Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3′-phosphomonoesterase and 3′-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn²⁺• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3′-deoxynucleotide, 3′-deoxynucleotide 3′-phosphate, or 3′ ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3′-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd²⁺) and inhibitory (Zn²⁺) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn²⁺.     

mwr Xer site-specific recombination is hypersensitive to DNA supercoiling

The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites.     

Susceptibility of sheep,human, and pig erythrocytes to haemolysis by the antimicrobial peptide Modelin 5

Modelin-5-CONH₂, a synthetic antimicrobial peptide, was used to gain an insight into species-selective haemolytic activity. The peptide displayed limited haemolytic activity against sheep (12 %), human (2 %), and pig (2 %) erythrocytes. Our results show that Modelin-5-CONH₂ had a disordered structure in the presence of vesicles formed from sheep, human, and pig erythrocyte lipid extract (<26 % helical) yet folded to form helices in the presence of a phosphatidylcholine (PC) membrane interface (e.g. >42 % in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine). Monolayer studies showed a strong correlation between anionic lipid content and monolayer insertion and lysis inducing surface pressure changes of 9.17 mN m^?1 for 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine compared with PC monolayers, which induced pressure changes of ca. 3 mN m^?1. The presence of cholesterol in the membrane is shown to increase the packing density as the PC:sphingomyelin (SM) ratio increases so preventing the peptide from forming a stable association with the membrane. The data suggests that the key driver for membrane interaction for Modelin-5-CONH₂ is the anionic lipid attraction. However, the key factors in the species-specific haemolysis level for this peptide are the differing packing densities which are influenced by the SM:PC:cholesterol ratio.     

Influence of the tritium labeling of calf thymus DNA by the Wilzbach method on its physical properties

The adsorption at the air-water interface of calf thymus H³-DNA labeled in the dry state by the Wilzbach method was studied by measuring surface concentration, surface tension, and surface potential. It was found that, correspondingly to the behavior of E. coli H³-DNA labeled by incorporation of thymine H³ and described in another paper, both the rate of adsorption and the amount of material adsorbed increased with increase in DNA concentration, salt concentration, or in the valency of the counterion. Surface pressure and potential did not change in the course of adsorption, and this is also in accordance with the properties found for E. coli DNA. However, while the surface concentration of the E. coli DNA corresponds approximately to monomolecular layer adsorption, the radiation from the adsorbed layer of the calf thymus H³-DNA indicated apparently multilayer adsorption. On comparing the physical properties of the H³-DNA labeled by the Wilzbach method to those of nonlabeled DNA, it is found that while the chemical composition and the bihelical structure is essentially maintained in the labeled material, exposure to tritium gas results in a reduction in molecular weight and produces random breaks in the strands of the bihelix. The H³-DNA produced by Wilzbach's method is not labeled homogeneously. The more the molecule is exposed to the gaseous tritium, the more efficient is the isotopic, exchange and the greater the alteration in physical properties. The defects in the labeled H³-DNA molecule make it more surface active, thus H³-DNA of higher specific radioactivity concentrates at the interface, conveying the impression of multilayer formation although actually the adsorbed layer is approximately monomolecular.     

Effects of specific versus nonspecific ionic interactions on the structure and lateral organization of lipopolysaccharides

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na⁺ ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca²⁺ ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na⁺ ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca²⁺ ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na⁺- and Ca²⁺ ions in the subphase, the screening effect of Na⁺ is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca²⁺ ions. The unspecific charge-screening effect of the Na⁺ ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.     

