Quenching of Hoechst 33258 fluorescence in erythroid precursors.

Quenching of the fluorescence of DNA-bound Hoechst 33258 in erythroid precursors was studied by flow cytometry and cytochemistry. This quenching artifact may affect the measurement of ploidy in specific cases. The bone marrow cells of two patients with hemolytic disease and active erythropoiesis contained subpopulations of cells with an apparent hypodiploid DNA content as measured by flow cytometry of paraformaldehyde-fixed cells stained with Hoechst 33258. No aneuploidy was detected in either of the two cases when cells were stained with mithramycin or 7-aminoactinomycin D. Cells exhibiting reduced Hoechst 33258 fluorescence expressed glycophorin A and low amounts of CD36, and were therefore erythroid precursors. In one case studied, the number of cells with reduced Hoechst 33258 fluorescence and glycophorin A expressed agreed well with the number of cells containing nuclear hemoglobin. In the other case, hemoglobin was present in a significant proportion of nucleated cells. Calculated values for the efficiency of resonance energy transfer from Hoechst 33258 to hemoglobin were in accordance with the observed levels of quenching (approximately 10%). However, the results could also be explained by hemoglobin reabsorption of Hoechst 33258 fluorescence. Nuclei stained with Hoechst 33258 showed uniform fluorescence, probably due to extraction of hemoglobin during the isolation procedure.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.     

Some Properties of New DNA-Specific Bisbenzimidazole Fluorochromes without a Piperazine Ring

Three new bisbenzimidazole (BBI) compounds, which differ from Hoechst 33258 mainly by substitution of a N-dimethylaminopro-pylcarboxamide group in place of the N-methyl-piperazine ring, were studied for their DNA- and AT-base pair specificity as well as for their ability to be quenched by incorporated 5-bromodeoxy-uridine (BrdU). Each of them had DNA binding specificity comparable to or greater than that of Hoechst 33258 and each had a greater specificity for AT-rich regions than did Hoechst 33258. The dependence of fluorescence of new dyes on the BrdU-incorporation into DNA is different from that of Hoechst 33258 and related compounds with piperazine ring. The quenching effect is much weaker, and two of the new compounds (BBI-1 and BBI-2) even show somewhat enhanced binding (fluorescence) at lower concentrations. Certain BBI dyes without piperazine ring may have some advantage over Hoechst for accurate DNA [AT-specific] measurements. The piperazine ring appears to play an important role in the yet unknown mechanism of Hoechst quenching by incorporated BrdU.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Summary Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-U.V irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.In honour of Prof. P. van Duijn     

Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

Recognition of RNA duplex by a neomycin–Hoechst 33258 conjugate

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin–Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.     

Hoechst 33258--poly(dG-dC).poly(dG-dC) complexes of three types

It was found recently that Hoechst 33258, a dsDNA fluorescent dye used in cytological studies, is an efficient inhibitor of the interaction of TATA-box-binding protein with DNA, DNA topoisomerase I, and DNA helicases. In addition it proved to be a radioprotector. Biological activity of Hoechst 33258 may be associated with dsDNA complexes of not only monomeric, but also dimeric type. In this work, the Hoechst 33258 interaction with poly(dG-dC).poly(dG-dC) was studied using UV-vis and fluorescent spectroscopy, circular and flow-type linear dichroism. It was found that Hoechst 33258 formed with poly(dG-dC).poly(dG-dC) complexes of three types, namely, monomeric, dimeric, and, apparently, tetrameric, and their spectral properties were studied. Complexes of monomeric and dimeric types competed with distamycin A, a minor groove ligand, for binding to poly(dG-dC).poly(dG-dC). We proposed that Hoechst 33258 both monomers and dimers form complexes of the external type with poly(dG-dC).poly(dG-dC) from the side of the minor groove.     

Note from the Biological Stain Commission: Certification of Wright Stain Solution

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

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.     

Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs.

