Effects of pingyanymycin on chromosomes: a possible structural basis for chromosome aberration

Pingyanymycin (PYM), and antitumor-antibiotic complex which belongs to the bleomycin family can induce "G2-free chromatin" and "uncompleted-packing-mitotic figures" (UPM) at increased frequency after treatment of cultured human lymphocytes. PYM can also induce an extraordinarily high frequency of chromosomal breaks but few sister-chromatid exchanges (SCE) in the same experiment, which is similar to the action of bleomycin. To solve this remarkable contradiction we presume that the UPM is related to a basic mechanism for producing chromosomal aberrations. Our results also show that various steps of the chromosomal cycle can be affected by certain chemical agents, and these treatments lead to chromosomal aberrations. Thus, other testing systems should be used in addition to the SCE system.     

Displacement of chromosomes in mitosis: a technique for assessing differential chromosome error

To assess whether displacement--the appearance of individual chromosomes inside of the ring of chromosomes attached to spindle fibers--is a valid method of analyzing chromosome misdivision, displacement data from six healthy female subjects were compared with data for mitotic and meiotic nondisjunction. Statistical analysis indicated that nondisjunction and displacement are equivalent methods of assessing differential chromosome error. Tests for homogeneity of the data sets showed no significant heterogeneity for the mitotic data, but chromosomes 1, 2, 11, 16, 17, 19, and 20 were responsible for significant heterogeneity in the meiotic data.     

High resolution mid-prophase human chromosomes induced by echinomycin and ethidium bromide

The antitumor antibiotic echinomycin (EM) was used in combination with ethidium bromide (EB) to induce high resolution mid-prophase chromosomes from amethopterin synchronized blood lymphocytes. With this combination of drugs, trypsin G-banded chromosomes showed over 1200 bands per haploid set in cells having a mid-prophase length of decondensation.     

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities ofEscherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg²⁺ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg²⁺ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.     

Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding

Ethidium bromide (EB) is known to intercalate between stacked base pairs without specific base-pair preference. Its use in cultured human lymphocytes and Burkitt's lymphoma cells resulted in the accumulation of cells in prophase and prometaphase stages. Inhibition of mitotic chromosome condensation as a possible mechanism involved in this phenomenon is discussed. A simple method for obtaining high-resolution banding patterns on elongated chromosomes was devised as follows: Human lymphocytes cultured for 3 days with phytohemagglutinin were exposed to EB (5-10 micrograms/ml) and Colcemid (0.02 micrograms/ml) simultaneously for 2 h and then routinely harvested for chromosome preparation. High-resolution G-bands were obtained by Giemsa staining following mild trypsin treatment.     

Application of ethidium bromide for high-resolution banding analysis of chromosomes from human malignant cells

Ethidium bromide was added to cultured human leukemic bone marrow and solid tumor cells to evaluate its inhibitory effect on mitotic chromosome condensation and its possible application to high-resolution banding analysis. In most experiments ethidium bromide treatment resulted in a high proportion of mitotic cells having elongated chromosomes, without remarkable reduction in either the mitotic index or quality of metaphase chromosomes. Optimal effect on chromosome length was obtained by adding 10 micrograms/ml of ethidium bromide during the final 2 hr of culture. Because of the simplicity and reproducibility of the technique involved, ethidium bromide can be used routinely to extend the length of chromosomes for fine-banding analysis of malignant cells.     

Induction of chromosome banding in early stages of spermatogenesis by ethidium bromide

Distinct chromosome banding of early meiotic prophase (leptotene through early pachytene) can he induced in male Chinese hamsters by injecting ethidium bromide and Actinomycin D intratesticularly for 4 hours and making acetic orcein squash preparations from minced testicular tissue. Zygotene pairing apparently starts from chromosome ends.Fellow, China Medical Board of New York, Inc.     

Study of mitosis in root-tip cells ofTriticum turgidum treated with the DNA-intercalating agent ethidium bromide

Summary This work examines mitosis in root-tip cells ofTriticum turgidum treated with the RNA synthesis inhibitor ethidium bromide, using tubulin immunolabeling and electron microscopy. The following aberrations were observed in ethidium bromideaffected cells: (1) incomplete chromatin condensation and nuclear-envelope breakdown; (2) delay of preprophase microtubule band maturation; (3) preprophase microtubule band assembly in cells displaying an interphase appearance of the nucleus; (4) prevention of the prophase spindle formation, caused by inhibition of perinuclear microtubule (Mt) formation and/or inability of the perinuclear Mts to assume bipolarity; (5) organization of an atypical metaphase spindle which is unable to arrange the chromosomes on the equatorial plane; (6) formation of an atypical perinuclear metaphase spindle in cells in which nuclear-envelope breakdown has been almost completely inhibited; (7) inhibition of the anaphase spindle formation as well as of anaphase chromosome movement; (8) disorganization of the atypical mitotic spindle during transition from mitosis to cytokinesis. The observations favor the following hypotheses. Nucleation of prophase spindle Mts is related to the mechanism that causes nuclear-envelope breakdown. The mitotic poles lack Mtnucleating and -organizing properties, and their function does not account for prophase and metaphase spindle assembly. The organization of the prophase spindle is not a prerequisite for the formation of the metaphase spindle; the metaphase spindle seems to be formed de novo by Mts nucleated on the nuclear envelope and/or in the immediate vicinity of chromosomes.Abbreviations 5-AU 5-aminouracil - EB ethidium bromide - EM electron microscopy - k-Mt kinetochore microtubule - Mt microtubule - MTOC microtubule-organizing center - NE nuclear envelope - NEB nuclear-envelope breakdown - PPB preprophase band of microtubules     

