Calculations on the effect of methylation on the electrostatic stability of the B- and Z-conformers of DNA

The three-dimensional Poisson–Boltzmann equation for the distribution of counterion charge density around double-helical DNA has been solved for solutions of .01M, .10M, and .20M monovalent salt. The polymers, poly[d(CpGp)] and poly[d(m⁵CpGp)], were studied in the B- and the Z-conformations. The effect of methylation on the relative stabilities of these conformers in solutions of different ionic strengths is known to favor the Z-form. Accumulation of charge density around the B- and the Z-conformers is compared in detail. The relative electrostatic stabilities of the B- and Z-conformers in .01M, .10M, and .20M solutions are compared and discussed in terms of the ion–DNA interactions and the self-energy of the structured ionic environment. The ion–DNA interaction energies, termed “phosphate screening,” monotonically decrease with ionic strength and are consistent with a B-to-Z conformation change induced in either polymer by increased electrolyte concentration. However, these calculated energies alone do not account for the fact that the ionic strength at the midpoint of the transition of the methylated polymer is substantially lower than that of its unmethylated analogues. The phosphate screening effect is counterbalanced by changes in the self-energy required for the creation of the structured counterion environment. This self-energy of the electrolyte environment monotonically increases with ionic strength. Methylation-induced shifts in the overall conformational equilibria depend on the relative changes of these competing effects. Increasing salt concentration is calcualted to favor the Z-conformer. The effect of methylation, lowering the ionic strength of the transition midpoint, is proposed to originate in minor structural changes in the Z-form of the polymer, making the groove more accessible to counterions in the G(3′ – 5′)C region. This allows a redistribution of counterion density and a lowering of the self-energy of the ionic environment, conferring added stability to the Z-conformation, as indicated by calculations of relative entropies. The experimentally observed temperature dependence of the B-to-Z transition, however, cannot be explained without assuming the release of bound water. Maps of the calculated three-dimensional structure at the counterion distribution near the surface of these molecules in both the B- and the Z-forms are also presented.     

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.     

Viscosity study of DNA. II. The effect of simple salt concentration on the viscosity of high molecular weight DNA and application of viscometry to the study of DNA isolated from T4 and T5 bacteriophage mutants

Polyelectrolyte expansion effects on high molecular weight bacteriophage DNA have been studied by examining the influence of simple salt concentration upon the intrinsic viscosity, [η]. The viscosity–molecular weight exponent a in the expression [η] = KM^a diminishes from 0.8 in 0.005M simple salt to a limiting value of 0.6 for salt concentrations greater than 0.6M at 25°C. The ε parameter of the N^1+ε hydrodynamic representation thus varies from approximately 0.2–0.07 over this range of salt concentration. The intrinsic, viscosity of DNA decreases slightly with increasing temperature at low and moderate salt concentrations but becomes independent of temperature at high salt concentrations. The expansion of the DNA molecular domain is linear in the reciprocal square root of the simple salt concentration. Viscosity differences among DNA's isolated from several bacteriophage T5 mutants reflect small differences in molecular weight which are in agreement, with sine determination by other techniques. The DNA's isolated from various rII mutants of T4 bacteriophage including some very large deletion mutations were found to be identically the same size in accord with current genetic ideas. Details of the representation and extrapolation of viscosity data are discussed and the sensitivity of the technique is evaluated.     

