New observations on the twisted arrangement of Dinoflagellate chromosomes

The Dinoflagellate Prorocentrum micans has been studied in classical and high voltage transmission electron microscopy, with the help of a goniometric stage. The general structure of the nucleus is analysed with special reference to the links observed between chromosomes and the nuclear envelope, the nucleoplasm and the nucleolus. The chromosomes present stacked series of nested arcs which are studied in detail. The sense of the arcs can be changed by a simple tilt of the section. These arcs do not correspond to DNA filaments with a genuine bend but to an illusion created by the overlap of layers of filaments whose orientation turns along the chromosome axis. — The transversal orientation of DNA and the examination of defects allow to rule out the polytenic hypothesis. It is clear that this hypothesis does not apply to bacterial nucleoids, which however can form series of nested arcs as in Dinoflagellate chromosomes. — The twisted arrangement of Dinoflagellate chromosomes is that of a liquid crystal of the cholesteric type. DNA is known to self assemble into cholesteric phases and this affords informations on the origin of the elongated shape of chromosomes and on the mechanisms of condensation and aggregation observed in this particular chromatin.     

Cholesteric organization of DNA in vivo and in vitro

In concentrated solutions DNA organizes spontaneously to form the "cholesteric" phase which is one type of liquid crystal. We have reproducibly obtained both continuous cholesteric phases and isolated cholesteric globules in equilibrium with the isotropic phase. A comparison is made between this in vitro cholesteric organization and dinoflagellate chromosomes which present the same organization in vivo. The observed defects are analyzed in the two cases. It appears that the cholesteric organization is due to self-assembly phenomena and that the shape of globules and chromosomes is due both to surface tensions and to the presence of defects.     

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.     

Chiral doping of nematic phases and its application to the determination of absolute configuration

Doping nematic liquid crystals with nonracemic chiral compounds induces a twisted nematic (cholesteric) phase. The ability of solutes to twist the nematic phase may be related to the overall shape of the chiral dopant and consequently to its absolute configuration. The cholesteric induction is therefore a powerful tool complementary to chiroptical techniques to obtain stereochemical information on chiral molecules.     

Chromosome separation and segregation in dinoflagellates and bacteria may depend on liquid crystalline states

The patterns characteristic of certain liquid crystals called 'twisted nematics' or 'cholesterics' have been observed in thin sections of both dinoflagellates and bacterial chromosomes. These liquid crystals have also been obtained in vitro in concentrated DNA solutions. A large part of DNA in prokaryotic chromosomes forms such a twisted liquid crystal, whilst the remainder consists of lateral loops and is less concentrated. These semi-ordered phases could help chromosome separation to occur during and after DNA replication. We suggest that, owing to chemical differences, one of the two replicated filaments is immiscible with the rest of DNA in this chromosome. This immiscibility occurs in the context of an ordered liquid, with the DNA closely layered by a regular twist, a situation proposed to strongly minimize entangling after replication and hence to facilitate segregation.     

