The acceleration of linear DNA during pulsed-field gel electrophoresis

The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur. For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s. T4 DNA behaves similarly, but more slowly. Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied. The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity. When the field strength increases, both the velocity and the alignment reach their peaks more quickly. For all field strengths and both molecular weights, the velocity peak occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length. A qualitative model is provided.     

Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels.

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.     

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.     

Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments

Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.     

Use of pulsed field gel electrophoresis to size the chromosome of the bacterial fish pathogen Yersinia ruckeri

Field inversion gel electrophoresis (FIGE) and contour-clamped homogeneous field (CHEF) electrophoresis were used to analyse the chromosome of Yersinia ruckeri. The 8 base-pair recognition endonucleases, NotI and SfiI, generated less than 47 DNA fragments whose size and distribution were appropriate for pulsed field separation. Each isolate displayed a characteristic restriction pattern, with about 20% of bands in common. Depending on the strain used, the estimated genome size for this bacterial fish pathogen ranged from 4460 to 4770 kilobase pairs.     

Dynamics of single-stranded DNA in polyacrylamide gels during pulsed field gel electrophoresis. A birefringence study

The study of the orientation of single-stranded DNA in polyacrylamide gels in denaturing conditions has been undertaken by electric birefringence in order to determine the mechanism involved in the electrophoretic transport. The presence of an overshoot in the birefringence signal, when applying the electric field, and the study of the influences of the electric field and of the gel concentration on the dynamics show that a mechanism of reptation with elongation of the molecule occurs in polyacrylamide gels with low T values. Therefore it is suggested that the use of pulsed fields in sequencing electrophoresis is possible and can lead to a large increase of the length of the fragments that can be sequenced in one single run.     

Orientational dynamics of T2 DNA during agarose gel electrophoresis: influence of gel concentration and electric field strength

The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration ("electrophoretic orientation") is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Akerman, and B. Nordén, (1988) Biopolymers 27, 381-414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23-kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3-2%), electric field strength (0-40 V/cm), and pulse characteristics. The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady-state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over- and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented. The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.     

Mitogen dose-dependent effect of weak pulsed electromagnetic field on lymphocyte blastogenesis

The effects of pulsed extremely low frequency electromagnetic fields on human peripheral blood lymphocyte mitogenesis induced by phytohaemoagglutinin, concanavalin A or calcium ionophore A23187 were studied. The dependence of the field effect on mitogen concentrations was investigated. Field exposure produced strong inhibition of DNA synthesis when optimal doses of mitogens were used, confirming our previous findings. Opposite effects were observed at suboptimal concentration of mitogens. Experiments performed by exposing cell cultures to the field for short periods indicated that a field application of at least 6 h is needed to influence irreversibly lymphocyte blastogenesis.     

Single-molecule DNA digestion by lambda-exonuclease.

We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained lambda-DNA attached to optically trapped beads. The setup has been described previously (Dapprich and Nicklaus: Bioimaging 6:25-32, 1998) and works by observing the change in position of a trapped bead depending on its viscous drag force during motion. The drag force of a naked bead increases with each attached DNA molecule to a characteristic level that depends on the length and the number of DNAs per bead. A single undigested DNA molecule on a bead will remain stable for extended periods and exhibit a constant drag force in flow. If lambda-exonuclease is added, the drag force decreases from the level for one strand of DNA on a bead to that of a naked bead in about 45 min. This result indicates that the digestion of native lambda-DNA by lambda-exonuclease occurs at an average rate of approximately 15-20 Hz.     

A tightly regulated inducible expression system utilizing the fim inversion recombination switch

The fim inversion system of Escherichia coli (E. coli) can behave as a unidirectional switch in an efficient manner. We have developed a new expression system for E. coli, comprising the arabinose-inducible fimE gene and the fim invertible DNA segment containing a constitutively active promoter. In this system, the target gene is cloned with the promoter in the OFF orientation, resulting in no transcribed product. When induced by arabinose, the active promoter is switched to the ON orientation via FimE-catalyzed DNA inversion, and the gene is expressed. Our expression system exhibited very tightly controlled basal expression and high induced expression, with simple induction by inexpensive arabinose. These characteristics make our system suitable for large-scale expression or for production of toxic proteins.     

