Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 60
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The Phosphates(P) join the 3' carbon atom of one deoxyribose(horizontal line) to the 5' carbon atom of theadjacent deoxyribose.what misleading. DNA is a dynamic molecule, constantly in motion, In some regions, the strands can separatebriefly and then come together again in the same conformation or in a different one. Although the right-handeddouble helix in Figure 5.4 is the standard form, DNA can form more than 20 slightly different variants of righthanded helices, and some regions can even form helices in which the strands twist to the left (called the Z form ofDNA). If there are complementary stretches of nucleotides in the same strand, then a single strand, separated fromits partner, can fold back upon itself like a hairpin. Even triple helices consisting of three strands can form inregions of DNA that contain suitable base sequences.5.3—What a Genetic Material Needs That DNA SuppliesNot every polymer would be useful as genetic material.
However, DNA is admirably suited to a genetic functionbecause it satisfies the three essential requirements of a genetic material. First, any genetic material must be able tobe replicated accurately, so that the information it contains is precisely replicated and inherited by daughter cells.The basis for exact duplication of a DNA molecule is the complementarity of the A–T and G-C pairs in the twopolynucleotide chains. Unwinding and separation of the chains, with each free chain being copied, results in theformation of two identical double helices (Figure 5.7).A genetic material must also have the capacity to carry all of the information needed to direct the organization andmetabolic activities of the cell.
As seen in Chapter 1, the product of most genes is a protein molecule—a polymercomposed of molecular units called amino acids. The sequence of amino acids in the protein determines itschemical and physical properties. A gene is expressed when its protein product is synthesized, and one requirementof the genetic material is that it direct the order in which amino acid units arePage 182Figure 5.7Watson-Crick model of DNA replication. Thenewly synthesized strands are in red. We willsee in Section 5–6 that the details of DNAreplication are considerably more complex, becauseany new DNA strand can be synthesized only inthe 5'-to-3' direction.added to the end of a growing protein molecule. In DNA, this is done by means of a genetic code in which groupsof three bases specify amino acids.
Because the four bases in a DNA molecule can be arranged in any sequence,and because the sequence can vary from one part of the molecule to another and from organism to organism, DNAcan contain a great many unique regions, each of which can be a distinct gene. A long DNA chain can direct thesynthesis of a variety of different protein molecules.A genetic material must also be capable of undergoing occasional mutations in which the information it carries isaltered. Furthermore, the mutant molecules must be capable of being replicated as faithfully as the parentalmolecule, so that mutations are heritable. Watson and Crick suggested that heritable mutations might be possible inDNA by rare mispairing of the bases, with the result that an incorrect nucleotide becomes incorporated into areplicating DNA strand.5.4—The Replication of DNAThe process of replication, in which each strand of the double helix serves as a template for the synthesis of a newstrand (Figure 5.7), is simple in principle.
It requires only that the hydrogen bonds joining the bases break to allowseparation of the chains and that appropriate free nucleotides of the four types pair with the newly accessible basesin each strand. However, it is a complex process with geometric problems requiring a variety of enzymes and otherproteins. These processes are examined in this section.The Basic Rule for the Replication of Nucleic AcidsThe primary function of any mode of DNA replication is to reproduce the base sequence of the parent molecule.The specificity of base pairing—adenine with thymine (replaced by uracil in RNA) and guanine with cytosine—provides the mechanism used by all genetic replication systems.
Furthermore,• Nucleotide monomers are added one by one to the end of a growing strand by an enzyme called a DNApolymerase.• The sequence of bases in each newly replicated strand, or daughter strand, is complementary to the basesequence in the old strand, or parental strand, being replicated. For example, whereverPage 183an adenine nucleotide is present in the parental strand, a thymine nucleotide will be added to the growing end of thedaughter strand.The following section explains how the two strands of a daughter molecule are physically related to the two strandsof the parental molecule.The Geometry of DNA ReplicationThe production of daughter DNA molecules from a single parental molecule gives rise to several problems thatresult from the helical structure and great length of typical DNA molecules and the circularity of many DNAmolecules.
