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1.
The azide analog of 14C-labeled ethidium bromide was mixed with yeast cells and when photolyzed by visible light, formed covalent complexes with all yeast cell organelles. The 14C counts were found in DNA, RNA and protein of yeast subcellular fractions, illustrating the complexity of binding of a drug which appears highly specific in its actions.  相似文献   

2.
Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.  相似文献   

3.
The azide analog of [14C]ethidium bromide was mixed with lymphocytes and photolyzed with visible light. The distribution of azide in the chromatin fraction was found to be 55% in DNA, 28% in protein and 16% in RNA. Label in the DNA portion was found to be almost exclusively in the region digestible with micrococcal nuclease. The parent compound, ethidium bromide, competed with azide for binding sites, illustrating that the azide analog mimics the action of ethidium bromide.  相似文献   

4.
Mutants of the petite-negative yeast, Kluyveromyceslactis, resistant to the inhibitors of oxidative phosphorylation, decamethylenediguanidine and octylguanidine were isolated from medium containing ethidium bromide. All mutants were resistant to ethidium bromide; some mutants were resistant to both alkylguanidines, some to one, others to neither. Both nuclear and cytoplasmic inheritance of resistance to decamethylenediguanidine and ethidium bromide was demonstrated by tetrad analysis.  相似文献   

5.
Petite induction of ethidium analogs was examined in both resting and growing yeast cells. All of the analogs used in these experiments were active in dividing cells of Saccharomyces cerevisiae; only the parent ethidium bromide was mutagenic under resting conditions. Incorporation of adenine into mitochondrial DNA appeared to be prevented completely by ethidium and partially inhibited by other analogs. Treatment of growing cells with analogs affected fragmentation of pre-existing DNA as seen by the loss of a mitochondrial antibiotic resistance marker. The rates of elimination of the marker were different; ethidium generated greater loss than the monoamino analogs (3-amino and 8-amino-); and the deaminated analog was least effective. However, in resting yeast the marker was partially eliminated only with treatment of the parent ethidium. The degradation of the mitochondrial DNA by exposure to ethidium compounds was confirmed by agarose gel electrophoresis. Electrophoretic patterns of the mitochondrial DNA treated with each of the analogs under growing conditions and only with ethidium under resting conditions showed degradation of the mitochondrial DNA.  相似文献   

6.
Two photoaffinity analogs of ethidium, 8-azido-3-amino, and 3-azido-8-amino-5-ethyl-6-phenylphenanthridinium chloride, have been used to probe the structure of mammalian chromatin and its interactions with the ethidium moiety. The monoazido analogs were established as suitable probes by comparing their interactions with chromatin and pure DNA prepared from chromatin to those of the parent ethidium bromide. Scatchard analysis of the binding data determined from spectrophotometric titrations showed that the analogs interacted with both nucleic acids in a manner similar to the parent compound. The effect of chromatin proteins on the interaction of the ethidium moiety with intact chromatin was investigated directly. By exposing the noncovalent complex to visible light, the monoazido analog was attached covalently in its interaction sites within chromatin, and the amount of drug bound covalently to DNA was determined for both protein-free DNA and chromatin. Using saturating concentrations of drug, DNA within intact chromatin was found to be associated with only half as much drug as DNA extracted from its protein prior to drug exposure. The distribution of drug bound within chromatin was determined following the attachment of the monoazido analog (by photoactivation) to chromatin that had undergone limited nuclease digestion. Several distinct populations isolated by size fractionation and quantitative measurements revealed that (1) both the core particles and the spacer-containing particles contained bound drug, reflecting high-affinity binding sites; and (2) chromatin particles containing 150 DNA base pairs (putatively nucleosome core structures) contained less total bound drug at high drug concentrations than those particles having intact spacer DNA.  相似文献   

7.
Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from Leptomonas sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg2+ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg2+ (Ki = 115 μM) and spermidine (Ki = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg2+ (Ki = 400 μM) but mixed for spermidine (Ki = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg2+ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the Leptomonas enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.  相似文献   

8.
Five N10‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.  相似文献   

9.
Two classes of inhibitors of histone methyltransferase I from calf thymus are reported. High concentrations (≧ 10 mM) of various alkyl or aralkyl amines and polyamines were inhibitory to the enzyme. Spermine and spermidine were among the most potent compounds in this group. The best monoamine inhibitor was 2-phenylethylamine, which gave 47% inhibition at 10 mM.The substituted phenanthridinium compound ethidium bromide was also an inhibitor of the enzyme. A number of analogs of ethidium bromide were tested, and the most potent compound (17) gave 50% inhibition at 0.125 mM. S-Adenosyl-l-ethionine (SAM) showed competitive inhibition of the enzyme as determined from a Lineweaver-Burke plot, while ethidium bromide was noncompetitive.  相似文献   

10.
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   

11.
Xu et al. [Biochem. Biophys. Res. Commun. 305 (2003) 941] reported that, when a mutant strain of Pseudomonas aeruginosa lacking its major multidrug efflux pump complex, MexAB-OprM, was incubated with 100 μM ethidium bromide, the fluorescence, caused by its binding to DNA following its entry into cells, decreased gradually. The authors concluded that the intracellular ethidium bromide “induced” either its degradation or its efflux through the assembly of unknown efflux pumps. We found, through quantitation of ethidium bromide by absorption spectroscopy, that the total amount of ethidium bromide in the system remained constant under these conditions, indicating the absence of its degradation. Furthermore, intracellular ethidium bromide kept increasing during the experiment, showing that the decrease of fluorescence was due to self-quenching, and that ethidium bromide is not pumped out by a newly assembled efflux system.  相似文献   

