Electron microscopic and renaturation kinetic analysis of mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Mitochondrial DNA isolated from a series of nine petite yeast strains and from the parent grande strain was characterized by electron microscopic and renaturation kinetic analysis. The mtDNA² from all strains contained a variety of branched molecules which may be intermediates of replication or recombination. Although no circles were observed in the grande mtDNA, all the petites contained circular mtDNA molecules. The size distribution of the circles conformed to an oligomeric series that was characteristic for each strain. In seven petites, the length series could be related to a single circle monomer size, ranging from 0.13 μm to 5.5 μm; and in two petites to two or more circular monomer lengths. In contrast to circular mtDNA, linear molecules showed no unique size distribution. Circle monomer lengths were linearly related to the kinetic complexity (κ₂ or $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of sheared total mtDNA in the seven petite strains that contained a predominant single series of circle lengths. Thus in each of these petite strains the circle monomer length defined the same DNA sequence present in the linear DNA molecules of non-unique length.     

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.     

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.     

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.     

Analysis of a 120-Kilobase Mitochondrial Chromosome in Maize

The organization of the mitochondrial genome in plants is not well understood. In maize mitochondrial DNA (mtDNA) several subgenomic circular molecules as well as an abundant fraction of linear molecules have been seen by electron microscopy. It has been hypothesized that the circular molecules are the genetic entities of the mitochondrial genome while the linear molecules correspond to randomly sheared mtDNA. A model has been proposed that explains the mechanism of generation of subgenomic circles (of a predictable size) by homologous recombination between pairs of large direct repeats found on a large (approximately 570 kb for the fertile (N) cytoplasm) master circle. So far the physical entities of the mitochondrial genome, as they exist in vivo, and the genes they carry, have not been identified. For this purpose, we used two gel systems (pulsed field gel electrophoresis and Eckhardt gels) designed to resolve large DNA. Large DNA was prepared from the Black Mexican Sweet (BMS) cultivar. We resolved several size classes of mtDNA circles and designate these as chromosomes. A 120 kb chromosome was mapped in detail. It is shown to contain the three ribosomal genes (rrn26, rrn18 and rrn5) plus two genes encoding subunits of cytochrome oxidase (Cox1 and Cox3); it appears to be colinear with the 570-kb master circle map of another fertile cytoplasm (B37N) except at the "breakpoints" required to form the 120-kb circle. The presence of the 120-kb chromosome could not have been predicted by homologous recombination through any of the known repetitive sequences nor is it a universal feature of normal maize mitochondria. It is present in mitochondria of BMS suspension cultures and seedlings, but is not detectable in seedlings of B37N. No master genome was detected in BMS.     

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.     

The curious history of yeast mitochondrial DNA

Forty years ago, soon after yeast mitochondrial DNA (mtDNA) was recognized, some animal versions of mtDNA were shown to comprise circular molecules. Supporting an idea that mitochondria had evolved from bacteria, this finding generated a dogmatic belief that yeast mtDNA was also circular, and the endless linear molecules actually observed in yeast were regarded as broken circles. This concept persisted for 30 years and has distorted our understanding of the true nature of the molecule.     

Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules.

Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed.     

Circular mitochondrial DNA species from Oenothera with unique sequences

Summary Closed circular DNAs have been isolated from Oenothera and analysed by gel electrophoresis. A number of different size classes could be observed in the supercoiled fraction from the density gradient. The five smallest size classes of 6.3, 7.0, 8.2, 9.9, and 13.5 kilobases linear length were purified and their restriction fragment sizes compared. These five mtDNA species appear to be distinct homologous populations with no detectable large homologies between thedifferent classes. Hybridization data confirm this result; the smallest species hybridizes to itself and very weakly to some of the larger DNA molecules indicating only very little common sequence arrangement between the different circular DNAs. A restriction map of the smallest size class was constructed and confirms the circular arrangement of this DNA.Abbreviations used kb kilobases or kilobasepairs - mtDNA mitochondrial DNA - EthBr Ethidium bromide - SSC 150 mM NaCl, 15 mM sodium citrate (pH 7.0)     

Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis

The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h ^-was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simply Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested.     

Moving pictures of DNA released upon lysis from bacteria,chloroplasts, and mitochondria

Summary Cells and organelles suspended in gelled agarose agarose were lysed with detergent and protease, stained with ethidium bromide and their DNA was observed by fluorescence microscopy. The migration of individual DNA molecules during electrophoresis on a microscope slide was recorded on video tape so that moving pictures could be analyzed. The DNA from lysed bacteria (Escherichia coli andAgrobacterium tumefaciens) appeared as a rosette of at least twenty loops of varying size, whereas that from bacterial spheroplasts (E. coli andPseudomonas aeruginosa) appeared as circular forms or rods with many fine filaments of RNA extending toward the anode. The DNA from chloroplasts of watermelon (Citrullus vulgaris) and pea (Pisum sativum) did not appear as a rosette of loops. Many or most of the chloroplast DNA molecules per lysed chloroplast were immobile in the electric field, as if in circular form hooked on agarose fibers. The amount of DNA-fluorescence per watermelon mitochondrial particle was much less than that found for either chloroplasts or bacteria. The appearance of the mitochondrial DNA during electrophoresis was that of linear molecules, no obviously circular forms were evident and no rosette structures were observed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - kb kilobase pairs - mtDNA mitochondrial DNA - PFGE pulsed-field gel electrophoresis     

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.     

