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.     

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     

Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite,Romanomermis culicivorax

Summary Physical characterization of the mitochondrial genome derived from the obligate mosquito parasite, Romanomermis culicivorax has generated some surprising physical properties regarding the molecular structure of nematode mitochondrial DNA (mtDNA). Restriction enzyme analysis of this mtDNA has revealed a mitochondrial genome size of approximately 26 kb, the largest metazoan mtDNA reported to date. Isofemale lineages are monomorphic for one of three size variants, differing by 500-1,000 base pairs, present in our original field population. Cloned hybridization probes derived from a single region exhibiting a 600 by size polymorphism share strong homology with several spatially separated sites distributed about the mtDNA. This suggests that the homology is a result of repeated DNA sequence elements contained within this mitochondrial genome that contribute to mtDNA size polymorphism.     

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.     

Electrophoretic analysis of the nuclear and organellar genomes in the ultra-small alga Cyanidioschyzon merolae

Electrophoretic analysis reveals that the nucleus of the ultra-small eukaryotic alga Cyanidioschyzon merolae contains approximately 11.7×10⁶ base pairs (11.7 Mb) of DNA. This compact genome is fragmented into 15 small chromosomes ranging in size from 410 to 1700 kb. The migratory behaviour of chloroplast DNA is consistent with the presence of a circular plastid genome of about 170 kb. The conformation of mitochondrial DNA resembles that in yeasts and fungi and is predominantly linear and heterogenous in size.     

In vivo conformation of mitochondrial DNA in fungi and zoosporic moulds

Summary Migratory behaviour of mitochondrial DNA (mtDNA) from fungal species belonging to Plectomycetes, Loculoascomycetes and the zoosporic moulds, Oomycetes, has been studied by Pulsed Field Gel Electrophoresis (PFGE). Electrophoretic profiles demonstrate that long, linear molecules of a heterogenous size are the preveailing in-vivo form of organelle DNA in all examined species. These profiles are consistent with the presence of the rolling-circle mode of mtDNA replication that occurs in all branches of true fungi and zoosporic moulds.     

Moving pictures and pulsed-field gel electrophoresis show linear DNA molecules from chloroplasts and mitochondria

Summary The structure of the cytoplasmic genomes in plants has been investigated by using fluorescence microscopy to make moving pictures of ethidium-stained DNA fractionated by pulsed-field gel electrophoresis (PFGE) and emerging from organelles lysed within gelled agarose. For watermelon chloroplasts, PFGE fractions contained linear molecules representing monomeric to tetrameric lengths of the unit genome (155 kilobase pairs, kb) and linear DNA of at least 1,200 kb, whereas circular molecules were clearly identified only with chloroplast agarose inserts. For pea, the oligomeric series extended only to the trimer. Most of the DNA from watermelon mitochondria was in the form of 50–100 kb linear molecules with some DNA of at least 1,200 kb, but no band was seen at the size of this genome (330 kb) and no circular molecules were identified in either PFGE fractions or mitochondria embedded in agarose. Most mitochondrial DNA from cauliflower also consisted of 50–100 kb linear molecules with some much longer linear forms, but no genome-sized (220 kb) PFGE band was evident. The possible relevance of the very long linear DNAs to previous cytological and genetic observations is discussed.     

Mitochondrial DNA of Endomyces (Dipodascus) magnusii

Mitochondrial DNA was isolated from a yeast-like microorganism, Endomyces (Dipodascus) magnusii. The mtDNA consisted of circular molecules 40.4 kb long. A restriction map was constructucted using the cleavage data of seven endonuclease. The arrangement of several genes within the mitochondrial genome of E. magnusii was established by specific hybridization with probes prepared from the mtDNA of Saccharomyces cerevisiae.     

On a model for the structure of circular mitochondrial genomes in higher plants

The mitochondrial genome of some higher plants consists of a discrete set of circular molecules. The heterogeneity found in the number of circles and their size is puzzling. In some plants evidence exists that indicates that these circular DNA molecules undergo intermolecular and intramolecular recombination events. The result of these events is that two small circles can combine to give one large circle or one large circle can break up into two small circles. Here we pursue the idea that such recombination events are responsible for the variability in genome composition. Treating the recombination events as chemical reactions we derive the equilibrium size distribution of circles. In Brassica campestris there are two basic subunits, circles A and B, containing 135 and 83 kilobases, respectively. By restriction-enzyme mapping one can determine in a multimeric circle, the number of interfaces between A- and B-derived DNA. Using a combinatorial argument we predict the frequency of such interfaces and compare our predictions with published data. A number of suggestions are made for additional experimental tests of the recombinational theory of genomic diversity.     

