Characterization of the structure and DNA complexity of mung bean mitochondrial nucleoids

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50–200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fastmoving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.     

The structure of human mitochondrial DNA variation

Summary Restriction analysis of mitochondrial DNA (mtDNA) of 3065 humans from 62 geographic samples identified 149 haplotypes and 81 polymorphic sites. These data were used to test several aspects of the evolutionary past of the human species. A dendrogram depicting the genetic relatedness of all haplotypes shows that the native African populations have the greatest diversity and, consistent with evidence from a variety of sources, suggests an African origin for our species. The data also indicate that two individuals drawn, at random from the entire sample will differ at approximately 0.4% of their mtDNA nucleotide sites, which is somewhat higher than previous estimates. Human mtDNA also exhibits more interpopulation heterogeneity (G_ST=0.351±0.025) than does nuclear DNA (G_ST=0.12). Moreover, the virtual absence of intermediate levels of linkage disequilibrium between pairs of sites is consistent with the absence of genetic recombination and places constraints on the rate of mutation. Tests of the selective neutrality of mtDNA variation, including the Ewens-Watterson and Tajima tests, indicate a departure in the direction consistent with purifying selection, but this departure is more likely due to the rapid growth of the human population and the geographic heterogeneity of the variation. The lack of a good fit to neutrality poses problems for the estimation of times of coalescence from human mtDNA data.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Evolutionary tinkering with mitochondrial nucleoids

Mitochondrial DNA (mtDNA) is organized in nucleoprotein particles called nucleoids. Each nucleoid, which is considered a heritable unit of mtDNA, might contain several copies of the mitochondrial genome and several different proteins. Some nucleoid-associated proteins, such as the high mobility group (HMG) box family, have well defined functions in mtDNA maintenance and packaging; others, such as Aco1 and IIv5, are bifunctional, fulfilling their roles in nucleoids in addition to well established metabolic functions. The fact that the HMG box mtDNA packaging proteins are of eukaryotic rather than bacterial origin and also that every organism seems to have a unique set of nucleoid-associated proteins suggests that evolutionary tinkering occurred to reinvent mitochondrial nucleoprotein during the evolution of mitochondrial genomes.     

The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway

Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid). We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number. In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number. The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination. Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control. Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.     

Ecm10p localizes in yeast mitochondrial nucleoids and its overexpression induces extensive mitochondrial DNA aggregations

Ecm10p was initially identified as a cell wall synthesis-related gene product [Genetics 147 (1997) 435] and also reported as a mitochondrial protein which was partially capable of compensating the phenotypic defect by SSC1 gene mutation [FEBS Lett. 487 (2000) 307]. Here we report that ecm10p is localized in mitochondrial nucleoids as its major component and the targeting signal resides between amino acid residues 161 and 240. Overexpression of ecm10p induces extensive mitochondrial DNA aggregations, which might be due to aberrant mitochondrial DNA cleavages through an altered endonuclease activity in mitochondrial nucleoids.     

Organization, dynamics and transmission of mitochondrial DNA: focus on vertebrate nucleoids

Eukaryotic cells contain numerous copies of the mitochondrial genome (from 50 to 100 copies in the budding yeast to some thousands in humans) that localize to numerous intramitochondrial nucleoprotein complexes called nucleoids. The transmission of mitochondrial DNA differs significantly from that of nuclear genomes and depends on the number, molecular composition and dynamic properties of nucleoids and on the organization and dynamics of the mitochondrial compartment. While the localization, dynamics and protein composition of mitochondrial DNA nucleoids begin to be described, we are far from knowing all mechanisms and molecules mediating and/or regulating these processes. Here, we review our current knowledge on vertebrate nucleoids and discuss similarities and differences to nucleoids of other eukaryots.     

The structure of Tetrahymena pyriformis mitochondrial DNA. II. The complex structure of strain GL mitochondrial DNA.

1. Isolated mtDNA from Tetrahymena pyriformis strain GL is a linear duplex molecule with an average molecular weight of 32.6 - 10(6) and without internal gaps or breaks. Denaturation of this DNA results in single strands with a duplex hairpin at one end. The length of this hairpin varies between 0 and 5 micrometer within one preparation. 2. Uder renaturation conditions the single strands with hairpins are able to circularize in two ways, depending on the length of the hairpin. Circularization is also observed after partial digestion with exonuclease III of native strain GL mtDNA. 3. All these data fit a model (see Fig.2) in which the DNA is heterogeneous in length at both ends. At the left end a 10-micrometer duplication-inversion is present; part of this duplication-inversion is complementary to a region at the right end of the molecule. 4. The analogy between the structural peculiarities of strain GL mtDNA and of some linear viral DNAs is stressed.     

Studies on mitochondrial structure and function in Physarum polycephalum. V. Behaviour of mitochondrial nucleoids throughout mitochondrial division cycle

The fine structure of mitochondria and mitochondrial nucleoids in exponentially growing Physarum polycephalum was studied at various periods throughout the mitochondrial division cycle by light and electron microscopy. The mitochondrial nucleoid elongates lingitudinally while the mitochondrion increases in size. When the nucleoid reaches a length of approximately 1.5 mum the mitochondrial membrane invaginates at the center of the mitochondrion and separates the mitochondrial contents. However, the nucleoid does not divide even when the mitochondrial sections are connected by a very narrow bridge. Just before division of the mitochondrion, the nucleoid divides by constriction of the limiting membrane of the dividing mitochondrion. After division, one end of the nucleoid appears to be associated with the inner mitochondrial membrane. The nucleoid then again becomes situated in the center of the mitochondrion before repeating these same processes.     

Effects of Fcj1-Mos1 and mitochondrial division on aggregation of mitochondrial DNA nucleoids and organelle morphology

Mitochondrial DNA (mtDNA) is packaged into DNA–protein complexes called nucleoids, which are distributed as many small foci in mitochondria. Nucleoids are crucial for the biogenesis and function of mtDNA. Here, using a yeast genetic screen for components that control nucleoid distribution and size, we identify Fcj1 and Mos1, two evolutionarily conserved mitochondrial proteins that maintain the connection between the cristae and boundary membranes. These two proteins are also important for establishing tubular morphology of mitochondria, as mitochondria lacking Fcj1 and Mos1 form lamellar sheets. We find that nucleoids aggregate, increase in size, and decrease in number in fcj1∆ and mos1∆ cells. In addition, Fcj1 form punctate structures and localized adjacent to nucleoids. Moreover, connecting mitochondria by deleting the DNM1 gene required for organelle division enhances aggregation of mtDNA nucleoids in fcj1∆ and mos1∆ cells, whereas single deletion of DNM1 does not affect nucleoids. Conversely, deleting F1Fo-ATP synthase dimerization factors generates concentric ring-like cristae, restores tubular mitochondrial morphology, and suppresses nucleoid aggregation in these mutants. Our findings suggest an unexpected role of Fcj1-Mos1 and organelle division in maintaining the distribution and size of mtDNA nucleoids.     

Strict paternal transmission of mitochondrial DNA of Chlamydomonas species is explained by selection against maternal nucleoids

Summary.  The non-Mendelian inheritance of organelle DNA is common in most plants and animals. Here we examined inheritance mechanisms involved in the transfer of mitochondrial DNA. We successively backcrossed (to F₅) two interfertile strains of the unicellular isogamous haploid algae Chlamydomonas reinhardtii and Chlamydomonas smithii to match nuclear backgrounds and examine transmission patterns of mitochondrial DNA by PCR analysis of cob gene sequences. Mitochondrial DNA was strictly transmitted paternally. To investigate the behavior of parental mitochondrial DNA, we used F₅ progeny to form zygotes and isolated single zygotes. The results showed selective disappearance of maternal mitochondrial nucleoids occurred between 3 and 6 h after zygote formation. Received July 11, 2002; accepted September 28, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.     

Polymorphism of human mitochondrial DNA

To date, a large data set on the mitochondrial DNA (mtDNA) sequence variation in human populations has been accumulated. The use of direct sequencing of the main noncoding region of mtDNA along with the RFLP analysis provide performance of complex analysis of mtDNA polymorphism in human populations. This approach proved to be effective for obtaining molecular genetic portraits of the world populations, as well as for the elucidation of the human evolutionary history and past migrations.     

Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

Summary The yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 m) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 m) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho^– cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho^– cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.     

Purification and identification of subunit structure of the human mitochondrial DNA polymerase.

The mitochondrial DNA polymerase of HeLa cells was purified 18,000-fold to near homogeneity. The purified polymerase cofractionated with two polypeptides that had molecular mass of 140 and 54 kDa. The 140-kDa subunit was specifically radiolabeled in a photoaffinity cross-linking assay and is most likely the catalytic subunit of the mitochondrial DNA polymerase. The purified enzyme exhibited properties that have been attributed to DNA polymerase gamma and shows a preference for replicating primed poly(pyrimidine) DNA templates in the presence of 0.5 mM MgCl2. As in the case of mitochondrial DNA polymerases from other animal cells, human DNA polymerase gamma cofractionated with a 3'----5' exonuclease activity. However, it has not been possible to determine if the two enzymatic activities reside in the same polypeptide. The exonuclease activity preferentially removes mismatched nucleotides from the 3' end of a duplex DNA and is not active toward DNA with matched 3' ends. These properties are consistent with the notion that the exonuclease activity plays a proofreading function in the replication of the organelle genome.