摘要:

AbstractGel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes.Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels.Junctions containing intramolecular “tethers” that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to moverelative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti‐parallel. In this article we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti‐parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over arange of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these

ISSN:0173-0835    

DOI:10.1002/elps.1150100512

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractCurved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved seguences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence‐specific DNA twist and wedge angle

ISSN:0173-0835    

DOI:10.1002/elps.1150100513

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe use of gel electrophoresis in studies of nucleic acid‐protein (especially DNA‐protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the “gel retardation” assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, “snakelike”, manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of theE. colicatabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild typelacbinding site is retained in complexes where RNA polymerase is bound simultaneously at thelacU

ISSN:0173-0835    

DOI:10.1002/elps.1150100514

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractNative gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein‐DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process Factors which affect the lifetimes and mobilities of protein‐DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA‐protein interac

ISSN:0173-0835    

DOI:10.1002/elps.1150100515

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractTemperature‐gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein‐nucleic acid interactions. A linear and highly reproducible temperature‐gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature‐gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand‐separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double‐stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (–)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch‐formation during the renaturation of (+)‐ and (−)strands from different clones. Examples are given for different viroid clones and HIV‐clones from one and the same patient. In another example, clones with point mutations from site‐directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor fromE. colion the thermal stability of the represser and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytic

ISSN:0173-0835    

DOI:10.1002/elps.1150100516

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe describe the use of denaturing gradient gel electrophoresis to screen for DNA sequence polymorphisms in the human factor VIII gene. DNA fragments that differ in sequence by only a single base pair can be separated on denaturing gradient gels due to changes in their melting behavior. Previous studies have demonstrated the use of denaturing gradient gels to detect sequence changes in human genomic DNA, including mutations in the β globin gene and polymorphisms on chromosome 20. We have begun to use denaturing gradient gels to look for polymorphisms within the human factor VIII gene. The DNA sequences of seven cloned fragments from introns in the human factor VIII gene were determined and used to predict a melting map for each fragment. The melting behavior of each cloned fragment was evaluated by electrophoresis into denaturing gradient gels. Appropriate fragments were then used as radioactive probes for hybridization to human DNA samples that had been digested with restriction enzymes. Heteroduplexes formed between the probe and genomic DNA samples were electrophoresed into denaturing gradient gels. The final positions of heteroduplex bands were determined by autoradiography. We describe a general approach for using denaturing gradient gel electrophoresis to find DNA polymorphisms, with particular emphasis on the predictive value of DNA sequence data. We compare the efficiency of polymorphism detection by denaturing gradient gel electrophoresis with detection by restriction fragment length polymorphism (RFLP) analysis. The factor VIII gene appears to have a low level of DNA sequence polymorphism. We detected only two rare sequence variants using seven different probes to screen almost 2500 base pairs from each of at least 12 different chromosomes. This low level of sequence polymorphism is consistent with other measurements suggesting that nucleotide variations occur less frequently on the X chromosome than on autosomes

