Microfluorometric analysis of cellular DNA following incorporation of bromodeoxyuridine.

Bromodeoxyuridine (BrdU) incorporation into cellular DNA has a differential effect on the cell-associated fluorescence of several DNA-specific dyes. After cells were treated with BrdU, flow microfluorometry was used to study the relative increase or decrease influorescence of stained cells. Bromodeoxyuridine incorporation into CHO cells increased the fluorescence of mithramycin-, olivomycin-, or chromomycin-stained cells, decreased that of propidium iodide-stained cells, and had little, if any, effect on the fluorescence of acriflavine Feulgen-stained cells. Changes in relative fluorescence of cell associated dyes are due to changes in the amounts of dye bound to cells with BrdU-substituted DNA. Colorimetric and absorbance measurement of DNA content showed that BrdU does not alter the diploid DNA content of CHO cells; however, BrdU induces perturbations in the distribution of cells about the cell cycle which cause an increase in average DNA content.     

Dual wavelength scanning cytophotometry (Bicoscan).

A computer program has been developed for stage-scanning cytophotometry of double-stained microscopical specimens. The program permits the simultaneous measurement of absorbance values at two wavelengths in each measuring spot. To account for overlap in the absorbance spectra of the two stained endproducts, the program incorporates correction of the measured data to compensate for the contribution of each chromophore to the absorbance measured for the other. The program will compute the corrected local absorbance values at specified wavelengths for each chromophore at each measuring spot and integrate these values over the total object to give separate totals for each stain. It is also possible to have integrated the absorbance values of one chromophore for all those measuring spots where the local corrected absorbance value of the other chromophore exceeds a preset minimum value. When this other chromophore is a nuclear DNA stain, it is possible to obtain an approximate measure of the content of any compound in the nuclear area which can be stained with a chromophore having an absorbance spectrum different from the DNA stain. The validity of the program was investigated on model preparations consisting of two (differently) coloured films of which the absorbance values could be measured either individually or in combination, by partially overlaying one film on the other. The program's potential has been demonstrated by using the combination of either Naphthol Yellow S or dinitrofluorobenzene as protein stains with the Feulgen-pararosaniline(SO2) procedure for DNA in chicken erythrocytes and rat liver cells.     

An improved flow microfluorometer for rapid measurement of cell fluorescence

An improved instrument for the making of high-speed fluorescent measurements on single cells has been constructed and characterized. Instrumental optimization was accomplished with Chinese hamser cells (line CHO) stained by the fluorescent Feulgen procedure using either auramine-0 or acriflavine. Contributions of instrumental resolution and artifacts to cellular DNA distributions have been determined. The instrument introduces a total coefficient of variation of less than 2% for the case of CHO cells stained with bright dyes such as acriflavine, where photon-statistical effects are minimal. The design of the instrument is such that any of a number of cellular constituents or properties can be studied and quantitated on a single-cell basis at a rate of 50 000 cells/min.     

Dual wavelength scanning cytophotometry (Bicoscan)

Summary A computer program has been developed for stage-scanning cytophotometry of double-stained microscopical specimens. The program permits the simultaneous measurement of absorbance values at two wavelengths in each measuring spot.To account for overlap in the absorbance spectra of the two stained endproducts, the program incorporates correction of the measured data to compensate for the contribution of each chromophore to the absorbance measured for the other.The program will compute the corrected local absorbance values at specified wavelengths for each chromophore at each measuring spot and integrate these values over the total object to give separate totals for each stain. It is also possible to have integrated the absorbance values of one chromophore for all those measuring spots where the local corrected absorbance value of the other chromophore exceeds a preset minimum value. When this other chromophore is a nuclear DNA stain, it is possible to obtain an approximate measure of the content of any compound in the nuclear area which can be stained with a chromophore having an absorbance spectrum different from the DNA stain. The validity of the program was investigated on model preparations consisting of two (differently) coloured films of which the absorbance values could be measured either individually or in combination, by partially overlaying one film on the other.The program's potential has been demonstrated by using the combination of either Naphthol Yellow S or dinitrofluorobenzene as protein stains with the Feulgen-pararosaniline(SO₂) procedure for DNA in chicken erythrocytes and rat liver cells.Supported by Grant Nr. 28-394 of the Praeventiefonds, The Hauge     

Versatile copurification procedure for rapid isolation of homogeneous RAR-RXR heterodimers.

