RESUMO
We quantified the Manning free (uncondensed) counterions fraction θ for dilute aqueous solutions of rodlike polyions: 150 bp DNA fragments, in the presence of a very low concentration of monovalent salt c(salt)<0.05 mM. Conductivity measurements of these solutions for DNA base pair concentration range 0.015≤c≤8 mM were complemented by fluorescence correlation spectroscopy (FCS) measurements of the DNA polyion diffusion coefficient D(p)(c). We observed a crossover in the normalized conductivity σ(c)/c that nearly halved across the c=0.05-1 mM range, while D(p)(c) remained rather constant, as we established by FCS. Analyzing these data we extracted θ(c)=0.30-0.45, and taking the Manning asymmetry field effect on polyelectrolyte conductivity into account we got θ(c)=0.40-0.60. We relate the θ(c) variation to gradual DNA denaturation occurring, in the very low salt environment, with the decrease in DNA concentration itself. The extremes of the experimental θ(c) range occur toward the highest, above 1 mM, and the lowest, below 0.05 mM, DNA concentrations and correspond to the theoretical θ values for dsDNA and ssDNA, respectively. Therefore, we confirmed Manning condensation and conductivity models to be valuable in description of dilute solutions of rodlike polyions.
Assuntos
DNA/química , DNA/ultraestrutura , Íons/química , Modelos Químicos , Modelos Moleculares , Sais/química , Água/química , Simulação por ComputadorRESUMO
The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.
Assuntos
Bacteriófagos/metabolismo , DNA Viral/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Capsídeo , Microscopia Crioeletrônica , DNA/química , Empacotamento do DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Genoma , Cinética , Modelos Biológicos , Osmose , Pressão , Ligação ProteicaRESUMO
The basic proteins, protamines and histones H1, are known to condense DNA in vivo. We examine here their ability to condense and solubilize in vitro linear DNA [and a synthetic polyanion, Poly(Styrene-Sulfonate) or PSS] at low ionic concentrations by varying the charge concentration ratio. Phase separation is observed in a very narrow range of ratios for short DNA and PSS; on both sides of this range, polydisperse and charged complexes are formed. A charge inversion is detected. For long DNA chains however, a different behavior is observed: the complexes are not soluble in excess of proteins.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Modelos Químicos , Modelos Moleculares , Sítios de Ligação , Simulação por Computador , Substâncias Macromoleculares/química , Ligação Proteica , Solubilidade , Eletricidade EstáticaRESUMO
The structure of polyamines-DNA precipitates was studied by x-ray diffraction. Precise measurements of the interhelix distance a(H) were obtained at different NaCl, polyamine, and DNA concentrations. Most of the results were obtained using spermine and few others using spermidine. The precipitates are liquid crystalline, either hexagonal and/or cholesteric, with an interhelical spacing that depends on the ionic concentrations and on the polyamine type. In our experimental conditions, the spacing varies from 28.15 to 33.4 angstroms. This variation is interpreted in terms of different ionic components that are present inside the precipitates and that are thought to regulate the value of the cohesive energy of DNA. These results are discussed in relation to the biological processes requiring a closeness of double helices and to the role played by polyamine analogs in cancer therapy.
Assuntos
DNA/química , Espermidina/análogos & derivados , Espermidina/química , Animais , Bovinos , Colesterol/química , Relação Dose-Resposta a Droga , Íons , Modelos Químicos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Nucleossomos/metabolismo , Fosfatos/química , Poliaminas/química , RNA/química , Sais/química , Cloreto de Sódio/química , Espermina/química , Timo/metabolismo , Fatores de Tempo , Difração de Raios XRESUMO
We present a phase diagram of the nucleosome core particle (NCP) as a function of the monovalent salt concentration and applied osmotic pressure. Above a critical pressure, NCPs stack on top of each other to form columns that further organize into multiple columnar phases. An isotropic (and in some cases a nematic) phase of columns is observed in the moderate pressure range. Under higher pressure conditions, a lamello-columnar phase and an inverse hexagonal phase form under low salt conditions, whereas a 2D hexagonal phase or a 3D orthorhombic phase is found at higher salt concentration. For intermediate salt concentrations, microphase separation occurs. The richness of the phase diagram originates from the heterogeneous distribution of charges at the surface of the NCP, which makes the particles extremely sensitive to small ionic variations of their environment, with consequences on their interactions and supramolecular organization. We discuss how the polymorphism of NCP supramolecular organization may be involved in chromatin changes in the cellular context.
Assuntos
Nucleossomos/metabolismo , Animais , Bovinos , Cristalografia por Raios X , Microscopia Eletrônica , Modelos Biológicos , Nucleossomos/ultraestrutura , Pressão OsmóticaRESUMO
Among the multiple effects involved in chromatin condensation and decondensation processes, interactions between nucleosome core particles are suspected to play a crucial role. We analyze them in the absence of linker DNA and added proteins, after the self-assembly of isolated nucleosome core particles under controlled ionic conditions. We describe an original lamellar mesophase forming tubules on the mesoscopic scale. High resolution imaging of cryosections of vitrified samples reveals how nucleosome core particles stack on top of one another into columns which themselves align to form bilayers that repel one another through a solvent layer. We deduce from this structural organization how the particles interact through attractive interactions between top and bottom faces and lateral polar interactions that originate in the heterogeneous charge distribution at the surface of the particle. These interactions, at work under conditions comparable with those found in the living cell, should be of importance in the mechanisms governing chromatin compaction in vivo.
Assuntos
DNA/metabolismo , Histonas/metabolismo , Íons/metabolismo , Nucleossomos/metabolismo , Animais , Bovinos , Galinhas , Cromatina/química , Cromatina/metabolismo , Microscopia Crioeletrônica , DNA/química , Histonas/química , Íons/química , Substâncias Macromoleculares , Nucleossomos/química , Nucleossomos/ultraestrutura , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.
Assuntos
Cátions Bivalentes/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Precipitação Química , Eletroforese , Magnésio/metabolismo , Conformação Molecular , Eletricidade Estática , TimoRESUMO
In human and other mammal sperm nuclei, DNA is packed in a highly condensed state, the structure of which remains unsolved. Cryoelectron microscopy of vitrified sections provides a first direct view of the local arrangement of the nucleoprotamine filament. DNA aligns in parallel in layers and its orientation rotates along a single-twist direction as in a cholesteric liquid crystal. The structure contains numerous defects, which introduce locally double-twist configurations. Destruction of the SS bonds with dithiotrehitol relaxes the twist and favors the extension of the hexagonal close packing of the filaments, though keeping constant their interfilament distance.
Assuntos
DNA/química , Cavalos/fisiologia , Espermatozoides/química , Animais , Cromatina/química , Microscopia Crioeletrônica , Cristalização , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/efeitos dos fármacos , Ditiotreitol/farmacologia , Humanos , Masculino , Modelos Moleculares , Conformação MolecularRESUMO
The contribution of counterions to macroscopic properties of isotropic DNA solutions has been studied using osmotic pressure measurements in low added salt condition. In the high DNA concentration range, the counterion contribution prevails and the associated osmotic coefficient is equal to 0.245+/-0.020. In the lower concentration range, the osmotic pressure may be exerted either by polymers or by ions, or due to a combination of both effects, depending on the added salt and DNA concentrations.
Assuntos
DNA/química , Pressão OsmóticaRESUMO
In concentrated solution and in the presence of high concentrations of monovalent cations, nucleosome core particles order into a discotic columnar mesophase. This phase is limited to finite-sized hexagonal germs that further divide into six coiled branches, following an iterative process. We show how the structure of the germs comes from the competition between hexagonal packing and chirality with a combination of dendritic facetting and double-twist configurations. Geometrical considerations lead us to suspect that the chirality of the eukaryotic chromosomes may originate from the same competition.
Assuntos
Nucleossomos/ultraestrutura , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Técnica de Fratura por Congelamento , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Microscopia de Polarização , Modelos Biológicos , Nucleossomos/química , EstereoisomerismoRESUMO
We have analyzed the conditions of aggregation or precipitation of DNA in four different states: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), mononucleosome core particles (NCP), and H1-depleted chromatin fragments (ChF) in the presence of the multivalent cation spermine (4+). In an intermediate regime of DNA concentration, these conditions are identical for the four states. This result demonstrates that the mechanism involved is general from flexible chains to rigid rods and quasi-colloidal states. It is dominated by local electrostatic attractions that are considered, for instance, by the "ion-bridging" model. The onset of precipitation does not require the electroneutrality of the DNA chains. Above a given spermine concentration dsDNA aggregates remain neutral, whereas NCP aggregates turn positively charged. The difference is thought to originate from the extension of the positively charged proteic tails of the NCP. This suggests that local fluctuations of polyamine concentrations can induce either positively or negatively charged chromatin domains.
Assuntos
Cromatina/efeitos dos fármacos , DNA/efeitos dos fármacos , Nucleossomos/efeitos dos fármacos , Espermina/farmacologia , Animais , Cromatina/fisiologia , Coloides , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Cinética , Nuclease do Micrococo , Nucleossomos/fisiologia , Ratos , Espalhamento de Radiação , Eletricidade Estática , Timo/fisiologia , Timo/ultraestruturaRESUMO
We investigate the effect of the addition of a trivalent cation, spermidine, to dilute solutions of nucleosome core particles (NCP). In the presence of spermidine, part of the NCP segregates from the initial homogeneous solution, forming dense aggregates. We follow this precipitation process over a wide range of spermidine and NaCl concentrations and determine the conditions of aggregation of the particles. The structure of the dense phases is analyzed by means of polarizing light microscopy and cryo-electron microscopy. We report the existence of multiple supramolecular organizations. According to the relative concentrations of spermidine, monovalent salt and NCP, the particles may aggregate into amorphous phases, stack into randomly oriented columns, or form liquid crystalline phases. Two discotic liquid crystalline phases are identified and analyzed: a columnar nematic corresponding to columns of NCP simply aligned in parallel, and a columnar hexagonal phase in which the columns order into a transversal 2D hexagonal array. We discuss the nature and origin of the interactions possibly involved in the formation and maintenance of these different types of order.
Assuntos
Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Espermidina/farmacologia , Animais , Bovinos , Precipitação Química , Microscopia Crioeletrônica , Cristalização , Relação Dose-Resposta a Droga , Microscopia de Polarização , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Concentração Osmolar , Cloreto de Sódio/farmacologia , Eletricidade EstáticaRESUMO
Conditions of double-stranded DNA precipitation by the polyamines spermidine and spermine have been determined experimentally and compared to theoretical predictions. The influence of the concentrations of DNA and added monovalent salt, and of the DNA length has been investigated in a systematic manner. Three regimes of DNA concentrations are observed. We clarify the dependence of these regimes on the monovalent salt concentration and on the DNA length. Our observations make possible a rationalization of the experimental results reported in the literature. A comparison of the precipitation conditions of different kinds of polyelectrolytes suggests a general process. Our experimental data are compared to the "ion-bridging" model based on short-range electrostatic attractions. By starting from the spinodal equation, predicted by this model, and using the limiting form of Manning's fractions of condensed counterions, analytical expressions of the precipitation conditions have been found in the three regimes. Experimental and theoretical results are in good agreement.
Assuntos
DNA/química , Espermidina , Espermina , Animais , Bovinos , Precipitação Química , Cromatina/química , Cinética , Modelos Químicos , Concentração Osmolar , Polidesoxirribonucleotídeos/química , Cloreto de SódioRESUMO
Macromolecular crowding conditions occurring inside the cell nucleus were reproduced experimentally with solutions of mononucleosome core particles to study their supramolecular organization. We report here that under these conditions, and over a large range of monovalent salt concentrations, mononucleosome core particles self-assemble to form a discotic liquid crystalline phase characterized in polarizing and freeze-fracture electron microscopy. Mononucleosomes are stacked on each other to form columns, which are themselves closely packed into an hexagonal array. The nucleosome concentration, estimated from the network parameters, falls in the range of values measured in cell nuclei. We suggest that these concentrated solutions, although their organization cannot be immediately compared to the organization of chromatin in vivo, may be used to investigate the nucleosome-nucleosome interactions. Furthermore, this approach may be complexified to take into account the complexity of the eucaryotic chromatin.
Assuntos
Nucleossomos/química , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Cromatina/química , Cromatina/ultraestrutura , Cristalização , Técnica de Fratura por Congelamento , Técnicas In Vitro , Substâncias Macromoleculares , Microscopia Eletrônica , Modelos Moleculares , Nucleossomos/ultraestruturaRESUMO
Using in parallel electron microscopy of ultrathin frozenhydrated sections and freeze-fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam-freezing at liquid helium temperature and high-pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam-freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high-pressure freezing does not preserve the structure of the liquid crystal: the long-range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high-pressure freezing process.
Assuntos
DNA/química , Congelamento , Cristalização , Técnica de Fratura por Congelamento , PressãoRESUMO
Conditions of formation of DNA aggregates by the addition of spermidine were determined with 146 base pair DNA fragments as a function of spermidine and NaCl concentration. Two different phases of spermidine-DNA complexes are obtained: a cholesteric liquid crystalline phase with a large helical pitch, with interhelix distances ranging from 31.6 to 32.6 A, and a columnar hexagonal phase with a restricted fluidity in which DNA molecules are more closely packed (29.85 +/- 0.05 A). In both phases, the DNA molecule retains its B form. These phases are always observed in equilibrium with the dilute isotropic solution, and their phase diagram is defined for a DNA concentration of 1 mg/ml. DNA liquid crystalline phases induced by spermidine are compared with the DNA mesophases already described in concentrated solutions in the absence of spermidine. We propose that the liquid crystalline character of the spermidine DNA complexes is involved in the stimulation of the functional properties of the DNA reported in numerous experimental articles, and we discuss how the nature of the phase could regulate the degree of activity of the molecule.
Assuntos
DNA/química , Espermidina/química , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Precipitação Química , Cromatina/química , Cromatina/isolamento & purificação , Cristalografia por Raios X , DNA/isolamento & purificação , Técnicas In Vitro , Cloreto de Magnésio , Microscopia de Polarização , Estrutura Molecular , Cloreto de Sódio , TermodinâmicaRESUMO
We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 microM/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions.
Assuntos
Cobalto/farmacologia , DNA/química , DNA/efeitos dos fármacos , Espermidina/farmacologia , Espermina/farmacologia , Animais , Composição de Bases , Bovinos , Cinética , Concentração Osmolar , Cloreto de Sódio/farmacologia , Solubilidade , Soluções , TimoRESUMO
Over a large range of salt and spermidine concentrations, short DNA fragments precipitated by spermidine (a polyamine) sediment in a pellet from a dilute isotropic supernatant. We report here that the DNA-condensed phase consists of a cholesteric liquid crystal in equilibrium with a more concentrated phase. These results are discussed according to Flory's theory for the ordering of rigid polymers. The liquid crystal described here corresponds to an ordering in the presence of attractive interactions, in contrast with classical liquid crystalline DNA. Polyamines are often used in vitro to study the functional properties of DNA. We suggest that the existence of a liquid crystalline state in spermidine-condensed DNA is relevant to these studies.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Espermidina , Cristalização , Concentração OsmolarRESUMO
Aqueous solutions of 146-base pair DNA fragments form a cholesteric liquid crystalline phase in the range of about 160-290 mg/ml. We present a structural analysis of this phase by comparing the data obtained from polarizing and electron microscopy. This phase shows multiple aspects or "textures" which are presented and interpreted. They mainly depend on the orientation of the structure relative to the observation plane and on the nature, distribution, and amount of defects present in the phase. These defects are then analyzed with the two methods, and the molecular orientations can be defined precisely in their core. The biological interest of such structural analyses is discussed in relation with the understanding of chromatin structure and function.
Assuntos
DNA/ultraestrutura , Fenômenos Biofísicos , Biofísica , Cromatina/química , Cromatina/ultraestrutura , Cristalização , DNA/química , Técnica de Fratura por Congelamento , Substâncias Macromoleculares , Microscopia Eletrônica , Microscopia de Polarização , Conformação de Ácido NucleicoRESUMO
Striking light-scattering behavior was observed from a marine dinoflagellate, Prorocentrum micans. Measurements of the angular dependence of the 16 Mueller matrix elements were performed on single cells with a polarization-modulation nephelometer by using a new method for cell immobilization. First the dinoflagellate cells were immobilized in a transparent silica gel containing alcohol, and then a second liquid was diffused into the gel to match the index of refraction of the gel network, thereby producing a transparent support medium that scatters less than one tenth the amount of light scattered by a single cell at 90 degrees . Measurements of scattering by a single cell revealed that all 16 matrix elements were significantly nonzero and different from each other. All matrix elements have an extremely rich, reproducible structure that is highly dependent on cell orientation. The matrix elements symmetrically across the diagonal were not equivalent. Striking features of the measurements are the large peak values of S(13), S(14), and other off-diagonal block elements. We believe that this is the first report of such scattering signals by single, suspended marine microorganisms.