Complete mitochondrial genomes of the three brown algae (Heterokonta: Phaeophyceae) Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis

We report the complete mitochondrial sequences of three brown algae (Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis) belonging to three phaeophycean lineages. They have circular mapping organization and contain almost the same set of mitochondrial genes, despite their size differences (31,617, 36,392 and 39,049 bp, respectively). These include the genes for three rRNAs (23S, 16S and 5S), 25–26 tRNAs, 35 known mitochondrial proteins and 3–4 ORFs. This gene set complements two previously studied brown algal mtDNAs, Pylaiella littoralis and Laminaria digitata. Exceptions to the very similar overall organization include the displacement of orfs, tRNA genes and four protein-coding genes found at different locations in the D. dichotoma mitochondrial genome. We present a phylogenetic analysis based on ten concatenated genes (7,479 nucleotides) and 29 taxa. Stramenopiles were always monophyletic with heterotrophic species at the base. Results support both multiple primary and multiple secondary acquisitions of plastids. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers AY494079, AY500368 and AY500367.     

The genes for cytochrome b,ND 4L,ND6 and two tRNAs from the mitochondrial genome of the locust,Locusta migratoria

We have cloned and characterized a 2,778-kb XbaI segment of the mitochondrial genome of the locust, Locusta migratoria. It harbours portions of the ND4 and the ND1 genes, the entire genes for ND6, ND4L and cytochrome b, and the genes for three mitochondrial tRNAs. The genes are arranged in an order which is conserved between orthopteran and dipteran insects. The analysis of the cytochrome b sequence, and its comparison with other systems, supports the current model structure for this polypeptide.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the ND4 gene encoding a subunit of NADH dehydrogenase

Summary The ND4 gene encoding a subunit of respiratory NADH dehydrogenase has been identified on the linear 15.8 kb mitochondrial DNA of Chlamydomonas reinhardtii. The gene maps downstream of ND5. The 1,332 bp nucleotide sequence presented is the first complete reported ND4 sequence from a photoautotrophic organism. The deduced protein of 443 amino acid residues shows 34%, 29% and 27% homology to the protein sequences of Aspergillus amstelodami, Drosophila yakuba and mouse, respectively. ND4 is the fifth and last mitochondrial gene of the NADH dehydrogenase complex on the 15.8 kb mitochondrial genome of C. reinhardtii.     

Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning,sequencing and chromosomal localization of its TRP1 gene

 The genome of the amylolytic yeast strain Lipomyces starkeyi NCYC 1436 was analysed using contour-clamped homogeneous electric field gel electrophoresis (CHEF). The banding pattern under a variety of running conditions indicating the presence of 11 different chromosome-sized DNA molecules. The sizes of these chromosome bands were determined by comparison with chromosomes from standard strains of Schizosaccharomyces pombe and Saccharomyces cerevisiae. The chromosomal bands were estimated to be within the range 0.7–2.8 Mb, with the genome (excluding mitochondrial DNA) estimated at 15 Mb. The molecular cloning of the TRP1 gene, isolated from a genomic library of this strain, is also reported: the gene was present on a 6.5-kb Sau3A DNA fragment, and complemented the trpC gene of E. coli. The DNA sequence was determined (EMBL accession No. Z68292) and compared to other tryptophan biosynthetic genes encoding N-(5′-phosphoribosyl) anthranilate isomerase (PRAI) activity. The gene was also used as a probe in hybridization studies, and by this means, its chromosomal location was identified. Received: 2 November 1995/5 January 1996     

Partial sequence of the mitochondrial genome of the crustacean Daphnia pulex

 A 4062-nucleotide (nt) fragment of the mitochondrial genome of the crustacean Daphnia pulex was sequenced and found to contain the complete genes for eight tRNAs and five proteins (ATP6, ATP8, COII, COIII, ND3) and the partial sequence of COI. In combination with data described previously, approximately 50% of the D. pulex mitochondrial genome has been sequenced. The gene order in this half of the genome is identical to that of Drosophila yakuba which differs from that of the other completely sequenced crustacean mitochondrial genome, Artemia franciscana. Comparison of seven mitochondrial proteins among D.␣pulex, A. franciscana, D. yakuba, Anopheles gambiae, Locusta migratoria and Apis mellifera showed that, with one exception, the D. pulex proteins are most similar in length and sequence to the proteins of the dipteran insects. Conversely, patterns of nucleotide bias at third codon positions in fourfold degenerate codon families are very similar in the two crustaceans but differed substantially from the insects. Received: 15 March / 23 September 1996     