盐生植物盐爪爪的耐盐生理特性探讨

当前土壤盐渍化日益严重,是限制植物生长的一个主要环境因子,然而在盐碱自然环境中生长着许多耐盐植物,为更好地了解盐生植物的耐盐机理,该文从无机离子Na+,K+,Ca2+含量、脯氨酸水平、水势变化、丙二醛含量和盐胁迫的表型等生理参数以及半定量RT-PCR检测脯氨酸合成关键酶基因(P5CS)的表达规律等方面探讨盐胁迫下盐爪爪的耐盐特性。结果表明:(1)随着盐浓度的升高,Na+在根和肉质化的叶中显著地富集,且叶中积累的Na+比根中更多;(2)在盐胁迫条件下,随着盐浓度的增加,脯氨酸的含量和脯氨酸合成关键酶基因的表达显著地增强;(3)Na+和脯氨酸是植物有效的渗透调节剂,可使处于低水势的植物细胞仍能从细胞外高浓度的盐溶液中吸收水分;(4)在0和700 mmol·L-1Na Cl处理下,盐爪爪肉质化叶中丙二醛的含量较其它处理高,这表明植物在这两个处理下可能受到了氧化胁迫;(5)从盐胁迫3个月的生长表型来看,低盐环境中生长的盐爪爪植株的生物量更多,肉质化的叶嫩且绿。综上所述,结合对野外生境的调查和实验室长期的盐胁迫表型结果表明盐爪爪的生长是需盐的,相对低的盐浓度环境对盐爪爪的生长是顺境,而无盐或高浓度盐环境对于盐爪爪的生长来说都是逆境。该研究结果为全面深入研究盐爪爪的耐盐特性,以及更好地利用盐爪爪的生物和基因资源改良土壤和提高作物和林木的耐盐性奠定基础。     

Left handed DNA in synthetic and topologically constrained form V DNA and its implications in protein recognition

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me⁵dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent Z ⇌ B transition it has been shown that a minor fluctuation in Na⁺ concentration at ambient temperature can bring about B to Z transition. Forthe first time, wehave observed a novel Z⇌B⇌Zuble transition in poly(dG-Me⁵dC) as the Na⁺ concentration is gradually increased. This suggests that a minor fluctuation in Na⁺ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pβG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (L _k = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pβG and pβR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.     

DNA polymorphism: spectroscopic and electro-optic characterizations of Z-DNA and other types of left-handed helical structures induced by Ni2+

CD and uv spectroscopy reveal that the synthetic polynucleotides poly(dG–dC) · poly(dG–dC) and poly(dG–m⁵dC) · poly(dG–m⁵dC) undergo a transition induced by small amounts of Ni⁺⁺ cation from a right-handed B-form to left-handed Z-type conformations. We describe the application of steady-state and transient electric birefringence to the characterization of the transition observed at very low ionic strength (10 mM Tris HCl, pH 7.4). Dialysis experiments show that the changes in spectroscopic and electro-optic properties upon addition of Ni⁺⁺ are completely reversible. The differences in shape of the inverted CD spectra suggest the existence of a family of left-handed conformations, depending on the polymer used and on the amounts of cation added. The stoichiometry required for inducing the Z-conformation of poly(dG–m⁵dC) is 1 cation/4 nucleotide phosphates. The transition is accompanied by a decrease in birefringence, an increase in length, and the more important contribution of a permanent or slowly induced dipole moment to the orientation mechanism. High concentrations of Ni⁺⁺ promote the Z → Z* transition. Upon increasing the Ni⁺⁺ concentration, poly(dG–dC) undergoes a biphasic transition, first to one intermediate conformation that is neither B- nor Z-like and then to a left-handed form that is probably different from Z*. These conversions are accompanied by regular decreases both in birefringence and in chain length, but no transient appears in the field-reversal experiments.     

Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC)

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA-dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by ¹⁹F-nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA-dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG-dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG-dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left-handed to a right-handed conformation.     

Salt effects on the denaturation of DNA. V. Preferential interactions of native and denatured calf thymus DNA in Na2SO4 solutions of varying ionic strength.

