TheMeselson–Stahl experiment is an experiment byMatthew Meselson andFranklin Stahl in 1958 which supportedWatson andCrick's hypothesis thatDNA replication wassemiconservative. In semiconservative replication, when the double-strandedDNA helix is replicated, each of the two new double-stranded DNA helices consisted of one strand from the original helix and one newly synthesized. It has been called "the most beautiful experiment in biology".[1] Meselson and Stahl decided the best way to trace the parent DNA would be to tag them by changing one of its atoms. Sincenitrogen is present in all of theDNA bases, they generated parent DNA containing a heavierisotope of nitrogen than would be present naturally. This altered mass allowed them to determine how much of the parent DNA was present in the DNA after successive cycles of replication.
Three hypotheses had been previously proposed for the method of replication of DNA.
In thesemiconservative hypothesis, proposed byWatson andCrick, the two strands of a DNA molecule separate during replication. Each strand then acts as a template for synthesis of a new strand.[2]
Theconservative hypothesis proposed that the entire DNA molecule acted as a template for the synthesis of an entirely new one. According to this model,histone proteins bind to the DNA, revolving the strand and exposing the nucleotide bases (which normally line the interior) for hydrogen bonding.[3]
Thedispersive hypothesis is exemplified by a model proposed byMax Delbrück, which attempts to solve the problem of unwinding the two strands of the double helix by a mechanism that breaks the DNA backbone every 10 nucleotides or so, untwists the molecule, and attaches the old strand to the end of the newly synthesized one. This would synthesize the DNA in short pieces alternating from one strand to the other.[4]
Each of these three models makes a different prediction about the distribution of the "old" DNA in molecules formed after replication. In the conservative hypothesis, after replication, one molecule is the entirely conserved "old" molecule, and the other is all newly synthesized DNA. The semiconservative hypothesis predicts that each molecule after replication will contain one old and one new strand. The dispersive model predicts that each strand of each new molecule will contain a mixture of old and new DNA.[5]
Nitrogen is a major constituent of DNA.14N is by far the most abundantisotope of nitrogen, but DNA with the heavier (but non-radioactive)15N isotope is also functional.
E. coli was grown for several generations in a medium containing NH4Cl with15N. When DNA is extracted from these cells and made to undergobuoyant density centrifugation on a salt (CsCl) density gradient, the DNA separates out at the point at which its density equals that of the salt solution. The DNA of the cells grown in15N medium had a higher density than cells grown in normal14N medium. After that,E. coli cells with only15N in their DNA were transferred to a14N medium and were allowed to divide; the progress of cell division was monitored by microscopic cell counts and by colony assay.
DNA was extracted periodically and was compared to pure14N DNA and15N DNA. After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. However, this result was consistent with both semiconservative and dispersive replication. Semiconservative replication would result in double-stranded DNA with one strand of15N DNA, and one of14N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of15N and14N DNA, either of which would have appeared as DNA of an intermediate density.
The authors continued to sample cells as replication continued. DNA from cells after two replications had been completed was found to consist of equal amounts of DNA with two different densities, one corresponding to the intermediate density of DNA of cells grown for only one division in14N medium, the other corresponding to DNA from cells grown exclusively in14N medium. This was inconsistent with dispersive replication, which would have resulted in a single density, lower than the intermediate density of the one-generation cells, but still higher than cells grown only in14N DNA medium, as the original15N DNA would have been split evenly among all DNA strands. The result was consistent with the semiconservative replication hypothesis.[6]
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