SCARABAEID BEETLE EXOCUTICLE AS AN OPTICAL ANALOGUE OF CHOLESTERIC LIQUID CRYSTALS

1. A review is given of the optical and architectural analogies between cholesteric liquid crystals and certain insect cuticles (Coleoptera: Scarabaeidae). Earlier observations on the optical properties (reflexion of circularly polarized light and high form optical rotation) are confirmed and extended. Both cholesteric liquid crystals and lamellate cuticle have helicoidal structure (Fig. i). Even though their chemistry and physical states are very different, we are justified in making the analogy, since their optical properties depend primarily on the pitch of their helicoidal architecture. 2. The unusual optical properties were located for the first time in the outer 5 to 20 μ of the exocuticle. This layer is transparent and has regular spacings in the range required for interference colours according to Bragg's law. Among Scarabaeid beetles which show interference colours, we distinguish two types of outer exocuticle. (i) Optically active cuticles which reflect circularly polarized interference colours; show high angles of form optical rotation in transmitted light; and anomalous form birefringence perpendicular to the cuticle surface (reversible by deproteinization). (2) Optically inactive cuticles which show none of the above properties and in which the form birefringence is parallel to the cuticle surface. In the electron microscope the ultrastructure of these two types of outer exocuticle is clearly different. 3. All of the optically active species reflect left hand circularly polarized light, irrespective of the wavelength of the reflected colour. They therefore appear dark when viewed through a right hand circular analyser. The sense of reflected circularly polarized light does not reverse at higher wavelengths as recorded by previous workers. (A simple treatment is given for combinations of various wavelengths with retardation plates of varying values, as used in circular analysers.) We confirm earlier reports that the sense of reflected circularly polarized light is of the opposite sense to the transmitted light. 4. Using monochromatic light we have measured the anomalous dispersion with wavelength of the magnitude of optical rotation for various optically active cuticles. The dispersion curves change from negative values at lower wavelengths to positive values at higher wavelengths, and cross the zero optical rotation axis at a wavelength (AQ) corresponding to the interference colour of each sample. There is reasonable agreement between A₀ and the interference colour calculated from ultrastructural evidence and by comparison with interference filters of known wavelength. A dispersion curve measured for a combined sample of two cuticles with different dispersion curves showed that the resultant is an algebraic summation of the two component curves. 5. We present the first experimental verification of existing mathematical treatments of anomalous form optical rotatory dispersion curves. Although these treatments were derived for cholesteric liquid crystals, they give a reasonable fit to our measured curves for cuticle. We have confirmed from our cuticle dispersion curves that a second zero value for optical rotation occurs at a wavelength higher than A₀, as predicted by the theory of Chandrasekhar and Rao (1968). This has not yet been observed in any cholesteric liquid crystal system. 6. Our evidence shows that in optically active cuticle, interference colour is determined by helicoid pitch. In Lomaptera interference coloration follows the bilateral symmetry of the insect. Hence helicoidal pitch is controlled in a bilaterally symmetrical manner. However, the sense of helicoid rotation is the same all over the beetle and is therefore bilaterally asymmetrical. This supports the view that helicoid pitch is under the local control of the epidermal cells which secrete the cuticle, whereas its sense of rotation may be determined by an extracellular self-assembly process. In view of the self-assembling properties of cholesteric liquid crystals, it is tempting to suggest that helicoidal cuticle could be formed by the stabilization of a liquid crystal. 7. We discuss in detail the differences between optically active and inactive cuticles. The constructive interference colours arising from both types are then briefly compared with other multiple layer reflecting systems in other animals. 8. A detailed comparison is made between the optics of cuticle and cholesteric liquid crystals. The optical analogy provides a two-way contact between cuticle biophysicists and liquid crystal physical chemists.     

Ordering of double-stranded DNA molecules in a cholesteric liquid-crystalline phase and in dispersion particles of this phase

The current notion of the organization of molecules in a cholesteric phase is fairly well substantiated in the case of low-molecular-weight compounds. However, this question is open to discussion in the case of double-stranded nucleic acids. In this work, an attempt to compare the well-known data on the structure of cholesteric phases formed by double-stranded DNA molecules and the results of experimental modeling obtained by the authors has been undertaken. The comparison brings leads to assumption regarding the high probability of the existence of both short-range (positional) and long-range (orientational) order in the arrangement of double-stranded DNA molecules in the liquid crystalline phase. The presence of the orientational order, i.e., the rotation of quasinematic layers of double-stranded DNA molecules through a small angle, determines the formation of a spatially twisted (cholesteric) structure with specific physical and chemical properties. In addition, these results prompt a suggestion on the mode of the ordering of dsDNA molecules in liquid-crystalline dispersion particles and allow these particles to be considered candidate biosensing units.     

The helicoidal plant cell wall as a performing cellulose-based composite

The helicoidal plant cell wall can be considered as a composite in which cellulose is the constant reinforcing fiber. In order to strengthen the analogy with cholesteric liquid crystals, and taking into account a range of data, we describe a progressive series showing that cellulosic helicoidal systems are versatile and multifunctional. The following examples were considered: a) the cellulose microfibrils, with their rigid backbone possibly coated with a plastifying matrix; b) actual cholesteric cellulosic derivatives, such as in vitro liquid crystals and in vitro cellulosic mucilages; c) viscoplastic. growing cell walls; d) consolidated “stony” cell walls with their adaptation to intercellular communications. The series shows a dramatic progression from a liquid construction to what is the hardest in the plant cells, i.e. the sclerified walls.     

