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V A Bloomfield

Publications and source records attributed to V A Bloomfield.

At least 73 records · Page 4Linked to original sources
PubMed1990

Effects of spermidine and hexaamminecobalt(III) on thymine imino proton exchange.

We examine the influence of spermidine and hexaamminecobalt(III) binding on DNA base pair dynamics. Proton NMR line-width measurements are used to monitor the exchange of the thymine imino proton in poly[dA].poly[dT] and in the alternating copolymer [d(AT)]15.[d(AT)]15. We employ the treatment by Benight et al. (1988) of diffusion effects in imino proton exchange to estimate catalytic rate constants and to extract apparent equilibrium constants for the interconversion between closed (nonexchanging) and solvent-accessible (exchangeable) states of the base pairs. The salt dependence of the spermidine-catalyzed imino proton exchange in poly[dA].poly[dT] is qualitatively described by this theory. van't Hoff analysis of the temperature dependence of the apparent equilibrium constants suggests that the binding of spermidine and hexaamminecobalt(III) lowers the energy difference between the nonexchanging and exchanging states of the AT base pair in both duplexes.

Base Composition↗
PubMed1990

Structural and electrostatic effects on binding of trivalent cations to double-stranded and single-stranded poly[d (AT)].

We have measured the thermal melting profile for poly[d(AT)].poly[d(TA)] as a function of concentration of three trivalent cations: spermidine, me8spermidine, and hexammine cobalt(III). Using McGhee's (1976) theory of DNA melting in the presence of ligands, we have estimated association constants Kh, Kc and binding site sizes nh, nc for binding to double-helical (h) and single-stranded (c) polynucleotide. The results are as follows: (table; see text) The binding parameters for spermidine and hexammine cobalt(III) to double helical molecules agree fairly well with direct equilibrium dialysis measurements, and are in reasonable accord with predictions of counterion condensation theory. However, despite their identical charges, the three ligands bind to single-stranded DNA with quite different affinities. Estimates of the charge spacing of single-stranded DNA suggest that poly[d(AT)] is less elongated in the presence of spermidine and hexammine cobalt(III) than it is when complexed with me8spermidine.

Binding Sites↗
PubMed1990

Condensation of DNA by trivalent cations. 1. Effects of DNA length and topology on the size and shape of condensed particles.

In vitro condensation of DNA by multivalent cations can provide useful insights into the physical factors governing folding and packaging of DNA in vivo. We have made a detailed study of hexammine cobalt (III) induced condensation of 2700 and 1350 base pair (bp) fragments of plasmid pUC12 DNA by electron microscopy and laser light scattering. The condensed DNA takes the form of toroids and rods. Both are present in all condensates, but the proportion of toroids is higher with the larger fragments. The intact, closed circular plasmid produces smaller particles than the linear fragments. The size of a particle is independent of DNA fragment length. Two hours after adding the condensing agent, a typical toroid is about 800 A in diameter; the outer radius (R1) is approximately 400 A, and the inner radius (R2) is approximately 140 A for both sets of fragments. These dimensions are relatively stable, but there is sufficient change in both R1 and R2 to produce approximately 50% increase in volume from 2 to 24 h. A typical rod at 2 h is about 1800 A long and 300 A wide. The distribution of rod lengths is similar to that of mean toroid circumferences pi (R1 + R2), and the distribution of rod widths is similar to that of toroidal widths (R1-R2). The 2700-bp fragments show a significantly higher ratio of toroids to rods than the 1350-bp fragments. Both types of particle are multimolecular. The average number of molecules/particle, calculated from the above dimensions, assuming hexagonally packed B-form DNA with a center-to-center spacing of 27 A, is 13 +/- 4 for condensates of 2700-bp fragments and 26 +/- 11 for those of 1350-bp fragments. Monomolecular condensates of much larger DNAs have similar dimensions, suggesting that size is governed primarily by the balance of attractive and repulsive intermolecular forces rather than by the entropic factors associated with incorporation of a number of small particles into a larger one. The similar dimensions and volumes of toroids and rods indicate that the free energy cost of continual bending in toroids, minus that gained by extra net attraction in a cyclic particle, is comparable to that of abrupt bending or kinking in rods. Although the condensed particles are multimeric, their distinct toroidal or rodlike shapes distinguish them from the random aggregates that would be generally expected from the multimolecular association of large, flexible polymers.

Cations↗
PubMed1990

Condensation of DNA by trivalent cations. 2. Effects of cation structure.

Electron microscopy is employed to examine DNA aggregates produced by three tripositively charged condensing agents. Spermidine, hexammine cobalt (III), and me8spermidine (in which the amine groups of spermidine are exhaustively methylated) all produce condensates. The predominant form of condensate observed is toroidal; however, me8spermidine produces a large fraction of rodlike condensates. Distributions of toroidal radii and estimated volumes suggest that the size of condensates depends on the condensing agent employed, its concentration, and the time elapsed after addition of condensing agent. While ligand charge seems to be the major factor in predicting condensing power, ligand structure influences the morphology and dimensions of the particles produced. The ability to form hydrogen bonds is not required to promote condensation, since me8spermidine has no NHs. There may be a kinetic barrier to condensation at low me8spermidine concentrations. The relative proportions of toroids and rods may depend on the energetic compensation between bending and binding in cyclic structures, or on rate-limiting formation of sharply bent or kinked regions in rods.

Cations↗
PubMed1990

Scanning tunnelling microscopy in biotechnology.

The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment.

Biotechnology↗
PubMed1990

Cation radius effects on the helix-coil transition of DNA. Cryptates and other large cations.

