Mitochondrial DNA rearrangements associated with mF plasmid integration and plasmodial longevity in Physarum polycephalum

Plasmodial cultures of Physarum polycephalum have defined life spans and undergo a reproducible decline (senescence) before losing the ability to be subcultured. Studies of the mtDNA of a long-lived Physarum strain, which does not undergo senescence, revealed a 7.9-kb insertion in its mtDNA. This insertion is derived from a mitochondrial plasmid which enhances mitochondrial fusion and mtDNA recombination. Four different recombination events are required to convert the parental mtDNA to the form found in the long-lived strain. An additional recombination event, which deletes a 2.4-kb region of the insert from the long-lived strain, has been identified in the mtDNA of a normally senescing strain. These observations imply a mitochondrial involvement in the process of plasmodial senescence and implicate a region of the DNA derived from the mitochondrial plasmid as being necessary for plasmodial longevity. Received: 11 August / 18 November 1997     

Early zygote-specific nuclease in mitochondria of the true slime mold Physarum polycephalum

The active, selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum polycephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucleoids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca²⁺-dependent, high-molecular-weight nuclease complex (about 670 kDa), and the other is a Mn²⁺-dependent, high-molecular-weight nuclease complex (440–670 kDa); the activity of the latter was detected as a Mn²⁺-dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn²⁺-dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn²⁺-dependent DNase activity matched the selective digestion of mtDNA.     

Rearrangements in the Physarum polycephalum mitochondrial genome associated with a transition from linear mF-mtDNA recombinants to circular molecules

Although mitochondrial DNA (mtDNA) is transmitted to progeny from one parent only in Physarum polycephalum, the mtDNAs of progeny of mF⁺ plasmodia vary in structure. To clarify the mechanisms associated with the mitochondrial plasmid mF that generate mtDNA polymorphisms, 91 progeny of four strains (KM88 × JE8, KM88 × TU111, KM88 × NG111, Je90) were investigated using RFLP analysis, PCR, and pulse-field gel electrophoresis (PFGE). Nine mtDNA rearrangement types were found, with rearrangements occurring exclusively in the mF regions. PFGE revealed that, in the groups containing rearranged mtDNA, the linear mF–mtDNA recombinants had recircularized. Sequencing the rearranged region of one of the progeny suggested that the mF plasmid and the mtDNA recombine primarily at the ID sequences, linearizing the circular mtDNA. Recombination between the terminal region of the mF plasmid and a region about 1 kbp upstream of the mitochondrial/plasmid ID sequence results in a rearranged circular mtDNA, with variations caused by differences in the secondary recombination region.     

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

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

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     

Rapid disappearance of one parental mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum

Summary Five haploid amoebal strains of the myxomycete Physarum polycephalum, each with a distinct mitochondrial genotype, were crossed in all pairwise combinations. The mitochondrial genotype in the diploid plasmodia resulting from these isogamous matings were found to be transmitted uniparentally. This uniparental inheritance could be arranged in a dominant hierarchical order. Time-course analysis of the presence of mitochondrial genotypes in the zygotes and young developing plasmodial genotypes is virtually completed during the first two nuclear cycles in the zygote/differentiating plasmodium. To our knowledge this is the first report indicating an active mechanism involving the degradation of mitochondrial genomes in sexual crosses.     

Complex terminal structure of a linear mitochondrial plasmid from Physarum polycephalum: three terminal inverted repeats and an ORF encoding DNA polymerase

The mitochondria of Physarum polycephalum have a linear plasmid (mF) which promotes mitochondrial fusion. To determine the terminal structure of the mF plasmid, restriction fragments derived from its ends were cloned and sequenced. The sequences showed that the mF plasmid has three kinds of terminal inverted repeats (TIRs). The most characteristic feature is a 144-bp repeating unit which exists between a 205-bp TIR at the extreme ends of the plasmid and another 591-bp TIR. All of the clones showed at least one of these 144-bp repeating units. The GC content of the 205-bp TIR (49%) was higher than those of the other TIRs and of another sequenced region (23%). This TIR can form three thermodynamically-stable hairpin structures based on complex internal palindromic components. Moreover, in the right terminal region of the mF plasmid, there is an open reading frame (ORF) which covers the entire 591-bp TIR and most of one of the 144-bp repeating units. This ORF encodes a 547-amino-acid polypeptide, ORF-547, and shows extensive homology with the polymerization domain of the putative DNA polymerases of linear mitochondrial plasmids from other sources.     

Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis,aging and age-related diseases

As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined.     

