In search of mitochondrial markers for resolving the phylogeny of cyclophyllidean tapeworms (Platyhelminthes,Cestoda) — a test study with Davaineidae

The most species rich order of tapeworms is the Cyclophyllidea and prior to wide-scale sampling of these worms for phylogenetics, we wished to develop reliable PCR primers that would capture fragments of mitochondrial (mt) DNA with phylogenetic utility across the order. Nuclear ribosomal RNA gene sequences are well-established and valuable markers for resolving flatworm interrelationships spanning a wide range of taxonomic divergences, but fail to provide resolution amongst recently diverged lineages. Entire mt genomes of selected cyclophyllidean tapeworms are available on GenBank, and we used these to design PCR primers to amplify mtDNA from cox1, rrnL and nad1 for a range of cyclophyllideans (7 davaineids, 1 hymenolepidid and 1 dilepidid) and selected outgroups (Tetrabothrius sp. and Mesocestoides sp.). A combined nuclear and mt gene data set was used to estimate a reference phylogeny and the performance of the individual genes was compared to this. Although nuclear and mt genes each contributed to the structure and stability of the phylogenetic estimate, strongest nodal support was provided by nuclear data amongst the basal lineages and by mt data amongst the most recently diverged lineages. The apparent complementarity afforded by combining nuclear and mt data was compromised by these data partitions providing conflicting signal at poorly supported nodes. Nevertheless, we argue for a combined evidence approach. PCR primers that amplify rrnL were designed and tested successfully against a diversity of cyclophyllideans; rrnL and nad1 appeared to be more informative than the fragment of cox1. The genus Raillietina was not supported by molecular evidence. The new primers will likely provide considerable resolution to estimates of cyclophyllidean interrelationships in future studies.     

The complete DNA sequence of the mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum

We report here the complete nucleotide sequence of the 30.9-kb mitochondrial genome of the dermatophyte fungus Epidermophyton floccosum. All genes are encoded on the same DNA strand and include seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxireductase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), three subunits of cytochrome oxidase (cox1, cox2, and cox3), apocytochrome b (cob), three subunits of ATP synthase (atp6, atp8, and atp9), the small and large ribosomal RNAs (rns and rnl), and 25 tRNAs. A ribosomal protein gene (rps5) is present as an intronic ORF in the large ribosomal subunit. The genes coding for cob and cox1 carry one intron and nad5 carries two introns with ORFs. The mtDNA of E. floccosum has the same gene order as Trichophyton rubrum mtDNA, with the exception of some tRNA genes. Maximum likelihood phylogenetic analysis confirms T. rubrum as a close relative of E. floccosum. This is the first complete mitochondrial sequence of a species of the order Onygenales. This sequence is available under GenBank accession number AY916130.     

Physical mapping and genome organization of mitochondrial DNA from Candida maltosa

Summary Mitochondrial (mt) DNA of the ascomycetous yeast Candida maltosa was isolated and characterized. The mtDNA is circular and the size estimated from restriction analysis performed with 7 endonucleases was 52 kb pairs. A restriction map was constructed, using the cleavage data of four endonucleases. Using mt genes from Saccharomyces cerevisiae, six structural genes (large rRNA, apocytochrome b, cytochrome c oxidase subunit I and subunit 11, ATPase subunit 6 and subunit 9) were located on the C. maltosa chondriome by cross hybridization experiments. The comparison between the mt genomes of C. maltosa and six other yeasts showed differences in the overall genome organization.     

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron

Summary The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8 826 bp and contains five exons (termed E1–E5) totalling 1 602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1–1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5a, and 1.5b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5a and K1 cox1.4/Sc cox1.5b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.EM BL Accession Number X57546     

Mitochondrial DNA of Chenopodium album (L): a comparison of leaves and suspension cultures

Summary The mitochondrial genome (mt genome) of Chenopodium album was analysed. It was found to have a size of about 270 kb and a GC content of 46%. The genomes of plant cells and suspension cultures were compared. Restriction fragment pattern analyses and hybridization experiments revealed quantitative and qualitative alterations. However, a comparison of the restriction fragment patterns of two independently established in vitro cultures did not reveal differences as far as the mt chromosomal DNA is concerned. Therefore, alterations in mtDNA induced by in vitro culture seem not to be caused by an entirely undirected process in Chenopodium. A plasmid-like DNA molecule was amplified in only one of the cultures investigated and not in the other or in the plant cells. This molecule has a length of 1,083 by and is referred to as Ca1 mp1.     

