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Semi-Conservative DNA Replication: Meselson and Stahl Experiment

#dnasemiconservative #dnareplication #meselsonsthal Meselson and Stahl’s Elegant Experiment Matthew Meselson and Franklin Stahl were well acquainted with these three predictions, and they reasoned that if there were a way to distinguish old versus new DNA, it should be possible to test each prediction. Aware of previous studies that had relied on isotope labels as a way to differentiate between parental and progeny molecules, the scientists decided to see whether the same technique could be used to differentiate between parental and progeny DNA. If it could, Meselson and Stahl were hopeful that they would be able to determine which prediction and replication model was correct. The duo thus began their experiment by choosing two isotopes of nitrogen—the common and lighter 14N, and the rare and heavier 15N (so-called "heavy" nitrogen)—as their labels and a technique known as cesium chloride (CsCl) equilibrium density gradient centrifugation as their sedimentation method. Meselson and Stahl opted for nitrogen because it is an essential chemical component of DNA; therefore, every time a cell divides and its DNA replicates, it incorporates new N atoms into the DNA of either one or both of its two daughter cells, depending on which model was correct. "If several different density species of DNA are present," they predicted, "each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species. In this way, DNA labeled with heavy nitrogen (15N) may be resolved from unlabeled DNA" (Meselson & Stahl, 1958). The scientists then continued their experiment by growing a culture of E. coli bacteria in a medium that had the heavier 15N (in the form of 15N-labeled ammonium chloride) as its only source of nitrogen. In fact, they did this for 14 bacterial generations, which was long enough to create a population of bacterial cells that contained only the heavier isotope (all the original 14N-containing cells had died by then). Next, they changed the medium to one containing only 14N-labeled ammonium salts as the sole nitrogen source. So, from that point onward, every new strand of DNA would be built with 14N rather than 15N. Just prior to the addition of 14N and periodically thereafter, as the bacterial cells grew and replicated, Meselson and Stahl sampled DNA for use in equilibrium density gradient centrifugation to determine how much 15N (from the original or old DNA) versus 14N (from the new DNA) was present. For the centrifugation procedure, they mixed the DNA samples with a solution of cesium chloride and then centrifuged the samples for enough time to allow the heavier 15N and lighter 14N DNA to migrate to different positions in the centrifuge tube. By way of centrifugation, the scientists found that DNA composed entirely of 15N-labeled DNA (i.e., DNA collected just prior to changing the culture from one containing only 15N to one containing only 14N) formed a single distinct band, because both of its strands were made entirely in the "heavy" nitrogen medium. Following a single round of replication, the DNA again formed a single distinct band, but the band was located in a different position along the centrifugation gradient. Specifically, it was found midway between where all the 15N and all the 14N DNA would have migrated—in other words, halfway between "heavy" and "light" (Figure 2). Based on these findings, the scientists were immediately able to exclude the conservative model of replication as a possibility. After all, if DNA replicated conservatively, there should have been two distinct bands after a single round of replication; half of the new DNA would have migrated to the same position as it did before the culture was transferred to the 14N-containing medium (i.e., to the "heavy" position), and only the other half would have migrated to the new position (i.e., to the "light" position). That left the scientists with only two options: either DNA replicated semiconservatively, as Watson and Crick had predicted, or it replicated dispersively. To differentiate between the two, Meselson and Stahl had to let the cells divide again and then sample the DNA after a second round of replication. After that second round of replication, the scientists found that the DNA separated into two distinct bands: one in a position where DNA containing only 14N would be expected to migrate, and the other in a position where hybrid DNA (containing half 14N and half 15N) would be expected to migrate. The scientists continued to observe the same two bands after several subsequent rounds of replication. These results were consistent with the semiconservative model of replication and the reality that, when DNA replicated, each new double helix was built with one old strand and one new strand. If the dispersive model were the correct model, the scientists would have continued to observe only a single band after every round of replication.