Nanomechanical behaviors of microcantilever-based single-stranded DNA chips induced by counterion osmotic effects

Experiments show that deflections of microcantilever-DNA chip can be induced by many factors, such as grafting density, hybridization efficiency, concentration, length and sequence of DNA molecules, buffer salt concentration, time, and temperature variation. However, there are few theoretical works on microcantilever-DNA chips. The present paper is aimed to study the influence of counterion effects of single-stranded DNA (ssDNA) polyelectrolyte solution on the nanomechanical behaviors of microcantilever-based ssDNA chips during packing process. First, the effect of osmotic pressure induced by ingress of counterions into DNA brush structures is studied with Hagan’s model for a cylindrical polyelectrolyte brush system on the basis of Poisson-Boltzmann distribution hypothesis. Second, Zhang’s two-variable method for a laminated cantilever is used to formulate a four-layer energy model for ssDNA chips with weak interactions. Third, the influence of grafting density, ssDNA chain length, and salt concentration on packing deflection is investigated using the principle of minimum energy. The predictive tendency is qualitatively similar to those observed in some related ssDNA chip experiments. The difference between the four-layer model and the simplified two-layer model for ssDNA chips is also discussed.     

Osmotic responses of maize roots

Water and solute relations of excised seminal roots of young maize (Zea mays L) plants, have been measured using the root pressure probe. Upon addition of osmotic solutes to the root medium, biphasic root pressure relaxations were obtained as theoretically expected. The relaxations yielded the hydraulic conductivity Lp _r) the permeability coefficient (P _sr), and the reflection coefficient (σ _sr) of the root. Values of Lp _r in these experiments were by nearly an order of magnitude smaller than Lp _r values obtained from experiments where hydrostatic pressure gradients were used to induce water flows. The value of P _sr was determined for nine different osmotica (electrolytes and nonelectrolytes) which resulted in rather variable values (0.1·10^-8–1.7·10^-8m·s^-1). The reflection coefficient σ _sr of the same solutes ranged between 0.3 and 0.6, i.e. σ _sr was low even for solutes for which cell membranes exhibit a σ _s≈1. Deviations from the theoretically expected biphasic responses occured which may have reflected changes of either P _sr or of active pumping induced by the osmotic change. The absolute values of Lp _r, P _sr, and σ _sr have been critically examined for an underestimation by unstirred layer effecs. The data indicate a considerable apoplasmic component for radial movement of water in the presence of hydrostatic gradients and also some solute flow byppassing root protoplasts. In the presence of osmotic gradients, however, there was a substantial cell-to-cell transport of water. Cutting experiments demonstrated that the hydraulic resistance for the longitudinal movement of water was much smaller than for radial transport except for the apical ends of the segments (length=5 to 20 mm). The differences in Lp _r as well as the low σ _sr values suggest that the simple osmometer model of the root with a single osmotic barrier exhibiting nearly semipermeable properties should be extended for a composite membrane model with hydraulic and osmotic barriers arranged in series and in parallel.     

X-ray diffraction studies on cation-collapsed DNA

The polyamines spermidine, spermine and putrescine are now known to induce tertiary collapse of DNA. In this collapsed state DNA assumes a compact toroidal conformation. However, the structural details of DNA in these compact particles and the forces that stabilize the collapsed state are not clear. We show here that the structural arrangement of DNA in this tertiary conformation is determined by the chemical structure of the agent used to collapse. We have used aliphatic triamines (NH₃⁺-(CH₂)₃-NH₂⁺-(CH₂)_n-NH₃⁺ with n = 3, 4, 5 and 8) and diamines (NH₃⁺-(CH₂)_x-NH₃⁺ with x = 2, 3, 4 and 6) to collapse DNA. We find that the Bragg spacing and the calculated interhelical spacing for a hexagonal packing model vary systematically with the length of the methylene bridge. We also find that the ionic strength of the solution has no effect on the Bragg spacing. This observation suggests that the arrangement of DNA strands in the complexes is determined by the structure of the polycation, and argues against suggestions that the structure of the collapsed state is maintained by the balance of long-range electrostatic repulsive and attractive forces. Instead we propose that DNA helices form a hexagonal array with counterions in the interstices between the helices resulting in a stable three-dimensional phase with high structural order. Arguments are presented favoring such a model in terms of stabilizing and destabilizing thermodynamic forces.     

