Intramolecular recombination of a mitochondrial minicircular plasmid-like DNA of date-palm mediated by a set of short direct-repeat sequences

A molecular clone containing the complete sequence of a mitochondrial circular plasmid-like DNA (the R plasmid) isolated from the date-palm variety V3DP was used as a probe in Southern analyses of mitochondrial DNA prepared from other varieties. Another circular structure (the S plasmid) was detected in some of these varieties, and sequenced from variety V2DP. It appears that the R plasmid could have arisen from the S plasmid by an intermolecular recombination event at a set of 26-bp imperfect short direct repeats.     

Differential changes in copy numbers of rice mitochondrial plasmid-like DNAs and main mitochondrial genomic DNAs that depend on temperature

The mitochondria of rice contain four kinds of circular plasmid-like DNAs, namely, B1, B2, B3 and B4, in addition to the main mitochondrial genomic DNAs. In order to examine the genetic stability of mitochondrial plasmid-like DNAs, changes in the amounts of plasmid-like DNAs and main mitochondrial genomic DNAs were analyzed in calli that had been cultured at various temperatures. The observed effect of temperature on the levels of plasmid-like DNAs was larger than that on the main mitochondrial genomic DNAs. A significant reduction in the copy number of plasmid-like DNAs was detected in calli cultured at 35 °C, as compared to 20 °C, 25 °C and 30 °C. The effect of temperature on DNA synthesis in isolated mitochondria was also analyzed. Synthesis of the main mitochondrial genomic DNAs occurred at all the temperatures examined, whereas synthesis of plasmid-like DNAs occurred only over a limited range of temperatures. The results of both in vivo and in vitro analyses suggest that plasmid-like DNAs are less stably maintained than the main mitochondrial genomic DNAs, which is consistent with the notion that the transmission of mitochondrial plasmid-like DNAs from one generation to the next may be unstable under unusual conditions. Received: 17 February 1997 / 24 April 1998     

Plasmid-like DNAs in the commercially important mushroom genus Agaricus

Summary Two unique plasmid-like DNA components were localized in isolated mitochondria of the commercially important mushroom genus Agaricus: pEM (7.35 ± 0.15 kilobases) and pMPJ (3.65 ± 0.15 kilobases). These DNA moieties were linear; pEM possessed regions of terminal inverted repeated sequences. No homology was detected between pEM or pMPJ DNA and the nuclear or mitochondrial genomes. No homology existed between pEM and pMPJ. This suggests independent replication of pEM and pMPJ. Restriction endonuclease digests indicated that pEM consisted of two components (pEM₁ and pEM₂) with uniquely different restriction sites and copy number.     

Nucleotide sequence and molecular characterization of plasmid-like DNAs from mitochondria of cytoplasmic male-sterile rice

Summary Two plasmid-like DNAs, B2 and B3, were isolated from mitochondria of the cytoplasmic malesterile rice, A-58 CMS. Molecular clones having their complete sequences were constructed and used as probes of mitochondrial and nuclear genomes by Southern hybridization. No evidence was found that integrated copies of either one exist in the main mitochondrial genome, but sequences homologous to both were present in the nuclear genome. The complete nucleotide sequences of B2 and B3 were established and compared to those of rice B1 and B4 and to the 1.9-and the 1.4-kbp plasmid-like DNAs of maize. Many of the sequences were common to both plant species.     

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.     

Characterization of a variant Neurospora crassa mitochondrial DNA which contains tandem reiterations of a 1.9 kb sequence

Summary We have identified a second variant of Neurospora mtDNA which contains tandem, head-to-tail repeats of sequences at the boundary of Eco RI-4 and 6. This region may contain a. major replication origin of Neurospora mtDNA.     

Hairpin and dimer structures of linear plasmid-like DNAs in mitochondria of Paramecium caudatum

The molecular structure of plasmid-like DNAs (designated type-II) which were isolated from mitochondria in the ciliated protozoan Paramecium caudatum was characterized. These type-II DNAs are always detected as a set of four kinds with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The DNAs of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomer molecules, respectively. Electron microscopic observations indicated configurations of a hairpin structure that had a protruding end of single-stranded DNA in one terminus and a loop in the other terminus. The monomers stick together with base-pairing in opposite directions at the protruding end to form the dimers, suggesting the presence of inverted repeats. These unusual dimers may have a role in replication of the DNAs in which the monomers can serve as a primer for each other.     

Analysis of nuclear sequences homologous to the B4 plasmid-like DNA of rice mitochondria; evidence for sequence transfer from mitochondria to nuclei

Summary Nuclear sequences homologous to the plasmid-like DNA, B4, were analyzed in the Japonica rice variety, Fujiminori. Homologous sequences existed at several positions in the nuclear genome, but each contained only a portion of the B4 sequence. It was impossible to reconstruct the entire sequence of B4 even by collating all the homologous sequences. Overlaps between some of the B4 sequences present in the nuclear genome resulted in parts of the sequence being represented more than once. These features indicate that nuclear sequences homologous to B4 are not the origin of B4 and that they have been transferred from mitochondria and integrated into the nuclear genome. Five other foreign sequences originating in the chloroplast or mitochondrial genome were found within 1 kb of the B4-homologous sequences. Structural analysis is consistent with the hypothesis that the DNA sequences were transfered via RNA.     

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.     

