DNA sequence analysis of the 24.5 Kilobase pair cytochrome oxidase subunit I mitochondrial gene from Podospora anserina: a gene with sixteen introns

Summary The DNA sequence of a 26.7 Kilobase pair (10³ base pairs = 1 Kb) region of the mitochondrial genomes of races s and A from Podospora anserina was determined. Within this region, the 24.5 Kb cytochrome oxidase subunit I gene was located and its exon sequences determined by computer analysis comparisons with other fungal genes. The Podospora COI gene was interrupted by two group II introns (one in race s) and fourteen group I introns ranging in size from about 2.2 Kb to 404 bp. Earlier studies on secondary structure analysis, as well as comparison of their open reading frames (ORFs), showed that the two group II introns were closely related. The fourteen group I introns were representatives of three subgroupings (IB, C and a new category, subgroup ID). Two of these group I introns were separated by just a single exon codon. The analysis of all these introns is discussed in comparison with other fungal introns as well as with the known Podospora anserina introns.     

Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron

Summary Recently, the nucleotide sequences for three mitochondrial plasmids associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian URF1 gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.     

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.     

The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene

Summary In the ascomycete Podospora anserina strain ageing (senescence) is caused by a mitochondrial plasmid. In juvenile mycelia it is an integral part of the mtDNA and becomes liberated during ageing. The nucleotide sequence of this plasmid and of its flanking regions was determined. It consists of 2,539 by and contains an un identified reading frame (URF) originating in the adjacent mtDNA upstream of excision point 1. Within the URF a putative 48 by autonomously replicating sequence (ars) was identified. At both excision sites of the plasmid there are two short nonidentical interrupted palindromes and a few base pairs apart from these palindromes, both upstream and downstream, two short inverted repeats are localised. The experimental data make it evident that the mt plasmid is an intron of the cytochrome c oxidase gene (subunit I) which may be excised at the DNA level and thus become the mobile infective agent causing senescence. The concept of this mobile intron and current hypotheses concerning the relationship between introns and transposons are stressed.     

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 linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

Genetic map of mitochondrial DNA in Podospora anserina

Summary In order to develop an eukaryotic vector with the Podospora plasmid, further characterization is required of the mitochondrial DNA into which this plasmid is integrated, a physical map (restriction sites) of the Podospora chondriome (size 95 kb) has been completed. As prerequisite for the establishment of a genetic (functional) map, 70% of the chondriome was cloned in E. coli vectors. Using mitochondrial genes from Saccharomyces cerevisiae, six structural genes were located on the Podospora chondriome by cross hybridization experiments. There is strong evidence that the plasmid is inserted into the cytochrome b gene. A comparison of the genetic map of the Podospora chondriome with those of Neurospora crassa and Aspergillus nidulans exhibits a rather good accordance with respect to the sequence of genes.     

Mitochondrial DNA amplification in senescent cultures of Podospora anserina: Variability between the retained,amplified sequences

Summary The non-nuclear DNA of a number of independent senescent cultures of Podospora anserina was extracted and studied. In all cases, a specific repetitive DNA (SEN-DNA) arranged in multimeric sets of circular molecules, was identified. Depending on the senescent culture, the SEN-DNA was found either in a band of about same density as the mitochondrial DNA from young mycelia (1.694 g/cm³) or in a band of higher density (1.699 g/cm³). Electron microscopy, restriction enzyme analysis and Southern hybridization experiments allowed us to establish that: (1) SEN-DNAs obtained from independent senescent cultures, both from the same strain and from different strains, can differ in the size of their monomer unit (from 2.5 to 6.3 kb). (2) All SEN-DNAs hybridize with mitochondrial DNA of a young culture and not with nuclear DNA. (3) These SEN-DNAs belong to two classes which hybridize with two non-overlapping regions of the mitochondrial chromosome.     

Transformation to senescence with plasmid like DNA in the ascomycete Podospora anserina

Summary In the ascomycete Podospora anserina senescence through strain aging is under nucleo-cytoplasmic control and inducible in juvenile mycelia by an infective principle transferred after cytoplasmic contact via anastomoses. A specific DNA called plasmid-like (pl) DNA, present exclusively in aging mycelia, was found to be identical with this infective principle, since it was possible to transform juvenile protoplasts to senescence by using purified p1DNA. Therefore a specific function may be attributed to this ccc DNA. Its direct involvement in a genetically programed senescence is confirmed and its development as a vector for transfer of genetic information in eukaryotes can be undertaken.     

