Meselson and Stahl Research Assignment

Matthew Meselson and Franklin Stahl are famous for their DNA replication experiment. Meselson and Stahl conducted the experiment which supported the hypothesis made by Watson and Crick that DNA replicates. The purpose of the experiment was to provide an explanation for James Watson and Francis Crick’s structure of DNA. The model represented DNA as two helical strands put together in a double helix that replicated semi-conservatively. The experiment came about when there was a discussion among scientists in the 1950s on the way DNA replicated.

In 1956 Meselson and Stahl began their experiment. Matthew and Franklin did many projects together including DNA replication. Most of their projects used a method created by Meselson in 1954: Density-Gradient Centrifugation. Density-gradient centrifugation separates molecules based on their densities depending on the molecular weight of the molecules. This technique was used in the experiment of DNA replication to separate DNA molecules in a solution.

On October 1957, Meselson and Stahl began the experiment with E. coli containing only the heavy isotope of nitrogen (15N) to give the parental DNA a higher than normal density. As bacteria grew, they duplicated which replicated their DNA in the process. They then added an excess of light isotopes of nitrogen (14N) to the heavy nitrogen environment. Meselson and Stahl grew E. coli in the 14N isotope environment for all following generations, so that any new DNA strands produced were of lower density than the original parent DNA. Before adding 14N nitrogen, the scientists pulled samples of E. coli out of the growth medium for testing; they centrifuged each sample for initial separation, and then they added salt to the bacteria so that the bacteria released its DNA contents, which allowed them to analyze the samples.

Next, they conducted density gradient centrifugation for each DNA sample to see they way the parental and daughter DNA distributed according to their densities. They added a small amount of each sample to a cesium chloride solution. The researchers centrifuged the DNA in an ultracentrifuge for twenty hours until the DNA reached equilibrium. Using ultraviolet light, the scientists photographed the resulting DNA bands, which showed peaks of DNA concentrations at different densities. The density of the DNA depended on the amount of the type of nitrogen present. Which means the more 15N atoms present, the denser the DNA. For the bacterial DNA collected before, the UV photographs showed only one band for DNA with 15N isotopes. This occurred because the DNA from the first sample grew in an environment with only 15N isotopes. For samples pulled during the first replication cycle, the photographs showed fainter 15N DNA bands. Also, a new DNA band formed representing half 15N DNA isotopes and half 14N DNA isotopes. Towards the end of the first replication cycle, the heavy DNA band disappeared. Only a dark half 15N and dark half 14N DNA band remained. The data from the first replication cycle showed some distribution of parental DNA because only parental DNA contained 15N nitrogen isotopes and only parental DNA could represent the 15N nitrogen isotopes in daughter DNA.

Similar trends continued in future DNA replication cycles. UV photographs consistently showed the band representing half 15N and half 14N DNA depleted. A new band- representing DNA containing only 14N isotopes- became the common DNA band in the sample. The depletion had occurred because Meselson and Stahl never again added 15N, so the amount of 15N DNA decreased. Meselson and Stahl mixed the samples pulled from different replication cycles and centrifuged them together. The UV photographs showed three bands of DNA with the half 15N half 14N DNA band at the midpoint between the 15N DNA band and 14N DNA band, making it an intermediate band. The result indicated that the half 15N half 14N DNA band had a density exactly between the 15N and 14N nitrogen DNA, showing that the DNA in the central band contained half of the 15N nitrogen and half of the 14N nitrogen isotopes which was predicted by the Watson and Crick model. The exact split between light and heavy made it semiconservative DNA replication.

Meselson and Stahl made three conclusions. First, they concluded that the nitrogen in each DNA molecule divided evenly between the two subunits of DNA, and that the subunits stayed intact throughout the replication cycles. Meselson and Stahl made that conclusion because the intermediate band had a density halfway between the heavy and light DNA bands. The second conclusion stated that each new DNA double helix contained one parental subunit, which supported semi-conservative replication. Assuming that DNA consists of two subunits, if a parent passes on one subunit of DNA to its offspring, then half of the parental DNA is present in the offspring DNA, and the other half is not. The third conclusion made stated that for every parental DNA molecule, two new molecules were made. Which means, the amount of DNA after each replication increases by a factor of two. Meselson and Stahl related their findings to the structure of DNA made by Watson and Crick.