THE REPLICATION OF DNA IN ESCHERICHIA COLI
MESELSON AND STAHL
See the paper here.
The DNA and nucleic acid structure had been modelled but was not proven by Watson and Crick in 1953. Previous studies about DNA has shown that the DNA carries hereditary information and it was capable to replicate itself. However, the replication mechanism was unknown.
Back in time, there were three models suggested for the DNA replication; conservative, semi- conservative and dispersive models. According to the conservative theory which was proposed by David P. Bloch; the entire DNA molecules act as template and synthase itself like as a photocopy machine. Based on this theory; the daughter molecules were newly synthesized from the parental DNA. The other theory was the semi-conservative theory which was proposed by Watson and Crick in a paper published called “Genetical implications of the structure of Deoxyribonucleic acid”. The paper was about the possible replication of the DNA which is called “semi- conservative replication theory”. According to this theory the DNA strands break and each strand was used as a template. According to this theory, the daughter molecule would have one newly synthesized strand and one old strand. The third theory was the dispersive theory proposed by Max Delbruck. According to this model, the DNA backbones are chopped into the little pieces and each piece acts as template, DNA is synthesized in short pieces where the daughter molecules would carry the mixture of one old and one newly synthesized pieces of the DNA.
Meselson and Stahl wanted to answer this question via measuring density and mass difference of the DNA molecules with the method called density gradient centrifugation by labeling the DNA. They decided to run an experiment which might prove that the DNA replication mechanism. They used Escherichia coli which was easy and rapid to grow.
Meselson and Stahl wanted to label the DNA of Escherichia coli with something heavy which would give a chance to separate the DNA molecules based on their density and weight.
Nucleic acids are the main and required elements of the DNA molecules to replicate itself. Each nucleotide has three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. These nitrogen containing bases are organic molecules with a nitrogen (N) atoms. The natural N has 14 isotopes (7 protons and 7 neutrons) with the isotopic mass of 14 atomic mass unit (amu). Meselson idea was to use heavy isotopes to determine the replication model of the DNA. He wanted to use radioisotopic labels thereby they used the isomer called N15 which has 7 protons and 8 neutrons. The atomic mass of this isomer was 15 amu which makes it heavier than N14.
So, they hypnotized that; The DNA which replicates itself under the heavy N15 presence would have a density increase which might permit the analysis the difference of the DNA mass and density. Based on this knowledge, Meselson and Stahl grew Escherichia coli in heavy N isotope (N15) containing medium and removed a sample of Escherichia coli colonies lysed and DNA was extracted and ultra-centrifuged. They moved some of the colonies from N15 into the N14 containing media and later they extracted the DNA from these various generations of colonies and ultra-centrifuged them too.
For the centrifugation; they added the extracted DNA molecules into the solution of cesium chloride (CsCL) and measured the band weights and tried to determine separation via density- gradient centrifugation method which allows to determine the small density differences between micro-molecules. The cesium chloride helped them to separate the DNA with different molecular weight if there were any. Based on this approach, DNA would form a band form at the place where the CsCL is equal to buoyant density. By doing this, they expected to see the DNA which was labeled with heavy N (N15) would separate from the light one which was labeled with N14. Based on this theory; they were able to observe the separation of the DNA molecules according to their density and mass. They were expecting to see some bands which are the belong to N15 labeled DNA molecules would possibly move closer to the bottom whereas some of the light (harboring N14) one would be on the top of that.
UV photograph of the DNA bands were taken during the centrifugation processes and analysis were done by microdensitometer.
They process 2 different experiments. The second experiment was like a confirmation of the first one. They published the graph of the DNA band from different generations of E.coli and compare them by mixing the generations. The picture was revealing that there were different bands located in various segments. Only semi- conservative model could cause such difference.
According to the semi- conservative model; the parental strands which were labeled with N15 would divide and match itself with light N14 nitrogen containing nucleotides, when they were transferred from N15 media to N14 media. Thereby in the second generation, the expected results were to see the entire daughter DNA molecules would have one old and one newly synthesized strands. This would lighten the DNA weight when compared with the parental strands. After the second generation. In the third generation; 50% of the daughter molecules would harbor only N14 where as other 50% would have one old and one newly synthesized strands. As a result of these replications and generations by time; the number of newly synthesized DNA molecules with the N14 would increase and the heavy N15 labeled DNA strands number would be stable. Since those could not synthesized themselves with the N15 anymore.
That was the results that Meselson and Stahl found. These finding were supporting the hypothesis of Watson and Crick. They were able to photograph these bands by the time of the generations and prove this hypothesis.
The finding were revealed 3 key points;
- The original parental molecules contain N15 because they initially grown into N15 rich media.
- Replicated daughter molecules will have N14
- DNA with different densities can be separated by centrifugation where they have a difference in mass.
In addition to these finding that I mentioned above, they also analyzed the heat effect on the denaturation of the DNA. When they heated the DNA of the labeled (N15) and unlabeled (N14) DNA in the CsCL centrifuging medium, they realized that the heating decreased the density of the DNA almost half that of unheated material. Upon these findings, they concluded that the two molecular sub-units were disassociated upon heating. When they tried to compare the molecular density of the salmon sperm DNA with E. coli DNA by heating at 100 °C, 30 min. The salmon sperm DNA weight did not decreased by heating. These findings they achieved revealed two conclusions. Their first suggestion was; the salmon DNA strands might had bound more tightly when compared with E. coli DNA. The second conclusion was; if the salmon DNA does not have the subunits that the E. coli DNA has, the salmon sperm DNA might be more complex than E. coli DNA.
As a result of these studies that they conducted in the California Research Institute of Technology, they had proven that the DNA uses the semi-conservative model to replicate itself and heat had an effect on the DNA structure.
Based on their conclusion of the heating effect on salmon sperm DNA and E. coli DNA, it was reasonable to conclude such ideas however, they could change the heating time and the temperature too see how various heating conditions might affect the salmon sperm DNA and E. coli DNA.