Torsional and bending rigidity of the double helix from data on small DNA rings

We have calculated the variance of equilibrium distribution of a circular wormlike polymer chain over the writhing number, [Wr)2), as a function of the number of Kuhn statistical segments, n. For large n these data splice well with our earlier results obtained for a circular freely jointed polymer chain. Assuming that [delta Lk)2) = [delta Tw)2) we have compared our results with experimental data on the chain length dependence of the [delta Lk)2) value recently obtained by Horowitz and Wang for small DNA rings. This comparison has shown an excellent agreement between theory and experiment and yielded a reliable estimate of the torsional and bending rigidity parameters. Namely, the torsional rigidity constant is C = 3.0.10(-19) erg cm, and the bending rigidity as expressed in terms of the DNA persistence length is a = 500 A. The obtained value of C agrees well with earlier estimates by Shore and Baldwin as well as by Horowitz and Wang whereas the a value is in accord with the data of Hagerman. We have found the data of Shore and Baldwin on the chain length dependence of the [delta Lk)2) value to be entirely inconsistent with our theorectical results.     

Microfabricated polymer chip for capillary gel electrophoresis

A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length.     

Separation of Different Conformations of Plant Mitochondrial DNA Molecules by Field Inversion Gel Electrophoresis

Mitochondrial (mt) DNA structure in higher plants is still unclear as to the circularity or linearity of the genome. We have developed a system to electrophoretically separate distinct populations of mtDNA, with some populations enriched for networked linear and circular DNA molecules. Using field inversion gel electrophoresis (FIGE) and electron microscopy (EM), we have identified four distinct populations of mtDNA from two Brassica species. Using FIGE, two slow migrating mtDNA populations ran faster than a 66 kbp Escherichia coli circular plasmid marker, while these same populations comigrated in the compression zone in contour-clamped homogeneous electrophoretic field (CHEF) gels. A fast-migrating mtDNA population was also resolved by FIGE as a diffuse band between 20 to 70 kbp when compared with linear lambda () markers. FIGE resolved the 66 kbp circular marker into several multimers, while CHEF resolved only open-circular monomers and linears. In agreement with FIGE results, EM analysis indicated the two slow migrating mtDNA populations contained circular (both supercoiled and relaxed circles) and free linear molecules of 10-60 kbp, and networked linear molecules of 45–140 kbp total size that may represent recombination intermediates. The fast migrating population consisted of 10–50 kbp linear molecules. Well-bound mtDNA showed only long linear molecules of 40–150 kbp with no detection of circles or complex/rosette molecules. This report shows that FIGE has clear advantages over CHEF for separating large DNA molecules with different conformations, and may be very useful for studies to characterize genome structure in complex systems such as plant mitochondria.     

Immobilization of S-methyltransferase

S-methyltransferase was solubilized from pig liver microsomes by treatment with N-dodecyl-N,N-dimethyl-3-ammonio-1-sulfonate (Zwittergent). The soluble enzyme was immobilized by covalent binding to agarose and by copolymerization with acrylamide. The specific activity for the agarose-bound enzyme towards the substrate ethane thiol was 0.87 nmol/min/mg and for the acrylamide-bound enzyme 0.55 nmol/min/mg. The specific activity of the soluble enzyme was found to vary with increasing chain length of the substrate molecules from 0.5 nmol/min/mg for methane thiol (C1) to 6.3 nmol/min/mg for n-heptane thiol (C7). After binding of the enzyme to agarose beads, the increase in specific activity towards substrates with increasing chain length was no longer detectable. Instead, a relatively constant specific activity of 1.1 nmol/min/mg was observed for the whole range of substrates tested from C1 to C7. The stability of the agarose immobilized enzyme at -20 degrees C is twice as good as the soluble enzyme. The acrylamide immobilized enzyme is less stable than the soluble enzyme.     

