Fig. 1 Dependence of solid state31 P NMR linewidth on DNA concentration suggesting multiple phase transitions. DNA fragments of average length 146 base pairs (~500 Å) and with a narrow distribution about this length were prepared by digestion with micrococcal nuclease of calf-thymus chromatin previously depleted of histone H1, and by subsequent deproteinization 16 . Short DNA molecules are studied as a preliminary step to understanding the natural behavior of DNA at in vivo concentrations. Such defined samples are useful because effects of relative molecular mass heterogeneity are minimized and phase transitions are sharp and kinetically rapid. Using DNA fragments of this size is appropriate because the driving forces are the same for ordering of high axial ratio, rod-like molecules and long semi-flexible polymers of identical composition 5,6 . Open circles: data taken at 30ºC; filled circles: data taken at 50ºC. Spectra were obtained on non-spinning samples at a phosphorus frequency of 61.3 MHz on the 'Seminole', an in-house constructed multinuclear Fourier transform (FT) spectrometer equipped with wide-bore superconducting solenoid and quadrature detection, modified for solid-state applications. Sweep width was ±25 kHz, pulse repetition times were 3 s, and 600 scans were added for each spectrum. Gated proton decoupling was applied during data acquisition.
The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.
Excellent electrophoretic separations of a variety of biological molecules can be accomplished by using uncharged, triblock copolymers as the ''gel'' media. These copolymers form uncrosslinked, lyotropic liquid crystalline phases of large micelles between which molecules must travel. Unlike crosslinked hydrogels in common use, these alternative media have highly ordered internal structures. Pluronic F127, representative of the copolymer class, contains poly(ethylene oxide) (EO) and poly(propylene oxide) (PO) units with an approximate molecular formula (EO) 106 (PO) 70 (EO) 106 . Concentrated (18-30%) solutions of Pluronic F127 are freely f lowing liquids at low temperature (0-5°C) but form gel-like, cubic liquid crystals of large, spherical micelles when warmed. The utility of these media is illustrated by separations of linear, double-stranded DNA up to 3,000 bp long by conventional electrophoresis, and of single-stranded DNAs from 4 to 60 nt long by capillary electrophoresis. Extraordinary separations of supercoiled DNAs were also obtained by capillary electrophoresis. The versatility, availability, and ease of use of Pluronic polymers offer major advantages over conventional media for preparative and high performance analytical separations of nucleic acids and other biomolecules. Mechanisms of molecular transport and separation operating in polymer liquid crystals must differ in fundamental ways from those in crosslinked gels. Lyotropic polymer liquid crystals are unique systems for elucidating mechanisms of macromolecule migration in ordered, dense media, and provide opportunities in separations science.Electrophoresis is an essential separation method of biochemistry and molecular biology that distinguishes between proteins or nucleic acids differing only slightly in size, charge, conformation, or degree of association. Gene mapping, DNA sequencing, and transcription factor identification stand out among recent advances uniquely attributable to this method.Conventional electrophoresis is usually conducted on crosslinked hydrogels such as polyacrylamide ''chemical'' gels, formed by solution polymerization of simple monomers; or agarose ''physical'' gels formed by hydrogen bonding between pre-existing polymer chains. These completely interconnected polymer networks provide mechanical support and quench convection, and separate molecules by sieving according to size and shape. Structural details such as pore shape, size distribution, and connectivity strongly influence separations. The structures of both gel classes have been studied, and there is a basic understanding of how globular proteins (1-5) and semirigid polymers such as DNA (6-9) migrate through gel networks, but understanding of the influence of gel structure on separations is still incomplete.Capillary electrophoresis (CE) with high-voltage gradients offers much higher resolution than conventional electrophoresis or HPLC (10-13) and has been proposed to speed DNA sequencing for the Human Genome Project (14, 15). CE is often performed in con...
Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.
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