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155

 In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155. Received: 19 September 1995/4 January 1996     

The mitochondrial genome of Nicotinia plumbaginifolia

Summary DNA was isolated from DNase-treated, osmotically shocked mitochondria from leaves and calli of Nicotiana plumbaginifolia. Electron microscopic analysis only revealed linear molecules, although the same technique yielded a sizable fraction of circular mitochondrial DNA molecules in the fission yeast Schizosaccharomyces pombe. Most of the plant mitochondrial DNA molecules could be assigned to six size classes of 3.11 ± 0.19, 7.08 ± 0.53, 11.2 ± 0.29, 14.10 ± 0.23, 18.13 ± 0.39, and 23.17 ± 0.51 m length (mean ± 95% confidence intervals). These results are compared with those obtained for mitochondrial genomes of other higher plants.     

Molecular analysis of mitochondrial DNA from tomato cell suspension cultures

Summary Mitochondrial DNA (mtDNA) from Lycopersicon esculentum was purified from cell suspension cultures. The DNA, isolated from mitochondria purified by two successive sucrose density gradients, was uncontaminated with nuclear DNA or DNA from proplastids. The total molecular weights of BamHI, BglI, and BglII fragments indicate a mitochondrial genome size of at least 270 kb. Cross hybridization between tomato mtDNA and cloned spinach plastid genes revealed some homology. In hybridization experiments using cloned mitochondrial rRNA genes and BamHI digested total mtDNA the presence of recombination repeats is demonstrated.     

Maintenance of chloroplast-derived sequences in the mitochondrial DNA of Gramineae

Evidence for the transfer of DNA from the chloroplast to the mitochondrion has been reported in many higher plants and, in most cases, the transferred chloroplast genes do not have the ability to encode functional products as a consequence of base substitutions and/or multiple rearrangements. We reported previously that the sequence of one end of a chloroplast-derived (ct-derived) fragment of DNA that contained the rps19 and trnH genes has been maintained in most gramineous plants and that its presence seems to be correlated with gene expression in this region. In the present study, we have investigated whether or not the ct-derived sequences in mitochondrial DNA (mtDNA) from some gramineous plants and species of Oryza are conserved, and whether or not such conservation is related to gene expression in these regions. We identified two junctions between ct-derived and mitochondrial sequences that were conserved among some gramineous plants. Around these regions, we found a ct-derived gene for tRNA and the promoter of a mitochondrial gene on the ct-derived sequences, respectively, and these regions were transcribed through the junctions. This result indicates that the junctions and/or regions that are transcribed and functional in mitochondria have been strongly conserved and maintained during their evolution. In Oryza, some junctions between ct-derived and mitochondrial sequences were conserved and other junctions were not. These variations seem to have been caused by deletions and/or rearrangements, and appear to be specific to the type of genome. In the case of Oryza, the timing of deletions and/or rearrangements of ct-derived sequences is likely to have coincided with the divergence of the various genome types. Received: 24 July / 13 August 1997     

Evidence for giant linear plasmids in the ascomycete Podospora anserina

In the extrachromosomal mutant AL2 of the ascomycete Podospora anserina longevity is correlated with the presence of the linear mitochondrial plasmid pAL2-1. In addition to this autonomous genetic element, two types of closely related pAL2-1-homologous molecules were detected in the high-molecular-weight mitochondrial DNA (mtDNA). One of these molecules is of linear and the other of circular structure. Both molecules contain pAL2-1 sequences which appear to be integrated at the same site in the mtDNA. Sequence analysis of a DNA fragment cloned from one of these molecules revealed that it contains an almost full-length copy of pAL2-1. At the site of plasmid integration a 15-nucleotide AT-spacer and long inverted mtDNA sequences were identified. Finally, two giant linear plasmid-like DNAs of about 50 kbp and 70 kbp were detected in pulsed-field gels of mutant AL2. These molecules are composed of mtDNA and pAL2-1-specific sequences and may result from the integration of mtDNA sequences into linear plasmid pAL2-1.     

On a Model for the Structure of Circular Mitochondrial Genomes in Higher Plants

The mitochondrial genome of some higher plants consists of adiscrete set of circular molecules. The heterogeneity foundin the number of circles and their size is puzzling. In someplants evidence exists that indicates that these circular DNAmolecules undergo intermolecular and intramolecular recombinationevents. The result of these events is that two small circlescan combine to give one large circle or one large circle canbreak up into two small circles. Here we pursue the idea thatsuch recombination events are responsible for the variabilityin genome composition. Treating the recombination events aschemical reactions we derive the equilibrium size distributionof circles. In Brassica campestris there are two basic subunits,circles A and B, containing 135 and 83 kilobases, respectively.By restriction-enzyme mapping one can determine in a multimericcircle, the number of interfaces between A- and B-derived DNA.Using a combinatorial argument we predict the frequency of suchinterfaces and compare our predictions with published data.A number of suggestions are made for additional experimentaltests of the recombinational theory of genomic diversity.     

