Добірка наукової літератури з теми "DNA toroids"

Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N=35cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6 ± 1.7%, mean ± SEM) when compared with the miscarriage (51.2 ± 6.6%) or nonpregnant groups (60.9 ± 4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R=0.32,P=0.04) and pregnancy outcome (pregnant cases 73.6 ± 1.7% versus nonpregnant 58.0 ± 3.9%,P=0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment.

It is well known that multivalent cations can cause DNA to condense from solution to form high-density nanometer scale particles. However, several fundamental questions concerning the phenomenon of DNA condensation remain unanswered. DNA condensation in vitro has been of interest for many years as a model of naturally occurring DNA packaging (e.g. chromatin, sperm head and virus capsid packing). More recently, DNA condensation has been of interest in optimizing artificial gene delivery, where packaging genes to an optimal size is essential to developing efficient uptake and delivery systems. The research presented in this dissertation provides an in depth biophysical study of the factors that control DNA condensate size and morphology. Millimolar changes in the ionic strength of the solution were found to alter the size of toroidal condensates. Variations in the order of addition of the counterions also significantly changed the size and morphology of the condensates. Studies were also performed to investigate the effects of static curvature and increased DNA flexibility on DNA condensation. These include the addition of static bending by sequence directed curvature, dynamic bending through protein-DNA interactions and reducing DNA persistence length by condensing single-stranded DNA. Several new models of DNA condensation are proposed based on the experimental data presented in this thesis.

Les tores d'ADN sont des structures cristallines liquides formées spontanément par la condensation de molécules d'ADN en solution par un agent de condensation tel que la spermine 4+. Ces tores servent de modèles pour comprendre le repliement des chromosomes dans certains virus à ADN double brin et pour leur potentiel en nano-ingénierie. La caractérisation détaillée de leur organisation tridimensionnelle reste limitée à un ordre hexagonal localisé. Cette thèse vise à élucider la structure fine et le mécanisme de formation des tores d'ADN, encore mal compris malgré de nombreuses études théoriques et simulations. Au Laboratoire de Physique des Solides (LPS), nous avons développé un protocole permettant de contrôler la courbure des tores sur une large gamme dimensionnelle, de quelques dizaines à plusieurs centaines de nanomètres. Ceci permet d'étudier la morphogenèse des tores par cryo-microscopie électronique à transmission (cryo-TEM), une technique désormais largement utilisée pour observer des structures biologiques dans leur état natif, après vitrification à basse température. Les images obtenues par cryo-TEM ont révélé un ordre hexagonal au sein des tores d'ADN, en accord avec les résultats connus. Nous avons identifié des corrélations entre les doubles hélices d'ADN, formant une "fermeture éclair" électrostatique. Notre étude révèle que l'optimisation des corrélations hélicoïdales est associée à des réarrangements ausein du tore au fur et à mesure de sa croissance, l'établissement de la corrélation étant suivi d'une mise en forme polygonale. En outre, une diminution locale du pas hélicoïdale de l'ADN est mesurée dans les régions à forte courbure. Nous démontrons une ségrégation ordre-désordre au sein des tores, avec des défauts structurels (extrémités de l'ADN et défauts de " pont ") concentrés dans un secteur spécifique du tore. Ce phénomène joue un rôle dans l'optimisation des interactions électrostatiques, y compris la fermeture éclair électrostatique. Enfin, nous avons initié la microscopie électronique en phase liquide, une technique émergente pour étudier la dynamique des processus biologiques à l'échelle nanométrique. Notre objectif est de suivre la formation des tores, depuis leur nucléation jusqu'à leur état final. Nous avons obtenu des images préliminaires sur des bactériophages, utilisés ici comme précurseurs du tore. Cette approche innovante ouvrirait de nouvelles perspectives pour comprendre la morphogenèse des tores d'ADN et pourrait potentiellement révéler des mécanismes fondamentaux qui sous-tendent leur formation et leur stabilité. Cette étude des tores d'ADN combine des approches expérimentales pour explorer leur structure, leur dynamique et leur mécanisme de formation. Ces résultats contribuent à notre compréhension fondamentale de la biophysique des états condensés de l'ADN
DNA toroids are liquid crystalline structures formed spontaneously by the condensation of DNA molecules in solution by a condensation agent such as spermine 4+. These toroids serve as models for understanding chromosome folding in certain double-stranded DNA viruses and for their potential in nano-engineering. Detailed characterisation of their three-dimensional organisation remains limited to a localised hexagonal order. This thesis aims to elucidate the fine structure and formation mechanism of DNA toroids, which are still poorly understood despite numerous theoretical studies and simulations.At the Solids Physics Laboratory (LPS), we have developed a protocol for controlling the curvature of toroids over a wide dimensional range, from a few tens to several hundreds of nanometres. This enables to study toroid morphogenesis by cryo Transmission Electron Microscopy (cryo-TEM), a now widely used technique for observing biological structures in their native state, after vitrification at low temperature.The images obtained by cryo-TEM revealed an hexagonal order within the DNA toroids, in agreement with previous results. We identified correlations between the DNA double helices, forming an electrostatic 'zipper'. Our study reveals that optimization of the helical correlations is associated with rearrangements within the toroid as it grows, with the establishment of correlation followed by polygonal shaping. In addition, a local decrease in the DNA helical repeat is measured in high curvature regions.We demonstrate an order-disorder segregation within toroids, with structural defects (DNA ends and “bridge” defects) concentrated in a specific sector of the toroid. This phenomenon plays a role in optimization of electrostatic interactions, including the electrostatic zipper.Lastly, we have initiated liquid phase electron microscopy, an emerging technique for studying the dynamics of biological processes at the nanoscale. We aim to follow toroid's formation, from their nucleation to their final state. We obtained preliminary images on bacteriophages, used here as a precursor of the toroid. This innovative approach would open up new perspectives for understanding the morphogenesis of DNA toroids and could potentially reveal fundamental mechanisms underlying their formation and stability.This study of DNA toroids combines experimental approaches to explore their structure, dynamics and formation mechanism. These results contribute to our fundamental understanding of the biophysics of condensed states of DNA