Dipole moment analysis of membrane proteins suggests role in orientation in the membrane

The position independent dipole membrane proteins need to be oriented in the membrane in order to function as channels, transporters or recognition systems. Membrane proteins can be broadly classified as either predominantly alpha helical or beta barrel in nature. All the different types of thirteen beta barrel membrane proteins (2OMF, 2POR, 1PRN, 1PHO, 1IIV, 1AF6, 1AOT, 2MPR, 1OSM, 1QJ8, 1BXW, 2FCP and 1FEP) and six alpha helical membrane proteins (1BL8, 1MSL, 1QLB, 1AR1, 1PSS and 1QHJ) from the Protein Data Bank were analyzed. Dipole moment was calculated for both classes of proteins. In all the oligomers, the orientation of the dipole was found to be parallel to direction of insertion that is perpendicular to the possible membrane layer. Monomers do not show a similar orientation. In all the alpha helical oligomers, the dipole points from the intra-cellular to the extra-cellular side. In the oligomeric beta barrel proteins, the direction of the dipole is from the extra-cellular to the intra-cellular side, except for OmpF from E.coli, Omp36 from Klebsiella pneumonia and LamB from E.coli where the situation is reversed. However, the dipole moments of the monomeric proteins and the monomers of the oligomers themselves are not oriented parallel to the molecular axis and the insertion orientation, but they are almost parallel to the membrane surface. It is possible that the quaternary oligomeric association is necessary for the correct orientation in the membrane and this is aided by the dipole orientation. The electrostatic potential surface calculated with all atoms, which also do not show clear separation of charge surfaces. Calculations suggest that backbone structure and oligomer are sufficient for providing the dipole orientation.     

The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

Bacteriophage lambda int gene is required for the integration of viral DNA into the chromosome of Escherichia coli. We have extensively purified the product of the int gene (Int) from a lysogen of E. coli that constitutively expresses this gene. Int was assayed by its ability to promote integrative recombination of supertwisted substrate DNA in vitro using a new method based on filter trapping of a recombinant product DNA. In order to catalyze integrative recombination, Int must be supplemented by other factors that can be extracted from bacterial host cells. By itself, purified Int does not demonstrate detectable endonuclease, exonuclease, or nicking-closing activities. However, Int does make stable complexes with double-stranded lambda-DNA containing an attachment site, the region at which recombination takes place. No stable complexes are observed between Int and lambda-DNA without an attachment site or between Int and DNA containing the bacterial site of integration. Int, therefore, appears to be a specificity element that relies on additional factor(s) to provide or activate the catalytic functions required for recombination.     

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.     

Field inversion gel electrophoresis with different pulse time ramps.

The influence of different pulse time ramps on the separation of yeast chromosomes with field inversion gel electrophoresis (FIGE) was investigated by the means of two dimensional gel electrophoresis. The problem of band inversion, which makes it difficult to distinguish DNA molecules of different size, has been solved by using double randomized pulse times. A major disadvantage of the field inversion technique is thereby overcome, making this system comparable to other pulsed field techniques.     

Growth control of mouse mammary epithelial cells by keratin antibody-targeted liposome containing oligonucleotides antisense to epidermal growth factor receptor

NMuMG cells were incubated with 17beta-estradiol (E)+progesterone (P)+epidermal growth factor (EGF), with or without various types of oligomers (21-mers) to the EGF receptor activity domain (amino acid residues 718 to 724). Sense or antisense oligomers were encapsulated in protein A-bearing liposomes. Uncoupled protein A and unencapsulated sense or antisense oligomers were separated from liposomes on a Sepharose 4B column (the encapsulation efficiency of oligomers in liposome-protein A was 0.8%). The addition of various concentrations of EGF to E+P showed an interaction between them during DNA synthesis (P<0.05). Antisense oligomers (1 microM) decreased DNA synthesis induced by E+P+EGF (65.0% inhibition, P<0.05). Sense oligomers also inhibited DNA synthesis induced by E+P+EGF (P<0.05). However, random-sequence oligomers did not inhibit EGF-induced DNA synthesis. We cannot rule out the possibility that sense oligomers match an unknown functional gene mRNA involved in cell growth, which causes their inhibitory effect. Cells were incubated with a keratin monoclonal antibody and then with dilutions of protein A-bound liposomes containing sense or antisense oligomers in the presence of E+P+EGF. Dose dependent inhibition of DNA synthesis was observed. The encapsulated oligomers in protein A-bound liposomes inhibited DNA synthesis at a 100-fold lower concentration than that of unencapsulated oligomers or the oligomer+liposome mixture. The tyrosine kinase activity domain has an important role in EGF regulation of mammary growth. The effect of a cytokeratin-targeted antibody on DNA synthesis in normal mouse mammary epithelial cells was marginal.     

