Presence Of Acridine Orange Research Articles

Infectious transfer of a fertility factor in Streptomyces coelicolor.

SUMMARYInitial Fertility (IF) strains ofStreptomyces coelicolorare able to convert recipient strains (UF) to the IF condition by contact, without concomitant transfer of chromosomal markers. The conversion is prevented by the presence of acridine orange in the medium of the mixed culture. Acridine orange is also moderately effective in inducing the formation of UF variants from IF-treated strains. No effect of the drug is observed on UF variant formation from Normal Fertility (NF) strains nor on the behaviour of the fertility factor in NF × UF mixed cultures. The hypothesis is put forward that the fertility factor works as an episome inS. coelicolor, fixed to the chromosome in the NF strains, free in the IF strains and missing in the UF strains.

Further fluorospectrophotometric studies on the binding of acridine orange with DNA. Effects of thermal denaturation of DNA and additions of spermine, kanamycin, dihydrostreptomycin, methylene blue and chlorpromazine

Fluorospectrophotometric studies on the binding of acridine orange (AO) with calf thymus DNA showed that the thermal denaturation of DNA reduced markedly the fluorescence of Complex II and the extent of this decrease depended on the temperature to which the DNA solutions were heated. The denaturation was carried out in the absence and presence of AO (methods A and B, respectively), and then fluorescence measurements of solutions were carried out at 23 °C. The fluorescence intensity-heating temperature curves obtained by methods A and B were similar in shape to the usual melting curves of DNA and AO-DNA solutions, respectively. The higher midpoint value obtained with method B indicates the stabilizing activity of AO against denaturation. These findings support an intercalation model for Complex II and an external self-association binding model for Complex I. A high concentration of ethylene diamine (EDA) restored the fluorescence of denatured Complex II to about 80% of the intensity value of native Complex II. The effects of spermine, kanamycin and dihydrostreptomycin were much stronger than that of EDA. Methylene blue (MB) and chlorpromazine (CP) reduced the fluorescence of native Complex II markedly. Since the analysis of the difference absorption spectra declared that MB and CP were intercalated without release of bound AO, the interacting MB and CP were considered to weaken the interaction between AO and DNA bases, that made AO more fluorescent. Free radical (CP·) of CP was prepared by a new method using H 2O 2, peroxidase, and ascorbic acid. Intercalated CP· showed a much stronger quenching effect on Complex II, indicating that unpaired electron spin contained in the costacking unit between CP· and DNA bases might affect the fluorescence of the adjacent AO molecule by paramagnetic perturbation.

Transfer among Erwinia spp. and other enterobacteria of antibiotic resistance carried on R factors.

Antibiotic resistance carried on R factors was transferred by conjugation from Escherichia coli B/r and Shigella flexneri 1a to Erwinia spp. Tetracycline resistance (TetR) carried on R factor R100 drd-56 was transferred from E. coli B/r to strains of Erwinia amylovora, E. aroideae, E. atroseptica, E. chrysanthemi, E. cytolytica, E. dissolvens, E. herbicola, E. nigrifluens, and E. nimipressuralis, but not to strains of Erwinia carotovora, E. carnegieana, E. dieffenbachiae, E. oleraceae, and E. quercina. Multiple antibiotic resistance (chloramphenicol, streptomycin, tetracycline; ChlR-StrR-TetR) carried on R factor SR1 was transferred from a clinical isolate of S. flexneri 1a to strains of E. aroideae, E. chrysanthemi, E. herbicola, and E. nigrifluens, but not to strains of other Erwinia spp. The frequency of this transfer was low with receptive cultures of Erwinia spp. and E. coli (F(-) strain). Antibiotic resistance in the exconjugants showed varying degrees of stability in the presence or absence of acridine orange, depending on the strain tested. The frequencies of segregation to drug susceptibility in the presence of acridine orange, though low, suggest that the elements exist as plasmids in the majority of the Erwinia exconjugants. Multiple antibiotic resistance (ChlR-StrR-TetR) was found to segregate into various resistance classes (ChlR-StrR, StrR-TetR, TetR, StrR, and none) in these exconjugants. The exconjugants of E. amylovora, E. herbicola, and E. nigrifluens, to which R100 drd-56 was transferred from E. coli B/r, were sensitive to the male (F)-specific phage M13. There was a positive correlation between the susceptibility of exconjugants to the F-specific phage M13 and their ability to transfer R100 drd-56 to the recipient cultures of Escherichia coli, Erwinia herbicola, Salmonella typhimurium, and Shigella dysenteriae. Exceptions were, however, noted with Erwinia dissolvens and E. nimipressuralis exconjugants harboring R100 drd-56; these exconjugants, although not susceptible to M13, transferred R100 drd-56 to the recipient cultures. The frequency of transfer of R100 drd-56 and the levels of resistance to tetracycline in Erwinia exconjugants were found to differ markedly depending upon the strain employed. Transfer of multiple antibiotic resistance (ChlR-StrR-TetR) from Erwinia exconjugants was not obtained in preliminary trials with an E. coli F(-) strain as the recipient culture.