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     

Properties and nucleotide sequence of a mitochondrial plasmid from sunflower

Summary The 1.413 circular supercoiled mitochondrial DNA plasmid P 1 from a fertile sunflower line was sequenced, and a series of 160 by tandemly repeated sequences was observed. The P1 plasmid was detected in both fertile and cytoplasmic male-sterile (CMS) lines, but in different quantities. Two other circular plasmids, P2 and P3, each 1.8 kbp in length, were shown to share common sequences with Pl. The mitochondrial plasmid P1 detected homologous sequences in the nuclear DNA of sunflower, but not in chloroplast DNA nor in main band mitochondrial DNA. RNA molecules of about 680 and 550 nucleotides were detected that were complementary to mt plasmid P1.     

Characterization of inverted repeats from plasmid-like DNAs and the maize mitochondrial genome

Summary Integrated inverted repeat (IR) sequences similar to those of the S plasmids have been isolated from the genomes of the normal and S type male-sterile cytoplasms of maize mitochondria. The nucleotide sequences of both the IRs and their flanking regions have distinguished and characterized several different types of repeats. The repeats may be involved in the recombinational process that occurs continuously in the mitochondrial genome. One cloned fragment, derived from a fertile revertant and containing sequences similar to S-2, does not appear to act as atypical transposable element during reversion. Several of the flanking regions examined contain a small repeat of 34 base pairs, in which a nonanucleotide segment is found with similarity to the yeast mitochondrial promoter.     

Electron microscopic investigation of mitochondrial DNA fromChenopodium album (L.)

DNA molecules from mitochondria of whole plants and a suspension culture ofChenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0.3–183 kb represented 23–26% of all mtDNA molecules in the preparations from the suspension culture and 13–15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rollingcircle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.     

Partial mitochondrial DNA sequence of the crustaceanDaphnia pulex

A 3667-base pair (bp) fragment of the mitochondrial genome of the crustaceanDaphnia pulex has been sequenced and found to contain the complete genes for the small subunit ribosomal RNA, ND2, seven tRNAs and the control region. This organization is identical to that found inDrosophila yakuba mtDNA yetD. pulex mtDNA exhibits several unique features when compared to other mitochondrial sequences. The sequenced fragment is only 62.6% A+T which is much lower than that of any other arthropod mtDNA sequenced to date.D. pulex mtDNA also exhibits length conservation having shorter coding and non-coding regions. The putative control region is 689 bp in length and includes a sequence that has the potential to fold into a hairpin structure with a perfect 20-bp pair stem and a 22-base loop.     

Physical and gene organization of mitochondrial DNA from the fertile cytoplasm of sugarbeet (Beta vulgaris L.)

We have constructed a complete physical map of the mitochondrial genome from the male-fertile cytoplasm of sugarbeet. The entire sequence complexity can be represented on a single circular master chromosome of 358 kb. This master chromosome contains three copies of one recombinationally active repeat sequence, with two copies in direct orientation and the other in inverted orientation. The positions of the rRNA genes and of 23 polypeptide genes, determined by filter hybridization, are scattered throughout the genome, with triplicate rrn26 genes located partially or entirely within the recombination-repeat elements. Three ribosomal-protein genes (rps1A, rps14 and rps19) were found to be absent from sugarbeet mtDNA. Our results also reveal that at least six regions homologous with cpDNA are dispersed in the mitochondrial genome.     

A rapid and simple method for extracting yeast mitochondrial DNA

Summary A rapid method for the extraction of yeast mitochondrial DNA (mtDNA) is described. In comparison with previous methods, it simplifies several steps, does not require either the isolation of mitochondria or phenol treatment and is less time consuming. This protocol gives a high yield of pure mtDNA (50–120 g from a 100-ml culture), which can be directly used in various molecular applications: restriction enzyme digestion, electrophoresis, blotting, labeling, cloning and sequencing.     

The regulation of mitochondrial DNA levels in Saccharomyces cerevisiae

Summary Mitochondrial DNA (mtDNA) synthesis can continue under conditions which block cell division and nuclear DNA (nDNA) synthesis, producing cells with several times the normal level of mtDNA. We have examined mtDNA synthesis in cultures recovering from such cell cycle blocks. Our results show that the rate of mtDNA synthesis is not affected either during a block of the cell cycle with -factor or during recovery from a perturbation in the amount of mtDNA/cell induced by blocking the cell cycle with -factor or cdc4. The normal mtDNA content was restored a period of several generations when permissive conditions were restored. These results suggest that mtDNA synthesis is coupled to cell growth.     

Large size and complex structure of mitochondrial DNA in two nonflowering land plants

Summary We report the first estimates of genome size and complexity for mitochondrial DNAs (mtDNAs) from nonflowering land plants. The mtDNA of Onoclea sensibilis (sensitive fern) is approximately 300 kb in size, while that of Equisetum arvense (common horsetail) is at least 200 kb. Sufficient mtDNA of Onoclea was available to permit an estimation of the copy number and a linkage analysis of nine mitochondrial genes. Six of these genes appear to be present in only one or two copies in the Onoclea genome, whereas three other genes are present in multiple copies. Five of the approximately ten genes encoding 26S rRNA are located on a large, >10kb, dispersed repeat that also contains closely linked genes for 18S rRNA and the alpha subunit of ATPase (atpA). The other 26S genes belong to a second dispersed repeat family of >8 kb whose elements do not contain any other identified genes. Because flowering plant mtDNAs are also large and contain dispersed, gene-containing, repeats, it appears that these features arose early in the evolution of land plants, or perhaps even in their green algal ancestors.