Precision density measurements were performed at 25°C on Na-DNA-Na₂SO₄ mixtures in the presence of either 0.005 m cacodylic acid buffer (pH 6.8) or in the presence of 0.1 m NaOH (pH 12.3). From measurements executed under equilibrium dialysis conditions, the so-called “density increments” (?ρ/?c₂)_μ⁰ for native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were evaluated as a function of Na₂SO₄ concentration. (?ρ/?c₂)_μ⁰ for native DNA was found to decrease almost linearly with ionic strength I^1/2 of the supporting electrolyte. The density increment for Na-DNA at pH 12.6, on the other hand, increases in more or less linear fashion with I^1/2. (?ρ/?c₂)_μ⁰ for heat-denatured DNA at pH 6.8 is not affected very much by increasing salt strength. From density measurements performed on the Na-DNA–Na₂SO₄ mixtures at fixed concentrations of diffusible components, the partial specific volumes ν ₂° of native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were determined as a function of Na₂SO₄ concentration. All ν ₂° values, irrespective of the secondary structure of the DNA, increase with increasing salt concentration although the increase for heat denatured DNA (pH 6.8) is barely noticeable. ν ₂° of both native and heat-denatured DNA (pH 6.8) extrapolates to a value of 0.50_o ml/g at vanishing salt concentration; ν ₂° of DNA in 0.1 m NaOH, on the other hand, assumes the value 0.2_o ml/g. Distribution coefficients of diffusible components, expressed in terms of preferential water and salt interaction, were evaluated from the (?ρ/?c₂)_μ⁰ data, solvent densities, and partial specific volumes of all solution components. All interaction parameters depend strongly on salt concentration and on the conformation of DNA. From data collected and from information available in the literature it is concluded that Na₂SO₄, for instance, displaces water of hydration from native DNA much more readily with increasing salt concentration than does NaCl. The solvation properties of the denatured forms of Na-DNA are rather complex but appear to be in harmony with whatever information can be gathered from the literature.     

Kinetics of the salt-induced B- to Z-DNA transition

The salt-induced B- to Z-DNA conformational transition is a cooperative- and time-dependent process. From a modified form of the logistic equation which describes an equilibrium between two states we have deduced a kinetic function to quantify the degree of the B to Z transition of a synthetic (dG-dC) ⋅ (dG-dC) polynucleotide. This function was obtained by introduction of time as a variable in the logistic function so that the equilibrium constant, K, is replaced by a new constant K _s , characteristic of the type of salt used. This constant is defined as the salt concentration needed to reach the B-Z transition-midpoint in the time unit. The equation fits the data obtained by circular dichroism (CD) for changes in molecular ellipticity of poly(dG-m⁵dC) ⋅ poly(dG-m⁵dC) and poly(dG-dC) ⋅ poly(dG-dC) incubated with various concentrations of mono-, di-, and trivalent salts at a constant temperature. The derived expression may be a very useful tool for studying the kinetics of the B- to Z-DNA transition. Received: 1 December 1997 / Revised version: 16 March 1998 / Accepted: 27 March 1998     

Salt-concentration dependence of melting profiles of lambda phage DNAs: evidence for long-range interactions and pronounced end effects.

Melting profiles of DNAs from wild-type λ phage and a deletion mutant phage λb2 were examined in a wide range of salt concentration. The fine structure of the melting profiles changed sharply with salt concentration, especially in the range [Na⁺] ? 10 mM. A comparison of the melting profiles between the wild-type and the deletion mutant DNAs provided good evidence for extremely high melting cooperativity under low salt conditions, which is clearly manifested as the long-range interactions and the pronounced end effects; a large melting peak appeared as a result of the b2 deletion without any inserted sequence in the salt range [Na⁺] ? 2.8 mM. It was also suggested that in the further reduced salt range [Na⁺] ? 2.0 mM, melting of a λ DNA molecule starts from its right end rather than the most (A + T)-rich central region. The molecular basis of the high melting cooperativity at low salt concentrations can be explained in terms of the increased free energy associated with loop formation in the double-helical structure of DNA.     

The role of chitinase from Lecanicillium antillanum B-3 in parasitism to root-knot nematode Meloidogyne incognita eggs

B-3 fungal isolate was isolated from soil samples of Gwangju in Korea. Based on morphological and phylogenetic analysis, it was designated as Lecanicillium antillanum B-3 (syn. Verticillium antillanum B-3). The fungus was a chitinolytic-nematophagous microorganism. B-3 chitinase activity from 0.5% swollen chitin broth medium reached the highest level on the sixth day and then plateaued until 12 days. B-3 isolate showed the high rate of parasitism on Meloidogyne incognita eggs with more than 90% infection rate on the third day after treatment. B-3 crude chitinase damaged the eggshell structures more than 78% based on lactoglycerol staining data at a final protein concentration of 14.6 µg mL^?1 on the fourth day following treatment. Partially purified chitinase with molecular 37 kDa from DEAE-Sephadex chromatography also showed damaging effect on the eggs. These results suggested that chitinase from B-3 isolate was responsible for degradation of M. incognita eggshell structures.     

On the application of polyelectrolyte "limiting laws" to the helix-coil transition of DNA. II. The effect of Mg++ counterions

The techniques of the previous article are here applied to the case for which the solution contains, in addition to excess uni–univalent salt, one equivalent of divalent counterions per mole nucleotide. In agreement with the melting temperature measurements of Dove and Davidson for Mg⁺⁺, it is predicted that a region of uni–univalent salt concentration then exists in which (dT _m/^d log m A +) is negative. It is further predicted, in accord with experiment, that in the presence of divalent counterions, the helical form of DNA is much more stable than in their absence.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Conformation of double-stranded polydeoxynucleotides in solution by proton two-dimensional nuclear Overhauser enhancement spectroscopy

Proton 2D-NOE spectroscopy has been used to investigate the three-dimensional conformations of several sonicated polydeoxynucleotides in solution. The observed pattern of cross peaks indicate that poly(dA-dT) · poly(dA-dT) in all salt concentrations studied (up to 6.6M CsF), and poly(dG-m⁵dC) · poly(dG-m⁵dC) in low salt (0.1M NaCl) are righthanded B-structures. Poly(dG-m⁵dC) · poly(dG-m⁵dC) in Mg²⁺ (3 mM) solution exhibits a pattern characteristic of the left-handed Z-form. These results for poly(dA-dT) · poly(dA-dT) are in contrast to suggestions that this copolymer exists as a left-handed form, either in low or high salt. We present pure absorption-mode 2D-NOE spectra that enable us to compare several distances and define the conformations of these polydeoxynucleotides in solution.     

Left-handed Z-DNA Helices in Polymers,Restriction Fragments,and Recombinant Plasmids

Abstract

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)_n·(dC-dG)_n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)_n·(dC-dG)_n forms left-handed, ψ(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)_n·(dC-dA)_n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG)·(dC-dG) and (dT-dG)·(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (σ≤ ?0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methyla- tion in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) S₁ and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of S₁ nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)_n·(dC-dA)_n polymer requires base modification and high salt conditions to undergo the R?L transition, supercoiling (σ ?0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG)·(dC-dA) sequences to undergo a R?L transition.     

An electrostatic model for the dielectric effects,the adsorption of multivalent ions,and the bending of B-DNA

We have studied electrostatic properties of DNA with a discrete charge model consisting of a cylindrical dielectric core with a radius of 8 Å and a dielectric constant D_i = 4, surrounded by two helical strings of phosphate point charges at 10 Å from the axis, immersed in an aqueous medium with dielectric constant D_w = 78.54. Eliminating the dielectric core makes potentials in the phosphate surface less negative by about 0.5 kT/e. Salt effects are evaluated for the model without a dielectric core, using the shielded Coulomb potential. Smearing the phosphate charges increases their potential by about 2.5 kT/e, due mostly to the self-potential of the smeared charge. Potentials in the center of the minor and major grooves vary less than 0.02 kT/e along their helical path. The potential in the center of the minor groove is from 1.0 to 1.7 kT/e, more negative than in the center of the major groove, depending on dielectric core and salt concentration. So multivalent cations and also larger cationic ligands, such as some antibiotics, are likely to adsorb in the minor groove, in agreement with earlier computations by A. and B. Pullman. Dielectric effects on the surface potential and the local potential variations are found to be relatively small. Bending of DNA is studied by placing a multivalent cation, M^Z+, in the center of the minor or major groove, curving DNA around it for a certain length, and calculating the free energy difference between the bent and the straight configuration. Boltzmann averaged bending angles, 〈β〉, are found to be maximal in 0.03M monovalent salt, for a length of about 50 or 25 Å of curved DNA when an M^Z+ ion is adsorbed in the minor or the major groove, respectively. When the dielectric constant of water is used throughout the calculation, we find maximal bends of 〈β〉 = 11° for M²⁺ and 〈β〉 = 16° for M³⁺ in the minor groove, 〈β〉 = 13° for M³⁺ in the major groove. The absence of bends in DNA adsorbed to mica in the presence of Mg salts supports the role of Mg²⁺ in “ion bridging” between DNA and mica. The treatment of the effective dielectric constant between two points outside a dielectric cylinder in water is appended. © 1998 John Wiley & Sons, Inc. Biopoly 46: 503–516, 1998     

Na2SO4和Na2CO3胁迫下苦楝幼苗的形态及光合生理特性

为探索苦楝应对盐胁迫的响应机制,该文以1年生苦楝(Melia azedarach)实生苗为材料,在盆栽条件下设置中性盐Na_2SO_4和碱性盐Na_2CO_33个盐浓度(200、400、600 mmol·L~(-1))处理40 d,研究苦楝的抗盐碱水平及在不同程度盐碱胁迫条件下的生长及光合生理变化。结果表明:随着盐浓度的提高,苦楝的苗高、地径和生物量的增长量均呈现下降趋势,且碱性盐胁迫条件下降程度更大,盐胁迫提高苦楝的根冠比。处理10 d时,苦楝幼苗的所有光合指标随中性盐和碱性盐浓度的提高呈相似的下降特征,碱性盐胁迫条件下的降低幅度显著大于中性盐胁迫,且随处理时间的增加,中性盐和碱性盐处理下苦楝幼苗的净光合速率和蒸腾速率显著降低。随着盐浓度的提高,苦楝的叶绿素含量呈现下降趋势,200 mmol·L~(-1)盐胁迫对叶绿素含量影响较小,400、600 mmol·L~(-1)盐胁迫均对叶绿素含量有显著影响。600 mmol·L~(-1)碱性盐胁迫条件下,苦楝叶片相对电导率和饱和水分亏缺最高,显著高于其余处理。同等浓度下,碱性盐胁迫的苦楝叶片相对电导率和饱和水分亏缺显著高于中性盐胁迫处理。综上结果认为,苦楝具有一定的耐盐碱能力,碱性盐比中性盐对苦楝幼苗的影响更大。     

Poly(dA-dT)•Poly(dA-dT) in Low Salt Appears to be a Left-Handed B-Helix Combined Use of Chemical Theory,Fiber Diffraction and NMR Spectroscopy

Abstract

Poly(dA-dT)?poly(dA-dT) can adopt the B- and D- forms in the fibrous state. Theoretical energy calculations and fiber diffraction analyses suggest that there can be three structural models of poly(dA-dT)?poly(dA-dT) in each of these two forms viz right and left-handed Watson Crick models and left-handed Hoogsteen—a total of six possible models. Fiber data for the polymer in the B- or the D-form or energy calculations cannot distinguish any one model from the other. However, a comparison of observed proton chemical shifts with the theoretically computed ones and the NOE studies on exchangeable and nonexchangeable protons suggest that poly(dA-dT)?poly(dA-dT) in low salt solution exists predominantly in the left-handed B-conformation.     

盐碱胁迫对海岛棉幼苗生物量分配和根系形态的影响

为探求海岛棉幼苗生物量分配和根系生长对混合盐碱胁迫的响应,探索海岛棉幼苗对混合盐碱胁迫的适应特点,以新疆阿克苏地区主栽海岛棉品种新海31号(XH31)、新海35号(XH35)、新海43号(XH43)及新海48号(XH48)为试材,采用NaCl、Na_2SO_4和NaCl、NaHCO_3以物质的量1∶1混合分别模拟中性盐混合盐(中性盐)和碱性盐混合盐(碱性盐)2种盐类型,总盐浓度(Na~+)0(CK)、120、180、240、300、360 mmol/L,对2种盐胁迫下不同浓度海岛棉幼苗生物量、根系长度、根系体积以及根系表面积等海岛棉幼苗根系形态特征指标变化差异以及不同茎级的根系形态特征进行了分析。结果表明,中性盐下,盐浓度在0—180 mmol/L时,对海岛棉幼苗的根系总长度、总表面积、总体积、总根尖数有显著的促进的作用或不显著的抑制作用,尤其盐浓度为120 mmol/L对海岛棉幼苗细根(0d0.5 mm)的长度有显著的促进作用;大于180 mmol/L后,随着盐浓度的增加各项根系参数均显著减小;碱性盐下,除新海31号的总根尖数、细根及中根根尖数在120 mmol/L的盐浓度下比CK增加,其余处理均随着盐浓度的增加再减小;地上部生物量、根系生物量及根冠比在两种盐下随着盐浓度的增加逐渐减小,减小程度也由小变大。低盐(120 mmol/L)环境能促进细根的伸长,使海岛棉幼苗可以更加多的吸收养分和水分,这是根系接触低盐环境时做出的响应,高盐环境对海岛棉幼苗造成较大的损伤。     

Cooperative and Anticooperative Effects in Binding of the First and Second Plasmid OsymOperators to a LacI Tetramer: Evidence for Contributions of Non-operator DNA Binding by Wrapping and Looping

The interaction oflacoperator DNA withlacrepressor (LacI) is a classic example of a genetic regulatory switch. To dissect the role of stoichiometry, subunit association, and effects of DNA length in positioning this switch, we have determined binding isotherms for the interaction of LacI with a high affinity (O^sym) operator on linearized plasmid (2500 bp) DNA over a wide range of macromolecular concentrations (10⁻¹⁴to 10⁻⁸M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into dimers (D), and binding of operator-containing plasmid DNA (O) to dimers and tetramers to form three distinct complexes, DO, TO, and TO₂. Over the range of con- centrations of repressor, operator, and salt (0.075 M K⁺to 0.40 M K⁺) investigated, we find no evidence for any significant thermodynamic effect of LacI dimers. Instead, all isotherms can be interpreted in terms of just two equilibria, involving only T and the TO and TO₂complexes. As a reference binding equilibrium, which we propose must approximate the DO binding interaction, we compare the plasmid O^symresults with our extensive studies of the binding of a 40 bp O^symDNA fragment to LacI. On this basis, we obtain a lower bound on the LacI dimer – tetramer equilibrium constant and values of the equilibrium constants for formation of TO and TO₂complexes.At a salt concentration of 0.40 M, the O^symplasmid binding data are consistent with a model with two independent and identical binding sites for operator per LacI tetramer, in which the binding to a site on the tetramer is only slightly more favorable than the reference binding interaction. Increasingly large deviations from the independent-site model are observed as the salt concentration is reduced; binding of a second operator to form TO₂becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.125 M). In addition, binding of both the first and second plasmid operator DNA molecules to the tetramer becomes increasingly more favorable than the reference binding interaction as [K⁺] is reduced from 0.40 M to 0.125 M. At 0.075 M K⁺, however, the strength of binding of the second plasmid operator DNA to the LacI tetramer is dramatically reduced; this interaction is much less favorable than binding the first plasmid operator DNA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO₂complexes with decreasing salt concentration. At low salt concentration, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator DNA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and 2:1 complexes at low salt concentration, and that looping stabilizes the 1:1 complex but competitively destabilizes the 2:1 TO₂complex at low salt concentration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytoplasmic [K⁺] varies in response to changes in extracellular osmolarity.