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.     

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.     

An adaptive response to alkylating agents in Aspergillus nidulans

Summary The analysis of mitochondria' DNA (mtDNA) from several strains of Candida parapsilosis and Candida rhagii by restriction endonucleases enabled us to discriminate between several groups within the C. parapsilosis species and to allocate laboratory strains to one of these. The mtDNAs isolated ranged in size from 20 to 31 kb. The mtDNA isolated from group 1 C. parapsilosis hybridises with both ATPase subunit 6 and 8 gene probes, the same restriction fragment hybridising with both probes.     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

Location,identity, amount and serial entry of chloroplast DNA sequences in crucifer mitochondrial DNAs

Southern blot hybridization techniques were used to examine the chloroplast DNA (cpDNA) sequences present in the mitochondrial DNAs (mtDNAs) of two Brassica species (B. campestris and B. hirta), two closely related species belonging to the same tribe as Brassica (Raphanus sativa, Crambe abyssinica), and two more distantly related species of crucifers (Arabidopsis thaliana, Capsella bursa-pastoris). The two Brassica species and R. sativa contain roughly equal amounts (12–14 kb) of cpDNA sequences integrated within their 208–242 kb mtDNAs. Furthermore, the 11 identified regions of transferred DNA, which include the 5 end of the chloroplast psaA gene and the central segment of rpoB, have the same mtDNA locations in these three species. Crambe abyssinica mtDNA has the same complement of cpDNA sequences, plus an additional major region of cpDNA sequence similarity which includes the 16S rRNA gene. Therefore, except for the more recently arrived 16S rRNA gene, all of these cpDNA sequences appear to have entered the mitochondrial genome in the common ancestor of these three genera. The mitochondrial genomes of A. thaliana and Capsella bursa-pastoris contain significantly less cpDNA (5–7 kb) than the four other mtDNAs. However, certain cpDNA sequences, including the central portion of the rbcL gene and the 3 end of the psaA gene, are shared by all six crucifer mtDNAs and appear to have been transferred in a common ancestor of the crucifer family over 30 million years ago. 1n conclusion, DNA has been transferred sequentially from the chloroplast to the mitochondrion during crucifer evolution and these cpDNA sequences can persist in the mitochondrial genome over long periods of evolutionary time.     

A tRNASer(UCN) gene in Artemia salina mitochondrial DNA: a case of mistaken identity

We have sequenced a segment of mitochondrial DNA (mtDNA) of a crustacean, the brine shrimp, Artemia salina, that includes 3 end-proximal regions of the genes for subunit 1 of the NADH dehydrogenase complex (ND1) and cytochrome b (Cyt b). From our data we conclude that in this mtDNA, as in the mtDNAs of Drosophila species, a tRNA^Ser(UCN) gene separates the ND1 and Cyt b genes. This is contrary to an earlier report that the A. salina ND1 and Cyt b genes are immediately adjacent to each other.     

High-resolution mtDNA evidence for the late-glacial resettlement of Europe from an Iberian refugium

The advent of complete mitochondrial DNA (mtDNA) sequence data has ushered in a new phase of human evolutionary studies. Even quite limited volumes of complete mtDNA sequence data can now be used to identify the critical polymorphisms that define sub-clades within an mtDNA haplogroup, providing a springboard for large-scale high-resolution screening of human mtDNAs. This strategy has in the past been applied to mtDNA haplogroup V, which represents <5% of European mtDNAs. Here we adopted a similar approach to haplogroup H, by far the most common European haplogroup, which at lower resolution displayed a rather uninformative frequency distribution within Europe. Using polymorphism information derived from the growing complete mtDNA sequence database, we sequenced 1580 base pairs of targeted coding-region segments of the mtDNA genome in 649 individuals harboring mtDNA haplogroup H from populations throughout Europe, the Caucasus, and the Near East. The enhanced genealogical resolution clearly shows that sub-clades of haplogroup H have highly distinctive geographical distributions. The patterns of frequency and diversity suggest that haplogroup H entered Europe from the Near East approximately 20,000-25,000 years ago, around the time of the Last Glacial Maximum (LGM), and some sub-clades re-expanded from an Iberian refugium when the glaciers retreated approximately 15,000 years ago. This shows that a large fraction of the maternal ancestry of modern Europeans traces back to the expansion of hunter-gatherer populations at the end of the last Ice Age.     

