Total intensity, Rayleigh light scattering has been used to measure the rms radius, second virial coefficient, persistence length, and excluded volume of homogeneous T7 bacteriophage DNA as a function of Na+ concentration (0.005 to 3.0 M). All parameters decrease sharply as [Na+] increases, and tend to level off at high Na+. The variation of persistence length with [Na+] is consistent with predictions from counterion condensation theory.
Functional genes can be introduced into mammalian cells in vitro by a variety of physical methods, including direct microinjection, electroporation, and co-precipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes into tissues of intact animals. In contrast, receptor-mediated gene transfer has been shown to successfully introduce DNA into suitable recipient cells, both in vitro and in vivo (1-12). This procedure involves the formation of a complex between DNA and a polycation (such as poly-L-lysine), which bears a covalently linked ligand moiety specific for a given receptor on the surface of cells in the target tissue. The gene is internalized by the tissue, transported to the nucleus, and expressed in the cell for varying lengths of time (1,3,6,11). The overall level of expression of the transgene in the target tissue is dependent on several factors, such as the stability of the DNA/ligand⅐poly-L-lysine complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-DNA/ligand interaction, endocytosis of the DNA complex and the efficiency of gene transcription in the nucleus of the target cells.DNA in the nucleus of a higher eukaryote is intimately associated with basic nuclear proteins rich in lysine (i.e. histones) or arginine (i.e. protamines). The interaction of DNA with these basic proteins is responsible for the control of the condensation process that occurs upon chromosome formation during metaphase and is thought to play a role in the regulation of gene expression. DNA condensation, which occurs naturally in viruses, bacteria, and eukaryote nuclei, has been extremely difficult to reproduce in the laboratory (13,14). Due to the high negative charge of the DNA phosphate backbone, an increase in the degree of charge neutralization of the DNA theoretically results in extensive condensation and the separation of the DNA phase in the form of insoluble compact structures (15, 16). We have found, however, that the structure and stoichiometry of DNA⅐polycation complexes in solution can be manipulated by means of the process by which DNA⅐cationic polypeptide complexes are formed.Specific complexes of DNA (⌿-DNA) are formed with cationic homo-polypeptides (poly-L-lysine, poly-L-arginine, or poly-L-ornithine) after "annealing" both components in a step-down dialysis from NaCl concentrations of 3 to 0.010 M (11, 16). In contrast, direct addition of basic polypeptides to DNA at physiological salt concentrations results in reversible molecular aggregation and the formation of precipitates (7,17,18). Shapiro et al. (16) elucidated changes in DNA secondary structure in DNA⅐poly-L-lysine complexes prepared by directly mixing poly-L-lysine and DNA. The physical properties of the resulting soluble complexes were investigated by circular dichroism (CD) and optical rotatory dispersion. A change in the magnitude of the molar residue rotation was found, with a characteristic red shift and a strong negative rotatory transition center...
Static light scattering measurements have been made at angles as low as 8 degrees on whole, half, and quarter molecules of native, T2 bacteriophage DNA in 0.195 M Na+. The fragments were obtained by high-speed stirring of the native DNA, and fractionated on methylated-albumin-kieselguhr columns. Accompanying measurements of sedimentation coefficients and intrinsic viscosities were made. Because linear extrapolations of light scattering data above 8 degrees for these samples were suspect, the measurements were analyzed by fitting curves calculated from the theory of wormlike coils to experimental curves at c = 0. Results showed that the excluded volume parameter, epsilon, must be used in analyzing the scattering curves; a reasonable value of epsilon was 0.08, in agreement with that found for T7 DNA (Harpst, J. A. 1980. Biophys. Chem. 11:295-302). The persistence length of all three DNAs in this paper was 50 +/- 5 nm, showed no dependence on molecular weight, but was somewhat below that reported previously for T7 DNA (60 nm). Theoretical curves calculated with the preceding parameters had a clear upward curvature in scattering envelopes below 8 degrees for quarter and half molecules, but such curvature was minimal for whole T2 DNA, so that linear extrapolations of experimental data above 8 degrees gave a molecular weight and root-mean-square radius which were nearly the same as those from theory. The molecular weight and radius for whole T2, derived from the comparison of theory and experiment, were 115 X 10(6) and 1,224 nm, respectively. The measurements on T2 DNA were clearly at the upper limit of current techniques.
SUMMARYAdenovirus type 5 'cores' prepared by heating in the presence of deoxycholate and partially purified on a glycerol density gradient could be visualized as roughly isometrical particles with a condensed centre from which twisted filaments or loops of DNA emanated. This compact structure was readily dispersed by spreading on distilled water or by treatment with EDTA, Nonidet, DNase or trypsin. Spreading with Nonidet was particularly effective in unfolding the cores and revealing long filaments about IOO A thick presumably of the virus nucleoprotein. Subunits (about 30 to 60 A in diam.) could be seen free in the DNase-treated cores, suggesting a particulate nature of one or both of the core proteins.
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