A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Electrophoresis of DNA in agarose gels. II. Effects of loading mass and electroendosmosis on electrophoretic mobilities

We have investigated several experimental factors which affect the accurate determination of electrophoretic mobilities of circular and linear DNAs in agarose gels. We demonstrate that: (1) The mobility of individual DNA species is affected by the total mass in the sample loaded. The increased mobility and band distortion observed become apparent when the DNA mass exceeds approximately 0.2 μg per 0.15 cm² of surface area in the loading well. (2) The migration velocity of a given DNA species depends on the coefficient of electroendosmosis (?m_r) of the agarose preparations used. In the range 0.081 ≤ ?m_r ≤ 0.441, the DNA migration velocity is proportional to (?m_r)^?0.5.     

Orientation of DNA in agarose gels.

An orientation of the lambda DNA during the electrophoresis in agarose gels was measured by a microscopic linear dichroism technique. The method involved staining the DNA with the dye ethidium bromide and measuring under the microscope the polarization properties of the fluorescence field around the electrophoretic band containing the nucleic acid. It was first established that the fluorescence properties of the ethidium bromide-DNA complex were the same in agarose gel and in a solution. Then the linear dichroism method was used to measure the dichroism of the absorption dipole of EB dye bound to lambda DNA. In a typical experiment the orientation of two-tenth of a picogram (2 x 10(-13)g) of DNA was measured. When the electric field was turned on, the dichroism developed rapidly and assumed a steady state value which increased with the strength of the field and with the size of DNA. A linear dichroism equation related the measured dichroism of fluorescence to the mean orientation of the absorption dipole of ethidium bromide and to an extent to which the orientation of this dipole deviated from the mean. The observed development of dichroism in the presence of an electric field was interpreted as an alignment of DNA along the direction of the field. The increase in the steady state value of dichroism with the rise in the strength of the field and with the increase of the size of DNA was interpreted as a better alignment of DNA along the direction of the field and as a smaller deviation from its mean orientation.     

Production of antibody against T-2 toxin.

Antibody against T-2 toxin was obtained after immunization of rabbits with bovine serum albumin-T-2 hemisuccinate conjugate. The antibody had greatest binding efficiency for T-2 toxin, less efficiency for HT-2, and least for T-2 triol. Cross-reaction of antibody with neosolaniol, T-2 tetraol, and 8-acetyl-neosolaniol was very weak. Diacetoxyscirpenol, trichodermin, vomitoxin, and verrucarin A essentially gave no cross-reaction with the antibody. The sensitivity of the binding assay for T-2 toxin detection was in the range of 1 to 20 ng per assay. Detailed methods for the preparation of the conjugate and the production of immune serum and methods for antibody determination are described.     

Brownian diffusion and electrophoretic transport of double‐stranded DNA in agarose gels

The field free diffusion constant and the electric field dependence of the electrophoretic mobility and molecular orientation of DNA samples from 5 to 164 kilobase pairs in agarose gels from 0.5 to 2% have been measured by fluorescence recovery after photobleaching and birefringence. In conditions where the reptation predictions hold for the field free diffusion, they partially fail for the DNA size dependence of the low field limit of the electrophoretic mobility. The linear field dependencies of the electrophoretic mobility and orientation factor seem to favor the biased reptation model with fluctuations over the standard biased reptation model, which predicts a quadratic field dependence. The quantitative analysis of the molecular parameters shows, however, that most experiments have been carried out at values of the field where the difference between the two models may be less conclusive. The pore size dependence of the different quantities has been given a particular attention and the role of the distribution of pore sizes in the departures from the reptation predictions is discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 45–59, 1999     

从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

New process for T-2 toxin production.

Strains of Fusarium produced high levels of T-2 toxin when cultured on certain media absorbed into vermiculite. Modified Gregory medium was nutritionally complex (2% soya meal, 0.5% corn steep liquor, 10% glucose) and, when inoculated with the appropriate fungal strain, yielded maximum T-2 toxin within 24 days of incubation at 19 degrees C. On Vogel synthetic medium N (H. J. Vogel, Microb. Genet, Bull. 13:42-43, 1956) supplemented with 5% glucose, optimal toxin levels were synthesized after incubation for 12 to 14 days at 15 degrees C. Fusarium tricinctum T-340 produced 714 and 353 mg/liter on modified Gregory medium and Vogel synthetic medium N plus 5% glucose, respectively. Improved analytical procedures were developed and involved aqueous methanol extraction, purification by liquid-liquid partitions, and gas-chromatographic quantitation.     

The influence of agarose--DNA affinity on the electrophoretic separation of DNA fragments in agarose gels

The effects of DNA concentration, buffer composition, added "carrier" DNA, and chemical modification of agarose on the electrophoretic separation of DNA restriction fragments in agarose gels were tested. Electrophoretic zones of migrating DNA were found to broaden by trailing as sample load was decreased, and this effect was found to be more pronounced for species of higher molecular weight. As DNA sample load was increased, DNA fragments were found to move faster in the direction of electrophoresis (front forward). Sharp, well-resolved electrophoretic zones were obtained at very low DNA loads only when a high-salt, high-pH, high-EDTA buffer was employed or when "carrier DNA" having a broad and uniform molecular weight distribution was included in the sample. Moreover, DNA in high concentration was found to displace DNA in low concentration from a given gel region. Unmodified agaroses were found to differ only slightly in their effectiveness in retarding DNA fragments at a given agarose concentration. However, hydroxyethylated agarose was much more effective in retarding DNA, at a given gel concentration, than the unmodified agaroses tested. These results show that it is useful to consider the agarose gel matrix as possessing the properties of both a molecular sieve and a chromatographic adsorbent when designing electrophoretic separation techniques for DNA. A model for these separations which includes the effects of DNA-agarose interaction and molecular sieving is discussed.     

