The mitochondrial genome of Moniliophthora roreri, the frosty pod rot pathogen of cacao

In this study, we report the sequence of the mitochondrial (mt) genome of the Basidiomycete fungus Moniliophthora roreri, which is the etiologic agent of frosty pod rot of cacao (Theobroma cacao L.). We also compare it to the mtDNA from the closely-related species Moniliophthora perniciosa, which causes witches' broom disease of cacao. The 94 Kb mtDNA genome of M. roreri has a circular topology and codes for the typical 14 mt genes involved in oxidative phosphorylation. It also codes for both rRNA genes, a ribosomal protein subunit, 13 intronic open reading frames (ORFs), and a full complement of 27 tRNA genes. The conserved genes of M. roreri mtDNA are completely syntenic with homologous genes of the 109 Kb mtDNA of M. perniciosa. As in M. perniciosa, M. roreri mtDNA contains a high number of hypothetical ORFs (28), a remarkable feature that make Moniliophthoras the largest reservoir of hypothetical ORFs among sequenced fungal mtDNA. Additionally, the mt genome of M. roreri has three free invertron-like linear mt plasmids, one of which is very similar to that previously described as integrated into the main M. perniciosa mtDNA molecule. Moniliophthora roreri mtDNA also has a region of suspected plasmid origin containing 15 hypothetical ORFs distributed in both strands. One of these ORFs is similar to an ORF in the mtDNA gene encoding DNA polymerase in Pleurotus ostreatus. The comparison to M. perniciosa showed that the 15 Kb difference in mtDNA sizes is mainly attributed to a lower abundance of repetitive regions in M. roreri (5.8 Kb vs 20.7 Kb). The most notable differences between M. roreri and M. perniciosa mtDNA are attributed to repeats and regions of plasmid origin. These elements might have contributed to the rapid evolution of mtDNA. Since M. roreri is the second species of the genus Moniliophthora whose mtDNA genome has been sequenced, the data presented here contribute valuable information for understanding the evolution of fungal mt genomes among closely-related species.     

Structural features and expression analysis of a linear mitochondrial plasmid in rapeseed (Brassica napus L.)

A linear plasmid molecule about 11 kb in length is present in the mitochondria of some varieties of rapeseed (Brassica napus L.). This plasmid can be inherited from the male parent, through the pollen, as well as by the usual maternal route, although the main mitochondrial genome is maternally inherited in rapeseed. We determined the complete nucleotide sequence of this plasmid DNA and clarified its genetic organization. The length of the linear plasmid is 11,640 bp. At the termini of the plasmid molecule are inverted repeats of 327 bp. The GC content of the plasmid DNA is 30.9%; thus, the plasmid is quite AT-rich compared to the main mitochondrial genome in higher plants. The plasmid has six ORFs, two of which encode a phage-type DNA polymerase and a phage-type RNA polymerase, respectively. RT-PCR analyses revealed that all six ORFs are transcribed, and all four ORFs on the minus strand are probably cotranscribed from a single promoter located in the terminal inverted repeat. We also show here that at least three of the six ORFs are translated into proteins in rapeseed mitochondria, and expressed at relatively high levels in flowers, as shown by Western analysis. These results suggest that this linear DNA molecule is able to replicate as an autonomous replicon and to express the genes it carries in rapeseed mitochondria.     

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.     

Complete nucleotide sequence of pLD-TEX-KL, a 66-kb plasmid of Legionella dumoffii TEX-KL strain

The complete nucleotide sequence of a large (66 kb) plasmid pLD-TEX-KL of Legionella dumoffii TEX-KL strain was determined. Of the 57 predicted open reading frames (ORFs), 39 (68%) encoded proteins similar to previously known proteins, five (9%) were assigned with putative functions, three (5%) encoded conserved hypothetical proteins, and 10 (18%) had no homology to any genes present in the current open databases. The ORFs with similar functions were organized in a modular structure; thus, transfer region was identified, as well as a putative heavy-metal ion transporter system (hel). The transfer region encoded homologs of the Salmonella entrica serovar Typhi conjugative system components involved in conjugation. In addition, we also found a potential protein that was analogous to the DNA polymerase III epsilon subunit. It is rarely found that plasmid encode the DNA polymerase.     

Complete sequence and genetic features of the mitochondrial genome of Pyropia tenera (Rhodophyta)

We determined the complete mitochondrial genome (mtDNA, mitogenome) of Pyropia tenera (Bangiales, Rhodophyta). Pyropia tenera mtDNA had a larger size (42,268 bp) than the mtDNA sequences of Porphyra and Pyropia reported previously, and encoded two ribosomal RNA genes [large subunit (rnl), small subunit (rns)], 24 transfer RNAs, four ribosomal proteins, and 17 genes involved in electron transport and oxidative phosphorylation. Moreover, four conserved open reading frames (ORFs) and six intronic ORFs (three in rnl and three in the cox1 gene) were also identified. In comparison with other Porphyra and Pyropia species, Py. tenera had four major structural changes in two gene loss/rearrangement regions [tRNA-Gln(uug)–tRNA-SeC(uca) and tRNA-Ala(ugc)–tRNA-Arg(ucu)] and two different patterns of exon, intron, and intronic ORFs (rnl and cox1). The unique features of Py. tenera mtDNA include the complete sequence of red algal mtDNA for investigating mtDNA evolution and developing molecular markers for species identification. In addition, red algal mtDNA can provide useful genetic information as a genetic reservoir for bioengineering.     