Responses of Atriplex spongiosa and Suaeda monoica to Salinity

The growth and tissue water, K⁺, Na⁺, Cl⁻, proline and glycinebetaine contents of the shoots and roots of two Chenopodiaceae, Atriplex spongiosa and Suaeda monoica have been measured over a range of external NaCl salinities. Both species showed some fresh weight response to low salinity mainly due to increased succulence. S. monoica showed both a greater increase in succulence (at low salinities) and tolerance of high salinities than A. spongiosa. Both species had high affinities for Na⁺ and maintained constant but low shoot K⁺ contents with increasing salinity. These trends were more marked with S. monoica in which Na⁺ stimulated the accumulation of K⁺ in roots. An association between high leaf Na⁺ accumulation, high osmotic pressure, succulence, and a positive growth response at low salinities was noted. Proline accumulation was observed in shoot tissues with suboptimal water contents. High glycinebetaine contents were found in the shoots of both species. These correlated closely with the sap osmotic pressure and it is suggested that glycinebetaine is the major cytoplasmic osmoticum (with K⁺ salts) in these species at high salinities. Na⁺ salts may be preferentially utilized as vacuolar osmotica.     

Re-entrant cholesteric phase in DNA liquid-crystalline dispersion particles

In this research, we observe and rationalize theoretically the transition from hexagonal to cholesteric packing of double-stranded (ds) DNA in dispersion particles. The samples were obtained by phase exclusion of linear ds DNA molecules from water-salt solutions of poly(ethylene glycol)—PEG—with concentrations ranging from 120 mg ml^?1 to 300 mg ml^?1. In the range of PEG concentrations from 120 mg ml^?1 to 220 mg ml^?1 at room temperature, we find ds DNA molecule packing, typical of classical cholesterics. The corresponding parameters for dispersion particles obtained at concentrations greater than 220 mg ml^?1 indicate hexagonal packing of the ds DNA molecules. However, slightly counter-intuitively, the cholesteric-like packing reappears upon the heating of dispersions with hexagonal packing of ds DNA molecules. This transition occurs when the PEG concentration is larger than 220 mg ml^?1. The obtained new cholesteric structure differs from the classical cholesterics observed in the PEG concentration range 120–220 mg ml^?1 (hence, the term ‘re-entrant’). Our conclusions are based on the measurements of circular dichroism spectra, X-ray scattering curves and textures of liquid-crystalline phases. We propose a qualitative (similar to the Lindemann criterion for melting of conventional crystals) explanation of this phenomenon in terms of partial melting of so-called quasinematic layers formed by the DNA molecules. The quasinematic layers change their spatial orientation as a result of the competition between the osmotic pressure of the solvent (favoring dense, unidirectional alignment of ds DNA molecules) and twist Frank orientation energy of adjacent layers (favoring cholesteric-like molecular packing).     

Extent and location of DNA synthesis associated with a class of Rec-mediated recombinants of bacteriophage lambda

The extent and location of DNA synthesis associated with Rec recombination of a lambda phage mutant has been determined approximately for recombinants arising under conditions that restrict DNA duplication. The mutant bio1 contains a substitution in its DNA, and nearly all phage maturing under these conditions have undergone a recombination event within a short region in or near the inserted DNA. Density labeled phage bio1 were used to prepare a lysate under these conditions and the extent of new DNA synthesis was determined by analyzing the density of the progeny phage. On the average, about 6% of the phage chromosome was resynthesized in such a cross.DNA was extracted from bio1 phage crossed under similar conditions in the presence of ³²PO₄. The position of incorporated ³²PO₄ was determined by cleaving the DNA with EcoRI restriction endonuclease and resolving the resulting fragments by electrophoresis on agarose gels. The fragment found to have the most newly synthesized DNA and the highest average amount of synthesis per nucleotide contains the bio1 insertion near its left end and the “hot spot” for Rec-mediated recombination near its center. It appears that in these crosses recombination-associated DNA synthesis is localized about the region of the Rec-mediated recombination event.     

