Yeast mutants resistant to ethidium bromide

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.     

Decreased rate of uridine and glycerin uptake in L cells resistant to ethidium bromide

A comparison was made among rates of uptake of 3H-uridine, 3H-glycerol and 3H-D-xylose into mouse fibroblasts of line L sensitive to ethidium bromide (EB), and into EB-resistant cells obtained from this line by selection. Constants of uridine transport and phosphorylation were determined. For EB sensitive L cells Kt was 162 +/- 27 microM, Vt was 7.5 +/- 0.7 microM/sec. For EB resistant cells Kt was 178 +/- 27 microM, and Vt was 4.6 +/- 0.2 microM/sec. Thus, the transport rate of uridine in resistant cells was twice lower than in EB sensitive cells. The rate of uridine phosphorilation in EB resistant cells was by three times lower than in EB sensitive ones. The uptake of glycerol into resistant cells was also lowered. There was no difference in transport of 3H-D-xylose between sensitive and resistant cells. The data obtained may suggest some changes in plasma membrane in the EB resistant cells.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus-transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X-100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G-50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.     

The release of transforming growth factors by cells resistant to ethidium bromide and growing on a serum-free medium

350sf and 625sf cells growing in serum free medium secrete transforming growth factors (TGFs) that induce NIH 3T3 indicator cells to form colonies in soft agar. The addition of 2 ng/ml of EGF increases twice the number of colonies of NIH 3T3 indicator cells. The TGFs secreted by 350sf and 625 sf cells do not compete with 125I EGF for binding to EGF receptors on human A-431 cells. The number of EGF receptors on 350 sf and 625 sf and 625 sf cells continuously grown in serum-free medium do not differ from that of EGF receptors on parental Lebr-350 and Lebr-625 cells continuously grown in the presence of 10% serum. These results suggest that TGFs produced by 350 sf and 625 sf cells are not alpha TGF. It is possible that cells secrete beta TGFs of yet unknown type.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 micrograms ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus-transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 micrograms ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a "normal" karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromosome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide-resistant cells. The mitochondria of the ethidium bromide-resistant mutants appear somewhat enlarged with a normal morphology. The effect of ethidium bromide on selected respiratory enzymes in normal and virus-transformed ethidium bromide-resistant baby hamster kidney cells was determined. Ethidium bromide-resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide-free media. Ethidium bromide-resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines. Purified mitochondria isolated from virus-transformed ethidium bromide-resistant cells exhibited a depression in cytochrome oxidase-specific activity, while the ethidium bromide-resistant control cells did not. All cell lines studied showed a depression in NADH-ferricyanide and NADH-cytochrome c reductase-specific activities relative to their parental BHK21/C13 cells. No increase was observed in virus-transformed ethidium bromide-resistant cells. Ethidium bromide-resistant control cells exhibited a two-fold increase in oligomycin-insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin-sensitive adenosine triphosphatase-specific activity except for the virus-transformed, dye-resistant mutant, whose activity was increased.     

Evolution in vitro: sequence and phenotype of a mutant RNA resistant to ethidium bromide

Ethidium bromide inhibits the in vitro replication of MDV-1 RNA (a small replicating RNA molecule) by reducing the rate of chain elongation. In a serial transfer experiment, in the presence of ethidium, a mutant RNA was selected that was more resistant to ethidium inhibition than is the wild-type MDV-1 RNA.The complete nucleotide sequence of the mutant RNA was determined and three nucleotides in the mutant sequence were found to be different from those in the wild type. The mixture of mutant and wild-type RNAs present in successive transfers was also sequenced. Each of the three point mutations occurred at a different time. These results show that the mutant RNA did not arise from a pre-existing strand present in the wild-type population, but rather, occurred de novo in the course of the experiment.It is probable that the chemical basis of resistance is the elimination of ethidium binding sites due to the specific alterations in the nucleotide sequence, since the mutant RNA was found to bind less ethidium than the wild-type molecules.     

Binding of ethidium bromide to fractionated chromatin

The binding of ethidium bromide (EB) to different chromatin preparations was tested. Scatchard plots showed that the slowly sedimenting fraction of sheared chromatin is enriched in dye-binding sites. Limited nuclease digestion of rat liver nuclei, which has been shown to preserve the subunit structure of chromatin, reduces the number of binding sites available for intercalation of the dye.     