Magnetic ordering of DNA liquid crystals

Sonicated calf thymus DNA with an average length of approximately 100 base pairs has been found to form a cholesteric liquid crystal at a concentration of approximately 250 mg of DNA/mL of solution. Immediately after preparation, small ordered domains of a few micrometers are formed, resulting in an opaque solution. This liquid crystal can readily be oriented in the magnetic field of an NMR magnet, resulting in a clear birefringent phase. The DNA molecules align with their helix axes perpendicular to the field so that the cholesteric pitch axis was parallel with the field. A pitch length of approximately 2.5 microns for the cholesteric phase was determined both from optical measurements (optical light rotation) and from NMR measurements (solvent diffusion). The observation that DNA molecules can be magnetically oriented opens up new possibilities for studying the structure and dynamics of the aligned DNA molecules.     

The evolution of unusual chromosomal systems in coccoids: extraordinary sex ratios revisited

Coccoids (scale insects) exhibit a wide variety of chromosomal systems. In many species, paternal chromosomes are eliminated from the male germline such that all of a male's sperm transmit an identical set of maternal chromosomes. In such species, an offspring's sex is determined by whether or not paternal chromosomes are inactivated in the egg's cytoplasm after fertilization. This paper presents a model of the evolution of paternal genome loss in coccoids from an ancestral system of XX-XO sex determination. The model is based on Hamilton's (1967) theory that different genetic elements within the genome have different unbeatable sex ratios. In this model (1) meiotic drive by the X chromosome in XO males causes female-biased sex ratios; (2) the maternal set of autosomes in males evolves effective sex linkage to exploit X-drive; and (3) genes expressed in mothers are selected to convert some of their XX daughters into sons. A similar model may explain the evolution of haplodiploidy.     

Cholesteric organization of DNA in the stallion sperm head

The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chromatin structure of these spermatozoa is that of a cholesteric liquid crystal. This structure resembles that of a plywood, made of superimposed layers of parallel filaments, but instead of having a right angle between two successive layers, there is a progressive rotation and similar orientation occurs at each 180 degrees rotation. The apparent lamellae result from cleavages due to freeze-fracture between levels of parallel filament orientation. The thickness of lamellae corresponds therefore to the half helicoidal pitch of the cholesteric liquid crystal. This model is consistent with our observations by polarizing microscopy. The lamellation is not visible in thin sections of stallion spermatozoa. There are however biochemical methods to decondense chromatin and we are able to observe this lamellation in sections normal to the flattening plane of sperm heads. The methods used classically to decondense the sperm chromatin lead to extremely varied aspects which are discussed, some of them being closely related to the structure of cholesteric liquid crystals.     

The evolution of unusual chromosomal systems in sciarid flies: intragenomic conflict and the sex ratio

A model is presented for the evolution of the sciarid chromosomal system. In this model, a driving X chromosome caused female-biased sex ratios. The drive was exploited by maternal autosomes that segregated with the X at spermatogenesis. Genes in mothers converted some of their XX daughters into sons by eliminating a paternal X from the embryonic soma. L chromosomes were derived from X chromosomes and favored male-biased sex ratios. An X' chromosome arose that suppressed the effects of L chromosomes. The 1:1 sex ratio is a stalemate between the X' and X'X mothers causing all-female broods and the L chromosomes in XX mothers causing all-male broods. Any element (such as an L chromosome) that is preferentially transmitted through one sex will be selected to bias the sex ratio towards this sex.     

