Alkaline Titrations of poly(dG-dC)·poly(dG-dC): Microemulsion Versus Solution Behavior

Abstract

PolyGC was titrated with a strong base in the presence of increasing concentrations of NaCl (from 0.00 to 0.60M) either in water solution or with the polynucleotide solubilized in the aqueous core of reverse micelles, i.e., the cationic quaternary water-in-oil microemulsion CTAB/n-hexane/n-pentanol/water. The results for matched samples in the two media were compared. CD and UV spectroscopies and, for the solution experiments, pH measurements were used to follow the course of deprotonation. In both media the primary effect of the addition of base was denaturation of the polynucleotide, reversible by back-titration with a strong acid.

In solution, the apparent pK_a of the transition decreases with increasing the salt concentration and a roughly linear dependence of pK_a on p[NaCl] has been found. A parallel monotonic decay with ionic strength has been found in solution for R_OH, defined as the number of hydroxyl ions required per monomeric unit of polyGC to reach half-transition. By contrast, in microemulsion, R_OH has been found to be independent of the NaCl concentration (and 10 to 50 times lower than in solution). This result is proposed as an indirect evidence of the independence of pK_a on the salt concentration in microemulsion, where the pH cannot be measured. A sort of buffering effect of the positive charges on the micellar wall and of their counter-ions on the ionic strength could well explain this discrepancy of behavior in the two media.     

Hydrolysis of triglyceride by solid phase lipolytic enzymes of Rhizopus arrhizus in continuous reactor systems

Continuous hydrolysis of triglyceride in organic solvent systems using Rhizopus arrhizus mycelia as a source of insolubilized lipase has been studied in packed-bed and stirred-tank reactors. Typically a packed bed reactor containing 1 g of mycelia fed at 1 mL/min with a solution of 2.5% (w/v) olive oil in di-isopropyl ether gave a fatty acid yield of 45% at 30°C. The optimum water concentration was found to be 0.17% (w/v) except under conditions of high oil feed concentration and high yield where no optimum was established. No temperature optimum was observed over the range 20–55°C. Calculated activation energies of 13–20 kJ/mol, depending on temperature, were lower, while K_m(app) values of 0.1–0.3M were higher than those for hydrolysis in conventional aqueous emulsion systems. No evidence of any significant diffusional limitation, which could account for these values, was obtained. The mycelia showed a loss of activity of 0.6–1.0%h at 30°C. The packed bed proved markedly superior to the stirred tank for this system.     

Electrochemical,UV-Visible and EPR studies on nitrofurantoin: Nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

Effect of ionic strength on the pKa of ligands bound to DNA

The pK_a of 3,8-diamino-6-phenyl-phenanthridine (DAPP), a nonquaternary analog of ethidium bromide, has been determined spectrophotometrically as a function of sodium ion concentration both free in solution and complexed to DNA. Unwinding angle determinations with this compound were determined with Col El DNA using ethidium bromide as a standard. The unwinding angle for DAPP was 24 ± 2° relative to 26° for ethidium, and this suggests that DAPP binds in a manner quite similar to ethidium and with no significant outside bound DAPP under these experimental conditions. Isobestic behavior was obtained on spectrophotometric pH titration above pH 5 as long as the ratio of DNA-phosphate to ligand was between 100 and 300 and the DNA phosphate concentration was approximately 0.01M or greater. The loss of isosbestic behavior which occurred below pH 5 is probably due to titration of the 8 amino group of the ligand complexed to DNA. To circumvent this problem, pK_a values and the extinction coefficient of the acidic species were both determined by a computer program using experimental data obtained above pH 5. The pK_a of the free compound has only a minor dependence on ionic strength, while the pK_a of the ligand bound to DNA in an intercalated complex depends strongly on the sodium ion concentration. The pK_a of the DAPP-DNA complex is a linear function of –log[Na⁺] as predicted by the ion-condensation theory of polyelectrolytes. It was determined that DAPP is essentially completely bound to DNA under the conditions of these experiments by (1) determination of apparent pK_a values as a function of total DNA concentration, (2) calculation of binding constants for the neutral species of DAPP, and (3) spectral analysis of the protonated and neutral species of DAPP bound to DNA relative to DAPP free in solution. These results support the ion-condensation theory; provide an independent method for measuring ψ*, the average number of counterions associated per phosphate of DNA in the intercalated conformation; and illustrate that there are no specific pH effects or absolute pK_a values for ligands bound to DNA, but only ionic-strength-dependent results.     

