Mitochondrial DNA recombination in Brassica campestris

The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants.     

Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures

Summary A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37–80 kb subpopulation, which was predominant in whole plants, to 18–34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5–10 kb significantly increased in suspension culture cells. In addition, a new peak (10–12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4–2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.     

Separation of Different Conformations of Plant Mitochondrial DNA Molecules by Field Inversion Gel Electrophoresis

Mitochondrial (mt) DNA structure in higher plants is still unclear as to the circularity or linearity of the genome. We have developed a system to electrophoretically separate distinct populations of mtDNA, with some populations enriched for networked linear and circular DNA molecules. Using field inversion gel electrophoresis (FIGE) and electron microscopy (EM), we have identified four distinct populations of mtDNA from two Brassica species. Using FIGE, two slow migrating mtDNA populations ran faster than a 66 kbp Escherichia coli circular plasmid marker, while these same populations comigrated in the compression zone in contour-clamped homogeneous electrophoretic field (CHEF) gels. A fast-migrating mtDNA population was also resolved by FIGE as a diffuse band between 20 to 70 kbp when compared with linear lambda () markers. FIGE resolved the 66 kbp circular marker into several multimers, while CHEF resolved only open-circular monomers and linears. In agreement with FIGE results, EM analysis indicated the two slow migrating mtDNA populations contained circular (both supercoiled and relaxed circles) and free linear molecules of 10-60 kbp, and networked linear molecules of 45–140 kbp total size that may represent recombination intermediates. The fast migrating population consisted of 10–50 kbp linear molecules. Well-bound mtDNA showed only long linear molecules of 40–150 kbp with no detection of circles or complex/rosette molecules. This report shows that FIGE has clear advantages over CHEF for separating large DNA molecules with different conformations, and may be very useful for studies to characterize genome structure in complex systems such as plant mitochondria.     

Organization of the large mitochondrial genome in the isopod Armadillidium vulgare.

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of approximately 15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.     

Circular mitochondrial DNA molecules from petite mutants of Saccharomyces cerevisiae: resolution by polyacrylamide gel electrophoresis

Mitochondrial DNA (mtDNA) from petite strain K45 ofSaccharomyces cerevisiae contains about 7% circular DNA molecules which comprise a simple oligomeric series based on a monomeric size of 1.7 kilobase pairs. Electrophoresis of K45 mtDNA on a polyacrylamide-agarose slab gel fractionates the mtDNA into a major band (containing linear DNA) and several faster running minor bands each containing particular size class of circular DNA molecules. From study of mtDNA from K45 and two other simple petites it was found that the mobility of circles is inversely proportional to the logarithm of the circle size. Polyacrylamide gel electrophoresis thus permits the separation of circular mtDNA from the linear mtDNA of simple petites, and physically resolves circles of different size from one another.     

Structure of the mitochondrial genome of Beta vulgaris L.

Summary The structure of mitochondrial DNA (mt-DNA) from sugarbeet (Beta vulgaris L.) has been studied by biochemical methods and electron microscopy. It was found to be complex multipartite consisting of two main classes of molecules: high molecules weight (HMW) mtDNA and low molecular weight (LMW) mtDNA. The HMW mtDNA consists of rosette-like structures and globules resembling chromomeres (150–200nm). A typical rosette has a protein core and radially stemming closed DNA loops (from 0.6-1.5 m). The number of loops in a rosette varies from 16–30. The bulk of HMW mtDNAs are represented by interconnected rosettes (total contour length about 130–160 m, 403–496 kbp). Such large circular DNAs may be evidence of the master chromosome arrangement of the sugarbeet genome. Globules and rosettes are interconnected by thick and thin DNA fibrils, along which nucleosome- and nucleomere-like structures are distributed. The LWM mtDNA is composed of two groups of supercoiled circular molecules, 0,2–1.5 m and 0.02–0.05 m in size. Electrophoretic analysis demonstrated that LWM mtDNA is represented by minicircle plasmid-like DNA molecules of 1.3, 1.4 and 1.6 kbp.     