线粒体能量代谢和DNA复制对小鼠卵子体外成熟、受精及发育的影响

目的 探讨胞质线粒体的氧化磷酸化（OXPHOS）活性或线粒体DNA复制在卵子成熟、受精和胚胎发育过程中的作用。方法 通过在小鼠体外成熟培养液中引入不同浓度的羰基氰4-(三氟甲氧基)苯腙 (FCCP, 10 nmol/L和100nmol/L)或2′,3′-双脱氧胞苷(ddC,10μmol/L和100μmol/L),抑制线粒体OXPHOS活性或线粒体DNA复制,统计分析各组卵子的体外生发泡破裂（GVBD）率、核成熟率、受精率及囊胚形成率,以分析线粒体功能抑制对卵子成熟、受精和胚胎发育的影响。 结果 线粒体OXPHOS活性和DNA复制功能在卵子和胚胎中所发挥的作用并不完全相同。FCCP抑制线粒体OXPHOS活性可显著降低卵子的核成熟率和囊胚形成率;但对卵子的GVBD的发生率和受精率无显著影响。而ddC抑制线粒体DNA复制不影响卵子的体外成熟和受精,但可显著抑制囊胚的形成。 结论 OXPHOS活性主要影响卵子成熟及胚胎发育;线粒体DNA复制则主要影响胚胎发育;而线粒体功能抑制不影响卵子的成熟启动和体外受精。     

Origins and genetic features of the Okhotsk people,revealed by ancient mitochondrial DNA analysis

In order to investigate the phylogenetic status of the Okhotsk people that were distributed in northern and eastern Hokkaido as well as southern Sakhalin during the fifth to the thirteenth centuries, DNA was carefully extracted from human bone and tooth remains excavated from archaeological sites. The hypervariable region 1 sequences of the mitochondrial DNA (mtDNA) control region were successfully amplified and 16 mtDNA haplotypes were identified from 37 individuals of the Okhotsk people. Of the 16 haplotypes found, 6 were unique to the Okhotsk people, whereas the other 10 were shared by northeastern Asian people that are currently distributed around Sakhalin and downstream of the Amur River. The phylogenetic relationships inferred from mtDNA sequences showed that the Okhotsk people were more closely related to the Nivkhi and Ulchi people among populations of northeastern Asia. In addition, the Okhotsk people had a relatively closer genetic affinity with the Ainu people of Hokkaido, and were likely intermediates of gene flow from the northeastern Asian people to the Ainu people. These findings support the hypothesis that the Okhotsk culture joined the Satsumon culture (direct descendants of the Jomon people) resulting in the Ainu culture, as suggested by previous archaeological and anthropological studies.     

Life span is related to the free energy of mitochondrial DNA

Broadly speaking, the mitochondrial theory of aging relates aging to the rate of damage to mitochondria. In this work, I concentrate on a DNA sequence property, the free energy, which can be interpreted as a factor in the susceptibility of mitochondrial DNA (mtDNA) to mutation. I show that life spans across a broad range of species are a function of the mtDNA free energy and are proportional to the probability of opening of bubbles of single-stranded mtDNA of approximately 20 base pairs in length, in agreement with the measured nucleation size of these bubbles. These transient separations of the mtDNA strands are a possible aging mechanism, through increased mtDNA mutations. In comparisons of species with similar life spans, avian mtDNA has more negative free energy than does mammalian mtDNA, suppressing the predicted probability of mtDNA bubble formation in birds by over 80% and thus protecting them against mutation. Based on these results I propose three hypotheses about the conflicting evolutionary forces that have acted on the free energy of mtDNA.     

Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter

Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase I protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria. We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase I into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome.     

Genetic background of people in the Dominican Republic with or without obese type 2 diabetes revealed by mitochondrial DNA polymorphism

People in the Dominican Republic are considered to be genetically heterogeneous owing to the post-Colombian admixture of Native American, African, and European populations. To characterize their genetic background, nucleotide sequences of the D-loop region of human mitochondrial DNA (mtDNA) were examined in 33 healthy women and 50 gender-matched patients with obese type 2 diabetes (OD) from the Dominican Republic. Phylogenetic analysis of 198 mtDNA lineages including Native Americans, Africans, and Europeans enabled us to assess relative genetic contributions of the three ancestral fractions to the two groups in the Dominican Republic. In the OD group, the majority (64.0%) of the mtDNA lineages were from African ancestry, whereas the Native American fraction was predominant (51.5%) in the healthy group, with both showing smallest amounts (14.0% and 9.1%, respectively) of European contribution. This difference in maternal genetic background between the two groups was similarly demonstrated by phylogenetic analysis at the population level based on net nucleotide diversities between populations. These findings may imply ethnic-specific predisposition to OD, a possible association of an unidentified factor from African ancestry with OD in the Dominican Republic population.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB174901–AB174983.     

