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Diverse experimental evidences, including atomic force microscopy and cryo-electron tomography results, suggest that the chromatin filament of each metaphase chromosome is self-organized into stacked thin layers oriented perpendicular to the chromosome axis. In this multilayered structure, each individual layer has the width corresponding to a mononucleosome sheet. This avoids the entanglement of genomic DNA and allows the interaction of nucleosomes in adjacent layers. This structure justifies the mechanical properties of metaphase chromosomes and is compatible with the transverse orientation of the cytogenetic bands, with the existence of very thin bands, and with band splitting; it is also compatible with the orthogonal orientation and planar geometry of the connection surfaces observed in sister chromatid exchanges and in chromosome translocations. Further structural and cytogenetic studies indicate that chromatin layers are maintained during interphase. This may facilitate DNA replication and repair, and it has been suggested that gene expression occurs in compartments in which chromatin layers are unstacked.

This perspective aims to identify the relationships between the structural and dynamic properties of chromosomes and the fundamental properties of soft-matter systems. Chromatin is condensed into metaphase chromosomes during mitosis. The resulting structures are elongated cylinders having micrometer-scale dimensions. Our previous studies, using transmission electron microscopy, atomic force microscopy, and cryo-electron tomography, suggested that metaphase chromosomes have a multilayered structure, in which each individual layer has the width corresponding to a mononucleosome sheet. The self-assembly of multilayer chromatin plates from small chromatin fragments suggests that metaphase chromosomes are self-organized hydrogels (in which a single DNA molecule crosslinks the whole structure) with an internal liquid-crystal order produced by the stacking of chromatin layers along the chromosome axis. This organization of chromatin was unexpected, but the spontaneous assembly of large structures has been studied in different soft-matter systems and, according to these studies, the self-organization of chromosomes could be justified by the interplay between weak interactions of repetitive nucleosome building blocks and thermal fluctuations. The low energy of interaction between relatively large building blocks also justifies the easy deformation and structural fluctuations of soft-matter structures and the changes of phase caused by diverse external factors. Consistent with these properties of soft matter, different experimental results show that metaphase chromosomes are easily deformable. Furthermore, at the end of mitosis, condensed chromosomes undergo a phase transition into a more fluid structure, which can be correlated to the decrease in the Mg2+ concentration and to the dissociation of condensins from chromosomes. Presumably, the unstacking of layers and chromatin fluctuations driven by thermal energy facilitate gene expression during interphase.

Experimental evidence indicates that the chromatin filament is self-organized into a multilayer planar structure that is densely stacked in metaphase and unstacked in interphase. This chromatin organization is unexpected, but it is shown that diverse supramolecular assemblies, including dinoflagellate chromosomes, are multilayered. The mechanical strength of planar chromatin protects the genome integrity, even when double-strand breaks are produced. Here, it is hypothesized that the chromatin filament in the loops and topologically associating domains is folded within the thin layers of the multilaminar chromosomes. It is also proposed that multilayer chromatin has two states: inactive when layers are stacked and active when layers are unstacked. Importantly, the well-defined topology of planar chromatin may facilitate DNA replication without entanglements and DNA repair by homologous recombination.

Early results showed the emanation of chromatin fibers from mitotic chromosomes and nuclei swollen with water. In contrast, under metaphase ionic conditions, it was found that chromatin from mitotic chromosomes is planar and forms multilayered plates. Here, we show that in buffers containing interphase cation concentrations, the chromatin emanated from disrupted nuclei also has a planar morphology. Furthermore, the chromatin fragments produced by micrococcal nuclease digestion of nuclei form the typical beads-on-a-string fibers in the absence of cations, but they self-assemble into plate-like structures in buffers containing magnesium. The plates from interphase nuclei do not form the thick multilayered structures observed in metaphase chromosomes, suggesting that they are more exposed to the medium to facilitate DNA replication and gene expression.