A new model for cellulose architecture in some plant cell walls

Summary A new model of rotating fibre components (helicoidal model) is proposed to explain the architecture of some plant cell walls. On the basis of tilting observations under the electron microscope, we establish the validity of this model for the cell wall ofChara vulgaris oospores. We suggest that this model explains the architecture seen in a number of published micrographs from a variety of different plant cell walls. Helicoidal architecture is shown to be distinct from the previously established crossed polylamellate architecture. The diagnostic features of helicoidal architecture are given. Morphogenesis of plant cell walls is discussed, with particular reference to self assembly in cholesteric liquid crystals.     

Double helical arrangement of spread dinoflagellate chromosomes

Dinoflagellate chromosomes observed in thin section show regular patterns which have been shown to correspond to a liquid crystalline helicoidal arrangement of DNA. Peripheral DNA filaments form a system of loops in the surrounding nucleoplasm. When such chromosomes (studied in Prorocentrum micans) are in presence of water, they extend considerably and form a double helical bundle. At the periphery of these bundles, one observes numerous filaments, which are smooth and devoid of nucleosomes; their diameter is constant.This study, in phase contrast and in electron microscopy, allows statistical measurements. A geometrical model is proposed and shows the simplest way to pass from the intact to the extended form. The liquid crystalline character of the chromosome is probably involved in the extension mechanisms.     

Enantioselective separations using chiral supported liquid crystalline membranes

Porous and nonporous supported liquid crystalline membranes were produced by impregnating porous cellulose nitrate supports with cholesteric liquid crystal (LC) materials consisting of 4-cyano-4'-pentylbiphenyl (5CB) mixed with a cholesterol-based dopant (cholesteryl oleyl carbonate [COC], cholesteryl nonanoate [CN], or cholesteryl chloride [CC]). The membranes exhibit selectivity for R-phenylglycine and R-1-phenylethanol because of increased interactions between the S enantiomers and the left-handed cholesteric phase. The selectivity of both phenylglycine and 1-phenylethanol in 5CB/CN membranes decreases with effective pore diameter while the permeabilities increase, as expected. Phenylglycine, which is insoluble in the LC phase, exhibits no transport in the nonporous (completely filled) membranes; however, 1-phenylethanol, which is soluble in the LC phase, exhibits transport but negligible enantioselectivity. The enantioselectivity for 1-phenylethanol was higher (1.20 in 5CB/COC and 5CB/CN membranes) and the permeability was lower in the cholesteric phase than in the isotropic phase. Enantioselectivity was also higher in the 5CB/COC cholesteric phase than in the nematic phase of undoped 5CB (1.03). Enantioselectivity in the cholesteric phase of 5CB doped with CC (1.1), a dopant lacking hydrogen bonding groups, was lower than in the 5CB/COC phases. Finally, enantioselectivity increases with the dopant concentration up to a plateau value at approximately 17 mol%.     

Formation of the regular, optical isotropic phase of low molecular weight DNA molecules modified by cis-dichlorodiammineplatinum

It is shown that condensation of DNA molecules of low molecular mass (less than 1 X 10(6) in NaClO4-containing solution of poly(ethylene glycol) brings about formation of cholesteric liquid crystal phase; pattern of this phase is presented. It has been found by means of X-ray analysis and polarization microscopy that at certain level of modification with cis-dichlorodiammineplatinum (II) the DNA molecules instead of cholesteric structure form an ordered optically isotropic phase. The problem about the causes of the formation of such phase and about the pattern of spatial organisation of adjacent DNA molecules in it remains open.     

