymorphic sites among the populations. Five molecular RFLP markers, located in the mtDNA COI and COII genes, were surveyed, and two different haplotypes were detected. Phylogenetic analysis based on COI/COII nucleotide sequences revealed genetic differentiation between samples, and the results are discussed in relation to the geographic distribution of the corn stalk borer in two Mediterranean countries.     

Evolution of multicellular animals as deduced from 5S rRNA sequences: a possible early emergence of the Mesozoa.

The nucleotide sequences of 5S rRNA from a mesozoan Dicyema misakiense and three metazoan species, i.e., an acorn-worm Saccoglossus kowalevskii, a moss-animal Bugula neritina, and an octopus Octopus vulgaris have been determined. A phylogenic tree of multicellular animals has been constructed from 73 5S rRNA sequences available at present including those from the above four sequences. The tree suggests that the mesozoan is the most ancient multicellular animal identified so far, its emergence time being almost the same as that of flagellated or ciliated protozoans. The branching points of planarians and nematodes are a little later than that of the mesozoan but are clearly earlier than other metazoan groups including sponges and jellyfishes. Many metazoan groups seem to have diverged within a relatively short period.     

The complete mitochondrial genome of the damsel bug Alloeorhynchus bakeri (Hemiptera: Nabidae)

The complete sequence of the mitochondrial DNA (mtDNA) of the damsel bug, Alloeorhynchus bakeri, has been completed and annotated in this study. It represents the first sequenced mitochondrial genome of heteropteran family Nabidae. The circular genome is 15, 851 bp in length with an A+T content of 73.5%, contains the typical 37 genes that are arranged in the same order as that of the putative ancestor of hexapods. Nucleotide composition and codon usage are similar to other known heteropteran mitochondrial genomes. All protein-coding genes (PCGs) use standard initiation codons (methionine and isoleucine), except COI, which started with TTG. Canonical TAA and TAG termination codons are found in eight protein-coding genes, the remaining five (COI, COII, COIII, ND5, ND1) have incomplete termination codons (T or TA). PCGs of two strands present opposite CG skew which is also reflected by the nucleotide composition and codon usage. All tRNAs have the typical clover-leaf structure, except the dihydrouridine (DHU) arm of tRNA(Ser (AGN))which forms a simple loop as known in many other metazoa. Secondary structure models of the ribosomal RNA genes of A. bakeri are presented, similar to those proposed for other insect orders. There are six domains and 45 helices and three domains and 27 helices in the secondary structures of rrnL and rrnS, respectively. The major non-coding region (also called control region) between the small ribosomal subunit and the tRNA(Ile )gene includes two special regions. The first region includes four 133 bp tandem repeat units plus a partial copy of the repeat (28 bp of the beginning), and the second region at the end of control region contains 4 potential stem-loop structures. Finally, PCGs sequences were used to perform a phylogenetic study. Both maximum likelihood and Bayesian inference analyses highly support Nabidae as the sister group to Anthocoridae and Miridae.     

Kinetoplast DNA minicircles encode guide RNAs for editing of cytochrome oxidase subunit III mRNA.

Guide RNAs (gRNAs) for the editing of sites 1-8 of COIII mRNA and an "unexpected" partially edited COIII mRNA are encoded in the variable regions of specific kinetoplast DNA minicircles. The gRNAs can form 37 and 44 nucleotide perfect hybrids (allowing for G-U base pairs) with edited mRNAs. The gRNAs were detected on Northern blots and shown to have unique 5' ends situated close to the beginning of the potential base pairing with the edited mRNAs. We suggest that kinetoplast DNA minicircle molecules in general may encode gRNAs for editing of cryptogene mRNAs by a mechanism similar to that previously proposed for editing by maxicircle-encoded gRNAs.     

Use of mitogenomic information in teleostean molecular phylogenetics: a tree-based exploration under the maximum-parsimony optimality criterion

We explored the phylogenetic utility and limits of the individual and concatenated mitochondrial genes for reconstructing the higher-level relationships of teleosts, using the complete (or nearly complete) mitochondrial DNA sequences of eight teleosts (including three newly determined sequences), whose relative phylogenetic positions were noncontroversial. Maximum-parsimony analyses of the nucleotide and amino acid sequences of 13 protein-coding genes from the above eight teleosts, plus two outgroups (bichir and shark), indicated that all of the individual protein-coding genes, with the exception of ND5, failed to recover the expected phylogeny, although unambiguously aligned sequences from 22 concatenated transfer RNA (tRNA) genes (stem regions only) recovered the expected phylogeny successfully with moderate statistical support. The phylogenetic performance of the 13 protein-coding genes in recovering the expected phylogeny was roughly classified into five groups, viz. very good (ND5, ND4, COIII, COI), good (COII, cyt b), medium (ND3, ND2), poor (ND1, ATPase 6), and very poor (ND4L, ND6, ATPase 8). Although the universality of this observation was unclear, analysis of successive concatenation of the 13 protein-coding genes in the same ranking order revealed that the combined data sets comprising nucleotide sequences from the several top-ranked protein-coding genes (no 3rd codon positions) plus the 22 concatenated tRNA genes (stem regions only) best recovered the expected phylogeny, with all internal branches being supported by bootstrap values >90%. We conclude that judicious choice of mitochondrial genes and appropriate data weighting, in conjunction with purposeful taxonomic sampling, are prerequisites for resolving higher-level relationships in teleosts under the maximum-parsimony optimality criterion.     

