Separation of Microbial Deoxyribonucleic Acids Into Complementary Strands

Abstract

Abstract.-DNA preparations from seven bacterial species and from the E. coli phage T4 can, after denaturation with alkali, be separated chromatographically into two distinct components (L and H) through intermittent gradient elution from methylated albumin kieselguhr columns. The direct chemical analysis of the L and H fractions isolated from DNA specimens of the AT type shows them to exhibit a high degree of complementarity; but despite a bias in the distribution of purines and pyrimidines, either fraction contains equimolar quantities of 6-amino and of 6-keto nucleotides. In the L and H components derived from DNA of the equimolar and GC types, the distribution bias appears limited to guanine and cytosine. It is suggested that the L and H fractions represent the complementary DNA strands.As we reported recently,1-3 denaturated DNA of Bacillus subtilis can be separated by a technique of intermittent gradient elution from a column of methylated albumin kieselguhr (MAK), into two fractions, designated, by virtue of their buoyant densities, as L (light) and H (heavy). Taking into account some of the requirements that genuine strands deriving from a native DNA duplex must fulfill,4 we concluded (on the basis of evidence bearing on the biological activity, nucleotide composition, and temperature-absorbance behavior) that the two fractions isolated from B. subtilis DNA represented indeed such complementary strands. They could be annealed together easily, thereby regaining considerable secondary structure as shown by transforming activity and hypochromicity; they were in their nucleotide composition strictly complementary so that the adenine content of fraction L was equivalent to the thymine content of fraction H, the guanine of L to the cytosine of H, etc.; the greater abundance of purines in fraction L was matched by that of pyrimidines in fraction H. As would be expected of genuine strands,4 the molar sums of A + T and of G + C did not vary in the two fractions5 and were identical with those found in the native DNA, and this was, therefore, also true of the dissymmetry ratio A + T/G + C; but the preparations also exhibited an unexpected regularity, which would not have been predicted for a single strand, namely, the equality of the 6-amino and the 6-keto nucleotides, A + C and G + T.We were naturally interested in the generality of these observations and in their biological meaning with respect to template functions, etc. While the latter problem will be taken up in a subsequent paper, we present here evidence that denatured DNA from seven microbial species and from bacteriophage T4, representing DNA varieties of the AT, GC, and equimolar types, can be separated on MAK columns into two distinct and reproducible chromatographic 152

Distribution of Pyrimidine Oligonucleotides in Strands L and H of Bacillus subtilis DNA

“…As shown in Fig. 3, the distribution of the cytosine oligonucleotides of chain length [1][2][3][4][5] in both strands closely follows the statistical expectations with the abrupt disappearance of longer clusters. In contrast, the distribution of thymine oligonucleotides in both strands deviates from the calculated statistical distributions.…”

“…The base composition of intact lambda strands (22) resembles the values obtained for the strands of the bacterial host E. coli (1), and the distribution of pyrimidine oligonucleotides in the strands of lambda DNA consistently shows a greater asymmetry in the number of cytosine-rich clusters (22). One may conclude that irrespective of the origin and nature of the modifications that brought about the chemical asymmetry between the strands, the principal residues involved must have been cytosine and guanine.…”

“…Fractionation of the L and H strands, after alkali denaturation, by chromatography on MAK columns has been described in detail (1,8 Fig. 1).…”

“…Direct chemical analyses (1) and nucleotide composition of RNA transcripts synthesized in vitro show that complementary strands of DNA from bacteria exhibit various degrees of chemical asymmetry, namely, that the L strand is purine-rich and the H strand is pyrimidine-rich. For example, Bacillus subtilis DNA represents a type in which the chemical asymmetry between the strands involves both purines and pyrimidines, while in Escherichia coli, the asymmetry is limited to only guanine and cytosine.…”

Action of DNA Polymerase I of Escherichia coli with DNA-RNA Hybrids as Templates

“…The separation of denatured DNA of B. megaterium into the complementary strand fractions L and H by chromatography on a methylalbumin-kieselguhr column (7) and the 398 base composition and other properties of the separated preparations have been described (6,8).…”