Meselson and Stahl
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Matthew Meselson and Franklin Stahl are two biologists who prove that DNA replication was semiconservative.
At the time, many strong evidences from experiments using bacterial viruses had already convinced most scientists that DNA was the molecule of heredity; however they knew little about the DNA replication process. After the dimensionally accurate model building by Watson and Crick, it was clear that the process of replication and information distribution have to use the DNA from parent cell as template to achieve an orderly flow of genetic inheritance to daughter cells. However the model of Watson and Crick still cannot fill the missing information about how DNA might accomplish replication.
Before the Meselson-Stahl experiment, many researches have been done on the problem of DNA replication by other scientists, but all of those efforts were ineffective or not directly relate to the replicate process. There were three hypotheses suggested; which are Semi-Conservative, Conservative and Dispersive. The first possibility is Conservative replication; one daughter cell receives the original DNA that was used as template, while the other daughter cell receives only newly replicated strand of DNA. The second possibility is Semiconservative replication; each daughter cell receives only one strand of DNA from the parent cell, while the complementary strand is newly replicated. The third possibility is Dispersive replication; each daughter cell receives a new double-strand that consists of distinct regions of DNA from either original or new strands interspersed throughout out the strand (Templin & Fetters 2002).
In 1956, Meselson and Stahl began to carry out an idea that Meselson had earlier had to investigate the problem by incorporating a heavy isotope into the DNA molecules of a microorganism and tracing the distribution of these atoms into progeny DNA by separating molecules of different density in a centrifuge. The plan of their landmark experiment on DNA replication was to grow the organisms in the heavy medium, transfer them to the light medium, withdraw samples at the time intervals representing successive cell generations, lyse the bacteria to release their DNA, and centrifuge the latter in the density gradient.
At the beginning of designing their experiment, they have used 5BU as the density label but after concerning about the deleterious effects of its mutagenicity and cellular toxicity, as well as problems in obtaining uniform labelling. Meselson and Stahl decided instead to switch from 5BU to nitrogen-15 (15N) to produce heavy DNA. They assumed that if parent cells were grown in a medium that contain heavy nitrogen as the density label (15N instead of commonly occurring 14N), then the DNA of these parent cells would be denser than usual (Hanawalt 2004).
The first stage of the experimental design was to test whether Escherichia coli would grow normally in a medium which using 15NH4Cl as the sole source of nitrogen (heavy nitrogen), and they found that bacteria had no trouble in such an environment.
In October 1957 they produced the experiment to test which method of replication cells actually perform. They chose to grow the bacteria first on the 15N and then switch them to 14N rather than the other way around, because that strategy minimized the use of expensive isotope required.
In the experiment, E. coli was grown generations in 15NH4Cl medium so that the DNA would be essentially labelled by the nitrogen isotope 15N. The DNA of the resulting cells had a higher density (heavier). They followed the growth of the culture by standard assay methods and microscopical cell counts. After the titer had reached 2 Д– 108, representing fourteen bacteria generations, they switched the bacteria abruptly to 14N by adding the medium containing 14NH4Cl.
Meselson started the ultracentrifuge up with the sample taken just the medium had been switched. Photographic film showed a single sharp band, representing “pure heavy” DNA. He then began a run with sample drawn during the bacterial generation following the switch. The result of that run yielded two bands: an intense heavy band and a fainter band just to its lighter side. And, finally with the anticipated single band of heavy —light DNA.
Since conservative replication would result in equal amounts of DNA of the higher and lower densities but no intermediate density, therefore 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 of 15N DNA, and one of 14N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of 15N and 14N DNA, either of which would have appeared as DNA of an intermediate density.
Therefore they started to run the sample taken from second