Buoyant density of DNA in CsCl gradients with Hoechst 33258 (bisbenzimide) was investigated as a function of guanine plus cytosine content of the DNA (%GC; in mole percent). A formula for calculating %GC from the refractive index (nD) of the isopycnic CsCl/Hoechst 33258 solution over the range of 0-75 %GC was established: %GC = 351762.28 X nD - 123778.66 X nD2 - 249789.47 (the coefficients must not be rounded off). The shape of this curve indicates that under these conditions, in contrast to dilute buffers, Hoechst 33258 binds to single AT base pairs on DNA. Resolution of DNA bands in CsCl/Hoechst 33258 gradients is 1.6 to 2.1 times better than comparative CsCl gradients without the dye. Potential application to %GC determination is discussed.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Influence of ionic strength on Hoechst 33258 binding with DNA

The binding of Hoechst 33258 with DNA at various ionic strengths of solution and different ligand concentrations has been investigated. Existence of more than one type of interactions of Hoechst 33258 with DNA has been revealed, which were very sensitive to the ionic strength. Hoechst 33258 doesn't show specificity to AT sequences of DNA at low ionic strength. High affinity binding mode becomes obvious at high ionic strength. The values of binding constants and binding site sizes for revealed strong and weak interactions have been determined.     

Vital DNA staining and cell sorting by flow microfluorometry

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.     

Factors involved in differential giemsa-staining of sister chromatids

Microspectrophotometric evaluation of differentially stained sister chromatids made it possible to analyse precisely the factors involved in the Giemsa methods. The concentration of Hoechst 33258, pH of the mounting medium, temperature during UV-exposure and the quality (wavelength) of UV-light influenced the differential staining. Exposure of blacklight of 10^–5 M Hoechst 33528-stained BrdU-labeled chromosome specimens mounted in McIlvaine buffer (pH 8.0) at 50° C reproducibly allowed differential staining of sister chromatids within 15 min. On the other hand, Korenberg-Freedlender's method using no Hoechst 33258 was also UV-light-dependent. Thus, photolysis of BrdU-substituted DNA was considered the basic mechanism of the Giemsa methods where the photosensitive Hoechst 33258 played a role as a sensitizer.     

Use of Electric Linear Dichroism and Competition Experiments with Intercalating Drugs to Investigate the Mode of Binding of Hoechst 33258, Berenil and DAPI to GC Sequences

Abstract

The drugs Hoechst 33258, berenil and DAPI bind preferentially to the minor groove of AT sequences in DNA Despite a strong selectivity for AT sites, they can interact with GC sequences by a mechanism which remains so far controversial. The 2-amino group of guanosine represents a steric hindrance to the entry of the drugs in the minor groove of GC sequences. Intercalation and major groove binding to GC sites of GC-rich DNA and polynucleotides have been proposed for these drugs. To investigate further the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences, we studied by electric linear dichroism the mutual interference in the DNA binding reaction between these compounds and a classical intercalator, proflavine, or a DNA-threading intercalating drug, the amsacrine-4-carboxamide derivative SN16713. The results of the competition experiments show that the two acridine intercalators markedly affect the binding of Hoechst 33258, berenil and DAPI to GC polynucleotides but not to DNA containing AT/GC mixed sequences such as calf thymus DNA Proflavine and SN16713 exert dissimilar effects on the binding of Hoechst 33258, berenil and DAPI to GC sites. The structural changes in DNA induced upon intercalation of the acridine drugs into GC sites are not identically perceived by the test compounds. The electric linear dichroism data support the hypothesis that Hoechst 33258, berenil and DAPI interact with GC sites via a non-classical intercalation process.     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Interactions of heteroaromatic compounds with nucleic acids. A - T-specific non-intercalating DNA ligands.

In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, "Hoechst 33258" and two acridines substituted with t-butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations. Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA. The main results from DNA binding studies indicate that the triphenylmethane dyes except p-fuchsin are bound with high preference to two adjacent A - T pairs while Hoechst 33258 seems to need three A - T pairs as the binding site. Model studies with synthetic polynucleotides revealed that not only a sequence of A - T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA. Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.     

Use of DNA fluorochromes for studying meiosis in the woody species Thryptomene calycina.

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.