DNA condensates have attracted the attention of biophysicists, biochemists and polymer physicists for more than thirty years. In the biological community, the quest to understand DNA toroid formation has been motivated by its relevance to gene packing in certain viruses and by the potential use of DNA toroids in artificial gene delivery (e.g. gene therapy). In the physical sciences, DNA toroids are appreciated as a superb model system for studying particle formation by the collapse of a semiflexible, polyelectrolyte polymer. The thesis includes an analysis of the kinetic and thermodynamic factors governing DNA condensate morphology in solution, and discusses implications for future applications of DNA condensation in vitro as a model system for testing theories of polyelectrolyte collapse. In addition, DNA condensation by folded bovine protamine, a naturally occurring multivalent oligopeptide responsible for packing genomic DNA in bovine sperm cells, has been studied as well. The analysis of morphology, size, DNA strand packing density, and the stability of structural integrity of DNA condensates obtained with folded bovine protamines suggests that we have reconstituted native sperm cell chromatin. The results of this study were used to model the local structure of bovine sperm cell chromatin.

By using cryo-electron microscopy, we analyzed the morphology and structure of long double-stranded DNA chains condensed upon addition of varying amounts of the tetravalent polycation spermine (polyamine). Experiments have been performed i) with chains diluted in the bulk and ii) with individual chains confined in a virus capsid.Bulk experiments have been done with lambda DNA (48.5 kbp) at low concentration (0.03 mM Ph) and in low salt conditions (10 mM Tris HCl, 1 mM EDTA, pH 7.6). We explored a wide range of spermine concentration, from the onset of precipitation (0.05 mM sp) up to above the resolubilization limit (400 mM sp). Sixteen min after mixing spermine and DNA, samples have been trapped in thin films and vitrified in liquid ethane to keep ionic conditions unchanged, and imaged at low temperature with low doses of electrons (cryoTEM). DNA chains mostly form large aggregates of toroids in which DNA chains are hexagonally packed with interhelical spacings of 2.93, 2.88, and 2.95 nm at 0.05, 1 and 100 mM spermine, respectively, in agreement with previous X-ray data. At higher spermine concentration (200 mM), hexagonal toroids are replaced by cholesteric bundles with a larger interhelical spacing (3.32 nm). We conclude that the shape and the structure of the liquid crystalline sp-DNA condensates are linked to the DNA interhelix spacing and determined by the ionic conditions i.e. by the cohesive energy between DNA strands. Outside of the precipitation domain (400 mM spermine), DNA chains form a soluble network of thin fibers (4-6 nm in diameter) that let us reconsider the state of these DNA chains in excess of spermine. We also designed experiments to visualize condensates formed 6-60 sec after mixing Lambda DNA with 0.05 mM spermine, under identical buffer conditions. Among multiple original shapes (not found after 16 min), the presence of stretched and helical elongated fibers seen only 9sec after addition of spermine let us propose that DNA chains are immediately stretched upon addition of spermine, relax into helical structures and finally form small toroids (containing in some cases less than one Lambda chain) that further grow and aggregate. We also analyzed the dimensions and structural details of the complete collection of toroids, and reveal the existence of geometric constraints that remain to be clarified. Since it was only exceptionally possible to prevent the aggregation of DNA in dilute solution, we used another approach to observe the collapse of single DNA chains. We handled a population of T5 viruses containing a fraction of their initial genome (12-54 kbp long). The Na-DNA chain, initially confined in the small volume of the capsid (80nm in diameter) is collapsed by the addition of spermine. Compared to the first set of experiments, we explored a higher DNA concentration range (0.45 mM Phosphates in the whole sample) and the spermine concentration was varied from 0.05 to 0.5 mM (which corresponds to much lower +/- charge ratios). Experiments are thus performed close to the precipitation line, in the coexistence region, between the region where all chains are in a coil conformation, and the region where all chains are collapsed into toroids. We describe the existence of intermediate states between the coil and the toroidal globule that were not reported yet. In these "hairy toroids", part of the DNA chain is condensed in the toroid and the other part stays uncondensed outside of it. The interhelical spacing was also measured; it is larger in these partly-condensed toroids than in the fully organized toroids formed at higher spermine concentration.These two series of experiments show the interest of cryoEM to analyze the structural polymorphism and local structure of spermine-DNA aggregates. We also demonstrated how the confinement interferes with DNA condensation and the interest to investigate such effects that are important in the biological context.