RAR-RXR heterodimeric complexes (RARalphaDeltaAB-RXRalphaDeltaAB) bound to cognate DR5 DNA response elements were purified to apparent structural and functional homogeneity using a novel versatile immobilized metal affinity chromatography (IMAC) copurification procedure. Dynamic light scattering studies indicated that the complexes were more than 85% monodisperse. By electron microscopy the negatively stained RAR-RXR-DNA complexes appeared homogeneous and corresponded to a dimeric arrangement of the molecules. Using heterodimers purified according to this procedure we demonstrate ligand binding of RXR in the context of the RAR-RXR heterodimer-DNA complex. The present copurification procedure is rapid and has yielded high quality heterodimer-DNA complexes suitable for both structural and biochemical studies.     

Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation

The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety.     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

Acriflavine Uptake and Resistance in Serratia marcescens Cells and Spheroplasts

Acriflavine uptake and resistance were investigated in red, sensitive Serratia marcescens cells and in orange, resistant mutant cells and their respective spheroplasts. Acriflavine-sensitive cells bound more acriflavine than acriflavine-resistant cells. Spheroplasts from sensitive and resistant cells were both resistant to and bound similar amounts of acriflavine. Sensitive cells were resistant to acriflavine in medium supplemented with 0.01 M MgSO(4) and 0.5 M sucrose. In the presence of 0.01 M MgSO(4) and 0.5 M sucrose, acriflavine binding by sensitive cells was reduced to the level of binding by resistant cells. Inhibition of metabolism by carbon starvation, chloramphenicol, As(2)O(3), nitrosoguanidine, and bromouracil did not affect the uptake of acriflavine by sensitive and resistant cells. Rapid temperature changes did not alter the acriflavine-binding capacity of the cells, and no temperature dependence of acriflavine uptake or release was observed at 0 and 30 C. Acriflavine uptake by both sensitive and resistant cells increased with increase in pH from 5.7 to 8.0. The logarithm of acriflavine uptake was a linear function of the logarithm of the acriflavine concentration in the binding medium.     

Acriflavine binding and DNA stability

The influence of acriflavine in the process of exposing DNA solution to gamma-radiation is studied. Acriflavine being actively bound to DNA is demonstrated not to protect DNA molecule from radiation damage. No radiation-induced variation in acriflavine-DNA binding degree is discovered. Acriflavine protective properties are revealed when the concentration of acriflavine is essentially high. This confirms our early results according to which only free ligands in solution protect DNA from radiation damage.     

Model system studies of staining procedures for lysine and arginine residues

Summary Using polyacrylamide films containing poly-lysine, polyargine and DNA as test models, a variety of reportedly specific staining procedures have been examined. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin while arginine will stain with fast green, if proteins containing both amino-acids are stained with the dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acid-arginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrochloric acid in a Feulgen-like procedure which stains DNA to the same level as the classical hydrochloric acid based procedure while also staining arginine present.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques.

Feulgen nuclear staining with pararosanilin-SO2 was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell. The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality between the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by B?hm et al. (1968) and a reaction with ninhydrin at 37 degrees C for 10 h. The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure. Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.     

The glucocorticoid receptor in homodimeric and monomeric form visualised by electron microscopy.

The purified glucocorticoid receptor (GR) from rat liver has been visualised by electron microscopy. The specimens were prepared by spreading on thin carbon support and negatively stained using uranyl acetate. Two forms of GR, the monomeric and the dimeric forms, were identified based on size, chromatographic distribution, and DNA binding properties. The GR monomer consists of two globular domains of slightly different size with a thinner connecting domain in between. In the absence of DNA the dimeric GR has a characteristic four-leaf clover structure. The size and appearance of this structure is consistent with two GR subunits arranged in a side-by-side fashion. Monomeric and dimeric GR specifically bound to DNA are also shown.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

Sequence specificity of 125I-labelled Hoechst 33258 in intact human cells

Using polyacrylamide/urea DNA sequencing gels, the DNA sequence selectivity of 125I-labelled Hoechst 33258 damage has been determined in intact human cells to the exact base-pair. This was accomplished using a novel procedure with human alpha RI-DNA as the target DNA sequence. In this procedure, after size fractionation, the alpha RI-DNA is selectively purified by hybridization to a single-stranded M13 clone containing an alpha RI-DNA insert. The sequence specificity of [125I]Hoechst 33258 was indistinguishable in intact cells from purified high molecular weight DNA; and this is surprising considering the more complex environment of DNA in the nucleus where DNA is bound to nucleosomes and other DNA binding proteins. The ligand preferentially binds to DNA sequences which have four or more consecutive A.T base-pairs. The extent of damage was measured with a densitometer and, relative to the damage hotspot at base-pair 94, the extent of damage was similar in both purified high molecular weight DNA and intact cells. [125I]Hoechst 33258 causes only double-strand breaks, since single-strand breaks or base damage were not detected. These experiments represent the first occasion that the sequence specificity of a DNA damaging agent, which causes only double-strand breaks, has been determined to the exact base-pair in intact cells.     

In vitro association of empty adenovirus capsids with double-stranded DNA.

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.     

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.     

Subclasses of simian-virus-40 large tumor antigen. Partial purification and DNA-binding properties of two subclasses of tumor antigen from productively infected cells

Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells. These subclasses can be distinguished by their sedimentation properties: one tumor antigen form sediments at 5-6S and the other at 14-16S. The DNA-binding properties of these subclasses were investigated by two different experimental procedures. In the first procedure, the DNA binding of subclasses of crude tumor antigen, separated by zone velocity sedimentation, were assayed by immunoprecipitation of the DNA-protein complexes. In the second procedure, the two tumor antigen forms were partially purified by column chromatography and DNA binding was tested in a filter binding assay. Both procedures gave comparable results. (a) The 5-6-S and the 14-16-S tumor antigen bound specifically to a DNA restriction fragment containing the viral genome control regions. (b) At low salt concentrations, both subclasses bound to specific and to nonspecific DNA sequences; competition experiments in the presence of nonspecific DNA showed, however, that the affinity of both tumor antigen forms for the viral genome control region was at least 10-fold higher than their affinity for nonspecific DNA sequences. (c) The binding of the 5-6-S subclass to viral control region DNA was optimal at 60-80 mM NaCl while specific DNA binding of the 14-16-S form was optimal at 150-200 mM NaCl; however, binding of the 14-16-S form to nonspecific DNA sequences was also more resistant to high salt concentrations than that of the 5-6S form. (d) Both tumor antigen forms bound well to specific and to nonspecific DNA at pH 6-6.5; with increasing pH values, binding to nonspecific DNA decreased while binding to specific DNA reached an optimum at pH 7-7.5. Binding of the 14-16-S form to viral origin DNA was more resistant to pH values above 7.5 than binding of the 5-6-S form.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

THE LOSS OF KINETOPLASTIC DNA IN TWO SPECIES OF TRYPANOSOMATIDAE TREATED WITH ACRIFLAVINE

The effects of acriflavine on two species of Trypanosomatidae, Crithidia luciliae and Trypanosoma mega, have been investigated. It has been observed that kinetoplastic (i.e. mitochondrial) DNA is lost in a high percentage of acriflavine-treated cells. Resting flagellates, from stationary-phase or hemin-deficient cultures, are considerably more resistant to the acridine than are flagellates from a log-phase culture. When the kinetoplast has retained some DNA and still remains visible in stained smears, it appears reduced in size, and its ultrastructure is extremely abnormal: the DNA fibrils, clearly visible in normal kinetoplasts, are condensed; they appear as an electron-opaque, apparently homogeneous mass, separated from the membranes by a space of low electron-opacity. Analyses of DNA extracts, with high speed centrifugation in CsCl density gradients, revealed that the satellite band, presumably kinetoplastic DNA, is lost by trypanosomes grown for 5 days in the presence of acriflavine. Radioautography was used to study the effects of acriflavine on thymidine-³H incorporation in C. luciliae. At the concentration which affects the kinetoplast specifically, the dye produces an 87% inhibition of thymidine incorporation in this organelle. The kinetics of this inhibition suggest a direct effect on replication. No decrease in incorporation occurs in the nucleus. These results lead to the conclusion that loss of kinetoplastic DNA is due to continued growth and cell division in the absence of kinetoplastic DNA replication. Several hypotheses are discussed concerning the specificity of the dye's action upon the replication of extrachromosomal DNA.     

The analysis and interpretation of DNA distributions measured by flow cytometry

A principal use of flow cytometers is for the measurement of fluorescence distributions of cells stained with DNA specific dyes. A large amount of effort has been and is being expended currently in the analysis of these distributions for the fractions of cells in the G1, S, and G2 + M phases. Several methods of analysis have been proposed and are being used; new methods continue to be introduced. Many, if not most, of these methods differ only in the mathematical function used to represent the phases of the cell cycle and represent attempts to fit exactly distributions with known phase fractions or unusual shapes. In this paper we show that these refinements probably are not necessary because of cell staining and sampling variability. This hypothesis was tested by measuring fluorescence distributions for Chinese hamster ovary and KHT mouse sarcoma cells stained with Hoechst-33258, chromomycin A3, propidium iodide, and acriflavine. Our results show that: a) single measurements can result in phase fraction estimates that are in error by as much as 40% for G2 + M phase and 15-20% for G1 and S phases; b) different dyes can yield phase fraction estimates that differ by as much as 40% due to differences in DNA specificity; c) the shapes of fluorescence distributions and their interpretation are very dependent on the dye being used and on its binding mechanism.