Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

A simple method for the fluorescence analysis of nucleic acid-dye complexes in cytological preparations

This communication describes a simple method for recording fluorescence emission spectra of cytological preparations using a conventional fluorescence spectrophotometer. The emission characteristics of "in situ" complexes between some basic fluorochromes (DAPI, 33258 Hoechst, acridine orange, pyronin Y, and ethidium bromide) and nucleic acid containing structures from smears of chicken blood and Ehrlich tumor cells (chromatin, basophilic cytoplasm) are briefly described.     

Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells

Friend leukemia cells from exponentially growing or differentiated (DMSO-induced) cultures were permeabilized and their DNA was stained with 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acridine orange, ethidium bromide, propidium iodide, quinacrine, 7-amino-actinomycin D, mithramycin, or chromomycin A3. Accessibility of DNA to each of the above fluorochromes was compared in differentiated and nondifferentiated cells before and after nuclear proteins, mostly histones, were extracted with 0.1N HCl. A decrease in the accessibility of DNA to several dyes, especially pronounced in the case of some intercalators, was observed in differentiated cells. After extraction of nuclear proteins with HCl there was an increase in DNA accessibility, of varying degree depending on the fluorochrome and the difference between differentiated and nondifferentiated cells was abolished for most of the intercalating dyes. The increase was the lowest for DAPI (45%), the highest for 7-amino-actinomycin D (13-fold), and in general was higher for the intercalating dyes that unwind DNA than for dyes binding externally to the double helix. The results are discussed in terms of the mode of interactions between DNA and the fluorochromes and factors associated with chromatin structure that may affect accessibility of DNA in situ in exponentially growing and differentiated cells.     

Caffeine dissociates complexes between DNA and intercalating dyes: application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry

BACKGROUND: Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. METHODS: HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. RESULTS: Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. CONCLUSION: Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.     

An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations

Several preparative techniques (detergent treatment, ethanol fixation, and hypotonic cell lysis), DNA fluorochromes, and methods of numerical analysis (planimetric or curve-fitting) were compared for the estimation of cell-cycle kinetic parameters (G1, S, G2 + M) by flow cytometry. In addition, coefficients of variation (CV), relative fluorescence, and G1/chicken erythrocyte (CRBC) ratios were measured and the effects of the proportion of cycling cells and cellular RNA content were examined. DNA fluorochromes were ranked by relative fluorescence: 4,6-diamidino-2-phenylindole > ethidium bromide/mithramycin > Hoechst 33342 > mithramycin > ethidium bromide > acridine orange approximately equal to propidium iodide. The first four (DNA-specific stains) gave lower CVs than the remainder (DNA intercalators). Detergent treatment also increased relative fluorescence and slightly lowered CVs. Comparable results were obtained for the kinetic parameters independently of stain or staining procedure; intercalating dyes with cells of a high RNA content not treated with RNAse and acridine orange being the exceptions. Of the two methods of numerical analysis, the planimetric technique was more consistant. Although highly consistant G1/CRBC ratios were obtained for any one stain, independently of staining procedures, variations between stains were noted. It is suggested that the detergent treatment in combination with DNA-specific stains provide optimal results.     

Interaction of chromosomal stains with DNA. An electrofluorescence study

A novel fluorescence procedure has been used to study the binding characteristics of DNA with three modern fluorochromes currently used in chromosome cytochemistry. The transient changes in the polarised components of fluorescence have been recorded for dye-tagged DNA solutions when subjected to short duration electric pulses. From these data, it has been inferred that, like ethidium bromide, berberine sulphate and quinacrine mustard both intercalate the DNA structure whilst the bi-benzimidazole derivative Hoechst 33258 binds with a distinctively different geometry, probably within the helical grooves.     

Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures.

A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences. The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei. Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines.     

Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding.

Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X-rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non-cycling cell fraction; and a distinctive, non-cycling G-/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst 33258-ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non-cycling cell fraction in the control culture to 29% (X-rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3-aminobenzamide, this aberrant cell population increased significantly after X-ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non-cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non-cycling and cycling cell fractions, this subpopulation with non-stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of non-cycling cells represents damaged cells with altered dye binding properties.     

Quantitation of DNA and RNA in crude tissue extracts by flow injection analysis.

An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 micrograms DNA and 0.10 micrograms RNA), automation, and a high sample throughput.     