The complete mitochondrial DNA sequence of the green alga Oltmannsiellopsis viridis: evolutionary trends of the mitochondrial genome in the Ulvophyceae

The mitochondrial genome displays a highly plastic architecture in the green algal division comprising the classes Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae (Chlorophyta). The compact mitochondrial DNAs (mtDNAs) of Nephroselmis (Prasinophyceae) and Prototheca (Trebouxiophyceae) encode about 60 genes and have been ascribed an ‘ancestral’ pattern of evolution, whereas those of chlorophycean green algae are much more reduced in gene content and size. Although the mtDNA of the early-diverging ulvophyte Pseudendoclonium contains 57 conserved genes, it differs from ‘ancestral’ chlorophyte mtDNAs by its unusually large size (96 kb) and long intergenic spacers. To gain insights into the evolutionary trends of mtDNA in the Ulvophyceae, we have determined the complete mtDNA sequence of Oltmannsiellopsis viridis, an ulvophyte belonging to a distinct, early-diverging lineage. This 56,761 bp genome harbours 54 conserved genes, numerous repeated sequences, and only three introns. From our comparative analyses with Pseudendoclonium mtDNA, we infer that the mitochondrial genome of the last common ancestor of the two ulvophytes closely resembled that of the trebouxiophyte Prototheca in terms of gene content and gene density. Our results also provide strong evidence for the intracellular, interorganellar transfer of a group I intron and for two distinct events of intercellular, horizontal DNA transfer.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The complete mitochondrial genome of the vascular wilt fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis>: a novel gene order for <Emphasis Type="Italic">Verticillium</Emphasis> and a diagnostic tool for species identification

The complete sequence (27,184 bp) of the mitochondrial (mt) genome of the phytopathogenic fungus Verticillium dahliae has been determined. It contains 14 protein-coding genes related to oxidative phosphorylation, two rRNA genes and a set of 25 tRNA genes. A single intron, that harbors an intronic ORF coding for a putative ribosomal protein (rps), is located within the large rRNA gene (rnl). Gene order comparisons of V. dahliae mtDNA and complete mt genomes of Pezizomycotina revealed four units of synteny for Sordariomycetes, namely rnl-trn _(11–12)-nad2-nad3, nad4L-nad5-cob-cox1, nad1-nad4-atp8-atp6 and rns-trn _(1–5)-cox3-trn _(1–5)-nad6-trn _(2–5). These four units, in different combinations, merged to single continuous unit in the orders of Hypocreales and Sordariales. V. dahliae (Phyllachorales) and all members of the genus showed a unique feature which is the translocation of the nad1-nad4-atp8-atp6-rns-cox3-nad6 region in between genes nad3 and atp9 of the Hypocreales mtDNA gene order. Analysis of mt intergenic sequences of Verticillium species permitted the design of a species-specific primer allowing the discrimination of V. longisporum against V. dahliae and V. albo-atrum. By considering the protein-coding gene sequences as one unit, a phylogenetic comparison with representatives of Ascomycota complete mtDNA was performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized usersNucleotide sequence data reported are available in the GenBank database under the accession numbers DQ351941–DQ351957.     

