On the Toroidal Condensed State of Closed Circular DNA

Abstract

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

On the possible permeation of water across the glucose transporter

The possibility that the glucose transporter may serve as water channel is explored with the help of theoretical and experimental arguments. A model for a pore is drawn based on a hypothetical water channel structure, subject to the constraints that: molecules will bind to the channel wall in successive rings, forming a hollow sleeve; an integer number of molecules will exist in each ring; the pore radius will not be large enough to allow water molecules along its center, but will be large enough to allow glucose molecules across. The only configurations that meet these conditions exhibit either 5 or 6 water molecules abreast in each ring, with pore radii of 4.1 and 4.5 Å, respectively. The kinetic characteristics of such pores are estimated and found to conform to available evidence.     

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin’s embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.     

Orientation of DNA Molecules in Agarose Gels By Pulsed Electric Fields

Abstract

The electric birefringence of DNA restriction fragments of three different sizes, 622,1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N^2.8, in reasonable agreement with reptation theories.     

Topology and replication of a nuclear episomal plasmid in the rodent malaria Plasmodium berghei

The rodent malaria Plasmodium berghei is one of a small number of species of Plasmodium that can currently be genetically transformed through experimentally controlled uptake of exogenous DNA by bloodstage parasites. Circular DNA containing a selectable marker replicates and is maintained under selection pressure in a randomly segregating episomal form during the first weeks after transformation. In this study, using pulsed field gel electrophoresis and ionising radiation, we show that in dividing asexual blood stage parasites the episomes are completely converted, within 2 weeks post-infection, into non-rearranged circular concatamers ranging in size between about 9 and 15 copies of the monomer. These occur as slow-moving aggregates held together by radiation-sensitive linkers consisting partly of single-stranded DNA. The process generating these complexes is not clear but 2D gel analysis showed that Cairns-type replication origins were absent and it seems most likely that the initial concatamerisation takes place using a rolling circle mechanism followed by circularisation through internal recombination. We propose a model in which continued rolling circle replication of the large circular concatamers and the recombinational activity of the tails of the rolling circles could lead to the formation of the large aggregates.     

From bicycle chain ring shape to gear ratio: Algorithm and examples

A simple model of the bicycle drive system with a non-circular front chain ring is proposed and an algorithm is devised for calculation of the corresponding Gear Ratio As a Function Of Crank Angle (GRAFOCA). It is shown that the true effective radius of the chain ring is always the perpendicular distance between the crank axis and the line through the chain segment between the chain ring and the cog. It is illustrated that the true effective radius of the chain ring at any crank angle may differ substantially from the maximum vertical distance between the crank axis and the chain ring circumference that is used as a proxy for the effective chain ring radius in several studies; in particular, the crank angle at which the effective chain ring radius is maximal as predicted from the latter approach may deviate by as much as 0.30 rad from the true value. The algorithm proposed may help in designing chain rings that achieve the desired GRAFOCA.     

A Temperature-Monitoring Vaginal Ring for Measuring Adherence

BackgroundProduct adherence is a pivotal issue in the development of effective vaginal microbicides to reduce sexual transmission of HIV. To date, the six Phase III studies of vaginal gel products have relied primarily on self-reporting of adherence. Accurate and reliable methods for monitoring user adherence to microbicide-releasing vaginal rings have yet to be established.MethodsA silicone elastomer vaginal ring prototype containing an embedded, miniature temperature logger has been developed and tested in vitro and in cynomolgus macaques for its potential to continuously monitor environmental temperature and accurately determine episodes of ring insertion and removal.ResultsIn vitro studies demonstrated that DST nano-T temperature loggers encapsulated in medical grade silicone elastomer were able to accurately and continuously measure environmental temperature. The devices responded quickly to temperature changes despite being embedded in different thickness of silicone elastomer. Prototype vaginal rings measured higher temperatures compared with a subcutaneously implanted device, showed high sensitivity to diurnal fluctuations in vaginal temperature, and accurately detected periods of ring removal when tested in macaques.ConclusionsVaginal rings containing embedded temperature loggers may be useful in the assessment of product adherence in late-stage clinical trials.     

MULTIPLE FtsZ RING FORMATION AND REDUPLICATED CHLOROPLAST DNA IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) UNDER PHOSPHATE‐ENRICHED CULTURE1

We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate‐limited (no β‐glycerophosphate) and phosphate‐enriched (2 mM β‐glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote‐derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate‐enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate‐enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate‐enriched than in phosphate‐limited culture and decreased in cells cultured in phosphate‐enriched medium containing 5‐fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate‐enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.     

