density gradient. These bands appear in new positions, because denatured DNA has a higher density than native DNA. In semiconservative replication, the single strands of denatured DNA should have half the molecular weight of the native hybrid molecule. In the case of the conservative model mentioned above, the strands of denatured DNA should have a molecular weight one-quarter that of the hybrid molecule. The experimental results favored again the semi- conservative model, but the experimental accuracy was not sufficiently high to make the results entirely clear-cut. However, put together, the evidence in favor of semiconservative replication.is sufficient to allow us to accept this model. = In this connection (see also later) we should mention that the denatured DNA can reform to the native form? in spite of the fact that in denatured DNA the two strands have come apart. We note in passing that there is relatively little DNA between the three band positions indicating that the extracted DNA has almost always the uensities of pure *®N-15N, pure '4N-'5N, or pure 14N-14N DNA. This observation implies that the replication process replicates a complete length of DNA before going on to the next molecule. However, this dramatic conclusion applies only to the relatively short lengths of DNA which were prepared for analysis by the methods available at the time of the Meselson-Stahl experiment (Fig. 2.10). Had the molecules survived intact, then such unsynchronized cell populations would have produced some DNA of intermediate buoyant densities, and this would have obscured the tesultant picture. Inspection of Fig. 2.7(b) makes the point clear. PROPERTIES OF DNA In the isolation of DNA a major difficulty is to keep such a long rigid molecule physically intact. It has been shown '°~?? that the shear forces set up when a solution of DNA is vigorously stirred are sufficient to break the long rigid DNA molecule. The molecules are first broken near the middle, then into quarters, and so on until a short-enough size is reached that the molecule will be relatively insensitive to breakage by shearing. It is difficult to extrapolate the molecular weight of DNA found in solution to the actual molecular weight that exists in vivo because of the great ease of degradation by shearing during extraction or by the inadvertent action of hydrolytic enzymes. Perhaps the best estimates are derived from radioauto- graphic work in which the DNA is labeled with tritium. For example, in E. co/i, Cairns! was able to detect DNA molecules up to 1.1 mm in length. This corresponds to a molecular weight of 2.8 x 109%. Similarly the DNA from T2 bacteriophage has an average length of 49 yu, which would correspond to a molecular weight of 108. A rather smaller bacteriophage, called 2, has a DNA 23 in length. But even in the most carefully prepared samples of extracted DNA there is always 0.1 to 0.2 percent of protein attached to the material. Therefore the possibility existed that a DNA molecule is actually made up of shorter lengths of DNA held together by protein or peptide-like materials (see review inReference 13). However, the successful synthesis in vitro'* by known reactions of the circular double-stranded replicative form of @X 174 DNA, capable of infecting host cells, makes it likely that such DNA molecules are composed only of nucleotides. A method for isolating DNA almost free from protein and RNA was published, for example, by Marmur.' The walls of bacterial cells are digested with the enzyme lysozyme or broken with a detergent. Sodium perchlorate is used to dissociate protein and nucleic acid, and the solution is deproteinized by shaking it with a mixture of chloroform and isoamyl alcohol. RNA is removed with ribonuclease or by centrifugation ina density gradient of cesium chloride. In this gradient, RNA, which has a much higher density than DNA, is pelleted at the bottom of the centrifuge tube. The DNA may be selectively precipitated with isopropyl alcohol. Attack by deoxyribonuclease can be minimized if the operations are conducted in the presence of chelating agents or in the presence ofa detergent such as sodium dodecyl sulfate. This procedure yields biologically active DNA such as, for example, transforming DNA. The DNA obtained however is somewhat degraded by the shear forces developed during shaking. An alternate procedure for isolating highly polymerized DNA from animal tissues is the phenol-extraction method of Kirby,’® in which proteins are denatured at the phenol-water interphase and the nucleic acids are extracted into the aqueous layer. RNA would be removed as mentioned above and the DNA precipitated. For examination in the electron microscope, solutions of DNA are sprayed onto the grid and dried rapidly. Native DNA cléarly gives long, stiff rod-like molecules, whereas the heat-denatured or otherwise-denatured (random-coil) molecules form “puddles.” Almost all the native DNAs in solution from a large variety of sources have the double-stranded structure as shown by the hyperchromic shift (see below) " that occurs when a DNA solution is heated. On the other hand, denatured DNA is a flexible, loosely coiled, polyelectrolyte chain, very dependent in its hydrodynamic properties on the ionic environment. In fact, the denatured DNA in solution is rather similar to high-molecular-weight RNA, which shows some, but by no means complete, secondary structure. Closely similar also are the single-stranded synthetic polyribonucleotides made by the enzyme poly- nucleotide phosphorylase.'? A naturally occurring example of randomly coiled single-stranded DNA is found in the small bacteriophage xX 174. In general, the secondary structure of native DNA depends on its content of the bases guanine and cytosine (G + C). The higher the G + C content, the Stronger the forces holding the two chains together. This is possible because guanine and cytosine can form three hydrogen bonds when paired in the DNA Structure, whereas adenine and thymine can only form two (see Fig. 2.3). The hAyperciiromic shift is the increase in absorbency observed when double-stranded DNA is denatured to the single-stranded form. In an analogous Case (Fig. 2.11), the two polyribonucleotides polyadenylic acid and polyuridylic acid when mixed together have a lower absorbency at 260 mye than that cal- culated for the two polymers measured separately. This is so because theseza Unapter 2. DNA Nucleotides === =Polymers separately Mixture of polymers Extinction coefficient X 10” 220° 240 260 280 . Wavelength, mz Fig. 2.11 Absorption spectra of equimolar quantities of polyadenylic acid and polyuridylic acid, The upper curve refers to the mononucleotides obtained by alkaline hydrolysis. 20 9% increase x Thymus DNA * Enzymatic product Time, hrs Fig. 2.12. Increase in ultraviolet-light absorption at 260 my of natural and synthetic DNA upon digestion with pancreatic deoxyribonuclease.