The isolation and biophysical characterisation of DNA from Proteus mirabilis <13/S suc+>)

Abstract

Deoxyribonucleic acid was extracted from Proteus mirabilis (13/S suc+ ) protoplasts with buffers containing sodium chloride, ethylenediaminetetra acetate, sodium lauryl sulphate and Tris (hydroxymethyl) amino methane. Different results were obtained when the sodium chloride concentration in these buffers was varied, while the concentrations of the other buffer components were kept constant. Deproteinisation was achieved with phenol, while the RNA was removed by RNase treatment, followed by adsorption of the digestion products on activated charcoal. Phenolphthalein diphosphate proved to be unsuccessful in removing the RNA. Extraction with buffers containing 1 M NaCl and 5 mM NaCl gave fairly good DNA yields, viz. 65. 43% and 68. 72%, respectively. Extraction with 0.15 M NaCl gave a very poor yield (8. 38%), the greatest loss occurring during the deproteinisation. The purity of the DNA preparations was determined according to chemical assay methods, while the homogeneity was determined by sucrose density gradient and sedimentation velocity centrifugation. All these results indicated pure, homogeneous preparations. The biophysical constants of the DNA were determined. The sedimentation and diffusion coefficients of the whole molecules were 62. 5S and 0. 253 x 10- 7cm 2/sec, respectively. The sedimentation coefficients were determined at 29,500 r.p.m. The diffusion coefficients were calculated from the boundary spreading at low centrifugal forces. In both cases ultra-violet optics were employed. The intrinsic viscosity (262. 5 dl/g) was determined at a shear stress of 1. 8 x 10-3dynes/cm2 in a rotating cylinder viscometer. The partial specific volume (0. 5608) was calculated from the solvent and solution densities, which were determined in density gradient columns. The molecular weight was calculated from the equations of Svedberg, Eigner and Doty, Rubenstein, Thomas and Hershey and according to the Crothers and Zimm equation. The values obtained from the above-mentioned equations for whole molecules were 13 x 106, 114. 3 x 106, 132 x 106 and 112 x 106, respectively. Half molecules were obtained when 1 M NaCl was the extractant while quarter molecules were produced when inadequate precautions were taken to avoid shear forces during the isolation procedures. The base ratio, determined from the melting temperature of the DNA was 37 .1 mole per cent of guanine plus cytosine.