Filamentous Biopolymers on Surfaces: Atomic Force Microscopy Images Compared with Brownian Dynamics Simulation of Filament Deposition

Nanomechanical properties of filamentous biopolymers, such as the persistence length, may be determined from two-dimensional images of molecules immobilized on surfaces. For a single filament in solution, two principal adsorption scenarios are possible. Both scenarios depend primarly on the interaction strength between the filament and the support: i) For interactions in the range of the thermal energy, the filament can freely equilibrate on the surface during adsorption; ii) For interactions much stronger than the thermal energy, the filament will be captured by the surface without having equilibrated. Such a ‘trapping’ mechanism leads to more condensed filament images and hence to a smaller value for the apparent persistence length. To understand the capture mechanism in more detail we have performed Brownian dynamics simulations of relatively short filaments by taking the two extreme scenarios into account. We then compared these ‘ideal’ adsorption scenarios with observed images of immobilized vimentin intermediate filaments on different surfaces. We found a good agreement between the contours of the deposited vimentin filaments on mica (‘ideal’ trapping) and on glass (‘ideal’ equilibrated) with our simulations. Based on these data, we have developed a strategy to reliably extract the persistence length of short worm-like chain fragments or network forming filaments with unknown polymer-surface interactions.     

Purification of plasmid DNA by an improved electrophoretic protocol

Plasmid DNA from Escherichia coli was isolated by electroelution carried out in an agarose gel that contains an incorporated dialysis membrane. As the relative mobility of circular plasmid DNA to linear chromosomal DNA increases when the agarose concentration is decreased, we were able to purify plasmids of up to 50 kbp in 0.3% agarose gel in Tris acetate buffer yielding 10-60 g DNA ml bacterial culture.     

Theory of gel electrophoresis of DNA

A theory of the electrophoresis of DNA through gels with large interfiber spacing, such as dilute agarose, is presented. We assume that the DNA molecule moves along its axis through a “tube” in a neutral gel under the influence of the electric field. The tube is random except for possible bias due to the effects of the field. When the field is small, we easily recover the inverse-length dependence of the mobility found previously by de Gennes and by Doi and Edwards. At higher fields, a new effect appears; the tube becomes oriented because the field biases the direction of the leading end of the chain as it moves to form an extension of the tube. This leads to an increase of the mobility with increasing field by adding a field-dependent but length-independent term to the mobility expression. In agreement with experiment, we find that the field effect can be important at fields as low as 1 V/cm and that the effect can seriously decrease the sensitivity of the mobility to chain length. We also examine the fluctuation of the migration distance, the degree of orientation induced by the field, and the transient effects occurring when the feld direction is rotated by a right angle.     

Algorithmic generation of freely jointed hard sphere chains and properties of their inertial tensors

A statistical algorithm, capable of generating a large number of freely jointed hard sphere chains, is presented. This is the first of a series of algorithms being developed to model unfolded proteins by different modes of hard sphere chains. The aim of these studies is to systematically investigate the effects of different factors, such as atomic radii, bond angles, torsion angles, chain length, etc., on the conformation of unfolded proteins and other random polymers. As continuous models, various types of hard sphere chains enable one to isolate the aforementioned factors one at a time for investigation and thus are advantageous over discrete lattice models. In particular, the freely jointed hard sphere chain model allows one to evaluate the excluded volume effect. As a first step in this endeavor, the average determinant D(N, r) and the average trace T(N, r) of the inertial tensor A of the random chains were calculated at various sphere radii r and chain lengths N. It is found that both the average determinant D(N, r) and the average trace T(N, r) scale linearly with chain length N after logarithmic transformation. However, the critical exponent of D(N, r) increases with r faster than that of T(N, r) as a result of the non-commutativity between the det operator and the average operator < >. The significance of the algorithm and the results obtained on understanding random polypeptide chains are discussed.     

Species of small DNA molecules found in mitochondria from sugarbeet with normal and male sterile cytoplasms

Summary Mitochondrial DNA and RNA were isolated from a range of normal and cytoplasmically male sterile sugarbeet varieties and breeding lines. When these nucleic acids were analysed by electrophoresis on agarose gels it was found that mitochondria from normal sugarbeet contain DNA species of sizes 1.3 kilobase pairs (kbp), 1.4 kbp and one of two other species of sizes 1.45 kbp and 1.5 kbp, in addition to their DNA of relatively much higher molecular weight. In contrast mitochondria from cytoplasmically male sterile sugarbeet contained only one of these DNA species, that of 1.5 kbp. Treatment with DNaseI, RNase and nuclease S1 showed that these species consisted of supercoiled circular DNA. It is not known whether the lack of the two smaller DNA species causes cytoplasmic male sterility, or whether the two traits are associated by chance.In addition it was found that mitochondria from some individual sugarbeets contained one or more types of high molecular weight RNA molecules which were probably double stranded. Also mitochondria from some sugarbeet lines and varieties contained a series of DNA molecules with molecular weights in the range 2 to 10 kbp. Neither these DNA molecules nor the RNA molecules were apparently correlated with cytoplasmic male sterility.     