Mitochondrial ATP synthase subunit 9 is not required for viability of the petite-negative yeast Kluyveromyces lactis

Specific mutations in nuclear MGI genes encoding the α, β and γ subunits of the mitochondrial inner membrane F₁-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K. lactis. In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal. These results imply that mtDNA encodes a gene that is essential. Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F₀ component. The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly^– 3.9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein. Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes. These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1. Received: 5 December 1996 / 6 March 1997     

Molecular phylogeny of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> from Central America (Guatemala) and a comparison with South American strains

Molecular phylogenetic analysis was carried out for 21 strains of Trypanosoma cruzi, nine of which were obtained from Guatemala and 12 from South America. Phylogenetic trees were constructed using the nucleotide sequences of two nuclear gene regions, dihydrofolate reductase–thymidylate synthase (DHFR-TS) and trypanothione reductase (TR), and contiguous portions of two mitochondrial genes, cytochrome oxidase subunit II (COII) and reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1). Possible genetic exchange between the rather divergent lineages of T. cruzi II from South America was suggested in the trees of the two nuclear genes. T. cruzi I strains obtained from Guatemala and Colombia were identical in all the genes examined, but other T. cruzi I isolates from South America were rather polymorphic in the DHFR-TS and mitochondrial genes. No genetic exchange was identified between T. cruzi I populations from Central and South America in the present study.     

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum

A 1 380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.     

The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.)

The mitochondrial atp6 gene in male fertile (N) and CMS (S) pepper has previously been compared and was found to be present in two copies (Kim et al. in J Kor Soc Hort Sci 42:121–127 2001). In the current study, these atp6 copies were amplified by an inverse PCR technique, and the coding region as well as the 5′ and 3′ flanking regions were sequenced. The atp6 copies in CMS pepper were detected as one intact gene and one pseudogene, truncated at the 3′ coding region. When the atp6 genes in pepper were compared to other plant species, pepper, potato, and petunia all possessed a sequence of 12 identical amino acids at the 3′ extended region, which was considered a hallmark of the Solanaceae family. Northern blot analysis showed differences in mRNA band patterns between CMS and restorer lines, indicating that atp6 gene is one of the candidates for CMS in pepper. GenBank accession number: DQ126682 (atp6-1 genomic sequence common to fertile and CMS pepper), DQ126681 (Fertile atp6-2 genomic sequence), DQ126680 (CMS pseudo-atp6-2 genomic sequence)     

Molecular characterization of <Emphasis Type="Italic">Atractolytocestus sagittatus</Emphasis> (Cestoda: Caryophyllidea), monozoic parasite of common carp,and its differentiation from the invasive species <Emphasis Type="Italic">Atractolytocestus huronensis</Emphasis>

Sequence structure of complete internal transcribed spacer 1 and 2 (ITS1 and ITS2) of the ribosomal DNA region and partial mitochondrial cytochrome c oxidase subunit I (cox1) gene sequences were studied in the monozoic tapeworm Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965) (Cestoda: Caryophyllidea), a parasite of common carp (Cyprinus carpio carpio L.). Intraindividual sequence diversity was observed in both ribosomal spacers. In ITS1, a total number of 19 recombinant clones yielded eight different sequence types (pairwise sequence identity, 99.7–100%) which, however, did not resemble the structure typical for divergent intragenomic ITS copies (paralogues). Polymorphism was displayed by several single nucleotide mutations present exclusively in single clones, but variation in the number of short repetitive motifs was not observed. In ITS2, a total of 21 recombinant clones yielded ten different sequence types (pairwise sequence identity, 97.5–100%). They were mostly characterized by a varying number of (TCGT)_n repeats resulting in assortment of ITS2 sequences into two sequence variants, which reflected the structure specific for ITS paralogues. The third DNA region analysed, mitochondrial cox1 gene (669 bp) was detected to be 100% identical in all studied A. sagittatus individuals. Comparison of molecular data on A. sagittatus with those on Atractolytocestus huronensis Anthony, 1958, an invasive parasite of common carp, has shown that interspecific differences significantly exceeded intraspecific variation in both ribosomal spacers (81.4–82.5% in ITS1, 74.4–75.2% in ITS2) as well as in mitochondrial cox1, which confirms validity of both congeneric tapeworms parasitic in the same fish host.     