The fluorescent staining of a mycobacteriophage (C2) nucleic acid.

By using the acridine orange staining technique a green fluorescence, characteristic of double-stranded nucleic acid, can be observed with purified preparations of mycobacteriophage C2 and its extracted nucleic acid. DNAse-treated samples do not show this fluorescence, which leads to the conclusion that this fluorescence is associated with phage DNA. Examination of preparations of phage grown in the presence of acridine orange supported these results.

Photoinactivation of Transfer RNA in the Presence of Acridine Orange

Photoinactivation of amino acid-acceptor ability of purified phenylalanine transfer RNA was investigated in the presence of acridine orange under various experimental conditions. The photoinactivation showed unique reaction kinetics: (1) The initial first-order reaction was followed by a reaction with a slower inactivation rate and (2) The relative amounts of the two components depended on dye preparation and concentration of Mg2+ in the system. Several possible mechanisms for these kinetics are discussed. Under the standard conditions the inactivation reaction was found to be independent of light intensity and almost independent of temperature over the range of 5-40°C. The oxygen dependence of the inactivation was rather sharp at low oxygen concentration and then leveled off at about 200 mm Hg. As the pH was raised, the photoinactivation increased gradually, reached a maximum around pH 9, and then decreased.

Studies on photodynamic action. II. The action on Escherichia coli cells.

The photodynamic inactivation of Escherichia coli strain 15T-, B/r and B was studied using acridine orange and methylene blue as sensitizers. The sensitivity was strongly dependent on the growth phase, namely, the cells at the stationary phase were extremely resistant. Parallel to the sensitivity to ultraviolet or X-rays, strain B/r was less sensitive than strain B. There was no recovery of strain 15T- in a liquid medium (M9) after illumination in the presence of acridine orange. When chloramphenicol was added to this medium, the inactivation proceeded further during the incubation. The syntheses of DNA, RNA and protein were greatly inhibited.

Some properties of the beta-lactamase genes in Staphylococcus epidermidis.

Eighty-three per cent of 200 different freshly isolated cultures of Staphylococcus epidermidis produced beta lactamase. Growth in the presence of acridine orange or ethidium bromide or growth at 44 C resulted in a high frequency of loss of the beta lactamase genes in some strains of S. epidermidis. The relationship between beta-lactamase production and resistance to mercuric, cadmium and arsenate ions differed from that observed in Staphylococcus aureus. It is postulated that the genes for beta lactamase in certain strains of S. epidermidis are on a plasmid.

Some Properties of the Beta-Lactamase Genes in Staphylococcus epidermidis

Eighty-three per cent of 200 different freshly isolated cultures of Staphylococcus epidermidis produced beta lactamase. Growth in the presence of acridine orange or ethidium bromide or growth at 44 C resulted in a high frequency of loss of the beta lactamase genes in some strains of S. epidermidis. The relationship between beta-lactamase production and resistance to mercuric, cadmium and arsenate ions differed from that observed in Staphylococcus aureus. It is postulated that the genes for beta lactamase in certain strains of S. epidermidis are on a plasmid.