Pulse time and agarose concentration affect the electrophoretic mobility of cccDNA during PFGE and FIGE [corrected].

Circular DNAs have been shown to migrate in an unusual manner during field inversion gel electrophoresis (FIGE) and orthogonal field alternating gel electrophoresis (OFAGE). We studied the effect of varying pulse time and agarose concentration on the electrophoretic mobility of supercoiled (ccc) DNAs ranging from 2 kbp to 16 kbp during FIGE and contoured homogeneous electric fields (CHEF). Both supercoiled and linear molecules display a minimum mobility as a function of pulse time in a CHEF apparatus. Linear and cccDNAs of the same size are differently affected by pulse time. Pulse-time dependence was observed for cccDNAs in both systems. Pulse-time dependence in FIGE is very small at a 1.0% agarose concentration, but is pronounced in 0.8% or 1.2% gels.     

Electrophoresis of DNA restriction fragments in poly-N-acryloyl-tris gels

Poly-N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) gels were evaluated as a matrix for DNA electrophoresis. The resolution of DNA restriction fragments in three poly(NAT)-N,N'-methylenebisacrylamide (Bis) gels (4, 5, and 6%) was compared with the resolution in polyacrylamide (AA)-Bis gels of the same percentage. Poly(NAT) gels were found to give a substantially improved separation of DNA fragments larger than 200 bp. In contrast to poly(AA) gels, DNA fragments of up to 4 kbp were well resolved in the new matrix. By pulse-field electrophoresis the useful separation range of poly(NAT) gels was expanded to at least 23 kbp. For DNA fragments below 10 kbp, the resolution was better than that in a 0.7% agarose gel. Thus poly(NAT) gels are most suitable for the electrophoretic separation of DNA molecules whose size is out of the optimal fractionation range of poly(AA) or agarose gels.     

Atypical sieving of open circular DNA during pulsed field agarose gel electrophoresis

Pulsed field agarose gel (PFG) electrophoresis, originally used to improve the resolution by length of linear DNA [Cantor et al. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 287-304], is found here to cause atypical sieving of 48.5-97.0-kb open circular DNA. Two procedures of PFG electrophoresis are used: rotating gel electrophoresis with rotation of 2 pi radians [2 pi RGE; Serwer, P., & Hayes, S.J. (1989) Appl. Theor. Electrophor. (in press)] and field inversion gel electrophoresis [FIGE; Carle, G.F., Frank, M., & Olson, M. V. (1986) Science 232, 65-68]. During 2 pi RGE at 6 V/cm, the electrophoretic mobility (mu) of 48.5-kb open circular DNA increases in magnitude as agarose percentage (A) increases from 0.4 to 1.5. The sieving revealed by this mu vs A relationship is highly atypical (possibly unique) for any particle. The extent of this atypical sieving increases as electrical potential gradient, DNA length, and pulse time increase. In some cases a maximum is observed in a plot of mu's magnitude vs A. The mu of open circular lambda DNA is smaller in magnitude than the mu of equally long linear lambda DNA. Atypical sieving has also been observed by use of FIGE. As pulse times used during FIGE decrease below those achievable by 2 pi RGE, the progressive loss of circular DNA's atypical sieving is accompanied by both a dramatic increase in mu's magnitude at the lower A values and a decrease in mu's magnitude at the higher A values. At the lower A values, open circular DNA sometimes migrates more rapidly than linear DNA of the same length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small-angle neutron scattering by a strongly denatured protein: analysis using random polymer theory.

Small-angle neutron scattering profiles are presented from phosphoglycerate kinase, in the native form and strongly denatured in 4 M guanidinium chloride (GdnHCl) solution. The data are interpreted using a model in which the excess scattering density associated with the protein is represented as a finite freely jointed chain of spheres. The similarity of the model-derived scattering function to experiment increases asymptotically with the number of spheres. The improvement of the fit obtained with more than approximately 200 spheres (i.e., two residues per sphere) is insignificant. The effects of finite size of the scattering units and of scattering length variation along the polypeptide chain are examined. Improved agreement with experiment is obtained when these effects are taken into account. A method for rapid calculation of the scattering profile of a full, all-atom configuration is examined. It is found that a representation of the chain containing two scattering units per residue, placed at the backbone and side-chain scattering length centroids, reproduces the full, all-atom profile to within 2%.     

