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Summary Yeast mutants resistant to ethidium bromide have been isolated among sensitive grande cells (+) for their ability to grow on glycerol in the presence of the dye. Mutant cells are also resistant to acriflavin and do not yield petites (-) when grown on galactose with the mutagen. Genetic analysis reveals that resistance to ethidium bromide is controlled by a cytoplasmic factor, carried by, or linked to, the determinant (mitochondrial DNA). The expression of resistance to ethidium bromide seems to be related to the presence in the cell of a product of mitochondrial protein synthesis. It is concluded that some mitochondrial DNA sequence is involved in the resistance to ethidium bromide of yeast mitochondria. 相似文献
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Replicative Deoxyribonucleic Acid Synthesis in Isolated Mitochondria from Saccharomyces cerevisiae
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The characteristics of a system for the in vitro synthesis of mitochondrial deoxyribonucleic acid (mtDNA) in mitochondria isolated from Saccharomyces cerevisiae are described. In this system the exclusive product of the reaction is mtDNA. Under optimal conditions the initial rate of synthesis is close to the calculated in vivo rate; the rate is approximately linear for 20 min but then decreases gradually with time. DNA synthesis proceeds for at least 60 min and the de novo synthesis of an amount of mtDNA equivalent to 15% of the mtDNA initially present is achieved. The rate and extent of synthesis observed with mitochondria isolated from grande and petite (rho(-)) strains were similar. The mode of DNA synthesis is semiconservative; after density labeling with 5-bromodeoxyuridine triphosphate, in vitro, the majority of labeled DNA fragments of duplex molecular weight, 6 x 10(6), are of a density close to that calculated for hybrid yeast mtDNA. The density label is incorporated into one strand of the duplex molecules. These properties indicate that the synthesis resembles replicative rather than repair synthesis. This system therefore provides a convenient method for the study of mtDNA synthesis in S. cerevisiae. The observation that mtDNA synthesis is semiconservative in vitro suggests that the dispersive mode of synthesis observed in S. cerevisiae in vivo labeling studies is the result of some other process, possibly a high recombination rate. 相似文献
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Dynamical change of mitochondrial DNA induced in the living cell by perturbing the electrochemical gradient. 总被引:3,自引:0,他引:3
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Digital-imaging microscopy was used in conditions that allowed the native state to be preserved and hence fluorescence variations of specific probes to be followed in the real time of living mammalian cells. Ethidium bromide was shown to enter into living cells and to intercalate stably into mitochondrial DNA (mtDNA), giving rise to high fluorescence. When the membrane potential or the pH gradient across the inner membrane was abolished by specific inhibitors or ionophores, the ethidium fluorescence disappeared from all mtDNA molecules within 2 min. After removal of the inhibitors or ionophores, ethidium fluorescence rapidly reappeared in mitochondria, together with the membrane potential. The fluorescence extinction did not result from an equilibrium shift caused by leakage of free ethidium out of mitochondria when the membrane potential was abolished but was most likely due to a dynamical mtDNA change that exposed intercalated ethidium to quencher, either by weakening the ethidium binding constant or by giving access of a proton acceptor (such as water) to the interior of mtDNA. Double labeling with ethidium and with a minor groove probe (4',6-diamino-2-phenylindole) indicated that mtDNA maintains a double-stranded structure. The two double-stranded DNA states, revealed by the fluorescence of mitochondrial ethidium, enhanced or quenched in the presence of ethidium, seem to coexist in mitochondria of unperturbed fibroblast cells, suggesting a spontaneous dynamical change of mtDNA molecules. Therefore, the ethidium fluorescence variation allows changes of DNA to be followed, a property that has to be taken into consideration when using this intercalator for in vivo as well as in vitro imaging studies. 相似文献
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Bérénice Ricard Manuel Echeverria Lisiane Christophe Simon Litvak 《Plant molecular biology》1983,2(4):167-175
DNA synthesis was studied using purified wheat embryo mitochondria as well as mitochondrial lysates deprived of endogenous DNA. The optimal conditions for DNA synthesis are very similar in both systems: ATP stimulates dramatically mitochondrial DNA synthesis and magnesium is a better co-factor than manganese, contrary to what has been reported in animal mitochondrial systems. Wheat mitochondrial DNA synthesis is resistant to aphidicolin and strongly inhibited by dideoxythymidine triphosphate and ethidium bromide. Thus, the DNA polymerase involved in this system seems to be the same as that previously purified and characterized from wheat embryo mitochondria (Christopheet al., Plant Science Letters 21: 181, 1981). Two different approaches: restriction endonuclease digestion followed by electrophoresis, and autoradiography and cesium chloride equilibrium centrifugation of mitochondrial DNA, where BrdUTP has been incorporated instead of TTP, show that long stretches of the mitochondrial genome have been synthesized. 相似文献
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Phillip Nagley John S. Mattick Ruth M. Hall Anthony W. Linnane 《Molecular & general genetics : MGG》1975,141(4):291-304