Variable abundance of a mitochondrial DNA fragment in cultured tobacco cells

Summary The relative abundance of a cloned 4.5 kilobase (kb) pair mitochondrial DNA sequence in two suspension cultures of tobacco (Nicotiana tabacum cv Turkish samsun and Nicotiana tabacum NT-1) has been examined. This sequence is 70-fold reduced in NT-1 relative to Turkish samsun; the reduction is correlated with an increase in supercoiled mitochondrial DNA. This sequence does not hybridize with mitochondrial DNA from watermelon, maize, or Saccharomyces cerevisiae, nor with several cloned mitochondrial genes and is thus probably not a gene. It may represent most of the plant mitochondria) genome thought to be non-essential for mitochondrial function. The sequence complexity of supercoiled mitochondrial DNA from NT-1 cells is about one-third that found for the entire mitochondrial genome and does not include the cytochrome oxidase subunit II gene.     

Physical and gene mapping of cauliflower (Brassica oleracea) mitochondrial DNA

Summary A physical map of the mitochondrial DNA isolated from B. oleracea (cauliflower) inflorescences was constructed with the restriction endonucleases Sall, Kpnl and Bgll. Physical mapping was made using the multi enzyme method with either unlabeled or labeled DNA fragments isolated by preparative electrophoresis and a clone bank prepared by inserting incomplete Sall restriction digests of mitochondrial DNA into a cosmid vector.The different mapping studies led to a circular map, about 217 kb in size, containing the entire sequence complexity of the genome. The 26S and 18S – 5S ribosomal RNA genes appeared to be separated by about 75 kb in this map. However, the particular cross-hybridization between several restriction fragments and the sequential diversity of some cosmids indicated that intra molecular recombination may occur naturally in higher plant mitochondria. Namely, one recombinational event resulted in the ribosomal RNA genes mapping closer together.Abbreviations mtDNA mitochondrial DNA - kb kilobasepairs - rRNA ribosomal RNA - LGT agarose low gelling temperature agarose     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

Role of sequence amplification in the generation of nematode mitochondrial DNA polymorphism

Summary Romanomennis culicivorax, an obligate parasitic nematode of mosquitos, possesses an unusually large mitochondrial genome. Individuals are monomorphic for one of several mitochondrial DNA (mtDNA) size variants ranging from 26–32 kb. In this report, we demonstrate that the mitochondrial genome size differential in three isofemale lineages is due to the presence of mtDNA sequences amplified to different copy numbers within each mtDNA molecule. Restriction enzyme analysis and DNA sequencing studies reveal that each mitochondrial genome contains one of two 3.0 kb repeat types that differ by approximately 30 bp. This difference is primarily due to a short (23 bp) imperfect tandem duplication present within the larger of two polymorphic repeating units. The 3.0 kb reiterated DNA sequences are present as direct, tandem repeats and as inverted portions of the same sequence located elsewhere in the genome. Based on mtDNA analysis of an independently reared R. culicivorax culture, we conclude that events resulting in mitochondrial genome rearrangement occurred in natural field populations prior to propagation within the laboratory.     

Assembly of the mitochondrial membrane system. Organization of yeast mitochondrial DNA in the Oli1 region

Summary The mitochondrial DNA (mtDNA) of a cytoplasmic petite mutant (DS401) of Saccharomyces cerevisiae genetically marked for the ATPase proteolipid, serine tRNA and varl genes has been characterized by restriction endonuclease analysis and DNA sequencing. The DS401 mtDNA segment is 5.3 kb long spanning the region between 79.1 and 86.8 units of the wild type genome. Most of the DS401 mtDNA consists of A+T rich sequences. In addition, however, there are ten short sequences with a high content of G+C and two sequences that have been identified as the ATPase proteolipid and the serine tRNA genes. The two genes map at 81 and 83 units and are transcribed from the same DNA strand. Even though there are other possible coding sequences in the DNA segment, none are sufficiently long to code for a gene product of the size of the varl protein. Based on the relative organization of the G+C rich clusters and genes, a model has been proposed for the processing of mitochondria) RNA. This model postulates the existence of mitochondrial double strand specific RNases that cleave the RNA at the G+C clusters.