Dark repair of acridine dye-sensitized photoeffects in [formula omitted] cells and bacteriophage

Bacteria and phages can be inactivated by visible light in the presence of sensitizing dyes and oxygen. The lesions, which are mainly due to effects in nucleic acid, are quite different from UV lesions not only in the physical-chemical changes brought about ( e.g. FREIFELDER and URETZ, 1966), but also with respect to the biological effects produced: they are neither photoenzymatically repairable nor multiplicity-reactivable ( WELSH and ADAMS, 1954). While recent work with E. coli and yeast cells suggest that the photodynamic lesions are also unaffected by enzymatic processes known to achieve extensive dark repair of UV lesions ( e.g. URETZ, 1964; FREIFELDER and URETZ, 1966; GEISSLER, 1966, 1967; PATRICK et al. , 1964 ), other investigations indicated the occurrence of dark repair of photodynamic damage in E. coli and Serratia marcescens ( RUPP, 1966; BRENDEL and KAPLAN, 1967). The results reported in this paper show that damage inflicted to bacterial or phage DNA by white fluorescent light in the presence of acridine orange or acriflavine is repaired at varying extents by both of the two dark repair systems operating on UV damage. However, the photodynamic lesions are not affected by two phage-controlled dark repair systems existing in T4.

Separation of lymphocytes of rat bone marrow by combined glass-wood filtration and dextran-gradient centrifugation.

SUMMARYThe two‐step separation of rat bone marrow involving glass‐wool filtration combined with dextran‐gradient centrifugation yields viable cell suspensions composed predominantly of lymphocytes with a minimum of contamination by granulocytes (less than 10 per cent). Because filtration of rat bone marrow through glass wool removes the larger marrow cells and most of the immature forms of the red cell series, it is useful as a first step in the separation of lymphocytes from bone marrow.Density‐gradient centrifugation of the glass‐wool filtrate serves to differentially settle out lymphocytes and granulocytes when dextran solutions of appropriate densities are used. The middle or layer 2 (density 1.0600) of the dextran gradient yields the greatest absolute numbers of lymphocytes. Small numbers of transitional and blast cells are also present in this layer.The separation procedure results in excellent preservation of cell morphology as evidenced by Jenner‐Giemsa staining. A high level of viability of the separated cells is indicated by dye exclusion, characteristic fluorescence in the presence of acridine orange, and capacity for oxygen consumption. In vivo viability of the separated cells is also indicated by their capacity to confer protection following their infusion into lethally irradiated rats.

Studies of the Interaction of Sodium Polyphosphates with Acridine Orange

Abstract The interaction of sodium polyphosphates with acridine orange has been studied by means of absorption spectra, fluorescence, and viscosity measurements. Polyphosphates with a variety of molecular weights were used in order to investigate the effect of the degree of polymerization (\barn) on the interaction. No spectral change was observed in the case of polyphosphates with \barn<5. In the case of \barn=9, an absorption band corresponding to the dye dimer was observed, whereas in the case of larger \barn values those corresponding to higher aggregates were also found. The maximum metachromasy occurred at P⁄D=3–6. Fluorescence quenching was observed, accompanied by an absorption change. Such behavior may be considered to be characteristic of the dye molecules bound to the long-chain polyphosphates; the bound-dye molecules seem to exhibit almost no fluorescence. The quenching constants of the fluorescence increased with the increase in \barn values. The binding energies were estimated to be 5–6 kcal/mol independent of the \barn values. In the presence of acridine orange, the reduced viscosity of Kurrol’s salt decreased remarkably, probably as a result of the neutralization of the charged phosphate groups. Further, its reduced viscosity decreased remarkably with time; this may be attributed to the catalytic action of the dye on the hydrolytic degradation of polyphosphates.

TRANSFER OF CLOVER INFECTIVITY OF RHIZOBIUM TRIFOLII TO RHIZOBIUM PHASEOLI AS MEDIATED BY AN EPISOMIC FACTOR

Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo (Received October 26, 1967) The transfer of host specificity by conjugation from one rhizobial species to the other was studied. Rhizobium trifolii and Rh. phaseoli were mixed-cultured in yeast extract-mannitol medium and then the former was eliminated completely from the culture by the action of Rh. trifolii-specific phage. Cells remained survived in the culture(strain W1, progeny of Rh. phaseoli) was examined for the clover infectivity as determined by the formation of infection threads in roothairs of clover plants. Strain W1 clearly showed the clover infectivity, but it retained various physiological and immunological characteristicsof Rh. phaseoli. When Rh. trifolii and strain W1 were cultured in the presence of acridine orange, which is known to eliminate episomic factors in bacterial cells, a marked decrease in infective activity on clover plants was demonstrated. The present observations strongly support the view that the host specificity in rhizobia is mediated by an episome-like factor just like as the drug resistance factor in bacteria, although the evidence is lacked in that the factor could be integrated into the chromosome.