Birefringence and DNA Condensation of Liquid Crystalline Chromosomes

DNA can self-assemble in vitro into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes—up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.DNA molecules are the indispensable genetic material of every organism. Apart from having the encoding power of a 4-base double-stranded polymer, DNA also has an enormous capacity to be condensed. It is probably this ability that allowed the evolution of increasing genome sizes in the eukaryotes. Histone-mediated nucleosome-based chromatin is the prevailing method for DNA packaging, but it is not the only way that DNA is condensed in the eukaryotes. Highly compact liquid crystalline DNA has been reported in several animal sperm nuclei (11, 28) and was also found in the nucleosomeless liquid crystalline chromosomes (LCCs) of dinoflagellates. Neither animal sperm nuclei nor dinoflagellate nuclei employ histones. In fact, the histone core octamer may hinder the attainment of ultrahigh levels of condensation due to its restrictive volume. In the sperm model, protamine is a sperm-specific DNA-binding protein (∼7 kDa) that is an order of magnitude smaller than the histone core octamer, and it adopts a structural role in the organization of the male gametic genome (1, 3). It is this very reduction in the protein-to-DNA ratio that may well enable the high DNA compaction into a liquid crystalline state. The dinoflagellate chromosomes are known to have a protein-to-DNA ratio even smaller than those of the prokaryotes (24). The typical eukaryotic genome has a protein/DNA ratio of 1:1, whereas the dinoflagellate genome has a ratio of 1:10 (23, 24).The dinoflagellates, counterintuitively, have the largest known genomes among all living organisms, with a DNA content per genome ranging from 1.5 pg to 200 pg per haploid cell (19, 25, 43). An extraordinarily high level of DNA condensation must be attained in order to sequester these genomes within the bounds of the nucleus, and this is achieved through the form of LCCs. The concentration of the DNA in the dinoflagellate nucleus was estimated to be ∼200 mg ml⁻¹ (up to 80 times more than a human cell) (24), falling well within the range observed for in vitro cholesteric liquid crystalline DNA formation (40). By observing ultrathin sections of dinoflagellate chromosomes using high-resolution transmission electron microscopy (TEM), plectonemic structures with nested series of arches and bands were seen (8, 12, 39). This architecture resembles the molecular architecture of thin-section TEM in vitro cholesteric liquid crystals (12, 39, 43).Knowledge of the condensation process would not only be of interest in terms of eukaryotic genome packaging, but would also provide new insights for the use of DNA as building blocks for nanotechnology applications. Self-assembly is increasingly understood to be of great significance in the regulation of various macromolecules in the crowded environment of living cells (7, 15, 16, 35, 36). Interestingly, it was also recently reported that nucleosomes and polynucleosomes themselves can be self-assembled (17), and in liquid crystalline form (33). The formation of LCCs probably also relies upon the associated chromosomal proteins, while self-assembly of DNA molecules via neutralization of the negatively charged polyelectrolytes by counterions and the crowding effects within the permanently closed nucleus must also contribute significantly (36, 49, 50). Most of the previous studies of dinoflagellate LCCs were focused on the chromosomes of Prorocentrum micans (27, 29). Though it has been reported that not all dinoflagellate chromosomes show the same degree of birefringence (5), the relationships of different species and their DNA contents, densities, or compaction ratios to birefringence have not been reported.The presence of two indices of refraction and optical anisotropy confer on the liquid crystalline DNA the property of birefringence, or double retardance. This optical phenomenon describes the phase differences between the two resultant light rays of a given light path that pass through the liquid crystal according to its refractive indices. For any given anisotropic material, the birefringence is a direct result of its nanoarchitecture. The property of birefringence and polarizing microscopy have long been employed to study the submicroscopic molecular organizations of different biological samples, such as mitotic spindles (5), filamentous actins (22), and microtubules (38), in living cells. Polarizing microscopy also allows the documentation of dynamic cellular behavior and measurements of cellular structures to be performed in a repeated noninvasive manner, even over extended periods of time (22). Taking advantage of this, we used a recently developed automatic rotating polarizing light microscope (the Metripol system) to study the relative amounts of birefringence and the ultrastructures of the chromosomes in several dinoflagellate species. Highly diverse chromosome structures were observed among the species, and surprisingly, not all dinoflagellates exhibited birefringent chromosomes, even though they are apparently constructed with similar liquid crystalline architectures, as seen in TEM ultrathin sections. The use of a semiautomatic Metripol polarizing microscope allowed the quantification of the retardance (20, 21, 47) of individual chromosomes of different dinoflagellate species in a fixative-free environment, and the relationships between the absolute retardance, DNA content, DNA condensation, and chromosome architecture in the dinoflagellate chromosomes are established in this study.     

Liquid crystalline ordering of procollagen as a determinant of three-dimensional extracellular matrix architecture

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly.