Bacteriophage T5 DNA ejection under pressure.

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.

Phase diagram of nucleosome core particles.

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.

Bilayers of nucleosome core particles.

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.

Aggregation of nucleosomes by divalent cations.

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.

DNA in human and stallion spermatozoa forms local hexagonal packing with twist and many defects.

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.

Chiral discotic columnar germs of nucleosome core particles.

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.

Spermine-induced aggregation of DNA, nucleosome, and chromatin.

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.

Spermidine-induced aggregation of nucleosome core particles: evidence for multiple liquid crystalline phases.

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.

Liquid crystalline ordering of nucleosome core particles under macromolecular crowding conditions: evidence for a discotic columnar hexagonal phase.

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.

Comparison of slam-freezing and high-pressure freezing effects on the DNA cholesteric liquid crystalline structure.

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.

DNA-DNA interaction in thin layer analysed by cryo-electron microscopy.

Thin vitrified films of 146 base pair double-stranded DNA fragments in semi-dilute solutions are observed by cryo-electron microscopy. Depending on the salt conditions of the buffer and the DNA concentration, the molecules form different characteristic arrangements: they are arranged either locally parallel to each other in random overall orientation or packed perpendicular to the film surface, or else are randomly oriented. These arrangements reflect the weak forces acting between the DNA molecules themselves, as well as between the molecules and the thin film surface.

HOROPLAN: computer-assisted nurse scheduling using constraint-based programming.

Nurse scheduling is a difficult and time consuming task. The schedule has to determine the day to day shift assignments of each nurse for a specified period of time in a way that satisfies the given requirements as much as possible, taking into account the wishes of nurses as closely as possible. This paper presents a constraint-based, artificial intelligence approach by describing a prototype implementation developed with the Charme language and the first results of its use in the Rouen University Hospital. Horoplan implements a non-cyclical constraint-based scheduling, using some heuristics. Four levels of constraints were defined to give a maximum of flexibility: French level (e.g. number of worked hours in a year), hospital level (e.g. specific day-off), department level (e.g. specific shift) and care unit level (e.g. specific pattern for week-ends). Some constraints must always be verified and can not be overruled and some constraints can be overruled at a certain cost. Rescheduling is possible at any time specially in case of an unscheduled absence.

Supramolecular ordering of DNA in the cholesteric liquid crystalline phase: an ultrastructural study.

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.

Cholesteric liquid crystalline DNA; a comparative analysis of cryofixation methods.

The ultrastructure of liquid crystalline phases of DNA raises numerous problems because of the structure itself which is fluid and which nature depends on the relative amount of DNA, water and ions. Different cryofixation methods were tested and compared after freeze-fracture of the specimen. A good ultrastructural preservation of the samples can be achieved without addition of any cryoprotectant by quick-freezing against a copper block cooled down to liquid helium temperature. Then, molecular orientations can be followed very accurately and the local disorder around a mean direction which exists in the liquid state is kept in the frozen structure.