Action of acridines on RNA and protein synthesis and on active transport in chick embryo cells

Acridine derivatives containing a primary amino group inhibit RNA and protein synthesis in chick-embryo cells without interfering with the labelling of the acid-soluble uridine derivatives by [ 14C]uridine. Acridines with a secondary or tertiary amino group or with a hydrazine group, and pyronine, strongly inhibit the formation of [ 14C]uridine phosphates. As with acridine orange, this effect is due to an inhibition of the active transport of [ 14C]-uridine into the cells. Penetration of actinomycin or actidione into the cells is not prevented by acridine orange, nor are [ 14C]uridine phosphates lost from pre-labelled cells in the presence of acridine orange. Acridine derivatives without an amino group, or compounds with two heteroatoms in the middle ring like methylene blue or neutral red, have no significant effect on RNA and protein synthesis and on the labelling of the acid-soluble pool.

OXYGEN EFFECT IN THE PROTECTION OF E. COLI AGAINST U.V. INACTIVATION AND MUTAGENESIS BY ACRIDINE ORANGE*

Abstract— Protection by acridine orange against ultraviolet light effects in resting cells of E. coli B/r/1, try‐ was studied with special reference to a possible oxygen effect. Dose‐response relationships were described by the function S= 1–(1 ‐ e‐kD)n where S is the surviving fraction and D is the u.v. dose in ergs/mm2. For cells suspended in 5 × 10‐‐6M acridine orange (AO) in air, the radiation sensitivity k was reduced from 0.010 (ergs/mm2))‐1 in the absence of the dye to 0.0053 (ergs/mm2)‐1 in the presence of the dye. Under anoxia at this AO concentration, k was further reduced to 0.0015 (ergs/mm2)‐1. The oxygen effect ratio, kO2/kN2, was 3.5 at this concentration of AO. Greater protection was observed in cells suspended in 2 × 10‐‐5M AO, the oxygen effect ratio was unchanged. No oxygen effect was detected in the u.v. response in the absence of the dye. The value of n was reduced from about 12 with no dye to about 5 at dye concentrations of 5 × 10‐‐6M AO or more when oxygen was present. Under anoxia, in the presence of AO, n was further reduced to about 1.3. Atebrin, an efficient u.v. protective agent but an inefficient photodynamic agent, had no oxygen effect for protection against u.v. inactivation. Acridine orange protected against u.v.‐induced reversion to tryptophan indepence in E. coli WP2 to about the same extent as it did for inactivation. A similar oxygen effect was observed for both inactivation and mutagenesis.

Kinetics ofF-curing by acridine orange in relation to the number ofF-particles inEscherichia coli

Acridine orange does not interfere with the distribution of theF-particles among the daughter cells but inhibits the multiplication of the episome.In growing cells the generation time of the episome in the presence of acridine orange is longer than the generation time of the cell.This lowers the number of episomes per cell to less than two copies at cell division.F-particles rapidly multiply after transfer until a relatively small fixed number (3–4) is reached.

Changes in immunodiffusion patterns and in nucleic acid content of Staphylococcus aureus grown in the presence of a nucleic acid fluorochrome.

Sonea, S. (University of Montreal, Montreal, Canada), J. de Repentigny, and A. Frappier. Changes in immunodiffusion patterns and in nucleic acid content of Staphylococcus aureus grown in the presence of a nucleic acid fluorochrome. J. Bacteriol. 84:1056-1060. 1962.-When grown in the presence of acridine orange, coagulase-positive Staphylococcus aureus exhibits changes in the number and the shape of its precipitin bands; these are more pronounced with commercial horse staphylococcal antitoxin than with normal human gamma-globulin. Simultaneously, there is an increase in the content of nucleic acids, especially deoxyribonucleic acid. Consequently, when grown in the presence of this nucleic acid fluorochrome, pathogenic staphylococci become similar to nonpathogenic strains in their antigenic structure and nucleic acid content. The bacterial population becomes a mixture of red and green fluorescing cells of different sizes. After centrifugation, most of the large and red fluorescing cells are found in the top layer, which shows more changes in antigenic composition and in nucleic acid content than the bottom layer. In subcultures grown in the absence of acridine orange, the cells revert to the original composition of the pathogenic strains.

