Gene organization of the mitochondrial DNA of yeasts: Kluyveromyces lactis and Saccharomycopsis lipolytica

Summary Restriction fragment maps have been constructed for the mitochondrial DNA from two petitenegative yeasts, Kluyveromyces lactis and Saccharomycopsis lipolytica (Candida lipolytica). On these circular genomes, we localized the sequences homologous to the S. cerevisiae mtDNA fragments carrying known genes. The arrangement of genes for ATPase subunit proteins, ribosomal RNA and 4S RNA shows a common feature with respect to S. cerevisiae mitochondrial genome.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA     

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.     

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.     

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).     

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

Identification and isolation of the cytochrome oxidase subunit II gene in mitochondria of the yeast Hansenula saturnus

Summary Mitochondrial DNA from the petite negative yeast Hansenula saturnus has been isolated and sized by digestion with restriction enzymes. The size of the mitochondrial genome is approximately 47 kb. The gene for subunit II of cytochrome oxidase was localized in the genome by Southern blotting using a [³²P]-labeled probe containing the subunit II gene of the yeast Saccharomyces cerevisiae. The probe hybridized to a 1.7 kb HindIII-BamHI fragment under stringent conditions (65°C), indicating a high degree of homology between the S. cerevisiae and H. saturnus mitochondrial DNA fragments. The 1.7 kb fragment from H. saturnus was cloned into pBR322 and physically mapped. The map was used to obtain the nucleotide sequence of the subunit II gene (Lawson and Deters presented in the accompanying paper).     

Mitochondrial DNA of Schizophyllum commune: restriction map,genetic map,and mode of inheritance

Summary Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4–40) is a circular molecule 49.75 kbp in lenght. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis.     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

Distribution of mitochondrial intron sequences among 21 yeast species

Summary The mitochondrial and nuclear genomes of 21 yeast species belonging to 12 genera have been tested for the presence of sequences similar to seven S. cerevisiae mitochondrial introns (Sc cox1.1,2,3,4,5c, Sc cob.4 and Sc LSU.1) and one K. lactis mitochondrial intron (Kl cox1.2). Some introns, (Sc cox1.4, Sc cob.4, Sc LSU.1 and Kl cox1.2-all group I type), are widely distributed and are found in species with either basidiomycete or ascomycete affinities. This distribution is suggestive of recent sequence transfer between species. The remaining S. cerevisiae introns cross react with an additional species but with no set pattern. Pulsed field gel electrophoretic studies confirm that none of the tested mitochondrial introns cross react with nuclear DNA. These introns are, therefore, mitochondria-specific. Seven strains of K. lactis exhibit striking variability in intron content. In contrast to all mitochondrial introns tested, two introns of nuclear genes (the K. lactis actin gene and the S. cerevisiae RP29B gene) are not detected beyond their source species.     

Functional co-operation between the nuclei of Saccharomyces cerevisiae and mitochondria from other yeast species

We elaborated a simple method that allows the transfer of mitochondria from collection yeasts to Saccharomyces cerevisiae. Protoplasts prepared from different yeasts were fused to the protoplasts of the ade2-1, ura3-52, kar1-1,ρ⁰ strain of S. cerevisiae and were selected for respiring cybrids on plates containing 5-fluoroorotic acid and a non-fermentable carbon source. The identity of putative cybrids was assessed by restriction analysis of mitochondrial DNA, pulse field electrophoresis and tetrad analysis. In the comprehensive screening, only mitochondrial genomes from synonymous species (S. italicus, S. oviformis, S. capensis and S. chevalieri) exhibited complete compatibility with S. cerevisiae nuclei. The closely related S. douglasii mitochondrial genome could also partially restore respiration-deficiency in ρ⁰ S. cerevisiae, whereas mitochondrial genomes from phylogenetically less related species could not. Received: 17 April 2000 / Accepted: 20 June 2000     

The gene for ribosomal protein S10 is present in mitochondria of pea and potato but absent from those of Arabidopsis and Oenothera

