Isolation of two DNA polymerases from a mitochondrial membrane-DNA complex of mouse cell cultures

A membrane-DNA complex isolated from the mitochondria of thymidine kinase deficient mouse cells could be shown to contain in addition to mitochondrial DNA two different DNA polymerases: (i) Mitochondrial DNA polymerase 1 exhibiting characteristics of the DNA polymerase described for HeLa cell mitochondria and (ii) mitochondrial DNA polymerase 2 showing properties comparable to those of the DNA polymerase isolated from mouse liver mitochondria.     

DNA Replication by a membrane-DNA complex from rat liver mitochondria

The results presented here indicate that mitochondrial DNA (mtDNA) synthesis occurs on the inner mitochondrial membrane and that a membrane-DNA complex, enriched in newly synthesized DNA, can be isolated. The complex is able to synthesize DNA in vitro. Enrichment studies demonstrated that mtDNA synthesis occurs on an intact membrane-DNA complex in vitro and that pulse-labeled mtDNA could be chased from the membrane-DNA complex to the top fraction of the discontinuous sucrose gradient. The membrane-DNA complex was also shown to carry out replicative synthesis of mtDNA in vitro. Replication was shown to be asynchronous with heavy-strand synthesis preceding light-strand synthesis. The progression of mtDNA replication by the membrane-DNA complex was shown to be from small fragments (<13 S) to larger fragments (14–24 S) liberated from closed circular molecules, to a heat-stable 27 S molecule, and finally to a 38 S heat-stable molecule. The time estimated to progress from small fragments to the 38 S molecule is 120 min.     

The distribution of DNA among dividing mitochondria of Tetrahymena pyriformis

A squash technique was developed for log phase Tetrahymena pyriformis which permitted the resolution of over 100 individual mitochondria from a single cell. Mitochondria incorporated thymidine at all stages of the cell cycle, even when nuclear DNA synthesis was not occurring. During the stage of macronuclear DNA synthesis, however, there was a significant increase in the extent of mitochondrial labeling. Low radioautograph background suggests that mitochondrial DNA is synthesized at the mitochondria themselves. All mitochondria incorporated thymidine-3H within one population-doubling time. Grain counts also showed that the amount of mitochondrial label was retained for four generations and that this label remained randomly distributed among all mitochondria during this time. The results are not consistent with any theory of de-novo or "microbody" origin of mitochondria, but do support the hypothesis that mitochondria are produced by the growth and division of preexisting mitochondria. The stability of the mitochondrial DNA and its distribution among daughter mitochondria satisfy two prerequisites for a genetic material. The possibility is discussed that some of the genetic information for the mitochondrion is contained in the DNA associated with this organelle.     

The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion

Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells.     

Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.     

The presence of intact mitochondrial DNA in HeLa cell nuclei.

Restriction analysis of DNA labelled with [32P]dCTP in an in vitro replication system with isolated nuclei from early S phase cells showed preferential labelling of restriction fragments derived from mitochondrial DNA (mtDNA) by a replication machinery distinct from that responsible for bulk nuclear DNA replication. Use of restriction nucleases with one recognition site in mtDNA gave rise to 16.5 kbp long fragments corresponding to full-length linearized mtDNA, indicating the presence of intact mtDNA in the isolated cell nuclei. Incorporation of dNTPs into mtDNA was not restricted to the S phase of the cell cycle. We were unable to increase the labelling of mtDNA by the addition of purified mitochondria or mtDNA to the nuclear replication system. These and other results presented is evidenced that the presence of mtDNA in the isolated nuclei was not due to uptake during preparation, thus indicating its presence in the cell nucleus in vivo.     

Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei.

RNAs that function in mitochondria are typically encoded by the mitochondrial DNA. However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion. It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import. We have identified and characterized mitochondrial precursor tRNAs in T. brucei. The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors. The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with [alpha-32P]CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P. We show that T. brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T. brucei tRNA precursors. Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis. The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.     

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.     