These problems and their solutions are described here.Semiconservative Replication of Double-Stranded DNA In the semiconservative mode of replication, eachparental DNA strand serves as a template for one new strand, and as each new strand is formed, it is hydrogenbonded to its parental template (Figure 5.7). As replication proceeds, the parental double helix unwinds and thenrewinds again into two new double helices, each of which contains one originally parental strand and one newlyformed daughter strand.In theory, DNA could be replicated by a number of mechanisms other than the semiconservative mode.
However,the reality of semiconservative replication was demonstrated experimentally by Matthew Meselson and FranklinStahl in 1958. The experiment made use of a newly developed high-speed centrifuge (an ultracentrifuge) thatcould spin a solution so fast that molecules differing only slightly in density could be separated. In theirexperiment, the heavy 15N isotope of nitrogen was used for physical separation of parental and daughter DNAmolecules.
DNA isolated from the bacterium E. coli grown in a medium containing 15N as the only available sourceof nitrogen is denser than DNA from bacteria grown in media with the normal 14N isotope. These DNA moleculescan be separated in an ultracentrifuge, because they have about the same density as a very concentrated solution ofcesium chloride (CsCl).When a CsCl solution containing DNA is centrifuged at high speed, the Cs+ ions gradually sediment toward thebottom of the centrifuge tube. This movement is counteracted by diffusion (the random movement of molecules),which prevents complete sedimentation.
At equilibrium, a linear gradient of increasing CsCl concentration—and ofdensity—is present from the top to the bottom of the centrifuge tube. The DNA also moves upward or downwardin the tube to a position in the gradient at which the density of the solution is equal to its own density. Atequilibrium, a mixture of 14N-containing (''light") and 15N-containing ("heavy") E.
coli DNA will separate into twodistinct zones in a density gradient even though they differ only slightly in density. DNA from E. coli containing14N in the purine and pyrimidine rings has a density of 1.708 g/cm3, whereas DNA with 15N in the purine andpyrimidine rings has a density of 1.722 g/cm3. These molecules can be separated because a solution of 5.6 molarCsCl has a density of 1.700 g/cm3.
When spun in a centrifuge, the CsCl solution forms a gradient of density thatbrackets the densities of the light and heavy DNA molecules. It is for this reason that the separation technique iscalled equilibrium density-gradient centrifugation.The result of the Meselson-Stahl experiment is shown in Figure 5.8. Prior to the experiment, bacteria were grownfor many generations in a 15N-containing medium. Therefore, at the beginning of the experiment, essentially all theDNA was uniformly labeled with 15N and had a heavy density.
The cells were then transferred to a 14N-containingmedium, and DNA was isolated from samples of cells taken from the culture at intervals and subjected toequilibrium density-gradient centrifugation. Each photograph in Figure 5.8 shows the image of a solution within acentrifuge tube taken in ultraviolet light of wavelength 260 nm (nanometers), which is absorbed by DNA. Thepositions of the DNA molecules in the density gradient are therefore indicated by the dark bands that absorb thePage 184Connection Replication by HalvesMatthew Meselson and Franklin W. Stahl 1958California Institute of Technology,Pasadena, CaliforniaThe Replication of DNA in Escherichia coliReplication of DNA by separation of its strands, followed by the synthesis of a new partner strand for eachseparated parental strand, is so fundamental a biological mechanism that it is easy to forget that it had to beproved experimentally somewhere along the way.
Although semiconservative replication is a simple and logicalway to proceed, biological systems might have found a different way to replicate their DNA. The Meselson-Stahlexperiment that demonstrated semiconservative replication made use of a novel technique, density-gradientcentrifugation, which was invented by the authors specifically for the purpose of separating DNA moleculesdiffering slightly in density. The use of cesium chloride was mandated, because in concentrated solution, it has adensity similar to that of DNA.
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