Cryo‐electron tomography and small‐angle X‐ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen‐hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X‐ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis., This work was supported in part by MINECO research grant BFU2010-18939, European Union (EU) and Horizon 2020 through grant West-Life (EINFRA-2015-1, Proposal: 675858), and a by a UAB-PIF predoctoral fellowship to AC. Cryo-ET experiments at Max-Planck-Institute of Biochemistry (Martinsried) and image processing at National Center of Biotechnology (Madrid) were funded by Instruct (PID250, PID2115), part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptions.

In a previous study, we found that metaphase chromosomes are formed by thin plates, and here we have applied atomic force microscopy (AFM) and friction force measurements at the nanoscale (nanotribology) to analyze the properties of these planar structures in aqueous media at room temperature. Our results show that high concentrations of NaCl and EDTA and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+) have a relatively high friction coefficient (μ ≈ 0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ ≈ 0.1). This lubricant effect can be interpreted considering the electrostatic repulsion between DNA phosphate groups and the AFM tip. Protease digestion increases the friction coefficient (μ ≈ 0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ ≈ 0.9), indicating that DNA is the main structural element of plates. Whereas nuclease-digested plates are irreversibly damaged after the friction measurement, native plates can absorb kinetic energy from the AFM tip without suffering any damage. These results suggest that plates are formed by a flexible and mechanically resistant two-dimensional network which allows the safe storage of DNA during mitosis.

In previous studies with partially denatured metaphase chromosomes, we detected platelike structures instead of the chromatin fibers currently considered in different structural models for chromosomes. Here we have observed that dilution of compact metaphase chromosomes with hyposmotic solutions can transform whole chromatids into extended plates formed by many layers. Since this treatment is soft and it does not change the ionic conditions, these observations indicate that native chromosomes are formed by stacked plates. This strengthens our hypothesis about the multilayer structure of chromosomes, which was originally based on results obtained using stronger denaturing conditions. We have investigated the structure of plates emanated from chromosomes using electron tomography. Our three-dimensional reconstructions demonstrate conclusively that the surface of the plates is very smooth and do not show repetitive structures supporting any regular organization of nucleosomes; even the nucleosomes in plate edges show irregular orientations. Furthermore, we have used polarizing microscopy for the study of whole chromosomes in metaphase cells in aqueous solution. Our results show that condensed chromosomes are not birefringent under structuring ionic conditions similar to those used with plates. This observation is incompatible with the existence of parallel columns of nucleosomes within chromosomes. In summary, we have not detected any regular orientation of nucleosomes, but at the same time, our results indicate that the bulk of chromatin in native chromosomes is organized forming very well-defined plates, in which the nucleosomes of the successive layers are interdigitated. Presumably, this dense structure is required for safe transfer of DNA to daughter cells.

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (approximately 6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young's modulus is approximately 0.2 GPa and the stress required for surface penetration is approximately 0.03 GPa in the presence of Mg(2+) (5-20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.

The three-dimensional organization of tightly condensed chromatin within metaphase chromosomes has been one of the most challenging problems in structural biology since the discovery of the nucleosome. This study shows that chromosome images obtained from typical banded karyotypes and from different multicolour cytogenetic analyses can be used to gain information about the internal structure of chromosomes. Chromatin bands and the connection surfaces in sister chromatid exchanges and in cancer translocations are planar and orthogonal to the chromosome axis. Chromosome stretching produces band splitting and even the thinnest bands are orthogonal and well defined, indicating that short stretches of DNA can occupy completely the chromosome cross-section. These observations impose strong physical constraints on models that attempt to explain chromatin folding in chromosomes. The thin-plate model, which consists of many stacked layers of planar chromatin perpendicular to the chromosome axis, is compatible with the observed orientation of bands, with the existence of thin bands and with band splitting; it is also compatible with the orthogonal orientation and planar geometry of the connection surfaces in chromosome rearrangements. The results obtained provide a consistent interpretation of the chromosome structural properties that are used in clinical cytogenetics for the diagnosis of hereditary diseases and cancers.