The mechanism of ion polarisation along DNA double helices

The orientation curves of short DNA fragments induced by electric field pulses are measured with high time resolution and analysed by efficient deconvolution techniques. A small, but clearly detectable delay of the 'on-field' orientation can be described accurately by the superposition of two exponential processes with opposite amplitudes. The time constant of the faster process is around 10 ns and the slower one in the range 50-1000 ns depending upon the electric field strength and chain length of the DNA fragment. The relation between amplitudes and time constants observed for each curve corresponds exactly to that expected for a convolution of two processes, where the first process is without optical response and becomes detectable only via the optical response of the second process. These results indicate that the first process reflects the polarisation of the ion atmosphere required for the second process of the orientation. Measurements at different ion concentrations c demonstrate that the reciprocal time constant of the fast process is a linear function of c and thus is consistent with an association reaction. The association rate constant evaluated from this dependence according to a simple bimolecular reaction model is 8 X 10(9) M-1 s-1 for a 95 base-pair fragment and is consistent with binding of Na+ to the helix, a reaction close to the limit of diffusion control. The association rate constant is almost independent of the electric field strength E, while the dissociation rate constant k- strongly increases with E, indicating dissociation of ions at high E values. The data suggest a linear correlation between log(k-) and E2 corresponding to a reaction driven by a dipole change. The apparent dipole change evaluated from this dependence is in the order of magnitude estimated for an elementary step of ion dissociation at one end of the helices. The combined results obtained from the polarisation and the orientation mechanism can be explained by dissociation of surprisingly few counterions biased towards one end of the helices. The experimental data obtained for a 76 base-pair fragment are analogous to those for the 95 base-pair fragment, whereas the 'slow' ion polarisation has not been detected for a fragment with 27 base-pairs. This result together with those obtained for the longer fragments at low field strengths indicate that there is a fast polarisation mechanism without 'ion dissociation' at low chain lengths and for low electric field strengths. This mechanism is replaced at high chain lengths and/or high electric field strengths by the ion dissociation mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

A computer program allows the separation of a wide range of chromosome sizes by pulsed field gel electrophoresis

The introduction of Pulsed Field Gel Electrophoresis techniques, which allow the separation of DNA molecules of molecular weights as high as chromosomes of lower eukaryotes, has given a powerful tool to geneticists. The resolution expected from these techniques is dependent on numerous parameters, among them pulse time and field strength. A given set of these parameters allows only a limited range of molecular weights to be resolved. To allow the separation of a broader molecular weight range on a single gel, we designed a computer program, driving a simple switching device, to take care of switching electrodes and power supplies in OFAGE migrations. This program has been designed to be used with any technique calling for periodic switching or inversion of the electric field, and/or variation of the electric field applied during electrophoresis. As an example, we show the results obtained with yeast genera in which chromosome sizes range from 260 to 9,000 kilobase pairs.     

Electric birefringence of polytetrafluoroethylene particles in agarose gels

Electric birefringence studies of strongly elongated, rod-like particles of polytetrafluoroethylene (PTFE) in agarose gels show that the negative effect observed by semi-diluted aqueous suspensions at low frequencies and at low electric field strengths (the so called "anomaly') disappears. The absolute value of the low frequency effect increases 3-4 times and the amplitude of modulation decreases faster compared to that of the suspensions. This together with decreased decay relaxation times in gels make the possibilty that the PTFE particles orientation in gels is not due to dipolar but to electrophoretic orientation mechanism quite probable. Similar change in the orientation mechanism could be expected also for suspensions of higher concentrations. The further elucidation of the orientation mechanism using fractions with lower polydispersity, broader ranges of experimental conditions (particle concentration, ionic strength and composition, electric field strengths, frequencies, etc.) could be interest for several fields: colloid electro-optics and especially that of concentrated colloids, pulsed field gel electrophoresis of DNA (and especially its sinusoidal biased field variant) and of nucleoprotein complexes and for the gel research.     

Excision of chromosomal DNA loops by treatment of permeabilised cells with Bal 31 nuclease

The specificity of long-range fragmentation of human nucleolar genes by Bal 31 nuclease was studied using fractionation of cleavage products by pulsed field gel electrophoresis, followed by Southern hybridization. It was found that limited treatment of permeabilised cells with this nuclease results in accumulation of DNA scissions in matrix attachment areas. Consequently, chromosomal DNA loops and their oligomers are released from the genome.