Most polyelectrolyte theories of the effect of ions on the thermal melting of DNA assume that the predominant influence of the cations comes through their charge. Ion size and structure are treated, for analytic convenience, as negligible variables. We have examined the validity of this assumption by measuring the melting temperature of calf thymus DNA as a function of salt concentration with four univalent cations of different hydrated radii. These are K+ (3.3 A), (n-Pr)4N+ (4.5 A), (EtOH)4N+ (4.5 A), and C222-K+ (5 A). C222-K+ is a complex of cryptand C222 with K+. With K+ as the sole cation, Tm varies linearly with the log of ionic strength over the range 0.001-0.1 M. With all the K+ sequestered by an equimolar amount of C222, Tm is depressed by 10-20 degrees C and the slope of Tm vs. ionic strength is lower. At low ionic strength, an even greater reduction in Tm is achieved with (n-Pr)4N+; but the similar-sized (EtOH)4N+ gives a curve more similar to K+. Theoretical modeling, taking into account cation size through the Poisson-Boltzmann equation for cylindrical polyelectrolytes, predicts that larger cations should be less effective in stabilizing the double helix; but the calculated effect is less than observed experimentally. These results show that valence, cation size, and specific solvation effects are all important in determining the stability of the double-helical form of DNA.

Bridged Bicyclo Compounds↗
PubMed1989

Scanning tunnelling microscopy of Z-DNA.

Scanning tunnelling microscopy (STM) has been used to map the surface topography of inorganic materials at the atomic level, and is potentially one of the most powerful techniques for probing biomolecular structure. Recent STM studies of calf thymus DNA and poly(rA).poly(rU) have shown that the helical pitch and periodic alternation of major and minor grooves can be visualized and reliably measured. Here we present the first STM images of poly(dG-me5dC).poly(dG-me5dC) in the Z-form. Both the general appearance of the fibres and measurements of helical parameters are in good agreement with models derived from X-ray diffraction.

DNA↗
PubMed1989

Scanning tunneling microscopy of nucleic acids.

The scanning tunneling microscope (STM) has been used to measure properties of poly(rA).poly(rU) and DNA, such as helical pitch, half-period oscillations that were interpreted as the alternation between the major and minor grooves, and interhelical spacing. Average pitches were measured by two-dimensional Fourier transforms and by topographic profiles along the fiber axes. Values were typically 7 percent less than standard dimensions of A-form RNA and B-form DNA fibers. This result is compatible with the mild dehydration that occurred under air-drying conditions. More extensive dehydration typically led to 19 percent shrinkage. Analysis of specific regions allowed local variations in helical pitch as small as 1 angstrom to be detected, thus demonstrating that the STM can visualize functionally significant modulations of nucleic acid structure.

DNA↗
PubMed1988

1H NMR study of the base-pairing reactions of d(GGAATTCC): salt and polyamine effects on the imino proton exchange.

Salts and polyamines have a variety of effects on the physical properties of DNA, including stabilization against thermal melting. We wished to gain greater insight into the mechanism of this stabilization by ascertaining its effect on the dynamics of base opening and closing reactions, as measured by NMR. Since the binding of spermidine(3+) is influenced by salt, and since spermidine may act as a base catalyst in proton exchange reactions, we have undertaken a study of salt and base catalyst effects on the imino proton exchange kinetics of a model oligomeric DNA. The selective longitudinal NMR relaxation rates of the hydrogen-bonded imino protons of the self-complementary octadeoxyribonucleotide d(GGAATTCC) monitor the rate of the base-catalyzed chemical exchange of these protons with solvent water. The exchange rates thus obtained provide a sensitive measure of the base-pair opening reactions of the DNA duplex. Under conditions of low pH and no added base catalyst, the NMR relaxation rates allow the determination of kd, the rate constant for the dissociation of the octameric duplex into single strands. Titration with the base catalyst tris(hydroxymethyl)aminomethane allows the determination of kop, the rate constant for the localized opening of individual base pairs, prior to dissociation. A significant Na+ concentration dependence is found for kd. From an analysis of this dependence, it is determined that 0.6 +/- 0.1 sodium ion is released during the dissociation event. The activation energy for helix dissociation (200 +/- 5 kJ/mol) is not dependent on the sodium ion concentration, indicating that the dissociation is entropically driven by the release of bound sodium ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1985

Toroidal condensation of Z DNA and identification of an intermediate in the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC).

Using a combination of spectroscopic techniques, quasi-elastic laser light scattering (QLS), and electron microscopy (EM), we have been able to show that the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC) is accompanied by extensive condensation of the DNA in both low and high ionic strength buffers. At low concentrations of NaCl (2 mM Na+), an intermediate rodlike form, which exhibits a circular dichroism (CD) spectrum characteristic of an equimolar mixture of B and Z forms, is observed. This is produced by the orderly self-association of about four molecules of the polymer after prolonged incubation of a concentrated solution at 4 degrees C. On addition of 5 microM Co(NH3)63+, the CD spectrum of the intermediate changes to that of the Z form, which is visualized as a dense population of discrete toroids on an EM grid stained with uranyl acetate. On the other hand, addition of NaCl to a solution of poly(dG-m5dC) X poly(dG-m5dC) in the absence of any multivalent ion condenses the polymer to toroidal structures at the midpoint (0.75 M NaCl) of the B to Z transition. Further addition of NaCl unfolds these toroids to rodlike structures, which show characteristic Z-form CD spectra. These results show that Z DNA can take up a variety of tertiary structural forms and indicate that its inverted CD spectrum is due to its left-handed helical sense rather than to differential scattering artifacts.

Circular Dichroism↗