Summary The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria.The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction. 相似文献
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Origin of the actinomycin D insensitive RNA species in Aedes albopictus cells. 总被引:2,自引:0,他引:2
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In the presence of actinomycin D or a combination of actinomycin D and either camptothecin or alpha-amanatin. Aedes albopictus cells synthesize a variety of single stranded RNA species. These actinomycin D resistant species are ethidium bromide sensitive and they are present in the cell cytoplasm in an RNase resistant structure which has the sedimentation and buoyant density characteristics of mitochondria. Twelve actinomycin D insensitive RNA species can be detected by electrophoresis in 7M urea and 11 of these bind to oligo(dT)-cellulose. An identical set of oligo(dT)-cellulose binding RNA species is obtained when A. albopictus cells are labeled in the presence of camptothecin alone. The actinomycin D insensitive RNA species which bind to oligo(dT)-cellulose hybridize to mitochondrial DNA. These data indicate that the actinomycin D insensitive RNA species have a mitochondrial origin and are not associated with the replication of an inapparent contaminating virus. 相似文献
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Phillip Nagley Elliot B. Gingold H.B. Lukins Anthony W. Linnane 《Journal of molecular biology》1973,78(2):335-350
This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ?ERr) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ?ERr clones, and in both the gene elimination rate is slower than in the parent ρ+ ERr strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes. 相似文献
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Replication of Coliphage M-13 II. Intracellular Deoxyribonucleic Acid Forms Associated with M-13 Infection of Mitomycin C-treated Cells 总被引:6,自引:4,他引:2
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Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F+, hcr−, thy−) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with 3H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated. 相似文献
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Alexandra Kukat Christian Kukat Jan Brocher Ingo Schfer Georg Krohne Ian A. Trounce Gaetano Villani Peter Seibel 《Nucleic acids research》2008,36(7):e44
Eukaryotic cells devoid of mitochondrial DNA (ρ0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ0 cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ0 cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK− the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK− ρ0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ0 mitochondria. 相似文献
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De novo synthesis of DNA in human platelets 总被引:1,自引:0,他引:1
G Soslau 《Archives of biochemistry and biophysics》1983,226(1):252-256
Platelets, incubated with radiolabeled thymidine and purified free of contaminating nucleated cells, were analyzed for their ability to synthesize DNA. The only DNA species isolated from these purified platelets was mitochondrial DNA. The CsCl gradient-purified platelet DNA was treated with the restriction endonucleases EcoRI, HindIII and HpaI yielding the expected pattern for human mitochondrial DNA. Nitrocellulose blots of the electrophoresed, restriction endonuclease-treated DNA were fluorographed. All of the DNA fragments generated by the restriction enzymes were labeled, indicating de novo synthesis. This was further substantiated by inhibition of DNA synthesis by ethidium bromide and 2',3'-dideoxythymidine. Platelet DNA appeared to become greatly fragmented after 4 to 7 days storage while all of the thymidine incorporated was observed in intact mitochondrial DNA. These results indicate a continuous degradation of platelet mitochondrial DNA with no apparent repair mechanism. The ability of platelets to synthesize DNA may be associated with the protein synthetic capacity of platelets previously described. 相似文献
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Akira Tanaka Yoshiaki Yamano Hideya Fukuzawa Kanji Ohyama Tohru Komano 《Bioscience, biotechnology, and biochemistry》2013,77(5):1239-1244
Lysate of chloroplasts prepared from liverwort Marchantia polymorpha L. cell suspension cultures incorporated [3H]-dTTP into acid insoluble materials when DNA was added exogenously as a template. The incorporation was highly dependent on the addition of template DNA, four deoxynucleoside triphosphates and magnesium ions (maximum incorporation at 5mM). Magnesium ions could be replaced by manganese ions. DNA synthesis inhibitors, N-ethylmaleimide (NEM) and ethidium bromide (EtBr), strongly inhibited the incorporation. Dideoxythymidine triphosphate (ddTTP), an inhibitor of DNA polymerases β and γ, inhibited the incorporation at the concentration of 50 μM (molar ratio of ddTTP/dTTP = 17). On the other hand, the incorporation by the chloroplast lysate was resistant to arabinofuranosyl cytosine triphosphate (araCTP) and aphidicolin as well as the RNA polymerase inhibitors, rifampicin and α-amanitin. The chloroplast lysate highly utilized denatured calf thymus DNA and bacteriophage ?X174 single-stranded DNA as templates when added exogenously, while a synthetic homopolymer, poly(rA)-oligo(dT)12 ~ 18, did not stimulate the incorporation at all. Autoradiographic analysis of DNA synthesized in isolated chloroplasts showed that the chloroplast DNA synthesis took place at several specific sites on the chloroplast DNA from cells of the liverwort, Marchantia polymorpha. 相似文献
15.