A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii

We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54±2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11–orf189–rps12–rps7–rpl2–rps19–orf425–orf1740–rpl16–rpl14–orf188–rps14–rps8–rpl6–rps13–orf127–orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. Received: 28 August 1997 / 2 January 1998     

Phylogeographic analysis of mitochondrial DNA haplogroup F2 in China reveals T12338C in the initiation codon of the ND5 gene not to be pathogenic

In this report, we studied on a homoplasmic T12338C change in mitochondrial DNA (mtDNA), which substituted methionine in the translational initiation codon of the NADH dehydrogenase subunit 5 gene (ND5) with threonine. This nucleotide change was originally identified in two mtDNAs belonging to haplogroup F2 by our previous complete sequencing of 48 mtDNAs. Since then, a total of 76 F2 mtDNAs have been identified by the variations occurring in the hypervariable segments and coding regions among more than 3,000 individuals across China. As the T12338C change was detected in 32 samples representing various sub-clades of the F2 haplogroup while not in 14 non-F2 controls, we believe that the T12338C change is specific to the F2 haplogroup. As F2 and its sub-clades were widely distributed in normal individuals of various Chinese populations, we conclude that T12338C is not pathogenic. In addition, based on the average distribution frequency, haplotype diversity and nucleotide diversity of haplogroup F2 in the populations across China, the T12338C nucleotide substitution seems to have been occurred in north China about 42,000 years ago. Our results provided a good paradigm for distinguishing a polymorphic change from a pathogenic mutation based on mtDNA phylogeny.Accession numbers and URLs for the sequence data of mtDNA control region (including HVS-I and HVS-II) of F2 types in this article are as follows: GenBank, (accession numbers: AY522667–AY522718).     

Transmission,recombination and conversion of mitochondrial markers in relation to the mobility of a group I intron in Chlamydomonas

Mitochondrial DNA transmission has been analyzed in diploids produced from sexual crosses or artificial fusions between Chlamydomonas strains which differ by several genetic markers: a group I intron (Cs cob.1 or intron), three restriction sites (Nh, Nc and H markers) located 0.5–5 kb from the insertion site of the intron, and a MUD2 point mutation (27 bp from the insertion site) conferring resistance to myxothiazol. Recombination between mitochondrial markers is a general property of all crosses and fusions analyzed. In crosses between two intron-containing (⁺) strains or two intron-less (^–) strains, the transmission is preferentially paternal (mt ^-), with a preoponderance depending on the nature of the parental genomes. In crosses between (⁺) and (^–) strains, the conversion of intron-less molecules into intron⁺ is frequent when the (⁺) parent is maternal (mt ⁺) and nearly absolute when the (⁺) parent is paternal (mt ^-). In 94% of cases, the conversion is accompanied by the co-conversion of the MUD2 marker. In both crosses and artificial fusions, the conversion of (^–) into (⁺) also influences the transmission of the more distant Nh, Nc and H markers. It is hypothesized that the more frequent transmission of the genome containing the intron results from the elimination of (^–) molecules, as a result of a double-strand cut which is induced by an endonuclease encoded by the intron.     

DNA degradation by the mixture of copper and catechol is caused by DNA-copper-hydroperoxo complexes, probably DNA-Cu(I)OOH

Free hydroxyl radicals (free (.)OH), singlet oxygen ((1)O(2)), or (. )OH produced by DNA-copper-hydroperoxo complexes are possible DNA-damaging reactive oxygen species (ROS) in the reaction system containing copper, catechol, and DNA. para-Chlorobenzoic acid (pCBA) degradation studies revealed that CuCl(2) mixed with catechol produced free (.)OH. In the presence of DNA, however, inhibition of the pCBA degradation suggested that another ROS is responsible for the DNA degradation. Of a series of ROS scavengers investigated, only KI, NaN(3), and Na-formate-all of the salts tested-strongly inhibited the DNA degradation, suggesting that the ionic strength rather than the reactivity of the individual scavengers could be responsible for the observed inhibition. The ionic strength effect was confirmed by increasing the concentration of phosphate buffer, which is a poor (.)OH scavenger, and was interpreted as the result of destabilization of DNA-copper-hydroperoxo complexes. Piperidine-labile site patterns in DNA degraded by copper and catechol showed that the mixture of Cu(II) and catechol degrades DNA via the intermediate formation of a DNA-copper-hydroperoxo complex. Replacement of guanine by 7-deazaguanine did not retard the DNA degradation, suggesting that the DNA-copper-hydroperoxo complexes do not bind to the guanine N-7 as proposed in the literature.