Biomedical subjects
Publications and source records attributed to L S Lerman.
The electrophoretic mobility of double helical DNA in agarose and polyacrylamide gels increases as a function of time after the electric field is applied to the gel and decreases after the field is terminated. The changes are large for long (more than 10 kb) molecules. The effects of other variables are indicated.
DNA molecules, several persistence lengths long in sedimentation equilibrium at speeds high enough to maintain fairly close packing, show a dense, sharply-bounded turbid phase and an isotropic phase (as with shorter fragments) and also an intermediate, somewhat turbid region. The concentration distribution in the isotropic phase is in satisfactory agreement with a simple extension of scaled particle theory in which semiflexible chains are equivalent to straight rods of the same length. The net intermolecular interactions, as inferred from the Zimm cluster integral, are purely repulsive. As in our previous study with short fragments, the results are compatible with a hard-core electrostatic radius, decreasing with increasing salt concentration. However, for the longer fragments it is necessary to infer either a slightly greater mass per unit length or a slightly smaller electrostatic radius for closest agreement with scaled particle theory. The properties of the solution at the boundary with the turbid, presumably strongly ordered phase are consistent with those found for shorter fragments and with theoretical scaling expectation for a hard, asymmetric particle.
The variation in electrophoretic mobility of DNA under conditions of marginal helix stability provides a useful means for investigation of the relation between the helix-random chain transition and base sequence in natural DNA and a powerful procedure for separation of DNA molecules according to sequence. The use of statistical mechanical theory for analysis of the transition equilibria together with new, simplified theoretical considerations on the effect of strand unravelling on mobility have shown that the gel behavior is predictable for known sequences. A number of the distinctive consequences of the theory and their correspondence with the properties of real molecules have been demonstrated. These include the extremely close cooperative linkage of large blocks of bases into domains, the existence of sharp boundaries between domains, the major role of nearest-neighbor interaction in determining stability, the dependence of domain structures on neighboring and more remote sequences, and the depression of domain melting temperature if the sequence lies at the end of a molecule. New and unusual applications derive from the possibility of separating DNA molecules by properties of their sequence. Exceedingly complex mixtures, such as the sum of all fragments produced by the action of a sixbase specific restriction endonuclease on a complete bacterial genome, can be resolved completely. Additional inserted sequences are easily discerned. The difference of a single base pair in a molecule permits detection and isolation of mutant sequences. The need for full sequential analysis of long molecules for characterization of mutants can be reduced by localizing a change within a small fragment.
The illustration that accompanied the review by C. C. Albritton, Jr., of W. H. Goetzmann and K. Sloan's Looking Far North (Viking, New York, 1982) in the issue of 10 December, page 1109, should have been credited to the Bancroft Library, University of California, Berkeley, as well as to the book under review.
DNA fragments 536 base pairs long differing by single base-pair substitutions were clearly separated in denaturing gradient gel electrophoresis. Transversions as well as transitions were detected. The correspondence between the gradient gel measurements and the sequence-specific statistical mechanical theory of melting shows that mutations affecting final gradient penetration lie within the first cooperatively melting sequence. Fragments carrying substitutions in domains melting at a higher temperature reach final gel positions indistinguishable from wild type. The gradient data and the sites of substitution bracket the boundary between the first domain and its neighboring higher-melting domain within eight base pairs or fewer, in agreement with the calculated boundary. The correspondence between the gradient displacement of the mutants and the calculated change in helix stability permits substantial inference as to the type of substitution. Excision of the lowest melting domain allows recognition of mutants in the next ranking domain.
Torsional thermal oscillations of the DNA double helix within the electron paramagnetic resonance (EPR) time scale (10(-10)-10(-3) s) as indicated by a rigid, intercalating probe are much smaller in the spacer segment between nucleosomes in chromatin than in long, free DNA molecules. Still smaller DNA oscillation is indicated in intact nuclei and yet smaller if the nuclei have been treated with glutaraldehyde. The values of EPR measurements are not affected by the loading density of probe. If the probe were capable of substantial oscillations or movement different from that of the helix, those oscillations would be expected to dominate the spectra when movement of the helix is restrained. We conclude that the correlation time for torsional movement of free DNA inferred from EPR spectra is characteristic of the double helix and that there is no significant independent motion of the probe. The correlation time for the DNA double helix in molecules longer than approximately 500 base pairs is close to 30 ns, corresponding to an elastic constant of 1.5 X 10(-19) ergs cm for deformation by twisting. The motions observed in chromatin are consistent with a model in which spheres of 50-60-A radius are connected by simple elastic rods with the length of spacer DNA and the same elastic constant. The spin-labeled ethidium probe has been characterized in detail by nuclear magnetic resonance, infrared, fluorescence, and visible light spectroscopy. The binding equilibria are consistent with the hypothesis that strongly immobilized probe molecules are preferentially bound to spacer DNA.
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The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule.
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When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60 degrees C in a concentration gradient of a urea-formamide mixture, each Eco RI fragment of lambda or E. coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment has been retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a two-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the Eco RI digest of E. coli DNA. Corresponding fragments from an isogenic lambda lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.
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In the presence of over-threshold concentrations of simple neutral polymers and salts, DNA undergoes a cooperative change in its solution structure. Sedimentation studies at low DNA concentrations show that phage DNA molecules collapse into particles approaching the compactness of the contents of phage heads. The interaction between DNA and polymers is thought to be nonspecifically replusive.
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