Distribution of mitochondrial introns in the species Schizosaccharomyces pombe and the origin of the group II intron in the gene encoding apocytochrome b

Summary The mitochondrial genome size of 26 different Schizosaccharomyces pombe strains varies between 17.6 and 24.6 kilobase pairs due to the presence or absence of introns. One of these is the group II intron in the gene encoding apocytochrome b (cob: intron cobI1 ). Partial DNA sequences of continuous cob genes from six strains (including strain EF1: Trinkl et al. 1985) revealed identical nucleotide sequence in the region where the group II intron is inserted in the mosaic form of the gene. In contrast, analysis of the mosaic cob, gene in strain UCD-Fstl revealed several base pair changes in the exon regions flanking the splice point, compared with the continuous genes and with the mosaic cob gene in strain 50 (Lang et al. 1985). The base pair differences between the exons of the two mosaic cob genes and the identity of exons in all continuous cob genes argue in favour of the two cob introns in strains 50 and UCD-FstI as independent later acquisitions of the genes, rather than loss of the intron from a common mosaic ancestor of all strains.Other introns present in some but not all strain include two group I introns without open reading frame in the gene encoding subunit 1 of cytochrome c oxidase (cox1: introns cox1I2a and cox1I3), and two group I introns with open reading frames in the same gene (introns cox1I1 and cox1I2b).     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe

Summary The three mutator strains ana ^r-8, ana ^r-14, and diu ^r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe. Abbreviations. MtDNA = mitochondrial DNA; S. pombe = Schizosaccharomyces pombe; cox1, cox2, and cox3 refer to the mt genes coding for the three subunits of cytochrome oxidase; ATPase 6 (oli2), ATPase 8 (aapl in Saccharomyces cerevisiae, urf a61 in HeLa) and ATPase 9 (olil) refer to the three respective subunits of ATP synthase; cob is thegene for apocytochrome b; urf a is the single intergenic unassigned reading frame in S. pombe; 1 rRNA and s rRNA refer to the large and small ribosomal RNA, respectively. Mut^– is a cytoplasmic mutator (the corresponding wild type allele is mut⁺). Mit^– are mitochondrially inherited respiratory deficient mutants with mitochondrial protein synthesis; RC = respiratory competent, RD = respiratory deficient.     

A mobile group II intron of a naturally occurring rearranged mitochondrial genome in Kluyveromyces lactis

Summary Mitochondrial intron content is variable in the yeast Kluyveromyces lactis. Strains can be divided into three classes depending on the structure of the cytochrome oxidase subunit 1 (COX1) gene: (1) those containing intron K1 cox1.1, (2) those containing K1 cox1.2, 3 and 4 and, (3) those that contain all four introns. In addition, strains belonging to the first class (designated Type B strains), have an altered mitochondrial gene order relative to strains from classes (2) and (3) (Type A, Hardy et al. 1989). Crossing experiments reveal that K1 cox1.1 (a group II intron) transfers at high frequency (89%) to mitochondrial genomes lacking this intron. By contrast, the mobility of the remaining introns (all group I) is of the order of 7%.     

Impact of a mutation in the mitochondrial <Emphasis Type="Italic">LSU rRNA</Emphasis> gene from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> on the activity and the assembly of respiratory-chain complexes

Two substitutions A1090G and A1098C (together called the m mutation) located in the conserved GTPase domain of the mitochondrial LSU rRNA gene were recently shown to weakly compensate for the phenotypical effect of a –1T frameshift mutation in the mitochondrial cox1 gene of C. reinhardtii. In order to analyze the impact of the m mutation on the mitochondrial translational machinery, a strain carrying the m mutation but wild-type for the cox1 gene was isolated. We found that the growth and the respiratory rate of the m mutant were affected and that the activities of complexes I, III, and IV, all containing mitochondria-encoded subunits, were lowered. In contrast the activities of complex II and of the alternative oxidase, both encoded exclusively by the nuclear genome, were not modified. The steady-state levels of complex I enzyme and of several components of the respiratory complexes I, III, and IV were also reduced in the mutant. We moreover showed that m did not suppress other frameshift or UGA stop mutations which affect mitochondrial genes.     

Interaction between mitochondrial DNA and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the IrRNA-gene

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pC1K1 and pClT5, to the upstream region of the large ribosomal RNA gene in the mtDNA of three strains (W3, T5 and K) has been investigated in detail to explore the widespread phenomenon of homology between mt plasmids and mtDNA in C. purpurea. Sequence comparison indicates that recombination between free plasmids and mtDNA is the cause of the observed homology. The process is similar to the integration of the structurally related adenoviruses into the mammalian genome. As in other fungi, palindromic sequences seem to be involved in this mitochondrial recombination process.     

Polymorphism of the mitochondrial L-RNA gene of the basidiomycete Coprinus cinereus

Summary Two unexpectedly small mitochondrial (mt) genomes of Coprinus cinereus, P and S, were compared with the H and J genomes we have described previously. H and J are 42 kb in size and differ in having alternative 1.23 kb insertions in or adjacent to the co-1 gene. P and S DNAs lacked both insertions and had an identical 4.4 kb deletion between the co-1 and L-RNA gene. P DNA contained a 700 by insertion and S DNA a 300 by deletion within a sequence coding the L-RNA gene. This was shown by Southern blot analysis using probes containing the 5 or the 3 exon sequences of the L-RNA gene of Neurospora crassa. These hybridisations showed also that the L-RNA gene and co-1 gene in the C. cinereus mt genome are oppositely orientated and must be transcribed from different DNA strands. No DNA homology was detected using probes containing intron sequences from the L-RNA genes of Saccharomyces cerevisiae or N. crassa. There was no evidence of respiratory deficiency in P and S strains and transfer of nuclei by dikaryon formation made it possible to recombine H nuclei with P and S mitochondria, S nuclei with H and P mitochondria and P nuclei with H mitochondria with no apparent detrimental effect on growth. We conclude that P and S mtDNAs represent naturally occurring variants of the C. cinereus mt genome.     