A novel group II intron has been identified in the pea (Pisum sativum) mitochondrial genome. The gene harbouring this intron is identified as rps10 (encoding protein S10 of the small ribosomal subunit) by similarity to its known homologues in bacteria and in the mitochondrion of the liverwort Marchantia polymorpha. The rps10 gene is transcribed in pea, the intron is removed, and RNA editing in the rps10 reading frame increases similarity to its homologue in the M. polymorpha mitochondrion. Contrary to the situation in bacteria and Marchantia, rps10 is not part of a ribosomal-protein gene cluster in pea. It is flanked upstream by the genes trnF and trnP, encoding phenylalanine-and proline-accepting tRNAs, and downstream by cox1, encoding subunit 1 of the cytochrome-c-oxidase. Southern hybridization shows that sequences homologous to rps10 exist in potato mitochondria but not in mitochondria of Oenothera berteriana and Arabidopsis thaliana. The pea rps10 intron is homologous to introns in rrn26 and cox3 in the Marchantia mitochondrial genome, while the Marchantia rps10 gene lacks an intron.     

Comparison of mitochondrial and chloroplast genome segments from three onion (Allium cepa L.) cytoplasm types and identification of a trans-splicing intron of cox2

To study genetic relatedness of two male sterility-inducing cytotypes, the phylogenetic relationship among three cytotypes of onions (Allium cepa L.) was assessed by analyzing polymorphisms of the mitochondrial DNA organization and chloroplast sequences. The atp6 gene and a small open reading frame, orf22, did not differ between the normal and CMS-T cytotypes, but two SNPs and one 4-bp insertion were identified in CMS-S cytotype. Partial sequences of the chloroplast ycf2 gene were integrated in the upstream sequence of the cob gene via short repeat sequence-mediated recombination. However, this chloroplast DNA-integrated organization was detected only in CMS-S. Interestingly, disruption of a group II intron of cox2 was identified for the first time in this study. Like other trans-splicing group II introns in mitochondrial genomes, fragmentation of the intron occurred in domain IV. Two variants of each exon1 and exon2 flanking sequences were identified. The predominant types of four variants were identical in both the normal and the CMS-T cytotypes. These predominant types existed as sublimons in CMS-S cytotypes. Altogether, no differences were identified between normal and CMS-T, but significant differences in gene organization and nucleotide sequences were identified in CMS-S, suggesting recent origin of CMS-T male-sterility from the normal cytotype.     

Genic rearrangements in Phytophthora mitochondrial DNA

Summary To assess evolutionary relationships among the oomycetous fungi we have constructed a physical and genic map of the mtDNA of a broad host range strain (695T) of Phytophthora megasperma. While, like other Phytophthora species, this 43.5 kb circular genome lacks the typical oomycete large inverted repeat, a short 0.5–0.9 kb inverted repeat has been identified. Comparison of the relative order of seven genic regions with host-specific Phytophthora strains reveals both a clustering of these loci within one-third of the host-specific genomes, and two genic inversion relative to the broad host range genome. The location of the short inverted repeat suggests that at least one of the inversions is a consequence of intramolecular recombination between repeat elements.Abbreviations atp6, atp9 genes for ATP synthase subunits 6 and 9 - cox1. cox2. cox3 genes for cytochrome c oxidase subunits I, II, and III - cob gene for apocytochrome b - rns, rnl genes for small and large mitochondrial rRNAs - mtDNA mitochondrial DNA - kb kilobase pairs     

The complete mitochondrial genome sequence of the liverwort <Emphasis Type="Italic">Pleurozia purpurea</Emphasis> reveals extremely conservative mitochondrial genome evolution in liverworts

Plant mitochondrial genomes have been known to be highly unusual in their large sizes, frequent intra-genomic rearrangement, and generally conservative sequence evolution. Recent studies show that in early land plants the mitochondrial genomes exhibit a mixed mode of conservative yet dynamic evolution. Here, we report the completely sequenced mitochondrial genome from the liverwort Pleurozia purpurea. The circular genome has a size of 168,526 base pairs, containing 43 protein-coding genes, 3 rRNA genes, 25 tRNA genes, and 31 group I or II introns. It differs from the Marchantia polymorpha mitochondrial genome, the only other liverwort chondriome that has been sequenced, in lacking two genes (trnRucg and trnTggu) and one intron (rrn18i1065gII). The two genomes have identical gene orders and highly similar sequences in exons, introns, and intergenic spacers. Finally, a comparative analysis of duplicated trnRucu and other trnR genes from the two liverworts and several other organisms identified the recent lateral origin of trnRucg in Marchantia mtDNA through modification of a duplicated trnRucu. This study shows that the mitochondrial genomes evolve extremely slowly in liverworts, the earliest-diverging lineage of extant land plants, in stark contrast to what is known of highly dynamic evolution of mitochondrial genomes in seed plants.     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