MITOCHONDRIAL GENOME CONFORMATION AMONG CW‐GROUP CHLOROPHYCEAN ALGAE1

Most green algal taxa have circular‐mapping mitochondrial genomes, whereas some have linear genome‐ or subgenomic‐sized mitochondrial DNAs (mtDNA). It is not clear, however, if the circular‐mapping genomes represent genome‐sized circular molecules, if such circular molecules and the linear forms are the predominant in vivo mtDNA structures, or if the linear forms arose only once or multiple times among extant green algal lineages. We therefore examined the DNA components detected with homologous mtDNA probes after pulsed‐field gel electrophoresis of total cellular DNA from the chlorophycean basal bodies displaced clockwise(CW)‐group taxa Chlamydomonas reinhardtii and Chlamydomonas moewusii. For C. reinhardtii, the 15.8‐kb linear mtDNA was the only DNA component detected, and there was no evidence of circular or large linear precursors of this DNA. In the case of C. moewusii, which is known to have a circular‐mapping 22.9‐kb mitochondrial genome, three DNA components were detected; these appeared to be circular (relaxed and supercoiled) and genome‐sized linear DNA molecules, the latter of which likely resulted from random double‐strand breaks in the circular forms during DNA isolation. In further studies, DNA from additional CW‐group taxa was examined using conventional gel electrophoresis and DNA‐filter blot analysis with C. reinhardtii and C. moewusii mtDNA probes. We conclude that all taxa from the “Volvox clade” (sensu Nakayama et al. 1996 of the CW‐group have genome‐ or subgenomic‐sized linear mtDNAs as their predominant mtDNA form and that these arose from a genome‐sized circular form in an ancestor that existed near the base of this clade.     

Physicochemical characterization of mitochondrial DNA from soybean

Mitochondrial DNA (mtDNA) of soybean (Glycine max L.) was isolated and its buoyant density was contrasted with that of nuclear (nDNA) and chloroplast (ctDNA) DNA. Each of the three DNAs banded at a single, characteristic buoyant density when centrifuged to equilibrium in a CsCl gradient. Buoyant densities were 1.694 g/cm³ for nDNA and 1.706 g/cm³ for mtDNA. These values correspond to G-C contents of 34.7 and 46.9%, respectively. Covalently closed, circular mtDNA molecules were isolated from soybean hypocotyls by ethidium bromide-cesium chloride density gradient centrifugation. Considerable variation in mtDNA circle size was observed by electron microscopy. There were seven apparent size classes with mean lengths of 5.9 μm (class 1), 10 μm (class 2), 12.9 μm (class 3), 16.6 μm (class 4), 20.4 μm (class 5), 24.5 μm (class 6), and 29.9 μm (class 7). In addition, minicircles were observed in all preparations. Partially denatured, circular mtDNA molecules with at least one representative from six of the seven observed size classes were mapped. In class 4, there appear to be at least three distinct denaturation patterns, indicating heterogeneity within this class. It is proposed that the mitochondrial genome of soybean is distributed among the different size circular molecules, several copies of the genome are contained within these classes and that the majority of the various size molecules may be a result of recombination events between circular molecules.     

High content,size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.)

Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknownfeature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22 800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.     

Larger rearranged mitochondrial genomes in Dekkera/Brettanomyces yeasts are more closely related than smaller genomes with a conserved gene order

Summary Mitochondrial genomes from yeasts in the Dekkera/Brettanomyces/Eeniella group vary in size from 28 to 101 kb. Mapping of genes has shown that the three smallest genomes, of 28–42 kb, have the same gene order, whereas the three larger mitochondrial DNAs of 57–101 kb are rearranged relative to the smaller molecules and between themselves. To examine the relationships between these genomes, a phylogenetic tree has been constructed by sequence comparison of the mitochondrialencoded cytochrome oxidase subunit gene (COX2) from the six species. Contrary to expectation, the tree shows that the larger rearranged genomes are more closely related than the smaller mtDNAs. This result indicates that the gene order of the smaller mtDNAs (28–42 kb) is ancestral and that larger mtDNA molecules (57–101 kb) are more prone to rearrangement than smaller forms.Offprint requests to: G.D. Clark-Walker     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

Rolling circle replication of DNA in yeast mitochondria.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.     

Molecular Cloning of the Fujinami Sarcoma Virus Genome and Its Comparison with Sequences of Other Related Transforming Viruses

Full-length proviral DNA of Fujinami sarcoma virus (FSV) of chickens was molecularly cloned and characterized. An analysis of FSV DNA integrated in mammalian cells showed that restriction endonuclease SacI has a single cleavage site on FSV DNA. Unintegrated closed circular FSV DNA obtained from newly infected cells was linearized by digestion with SacI and cloned into λgtWES·λB. The following three different molecules were isolated: FSV-1 (4.4 kilobases [kb]) and FSV-2 (4.7 kb), which appeared to be full-length FSV DNA molecules containing either one or two copies of the long terminal repeat structure, and FSV-3 (6 kb), which consisted of part FSV DNA and part DNA of unknown origin. An analysis of the structure of cloned FSV-1 and FSV-2 DNA molecules by restriction endonuclease mapping and hybridization with appropriate probes showed that about 2.6 kb of the FSV-unique sequence called FSV-fps is located in the middle of the FSV genome and is flanked by helper virus-derived sequences of about 1.3 kb at the 5′ end and 0.5 kb at the 3′ end. The long terminal repeats of FSV were found to have no cleavage site for either EcoRI or PvuI. Upon transfection, both FSV-1 DNA and FSV-2 DNA were able to transform mammalian fibroblasts. Four ³²P-labeled DNA fragments derived from different portions of the FSV-fps sequence were used for hybridization to viral RNAs. We found that sequences within the 3′ half of the FSV-fps gene are homologous to RNAs of PRCII avian sarcoma virus and the Snyder-Theilen strain of feline sarcoma virus, both of which were previously shown to contain transforming genes related to FSV-fps. These results suggest that the 3′ portion of the FSV-fps sequence may be crucial for the transforming activity of fps-related oncogenic sequences.     

Linear mitochondrial deoxyribonucleic acid from the yeast Hansenula mrakii.

The mitochondrial deoxyribonucleic acid (mtDNA) from a petite-negative yeast, Hansenula mrakii, was studied. A linear restriction map was constructed with 11 restriction enzymes. The linearity of the genome was confirmed by direct end labeling of the molecule, followed by restriction analysis. The molecular weight of the DNA was found to be 55,000 base pairs. This is the first linear mtDNA found in yeast species. Using specific gene probes obtained from Saccharomyces cerevisiae mtDNA, we have constructed a gene map of H. mrakii mtDNA. The arrangement of genes in this linear genome was very different from the circular mtDNA of other known yeasts.     

Mitochondrial DNA analysis of Sporothrix schenckii clinical isolates from China

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP)patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in China. In addition to 23 mtDNA types (Types 1–23) so far reported, a new mtDNA type (Type 24) was found in this study. Type 24 was divided into two subtypes, Subtype 24A and 24B based on RFLP with EcoRV. Sixty-seven isolates in China consisted of 58 isolates of Type 4, 5 of Type 6, 1 of Type 5, 1 of Type 20 and 2 of Type 24. Based on the phylogeny of the mtDNA types (Types 1–24) constructed by estimating sequence divergences of mtDNA, mtDNA types clustered into two groups: Group A (Types 1–3, Type 11, Types 14–19 and Types 22–23) and Group B (Types 4–10, Types 12–13,Types 20–21 and Type 24). These results suggest that mostS. schenckii isolates in China belong to Group B.This revised version was published online in October 2005 with corrections to the Cover Date.     

Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization

The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100-200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly-L-lysine. The intact circular BAC molecules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.     

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

Restriction endonuclease analysis of mitochondrial DNA of three farm animal species: Cattle, sheep and goat

Five restriction endonucleases (HindIII, BgIII, EcoRI, EcoRV and BamHI) were employed to analyse mitochondrial DNA of cattle, sheep and goat. The results showed completely different restriction patterns of mtDNA among the three bovid species. A total of 11, 16, and 17 restriction fragments in cattle, sheep and goat respectively, were detected by the five restriction endonucleases. Average total sizes of mtDNA of cattle, sheep and goat were found to be 16.49 ± 0.18, 16.30 ± 0.25 and 16.44 ± 0.08 kb, respectively. The mtDNA cleavage patterns were identical for all seven individuals belonging to two cattle breeds and for 10 individuals from one sheep breed.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

The Male Sterility-Associated Pcf Gene and the Normal Atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.