Agarose gel electrophoretic evidence for domains of nuclear DNA linked with bonds cleavable with sulfhydryl molecules

Complexes of intact nuclear DNA with proteins undissociable by 2.0 M NaCl and nonionic detergents were analyzed by agarose gel electrophoresis following physical or enzymatic fragmentation. Sulfhydryl molecules converted these DNAs (but not the bacteriophage lambda DNA) into smaller-Mr forms. Following limited restriction endonuclease digestion of complexes with PstI most of the nuclear DNA formed a high-molecular-mass band in the 60-110 kbp range. These 60-110 kbp fragments, releasable from the rest of nuclei by sulfhydryl molecules, have similar sizes to nuclear DNA loops detected by other techniques and may derive from supranucleosomal organizational units in the chromatin complex.     

Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Molecular Stretching of Long DNA in Agarose Gel Using Alternating Current Electric Fields

We demonstrate a novel method for stretching a long DNA molecule in agarose gel with alternating current (AC) electric fields. The molecular motion of a long DNA (T4 DNA; 165.6 kb) in agarose gel was studied using fluorescence microscopy. The effects of a wide range of field frequencies, field strengths, and gel concentrations were investigated. Stretching was only observed in the AC field when a frequency of ∼10 Hz was used. The maximal length of the stretched DNA had the longest value when a field strength of 200 to 400 V/cm was used. Stretching was not sensitive to a range of agarose gel concentrations from 0.5 to 3%. Together, these experiments indicate that the optimal conditions for stretching long DNA in an AC electric field are a frequency of 10 Hz with a field strength of 200 V/cm and a gel concentration of 1% agarose. Using these conditions, we were able to successfully stretch Saccharomyces cerevisiae chromosomal DNA molecules (225-2,200 kb). These results may aid in the development of a novel method to stretch much longer DNA, such as human chromosomal DNA, and may contribute to the analysis of a single chromosomal DNA from a single cell.     

Torsional and Bending Rigidity of the Double Helix from Data on Small DNA Rings

Abstract

We have calculated the variance of equilibrium distribution of a circular wormlike polymer chain over the writhing number, ?(Wr)²?, as a function of the number of Kuhn statistical segments, n, For large n these data splice well with our earlier results obtained for a circular freely jointed polymer chain. Assuming that ?(ΔLk)²? = ?(ΔTw)²? + ?(Wr)²? we have compared our results with experimental data on the chain length dependence of the ?(ΔLk) ²? value recently obtained by Horowitz and Wang for small DNA rings. This comparison has shown an excellent agreement between theory and experiment and yielded a reliable estimate of the torsional and bending rigidity parameters. Namely, the torsional rigidity constant is C = 3.0·10^?19 erg cm, and the bending rigidity as expressed in terms of the DNA persistence length is a = 500 A. The obtained value of C agrees well with earlier estimates by Shore and Baldwin as well as by Horowitz and Wang whereas the a value is in accord with the data of Hagerman. We have found the data of Shore and Baldwin on the chain length dependence of the ?(ΔLk) ²? value to be entirely inconsistent with our theoretical results.     

Human ureaplasmas show diverse genome sizes by pulsed-field electrophoresis.

Contour clamped homogeneous field (CHEF) agarose gel electrophoresis (AGE), ramped to give linear separation of DNA molecules of 600-1600 kilobase pairs (kbp), was used to determine mobilities for full-sized genomic DNA of the serotype standard strains of the human genital mollicutes, Ureaplasma urealyticum relative to yeast chromosomal DNA markers. Indicated genome sizes (in kbp) were 760 for the four biotype 1 strains and 840-1140 for eleven biotype 2 strains. Other estimates were: 720 for Mycoplasma hominis, 1070 for Mycoplasma hyopneumoniae, 890 for Mycoplasma flocculare, 1180 and 1350 for Mycoplasma mycoides subsp. mycoides Y and GC1176-2, respectively, and 1650 and 1580 for Acholeplasma laidlawii B and PG 8, respectively. These data supplement previous evidence from CHEF AGE that the genomes of the Mycoplasmataceae are diverse in size with some larger than previously estimated from DNA renaturation kinetics.     

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.