We have performed a very extensive electron microscopy investigation of the chromatin structures extruded from partially denatured metaphase chromosomes from HeLa cells under a wide variety of conditions. Denatured chromosomes having fibres as the dominant structural element are obtained in the presence of buffers of very low concentration or after incubation with water. At slightly higher ionic concentrations, metaphase chromosomes become granulated. The most frequently observed granules have a diameter of about 35 nm and show the same structural characteristics as the compact cylindrical chromatin bodies previously found in our laboratory in studies performed using small chromatin fragments. Our results suggest that fibres are formed by the face-to-face association of 35-nm chromatin bodies. We have observed a very compact morphology of chromosomes in solutions containing intracellular concentrations of monovalent cations and the Mg2+ concentration found in metaphase. The most abundant structural elements observed in chromatin extruded from partially denatured compact metaphase chromosomes are multilayered plate-like structures. This is the first time that these planar structures have been reported. The observation of the irregular plates found in some preparations and of the small planar structures seen in aggregates of small chromatin fragments suggests that plates are formed by side-by-side association of compact chromatin bodies.

The lengths of the DNA molecules of eukaryotic genomes are much greater than the dimensions of the metaphase chromosomes in which they are contained during mitosis. From this observation it has been generally assumed that the linear packing ratio of DNA is an adequate measure of the degree of DNA compaction. This review summarizes the evidence suggesting that the local concentration of DNA is more appropriate than the linear packing ratio for the study of chromatin condensation. The DNA concentrations corresponding to most of the models proposed for the 30–40 nm chromatin fiber are not high enough for the construction of metaphase chromosomes. The interdigitated solenoid model has a higher density because of the stacking of nucleosomes in secondary helices and, after further folding into chromatids, it yields a final concentration of DNA that approaches the experimental value found for condensed chromosomes. Since recent results have shown that metaphase chromosomes contain high concentrations of the chromatin packing ions Mg2+and Ca2+, it is discussed that dynamic rather than rigid models are required to explain the condensation of the extended fibers observed in the absence of these cations. Finally, considering the different lines of evidence demonstrating the stacking of nucleosomes in different chromatin complexes, it is suggested that the face-to-face interactions between nucleosomes may be the driving force for the formation of higher order structures with a high local concentration of DNA.Key words: chromosomes, metaphase chromosomes, chromatin, chromatin higher order structure, DNA.

The fluorescent hydrophobic dye Nile red allows the rapid, sensitive, and general staining of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Nile red staining does not preclude further electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes. The resulting Western blot can be stained with the covalent fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) using a simple procedure. MDPF staining allows further N-terminal microsequencing and immunodetection of specific bands. This review considers the physicochemical, structural, and analytical studies that have led to the development of Nile red and MDPF staining methods. The usefulness of these procedures is discussed in comparison to other currently available fluorescent and nonfluorescent protein detection methods.

The excitation spectra of Nile red and SYPRO red, two currently used dyes for the fluorescent staining of protein bands in sodium dodecyl sulfate (SDS)-polyacrylamide gels, show an excitation peak in the UV region and another in the visible region (maximum at about 550 nm). Ethidium bromide and other intercalating dyes, e.g. propidium iodide, ethidium dimers, and benzoxazolium-4-quinolinium dimer-3 (YOYO), used for the fluorescent staining of DNA bands in agarose gels also show an excitation peak in the same region of the visible spectrum. We have designed and constructed a green-light transilluminator with an emission maximum at 542 nm. This visible transilluminator allows the detection of protein bands stained with Nile red and SYPRO red with the same sensitivity obtained with a 300 nm UV transilluminator. The green-light transilluminator also allows the detection of about 2 ng of DNA per band in gels stained with ethidium bromide and the other intercalating dyes indicated above. In contrast to the UV transilluminators, the green-light transilluminator does not produce photodamage of DNA even after long exposures (10 min). This makes this transilluminator very useful for preparative work. Furthermore, the green-light transilluminator does not require UV safety equipment and, consequently, it can be very convenient for teaching laboratories.