Intramolecular cross-overs generate deleted mitochondrial DNA molecules in Podospora anserina

The unavoidable senescence process that limits the vegetative growth of Podospora anserina is always associated with an accumulation of various classes of circular, tandemly arranged, defective mitochondrial DNA molecules (senDNAs). The monomers of the senDNAs belonging to the so-called β class share a common core, but differ in both their length and termini. To understand the mechanism leading to their formation, we have determined the junction sequence of 36 senDNA β monomers present in various senescent cultures. In most cases, we observe that: (1) short direct repeats precisely bound the senDNA β termini and (2) one copy of the repeats is retained in the senDNA sequence. Moreover, PCR analysis of the mitochondrial DNA of some of the senescent cultures, has allowed us to detect another genome which is exactly lacking the sequence of the senDNA β found in the culture. These results demonstrate that an intramolecular unequal cross-over occurring between short direct repeats can generate deleted mtDNA molecules in P. anserina. In addition, the polymorphism displayed by one pair of repeats allows us to establish that this cross-over may be associated with a short conversion tract spanning a few (about 15) nucleotides. Received: 16 May / 11 November 1996     

Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome

Summary The nuclear gene coding for the mitochondrial subunit 9 of the F₀F₁-ATP synthase complex was isolated from a genomic library of Podospora anserina. Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid. The P. anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger. In contrast to the situation in Saccharomyces cerevisiae, N. crassa and A. nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P. anserina could be detected. Thus, in P. anserina this gene appears to be exclusively encoded by the nuclear genome.     

De-novo generation of mitochondrial DNA plasmids following cytoplasmic transmission of a degenerative disease in Ophiostoma novo-ulmi

A mitochondrial DNA plasmid was detected in an isolate of Ophiostoma novo-ulmi with a degenerative disease. The DNA plasmid was shown to be derived from the mitochondrial DNA and to map to a region corresponding to the large ribosomal RNA coding region. The DNA plasmid was not transmitted into sexual (ascospore) progeny, irrespective of whether the diseased isolate acted as the female or male parent. Transmission of the disease to healthy, plasmid-free, recipient isolates by hyphal anastomosis was not accompanied by transfer of mitochondrial DNA or DNA plasmid from the diseased donor isolate, but resulted in de-novo generation of different plasmids, derived from the recipient's mitochondrial DNA.     

Behaviour of a replicating mitochondrial DNA sequence fromAspergillus amstelodami inSaccharomyces cerevisiae andAspergillus nidulans

Summary An amplified sequence of mitochondrial DNA from a ragged (rgd) mutant ofAspergillus amstelodami has been shown to exist in multimeric circular form, suggesting that it is excised from the genome and can exist independently of it. This sequence has replicative (ARS) activity inSaccharomyces cerevisiae, and a subfragment responsible for this activity has been identified and sequenced. A homologous sequence fromAspergillus nidulans mtDNA also has ARS activity inS. cerevisiae. BothA. amstelodami andA. nidulans ARS elements have been incorporated into the integrative transformation vector pDJBI and the derived vectors used to transformA. nidulans. Inclusion of theA. nidulans ARS element enhanced the transformation frequency 5-fold relative to pDJBI. No increase in transformation frequency was evident with the ARS element fromA. amstelodami. The stability of transformants was variable but in comparison to pDJBI, ARS-containing plasmids were mitotically unstable inA. nidulans. Although plasmid DNAs could be rescued inEscherichia coli from undigested transformant DNA, no freely replicating plasmids were detected by Southern hybridisation.     

Electrophoretic karyotype of the ascomycete Podospora anserina

Summary Fractionation of intact chromosomes of the ascomycete Podospora anserina by contour-clamped homogenous electric field (CHEF) gel electrophoresis resulted in the resolution of five distinct chromosomal bands. Two of these bands migrated as doublets. Using chromosomal standards from Schizosaccharomyces pombe we estimated the size of the P. anserina genome to about 33.4 megabases (Mb). By heterologous hybridization of fractionated chromosomes the rDNA locus was identified on one of the two chromosomes migrating at about 4.9 Mb.     

DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame

Summary The 5,969 by (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region. This gene contained a 2,499 by subgroup IB and a 1,306 by subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s. The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences. All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns. We detected a new family of intronic ORFs comprising seven P. anserina introns, several N. crassa introns, as well as the T4td bacteriophage intron. Sequence similarities to intron-encoded endonucleases were noteworthy. The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA.     

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     

Linear mitochondrial genome organization in vivo in the genus Pythium

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.