Comments on the Significance of Stone Circle Excavations in Alberta

Abstract

A number of recently excavated stone circle sites “tipi rings” in Alberta are used to document the continuing necessity for detailed investigations into this site type. The excavations revealed quantities of cultural material, significant information and considerable time depth which are contrary to many previous assumptions.     

Inference is bliss: Simulation for power estimation for an observational study of a cholera outbreak intervention

BackgroundThe evaluation of ring vaccination and other outbreak-containment interventions during severe and rapidly-evolving epidemics presents a challenge for the choice of a feasible study design, and subsequently, for the estimation of statistical power. To support a future evaluation of a case-area targeted intervention against cholera, we have proposed a prospective observational study design to estimate the association between the strength of implementation of this intervention across several small outbreaks (occurring within geographically delineated clusters around primary and secondary cases named ‘rings’) and its effectiveness (defined as a reduction in cholera incidence). We describe here a strategy combining mathematical modelling and simulation to estimate power for a prospective observational study.Methodology and principal findingsThe strategy combines stochastic modelling of transmission and the direct and indirect effects of the intervention in a set of rings, with a simulation of the study analysis on the model results. We found that targeting 80 to 100 rings was required to achieve power ≥80%, using a basic reproduction number of 2.0 and a dispersion coefficient of 1.0–1.5.ConclusionsThis power estimation strategy is feasible to implement for observational study designs which aim to evaluate outbreak containment for other pathogens in geographically or socially defined rings.     

Modeling electroporation in a single cell

Electroporation uses electric pulses to promote delivery of DNA and drugs into cells. This study presents a model of electroporation in a spherical cell exposed to an electric field. The model determines transmembrane potential, number of pores, and distribution of pore radii as functions of time and position on the cell surface. For a 1-ms, 40 kV/m pulse, electroporation consists of three stages: charging of the cell membrane (0-0.51 micros), creation of pores (0.51-1.43 micros), and evolution of pore radii (1.43 micros to 1 ms). This pulse creates approximately 341,000 pores, of which 97.8% are small ( approximately 1 nm radius) and 2.2% are large. The average radius of large pores is 22.8 +/- 18.7 nm, although some pores grow to 419 nm. The highest pore density occurs on the depolarized and hyperpolarized poles but the largest pores are on the border of the electroporated regions of the cell. Despite their much smaller number, large pores comprise 95.3% of the total pore area and contribute 66% to the increased cell conductance. For stronger pulses, pore area and cell conductance increase, but these increases are due to the creation of small pores; the number and size of large pores do not increase.     

SPHEROIDAL AND RING NUCLEOLI IN AMPHIBIAN OOCYTES : Patterns of Uridine Incorporation and Fine Structural Features

In maturing oocytes of the newt Triturus viridescens, the nucleoli undergo a series of morphological changes that are very similar to those described by Callan for the axolotl, Ambystoma mexicanum. The nucleoli first assume the form of spheroids which then become extended into ring or necklace shapes that are DNase-sensitive; in mature oocytes the nucleoli revert to a spheroidal form. Short term in vitro incorporation studies with uridine-³H on both species show that RNA synthesis occurs in a restricted, eccentric portion of the spheroidal nucleoli, thereby producing an asymmetrical pattern of labeling. In the ring forms, however, the localization of the radioactivity suggests that synthesis takes place symmetrically throughout their entire length. The changes in nucleolar morphology apparently reflect the fact that the component DNA has undergone a redistribution from a localized region in the spheroidal nucleoli to an extended circle in the rings; the patterns of uridine-³H incorporation, therefore, parallel the distribution of DNA in both the spheroidal and the ring nucleoli. Ultrastructurally, the nucleoli contain a fibrillar component that corresponds in position to that of the DNA. The typical spheroidal nucleolus consists of a fibrillar core situated eccentrically and surrounded by a hull of granular, ribonucleoprotein material. The ring nucleoli are composed of a central fibrous region that is ensheathed all around its circumference by a layer of similar granular material. This granular substance is thicker at intervals along the length of the rings, representing the "beads" of the necklaces.     

Chromatin reconstitution on small DNA rings. II. DNA supercoiling on the nucleosome

DNA supercoiling on the nucleosome was investigated by relaxing with topoisomerase I mono- and dinucleosomes reconstituted on small DNA rings. Besides 359 base-pair (bp) rings whose linking differences were integers, two additional series of rings with fractional differences, 341 and 354 bp in size, were used. Mononucleosomes reconstituted on 359 bp rings were found to relax into a single mononucleosome form. In contrast, 341 and 354 bp mononucleosomes relaxed into a mixture of two forms, corresponding to two adjacent topoisomers. The observation that the ratio between these two forms was, within each ring series, virtually independent of the initial linking number of the topoisomer used for the reconstitution suggested that each partition reflected an equilibrium. Comparison with the equilibria observed for the same rings in the absence of histones showed that the formation of a single nucleosome is associated with a linking number change of -1.1(+/-0.1) turn. Dinucleosomes, in contrast, were not relaxed to completion and do not reach equilibria. The corresponding linking number change per nucleosome was, however, estimated to be similar to the above figure, in agreement with previous data from the literature obtained with circular chromatins containing larger numbers of nucleosomes. DNA structure in mononucleosomes was subsequently investigated by means of high-resolution electron microscopy and gel electrophoresis. It was found that the above linking number reduction could be ascribed to a particle with a large open extranucleosomal DNA loop and with no more than 1.5 turns of a superhelix around the histone core. A theoretical model of a nucleosome on a small ring was constructed in which one part of the DNA was wrapped around a cylinder and the other part was free to vary both in torsion and flexion. The linking number reduction predicted was found to be most consistent with experimental data when the twist of the DNA in the superhelix was between 10.5 and 10.65 pb per turn, suggesting that wrapping on the nucleosome does not alter the twist of the DNA significantly. A lower estimate of the linking number reduction associated with a two-turn nucleosome was also derived, based on an analysis of recent data obtained upon treatment of reconstituted minichromosomes with gyrase. The value, 1.6 turns, set a lower limit of 10.44 bp per turn for the twist of nucleosomal DNA, in agreement with the above estimate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling

By analyzing the Boltzmann populations of DNA topoisomers that differ only in their linking numbers, the dependence of the free energy delta G tau of DNA supercoiling on the linking number alpha has been determined for DNA rings as small as 200 base-pairs (bp) in length. All experimental data can be fitted by the relation delta G tau = K (alpha-alpha)2, where alpha is a constant for a given DNA at a given set of conditions and K is a DNA length-dependent proportionality constant. For DNA rings with length N larger than 2000 bp, K is inversely proportional to N and the product NK is nearly a constant around 1150 RT X bp. For rings smaller than 2000 bp NK increases steadily with decreasing N; for a 200 bp ring NK is 3900 RT X bp. The increase in NK when N decreases can be interpreted as a result of the decrease in the contribution of the fluctuation in the writhing number to the equilibrium distribution in alpha. Assuming that the writhing contribution approaches zero for DNA rings 200 bp in size, the torsional rigidity of the DNA double helix is calculated to be 2.9 X 10(-19) erg cm. In addition, the large value of K for the small circles allows precise calculation of the helical repeat of DNA. For the 210 bp rings, the repeat is measured to be 10.54 bp.     

Estimating the time course of pore expansion during the spike phase of exocytotic release in mast cells of the beige mouse

Our objective is to determine the time course of exocytotic fusion pore opening (P) in mast cells of the beige mouse from the measured efflux of the spike phase of exocytotic release (J). We show that a pore whose meridian or radius grows linearly with time cannot reproduce the efflux. We also show that a pore that opens very quickly [relative to the diffusivity of 5-hydroxytryptamine (5-HT)] and completely (P = π) also does not mimic the experimental efflux, and estimate maximum pore angles of 70(±20)°. We show that a larger class of opening functions reproduces the rising phase and part of the decay phase and calculate pore expansion rate, pore radius and pore angle, none of which can be readily measured. In the initial stages of the spike phase (50–200 ms) when the gel matrix has not expanded significantly, this model suggests that the pore radius increases exponentially with a time constant of 82(±62) ms with pore expansion reaching its maximum velocity of 20(±7) nm ms⁻¹. We conclude that the release process is dynamic and suggest that the velocity of pore opening (V) and the diffusivity of 5-HT (D), in addition to the size of the vesicle (R, radius), vary with time. We discuss assumptions and improvements to the model and propose that this methodology is applicable for determining P from measured J in other endocrine cells and neurons when D within the secretory vesicle is much less than D within the pore neck.     