The local concentration of DNA in metaphase chromosomes of different organisms has been determined in several laboratories. The average of these measurements is 0.17 g/mL. In the first level of chromosome condensation, DNA is wrapped around histones forming nucleosomes. This organization limits the DNA concentration in nucleosomes to 0. 3-0.4 g/mL. Furthermore, in the structural models suggested in different laboratories for the 30-40 nm chromatin fiber, the estimated DNA concentration is significantly reduced; it ranges from 0.04 to 0.27 g/mL. The DNA concentration is further reduced when the fiber is folded into the successive higher order structures suggested in different models for metaphase chromosomes; the estimated minimum decrease of DNA concentration represents an additional 40%. These observations suggest that most of the models proposed for the 30-40 nm chromatin fiber are not dense enough for the construction of metaphase chromosomes. In contrast, it is well-known that the linear packing ratio increases dramatically in each level of DNA folding in chromosomes. Thus, the consideration of the linear packing ratio is not enough for the study of chromatin condensation; the constraint resulting from the actual DNA concentration in metaphase chromosomes must be considered for the construction of models for condensed chromatin.

— We have examined the ability of different fluorescent DNA dyes to become chemically excited by the peroxyoxalate chemiluminescent reaction. The intercalating dyes ethidium bromide and propidium iodide, and the bis-intercalating dyes ethidium homodimer-1, benzoxazolium-4-pyridinium dimer-1 and benzoxazolium-4-quinolinium dimer-1, exhibit an intense Chemiluminescence when they are excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction in the absence of DNA. However, the Chemiluminescence of these dyes is very low when they are bound to double-stranded DNA (dsDNA). In contrast, the minor groove-binding dye Hoechst 33258 excited by the TCPO-H2O2 reaction shows approximately the same Chemiluminescence intensity when it is free in solution or complexed with dsDNA. Structural alterations or partial dissociation of dsDNA-bis-intercalating dye complexes produced by the addition of acetone, NaCl, MgCl2 or the cationic surfactant cetyltrimethylammonium bromide increases the Chemiluminescence intensity. A moderate Chemiluminescence intensity is observed when bis-intercalating dyes are complexed with single-stranded DNA. Our results indicate that the energy from the intermediates produced in the peroxyoxalate chemiluminescent reaction cannot be efficiently transferred to fluorescent dyes complexed with DNA; chemiexcitation is almost completely inhibited when dyes are buried in the dsDNA structure by intercalation between the base pairs.

The fluorogenic dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge-coupled device camera. Electrophoretic bands containing 5-10 ng of protein can be detected on the MDPF-stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate-polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625-626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N-terminal microsequencing and immunodetection of specific bands with polyclonal antibodies.

We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg2+, chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg2+. In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg2+ and form cylindrical structures with a diameter of ˜33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.

Structural analysis by cryo-electron microscopy and small-angle X-ray scattering of ten sodium dodecyl sulfate/protein complexes in 25 mM Tris/HCl, 0.192 M glycine, pH 8.3, showed necklace-like structures of spherical micelles dispersed along the unfolded peptide chain. The micelles of most SDS/protein complexes had a constant diameter (≈ 6.2nm), slightly larger than pure SDS micelles (≈ 5.7 nm), all micelles possessing a degree of surface roughness. The micelle-associated polypeptide is mostly situated at the interface of the sulfate head groups and hydrocarbon core, intruding into the core rather than outward from the surface. Proteins with a molecular mass less than about 20 kDa formed complexes with a single SDS micelle. Multi-micellar SDS/protein complexes had centre-to-centre intermicellar distances in the range 7.0–12.0 nm. Our findings on the constancy of micellar size, number of micelles/complex, and the relationship between the degree of occupancy of micelles and a polypeptide's molecular mass, have enabled us to speculate on the correlation between the electrophoretic mobility of a polypeptide in SDS/PAGE and its molecular mass. The anomalous electrophoretic behaviour observed for the sodium dodecyl sulfate/histone H5 complex is accounted for by the large micelle of its complex.