Analysis of genetic variability within Argulus japonicus from representatives of Africa, Middle East, and Asia revealed by sequences of three mitochondrial DNA genes

This study investigated the genetic variability within fish louse Argulus japonicus (Crustacea: Branchiura) from Africa, Middle East, and Asia by polymerase chain reaction in three mitochondrial DNA (mtDNA) regions, namely, cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4). Six different sequences from a portion of the cox1 gene (pcox1) and a portion of the nad1 and nad4 genes (pnad1 and pnad4) for ten adult specimens from infected fish in China, Egypt, and Syria were amplified separately from individual and the amplicons were subjected to direct sequencing. A + T percentages were 68.8–69% for pcox1, 77.1–77.6% for pnad1, and 60.4–60.9% for pnad4. Among all the collected parasites, A. japonicus sequence variations were 0.0–1.9% for cox1, 0.0–2.3% for nad1, and 0.0–0.8% for nad4. In rivers, sequence variations among all individuals were 0.4–0.8% for cox1, 1.0–2.3% for nad1, and 0.4–0.8% for nad4, while sequence variations among all the collected parasites in fish farms were 0.6–1.9% for cox1, 0.0–1.7% for nad1, and 0.2–0.6% for nad4. The nad1 was the most variable gene among selected markers, while nad4 was a more conserved gene than cox1. All isolates of A. japonicus were sister to Argulus americanus in phylogenetic tree and they grouped together in one sub-clade, while isolates from China and Egypt fish farms were closely clustered together. However, moderate genetic drift and slight mutation could be observed among A. japonicus individuals. These findings demonstrated the convenience and attributes of the three selected mtDNA sequences for population genetic studies of A. japonicus where nad1 is a new and reliable marker to detect the sequence variation among A. japonicus individuals.     

Deletion of the <Emphasis Type="Italic">cpku80</Emphasis> gene in the chestnut blight fungus, <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>, enhances gene disruption efficiency

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal–plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Δcpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Δcpku80 strain, a significant increase over the 2–5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The authors Xiuwan Lan and Ziting Yao contributed equally to this work.     

Small interspersed sequences that serve as recombination sites at the cox2 and atp6 loci in the mitochondrial genome of soybean are widely distributed in higher plants

Mitochondrial DNA (mtDNA) fragments that contain cox2 and atp6 were cloned from a wild soybean (Glycine soja, accession `B09002') and from a cultivated soybean (G. max, `Harosoy'). Comparison of these DNAs revealed that two sets of repeated sequences, namely, 299 bp and 23 bp, were present in the 5′ regions of cox2 and atp6. The 299-bp and 23-bp repeats were present close to each other on the 5′ flanking region and the 5′ part of the coding region of cox2 in both `Harosoy' and `B09002', as well as on the 5′ flanking region of atp6 in `Harosoy', while these two repeats were separated by a 706-bp nucleotide sequence that contained a truncated sequence of nad3 at the 5′ flanking region of atp6 in `B09002'. The mtDNA configurations upstream from atp6 and cox2 found in `Harosoy' appeared to have been generated from configurations of cox2 and atp6 found in `B09002' via recombination across the 299-bp or 23-bp repeated sequences, or vice versa, in the mitochondrial genome of the hypothetical progenitor of these plants. The 299-bp sequence was found to be interspersed in the mitochondrial genome. Eight loci were identified that include mtDNA configurations that are inter-convertible with each other via recombination across this sequence in `B09002'. Various loci on the mitochondrial genomes of higher plants that harbor segments of the 299-bp repeats in Glycine were identified. Received: 16 August / 15 December 1997     

Molecular characterization of Citrus tristeza virus isolates from the Northeastern Himalayan region of India

Citrus tristeza virus (CTV) isolates from the Darjeeling hills of the Northeastern Himalayan region of India were characterized by biological indexing, multiple molecular marker (MMM) analysis, heteroduplex mobility assay (HMA) and sequence analysis. Variability was studied using the CP gene and a 5′ ORF1a fragment of the CTV genome. HMA and sequence analysis of the 5′ ORF1a fragment classified Darjeeling isolates into two groups, whereas CP gene analysis provided evidence for three different groups. Darjeeling CTV isolates shared nucleotide sequence identities of 89–97 and 91–92% in the 5′ ORF1a fragment and CP gene, respectively, suggesting extensive diversity among CTV isolates from this Indian region.