Isolation and characterization of mitochondrial DNA of the oomycetous fungus Phytophthora infestans

Summary Mitochondrial DNA (mtDNA) of Phytophthora infestans has been isolated and preliminarily characterized. It has a low GC content of about 22.4% which is distinctly different from that of nuclear DNA (51 %). This property has been used to separate both DNA species in the presence of 4,6-diamidine-2-phenylindole (DAPI) in CsCl density gradients. The use of cetyl triammonium bromide (CTAB) for extraction of DNA significantly reduced its degradation. The base distribution of the mtDNA shows a limited intramolecular heterogeneity. The molecule contains 36.2 ± 0.3 kb as revealed by endonuclease digestion and seems to be circular as shown by restriction mapping. No differences were found in restriction patterns between mtDNAs from various pathotypes.     

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.     

An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera

Summary Sequences hybridzing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana. Sequence order for the 34.5 kbp mtDNA of E. nana is identical to that for mtDNAs from B. custersianus (28.5 kbp) and B. naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species. A closer relationship is suggested for D. intermedia and D. bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites. Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S. cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species. An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence. Likewise mtDNA from B. anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence. By contrast the largest mtDNA (101.1 kbp) from B. custersd has the cytochrome b and LrRNA sequences in the same orientation. In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites. This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues.     

Mitochondrial DNAs of the fungus Armillaria ostoyae: restriction map and length variation

A restriction-enzyme-site map is presented for the 147-kb mtDNA of North American Armillaria ostoyae. The locations of five structural genes, atp6, atp8, coxI, coxIII, and cob, along with the location and orientation of the large and small ribosomal RNA genes, were determined through Southern hybridizations with cloned genes from other fungal mtDNAs. Based on this map, the variation in mtDNA suggested geographic structure at two different levels. On a large geographic scale, 17 mtDNA types from North America were distinct, with respect to both size and restriction maps, from three mtDNA types from Europe. At the local scale, identical mtDNA types were evident among several different genetic individuals located no more than 1 km apart at a site in Michigan. No mtDNA type occurred more than once among genetic individuals from different regions of North America, although the occurrence of similar mtDNAs in isolates from distant regions suggested that this may occur at a low frequency with large sample sizes. Among the North American mtDNA types, analysis of discrete length variants was inconsistent with the hypothesis that the mtDNA of A. ostoyae evolves as a clonal lineage in which each length mutation represents a unique event. The two remaining hypotheses, that similar mutational events have occurred independently and that genetic exchange and recombination occurs among mtDNAs in natural populations of this species, remain to be tested.     

Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes

  The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast. An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE. Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome. Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50–100 kb, but weakly to the larger DNA from mitochondria of these two strains. For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50–100 kb, but a smear without bands was found for the circular-mapping strain. Received: 28 June 1995 / 15 January 1996     

R-type plasmids in mitochondria from a single source of Zea luxurians teosinte

Two linear DNA plasmids resembling the R1 and R2 plasmids that are present in the mitochondria of several South American strains of maize were found in mitochondria from a single source of Zea luxurians collected by L. Mazoti. The Mazoti mtDNA is closely related to mtDNAs of other Z. luxurians, but mitochondria derived from the other Z. luxurians sources lack the plasmids. The larger plasmid from Mazoti mitochondria, M1, was cloned and large portions of it were sequenced. Restriction mapping and sequence comparisons showed that approximately 4.9 kb is similar to the S1 plasmid of maize and an additional 2.6 kb is related to R1 sequences integrated into the main mitochondrial genome of N cytoplasm. Therefore, the M1 plasmid appears to be very similar to the R1 plasmid. The inverted repeats at the ends of the M1 plasmid are not identitical. The left end IR is similar to the S-TIRs found at the termini of the S plasmids. The right end IR more closely resembles the integrated R1 sequences, including the variant region of the TIR. Whereas the variant region contains 13 bp in the S-TIRs and 15 bp in an integrated version of R1, it is 16 bp long in M1. The region of M1 that has no homology to the S1 plasmid is expressed at very low levels in Mazoti and RU cytoplasms, but at much higher levels in CMS-S mitochondria, where part of it is present in the main mitochondrial genome.     