A single DNA molecule is a long and flexible biopolymer that contains the genetic code. Building upon the discovery of the iconic double helix over 50 years ago, subsequent studies have emphasized how its biological function is related to the mechanical properties of the molecule. A remarkable system which high-lights the role of DNA bending and twisting is the packing and ejection of DNA into and from viral capsids. A recent 3D reconstruction of bacteriophage φ29 reveals a novel toroidal structure thought to be 30–40 bp of highly bent/twisted DNA contained in a small cavity below the capsid. Here, we extend an elastic rod model for DNA to enable simulation of the toroid as it is compacted and subsequently ejected from a small volume. We compute biologically-realistic forces required to form the toroid and predict ejection times of several nanoseconds.

Abstract Exploration is in our DNA! It is this spark of curiosity that has taken us to the moon and beyond. It is not easy to get into orbit. The rockets that we build today are quite sophisticated. Although technology will improve, these massive machines will increasingly be complicated to play with. One big reason being the ‘tyranny of rocket equation.’ As of now, we do not have any technology that will propel us out into space without using rockets. We are constantly finding ways to make rockets more efficient and launch more meaningful payloads into orbit. This is done by intelligently choosing the propellants, radical change in the design of rocket nozzles, applying different rocket engine cycles and improving the manufacturing process. Quite recently, we find a range of rockets being developed. The most commonly used engine cycle is the gas generator cycle (open cycle). Another way is to use electric powered turbo pumps. This cycle is far simpler than a gas generator cycle as it uses batteries to directly power the pumps. However, unlike propellant tanks with fuel, these energy powerhouses (batteries), do not reduce their weight during flight. Hence they represent dead weight. This technology is preferred for smaller rockets. There is another, not so often used engine cycle, called the full flow combustion stage or closed cycle. This cycle is the most complicated cycle and was considered almost impossible to build. Here, the exhaust from the turbine is fed into the combustion chamber, turning it into useful thrust. Apart from engine cycles, various engine nozzles have also been researched on. The conventional bell shaped nozzle, although widely used, is designed for a specific altitude. This means that the rocket needs to be multi-staged. The aerospike nozzle however, is an altitude compensatory nozzle. Although an aerospike has never flown to space, it has been rigorously tested. Here in, is a concept design of a prototype aerospike rocket engine. The intention of the design is to solve the engineering complexity involved in making efficient rocket engines. From the research carried out over a period of time, the following problems were noticed in an aerospike: • Near full combustion of propellant was not observed. • Overall heating of the spike increased. • Thrust Vector Control was difficult. The suggested design concept aims to tackle the above mentioned problems. The key technology used here is additive manufacturing. Additive manufacturing provides great flexibility in design and manufacturing. The complexity involved in manufacturing the aerospike can be tackled with this. The exhaust from the turbine can be used to create additional thrust by letting it out from the bottom of the toroidal spike. Near full combustion of the fuel-oxidizer mixture can be achieved by a dedicated combustion chamber rotated around the exhaust pipe of the turbine; unlike previous aerospikes which didn’t. The outer shape of the combustion chamber will be cylindrical, which will house traditional thrust vector control assembly. The heating of the nozzle can be reduced by using high grade graphite, tungsten and aluminum alloys with composite and ceramic materials. Also, for the rocket to be fuel efficient, the initial momentum to the turbines used will be given by permanent magnets mounted on the shaft, surrounded by windings and powered by supercapacitors. Once a desired rpm is achieved, a very small amount of fuel is used to maintain the same.