A method of DNA quantitation for localization of DNA in metrizamide gradients.

A quantitative assay for DNA is described that involves the binding of the cytologicalstain, methyl green, to DNA. At pH 7.9, solutions containing free methyl green undergo complete fading whereas solutions containing DNA-bound methyl green retain color in proportion to the amount of DNA present. The procedure allows for the quantitation of DNA in the presence of urea, sucrose, EDTA, protein, dithiothreitol, metrizamide, and low-concentration salts. This method is also applicable to the quantitation of DNA in chromatin.     

Methyl green-pyronin; stoichiometry of reaction with nucleic acids

Study of the stoichiometry of the DNA-methyl green reaction by dialysis, precipitation of stain-nucleic acid mixtures, and the staining of nuclei of known DNA content, indicate that the compound consists of one dye molecule per 10 P. The significance of this result was discussed in the preceding paper (1). Histone and lanthanum (and probably other multivalent cations (3)) compete with the dye for the nucleic acid molecule, indicating a common site of attachment, presumably the phosphoric acid groups. With care in the avoidance of procedures which might depolymerize DNA, and the use of a buffer at about pH 4.1, a quantitative histochemical method for DNA by the use of methyl green is possible. Pyronin staining appears to be of qualitative significance only. Slight differences in degree of polymerization, as between the shad and mammalian DNA appear to have no effect on methyl green staining. It may be that a critical level of polymerization for DNA staining exists. This level must exceed 20 nucleotides to account for the 10 P to 1 dye molecule and the effect on the methyl green absorption spectrum; but it may be considerably greater. Beyond this critical level, whatever it may be, further polymerization probably has no influence on staining.     

Methyl green and its analogues bind selectively to AT-rich regions of native DNA.

Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30-90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30–90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Methyl green. III. Reaction with desoxyribonucleic acid,stoichiometry, and behavior of the reaction product

1. Methyl green ("ethyl green") C. I. Number 685 was examined and found to behave identically with methyl green C. I. Number 684 (no longer available) in respect to molar extinction coefficient, effect of combination with polymerized DNA, failure to react with depolymerized DNA, and effect of pH. 2. The mass law permits the calculation of P/dye. This is found to be 13 P/dye. The same value is obtained when an excess of methyl green is caused to fade by adjusting the pH to 7.5. 3. The compound formed by methyl green with DNA has the same maximum absorption at 642.5 to 645 mµ in the pH range 3.5–7.8, whereas the free dye fades markedly above pH 5.0.     

Methyl green staining and highly repetitive DNA in polytene chromosomes

Fixed polytene chromosomes of Rhynchosciara angelae were stained with methyl green-pyronin after different pretreatments. Methyl green affinity for chromatin in the polytene chromosomes is lost after acid, alkali and heatformaldehyde treatments as a consequence of DNA denaturation, but reappears if conditions for DNA strand reassociation are provided. When such conditions are absent, only some chromosomal regions recover their affinity for methyl green. The resulting differential staining pattern is described and its possible relation to highly repetitive DNA sequences is discussed.     

EVIDENCE OF DIFFERENCES IN THE DESOXYRIBONUCLEOPROTEIN COMPLEX OF RAPIDLY PROLIFERATING AND NON-DIVIDING CELLS

1. Quantitative cytophotometric analysis of the interphase cells of a rapidly proliferating differentiated tissue such as liver of new born rat, indicates that these cells can be separated into two groups on the basis of their staining characteristics after methanol fixation. 2. These groups are thought to correspond to two stages of interphase. The first, called "autosynthetic interphase," comprises cells which are duplicating chromosomal material in preparation for mitosis, and shows parallel increases in the methyl green and Feulgen staining of DNA and the fast green staining of histone from the diploid (2 C) to double these values (4 C). 3. The second group is designated the "heterosynthetic interphase," during which the cell ceases proliferating and functions in a manner commensurate with its state of differentiation. In this stage Feulgen staining indicates the diploid chromosomal complement, but there is a decreased capacity of the DNA to bind methyl green and of the histone to bind fast green. 4. The difference between the methyl green binding of the heterosynthetic and autosynthetic 2 C cells is due to the presence of a protein in the former which presumably inhibits staining by competing with the dye for binding sites on the DNA. The effect of this inhibition can be removed by extracting the protein, or by blocking the protein basic groups. 5. The decreased fast green staining of histone in the heterosynthetic cells can be minimized by prolonged fixation with formaldehyde. It is thought to stem either from a similar type of inhibition, or from an increased susceptibility of the histone to loss from the cell during this stage. 6. While histone inhibits methyl green staining of DNA in all cells, the differences between the staining properties of the autosynthetic and heterosynthetic interphase cells are believed to be due to another protein, whose properties appear similar to those of the chromosomal "residual protein." It is concluded that a complex of DNA and residual protein existing during the heterosynthetic interphase is dissociated during the autosynthetic interphase.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time,dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.     

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.     

Influence of cationic triphenylmethane dyes upon DNA polymerization and product hydrolysis by Escherichia coli polymerase I.

The cationic triphenylmethane dyes crystal violet, methyl green, and malachite green inhibited DNA synthesis catalyzed by Escherichia coli B polymerase I (polymerase I). Lower concentrations of the dyes inhibited DNA replication as a direct function of the proportion of AT to GC in the DNA of Clostridium perfringens, Escherichia coli, and Micrococcus lysodeikticus. When the intercalant proflavin was employed, the GC-rich micrococcal DNA was most severely inhibited. In addition, both the triphenylmethanes and proflavin inhibited product hydrolysis catalyzed by polymerase I.     

On the mechanism of the methyl green-pyronin stain for nucleic acids

Summary The combination of pyronin, methyl green, malachite green, crystal violet, and Alcian Blue with a large number of polynucleotides and acidic polysaccharides has been investigated. The critical electrolyte concentration approach has been used to provide a measure of affinity between dye and substrate.The interaction of methyl green with DNA, RNA and heparin has been examined spectroscopically.Previously published results are re-examined, and with the new experiments permit consistent interpretations of the specificities of dye binding in terms of modern ideas of nucleic acid and dye structure.All dyestuffs except Alcian Blue bind more strongly to polynucleotides than would be expected if solely electrostatic bonds were present. Pyronin and planar monovalent cationic dyes interact best with polynucleotides in which purine and pyrimidine bases are freely accessible, as in single stranded molecules without extensive secondary structure, such as RNA, denatured DNA, etc. Non-planar triphenylmethane dyes, e.g. methyl green, malachite green etc. bind less strongly to such substrates, but because of their shape they fit well into the secondary structure of native DNA. Tumour RNA and DNA did not differ from normal RNA and DNA.By varying the electrolyte concentration, pyronin-methyl green selectivity e.g. for DNA or RNA, can be controlled, and non-nucleotide staining suppressed. The relevance of the new interpretation to the ribonuclease-pyronin technique is discussed.     

A Differential Stain for Nucleic Acids

An easily used trichrome stain consisting of orange G, methyl green, and toluidine blue is proposed as a method of differentiating desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells. Carnoy's acetic-alcohol is the fixative of choice, though cold acetone is also satisfactory. Photomicrographs taken with ultraviolet and visible light show that the structures containing nucleic acid are exactly those which stain with methyl green and toluidine blue. Studies with nucleases and extraction of nucleic acids with cold and hot perchloric acid further indicate a specificityy of the dyes for DNA and RNA. Present experiments are directed toward using the stain for quantitative estimation of the nucleic acids.     

Visualization of viral DNA assembly using nonfluorescent staining

ABSTRACT

We present an easy test for rapid visualization of viral DNA assemblies in infected cell cytoplasm. We selected the best stains for nuclear staining: Nile blue A, Bismarck brown, gallocyanin chrome alum, methyl green pyronin and azure II. None of the staining techniques is fluorescent, which facilitates their use in everyday experiments. Methyl green is most promising for routine detection of viral DNA assemblies in the cytoplasm; the procedure enables ready detection of viral DNA accumulation in the cytoplasm.     

A new rapid method for selective extraction of RNA from fixed mammalian tissues.

Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.     

A comparison of four quantitative cytochemical methods directed toward demonstration of DNA.

Populations of nuclei isolated from mouse brain tissue were stained by the following cytochemical methods considered stoichiometric for DNA: (1) the Feulgen reaction; (2) gallocyanin-chromalum after RNase; (3) pH 4.0 methylene blue after RNase; and (4) methyl green used in the presence of 2M magnesium chloride. Replicate preparations to be stained with gallocyanin-chromalum, methylene blue, and methyl green were acetylated prior to staining. All of these groups were examined by high-resolution scanning microspectrophotometry. The results indicated that of the methods examined, the Feulgen reaction, gallocyanin-chromalum used without prior acetylation, and methylene blue used with prior acetylation were the most useful in revealing differences attributable to variability in chromatin organization. The greatest variability in total extinction measurements was observed in acetylated, methylene blue-stained nuclei, while the least variability was observed in nuclei stained with methyl green in the presence of 2 M magnesium chloride. Acetylation produced different effects on dye-binding in different groups. It greatly increased binding in nuclei stained with methylene blue; it reduced binding in the methyl green-2 M magnesium chloride series.     

Detection of Ribonucleic Acid with Pyronin

Pyronin, when used in the methyl green-pyronin stain, is useful in localizing ribonucleic acid (RNA). That it has rarely been used alone is perhaps a result of the observation (Kurnick 1955) that pyronin stains deoxyrobonucleic acid (DNA) of animal tissue when not competitively inhibited by methyl green. The tests described in this note indicate that pyronin alone can be used to demonstrate RNA in fixed plant tissues.     

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing.

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.     

Evidence in Escherichia coli that N3-methyladenine lesions and cytotoxicity induced by a minor groove binding methyl sulfonate ester can be modulated in vivo by netropsin

The use of DNA equilibrium binding molecules to transfer alkyl groups to specific positions on DNA is an approach to generating cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer. We have previously reported that in vitro a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in >90% yield relative to the formation of other adducts. While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate. We report herein the relationship between the in vivo formation of N3-methyladenine and toxicity in wild-type and base excision repair defective mutant Escherichia coli. In addition, it is demonstrated that both N3-methyladenine adduction and cytotoxicity can be inhibited in vivo with netropsin, a potent competitive inhibitor of binding of lex to DNA. The results show a clear relationship between the levels of N3-methyladenine and toxicity in an alkA/tag glycosylase mutant that cannot remove the adduct from its genome. For methyl methanesulfonate, which does not sequence selectively methylate DNA, a relationship between the formation of N3-methyladenine and toxicity is also observed. However, netropsin affects neither the level of N3-methyladenine nor the toxicity of methyl methanesulfonate in E. coli.