ISSN:0173-0835    

DOI:10.1002/elps.1150100517

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe hydroxyl radical has been used as a chemical probe to study in solution the structure of DNA and DNA‐protein complexes. The hydroxyl radical abstracts a deoxyribose hydrogen atom, cleaving one strand of the DNA. The cutting pattern, visualized by separating the cleavage products using gel electrophoresis, shows the reactivity of each backbone position toward the radical. This method has been applied to studies of DNA bending and helical twist. Phased runs of adenines (adenine tracts) cause sequence‐directed DNA bending. The hydroxyl radical cleavage of a bent DNA fragment containing short adenine tracts phased with the helix screw gives rise to an unusual cutting pattern. The hydroxyl radical cleavage rate decreases in the 5′ to 3′ direction along each adenine tract, with a minimum at the 3′ end of each adenine tract. The cleavage of the matching thy mine tract is similar, but the minimum in the pattern is offset in the 3′ direction. This pattern on the autoradiograph of the gel is interpreted to indicate that bending is accompanied by a narrow minor groove in the DNA molecule. Furthermore, hydroxyl radical cleavage results in different cutting patterns for two similar sequences, (CGA4T4)5and (CGT4A4)5, which have been shown to be bent and relatively straight, respectively. The hydroxyl radical method has also been used to determine the helical repeat of the metallothionein IIAgene to be about 10.5 base pairs per turn. Methods of optimizing the hydroxyl radical reaction for DNA‐protein footprinting are discussed. Because individual gel bands give information about cutting frequency at particular positions in the backbone, gel resolution and clear autoradiographs are important to this work. We discuss two problems in these areas which our lab has encountered and solutions we have found for them. Band compressions often occur in G/C‐rich sequences. This can be avoided by adding 25–40 % formamide to 50 % urea denaturing polyacrylamide gels. Drying gels affords clearer autoradiographs of gels, yet higher percentage polyacrylamide gels are difficult to dry or even to remove from the plates. A method has been developed for drying 20–25 % polyacrylamide gels that involves using plastic wrap, instead of filter paper, to remove the gel fr

ISSN:0173-0835    

DOI:10.1002/elps.1150100518

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractQuantitative footprinting studies involving a 139‐base pair restriction fragment from pBR322 DNA, a lexitropsin ligand and two different DNA cleavage agents, the enzyme DNase I and the footprinting reagent Fe(III) methidium‐propyl‐EDTA (Fe‐MPE), are described. The autoradiographic data showed that the ligand, an analogue of netropsin possessing twoN‐methylimidazole groups, binds to four regions on the 139‐mer which are rich in GC. Analysis of the data leading to individual binding constants for each of the four loading events on the 139‐mer revealed that Fe‐MPE and DNase I report the same binding constants for the lexitropsin bound to its interaction sequences. The fact that the data from both probes can be analyzed using a common model indicates that the DNA cleavage specificity of the probe and not its binding/ cleavage mechanism is the important factor in reporting of site loading information in the footprinting experiment. The study also showed that under certain conditions it is possible to gain information on the density of ligand binding sites on carrier DNA by monitoring site loading events on the l

ISSN:0173-0835    

DOI:10.1002/elps.1150100519

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractChromosome‐size DNA molecules can now be separated using a variety of pulsd field gel electrophoresis techniques. In this article, we study the predictions of the biased reptation model concerning the effect of two pulsed fields, making an arbicray angle, on the power of separation of gel electrophoresis. Separation is predicted to be largely enhanced for obtuse angles, in agreement with experiments. Interestingly very large molecules, which are not separated by pulsed fields, are predicted not to migrate along the gel diagonal for fairly long periods of time. Finally, we discuss the optimization of these techniques using the results of the theory, and the limitations of the latter when fluctuations and intramolecular modes probably dominate the syste

ISSN:0173-0835    

DOI:10.1002/elps.1150100520

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe apply the concepts of tube and reptation to the pulsed electrophoresis of DNA, considering both biased reptation and “breathing” modes (internal modes of the chain). Using suitable preaveraging approximations, analytical expressions are derived which relate displacement in crossed field electrophoresis to molecular weight, field strength, field period, pore size of the gel, and the angle between the field. These expressions provide scaling laws for the change of mobility when one (or more) of the parameters is varied as well as “universal” velocityversusmolecular weightversuspulse time curves. These results are quantitatively compared with experiments. At some point which depends on field angle, field strength and chain length, however, we predict a failure of this model due to symmetry breakdown and loss of ergodicity. Qualitatively, this should lead to considerable band spreading and/or splitting of the highest DNA bands into two bands migrating sideways from the diagonal. The case of field inversion is also investigated. It is shown that only breathing modes can explain the strong differences in mobility experienced by chains of different length when opposite fields of equal amplitude are applied: the “trapping” of chains in conformations of low mobility is associated with an antiresonance‐like coupling between the external field and the i

ISSN:0173-0835    

DOI:10.1002/elps.1150100521

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY