Flow birefringence of T2 bacteriophage DNA

The streaming birefringence and extinction angles of DNA from T2 bacteriophage in neutral aqueous buffers have been determined over a range of concentrations from 5 to 44 μg./ml. and of velocity gradients from 5 to 50 sec.^?1. Although the extinction angles are relatively small even at the lowest velocity gradients and concentrations studied, a lower limit estimate of the limiting extinction angle to shear rate ratio can be made, and this value is interpreted in terms of available dynamical theory. The statistical segment length has been estimated from the birefringence data as ～ 1100 A. This figure is in satisfactory agreement with the results of independent calculations by others.     

Optico-hydrodynamic properties of high molecular weight DNA from steady-state flow birefringence and viscosity at extremely low velocity gradients

The flow birefringence, extinction angles, and shear-dependent viscosity over a velocity gradient range of approximately 0.1–3 sec^?1 have been obtained for T2 bacteriophage DNA at low concentration in neutral aqueous buffer. The data are found to be interpretable and self-consistent in terms of subchain dynamical theory, including hydrodynamic, interactions and excluded volume, and the parameters characterizing these phenomena are in good agreement with the results of other hydrodynamic experiments and theoretical calculations. No necessity for modification of the subchain model in terms of limited extensibility or internal viscosity is found for high molecular weight DNA at the velocity gradients studied, although the latter cannot be ruled out on the basis of the present data. The Kuhn statistical segment length is determined from the intrinsic optical anisotropy and is estimated as 930 Å. Implications of these findings and their relation to appropriate dynamical models for DNA are discussed.     

Steady-state opticohydrodynamic properties of DNA: Molecular weight dependence and the internal viscosity problem

Extinction angles, flow birefringence, and intrinsic viscosities are compared for linear, bihelical DNAs from viral and other sources that span a range in molecular weight from ～10⁵ to 1.3 × 10⁸. This range effectively spans the region over which transition from rigid-rod to expanded-coil hydrodynamic property behavior occurs. All DNAs are in identical, phosphate–EDTA, neutral-pH buffers, 0.1M in NaCl. The extinction angle is a hydrodynamic property only and is thus particularly sensitive to effects of kinetic chain rigidity or internal viscosity. Our extinction angle results cannot be interpreted by any simple, single-function theoretical expression. Rather, they must be divided into distinct high- and low-molecular-weight domains. The low-molecular-weight region is typical of rigid-particle opticohydrodynamic property behaviour characterized primarily by particle orientation. The high-molecular-weight domain shows evidence for a finite internal viscosity effect, however, which can be interpreted as very nearly Kuhnian using Cerf's amplification of the Gaussian subchain model to include internal viscosity. It is found that the high-molecular-weight, monodisperse viral DNAs from T7, T5, and T2 bacteriophage show an internal viscosity contribution to the limiting extinction angle–shear rate ratio of ～3 × 10^?3 s. An effect of this magnitude may be marginally important in interpreting extinction angle and certain other hydrodynamic property data for high-molecular-weight DNA systems. Internal viscosity effects do not appear to be manifest in the ratio of flow birefringence to intrinsic viscosity, however, and the persistence length of the high-molecular-weight DNAs is found to be independent of molecular weight to within estimated experimental uncertainty.     

Optico-hydrodynamic properties of high molecular weight DNA. II. Effect of aqueous glycerol solvents

The optical and hydrodynamic properties of T2 bacteriophage DNA have been determined by steady-state flow birefringence and viscosity in glycerol–aqueous buffer solvents at 25°C. Flow birefringence and extinction angle data were obtained over a velocity gradient range of 0.1 to 5 sec^?1 and at concentrations from 3 to 55 μg/ml in solvents containing approximately 30, 42, and 48 vol-% glycerol. Large optical backgrounds were observed in the mixed solvent flow birefringence studies which presented special experimental difficulties; these are described and their effect upon the flow birefringence data are discussed. The data on extinction angle provide no evidence for an internal viscosity effect on the stationary-state hydrodynamic properties of high molecular weight DNA over a range of solvent viscosity from 0.9 to 4.6 cP. Both the optical and hydrodynamic properties under present conditions of measurement appear to be self-consistent in terms of the values for these quantities in neutral aqueous buffer solution. Interpretation of the birefringence is complicated by uncertainties inherent in calculating the form anisotropy of DNA in non-aqueous solvents, but the data imply no large changes in helical structure with increasing glycerol concentration. Both intact and slightly degraded DNA samples were investigated, and no significant polydispersity effects were observed under the experimental conditions described.     

Flow dichroism of DNA: a new apparatus and further studies

A new apparatus for the study of flow dichroism of macromolecules is described. The flow is down a long, narrow channel and an unpolarized light beam propagates along the flow direction. For a molecule such as DNA, in which the transition moments of the chromophores are perpendicular to the axis of orientation, an increase of absorbance is observed during flow. The apparatus is best suited for macromolecules which are readily orientable or at high shear gradients so that the extinction angle is close to 0°. The apparatus has the following advantages: dilute macromolecule solutions can be used; high shear gradients are easily obtained; only small volumes of solution are needed. The flow can be stopped rapidly so that relaxation times for disorientation can be studied. The flow dichorism of native, two-stranded DNA has been measured for the molecular weight range of 0.6 × 10⁶ to 125 × 10⁶, and for the shear gradient range (in aqueous solution at 25°C) from 200 sec^?1 to 21000 sec^?1. At a fixed gradient the dichroism increases with molecular weight, but the curve is concave downwards. At a given molecular weight the dichroism increases with increasing shear gradient, but the curve is concave downwards. When the solvent viscosity and temperature are varied, the dichroism is a function of η〈G〉/T showing that the orientation is due to hydro-dynamic shear stress and that the flexibility of DNA in a flow field is not due to local denaturation. The Zimm-Rouse theory with no parameters taken from flow optical data predicts the correct order of magnitude of the dichroism but the experimentally observed shear gradient and molecular weight dependence do not fit the theory. This is an expected result, since the theory is believed to be applicable only at small distortions and extensions of the macromolecule.     

The molecular parameters of monomeric and acid-soluble collagens. Low shear gradient viscosity and electric birefringence

The molecular parameters of pronase-treated acid-soluble bovine skin collagen (P-ASC) were determined from low-shear gradient viscosity, electric birefringence, and electron microscopic data in order to determine the shear gradient range in which viscosity studies yield data which can be correctly interpreted by use of the various hydrodynamic equations for prolate ellipsoids of revolution. The P-ASC solutions could be characterized by a single relaxation process in electric briefringence with rotary diffusion coefficient θ of 810 sec^?1 and a corresponding molecular length of 2850 Å. Viscosity data were found to be shear gradient dependent and only the extrapolated zero-shear value [η]D = 0 could be used with the viscosity hydrodynamic equations to provide a correct value of molecular length. Intrinsic viscosities obtained at shear gradients >250 sec^?1 are nearly 30% lower than the zero-shear value. Untreated acid-soluble collagen (ASC) solutions contain aggregates and these appear, from electric birefringence data, to be of endlinked character. ASC solutions show a much more marked shear gradient dependence than P-ASC. For example, at D～500sec^?1,[η] = 22 dl/g, whereas the extrapolated zero-shear value of[η] was found to be 44 dl/g. Thus, the shear gradient dependence of native collagen solutions is much more marked than previously assumed and, in contrast to the usual practice, only viscosities measured near zero shear can be interpreted in terms of molecular parameters for collagen solutions containing aggregates.     

Flow birefringence and intrinsic viscosity of a T2 bacteriophage DNA–methylated bovine serum albumin complex

The flow birefringence, extinction angles, and intrinsic viscosity have been determined at low velocity gradients for a complex of T2 bacteriophage DNA and methylated serum albumin prepared in dilute solution to a stoichiometry of approximately 90 proteins per DNA molecule. Comparative data upon equivalent solutions of pure uncomplexed T2 DNA are also presented, and these data are completely in accord with the results of previous study. The experimental data are interpreted in terms of current dynamical theory and indicate that the complex has an essentially linear chain structure, consisting of approximately two DNA molecules, which is hydrodynamically indistinguishable from the pure DNA and that extensive internal or intramolecular binding in the complex does not occur. Although interpretation of the results is hampered by an apparent moderate degree of polydispersity in the complex preparations and by relatively large shear extrapolations, the data for both DNA and the complex are substantially in accord with dynamical theory for a nondraining bead subchain model having high kinetic segmental rigidity.     

The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's

Anomalous sedimentation behavior has been observed for high molecular weight duplex DNA's in sucrose gradients. The sedimentation rate of DNA's having molecular weights of 10⁸ or higher is influenced by high centrifugal fields. The change in the sucrose sedimentation coefficient due to this effect, S^RPM_suc-S⁰_suc, is equal to 1 × 10^?48M^3.65( The anomalous behavior is not influenced by DNA concentration at sufficiently low concentrations. Because of the smallness of the coefficient this effect has not been previously detected for DNA's the size of T2 or smaller at rotor speeds below 40000 RPM. For example, the relative sedimentation coefficient of T2 DNA at 65 000 RPM is only 9% less than at 10000 RPM. However, the sedimentation profile of heterogeneous high molecular weight [(100 – 350) × 10⁶] E. coli DNA is severely altered even at moderate rotor speeds (37000 RPM). Therefore, it seems advisable to use low rotor speeds when sedimenting high molecular weight DNA's.     

Mammalian Metaphase Chromosomes with High Molecular Weight DNA isolated at <Emphasis Type="Italic">p</Emphasis>H 10.5

MAMMALIAN metaphase chromosomes may be isolated either at an acid pH^1–4 or at nearly neutral pH^5–7. The only exception is the method of Corry and Cole⁸, performed at pH 9.5. We used alkaline sucrose velocity gradients to determine the size distributions of DNA (molecular weight) in metaphase chromosomes in an attempt to understand their arrangement. Initial experiments yielded 1 × 10⁶ molecular weight DNA (single stranded) pieces regardless of chromosome size. When metaphase cells are lysed directly on the gradient a high molecular weight (1 × 10⁸ for single stranded) is obtained. We therefore examined a large number of chromosome isolation procedures and cytological conditions to determine their effects on the molecular weight of the DNA.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Dynamic mechanical and rheo-optical studies of collagen and gelatin

The frequency dependence of dynamic mechanical properties of rat tail tendon (RTT), enzyme-solubilized collagen membranes (ESC), AKM-23 dialysis membranes, and gelatin film have been measured at 110, 11, and 3.5 Hz from - 160 to 220°C. RTT and AKM-23 are devoid of a rubbery region; there are as many as six mechanical loss transitions. Gelatin and ESC membranes behave as rubbery materials above room temperature; only three tan δE peaks can be resolved for these materials. Strain birefringence was used to measure the crystalline and amorphous contribution of orientation induced by strain. Both the birefringence and the strain optical coefficient are sensitive to the amount of water in a sample. The effect of chemical swelling agents and of annealing on birefringence are described. Stress relaxation data on gelatin film were analyzed with the rubber elasticity theory to give the average number of chains per unit volume, the specific polarizability, the stress-birefringence ratio and the average molecular weight between hydrogen bonds were calculated. The intrinsic amorphous birefringence for “wet” gelatin film is 1.25 × 10^?2; it is estimated to be about 6 × 10^?2 for “dry” gelatin film.     

Extractive partition of L-aspartase and fumarase fromEscherichia coli using aqueous polyethylene glycol-ammonium sulfate systems

Summary Inorganic sulfate salts are used to form aqueous two-phase systems with polyethylene glycol (PEG) for enzyme purification. Two enzymes, L-aspartase and fumarase produced byEscherichia coli are efficiently separated into different phases in spite of the high degree of similarity in molecular weight and amino acid sequence between them. The ratio of L-aspartase to fumarase in the PEG-rich phase is more than sixty (60) times the ratio before extraction. A high degree of purification in a single extraction step can be achieved by careful selections of PEG molecular weight, pH, cation of the salts, and sodium chloride levels. Cations of sulfate-containing salts in the following order: NH ₄ ⁺ >Na⁺>Mg²⁺ tend to increase the partition of L-aspartase in the PEG-rich phase. The maximal degree of enzyme purification is obtained by using PEG 4000 and ammonium sulfate as a phase system at a stable pH for both enzymes.     

A study of dextran hydration in dilute,aqueous solution by the proton magnetic relaxation method

The times of proton magnetic relaxation in dilute (<1%), aqueous solutions of dextrans, having a molecular weight range of 17 x 10³?500 x 10³, are highly sensitive to the temperature—time prehistory of the samples investigated. Reliable results have been obtained only after preliminary heating of the solutions at 100° for 30 min. On the basis of the model of “two states of water in a solution”, the dependence of the degree of hydration of a dextran on its molecular weight has been obtained. In the molecular weight range 17 x 10³?110 x 10³, only a fraction of the D-glucose residues are hydrated, the degree of hydration increasing with the molecular weight. The data obtained are considered to be a consequence of intersegmentary interaction in a dextran macromolecule.     

Synthesis of high molecular weight DNA strands during S phase

The organization of the mammalian S phase was studied in synchronized mouse embryo cells in terms of the spatial relationship between replication units whose synthesis is initiated at different times in S phase and the rate of assimilation of replication units into high molecular weight DNA strands.The formation of high molecular weight nascent DNA strands several replication units in length was analyzed by velocity sedimentation in alkaline sucrose gradients and by isopycnic centrifugation in alkaline Cs₂SO₄/CsCl gradients. Differential labeling with an isotopic and a density label shows that replication units synthesized at different stages of the S phase are not found within the same high molecular weight polynucleotide strand. It is thus concluded that replication units duplicated at different stages of the S phase are spatially organized in clusters along the mammalian genome.The rate of formation of high molecular weight nascent DNA strands is at least 4 to 8 times slower than that predicted from the spatial organization of replication units and the rate of chain growth within replication units. It is concluded that the process of joining of the completed nascent strands of adjacent replication units plays a major role in the rate of completion of high molecular weight strands.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

Dansyl labeled proteins: Determination of extinction coefficient and number of bound residues with radioactive dansyl chloride

A method is described for preparing fluorescent conjugates of proteins labeled by reaction with methyl-C¹⁴-dansyl chloride and for determining their radioactivity by scintillation counting. The extinction coefficient of the bound dye varies in conjugates of different proteins and is considerably lower than the value generally employed to calculate degree of labeling. For assays of dansyl groups using absorption spectroscopy, a value of ? = 3.4 × 10³M^?1 cm^?1 will probably be approximately correct. However, the radioactive dye technique allows much more accurate determinations of degree of labeling, as well as of the extinction coefficient, which may be of interest in itself.     

Rapidly-labelled RNA species isolated from cell suspensions ofDaucus carota

Summary Carrot cells in suspension culture were incubated during the log-phase of the culture transfer cycle for different periods with one of the following precursors of nucleic acid synthesis: [^32P]-orthophosphate, [5,6-³H]-uridine, and [2-¹⁴C]-uridine. Cells were gently broken by a short period of sonication, and the total RNA of the cells was extracted by a phenoldetergent method at pH 9.0. Subsequently, crude RNA was purified from contaminating substances like carbohydrates and nucleotides, and the pure RNA preparations were characterized by MAK-chromatography and constant velocity sedimentation in isokinetic sucrose gradients.Rapidly-labelled RNA-fractions were detected in the radioactive profiles obtained with both separation methods. These RNA-fractions showed a high specific incorporation rate, but almost no detectable UV-absorbance,i.e., they are RNA species with a high turnover rate and represent only a small part of the total RNA of the cell. With increasing periods of labelling and in a series of pulse-chase experiments high molecular weight RNA-fractions released by high-salt washing of MAK-columns exhibited a shift of the incorporated radioactivity from fractions with higher to those of lower molecular weights. Furthermore, in sucrose gradients a similar shift was observed for RNA-fractions with estimated sedimentation coefficients of 50 S, 40 S, 34 S and 22 S; the radioactivity was converted from these high to the low S-values of the 26 S and 18 S rRNAs, respectively. This parallel in the behaviour of the high molecular weight RNA-fractions from both separation methods indicates their putative role as precursors of rRNA-synthesis. Moreover, there is evidence that the high molecular weight RNA-fractions from the MAK-columns which were eluted after the 26 S rRNA consist not only of the precursors of rRNAs, but also of polydisperse RNA-fractions with S-values smaller than 18 S. These probably contain fractions of HnRNA and mRNA.     