Dye-sensitized photoinactivation of yeast in relation to the x-ray oxygen effect.

It is now well established for a wide variety of biological systems that the amount of dissolved present in cells during X-irradiation is a crucial parameter in determining what fraction of the initial molecular changes produced by the ionizations is ultimately expressed in detectable form.l-3 The shape of the survival curves for various cells and microorganisms, using inhibition of colony formation as an assay for lethality, is often found to be independent of whether the radiation is delivered in the presence or absence of oxygen;2, 3 that is, the curves obtained under different tensions can be made to superimpose by a suitable linear adjustment of the dose axis, and so it is convenient to describe the increased effectiveness of the radiation in the presence of in terms of an oxygen enhancement ratio. The enhancement ratio for cellular inactivation has been found to depend on certain details of the biological assay such as the type and richness of the plating media, even for a given organism.4 However, for a given assay procedure, a fundamental question in any theory of radiation injury is whether or not there are any differences in the nature or pattern of the lesions relevant to this end point produced in cells following exposure to X rays under aerobic as opposed to anaerobic conditions. In a search for further criteria of ionizing radiation damage, especially any which might distinguish between effects produced in the presence and absence of oxygen, we have recently found that yeast cells which survive anaerobic X-irradiation are more sensitive to subsequent dye-sensitized photoinactivation than are the survivors of comparable aerobic X-irradiation.5 The results show that, under anaerobic conditions, X rays inflict latent or unexpressed damage that can be detected by subsequent photoinactivation, but which is not present after X-irradiation under aerobic conditions. There is no such sensitization by preliminary anaerobic X-irradiation to subsequent ultraviolet inactivation. Thus, conditions which appear to protect cells from the effects of X rays (e.g., anoxia) may, at the same time, serve to promote the formation of X-ray induced lesions that sensztize them to other agents. Materials and Methods.-The results reported in this paper were all derived from radiation interaction experiments on starved suspensions of the haploid and diploid yeasts, Saccharomyces cerevisiae (SC-7, SC-6). The use of such experiments to determine the extent to which X rays and ultraviolet light exert their lethal effects on these cells through common sensitive sites has already been described.6 In the present experiments, both fully oxygenated and anoxic suspensions of cells were inactivated with X rays down to a given survival level (usually 10%), and then further inactivated either by visible light in the presence of acridine orange' or by ultraviolet light. The percentage of cells surviving any given radiation dose was determined from visible

Damage by Visible Light to the Acridine Orange-DNA Complex

Salmon DNA has been irradiated with visible light in the presence of acridine orange. If the dye is bound to the DNA, there results: (a) a decrease in sedimentation coefficient, (b) a lowering of viscosity, and (c) a decrease in the thermal denaturation temperature. CsCl banding experiments show that the first two effects reflect depolymerization of the DNA. Depolymerization apparently occurs by single-strand scission although some double-strand scission is not excluded. The destabilization of secondary structure results probably from chemical attack on the components of the individual strands.

Cytological effects of phenylnitrosamines—I. The production of structural chromosome changes in the presence of light and acridine orange

Twenty-eight different agents were tested for their ability to activate the production of chromosomal aberrations in the root-tips of Vicia faba by the light-acridine orange system. Of these agents only sodium nitrite and the phenylnitrosamines diphenylnitrosamine and N-methylphenylnitrosamine were active. The effect of methylphenylnitrosamine in the light-acridine orange system was compared with that of cupferron. Oxygen was found to counteract the effects of both these phenylnitrosamines. The effect of methylphenylnitrosamine was independent of the pH of the treatment solution, whereas the effect of cupferron decreased when the pH increased. The nature and mode of action of the active agents is discussed. Nitric oxide and cupferron were not able to replace oxygen in the photodynamic inactivation of coliphage T3 in the presence of acridine orange.