A molecular dynamics simulation study of polyamine– and sodium–DNA. Interplay between polyamine binding and DNA structure

Four different molecular dynamics (MD) simulations have been performed for infinitely long ordered DNA molecules with different counterions, namely the two natural polyamines spermidine(3+) (Spd³⁺) and putrescine(2+) (Put²⁺), the synthetic polyamine diaminopropane(2+) (DAP²⁺), and the simple monovalent cation Na⁺. All systems comprised a periodical hexagonal cell with three identical DNA decamers, 15 water molecules per nucleotide, and counterions balancing the DNA charge. The simulation setup mimics the DNA state in oriented DNA fibers, previously studied using NMR and other experimental methods. In this paper the interplay between polyamine binding and local DNA structure is analyzed by investigating how and if the minor groove width of DNA depends on the presence and dynamics of the counterions. The results of the MD simulations reveal principal differences in the polyamine–DNA interactions between the natural [spermine(4+), Spd³⁺, Put²⁺] and the synthetic (DAP²⁺) polyamines.Abbreviations DAP diaminopropane - DDD Drew–Dickerson dodecamer - MD molecular dynamics - Put putrescine - RDF radial distribution function - Spd spermidine - Spm spermine     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Influence of diet composition on feeding and water excretion by the tsetse fly, Glossina morsitans

Adult Glossina morsitans fed on aqueous salt solutions containing phagostimulant ATP in an in vitro feeding system gave an optimal feeding response only over a narrow pH range equivalent to that of vertebrate blood. There was much less discrimination on the basis of molar concentration.The rate and extent of water excretion by the fly was found to depend on the concentration of Na⁺ ions in the food medium: an active transport mechanism is indicated which enables water to pass from the meal through the anterior midgut wall and into the haemocoele. A favourable osmotic gradient assisted water transport in the presence of Na⁺ ions: the system could not operate efficiently in the presence of Na⁺ ions if the osmotic pressure of the food medium was higher than that of vertebrate blood, nor could it operate efficiently in any solution lacking Na⁺ ions.Normal transfer of a meal from the crop to the anterior midgut occurred only when the food medium was isotonic with vertebrate blood or in the presence of Na⁺ ions if hypotonic. Normal transfer of isotonic solutions was prevented in the presence of excess K⁺ ions, and hypertonic solutions were not transferred normally even in the presence of Na⁺ ions. Thus the rate of water excretion was reduced.Tsetse flies fed on blood in an in vitro feeding system excreted water at a significantly lower rate than flies fed on a living animal. Evidence suggests that this is due to a combined effect of changes in viscosity, effective ionic composition, and osmotic pressure, upon the normal rate and extent of food uptake and manipulation of the meal prior to digestion. The implications of this are discussed in terms of future developments of in vitro feeding techniques for haematophagous insects.     

