Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

In an attempt to achieve accurate quantification of DNA levels in cell nuclei, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the original level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

Summary We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome.The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

Decrease of DNA per cell during development of the lens in chickens

Summary Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.     

Characterization of the fluorescence of the protamine thynnine and studies of binding to double-stranded DNA

The protamine thynnine is an arginine-rich protein approximately 30 amino acids long with a tyrosine in the middle of its sequence. Its fluorescence decay kinetics can be described by a biexponential function with lifetimes of 0.52 and 2.1 ns, with almost equal preexponential factors. The fluorescence quencher CsCl does not affect the short lifetime but shifts the equilibrium between the long and short lifetime toward the short one and reduces the long lifetime. In nature, thynnine is found complexed with chromosomal DNA. In vitro complexes of thynnine with double-stranded (ds) DNA are stable at physiologic ionic strength but dissociate at high NaCl concentration. This dissociation can be monitored by steady-state fluorescence. From the salt concentration dependence of the dissociation of the complex of thynnine with ds-DNA 145 bp long, it can be concluded that only 4 of 21 possible full electrostatic bonds are involved in thynnine-DNA binding. In addition, the binding constant at 1M NaCl is of the order of 10⁶, indicating a strong nonelectrostatic component in arginine-DNA binding.     

Decrease of DNA per cell during development of the lens in chickens.

Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.     

Dynamic structures of intact chicken erythrocyte chromatins as studied by 1H-31P cross-polarization NMR.

The dynamic properties of DNA in intact chicken erythrocyte cells, nuclei, nondigested chromatins, digested soluble chromatins, H1, H5-depleted soluble chromatins and nucleosome cores were investigated by means of single-pulse and 1H-31P cross-polarization NMR. The temperature dependence of the phosphorus chemical shift anisotropy was identical for the former three in the presence of 3 mM MgCl2, suggesting that the local higher order structure is identical for these chromatins. The intrinsic phosphorus chemical shift anisotropy of the nucleosome cores was -159 ppm. The chemical shift anisotropy of DNA in the chromatins can be further averaged by the motion of the linker DNA. The spin-lattice relaxation time in the rotating frame of the proton spins (T1p) of the nondigested chromatins was measured at various locking fields. The result was analyzed on the assumption of the isotropic motion to get a rough value of the correlation time of the motion efficient for the relaxation, which was eventually ascribed to the segmental motion of the linker DNA with restricted amplitude. The 30 nm filament structure induced by NaCl was shown to be dynamically different from that induced by MgCl2. Side-by-side compaction of 30-nm filaments was suggested to be induced in the MgCl2 concentration range higher than 0.3 mM. Biological significance of the dynamic structure was discussed in connection with the results obtained.     

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer

BACKGROUND: DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. METHODS: We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). RESULTS: The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. CONCLUSIONS: Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.     

Fluorescence anisotropy decay of ethidium bound to chromatin

The fluorescence anisotropy decays of the chromatin ethidium complexes have been measured in solutions in which the dye was bound to the high affinity sites of the nucleosome DNA. Energy transfers between chromatin-bound ethidium molecules cause an increase of the anisotropy decay rate for much smaller values of the concentration ratio of dye to nucleotide than in the case of nacked DNA-ethidium complexes. This result implies that the high affinity sites are clustered on a short nucleosomal DNA segment. Quantitative analysis of the experimental data by computer simulations of the energy transfer process, shows that these sites are gathered on a single nucleosomal DNA segment, 28 base pairs long. Such a segment probably belongs to the nucleosome “linker”, contributing about half of it.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Summary Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

Summary A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-P was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D=6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 g/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Evidence against the specific initiation of Okazaki fragments at the internucleosomal linkers

We have digested nuclei, isolated from [³H] thymidine pulse labelled cells, with nuclease S₁. Short pulse labelled DNA fragments were excised by the enzyme and released upon subsequent treatment with 2 M NaCl. Only a small fraction of the label was released from the S₁ digested nuclei by 0.5 M NaCl indicating that the cleavage sites were located in the DNA of the nucleosome cores. The results are not compatible with the hypothesis that the initiation of the Okazaki fragments occurs at the internucleosomal linkers.     

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Summary The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.Abbreviations A-T nucleic bases adenine and thymine - CRBC chicken red blood cell - FALS forward-angle light scatter - G-C nucleic bases guanine and cytosine - SEIM sorbitol enzymatic incubation medium - SWIM sea water incubation medium - Tm thermal denaturation temperature of DNA     

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-p was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D = 6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 micrograms/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 1. Rotational diffusion indicates an extended structure at low ionic strength

The fluorescence decay of ethidium intercalated into the DNA of nucleosome core particles increases in average lifetime from about 22 ns in H2O to about 39 ns in D2O. This increase, combined with the acquisition of large amounts of data (on the order of 10(8) counts per decay), allows measurement of anisotropy decays out to more than 350 ns. The overall slow rotational motions of the core particle may thereby be more clearly distinguished from the faster torsional motions of the DNA. In 10 mM NaCl at 20 degrees C, we recover a long correlation time of 198 ns in D2O (159 ns when corrected to a viscosity of 1.002 cP), in agreement with the value of 164 ns obtained in H2O. These values are consistent with hydrodynamic calculations based on the expected size and shape of the hydrated particle. To support our conclusion that this long correlation time derives from Brownian rotational diffusion, we show that the value is directly proportional to the viscosity and inversely proportional to the temperature. No significant changes in the rotational correlation time are observed between 1 and 500 mM ionic strength. Below 1 mM, the particle undergoes the "low-salt transition" as measured by steady-state tyrosine fluorescence anisotropy. However, we observe little change in shape until the ionic strength is decreased below approximately 0.2 mM, where the correlation time increases nearly 2-fold, indicating that the particle has opened up into an extended form. We have previously shown that the transition becomes nonreversible below 0.2 mM salt.     

The association of a magnesium-dependent endonuclease activity with a nucleosome fraction from rat-liver nuclei

The nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35 M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35 M NaCl buffer and then fractionated in a sucrose gradient containing 0.35 M NaCl, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35 M NaCl buffer, the nuclease activity was eluted at the position of 36,000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and double-strand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36,000 daltons is associated with the nucleosome particle(s) in rat-liver nuclei.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.     

Ion-induced DNA structure change in nucleosomes

Physical methods have been used to study calcium binding to the nucleosome core particle. Equilibrium dialysis of Ca2+ and spectroscopic analysis of a Ca2+ analogue show that the ion binds tightly to the particles, resulting in a significant change of DNA circular dichroism. This suggests that base stacking may be altered as a result of Ca2+ binding. In the presence of Ca2+, the absorbance and fluorescence properties of methylene blue (MB), a DNA-specific intercalator, confirm that the dye binds tightly to nucleosomes by intercalation. However, secondary changes occur which suggest that the MB binding site is altered as a result of Ca2+ binding. Triplet state anisotropy decay and triplet lifetime quenching both show that in the Ca2+-nucleosome complex, methylene blue is capable of wobbling over a substantial angular range at its binding site. To explain these data, it is proposed that Ca2+ binding to nucleosomes causes DNA to fold by means of a series of sharp bends (kinks). The properties of bound MB are best explained if it is presumed that the intercalator binds tightly to such kinked sites in the nucleosome. On the basis of these observations, we discuss the possibility that multivalent ion concentration in the nucleus is high enough that the smooth to kinked helix equilibrium may be near to its midpoint. Near such a midpoint, the secondary structure of DNA in the nucleosome might prove to be sensitive to effector molecule binding and to site-specific variation of DNA or histone composition within genes.