The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. Received: 5 July 1999 / Accepted: 17 January 2000     

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.     

Bioinformatic analysis of the Acinetobacter baumannii phage AB1 genome

As one of the pathogens of hospital-acquired infections, Acinetobacter baumannii poses great challenges to the public health. A. baumannii phage could be an effective way to fight multi-resistant A. baumannii. Here, we completed the whole genome sequencing of the complete genome of A. baumannii phage AB1, which consists of 45,159bp and is a double-stranded DNA molecule with an average GC content of 37.7%. The genome encodes one tRNA gene and 85 open reading frames (ORFs) and the average size of the ORF is 531bp in length. Among 85 ORFs, only 14 have been identified to share significant sequence similarities to the genes with known functions, while 28 are similar in sequence to the genes with function-unknown genes in the database and 43 ORFs are uniquely present in the phage AB1 genome. Fourteen function-assigned genes with putative functions include five phage structure proteins, an RNA polymerase, a big sub-unit and a small sub-unit of a terminase, a methylase and a recombinase and the proteins involved in DNA replication and so on. Multiple sequence alignment was conducted among those homologous proteins and the phylogenetic trees were reconstructed to analyze the evolutionary courses of these essential genes. From comparative genomics analysis, it turned out clearly that the frame of the phage genome mainly consisted of genes from Xanthomonas phages, Burkholderia ambifaria phages and Enterobacteria phages and while it comprises genes of its host A. baumannii only sporadically. The mosaic feature of the phage genome suggested that the horizontal gene transfer occurred among the phage genomes and between the phages and the host bacterium genomes. Analyzing the genome sequences of the phages should lay sound foundation to investigate how phages adapt to the environment and infect their hosts, and even help to facilitate the development of biological agents to deal with pathogenic bacteria.     

The complete mitochondrial genome of Pseudochauhanea macrorchis (Monogenea: Chauhaneidae) revealed a highly repetitive region and a gene rearrangement hot spot in Polyopisthocotylea

The complete mitochondrial genome of Pseudochauhanea macrorchis was determined and compared with other monogenean mitochondrial genomes from GenBank. The circular genome was 15,031 bp in length and encoded 36 genes (12 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs) typically found in flatworms. Structures of the mitochondrial genome were mostly concordant with that known for Microcotyle sebastis and Polylabris halichoeres, but also contained two noted features-a gene rearrangement hot spot and the highly repetitive region (HRR) in major non-coding region (NCR). The gene rearrangement hot spot located between the cox3 and nad5 genes, including a cluster of tRNA genes, nad6 gene and one major NCR. The HRR seemed to be a unique feature of the polyopisthocotylean mitochondrial genomes. In conclusion, the present study provided new molecular data for future studies of the comparative mitochondrial genomics and also served as a resource of markers for the studies of species populations and monogenean phylogenetics.     

Unusual location of a mitochondrial gene. Subunit III of cytochrome C oxidase is encoded in the nucleus of Chlamydomonad algae

The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.     

The complete mitochondrial genome sequence of Whitmania pigra (Annelida, Hirudinea): the first representative from the class Hirudinea

The mitochondrial genome is a significant tool for investigating the evolutionary history of metazoan animals. The currently available mitochondrial genome data in GenBank is limited to understand the detail evolutionary relationship among the metazoan animals, especially in the phylum Annelida. Here we present the mitochondrial gene organization, gene order and codon usage of the leech Whitmania pigra (Annelida), which is the first representative from the class Hirudinea. It is a circular molecule of 14,426bp, and encodes 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. All 37 genes of W. pigra mitochondrial genome are transcribed from the same strand, which is identical to studied annelids, two echiurans, two sipunculans and many other lophotrochozoans. Five conserved gene clusters can be found in mitochondrial genomes of nine studied annelids, including (1) cox1-N-cox2; (2) cox3-Q-nad6-cob-W-atp6; (3) H-nad5-F-E-P-T-nad4L-nad4; (4) srRNA-V-lrRNA; and (5) nad3-S(1)-nad2. Compared with that of other studied annelids, translocations of transfer RNAs were found in the gene arrangement of W. pigra mitochondrial genome. Phylogenetic analysis strongly support that the species from Hirudinina and Oligochaeta form a monophyletic group Clitellata (BPM=100, BPP=100), which is consistent with previous research based on morphological and other molecular data. Both gene order data and amino acid sequences reveal that echiurans are derived annelids and sipunculans should be clustered with annelids and echiurans.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.