Apoplastic transport across young maize roots: effect of the exodermis

The uptake of water and of the fluorescent apoplastic dye PTS (trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate) by root systems of young maize (Zea mays L.) seedlings (age: 11–21 d) has been studied with plants which either developed an exodermis (Casparian band in the hypodermis) or were lacking it. Steady-state techniques were used to measure water uptake across excised roots. Either hydrostatic or osmotic pressure gradients were applied to induce water flows. Roots without an exodermis were obtained from plants grown in hydroponic culture. Roots which developed an exodermis were obtained using an aeroponic (=mist) cultivation method. When the osmotic concentration of the medium was varied, the hydraulic conductivity of the root (Lp _r in m³ · m⁻² · MPa⁻¹ · s⁻¹) depended on the osmotic pressure gradient applied between root xylem and medium. Increasing the gradient (i.e. decreasing the osmotic concentration of the medium; range: zero to 40 mM of mannitol), increased the osmotic Lp _r. In the presence of hydrostatic pressure gradients applied by a pressure chamber, root Lp _r was constant over the entire range of pressures (0–0.4 MPa). The presence of an exodermis reduced root Lp _r in hydrostatic experiments by a factor of 3.6. When the osmotic pressure of the medium was low (i.e. in the presence of a strong osmotic gradient between xylem sap and medium), the presence of an exodermis caused the same reduction of root Lp _r in osmotic experiments as in hydrostatic ones. However, when the osmotic concentration of the medium was increased (i.e. the presence of low gradients of osmotic pressure), no marked effect of growth conditions on osmotic root Lp _r was found. Under these conditions, the absolute value of osmotic root Lp _r was lower by factors of 22 (hydroponic culture) and 9.7 (aeroponic culture) than in the corresponding experiments at low osmotic concentration. Apoplastic flow of PTS was low. In hydrostatic experiments, xylem exudate contained only 0.3% of the PTS concentration of the bathing medium. In the presence of osmotic pressure gradients, the apoplastic flow of PTS was further reduced by one order of magnitude. In both types of experiments, the development of an exodermis did not affect PTS flow. In osmotic experiments, the effect of the absolute value of the driving force cannot be explained in terms of a simple dilution effect (Fiscus model). The results indicate that the radial apoplastic flows of water and PTS across the root were affected differently by apoplastic barriers (Casparian bands) in the exodermis. It is concluded that, unlike water, the apoplastic flow of PTS is rate-limited at the endodermis rather than at the exodermis. The use of PTS as a tracer for apoplastic water should be abandoned. Received: 9 October 1997 / Accepted: 5 February 1998     

Osmotic Pressure of DNA Solutions and Effective Diameter of the Double Helix

Abstract

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col El plasmid DNA solutions in the concentration range of 1–4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A ₂ of the solution of DNA segments and the effective chain diameter d _eff in the ionic strength range of 10^?2-0.1 M, As the ionic strength is lowered from 0.1 to 10^?2 M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.     

Isolation of replicating DNA segments from Chinese hamster cells by density equilibrium centrifugation

When DNA is extracted from Chinese hamster cells grown in the presence of 5-bromodeoxyuridine from the beginning of the S (synthesis) phase until the middle of the first replication round, a significant fraction of total replicated DNA bands at intermediate densities between light-light and light-heavy DNA, in a CsCl gradient. Incomplete bromodeoxyuridine substitution compared with light-heavy DNA justifies the displaced banding of these molecules. Since “intermediate DNA” following alkaline or thermal denaturation gives rise to unsubstituted and fully substituted single strands, its particular density in neutral gradients cannot be ascribed to a uniformly reduced degree of bromodeoxyuridine substitution nor to covalently joined light and heavy strands. The segregation of DNA of intermediate densities into light-light and light-heavy components after shearing suggests that it includes at least one junction between replicated and still unreplicated segments, i. e. one replication fork that may or may not have lost one of its prongs. DNA of intermediate density specifically contains one to two sites sensitive to breakage by Neurospora crassa endonuclease.When a two-minute pulse of tritiated bromodeoxyuridine is given during replication in unlabelled heavy medium, the DNA fragments (mol. wt 35 × 10⁶) containing labelled segments band essentially at intermediate positions and are progressively converted to light-heavy molecules, with increasing duration of chase. The half-life of this pulse-labelled intermediate DNA (about 25 min) is consistent with the proportion of total replicated DNA found at displaced densities (10 to 15%) and, together with the distribution of the intermediate radioactivity, is compatible with the existence of adjacent growing replicons.If DNA is labelled and extracted during the second replication round in the presence of bromodeoxyuridine, “intermediate DNA” with similar properties is found between the light-heavy and heavy-heavy peaks.     