A simple method for the fluorescence analysis of nucleic acid-dye complexes in cytological preparations

Summary This communication describes a simple method for recording fluorescence emission spectra of cytological preparations using a conventional fluorescence spectrophotometer. The emission characteristics of in situ complexes between some basic fluorochromes (DAPI, 33258 Hoechst, acridine orange, pyronin Y, and ethidium bromide) and nucleic acid containing structures from smears of chicken blood and Ehrlich tumor cells (chromatin, basophilic cytoplasm) are briefly described.     

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.     

Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.     

A cytochemical and immunocytochemical study of DNA distribution in spermatid nuclei of mouse,rabbit, and bull

Summary DNA distribution in mouse, rabbit and bull spermatids was analyzed by electron microscopy, after using a Feulgen-like HCl-osmium ammine procedure, and after immunocytochemistry with anti-DNA antibodies. In addition, nucleic acids were visualized with the intercalating dye ethidium bromide and phosphotungstic acid. The parts of DNA displaying a beta helix configuration (possibly A-T rich parts) were identified by epifluorescence microscopy after staining with Hoechst 33258. In all 3 species, young spermatid nuclei were seen to have large areas poor in DNA, as well as DNA-rich areas, which were mostly concentrated into a peripheral layer close to the acrosome and into one or several masses, displaying species-specific locations. These DNA-rich areas were stained with Hoechst 33258. Elongating spermatid nuclei contained homogeneously distributed DNA, and this was evident following both immunocytochemistry and nucleic acid histochemistry in all 3 species. However, the distribution appeared more heterogeneous after the Feulgen-like procedure, and was accompanied by a disappearance of Hoechst-fluorescence. In fully elongated spermatids, all nuclear areas stained with Hoechst 33258, while the 3 other techniques labeled either all or species-specific parts of the condensed chromatin. The reasons for these variable reactions are discussed in terms of technique specificities, DNA configuration and nucleoprotein moiety replacements.     

Measurement of efflux from G1-phase in a growth factor dependent cell line

In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the Gl-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.     

Combination of Budr-Quenched Hoechst Fluorescence With Dna-Specific Ethidium Bromide Fluorescence For Cell Cycle Analysis With A Two-Parametrical Flow Cytometer

Stimulated or asynchronous L-cells were grown in a BUdR-medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a FACS II cell sorter. the u.v. laser line served as a light source for exciting the Hoechst fluorescence, the ethidium bromide fluorescence being excited mainly by energy transfer from the Hoechst dye. the quenched Hoechst fluorescence was analysed between 410 nm and 480 nm, the DNA specific EB fluorescence at beyond 630 nm. Thus, not only the actual location of each cell in the cycle could be determined, but also its initial location at time 0 of the experiment, together with its moment of division (BUdR-quenched Hoechst fluorescence). This method could become a powerful tool in many investigations dealing with cell cycle perturbations in culture.     

Combination of BUdR-quenched Hoechst fluorescence with DNA-specific ethidium bromide fluorescence for cell cycle analysis with a two-parameter flow cytometer

Stimulated or asynchronous L-cells were grown in a BUdR-medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a FACS II cell sorter. The u.v. laser line served as a light source for exciting the Hoechst fluorescence, the ethidium bromide fluorescence being excited mainly by energy transfer from the Hoechst dye. The quenched Hoechst fluorescence was analysed between 410 nm and 480 nm, the DNA specific EB fluorescence at beyond 630 nm. Thus, not only the actual location of each cell in the cycle could be determined, but also its initial location at time 0 of the experiment, together with its moment of division (BUdR-quenched Hoechst fluorescence). This method could become a powerful tool in many investigations dealing with cell cycle perturbations in culture.     

Detection of A + T-rich DNA in gels by differential fluorescence

The fluorochrome Hoechst 33258 preferentially forms complexes with A + T-rich duplex DNA, whereas ethidium bromide binds nucleic acids independent of base composition. Both compounds can be conveniently used to visualize DNA fractionated by gel electrophoresis. Determination of fluorescence emission from Hoechst 33258-stained restriction fragments normalized to fluorescence derived from the same sample after ethidium bromide staining provides a measure of emission due to A + T content, and allows easy identification of A + T-rich restriction fragments. To demonstrate the utility of this procedure, an A + T map of bacteriophage lambda DNA was constructed and found to be comparable to similar maps derived by alternate techniques. Analysis of recombinant plasmid DNAs with established nucleotide sequences demonstrated that the A + T content of individual restriction fragments could be estimated to within an accuracy of 5%.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.