Reconstructing ancient mitochondrial DNA links between Africa and Europe

Mitochondrial DNA (mtDNA) lineages of macro-haplogroup L (excluding the derived L3 branches M and N) represent the majority of the typical sub-Saharan mtDNA variability. In Europe, these mtDNAs account for <1% of the total but, when analyzed at the level of control region, they show no signals of having evolved within the European continent, an observation that is compatible with a recent arrival from the African continent. To further evaluate this issue, we analyzed 69 mitochondrial genomes belonging to various L sublineages from a wide range of European populations. Phylogeographic analyses showed that ~65% of the European L lineages most likely arrived in rather recent historical times, including the Romanization period, the Arab conquest of the Iberian Peninsula and Sicily, and during the period of the Atlantic slave trade. However, the remaining 35% of L mtDNAs form European-specific subclades, revealing that there was gene flow from sub-Saharan Africa toward Europe as early as 11,000 yr ago.     

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.     

Codon usage,genetic code and phylogeny of Dictyostelium discoideum mitochondrial DNA as deduced from a 7.3-kb region

We have sequenced a region (7 376-bp) of the mitochondrial (mt) DNA (54 kb) of the cellular slime mold, Dictyostelium discoideum. From the DNA and amino-acid sequence comparisons with known sequences, genes for ATPase subunit 9 (ATP), cytochrome b (CYTB), NADH dehydrogenase subunits 1, 3 and 6 (ND1, ND3 and ND6), small subunit rRNA (SSU rRNA) and seven tRNAs (Arg, Asn, Cys, Lys, f-Met, Met and Pro) have been identified. The sequenced region of the mtDNA has a high average A+T-content (70.8%). The A+T-content of protein-genes (73.6%) is considerably higher than that of RNA genes (61.3%). Even with the strong AT-bias, the genetic code employed is most probably the universal one. All seven tRNAs are able to form typical clover leaf structures. The molecular phylogenetic trees of CYTB and SSU rRNA suggest that D. discoideum is closer to green plants than to animals and fungi.     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Mitotic segregation of mitochondrial dnas in human cell hybrids and expression of chloramphenicol resistance

The relationship between the chloramphenicol (CAP)-resistant phenotype and the mtDNA genotype was investigated in segregating human, HeLa × HT1080, somatic cell hybrids. The parental mtDNAs were quantitated in heteroplasmic cells by using restriction fragment length polymorphisms (RFLPs) detected in Southern blots. CAP-resistant (R) × CAP-sensitive (S) hybrids selected and grown in CAP for brief periods had as little as 25% CAP-R mtDNA. With prolonged selection, the CAP-R mtDNA increased to 90–95%. Hybrids selected and passaged without CAP either retained both mtDNAs or progressively lost one mtDNA (mitotic segregation). The CAP-resistance phenotype of these hybrids changed abruptly when the proportion of CAP-R mtDNAs fluctuated around approximately 10% (threshold effect). Hybrids with greater than 25% HT1080 mtDNA had an additional characteristic. They cloned better with CAP than without. The cloning efficiency in CAP of hybrids having 90% HT1080 mtDNA was more than fivefold greater than the control.     

Maize mitochondrial DNA rearrangements between the normal type,the Texas male sterile cytoplasm,and a fertile revertant cms-T regenerated plant

Summary The maize mitochondrial DNA (mtDNA) from the cytoplasmic male sterile type T (cms-T) contains a 6.6 kb XhoI fragment not found in the normal type (N) or the fertile revertant (V3). Analysis of the fragment in cms-T mtDNA and homologous regions in N and V3 mtDNAs reveals that a number of events are necessary to explain the formation of the 6.6 kb XhoI fragment in T mtDNA and its subsequent loss in V3 mtDNA. This includes the formation and the loss of a 4 kb repeat and various recombinational events. It is interesting to note that a recombinational event that has occurred in the fertile revertant genome reconstitutes a sequence arrangement identical to one found in the normal genome.