We have used a linear DNA template (239 bp) containing a nucleosome positioning sequence (NX1) downstream of the T7 RNA polymerase promoter to study the mechanism of transcription elongation through a nucleosome. Under ionic strength approaching physiological conditions we have observed that transcription causes nucleosome dissociation and histone redistribution within the template. We have examined the role of the different elements that, in principle, could induce nucleosome dissociation during transcription. The high affinity of histones for single-stranded DNA observed in titration experiments performed using the purified (+) and (-) strands of the NX1 fragment suggests that nucleosome dissociation is not due to the formation of segments of single-stranded DNA by RNA polymerase in the elongation process. Furthermore, our results show that although RNA can interact with core histones, the synthesized RNA is not bound to the histones dissociated by transcription. Our results indicate that core histones released during transcription can be bound to naked DNA and chromatin (with or without histones H1-H5). From the dynamic properties of excess histones bound to chromatin, we suggest a nucleosome transcription mechanism in which displaced histones are transiently bound to chromatin and finally are reassembled with DNA after the passage of the polymerase.

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.

The folding of the chromatin filament and, in particular, the organization of genomic DNA within metaphase chromosomes has attracted the interest of many laboratories during the last five decades. This review discusses our current understanding of chromatin higher-order structure based on results obtained with transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and different atomic force microscopy (AFM) techniques. Chromatin isolated from different cell types in buffers without cations form extended filaments with nucleosomes visible as separated units. In presence of low concentrations of Mg(2+), chromatin filaments are folded into fibers having a diameter of ∼ 30 nm. Highly compact fibers were obtained with isolated chromatin fragments in solutions containing 1-2mM Mg(2+). The high density of these fibers suggested that the successive turns of the chromatin filament are interdigitated. Similar results were obtained with reconstituted nucleosome arrays under the same ionic conditions. This led to the proposal of compact interdigitated solenoid models having a helical pitch of 4-5 nm. These findings, together with the observation of columns of stacked nucleosomes in different liquid crystal phases formed by aggregation of nucleosome core particles at high concentration, and different experimental evidences obtained using other approaches, indicate that face-to-face interactions between nucleosomes are very important for the formation of dense chromatin structures. Chromatin fibers were observed in metaphase chromosome preparations in deionized water and in buffers containing EDTA, but chromosomes in presence of the Mg(2+) concentrations found in metaphase (5-22 mM) are very compact, without visible fibers. Moreover, a recent cryo-electron microscopy analysis of vitreous sections of mitotic cells indicated that chromatin has a disordered organization, which does not support the existence of 30-nm fibers in condensed chromosomes. TEM images of partially denatured chromosomes obtained using different procedures that maintain the ionic conditions of metaphase showed that bulk chromatin in chromosomes is organized forming multilayered plate-like structures. The structure and mechanical properties of these plates were studied using cryo-EM, electron tomography, AFM imaging in aqueous media, and AFM-based nanotribology and force spectroscopy. The results obtained indicated that the chromatin filament forms a flexible two-dimensional network, in which DNA is the main component responsible for the mechanical strength observed in friction force measurements. The discovery of this unexpected structure based on a planar geometry has opened completely new possibilities for the understanding of chromatin folding in metaphase chromosomes. It was proposed that chromatids are formed by many stacked thin chromatin plates oriented perpendicular to the chromatid axis. Different experimental evidences indicated that nucleosomes in the plates are irregularly oriented, and that the successive layers are interdigitated (the apparent layer thickness is 5-6 nm), allowing face-to-face interactions between nucleosomes of adjacent layers. The high density of this structure is in agreement with the high concentration of DNA observed in metaphase chromosomes of different species, and the irregular orientation of nucleosomes within the plates make these results compatible with those obtained with mitotic cell cryo-sections. The multilaminar chromatin structure proposed for chromosomes allows an easy explanation of chromosome banding and of the band splitting observed in stretched chromosomes.

This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described later, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.

In a previous work (J.-R. Daban, M. Samso, and S. Bartolome, Anal. Biochem. 199, 162–168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[α]phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 m Tris and 0.192 m glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680–685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein-SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethylrhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass.

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.

It is currently considered that metaphase chromosomes are formed by folded chromatin fibers [1]. Nevertheless, chromatin fibers are only observed when metaphase chromosomes are treated with solutions of very low ionic strength; in metaphase ionic conditions, chromosomes are very dense and chromatin is apparently folded forming plate-like structures [2]. Metaphase chromosomes from HeLa cells were purified on sucrose gradients containing 5 mM Mg2+, dialyzed extensively to remove sucrose and finally mechanically disrupted by rapid passage through a syringe needle. A TEM micrograph corresponding to typical chromatin plates from metaphase chromosomes prepared following this procedure is presented in Figure 1A. When the plates spread on carbon-coated grids were treated with high concentrations of NaCl, before crosslinking with glutaraldehyde, we observed denaturation of these planar structures leading to different levels of unfolding; in some micrographs we observed small granules having the diameter corresponding to single nucleosomes or aggregates that may contain few nucleosomes (see Figure 1B). AFM imaging was performed using an Asylum MFP-3D system working in AC mode. Uncrosslinked samples were adsorbed on freshly cleaved mica and observed in the presence of a solution containing 5 mM Mg2+. The best images were obtained under a low resonant frequency (∼12 kHz) and increasing the setpoint after engaging, in order to retract the piezo at maximum without losing the interaction between tip and plate. An example of an AFM image of chromatin plates is presented in Figure 2A. After the addition of approximately 1 volume of 4 M NaCl, the same structures can be seen in Figure 2B completely denatured.

Previous studies showed that during mitosis chromatin filaments are folded into multilayer plates (1). These structures can be self-assembled from chromatin fragments obtained by micrococcal nuclease digestion of metaphase chromosomes (2). Chromosomes of different animal and plant species show great differences in size (which are dependent on the amount of DNA that they contain), but in all cases chromosomes are elongated cylinders that have relatively similar shape proportions (the length to diameter ratio is approximately 13). It is possible to explain this morphology by considering that chromosomes are self-organizing supramolecular structures formed by stacked layers of planar chromatin having different nucleosome-nucleosome interaction energies in different regions (3). The nucleosomes in the periphery of the chromosome are less stabilized by the attractive interactions with other nucleosomes and this generates a surface potential that destabilizes the structure. Chromosomes are smooth cylinders because this morphology has a lower surface energy than structures having irregular surfaces. The symmetry breaking produced by the different values of the surface energies in the telomeres and in the lateral surface explains the elongated structure of the chromosomes. The results obtained by other authors in nanomechanical studies of chromatin and chromosome stretching have been used to test the proposed supramolecular structure. It is demonstrated quantitatively that internucleosome interactions between chromatin layers can justify the work required for elastic chromosome stretching. Chromosomes can be considered as hydrogels with a lamellar liquid crystal organization. The good mechanical properties of this structure may be useful for the maintenance of chromosome integrity during mitosis. Furthermore this chromatin organization avoids random entanglement of the extremely long genomic DNA molecules in chromosomes.(1) Daban (2011) Micron 42:733-750.(2) Milla and Daban (2012) Biophys J 103:567-575.(3) Daban (2014) J. R. Soc. Interface 11:20131043.

We have performed a very extensive investigation of chromatin folding in different buffers over a wide range of ionic conditions similar to those found in eukaryotic cells. Our results show that in the presence of physiological concentrations of monovalent cations and/or low concentrations of divalent cations, small chicken erythrocyte chromatin fragments and chromatin from HeLa cells observed by transmission electron microscopy (TEM) show a compact folding, forming circular bodies of approximately 35 nm in diameter that were found previously in our laboratory in studies performed under very limited conditions. Since TEM images are obtained with dehydrated samples, we have performed atomic force microscopy (AFM) experiments to analyze chromatin structure in the presence of solutions containing different cation concentrations. The highly compact circular structures (in which individual nucleosomes are not visible as separated units) produced by small chromatin fragments in interphase ionic conditions observed by AFM are equivalent to the structures observed by TEM with chromatin samples prepared under the same ionic conditions. We have also carried out experiments of sedimentation and trypsin digestion of chromatin fragments; the results obtained confirm our AFM observations. Our results suggest that the compaction of bulk interphase chromatin in solution at room temperature is considerably higher than that generally considered in current literature. The dense chromatin folding observed in this study is consistent with the requirement of compact chromatin structures as starting elements for the building of metaphase chromosomes, but poses a difficult physical problem for gene expression during interphase.

The high-energy intermediates generated in the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with H2O2 can excite electronically different fluorophores with a high quantum yield in organic solvents. We have previously applied this peroxyoxalate chemiluminescent reaction to the detection of proteins labeled with the fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. In this work, we have investigated the possibility to enhance the sensitivity of this detection method using specially designed cells in which the reagents TCPO and H2O2 in acetone are continuously renewed. In the flow cell, two syringes are used to renew the reagents in the reaction chamber containing the PVDF membrane with blotted proteins labeled with MDPF. In the evaporation cell, a fresh solution of reagents continuously replaces the volume of acetone evaporated in the reaction chamber. Both cells show a low emission background but the observed elution of proteins from the membrane produced by the flow of reagents in acetone limits the maximum sensitivity attainable with these cells. The best result (detection of 1 ng of MDPF-labeled protein) has been obtained with the evaporation cell.

We have previously shown that the bis (2,4,6-trichlorophenyl)oxalate (TCPO)–H 2 O 2 chemiluminescent reaction in acetone can be used for the detection of proteins labeled with the fluorescent reagent 2-methoxy-2,4-diphenyl-3(2 H )-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. To improve this method, in this work we have designed and constructed a cell that allows us to perform this chemiluminescent reaction on PVDF membranes with a homogeneous distribution of the reagents. Using this cell we have examined the analytical properties of several recently developed fluorescent protein dyes chemically different from MDPF. We have found that the metal chelate dye SYPRO Ruby can also be excited by the high-energy intermediate produced in the TCPO–H 2 O 2 reaction.

The measurement of the dimensions of metaphase chromosomes in different animal and plant karyotypes prepared in different laboratories indicates that chromatids have a great variety of sizes which are dependent on the amount of DNA that they contain. However, all chromatids are elongated cylinders that have relatively similar shape proportions (length to diameter ratio approx. 13). To explain this geometry, it is considered that chromosomes are self-organizing structures formed by stacked layers of planar chromatin and that the energy of nucleosome–nucleosome interactions between chromatin layers inside the chromatid is approximately 3.6 × 10−20J per nucleosome, which is the value reported by other authors for internucleosome interactions in chromatin fibres. Nucleosomes in the periphery of the chromatid are in contact with the medium; they cannot fully interact with bulk chromatin within layers and this generates a surface potential that destabilizes the structure. Chromatids are smooth cylinders because this morphology has a lower surface energy than structures having irregular surfaces. The elongated shape of chromatids can be explained if the destabilizing surface potential is higher in the telomeres (approx. 0.16 mJ m−2) than in the lateral surface (approx. 0.012 mJ m−2). The results obtained by other authors in experimental studies of chromosome mechanics have been used to test the proposed supramolecular structure. It is demonstrated quantitatively that internucleosome interactions between chromatin layers can justify the work required for elastic chromosome stretching (approx. 0.1 pJ for large chromosomes). The high amount of work (up to approx. 10 pJ) required for large chromosome extensions is probably absorbed by chromatin layers through a mechanism involving nucleosome unwrapping.

We have found previously that different fluorescent dyes cannot be efficiently excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H2O2 reaction when they are intercalated between the DNA bases or bound to the minor groove of the double helix. Here we show that the fluorescent dye Texas red, covalently bound to the 3′ ends of double-stranded DNA molecules, exhibits a high emission intensity when excited by the TCPO–H2O2 reaction. In this case, the charge transfer between the intermediate produced in the peroxyoxalate chemiluminescent reaction and Texas red can take place because this fluorophore is not buried inside the DNA structure. We describe the application of this chemiluminescent reaction to the detection of blotted DNA on nylon membranes. Copyright © 2001 John Wiley & Sons, Ltd.

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.

The effect of histones on the reaction of reassociation of the two complementary strands of DNA from different sources has been investigated. The reassociation rate of denatured linear DNA from bacteriophage M13 monitored spectrophotometrically and using nuclease S1 is roughly the same in the presence and absence of core histones at physiological ionic strength. Electron microscopy reveals that in the samples containing histones a large network of duplex DNA is produced. Nevertheless, closed circular M13 DNA and a cloned DNA fragment (158 bp) from nucleosomal origin are entirely renatured in the presence of histones as demonstrated by the well-defined double-stranded DNA bands seen in electrophoretic gels. Various experiments performed using the purified (+) and (-) strands of the cloned nucleosome DNA fragment at low ionic strength indicate that core histones initially bound to one or even to the two strands allow the formation of duplex DNA. These findings and the results obtained with partially denatured closed circular M13 DNA allow us to conclude that core histones neither prevent the nucleation nor inhibit the rapid zippering reactions leading to the formation of double-stranded DNA. The mechanism that allows the renaturation of DNA in the presence of histones may also participate in biological processes involving the pairing of complementary nucleotides.

It has been previously found using different physicochemical techniques [Aragay, A., Diaz, P., & Daban, J.-R. (1988) J. Mol. Biol. 204, 141-154] that histones H2A,H2B in the absence of H3,H4 can associate with nucleosome core DNA (146 base pairs). Here we describe a synchrotron X-ray scattering study of core DNA-(H2A,H2B) complexes in solution. Our results obtained using different histone to DNA weight ratios and ionic conditions ranging from very low ionic strength to 0.2 M NaCl show that histones H2A,H2B are unable to fold core DNA. Model calculations indicate that histones H2A,H2B produce very elongated structures even when the reconstituted complexes are prepared at physiological ionic strength. In contrast, our scattering data indicate that the reconstituted complexes prepared at physiological salt concentration either with the four core histones or with histones H3,H4 without H2A,H2B are completely folded particles with a radius of gyration similar to that corresponding to the native nucleosome core (4.2 nm). Furthermore, our results show that the DNA of the extended complexes containing histones H2A,H2B becomes completely folded after the histone pair exchange reaction that occurs spontaneously between preformed DNA-(H2A,H2B) and DNA-(H3,H4) complexes. These observations, together with our previous studies, suggest that the open conformation of DNA-(H2A,H2B) complexes facilitates the involvement of this structure as a transient intermediate in the reaction of nucleosome formation at physiological ionic strength.

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 μg of histone per band.

The nuclear cycle kinetics of Physarum polycephalum plasmodia were examined using flow cytofluorometry. The dyes Hoechst 33342 and propidium iodide were used to stain the DNA of isolated nuclei. In asynchronously growing microplasmodia. S phase consists of 13--15% of the nuclear division cycle time. Nuclei isolated from individual macroplasmodia, which have previously been demonstrated to divide in synchrony, were shown to be less synchronized during late S phase than during mitosis. The results obtained demonstrate the feasibility of flow cytometric measurement of the properties of nuclei isolated from a single cell.

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 m ). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 m -NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.

Micrococcal nuclease, DNase I, and trypsin have been employed to study the kinetics of core particle self-assembly by salt jump from 2.0 to 0.2 M NaCl. A few seconds after the initiation of the reassociation reaction, the bulk of core particle DNA becomes protected from digestion by micrococcal nuclease, whereas free DNA, under the same conditions, is completely hydrolyzed. The central and C-terminal regions of core histones are also protected from trypsin digestion immediately after the 2.0-0.2 M NaCl salt jump. Moreover, the extent of degradation produced by trypsin is the same for samples digested a few seconds after the salt jump and for samples digested 20 min after the salt jump. With DNase I, minor structural differences have been detected between samples obtained at different times during the reaction. However, even in this case our results indicate that many of the characteristic histone-DNA contacts within the core particle are made a few seconds after the initiation of the self-assembly reaction. Furthermore, core particles have been labeled with the fluorescent reagent N-(1-pyrenyl)maleimide (NPM), which was previously used as a sensitive probe for nucleosome conformation. Extensive DNase I or trypsin digestion of NPM-labeled core particles in 0.2 M NaCl does not produce significant changes in excimer fluorescence. This allows us to conclude that the covalent continuity of DNA is not required for the maintenance of the folded conformation of the core particle and that the trypsin-resistant domains of core histones play a fundamental role in the stabilization of this structure.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3·H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and H4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3·H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1–0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.