Cyclic adenosine monophosphate and the enzyme activity of its metabolism in L cells sensitive and resistant to the cytotoxic action of ethidium bromide

Using L-cells both sensitive and resistant to cytotoxic action of ethidium bromide (EB), a study was made of the intracellular level of cAMP, activities of adenylcyclase, phosphodiesterase and cAMP, liberated from cells into the surrounding medium. In EB resistant L-cells compared to EB sensitive ones, the higher level of cAMP with a decreased activity of adenylcyclase and an increased activity of the phosphodiesterase was shown to be associated with an impeded exit of cAMP from cells. It is suggested that the differences in cAMP levels in the EB sensitive and resistant cells are associated with the properties of cAMP-dependent protein kinases of these cells.     

Studies of the binding of ethidium bromide to cells before and after enzyme treatment

Summary Binding of ethidium bromide (EB) to cells before and after HCl, pepsin and RNase treatment was investigated by spectrophotometric and fluorimetric methods. Binding isotherms, calculated with the McGheevon Hippel equation, taking EB as a non-interacting ligand, revealed the influence of these treatments on the fluorescence characteristics of the cells which were measured by flow-through cytofluorimetry. Thus pepsin- and RNase-treated cells have a reduced intercalation capacity due to the loss of cytoplasmic RNA and RNA hydrolysis, respectively. HCl alone, or in association with pepsin, increased the equilibrium constant K considerably. Thus at low free EB concentrations the enhanced EB affinity of acid-pretreated cells generates a high fluorescence intensity, by comparison with treatments at neutral pH. This result contradicts the interpretation of high EB binding to acid pretreated cells which is commonly believed to be due to hydrolytic histone removal from potential intercalation sites.With increasing free EB concentrations the fluorescence intensities of RNase- and pepsin-treated cells culminate at the same level due to their almost identical intercalation capacities. Consequently, quantitative DNA analysis of pretreated cell suspensions with EB can only be performed if the alteration, induced by the pretreatment, has previously been studied.     

Studies of the binding of ethidium bromide to cells before and after enzyme treatment.

Binding of ethidium bromide (EB) to cells before and after HCl, pepsin and RNase treatment was investiaged by spectophotometric and fluorimetric methods. Binding isotherms, calculated with the McGheevon Hippel equation, taking EB as a non-interacting ligand, revealed the influcence of these treatments on the fluorescence characteristics of the cells which were measured by flow-through cytofluorimetry. Thus pepsin- and RNase-treated cells have a reduced intercalation capacity due to the loss of cytoplasmic RNA and RNA hydrolysis, respectively. HCl alone, or in association with pepsin, increased the equilibrium constant K considerably. Thus at low free EB concentrations the enchanced EB affinity of acid-pretreated cells generates a high fluorescence intensity, by comparison with treatments at neutral pH. This result contradicts the interpretation of high EB binding to acid pretreated cells which is commonly believed to be due to hydrolytic histone removal from potential intercalation sites. With increasing free EB concentrations the fluorescence intensities of RNase- and pepsin-treated cells culminate at the same level due to their amost identical intercalation capacities. Consequently, quantitative DNA analysis of pretreated cell suspensions with EB can only be performed if the alteration, induced by the pretreatment, has previously been studied.     

Location and molecular characteristics of fluorescent complexes of ethidium bromide in the cell

Ethidium bromide is taken up rapidly by bovine kidney cells and yeast. This compound complexes specifically with nucleic acids. Its fluorescence is thereby increased, and characteristic properties of the fluorescence such as polarization, excitation spectrum and decay time depend on the type of nucleic acid and its molecular environment. These characteristics have been measured in ethidium-labeled cells. Polarization values from 0.30 to 0.36 and decay times from 17 to 23 nsec have been found for different cells and conditions. Fluorescence from the cells is characteristic of RNA with intercalated EB. Different fluorescence characteristics are found, depending on the complexing of ethidium with free RNA, RNA in ribosomes or associated with the cell membrane.