The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene

Summary As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to lie in its single G,C rich cluster, which differs in just one nucleotide from the standard ori ^s sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergencies (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.     

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 cytoplasmic male-sterility (CMS) determinant of common bean is widespread in Phaseolus coccineus L. and Phaseolus vulgaris L.

To identify regions of the mitochondrial genome that potentially could specify cytoplasmic male sterility (CMS) in Phaseolus coccineus (including P. polyanthus), and to define differences amongst P. coccineus lines, mitochondrial (mt)DNA restriction patterns and Southern blots of total DNA from sterile and fertile lines were analysed. By restriction endonuclease mapping we isolated a region which was specific to CMS lines flanking an F1-ATPase -subunit (atpA) gene. DNA sequence analysis of this region showed 99.9% homology to the region previously isolated from P. vulgaris CMS Sprite. A high frequency of plants carrying the CMS-fragment was observed in a wild Phaseolus population, perhaps explaining the occurrence of inter- and intraspecific gene flow observed in the autogamous species P. vulgaris.     

Physical mapping of the mitochondrial genome of the cultivated mushroom Agaricus brunnescens (= A. bisporus)

Summary Mitochondrial (mt) DNA from the commercial mushroom Agaricus brunnescens Peck [= A. bisporus (Lange) Imbach] was purified by cesium chloride/bisbenzimide gradient centrifugation. A physical map of the mtDNA fragments produced by BamHI, EcoRl, and PvuII digestion was generated by filter hybridizations with radiolabelled BamHI mtDNA probes. The A. brunnescens mtDNA was a circular molecule 136 kilo-basepairs (kbp) in length and contained an inverted repeat between 4.6 and 9.2 kbp in size. Orientational isomers of the mitochondrial genome were not detected. The positions of six genes were located on the A. brunnescens mtDNA map by heterologous hybridization. No coding function has yet been ascribed to the inverted repeat. The large rRNA gene was located on the smaller single copy region. The genes for cytochrome b, cytochrome oxidase (subunit III), ATPase (subunits 8 and 6) and the small rRNA were located on different regions of the larger single copy region.     

The radish (Raphanus sativus L.) mitochondrial cox2 gene contains an ACG at the predicted translation initiation site

The mitochondrial cox2 gene has been sequenced from radish (Raphanus sativus L.). The gene is interrupted by a 1346-bp group-II intron and contains an ACG codon as the predicted translation initiation site. Analysis of cox2 cDNAs indicates that the ACG codon is not converted to an AUG codon in the mRNA, although 15 other RNA editing sites were identified. The cox2 gene from Raphanus raphanistrum, and other varieties of R. sativus, also contain an ACG as the predicted start codon; plants in the closely related genus, Brassica, do not. Western-blot analyses indicate that cox2 proteins in radish mitochondria are the same size as those found in Brassica mitochondria and different from cox2 proteins in plants where cox2 is nuclear-encoded. This finding, along with the observation that cox2 sequences are not present in the nuclear genome of radish, suggests that ACG is utilized as the radish cox2 initiation codon. Received: 4 February / 3 May 1998     

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.     

Genetic differentiation of cercariae infrapopulations of the avian schistosome <Emphasis Type="Italic">Trichobilharzia szidati</Emphasis> based on RAPD markers and mitochondrial <Emphasis Type="Italic">cox1</Emphasis> gene

Avian schistosome Trichobilharzia szidati is a member of the largest genus within the family Schistosomatidae (Trematoda). Population genetic structure of Trichobilharzia spp. schistosomes, causative agents of cercarial dermatitis in humans, has not been studied yet. The knowledge of the genetic structure of trichobilharzian populations is essential for understanding the host–parasite coevolutionary dynamics and epidemiology strategies. Here we examined genetic diversity in three geographically isolated local populations of T. szidati cercariae inhabiting Russia based on nuclear (randomly amplified polymorphic DNA, RAPD) and mt (cox1) markers. We analyzed T. szidati cercariae shed from seven naturally infected snails of Lymnaea stagnalis. Using three random primers, we demonstrated genetic variation among populations, thus posing genetic structure across geographic sites. Moreover, T. szidati cercariae have been genetically structured among hosts (infrapopulations). Molecular variance analysis was performed to test the significance of genetic differentiation within and between local populations. Of total parasitic diversity, 18.8% was partitioned between populations, whereas the higher contribution (48.9%) corresponds to the differences among individual cercariae within infrapopulations. In contrast to RAPD markers, a 1,125-bp fragment of cox1 mt gene failed to provide any significant within-species structure. The lack of geographic structuring was detected using unique haplotypes which were determined in the current work for Moscow and Western Siberian local populations as well as obtained previously for European isolates (Czech Republic and Germany). All T. szidati/Trichobilharzia ocellata haplotypes were found to be mixed across their geographical origin.