Most chloroplast DNA of maize seedlings in linear molecules with defined ends and branched forms

We used pulsed-field gel electrophoresis, restriction fragment mapping, and fluorescence microscopy of individual DNA molecules to analyze the structure of chloroplast DNA (cpDNA) from shoots of ten to 14 day old maize seedlings. We find that most of the cpDNA is in linear and complex branched forms, with only 3-4% as circles. We find the ends of linear genomic monomers and head-to-tail (h-t) concatemers within inverted repeat sequences (IRs) near probable origins of replication, not at random sites as expected from broken circles. Our results predict two major and three minor populations of linear molecules, each with different ends and putative origins of replication. Our mapping data predict equimolar populations of h-t linear concatemeric molecules differing only in the relative orientation (inversion) of the single copy regions. We show how recombination during replication can produce h-t linear concatemers containing an inversion of single copy sequences that has for 20 years been attributed to recombinational flipping between IRs in a circular chromosome. We propose that replication is initiated predominantly on linear, not circular, DNA, producing multi-genomic branched chromosomes and that most replication involves strand invasion of internal regions by the ends of linear molecules, rather than the generally accepted D-loop-to-theta mechanism. We speculate that if the minor amount of cpDNA in circular form is useful to the plant, its contribution to chloroplast function does not depend on the circularity of these cpDNA molecules.     

Circular DNA of Entamoeba histolytica encodes ribosomal RNA

The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis. DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules. Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.     

Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA

Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.     

Size of the intracellular circular Epstein-Barr virus DNA molecules in infectious mononucleosis-derived human lymphoid cell lines.

The size of non-integrated circular Epstein-Barr virus (EBV) DNA molecules isolated from seven different human lymphoblastoid cell lines of infectious mononucleosis origin has been determined by sedimentation analysis and by direct contour length measurements on electron micrographs. Six lines had intracellular circular EBV genomes of the same size as linear virion DNA molecules. The seventh line, established with the B95-8 strain of EBV, was the only one found to have circular EBV DNA molecules significantly smaller than virion DNA. The data show that intracellular EBV DNA circles of reduced size do not generally occur in infectious mononucleosis-derived cell lines.     

Circular DNA of Entamoeba histolytica Encodes Ribosomal RNA

. The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis, DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules.
Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.     

Size and Structure of Replicating Mitochondrial DNA in Cultured Tobacco Cells

The BY-2 tobacco cell line was used to study the size and structure of replicating mitochondrial DNA (mtDNA). Approximately 70 to 90% of the newly synthesized mtDNA did not migrate during pulsed-field gel electrophoresis. Moving pictures of the fluorescently labeled molecules showed that most of the immobile well-bound DNA was in structures larger than the size of the BY-2 mitochondrial genome of ~270 kb. Most of the structures appeared as complex forms with multiple DNA fibers. The sizes of the circular molecules that were also observed ranged continuously from ~20 to 560 kb without prominent size classes. Pulse-chase and mung bean nuclease experiments showed that the well-bound DNA contained single-stranded regions and was converted to linear molecules of between 50 and 150 kb. MtDNA replication in plants may be initiated by recombination events that create branched structures of multigenomic concatemers that are then processed to 50- to 150-kb subgenomic fragments.     

Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases.

DNA molecules isolated from bacteriophage P4 are mostly linear with cohesive ends capable of forming circular and concatemeric structures. In contrast, almost all DNA molecules isolated form P4 tailless capsids (heads) are monomeric DNA circles with their cohesive ends hydrogen-bonded. Different form simple DNA circles, such P4 head DNA circles contain topological knots. Gel electrophoretic and electronmicroscopic analyses of P4 head DNA indicate that the topological knots are highly complex and heterogeneous. Resolution of such complex knots has been studied with various DNA topoisomerases. The conversion of highly knotted P4 DNA to its simple circular form is demonstrated by type II DNA topoisomerases which catalyze the topological passing of two crossing double-stranded DNA segments [Liu, L. F., Liu, C. C. & Alberts, B. M. (1980) Cell, 19, 697-707]. The knotted P4 head DNA can be used in a sensitive assay for the detection of a type II DNA topoisomerase even in the presence of excess type I DNA topoisomerases.