In vitro metabolism of T-2 toxin in rats.

T-2 toxin was rapidly converted in the 9,000 X g supernatant fraction of rat liver homogenate into HT-2 toxin, T-2 tetraol, and two unknown metabolites designated as TMR-1 and TMR-2. TMR-1 was characterized as 4-deacetylneosolaniol (15-acetoxy-3 alpha, 4 beta, 8 alpha-trihydroxy-12,13-epoxytrichothec-9-ene) by spectroscopic analyses. Since the same metabolites were also obtained from HT-2 toxin used as substrate, it was concluded that T-2 toxin was hydrolyzed preferentially at the C-4 position to give HT-2 toxin, which was then metabolized to T-2 tetraol via 4-deacetylneosolaniol. In addition to HT-2 toxin, 4-deacetylneosolaniol and T-2 tetraol, a trace amount of neosolaniol was transformed from T-2 toxin by rat intestinal strips. In vitro metabolic pathways for T-2 toxin in rats are proposed.     

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.     

T-2 toxin degradation by micromycetes

The biodegradation of T-2 toxin was studied by strains of micromycetes which were isolated from the environment. The 26 tested strains were divided into three groups. Group contains strains which degraded T-2 toxin very fast. This toxin could not be chromatographically determined in the medium even after 48 hours of incubation and the antifungal activity of residua against Kluyveromyces fragilis CCY-51-1-2 was low or zero. There were strains of Alternaria sp., Ulocladium sp., Aspergillus candidus, Cladosporium cladosporioides, Rhodotorula sp., Aspergillus flavus and Cladosporium macrocarpum. Group II contains with a low activity and in group III the results were variable and non stable.     

Detection of T-2 toxin by an improved radioimmunoassay.

T-2 toxin in serum, urine, and saline was analyzed by a modified radioimmunoassay procedure. The specimens were added directly to the assay tubes without extraction steps. The reaction between antibody and ligands was optimal at 1 h. Albumin-coated charcoal was used to separate bound from free radioactivity. Quenching, which occurred with hemolyzed specimens, was corrected by a wet oxidation process with 60% perchloric acid and 30% hydrogen peroxide. The shorter incubation times resulted in an assay that takes less than 6 h to complete. The average affinity constant of the antibody (Km) was 1.75 X 10(10) liters/mol. The sensitivity was 1 ng per assay or 10 ng/ml. Among the other trichothecenes tested, only H-T-2 cross-reacted significantly (10.3%).     

Electrophoresis and orientation of F-actin in agarose gels.

F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase activity of HMM. The mobility of F-actin increased with the rise in pH. Acto-S-1 complex was also able to migrate in agarose at basic pH, but at a lower rate than F-actin alone. The orientation of fluorescein labeled F-actin and of fluorescein labeled S-1 which formed rigor bonds with F-actin was measured during the electrophoresis by the fluorescence detected linear dichroism method. The former showed little orientation, probably because the dye was mobile on the surface of actin, but we were able to measure the orientation of the absorption dipole of the dye bound to S-1 which was attached to F-actin, and found that it assumed an orientation largely parallel to the direction of the electric field. These results show that actin can migrate in agarose gels in the F form and that it is oriented during the electrophoresis.     

Metabolism of T-2 toxin in Curtobacterium sp. strain 114-2.

The metabolic pathway of T-2 toxin in Curtobacterium sp. strain 114, one of the T-2 toxin-assimilating soil bacteria, was investigated by thin-layer and gas-liquid chromatographic analyses. T-2 toxin added to the basal medium as a single carbon and energy source was biotransformed into HT-2 toxin and an unknown metabolite. Infrared, mass spectrum, proton magnetic resonance, and other physico-chemical analyses identified this new metabolite as T-2 triol. T-2 toxin was first deacetylated by the bacterium into HT-2 toxin, and this metabolite was then biotransformed into T-2 triol without formation of neosolaniol and T-2 tetraol. No trichothecenes remained in the culture medium after prolonged culture. Some properties of T-2 toxin-hydrolyzing enzymes were observed with whole cells, the cell-free soluble fraction, and the culture filtrate. Besides T-2 toxin, trichothecenes such as diacetoxyscirpenol, neosolaniol, nivalenol, and fusarenon-X were also assimilated by this bacterium.     

Metabolic effects of trichothecene T-2 toxin

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.     

Electrophoretic mobility of DNA in gels. II. Systematic experimental study in agarose gels

A systematic study has been undertaken to prove or disprove the predictions of a revised reptation model, biased reptation with fluctuations (BRF). Our data, which scan about two orders of magnitude of DNA sizes and of electric fields, and a fourfold range of gel concentrations, are in qualitative and quantitative agreement with the model and support the applicability of this theory to DNA gel electrophoresis. In particular, we show that the mobility in the compression zone scales as the first power of the electric field, and that the limit of separation scales as the inverse first power of the electric field, for low enough fields. © 1994 John Wiley & Sons, Inc.     

Biosynthesis of radiolabeled T-2 toxin by Fusarium tricinctum.

Incubation of Fusarium tricinctum NRRL 3299 on a solid rice medium in the presence of [1-14C]sodium acetate, [2-3H]mevalonic acid, [2-14C]mevalonic acid, or [5-3H]mevalonic acid yielded preparations of radiolabeled T-2 toxin with specific activities of 1.008, 1.64, 0.656, and 7.35 muCi/mmol, respectively.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

T-2 toxin inhibits mitochondrial function in yeast

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.