Nonlinear electric properties of DNA solutions.

DNA solutions are shown to present a nonlinear electric behavior. This property is measured through the third harmonic current intensity, which appears when the solution is placed in a sinusoidal electric field of moderately high strength (about 100 V·cm^?1). The influence of different parameters has been examined: fundamental frequency, field strength, concentration, molecular weight, and conformation. By progressive sonication, it is shown that the harmonic current is linearly proportional to the DNA molecular weight, but under an M_r of approximately 10⁶, the nonlinear electrical property decreases sharply and its spectrum shows a drastic change. It is thought that the harmonic current is not related to an orientational phenomenon; an explanation based on the electrical deformation of the molecule is suggested.     

Über die Adsorption von DNS in der Grenzfläche Quecksilber–Elektrolyt

The adsorption of deoxyribonucleic acid (DNA) in the mercury–electrolyte interface has been investigated. The effect of this adsorption on the differential capacity of the electrical double layer between a polarized mercury surface and an 0.15M NaCl solution containing DNA was measured by means of the alternating current polarography (Breyer polarography). The effective alternating current ? under actual conditions (adsorption processes only, small electrolytic resistance, small alternating current frequency, and alternating current amplitude) is directly proportional to the differential double layer capacity. The combination of this method with the application of a stationary mercury drop electrode allows the coverage of the electrode to be followed, continuously in the range 0.2 sec, to about 60 sec. The diffusion is the rate-controlled step of the adsorption kinetics. Therefore the lowering of the alternating current ? by the adsorbed DNA is proportional to the surface concentration for partly covered surface and reaches a constant value after the surface becomes fully covered. Adsorption of further layers does not affect the differential capacity. This makes it possible to determine the maximum surface concentration of the DNA. For that it is necessary to determine the diffusion coefficient of DNA. This was done directly by Strassburger and Reinert in our institute. The surface concentrations of the native DNA and the relative surface concentrations of the denatured DNA in dependence on the potential of the polarized mercury surface was estimated. Both surface concentrations show a pronounced dependence on the potential with a minimum of the surface concentration around ?0.4 V with respect to the normal calomel electrode. This property may be caused by the structure of the adsorption layer depending on the potential. That means that only several segments at the rigid DNA molecules are adsorbed and the other ones remain in the solution near the surface. The adsorption in the neighborhood of the electrocapillary zero potential at ?0.4 V is strongest, and therefore the fraction of the adsorbed segments has a maximum. At these potentials consequently the maximum coverage is already reached at relatively low surface concentrations. Opposite to this is Miller's hypothesis, that native DNA preserves its double helical structure when adsorbed on a negatively charged mercury surface, whereas unfolding occurs on a positively charged mercury surface. Miller's hypothesis is supported by facts that the surface concentration of the denatured DNA should be independent of the potential and should be equal to the surface concentration of the native DNA at a positively charged mercury surface. But an evaluation of Miller's diagrams by no means gives an independence on the potential of the surface concentration of the denatured DNA and no accordance between the surface concentrations of denatured and of native DNA's at the positively charged mercury surface. Moreover Miller compared different DNA samples with different moleculer weights and possibly with different molecular weight distributions. Both the molecular weight and the molecular weight distribution have a pronounced influence on the surface concentration. Therefore this accordance mentioned above is not evident. The critical inspection of Miller's work and the own investigation lead to the conclusion that an unfolding or denaturation of native DNA does not take place in the mercury–electrolyte interface.     

