Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 61
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The method of cesium chloride density centrifugation is still important in modernmolecular biology; it is used in the isolation of DNA molecules.Studies of bacterial transformation and bacteriophage infection strongly indicate that deoxyribonucleic acid (DNA)can carry and transmit hereditary information and can direct its own replication.
Hypotheses for the mechanism ofDNA replication differ in the predictions they make concerning the distribution among progeny molecules of atomsderived from parental molecules . . .. We anticipated that a label which imparts to the DNA molecule an increaseddensity might permit an analysis of this distribution by sedimentation techniques. To this end, a method wasdeveloped for the detection of small density differences among macromolecules . . .. A small amount of DNA in aconcentrated solution of cesiumThe results of the present experiment are in exact accord with the expectation of the WatsonCrick model for DNA duplication.chloride is centrifuged until equilibrium is closely approached.
The opposing processes of sedimentation anddiffusion have then produced a stable concentration gradient of the cesium chloride, with a continuous increase indensity along the direction of centrifugal force. The macromolecules of DNA present in this density gradient aredriven by the centrifugal field into the region where the solution density is equal to their own buoyant density . . ..Bacteria uniformly labeled with N15 were abruptly changed into N14 medium .
. .. Samples were withdrawn fromthe culture immediately and afterward at intervals for several generations . . .. Until one generation time haselapsed, half-labeled molecules accumulate. One generation after the switch to N14, these half-labeled or "hybrid"molecules alone are observed. Subsequently, only half-labeled DNA and completely unlabeled DNA are found.When two generation times have elapsed, half-labeled and unlabeled DNA are present in equal amounts.
Theseresults permit the following conclusions to be drawn: 1. The nitrogen of a DNA molecule is divided equallybetween two subunits which remain intact through many generations . . .. 2. Following replication, each daughtermolecule has received one parental subunit . . .. 3. The replicative act results in a molecular doubling . . .. Amolecular structure for DNA has been proposed by Watson and Crick that .
. .. suggested to them a definite andstructurally plausible hypothesis for the duplication of the DNA molecule . . .. The results of the present experimenare in exact accord with the expectation of the Watson-Crick model for DNA duplication . . .. The results presentedhere direct our attention to [other important problems]. What are the molecular structures of the subunits of DNAwhich are passed on intact to each daughter molecule? What is the relationship of these subunits to each other in aDNA molecule? What is the mechanism of the synthesis and dissociation of the subunits in vivo?Source: Proceedings of the National Academy of Sciences of the USA 44:671–682light.
Each photograph is oriented such that the bottom of the tube is at the right and the top is at the left. To the righof each photograph is a graph showing the absorption of the ultraviolet light from the top of the centrifuge tube to thbottom. In each trace, the peaks correspond to the positions of the bands in the photographs, but the height and widthof each peak make it possible to quantify the DNA in each band.At the start of the experiment (time 0), all of the DNA was heavy (15N).
After the transfer to 14N, a band of lighterdensity began to appear, and it gradually became more prominent as the cells replicated their DNA and divided. Afteone generation ofPage 185growth (one round of replication of the DNA molecules and a doubling of the number of cells), all of the DNA hada "hybrid" density exactly intermediate between the densities of 15N-DNA and 14N-DNA. The finding of moleculeswith a hybrid density indicates that the replicated molecules contain equal amounts of the two nitrogen isotopes.After a second generation of replication in the 14N medium (1.9 generations in the original experiment), half of theDNA had the density of DNA with 14N in both strands ("light" DNA) and the other half had the hybrid density.After three generations, the ratio of light to hybrid DNA was approximately 3 : 1, and after four generations (4.1 inthe original experiments), it was approximately 7 : 1 (Figure 5.8).
This distribution of 15N atoms is precisely theresult predicted from semiconservative replication of the Watson-Crick structure, as illustrated in Figure 5.9.Subsequent experiments with replicating DNA from numerous viruses, bacteria, and higher organisms haveindicated that semiconservative replication is universal.Figure 5.8The DNA replication experiment of Meselsonand Stahl. Cells whose DNA contained heavynitrogen were transferred into growth mediumcontaining only light nitrogen. Samples weretaken at intervals, and the DNA was subjectedto equilibrium density-gradient centrifugationin a solution of CsCl.
During centrifugation,each type of DNA molecule moves until itcomes to rest at a position in the centrifuge tubeat which its density equals the density of theCsCl solution at that position. Photographs ofthe centrifuge tubes taken with ultraviolet lightare shown at the left. The top of the tube is atthe left, the bottom at the right. The smoothcurves show quantitatively the amount ofabsorption of the ultraviolet light across thetube.[Photograph courtesy of MatthewMeselson. From M. Meselson and F. Stahl, Proc.Natl.
Acad. Sci. USA 1958. 44:671.]Page 186Figure 5.9Interpretation of the data in Figure 5.8 on the basis of semiconservative replication. The diagrams show thecomposition of the DNA duplexes after 0, 1, 2 and 3 rounds of replication. DNA strands labeledwith 15N are shown in red, those labeled with 14N in blue. The diagrams at the right show the positionsat which bands are expected in the CsCl gradient.Geometry of the Replication of Circular DNA Molecules In the Meselson-Stahl experiment, E. coli DNA wasextensively fragmented when isolated, so the form of the molecule was unknown. Later, the isolation of unbrokenmolecules and their examination by two techniques—autoradiography and electron microscopy—showed that theDNA in E.
coli cells is circular.The first proof that E. coli DNA replicates as a circle came from an autoradiographic experiment. (Geneticmapping experiments to be described in Chapter 8 had already suggested that the bacterial chromosome iscircular.) Cells were grown in a medium containing radioactive thymine (3H-thymine) so that all DNA synthesizedwould be radioactive. The DNA was isolated without fragmentation and placed on photographic film. Eachradioactive decay caused a tiny black spot to appear in the film, and after several months there were enough spotsto visualize the DNA with aPage 187microscope; the pattern of black spots on the film located the molecule.
One of the now-famous autoradiogramsfrom this experiment is shown in Figure 5.10. The actual length of the E. coli chromosome is 1.6 mm (4.7 millionbase pairs). On a smaller scale, Figure 5.11 shows a replicating circular molecule of a plasmid found inside certainbacterial cells. The total length of the molecule is only 0.01 mm (3000 base pairs). The replicating circle isschematically like the Greek letter θ (theta), and so this mode of replication is usually called θ replication.The circularity of the replicating molecules in Figures 5.10 and 5.11 brings out an important geometric feature ofsemi-conservative replication. There are about 400,000 turns in an E. coli double helix, and because the two chainsof a replicating molecule must make a full rotation to unwind each of these gyres, some kind of swivel must existto avoid tangling the entire structure (Figure 5.12). The axis of rotation for unwinding is provided by nicks made inthe backbone of one strand of the double helix during replication, nicks that are rapidly repaired after unwinding.Enzymes capable of making such nicks and then rapidly repairing them have been isolated from both bacterial andmammalian cells; they are called topoisomerases.
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