Direct Measurements of the Pressure and Flow in the Xylem Vessels of Nicotiana tabacum and their Dependence on Flow Resistance and Transpiration Rate

The relationships between xylem tension, velocity of water ascending and transpiration in tobacco plants were measured by means of the “xylem pressure probe technique” (Balling, A. and Zimmermann, U., Planta 182, 325–338, 1990). The flow velocity was determined by suction or injection of fluorescein (or FITC-labelled dextrans of various molecular weights) from the microcapillary of the pressure probe into the punctured xylem vessel, followed by serial-sectioning of the stem after a given propagation time. The distance travelled was defined as the distance from the injection point to the uppermost xylem section in which the dye could be detected. For a transpiration rate of 0.52 ± 0.12 ml . h^?1, a linear dependence between the flow velocity and the tension gradients was found as expected from the Hagen-Poiseuille law. The slope of the straight lines decreased with increasing molecular weight of the fluorescent labelled compound, presumably because of (partial) plugging of the pit membranes. The average value of the flow velocity (2.5 . 10^?4 ± 0.9 . 10^?4 m . s^?1) was one magnitude smaller than the value estimated from the geometric dimensions of the xylem vessels, but agreed well with the literature value of 2.8 . 10^?4 m . s^?1 for herbs (determined by the heat pulse technique; Huber, B. Ber. deutsch. bot. Ges. 50, 89–109, 1932). The average pressure gradient was determined to be 0.39 ± 0.23 MPa . m^?1, in agreement with the literature (Begg, J. E. and Turner, N. C. Plant Physiol. 46, 343–346, 1970). The first response of xylem pressure (or tension) and of flow velocity to a reduction of the transpiration rate (0.14 ± 0.06 ml . h^?1) occurred after about 24 h, when an increase of the xylem pressure towards higher values associated with a decrease in flow velocity was observed. In contrast, re-establishment of the normal transpiration rate brought the pressure (or tension) and the flow velocity back to normal values within half an hour. Similary, introduction of a transverse cross-sectional cut into the stem did not lead during the first 10 h to a change in xylem tension (or velocity). However, during the following day the pressure fell to relatively low values (about ?0.13 MPa). The velocity increased 10-fold. In the next two days the xylem pressure increased again to normal values (average +0.03 MPa), whereas the flow velocity assumed higher values than normal. The data are discussed in terms of the water status and storage of the adjacent tissue cells.     

Atomistic Simulations of Rare Gas Transport through Breathable Single-wall Nanotubes with Constrictions and Knees

We present the results of molecular dynamics (MD) computer simulations of rare gas diffusion through breathable nanotubes with pentagon–heptagon pair defects resulting in constrictions and knees. Diffusion involves interrupted high speed “choppy” motion with intermittent reversal in velocity direction. Single atoms exhibit a spiral-like path, in contrast to atoms traveling in groups. Considerable resistance to flow appears to reside in the upstream section of the nanotube where density gradients are small, prior to the constriction. Subsequently, considerable density gradients are present and speeds increase, becoming greatest at the tube exit. For the nanotubes examined, Kr and Xe diffusion was too hindered to provide reliable results. Diffusion of He through the nanotubes with knees occurs in a single-file fashion nearly along the center of the tube and the knee has no detectable effect on the diffusion kinetics. Transport diffusion coefficients are in the order of 10^-4–10^-2?cm²/s.