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.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Nuclear proteins : IV. Deficiency of non-histone proteins in condensed chromatin of Drosophila virilis and mouse

Drosophila virilis egg nuclei were fractionated by a technique of multiple sonication and centrifugation in an isotonic buffer containing 0.15 M NaCl, Mg²⁺ and Ca²⁺. This allowed the condensed chromatin to remain tightly condensed. By Hoechst 33258 staining this procedure resulted in brightly fluorescing and poorly fluorescing fractions. The brightly fluorescing fraction was enriched in satellite DNA. Examination of the non-histone proteins by SDS slab gel electrophoresis showed that this fraction was markedly deficient in non-histone proteins and contained no unique major non-histone proteins. The poorly fluorescing fractions were enriched in non-histone proteins. Similar results were obtained with mouse liver nuclei. Comparison of the non-histone proteins of D. virilis (40% satellite DNA), D. americana (35% satellite DNA), and D. ezoana (no satellites) confirmed the absence of major, satellite specific, non-histone proteins. These results, suggesting condensed chromatin is primarily a DNA-histone complex, agree with published cytochemical studies.     

Characterization of Drosophila heterochromatin

The C- and N-banding patterns of D. melanogaster, D. simulans, D. virilis, D. texana, D. ezoana and D. hydei were studied in comparison with quinacrine and Hoechst banding patterns. In all these Drosophila species the C bands correspond to the heterochromatin as revealed by the positive heteropycnosis in the prometaphase chromosomes. The N bands have the following characteristics: 1) they are always localized on the heterochromatin and generally do not correspond to the C bands; 2) they do not correspond to the nucleolar organizing regions; 3) they are inversely correlated with fluorescence, i.e., they correspond to regions which are scarcely, if at all, fluorescent after Hoechst 33258 or quinacrine staining; 4) they are localized both on regions containing AT rich satellite DNA and on those containing GC rich satellite DNA.     

Non-random position of the A-T rich DNA sequences in early embryos of Drosophila virilis

Examination of early embryos of Drosophila virilis by light and electron microscopy has shown that the A-T rich satellite DNA sequences have a non-random distribution within the nuclei. As observed by 33258 Hoechst staining and fluorescent microscopy, these sequences are consistently found to be located on the sides of the nuclei nearest to the vitelline membrane. This arrangement of the A-T rich sequences has been observed from the syncytial balstoderm stage into the gastrula stage where, in each nucleus, the satellite DNA sequences remain at a point nearest the topological outside of the organism.     

Quantitative variation of “Mus musculus-like” constitutive heterochromatin and satellite DNA-sequences in the genus Mus

The extent of conservation of constitutive heterochromatin in three species of Mus viz. M. musculus, M. booduga and M. dunni, with shared cytological properties and homologous DNA sequences has been studied. The cytological properties were investigated by doing fluorescence staining and condensation inhibition of their chromosomes with Hoechst 33258. Both the parameters indicate the occurrence of a reduced quantum of M. musculus like heterochromatin at specific sites in the other two genomes. In situ hybridization of the nick translated ³H-labelled M. musculus satellite DNA with M. booduga and M. dunni chromosomes, also corroborates our Hoechst 33258 findings and comparable variation in the amount and site of occurrence of sequences homologous to M. musculus satellite DNA in these species are noticed. The study thus provides a good example of a gradual quantitative variation of a particular type of heterochromatin and in turn of the repetitive DNA constituting it in different related species. Further since the heterochromatin in M. booduga and M. dunni is expected to contain different repetitive DNA sequences in addition to those homologous to M. musculus satellite DNA, it is proposed that a change in the balance between two or more repetitive sequences in heterochromatin may be more crucial in its evolutionary consequences rather than a mere increase or decrease of a homogeneous repetitive sequence.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

Molecular basis of chromosome banding

The effects of mouse satellite, main band and total DNA on the fluorescence intensity of quinacrine and of the bibenzimidazole derivative Hoechst 33258 were tested in solution. No significant differences were noticed between the double-stranded DNAs in spite of the 5% difference in AT-content between satellite and main band DNA. Single-stranded DNAs enhanced the fluorescence intensity of Hoechst 33258 far less than double-stranded DNAs. Having been denaturated and then reassociated the DNA fractions were intermediate in their enhancing effects on the fluorescence intensity of Hoechst 33258, the differences presumably being due to different degrees of reassociation. The effect of denatured and subsequently reassociated satellite DNA on the fluorescence intensity of quinacrine was similar to that of the native DNAs. Main band and total DNA quenched the fluorescence intensity of quinacrine more after denaturation-reassociation than it did when native. In the discussion the results are related to known cytological data.     

Effect of different fluorescent dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon Aeropyrum pernix

The interactions of DNA-binding dyes (Hoechst 33258, DAPI, acridine orange) and DiBAC₄(3) with hyperthermophilic archaeon Aeropyrum pernix cells were investigated by the combination of calorimetric, spectroscopic and microscopic techniques. All of the dyes, studied here, affect the thermal stability of DNA in vivo and in vitro. Hoechst 33258 is highly DNA-specific probe, which does not affect the thermal transitions of other cellular components as can be detected by differential scanning calorimetry (DSC). Due to this unique property, it can be used as a potential DNA marker for in vivo DSC studies. The localization of the dyes in the cells and viability assay was revealed by fluorescence microscopy. Hoechst 33258, DAPI and acridine orange did not distinguish between viable and non-viable cells of Aeropyrum pernix. Only with the commercially available Live/Dead BacLight^TM kit we were able to discriminate viable and non-viable Aeropyrum pernix cells.     

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.     

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).     

Generation of Five High-Complexity Painting Probe Libraries from Flow-Sorted Mouse Chromosomes

Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.     

Microfluorometric detection of asymmetry in the centromeric region of mouse chromosomes

A lateral asymmetry in the centromeric region of mouse chromosomes is revealed in studies involving the BUdR quenching of 33258 Hoechst fluorescence. This cytologically detected asymmetry may reflect the unequal distribution of thymidine between the two chains of mouse satellite DNA.     

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

Summary Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively. The work was supported by grant AI-15748 from the National Institutes of Health, Bethesda, MD     

DNA binding affinity of a macrocyclic copper(II) complex: Spectroscopic and molecular docking studies

The interaction of a novel macrocyclic copper(II) complex, ([CuL(ClO₄)₂] that L is 1,3,6,10,12,15-hexaazatricyclo[13.3.1.1^6,10]eicosane) with calf thymus DNA (ct-DNA) was investigated by various physicochemical techniques and molecular docking at simulated physiological conditions (pH = 7.4). The absorption spectra of the Cu(II) complex with ct-DNA showed a marked hyperchroism with 10 nm blue shift. The intrinsic binding constant (K_b) was determined as 1.25 × 10⁴ M^?1, which is more in keeping with the groove binding with DNA. Furthermore, competitive fluorimetric studies with Hoechst33258 have shown that Cu(II) complex exhibits the ability to displace the ct-DNA-bound Hoechst33258 indicating that it binds to ct-DNA in strong competition with Hoechst33258 for the groove binding. Also, no change in the relative viscosity of ct-DNA and fluorescence intensity of ct-DNA-MB complex in the present of Cu(II) complex is another evidence to groove binding. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the binding reaction. The experimental results were in agreement with the results obtained via molecular docking study.     

Fluorometric properties of the bibenzimidazole derivative hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA

A new fluorescent probe of chromosomal DNA structure in situ, the bibenzimidazole derivative Hoechst 33258, shows enhanced fluorescence with both AT- and GC-rich DNA; however, enhancement by AT-rich DNA is greater than enhancement with GC-rich DNA. When this compound is used as a probe, it produces localized fluorescence which can be correlated with AT concentration in specific chromosome regions. By the use of 33258, Hilwig and Gropp (1972) were able to demonstrate the relatively AT-rich DNA present in centric regions of mouse chromosomes; these regions do not fluoresce brightly when treated with quinacrine because of the presence of guanine residues which are spaced with high periodicity and which therefore efficiently quench quinacrine fluorescence. The data obtained in this study with DNA polymers of defined structure or composition, as test model compounds, suggest that 33258 is a useful cytochemical reagent for generally identifying all types of AT-rich regions in chromosomes, including those which are not demonstrable with quinacrine.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC-Banding)

Hoechst 33258 fluorescent staining of bromodeoxyuridine substituted chromosomes provided a high resolution technique for following the segregation of replicated chromosomal DNA (Latt, 1973). Modifications have produced the same results after Giemsa staining (Wolff and Perry, 1975). Since this does not necessarily require Hoechst (Korenberg and Freedlander, 1975), we call this bromodeoxyuridine-Giemsa banding (BG-banding). We here describe a further modification which allows one to follow the T-rich strand of the AT-rich satellite DNA of C-band heterochromatin. We call this TC-banding. This technique was used to examine metacentric marker chromosomes found in mouse L-cells that contain many interstitial blocks of centromeric-type heterochromatin in each arm plus the usual two blocks of centromeric heterochromatin. One of the advantages of this technique for such chromosomes is that it is possible to distinguish first from second cell cycle sister chromatid exchange and unambiguously detect centromeric sister chromatid exchange. We found some chromosomes to have high rates of centromeric sister chromatid exchange. After one cycle in bromodeoxyuridine we could examine the satellite polarity of the heterochromatic DNA. Since there was no change in satellite polarity in any of the heterochromatic blocks, marker chromosomes could not have been formed by paracentric inversions, inverted insertions or inverted translocations. These results allow the formulation of several rules of chromosome organization.     

New observations of the nuclear division in Entamoeba histolytica. The chromosomes

The morphologic organization of the nucleus and DNA during the nuclear division of Entamoeba histolytica was examined. The DNA of dividing amebic trophozoites was visualized with the fluorescent probe, Hoechst 33258 for light microscopy, and a DNA-specific antibody and phosphotungstic acid for electron microscopy. These techniques demonstrated features of the dividing amebic nuclei and the presence of spherical DNA-containing bodies corresponding to the condensed chromosomes. Based on light microscopy observations the number of chromosomes in E histolytica is five. Microtubules (MT) radiating from the microtubule organizing center (MTOC) were observed attached to the putative chromosomes.     

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.