12.
Frameshift mutations have been produced in specific repair-negative Salmonella tester strains by photoaffinity labeling technique using ethidium azide. Reversions requiring a +1 addition or a ?2 deletion were especially sensitive. Mutagenesis was reduced by the simultaneous addition of non-mutagenic ethidium bromide, and was prevented by photolysis of the azide prior to culture addition. Identical tester strains active in DNA excision repair were not mutagenized by the azide. These results are consistent with the interpretation that photolysis of the bound ethidium analog converts the drug from its noncovalent mode of binding (presumably intercalation) to a covalent complex with consequent production of frameshift mutations. Such photoaffinity labeling by drugs which bind to DNA not only confirms the importance of covalent drug attachment for frameshift mutagenesis, but also provides powerful techniques for studying the molecular details of a variety of genetic mechanisms.  相似文献   

13.
The binding of eight ethidium derivatives to short (approximately 35 base-pair), random sequence DNA has been investigated using 1H-NMR. At 35 degrees C, all drugs cause upfield shifts of the DNA imino proton resonances characteristic of intercalative binding to DNA, but the line shapes vary significantly with the nature of the drug. The results confirm our previous proposal that removal of the amino group at position-3, but not at position-8, on the parent ethidium shortens the lifetime of the intercalative state (less than 1-2 ms at 35 degrees C). These results suggest that hydrogen-bonding interactions with the 3-NH2 group are involved in stabilization of the drug-DNA complex or that changes in charge distribution that accompany removal of the 3-NH2 group reduce the complex stability. The magnitude of the shift of the drug-DNA spectra indicates a slight preference for binding of the drugs adjacent to G X C base-pairs.  相似文献   

14.
It was shown that petite induction in growing cells of Saccharomycescerevisiae by ethidium was strongly stimulated by the presence of propidium, a phenanthridinium dye of similar structure to ethidium. Propidium itself also induced petites in growing but not in resting cells. Furthermore, propidium could prevent petite induction in resting cells and caused recovery from ethidium induction with prolonged incubation. A possible mode of action of propidium in the ethidium-induced petite mutagenesis is discussed.  相似文献   

15.
In order to elucidate the intramolecular photoinduced electron-transfer or energy-transfer mechanisms of the zinc myoglobin (ZnMb) dyad and to construct a photoreaction system within a Mb–DNA complex, we newly prepared ZnMb appending an ethidium ion (Et+). The steady-state fluorescence of ZnMb–Et+ at 600 nm and its lifetime (2.2 ns) indicate that the excited singlet state of 1(ZnMb)* is not quenched by the Et+ moiety, whereas the lifetime of the excited triplet state of 3(ZnMb)*–Et+ was shorter (τ = 4.3 ms) than those of ZnMb and the intermolecular (ZnMb + ethidium) system. Upon photoirradiation of Et+, fluorescence studies indicated the intramolecular quenching reactions from the excited singlet state, 1(Et+)*, to ZnMb, the process of which is likely the photoinduced energy-transfer reaction via a through-space mechanism. We also demonstrate the photophysical and spectroscopic properties of ZnMb–Et+ in the presence of calf thymus (CT) DNA. The changes in the absorption and fluorescence spectra of ZnMb–Et+ on the addition of CT-DNA up to 15 equiv were very small, indicating that there are no major changes in the heme pocket. However, we observed a longer lifetime of 3(ZnMb)*–Et+ in the presence of CT-DNA (τ = 5.3 ms) by single flash photolysis. This was induced by noncovalent interactions between Et+ and CT-DNA, followed by a conformational change of Et+ at the surface of ZnMb, where the donor–acceptor distance was probably elongated by CT-DNA. The synthetic manipulation at the Mb surface, by using a DNA intercalator coupled with photoinduced reaction, may provide a sensitive transient signal for DNA and valuable information to construct new Mb–DNA complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

16.
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   

17.
An increase in temperature from 20 to 50° C results in the complete transition from the Z to B form of poly(d(G-C)], dissolved in a 55% ethanol-water solution. The transition is fully reversible and displays a slow kinetics. The transition profiles for the free polynucleotide and for that in the presence of ethidium bromide, which is known to stabilize the B form, are obtained by circular dichroism. Based on these data the enthalpy value for the B-Z transition in our conditions is estimated to be ΔHBZ = ?0.7 kcalmol.  相似文献   

18.
Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by Neurosporacrassa nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the Neurosporacrassa nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.  相似文献   

19.
The azide analog of 14C-labeled ethidium bromide was mixed with yeast cells and when photolyzed by visible light, formed covalent complexes with all yeast cell organelles. The 14C counts were found in DNA, RNA and protein of yeast subcellular fractions, illustrating the complexity of binding of a drug which appears highly specific in its actions.  相似文献   

20.
The fluorescent dye ethidium bromide binds to the acetylcholine receptor with an apparent Kd of 3 μM and a stoichiometry of 1 molecule of ethidium per α-bungarotoxin site. Time dependent fluorescent increases were observed upon addition of carbamylcholine, the amplitude and half-time of which were dependent on the Carb1 concentration. It appeared that these fluorescence increases resulted from a lowering of the Kd for ethidium as the AcChR-Carb complex underwent an isomerization from low to high affinity form(s) for carb, and more ethidium was bound. Titration with the local anesthetic procaine led to ethidium fluorescence increases at low procaine concentrations, followed by a fluorescence decrease at higher procaine concentrations to that level induced by saturating α-bungarotoxin. Thus it appeared that the ethidium binding site either interacted with or was identical with local anesthetic binding site(s).  相似文献   

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