The apocytochrome-b gene in Chlorogonium elongatum (Chlamydomonadaceae): an intronic GIY-YIG ORF in green algal mitochondria

The mitochondrial cob gene from the green alga Chlorogonium elongatum (Chlamydomonadaceae) is interrupted by two group-I introns each containing an open reading frame in-phase with the upstream exon. One of these ORFs belongs to the LAGLI-DADG family, the other to the GIY-YIG family. The latter has not yet been identified in any mitochondrial genome except those from fungi. The Chlorogonium ORFs are similar to ORFs encoded by fungal introns that are located at an identical position within the gene, and to the ORF encoded by the mobile intron in the Chlamydomonas smithii cob gene. Received: 8 November 1996 / 11 February 1997     

Complete sequence of the mitochondrial genome of a diatom alga Synedra acus and comparative analysis of diatom mitochondrial genomes

The first two mitochondrial genomes of marine diatoms were previously reported for the centric Thalassiosira pseudonana and the raphid pennate Phaeodactylum tricornutum. As part of a genomic project, we sequenced the complete mitochondrial genome of the freshwater araphid pennate diatom Synedra acus. This 46,657 bp mtDNA encodes 2 rRNAs, 24 tRNAs, and 33 proteins. The mtDNA of S. acus contains three group II introns, two inserted into the cox1 gene and containing ORFs, and one inserted into the rnl gene and lacking an ORF. The compact gene organization contrasts with the presence of a 4.9-kb-long intergenic region, which contains repeat sequences. Comparison of the three sequenced mtDNAs showed that these three genomes carry similar gene pools, but the positions of some genes are rearranged. Phylogenetic analysis performed with a fragment of the cox1 gene of diatoms and other heterokonts produced a tree that is similar to that derived from 18S RNA genes. The introns of mtDNA in the diatoms seem to be polyphyletic. This study demonstrates that pyrosequencing is an efficient method for complete sequencing of mitochondrial genomes from diatoms, and may soon give valuable information about the molecular phylogeny of this outstanding group of unicellular organisms.     

Mitochondrial DNA in the aquatic fungus Allomyces

Summary The mitochondrial DNA from seven species of the aquatic phycomycete Allomyces has been isolated and characterized by restriction enzyme analysis. Comparison of the mitochondrial DNA restriction enzyme fragmentation patterns showed pronounced differences not only among species but also among four isolates of A. arbuscula. The mitochondrial DNAs range in size from 39 kbp in A. neo-moniliformis to 56 kbp in A. macrogynus.A physical map of the mitochondrial DNA of Allomyces arbuscula strain Costa Rica 21 has been constructed. The genome is circular and has a size of 49.2 kbp. The genes coding for the small and large ribosomal RNAs, cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, and ATPase subunits 6 and 9 were localized in the mitochondria) DNA by heterologous hybridization with specific mitochondria) gene probes from Saccaromyces cerevisiae and Neurospora crassa. Comparison of the gene map of the closely related species Blastocladiella emersonii with that of A. arbuscula indicates a similar gene order in the two organisms.     

Distribution of mitochondrial r1-type introns and the associated open reading frame in the yeast genus Kluyveromyces

Summary We have sequenced the intron in the large subunit ribosomal RNA gene from the mitochondrion of Kluyveromyces lactis. It is a typical group I intron but, unlike the corresponding intron (r1) in Saccharomyces cerevisiae, it does not contain an open reading frame. This intron is widespread in the genus Kluyveromyces although intron-less strains were also found in some species of this genus. Sequences homologous to the open reading frame of the S. cerevisiae ribosomal intron were detected in some strains of K. waltii, K thermotolerans and K. africanus.