Interaction Between Denatured Dna, Polyribonucleotides, and Ribosomal Rna: Attempts at Preparative Separation of the Complementary Dna Strands

The buoyant density of DNA in the CsCl equilibrium density gradient depends on its composition and secondary structure. Denatured DNA's usually form a single band (Fig. 1B and F) at a density 0.013 (high % G + C) to 0.017 (low % G + C) gm/cm3 higher than that of the same DNA in its native helical form (Fig. 1A and E). Experience with Escherichia coli (Fig. 1F and H) and several other organisms had indicated that the band profile and density of denatured and rapidly cooled DNA are only slightly altered, if at all, by the presence of RNA during the denaturation and centrifugation procedures. It was somewhat surprising, therefore, to observe, in addition to the denaturedlike DNA band (dN'), a DNAand RNA-containing heavy band (DR) during CsCl gradient centrifugation of a thermally denatured and rapidly cooled DNA + RNA mixture extracted from Bacillus subtilis (Fig. 1D). Earlier2 and current studies on the origin, properties, and composition of the materials contained in the DR and dN' bands are the subject of this communication. Materials and Methods.-Nucleic acids extracted from the wild type and several mutant strains of B. subtilis,3 Clostridium perfringens,4 and E. coli strain B were chiefly employed. The DNA + RNA extract was prepared by lysis of the cells with lysozyme (100 ,g/ml) and sodium lauryl sulfate (1%) followed by exhaustive deproteinization with a chloroform + butanol mixture (4:1), precipitation with 1.5 volumes of ethanol, and prompt solution of the precipitate in SSC. Pure DNA was obtained from this mixture by treatment with RNase (100 Ag/ml, 30 min, 37?C), deproteinization, and selective isopropanol precipitation.5 The molecular weight of the DNA thus prepared was usually above 30 X 106 daltons. RNA was extracted from a centrifuged pellet of exponentially growing cells by treatment with lysozyme and sodium lauryl sulfate, and grinding in Tris buffer (0.01 M Tris, 0.1 M NaCl, 0.01 mg MgCl2, pH = 7.0) with bentonite (1%), sand, DNase (50 aLg/ml) and liquefied phenol (Mallinckrodt, A. R.); all these operations, including subsequent phenol deproteinization, were carried out at 0?C. Ribosomal RNA fractions (16S and 23S) were isolated by chromatography at 350C on a methylesterified albumin-kieselguhr column,6 or by the sucrose gradient centrifugation procedure. Poly A, poly C, poly G, poly I, and poly U are commercial products of the Miles Chemical Company, Elkhart, Ind. Poly G was purchased as a copolymer of inosinic and guanylic acids (1:1), but upon analysis was found to contain less than a few per cent inosinic acid residues; thus it will be referred to as poly G. Analytical or preparative CsCl or Cs2S04 density-gradient centrifugation was employed for the identification and isolation of the DNA, RNA, and DR and dN' bands.' Stepwise chromatography on Dowex-1 resin served to determine the