NMR study on molecular mobility of DNA molecules in solution

The molecular mobility of calf thymus DNA molecules in solution has been discussed in terms of correlation time τ calculated from measurements of longitudinal T₁ and transverse T₂ magnetic relaxation times. The influence of DNA concentration and ionic strength of the solution upon freedom of movement of DNA molecules was studied for native and denatured DNA and also during thermal helix-coil transition. The dependence of τ values on temperature was carried out by comparing the values of correlation times τ_tat given temperature with the correlation time τ₂₀ at 20°C. The molecular rotation of DNA at 20°C and at higher ionic strength at 0.15 and 1.0.M NaCl is described by τ values of the order of 1.0–1.2 × 10^?8 and was reduced slightly with increase of temperature below the helix-coil transition. The molecular rotation of DNA in 0.02MNaCl was lower at 20°C as compared to DNA in solvents with higher NaCl concentrations and increases rapidly with increase of temperature in the range 20–60°C. The values of correlation time are characterized by fast increase at temperatures above the spectrophotometrically determined beginning of melting curve. The beginning of this increase is observed at about 65, 80, and 85°C for DNA in 0.02, 0.15, and 1.0MNaCl, respectively. Values of correlation time for denatured DNA are in all cases about 1.1–1.4 times that for native DNA. The obtained results are discussed in terms of conformation of DNA molecules in solution as well as in terms of water dipole binding in DNA hydration shells.     

Protamine Sulfate Provides Enhanced and Reproducible Intravenous Gene Transfer by Cationic Liposome/DNA Complex

Abstract

A novel lipid/polycation/DNA (LPD) formulation has been developed for in vivo gene transfer. It involves the condensation of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complex, LPD offers greater protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse, at which concentration approximately 10 ng luciferase protein per mg extracted tissue protein could be detected in the lung. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h, and declined thereafter. Using LacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be non-toxic and only weakly immunogenic in humans, this novel vector may be useful for the clinical use of gene therapy.     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].     

A rapid,solid phase radioimmunoassay for prostaglandin F2α and its main circulating metabolite

Antibodies directed against prostaglandin F₂α and its main circulating metabolite, 13,14-dihydro-15-keto-prostaglandin F₂α, were insolubilized by reaction with ethylchloroformate. Suspensions of insolubilized antibodies retained immunogenic activity and were useful for the development of a simple and rapid solid phase radioimmunoassay for these two prostaglandins. Using this method, suppression of prostaglandin levels by the influence of indomethacin in vivo has been observed. Advantages of solid phase radioimmunoassay over current procedures are discussed.     

Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

Crystallographic and Spectroscopic Studies of d(CCGGTACCGG)

The decanucleotide sequence d(CCGGTACCGG) crystallizes as a four-way junction at low cobalt ion concentrations (i.e., 1 mM). When the cobalt concentration in the crystallization solution is increased to 5 mM, the sequence crystallizes as resolved B-DNA duplexes. Gel retardation studies of the decamer show both a faint slow-moving band and a much thicker fast-moving band at low cobalt ion concentrations, and only the intense fast-moving band at higher ion concentration. Circular dichroism (CD) spectroscopy of the decamer indicates a structural transition as the cobalt ion concentration in the solution is increased, probably from B-type to A-type DNA. These studies revealed that the oligomer sequence has several conformations and structures accessible to it, in a manner dependent on sequence, ion concentration, and DNA concentration.

[Supplementary materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids for the following free supplemental resources(s): Supplementary Figures 1, 2, and 3.]     

Mineral relationships and ecological distribution of Fissidens cristatus Wils.

Abstract

Studies carried out in the laboratory indicate that Fissidens cristatus Wils., a moss of chalk and limestone grassland, requires the following conditions for optimum growth: pH, 8.0; calcium, 10 mg/1; potassium, 10 mg/1; magnesium, 50–80 mg/1; iron, less than 0.1 mg/1; It has also been demonstrated that calcium inhibits the short-term uptake of potassium from the culture solution.