Repair of lesions induced by bruneomycin in DNA of isolated mitochondria from the mature eggs of the teleost fish Misgurnus fossilis

Addition of a radiomimetic antibiotic bruneomycin (Streptonigrin) to isolated mitochondria from mature quiescent oocytes of the teleost fish loach Misgurnus fossilis leads to the induction of unscheduled synthesis of mitochondrial DNA. Most of the newly synthesized DNA has the sedimentation properties of open circles and up to 15% of the label is present in the fraction of the covalently closed-circular molecules. The size of the newly synthesized DNA stretches determined from the bouyant shift of DNA labeled with 5-bromouracil and [3H]dAMP and sonicated to fragments of different molecular weight, was found to be equal to about 1000 nucleotides for the labeled covalently closed circles and to about 2000 nucleotides for the labeled open-circular DNA. Experiments with the centrifugation of non-sheared and sonicated 5-bromouracil and [3H]dAMP-labeled mitochondrial DNA (mtDNA) in alkaline CsCl density gradients provided evidence of a covalent linkage between newly-synthesized stretches and the parental DNA strands. It is concluded from these data that the unscheduled mtDNA synthesis induced by bruneomycin does at least in part represent mtDNA repair synthesis.     

Isolation of mitochondrial DNA, purified of nuclear DNA, from animal tissues (degree of methylation and level of pyrimidine nucleotide clustering--criteria of purity)]

A method of preparation of mitochondria free of nuclear DNA and its fragments by treatment of mitochondria with DEAE-cellulose has been developed. This method is based on binding nuclear nucleic acids and nucleoproteins to DEAE-cellulose particles in the media used for isolation of mitochondria. Treatment with DEAE-cellulose under the conditions described does not induce any visible degradation of mitochondria and mitochondrial DNA. The mitochondrial DNA preparations obtained from beef and rat liver are represented with closed circular molecules of contour length about 5.5 mu. The 5-methylcytosine content in beef and rat mitochondrial DNA (3.03 and 2.0 mole %, respectively) is twice as much as in corresponding nuclear DNA. Besides, mitochondrial DNA strongly differs from nuclear ones by a lower degree of pyrimidine clustering: the amount of mono- and dipyrimidine fragments (about 32 mole %) in mitochondrial DNA is 1.5 times as large and the content of long pyrimidine clusters (hexa- and others) is 2--4 times as low as those in nuclear DNA. The methylation level and the pyrimidine clustering degree may be used as criteria for the purity of mitochondrial DNA from nuclear DNA.     

DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation]

DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart. Adenine modifying DNA-methylases in mitochondria and nuclei were not found. DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated. Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity. After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme). Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree. In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E. coli B DNA about four times more methyl groups as compared to the nuclear one. After the methylation of E. coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments. Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins. The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase. All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Ribosomal-type ribonucleic acid from rodent mitochondria

1. Highly purified mitochondria containing 3.0mug of RNA/mg of mitochondrial protein were prepared from rat liver by differential centrifugation. 2. RNA, labelled with [(32)P]P(i) or [(3)H]orotate, was isolated from these mitochondria by a phenol extraction method. The RNA sedimented at 15S and 13S on sucrose density gradients. Its nucleotide composition was 23% uridylate, 30% adenylate, 22% guanylate and 25% cytidylate. 3. RNA from mouse L cells was labelled with [(3)H]-uridine in the presence of 0.1mug of actinomycin D/ml to suppress the synthesis of cytoplasmic rRNA. The RNA isolated from crude L-cell mitochondria by a cold-phenol-sodium dodecyl sulphate method had components sedimenting at 15S and 12.5S. These components had an electrophoretic mobility on agarose-acrylamide gels of 21 and 12S(E) compared with 28 and 18S(E) for cytoplasmic rRNA. The nucleotide composition was 26% uridylate, 34% adenylate, 18% guanylate and 22% cytidylate. 4. RNA extracted from crude L-cell mitochondria by a hotphenol-sodium dodecyl sulphate method had an additional component sedimenting at 21S and having an electrophoretic mobility of 18S(E). It was probably DNA because of its sensitivity to deoxyribonuclease and its insensitivity to ribonuclease and alkali. It was present in nuclear fragments contaminating the crude mitochondrial fraction and could be removed by deoxyribonuclease or isopycnic-gradient centrifugation.     

