The mitochondrial genome of the aquatic phycomycete Blastocladiella emersonii

Summary Mitochondrial DNA from the aquatic fungus Blastocladiella emersonii Cantino and Hyatt has been isolated and characterized. By restriction enzyme analysis the size of the mitochondrial genome was found to be 35.5 kb pairs. A restriction site map was constructed using the cleavage data for 6 endonucleases which showed the mitochondrial genome to be circular. The genes for the small and large ribosomal RNA, the ATPase subunits 6 and 9, the cytochrome c oxidase subunits 1, 2, and 3, and the apocytochrome b were located in the mitochonridal genome of B. emersonii by hybridizations with mitochondrial DNA probes from Saccharomyces cerevisiae and Neurospora crassa     

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.     

The mitochondrial DNA of the yeast Hansenula petersonii: genome organization and mosaic genes

Summary The mitochondrial genomes of yeasts are circular DNA molecules that vary greatly in size in different species. The mitochondrial DNA of the yeast H. petersonii is about 42 kbp in length, about one half the size of the corresponding genome in S. cerevisiae. Sequences homologous to protein-encoding genes from S. cerevisiae have been identified and localized on this genome by hybridization with DNA from petite mutants. The comparison between the mitochondrial genomes of H. petersonii and S. cerevisiae showed differences in the overall genome organization, but both include genes with mosaic organization. In fact, sequences homologous to the first intron of the S. cerevisiae cob short gene are found in (or adjacent to) the cob and cox1 genes present in the genome of H. petersonii. Moreover, an intron homologous to that present in the 21S rRNA gene of S. cerevisiae seems to have been conserved in the large ribosomal RNA gene of H. petersonii, in a similar position.     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

Anatomy of amplified mitochondrial DNA in “Ragged” mutants of Aspergillus amstelodami: Excision points within protein genes and a common 215 bp segment containing a possible origin of replication

Summary The extranuclearly-inherited ragged growth phenotype (Rgd) of Aspergillus amstelodami is always accompanied by excision and head-to-tail amplification of mtDNA sequences. In one mutant strain (Rgd1) the amplified mtDNA segment (rgd1 DNA, monomeric length 0.9 kb) maps downstream of the large subunit ribosomal RNA gene (Region 1), whereas in all other strains analyzed the amplified sequences (rdg3-7DNA) are located in Region 2 between genes coding for cytochrome b and ATPase subunit 6. The various region 2 sequences differ in lengths (1.5 to 2.7 kb) but have in common a 215 bp sequence mapping between an. unidentified protein gene (corresponding to URF4 of human mtDNA) and an arginine tRNA gene. This common sequence may contain an origin of replication, because a looped-out hairpin structure similar to that of yeast and human mitochondrial origin sequences can be formed. Furthermore, Region 2 DNA suppresses replication of Region 1 DNA, indicating that the former group of molecules contains the more efficient origin. The nucleotide sequence of the rgd6 repeat unit starts and ends within protein genes of mtDNA, and no homologies were found between heads and tails or their flanking sequences.Abbreviations mtDNA DNA isolated from DNase — treated mitochondria - Rgd ragged mutant strain - rgdDNA highly-reiterated DNA sequences isolated simultaneously with the wild-type genome from mitochondria of ragged mutants - bp base pairs - kb kilobase pairs - URF unassigned reading frame     

High-frequency inter-parental recombination between mitochondrial genomes of rice cybrids

Analyzing more than 100 independent rice cybrids, we found evidence for inter-molecular recombination between parental mitochondrial genomes occurring at high frequency soon after protoplast fusion. The structure of the region around the atp6 gene showed extensive polymorphism among Indica (MTC-5A), Japonica (Nipponbare), and wild abortive (IR58024A) mitochondrial genomes. Recombination between the mitochondrial genomes of IR58024A and MTC-5A around the atp6 gene was detected by Southern-blot analysis of cybrid plants. Such recombinant mitochondrial molecules were also cloned from IR58024A/Nipponbare cybrid callus. PCR analysis around the atp6 gene demonstrated that inter-parental recombination occurs in practically all cybrid calli within 2 weeks after protoplast fusion. At this point, parental and recombinant mitochondrial genomes coexisted within the callus. Over the course of further cultivation, however, mitochondrial genome diversity decreased as parental and/or recombinant genomes segregated out.     

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.     

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     

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.     

MboI,ThaI and HinfI endonuclease cleavage maps of the yeast mitochondrial DNA

Summary MboI, HinfI and ThaI cleavage maps have been constructed for the region of the mitochondrial DNA from S. cerevisiae where transfer RNA genes are principally located. About 40 cleavage sites have been localized between the C and P genetic markers. The MboI map covers about 50% of the total mitochondrial genome. For constructing maps we have used a series of rho^– deletion mutants whose mitochondrial DNAs have a typical single deletion structure as judged by previous genetic and physical analyses. The mutant DNAs carry known transfer RNA genes and genetic markers and, therefore, the comparison between genetic and restriction maps has allowed us to localize individual transfer RNA genes within defined physical segments.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA - ThaI formerly TacI     

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.     

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.     

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones

Summary Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.     

Cloning and sequence analysis of an XbaI fragment of rainbow trout mitochondrial DNA

Summary A 2.4 kbp Xbal fragment of rainbow trout mitochondrial DNA was cloned into pTZ18R. DNA sequence analysis reveals that this segment of the genome encodes URF3, tRNA_Arg, URF4L and URF4 in the same orientation as other vertebrate mitochondrial genomes. Comparison of these segments of the rainbow trout mitochondrial genome with the corresponding sequences in human mitochondrial DNA shows that approximately 60% of the nucleotides are the same in both species.     

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     

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.     

Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.