2 micrometer covalently closed non-mitochondrial circular DNA in the petite-negative yeast Schizosaccharomyces pombe.

Summary A population of small covalently closed non-mitochondrial circular DNA molecules was isolated from the petite-negative yeast Schizosaccharomyces pombe. The mean length of these molecules, possessing the same density as nuclear DNA (1.695 g/ cm³) is 1.95±0.18 m. The presence of these minicircles in crude mitochondrial preparations indicates their tight association with mitochondrial particles. Their disappearance after DNase treatment of mitochondria demonstrates their extramitochondrial location.     

Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and Homalodisca liturata for use in predator gut content examinations

To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy‐winged sharpshooter, Homalodisca coagulata (Say), and smoke‐tree sharpshooter, Homalodisca liturata (Ball) (Homoptera: Cicadellidae). Two different types of markers were compared, those targeting single‐copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multicopy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed, two SCAR and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata specific and HcF6/HcR9 was H. coagulata/H. liturata specific. COI (HcCOI‐F/R) and COII (HcCOII‐F4/R4) markers were H. coagulata specific, COII (G/S‐COII‐F/R) marker was H. coagulata/H. liturata specific, and lastly, COII marker (Hl‐COII‐F/R) was H. liturata specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66‐fold more sensitive than marker Hl‐COII‐F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2‐fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we describe the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing the polymerase chain reaction (PCR). This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.     

Differentiation of somatic mitochondria and the structural changes in mtDNA during development of the dicyemid <Emphasis Type="Italic">Dicyema japonicum</Emphasis> (Mesozoa)

Dicyemids (Mesozoa) are extremely simple multicellular parasites found in the kidneys of cephalopods. Their mitochondria are known to contain single-gene minicircle DNAs. However, it is not known if the minicircles represent the sole form of mitochondrial genome in these organisms. Here we demonstrate that high-molecular-weight (HMW) mtDNA is present in dicyemids. This form of mtDNA is probably limited to germ cells, and has been analyzed by PCR and Southern hybridization. In situ hybridization revealed that mtDNA is initially amplified during early embryogenesis, and then gradually decreases in copy number as larval development proceeds. Furthermore, we demonstrated using BrdU as a tracer that many of the mitochondria in terminally differentiated somatic cells no longer support DNA synthesis. Taking these observations into account, we propose an amplification-dilution model for mesozoan mtDNA. Stem mitochondria in the germ cells (1) amplify the HMW form of mtDNA in early embryos, followed by minicircle formation via DNA rearrangement, or (2) selectively replicate minicircles from the HMW DNA, concomitantly with the differentiation of the soma. Minicircle formation may itself lead to the loss of replication origins. Thereafter, the minicircles are simply distributed to daughter mitochondria without replication, resulting in the somatic mitochondria, which have lost the replicative form of the HMW mtDNA. The change in mtDNA configuration is discussed in relation to mitochondrial differentiation.     

Mitochondrial Genes Collectively Suggest the Paraphyly of Crustacea with Respect to Insecta

Complete sequences of seven protein coding genes from Penaeus notialis mitochondrial DNA were compared in base composition and codon usage with homologous genes from Artemia franciscana and four insects. The crustacean genes are significantly less A + T-rich than their counterpart in insects and the pattern of codon usage (ratio of G + C-rich versus A + T-rich codon) is less biased. A phylogenetic analysis using amino acid sequences of the seven corresponding polypeptides supports a sister-taxon status for mollusks–annelid and arthropods. Furthermore, a distance matrix-based tree and two most-parsimonious trees both suggest that crustaceans are paraphyletic with respect to insects. This is also supported by the inclusion of Panulirus argus COII (complete) and COI and COIII (partial) sequence data. From analysis of single and combined genes to infer phylogenies, it is observed that obtained from single genes are not well supported in most topologies cases and notably differ from that of the tree based on all seven genes. Received: 25 August 1998 / Accepted: 8 March 1999