M.R. Hall W. Meinke D.A. Goldstein R.A. Lerner 《Biochemical and biophysical research communications》1974,60(1):96-102
The cellular functions necessary for transport of cytoplasmic membrane associated DNA from nucleus to cytoplasm have been investigated utilizing inhibitors of macromolecular synthesis. Hydroxyurea, fluorodeoxyuridine, cytosine arabinoside, and ethidium bromide did not prevent transport of cytoplasmic membrane associated DNA to the cytoplasm. In contrast, rifampicin and N-demethyl rifampicin totally inhibited the appearance of newly synthesized DNA on cytoplasmic membranes, while dimethyl-benzyl-demethyl rifampicin was partially inhibitory. 相似文献
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The effect of undernutrition on the incorporation of [methyl-3H]thymidine into DNA and of 5-[3H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal. 相似文献
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Ethidium bromide (23 nmol/mg of protein) was found to be a potent inhibitor of oxidative phosphorylation, as determined by loss of respiratory control through the inhibition of the ADP-induced state-3 rate of oxygen uptake. A time latency for complete loss of respiratory control was noted, after which 2,4-dinitrophenol (DNP) was ineffective in overcoming this inhibition. In the absence of EDTA, ethidium bromide produced an apparent uncoupling, as evidenced by an increase of state-4 rates of oxygen uptake and loss of respiratory control. As low as 8 nmol of ethidium bromide/mg of protein stimulated mitochondrial adenosine triphosphatase (ATPase) for 5 min. Two to three times this amount of ethidium bromide reduced the amount Pi released. Preincubation of mitochondria with ethidium bromide prevented subsequent release of Pi during incubation with ATP. Likewise, preincubation inhibited the DNP-activated ATPase. The uptake of low levels of [14C]ADP preincubated with ethidium bromide (14 nmol/mg of protein) and succinate or α-ketoglutarate could apparently be reversed, with loss of radioactivity beginning several minutes after addition of the radioactive nucleotide. Inhibition of oxidative phosphorylation by ethidium bromide may be due to modification of the adenine nucleotide transport system in mitochondria. The production of apparently swollen mitochondria treated in vitro with ethidium bromide and substrates necessary for oxidative phosphorylation, as seen in electron micrographs, further indicates that the compound is capable of acting directly upon mouse liver mitochondrial function and structure. 相似文献
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Summary Germinating spores of the fungus Botryodiplodia theobromae incorporated guanine-8-C14 into both the nuclear DNA and mitochondrial DNA fractions. Ethidium bromide inhibited the synthesis of mitochondrial DNA without having a significant effect on nuclear DNA synthesis or on the rate and extent of spore germination. Rates of leucine and uracil incorporation and of oxygen uptake were not significantly affected by ethidium bromide until germination was nearly completed. Mitochondrial DNA synthesis is apparently not required for germination of the spores of B. theobromae but is probably essential to continued vegetative growth.Abbreviations DNA
deoxyribonucleic acid
- mit-DNA
mitochondrial DNA
- nuc-DNA
nuclear DNA
- RNA
ribonucleic acid
- EB
ethidium bromide
- Tris
tris (hydroxymethyl)aminomethane
Published with the approval of the Director as Paper No. 3331, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project No. 21-17. Paper No. 7877, Scientific Journal Series, Minnesota Agricultural Experiment Station. 相似文献
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《Mutation Research/Genetic Toxicology》1979,66(4):385-390
Ethidium mono- and diazide analogs have been used as photoaffinity probes to study the mechanism of the ethidium-induced petite mutation in yeast [7,10,14]. The azide moiety when exposed to light is converted to a reactive nitrine intermediate. The diradical nitrene effects a covalent attachment to sites of interaction bound reversibly by the drug. Ethidium azide photoaffinity labeling has been used to verify the prerequisite covalent attachment of ethidium to mitochondrial DNA to bring about the petite mutation in yeast [6]. Bastos has also reported a specific photoattachment of ethidium azide to a polypeptide (subunit 9) of the membrane bound ATPase in yeast mitochondria [1].Isolated DNA from yeast cells treated in vivo with [14C]ethidium monoazide plus light contained covalent adducts on both nuclear and mitochondrial DNA, although the specific radioactivity of mitochondrial DNA was more than 10 times higher than nuclear DNA [11]. Drug distribution studies of [14C]ethidium monoazide have indicated that greater than 50% of the covalent adducts in the nuclear centrifugation fraction (2000 × g) reside on nuclear proteins [3]. Nuclear damage in yeast by the photolysis of ethidium azides is apparent from the increased killing effect in cells treated with ethidium azide derivatives plus light as opposed to dark-treated cells [7,10,11]. This communication reports a concomitant increase in nuclear mutations and genetic activity from the photoactivated covalent attachment of ethidium azides to nuclear components. 相似文献
20.
Absence of ethidium bromide induced nicking and degradation of mitochondrial DNA in mouse L-cells.
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C A Smith 《Nucleic acids research》1977,4(5):1419-1427
The in vivo effects of ethidium bromide on the integrity of mitochondrial DNA have been studied in a mouse L-cell system in which this DNA may be nearly exclusively radiolabelled. This allows the detection of mitochondrial DNA in the presence of contaminating nuclear DNA and eliminates the need for extensive purification of mitochondria or the use of deoxyribonuclease. The mitochondrial DNA in treated cells rapidly attains a high negative superhelix density and is not substantially nickel or degraded over the course of several days. 相似文献