Proteins associated with mitochondrial DNA protect it against the action of X-rays and hydrogen peroxide

It has been suggested in a number of investigations that the high vulnerability of mitochondrial DNA to reactive oxygen species and other damaging agents is due to the absence in mitochondria of histones complexed with DNA. In the present study it was shown that DNA-binding proteins of mitochondrial nucleoids were able to shield mitochondrial DNA from X-ray radiation and hydrogen peroxide, as nuclear histones did. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver cells. The degree of damage to or protection of mitochondrial DNA was assessed from the yield of its PCR amplification product. The in vitro experiments demonstrated that mouse mitochondrial DNA, when in complex with mitochondrial nucleoids or nuclear histones, was damaged much less by radiation and/or hydrogen peroxide than in the absence of these proteins and histones. No significant difference between mitochondrial nucleoid proteins and nuclear histones was revealed in their efficiency to protect mitochondrial DNA from the damaging effect of radiation and hydrogen peroxide. It is likely that the nucleoid proteins in the mitochondria shield mitochondrial DNA against the attack of reactive oxygen species, thus significantly decreasing the level of the oxidative damage to mitochondrial DNA.     

Hyperactive recombination in the mitochondrial DNA of the natural death nuclear mutant of Neurospora crassa.

In Neurospora crassa, a recessive mutant allele of a nuclear gene, nd (natural death), causes rapid degeneration of the mitochondrial DNA, a process that is manifested phenotypically as an accelerated form of senescence in growing and stationary mycelia. To examine the mechanisms that are involved in the degradation of the mitochondrial chromosome, several mitochondrial DNA restriction fragments unique to the natural-death mutant were cloned and characterized through restriction, hybridization, and nucleotide sequence analyses. All of the cloned DNA pieces contained one to four rearrangements that were generated by unequal crossing-over between direct repeats of several different nucleotide sequences that occur in pairs and are dispersed throughout the mitochondrial chromosome of wild-type Neurospora strains. The most abundant repeats, a family of GC-rich sequences that includes the so-called PstI palindromes, were not involved in the generation of deletions in the nd mutant. The implication of these results is that the nd allele hyperactivates a general system for homologous recombination in the mitochondria of N. crassa. Therefore, the nd+ allele either codes for a component of the complex of proteins that catalyzes recombination, and possibly repair and replication, of the mitochondrial chromosome or specifies a regulatory factor that controls the synthesis or activity of at least one enzyme or ancillary factor that is affiliated with mitochondrial DNA metabolism.     

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     

Deoxyribonucleic Acid Synthesis in Permeabilized Spheroplasts of Saccharomyces cerevisiae

Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.     

Isolation and characterization of a mitochondrial RNA polymerase from Drosophila melanogaster

A DNA-dependent RNA polymerase was solubilized from sucrose gradient isolated, DNase-treated mitochondria of Drosophila melanogaster. The isolated mitochondria were not detectably contaminated with nuclear DNA as shown by CsCl gradient centrifugation and polylysine Kieselguhr chromatography. The detergent-solubilized RNA polymerase was sensitive to rifampicin, resistant to alpha-amanitin, had an apparent molecular mass of about 60 kilodaltons, and displayed a tendency to aggregate, both in crude extracts or when purified. The mitochondrial RNA polymerase could be distinguished from nuclear RNA polymerases on the basis of size, salt optima, rifampicin sensitivity, and alpha-amanitin resistance.     

The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I.

BACKGROUND: The yeast CDC9 gene encodes a DNA ligase I activity required during nuclear DNA replication to ligate the Okazaki fragments formed when the lagging DNA strand is synthesised. The only other DNA ligase predicted from the yeast genome sequence, DNL4/LIG4, is specifically involved in a non-homologous DNA end-joining reaction. What then is the source of the DNA ligase activity required for replication of the yeast mitochondrial genome? RESULTS: We report that CDC9 encodes two distinct polypeptides expressed from consecutive in-frame AUG codons. Translational initiation at these two sites gives rise to polypeptides differing by a 23 residue amino-terminal extension, which corresponds to a functional mitochondrial pre-sequence sufficient to direct import into yeast mitochondria. Initiation at the first AUG codon results in a 755 amino-acid polypeptide that is imported into mitochondria, whereupon the pre-sequence is proteolytically removed to yield the mature mitochondrial form of Cdc9p. Initiation at the second AUG codon produces a 732 amino-acid polypeptide, which is localised to the nucleus. Cells expressing only the nuclear isoform were found to be specifically defective in the maintenance of the mitochondrial genome. CONCLUSIONS: CDC9 encodes two distinct forms of DNA ligase I. The first is targeted to the mitochondrion and is required for propagation and maintenance of mitochondrial DNA, the second localises to the nucleus and is sufficient for the essential cell-division function associated with this gene.