Dendrochronology In Plains Prehistory: An Assessment

Abstract

Chronologies and climatological interpretations based upon annual growth rings of trees have been available to prehistorians within the Plains for more than half a century. Until the advent of radiocarbon dating, tree rings provided the only “absolute” chronology for most archeological complexes and subsequently, continued as an important adjunct to radiometric methods. Nonetheless, the validity of tree ring dates in the Plains must be questioned. Continuity of research has been lacking, there have been serious methodological problems, and the provenience of many specimens is in doubt. Moreover, there are significant conflicts between tree ring and recent radiocarbon dates.     

Ring theory

In what follows we demonstrate that the minimum requirement for the formation of a DNA ring is a pair of ordinary (ABC … ABC) or inverted (ABC … C′B′A′) repetitions. DNA fragments that are partly degraded from their ends by a 3′ (or 5′) specific exonuclease such as exonuclease III (or λ exonuelease) produce resected fragments that can only form rings by virtue of ordinary repetitions.Next we analyze how random fragments cut from DNA molecules containing ordinary repetitions would be expected to form rings. Since longer fragments (>5 to 10 μm) cyclize less efficiently than do shorter ones (2 μm), we are led to the view that the chromatid is composed of thousands of distinctive regions, called g-regions, within which characteristic repetitious sequences are clustered in an intermittent or tandem fashion. Mathematical expressions are derived that allow one to measure the length and number of these g-regions from the ring frequency, R, and its dependence on the length of the fragment.The interior organization of the g-regions is considered in terms of two models and their variants: intermittent repetition and tandem repetition. These are depicted in Figure 2. The objective of this effort is to calculate the frequency of rings that can be generated from these two models, and to explain the “shortside fall-off”, that is, the decrease in ring frequency as the fragment length becomes shorter. This could not be due to the stiffness of the DNA double helix and must reflect a distribution of spacing of the repetitious sequences within the g-regions. Mathematical expressions are obtained that allow one to estimate the average values of the repetitive or partly repetitive unit. These estimates may be obtained from the dependence of ring frequency on the extent of resection, and from the dependence of ring frequency on the length of shorter fragments.The mathematical expressions derived here are employed in the previous papers of this group, and lead to the conclusion that the g-regions are composed of tandemly repeating sequences.     

核孔复合体的结构及其功能

核孔复合体(Nuclear pore complexes, NPCs)镶嵌在核膜上,是细胞核与细胞质之间的唯一通道。冷冻电子X射线断层扫描将环状NPCs分为三个环,分别称为胞质环、内环和核质环,胞质环上附有胞质纤丝,核质环上附有核篮。由于物种不同,NPCs由30～50多种不同的核孔蛋白(nucleoporins, Nups)组成,但结构和功能高度保守。根据其结构、氨基酸序列,NPCs定位和功能,Nups被分为跨膜Nups、屏障Nups、骨架Nups、胞质纤丝Nups和核篮Nups。相互间作用稳定、紧密连接的数个Nups可组成亚复合体。为了应对不同生理需要,NPCs处于高度动态变化中,间期和有丝分裂期均可通过组装和去组装改变核孔数量和功能。NPCs的主要功能是调控核质转运,小分子物质可自由扩散,大分子物质则需在核转位信号和转运载体的介导下以主动运输的方式进行转运。除了核质转运这一主要功能外,Nups还能以一个独立于转运的方式影响基因组功能。通过影响染色质结构和影响转录调控元件对靶基因的访问,Nups促进或抑制转录。在酵母,Nups介导的基因调控主要由位于NPCs中的Nups执行;在多细胞生物,不仅NPCs中的Nups,核质内游离的Nups也具有基因调控功能。此外,Nups还能通过参与形成染色质边界和形成转录记忆对基因进行调控。在增殖细胞, Nups通过与DNA修复机器相互作用,参与DNA损伤修复,保护基因组完整性。有丝分裂时,Nups协助核膜解体和中心体迁移,并通过作用于着丝粒来控制有丝分裂组件的空间定位与活性,稳定它们与微管之间的相互作用,保证纺锤体正常组装和染色体准确分离。总之,NPCs与生物分子的核质转运、基因表达和细胞周期密切相关,它的结构和功能的稳定是真核细胞生长、增殖、分化等生命活动的基本保证。