Morphological basis for multiple interactions of ethidium bromide (EB) with yeast Candida utilis

Exposure of yeast cells to EB produced multiple effects on the cellular organelles: changes in the plasma membrane characterized by 75 to 110 nm deep pits; polymorphisms of the mitochondria ranging from cup-shaped, ring-shaped, ribbon-shaped, dumbbell-shaped structures to finally the formation of very elongated mitochondria (up to 4.5 micron in length); an increase in the length and number of endoplasmic reticulum; an increase in the number of cytoplasmic vesicles whose diameter varied between 25 to 45 nm. Furthermore, EB inhibited cytochrome c oxidase and cytochrome b biosynthesis, stimulated cytochrome c biosynthesis and uncoupled oxidative phosphorylation.     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.     

Effects of ethidium bromide on the mitochondrial adenosine triphosphatase from Trypanosoma cruzi

Culturing of Trypanosoma cruzi in the presence of ethidium bromide (EB; 1.0-2.5 microM), for 5 days, affected the biogenesis of the Fo/F1-ATPase complex, presumably by EB interference with the expression of the putative Fo-sector subunits encoded by maxicircle DNA. In vitro, EB inhibited ATP hydrolysis by the Fo/F1-ATPase complex and also by soluble F1, the former preparation being the most strongly affected (EB I50, 250 microM). Analysis of EB effect at increasing MgATP concentrations, as well as combination of EB and p-chloromercuribenzoate inhibitions, indicated that the dye interacts with ATPase at sites other than the catalytic ones. No correlation was found between the in vitro inhibition of ATPase and EB effect on parasite growth.     

Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems

In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used. Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems. Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems. The effect remained unchanged despite increasing the PCR cycles to 40. However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost. In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C). It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations. It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile.     

Effects of ethidium bromide,tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

Summary Several investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.     

Intercalation of ethidium bromide into a triple-stranded oligonucleotide.

We have examined the ability of a cationic planar chromophore, ethidium bromide, to intercalate into a short, defined triple helix. Using UV absorption, fluorescence spectroscopy and a gel retardation assay we demonstrate that ethidium bromide is able to bind to a triple helix with a lower affinity than to the corresponding duplex. Energy transfer from base triplets to ethidium shows that ethidium is intercalated into the triple helix. The spectroscopic characteristics of ethidium intercalated into a triplex are similar to those observed for intercalation into duplex DNA.     

Photophysics of ethidium bromide complexed to ct-DNA: a maximum entropy study

Time-integrated and time-resolved fluorescence spectroscopies have been used to probe the photophysical properties of ethidium bromide (Eb) complexed to calf thymus DNA (ct-DNA). Fluorescence decay profiles are obtained using the technique of time-correlated single photon counting (TCSPC), and subsequently analysed using conventional sum-of-exponential (SOE) routines and also the maximum entropy method (MEM). Through use of these methods and simulated decay data, it is demonstrated that the kinetics of Eb in the presence of ds-DNA are best described by a generic model consisting of three exponential terms. At all DNA:Eb ratios and NaCl concentrations studied, free Eb is detected. Furthermore, Eb is found to interact with ds-DNA through two mechanisms, each distinguishable by its fluorescence decaytime. Eb is shown to interact with DNA through classic intercalation, and also through binding at secondary sites. The component decaytimes are shown to be a function of NaCl concentration but independent of DNA:Eb molar ratio.     

Effects of ethidium bromide on growth and on loss of the penicillinase plasmid of Staphylococcus aureus

Ethidium bromide (EB) was more efficient than ethyl violet or rifampin as a curing agent for the penicillinase plasmids of Staphylococcus aureus strains. The effects of EB on growth and on the loss of the penicillinase plasmid of PS 81 were studied in detail. The growth rates of PS 81 and an EB-cured derivative were identical in broth, but the cured derivative had a shorter lag in the presence of added 6 x 10(-6)m EB. The shortened lag was due to prior exposure to EB as the cured derivative and an EB-treated but uncured strain of PS 81 gave identical growth lag and growth rates in the presence of EB. The curing of PS 81 by EB occurs in three phases. After a 4 to 5 hr lag, there is a 100-fold increase in the number of penicillinase-negative cells, and the proportion of cured cells continues to rise until 10 to 12 hr. Thereafter, the population becomes refractory to further curing, and the proportion of penicillinase-negative cells remains constant at about 20% of the total. Penicillinase-positive survivors of EB treatment showed increased EB resistance and were cured at lower rates upon subsequent EB treatment. Isolated colonies of the parental strain PS 81 were heterogeneous in their EB sensitivity. Thus, EB does not competitively favor spontaneously cured penicillinase-negative cells but appears to act in a manner analogous to acridine orange on the plasmids of enteric bacteria.