Water transport in maize roots : measurement of hydraulic conductivity, solute permeability, and of reflection coefficients of excised roots using the root pressure probe

A root pressure probe has been used to measure the root pressure (P_r) exerted by excised main roots of young maize plants (Zea Mays L.). Defined gradients of hydrostatic and osmotic pressure could be set up between root xylem and medium to induce radial water flows across the root cylinder in both directions. The hydraulic conductivity of the root (Lp_r) was evaluated from root pressure relaxations. When permeating solutes were added to the medium, biphasic root pressure relaxations were observed with water and solute phases and root pressure minima (maxima) which allowed the estimation of permeability (P_Sr) and reflection coefficients (σ_sr) of roots. Reflection coefficients were: ethanol, 0.27; mannitol, 0.74; sucrose, 0.54; PEG 1000, 0.82; NaCl, 0.64; KNO₃, 0.67, and permeability coefficients (in 10⁻⁸ meters per second): ethanol, 4.7; sucrose, 1.6; and NaCl, 5.7. Lp_r was very different for osmotic and hydrostatic gradients. For hydrostatic gradients Lp_r was 1·10⁻⁷ meters per second per megapascal, whereas in osmotic experiments the hydraulic conductivity was found to be an order of magnitude lower. For hydrostatic gradients, the exosmotic Lp_r was about 15% larger than the endosmotic, whereas in osmotic experiments the polarity in the water movement was reversed. These results either suggest effects of unstirred layers at the osmotic barrier in the root, an asymmetrical barrier, and/or mechanical effects. Measurements of the hydraulic conductivity of individual root cortex cells revealed an Lp similar to Lp_r (hydrostatic). It is concluded that, in the presence of external hydrostatic gradients, water moves primarily in the apoplast, whereas in the presence of osmotic gradients this component is much smaller in relation to the cell-to-cell component (symplasmic plus transcellular transport).     

DNA like-charge attraction and overcharging by divalent counterions in the presence of divalent co-ions

Strongly correlated electrostatics of DNA systems has drawn the interest of many groups, especially the condensation and overcharging of DNA by multivalent counterions. By adding counterions of different valencies and shapes, one can enhance or reduce DNA overcharging. In this paper, we focus on the effect of multivalent co-ions, specifically divalent co-ions such as SO\(_{4}^{2-}\). A computational experiment of DNA condensation using Monte Carlo simulation in grand canonical ensemble is carried out where the DNA system is in equilibrium with a bulk solution containing a mixture of salt of different valency of co-ions. Compared to systems with purely monovalent co-ions, the influence of divalent co-ions shows up in multiple aspects. Divalent co-ions lead to an increase of monovalent salt in the DNA condensate. Because monovalent salts mostly participate in linear screening of electrostatic interactions in the system, more monovalent salt molecules enter the condensate leads to screening out of short-range DNA–DNA like charge attraction and weaker DNA condensation free energy. The overcharging of DNA by multivalent counterions is also reduced in the presence of divalent co-ions. Strong repulsions between DNA and divalent co-ions and among divalent co-ions themselves lead to a depletion of negative ions near the DNA surface as compared to the case without divalent co-ions. At large distances, the DNA–DNA repulsive interaction is stronger in the presence of divalent co-ions, suggesting that divalent co-ions’ role is not only that of simple stronger linear screening.     

Physiological Control of Chloride Transport in Chara corallina: II. THE ROLE OF CHLORIDE AS A VACUOLAR OSMOTICUM

The extent to which Cl⁻ is replaceable as the major anionic constituent of the vacuole of Chara corallina was investigated. It was found that external Cl⁻ is not essential in order for nongrowing cells to increase internal osmotic pressure. After growth of cells in low (9 micromolar) Cl⁻, the vacuolar Cl⁻ concentration is one-half that of cells grown at normal external Cl⁻ concentration (850 micromolar). In contrast, both internal osmotic pressure and total concentration of the major cations, K⁺ and Na⁺, in the same cells were found to be only slightly sensitive to the external Cl⁻ concentration. Thus, it is proposed that, at limiting external Cl⁻ concentration, the cell is able to transport or synthesize another anion for vacuolar use rather than utilize a neutral solute.