Within the shoots of specimens collected from the natural habitat, the potassium content was always higher in the current growth segments, whereas that of calcium was always lower. Seasonal variations in the total content of calcium, potassium and magnesium were observed.

The need to wash material collected in the field prior to chemical analysis has been established and the results are discussed in relation to the distribution and local environmental contamination of F. cristatus.     

Polyacrylamide gel as a medium for DNA dissociation and reassociation

Several properties of thermal denaturation and renaturation of DNA in polyacrylamide gels were investigated: (1) Following electrophoresis the DNA band was scanned and shown to increase in absorbance with increasing temperature. The increase was proportioned to DNA concentration across the peak. (2) The dependence of theT _m on salt concentration over a hundred fold range was similar to that found for DNA in free solution. (3) Denaturation of several DNA samples ranging in G+C content from 26 to 71% was compared in gels and free solution. The relationship betweenT _m and % G+C was virtually identical for both sets of DNAs. (4) The kinetics of DNA renaturation in the gel was followed. Reassociation of bacteriophageT ₄ DNA was 2nd order and proceeded more rapidly in polyacrylamide gels than in free solution.     

A Computer Graphics Study of Sequence-Directed Bending in DNA

Abstract

We present theoretical results to account for the unusual physical properties of a 423 bp DNA restriction fragment isolated from the kinetoplast of the trypanosomatid Leishmania tarentolae. This fragment has an anomalously low electrophoretic mobility in Polyacrylamide gels and a rotational relaxation time smaller than that of normally-behaved control fragments of the same molecular weight. Our earlier work (Proc. Natl. Acad. Sci. USA 79, 7664, 1982) has attributed these anomalies to the highly periodic distribution of the dinucleotide ApA in the DNA sequence. As originally proposed by Trifonov and Sussman (Proc. Natl. Acad. Sci. USA 77, 3816,1980) local features of the DNA structure such as a small bend at ApA, if repeated with the periodicity of the helix, will cause systematic bending of the molecule.

Computer graphics representations of DNA chain trajectories are presented for different structural models. It is shown that the structural model of Calladine (J. Mol. Biol. 161, 343, 1982) which is based on crystallographic data, is unsuccessful in predicting the systematic bending of DNA in solution.     

Index to Subjects,Volume 5

Abstract

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors.

Analysis of the binding data by the model of Schwarz and Watanabe (J. Mol. Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 μM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range.

DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A° and TbEDTA^? chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

The Pore-Forming Properties of SsoHel308 Helicase from Saccharolobus solfataricus

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein embedded in rabbit erythrocyte membranes may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a planar artificial bilayer membrane and acts as a transformer. After embedding this enzyme into biolayer lipid membranes, the membrane conductivity is altered. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell. The possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

     

DNA Quadruplexes Assembled by Simple Peptide: Effects of DNA Homology and Peptide Removal

Abstract

Formation of heterologous (calf thymus dsDNA) and homologous (linearized pBR322 plasmid dsDNA) quadruplexes upon binding with the simple aliphatic tripeptide derivative (L- Val)₃?N₂H₂?DNS CF₃COOH—;DHTV) was examined by fluorimetry, flow linear (LD), circular dichroism (CD), and electron microscopy (EM). The morphology of the rod-like compact particles formed due to the association of dsDNA segments proved to be the same for both DNAs, whereas the stability of the compact DNA structure upon tripeptide removal from the complex with DNA differed substantially for homologous versus non-homologous dsDNA used. The increase in NaCl concentration in the solution up to 30 mM removes the peptide from both types of the complexes completely. At the same time at 20 mM NaCl calf thymus DNA quadruplexes readily dissociate, whereas the structures formed by plasmid DNA retain their morphology in the solution containing NaCl with concentrations up to 40 mM and are only partially disrupted at even higher NaCl concentration. These results provide an analogy between trivaline-DNA model complexes and RecA-DNA binding.