DNA genetica

Density gradient. These bands appear in new positions, because denatured DNA has a higher density tan native DNA. In semiconservative replication, the single strands of denatured ADN should have half the molecular weight one-quarter that of the hybrid molecule. The experimental results favored again the semiconservative model, but the experimental accuracy was not sufficiently high to make the result 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 thaht 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 densities of pure N-N, pure N-N, or pore N-N DNA. This observation implies that the replication process replicates a complete length of DNA before going on to the next molecule. However, this dramatic conclusión applies the methods available at the time of the Meselson-Stahl experiment. 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 resultant picture. Inspection of figure … 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 qurters, and so on until a short-enoungh 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 geat ease of degradation by shearing during extraction or by the inadvertent action of graphic work in which the DNA is labeled with tritium. For example, in E. coli, Cairns was able to delect DNA molecules up to 1.1 mm in length. This corresponds to a molecular weight of 2.8X10. similarly the DNA from T2 bacteriophage has an average length of 49 , which would correspond to a molecular weight of 10. A rather smaller bacteriophage, called lamda 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 melecule is actually made up of shorter lengths of DNA help together by protein or peptide-like materials (see review in Roference 13). However, the successful synthesis in vivo by known reactions of the circular doublé-straded replicative form of oX174 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 nucleica cid, and the solution is deproteinized by shaking it with a mixture of chloroform and isoamyl alcohol. RNA is removed with ribonuclease or by centrifugation in a density gradient of cesium chloride. In this gradient, RNA, wich has a much higher density that DNA, is pelleted at the bottom of the centrifuge tuve. 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 of a 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 iolating highly polymerized DNA  from animal tissues is the phenol-extraction method of Kirby, in which proteins are denature 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 clearly gives long, stiff rod-like molecules, whereas the heat-denatured or otherwise-denatured (random-coil) molecules form “puddles”

Almost all the native DNA in solution from a large variety of sources have the doublé-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 polynucleotide phodphorylase. A naturally occurring example of randomy coiled single-stranded synthetic polyribonucleotides made by the enzyme polynucleotide phosphorylase. A naturally occurring example of rndomly coiled single- stranded DNAis found in the small bacteriophage oX174.

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 stroger the forces holding the two chains togethe. This is posible because guanine the forces holding the two chains together. This is posible because guanine and cytosine can form three hydrogen bonds when pired in the DNA structure, whereas adenine and thymine can only form two.

The hyperchromic shift is the increase in absorbency observed when doublé-stranded DNA is denatured to the single-stranded form. In n analogous case, the two polyribonucleotides polyadenylic at 260 micrometros than that claculated for the two polymers measured separately. This is so because these polyribonucleotides can form base pairs A-U polydeoxyribonucleotides can be expected to behave in a similar manner. Another way of observing the hyperchromic shift is by destroying the doublé-stranded helical structure through the hydrolytic action of a nuclease.

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