Meselson and Stahl: 67 years later.

When Matthew Meselson and Franklin Stahl proposed the semi-conservative replication model of DNA, could they possibly have comprehended that their paper would still be quoted almost 70 years later?

Every biology A-level student in 2025 is taught the work of Meselson and Stahl. I remember learning it myself, and now I teach it to my students when I tutor. It is easy to forget just how important this experiment was in the advancement of genetics.

When the Watson-Crick model was released in 1953, it raised questions on how the double-helix model replicates. Meselson and Stahl aimed to conduct an experiment that could provide evidence on how DNA was able to do so.

One theory was conservative replication, suggested by Gunther Stent. Stent's theory was that the DNA helix creates two helixes; one is made up of two completely new DNA strands, whilst the second helix is made up of DNA strands that are completely the same. 

Another theory was dispersive replication. This basically suggests that the original DNA chains break apart and then recombine with new segments to create DNA that is a patchwork of old and new DNA.

Finally, the semi conservative replication theory that Meselson and Stahl found to be correct, was suggested by Watson and Crick themselves. It suggested that DNA is comprised of one parent strand and one daughter strand. 

 

Credit to IGenetics, Steven M Carr

 

To fully understand how Meselson and Stahl proved semi conservative replication, a basic understanding of radioactivity is needed- in particular, radioactive nitrogen. 

Nitrogen is known as nitrogen 14 and contains 7 neutrons and 7 protons. It is an example of an ‘isotope’ which is means that is one form that an element can take.

 Nitrogen 15 however, is a different isotope of nitrogen and contains 8 neutrons. It is rarer than nitrogen 14 and is also heavier-due to the presence of the extra neutron. 

Meselson and Stahl selected nitrogen due to it being a component of DNA. In a process called isotopic labelling, E. coli DNA was exposed to nitrogen-15. This meant that the two would be able to distinguish between parental and daughter DNA based on whether heavy or lighter nitrogen was present.

The below image shows two bands formed because of conducting this technique on equal amounts of Nitrogen 15 and Nitrogen 14 E. coli DNA:

 

Meselson and Stahl also devised a new technique to prove semi-conservative replication - Density-Gradient Centrifugation. This, along with nitrogen 15, was crucial to the experiment, as it allowed for the two nitrogen isotopes to be distinguished from each other: allowing Meselson and Stahl to prove that the daughter DNA strands contained the nitrogen 15- meaning that the DNA had to be comprised of a parental strand.

Meselson and Stahl used a concentrated solution of Cesium chloride (CsCl). In simple terms, extracted DNA was placed in this solution, and a stable concentration gradient was produced by applying the opposing processes of sedimentation and diffusion. This forced the DNA to move along the centrifugal force produced until it reached a point where its own density matched the density of the solution.

So, Meselson and Stahl exposed the first generation of E. coli only to nitrogen 15 and then transferred the E. coli to a growth medium that did not contain nitrogen 14. After growing E. coli for 15 generations, and conducting density-gradient centrifugation they were left with this image:

 

The density of the CsCl solution increases to the right, with each horizontal position representing the same density on each photograph. The right band (the only band in the topmost photo) represents Nitrogen 15. So, the only way the nitrogen 15 could be present in the later generations was if a strand of DNA from the beginning had been incorporated into the molecule. These images were taken using ultraviolet absorption. 

If the conversative model was correct, the first generation would have shown two completely different bands- one band for the molecule made up with the Nitrogen 14 and one band for the old molecule made up with the Nitrogen 15.

The first generation however fit with both the dispersive and the semi-conversative model, as it showed a hybrid. 

By generation 2 though, you can see two bands- one a hybrid weight and one corresponding to Nitrogen 14. This shows that DNA consisting of only Nitrogen 14 was being created. This meant that dispersive could not be the right model.

Arguably, the new age of molecular biology began with the DNA model in 1953, that Watson and Crick proposed with invaluable contributions from Maurice Wilkins and Rosalind Franklin. However, it is possible to argue that Meselson and Stahl really began the new age of molecular biology with their proof of semi-conservative replication.

Semi conservative replication answered a question that had been plaguing molecular biologists since the proposal of the Watson and Crick DNA model 5 years earlier; E. coli DNA replicates semi-conservatively. This suggested that eukaryotic DNA may replicate in a similar way.

 

 

Credit to OpenStax College, Biology

 

 

What came next was a series of experiments and discoveries, with only a few examples listed below:

Hot on the heels of Meselson and Stahl was Arthur Kornberg and his discovery of DNA polymerase I. In 1958, with postdoctoral fellows Maurice J Bessman and Robert I. Lehman, Kornberg purified DNA polymerase from E.coli , reported that DNA polymerase, magnesium ions and the four deoxynucleoside triphosphates were all needed to enable DNA synthesis and hypothesised that DNA polymerase was needed to act as a template 

Helicase, the enzyme needed to separate the stands of DNA, was not discovered until 1976 by Hoffman-Berling and Mackay and Linn. Meanwhile, Gyrase or Topoisomerase, the enzyme needed to stop DNA from overwinding during replication was first found in E. coli by Jim Wang in 1971.

In the 1960s, John Cairns performed a follow up to the Meselson and Stahl experiment to prove what he called ‘theta replication’- or how semi conservative replication occurs in E. coli. By growing E. coli bacteria in the presence of radioactive nucleotides and allowing DN loop A to replicate, he created DNA with one radioactive strand. The resulting electron micrograph showed that the circular chromosomes of the E. coli first unwind at a single spot, which is now termed as the replication origin. The double helix will then continue to unwind, resulting a loop- the replication bubble.

This highlights that whilst the experiment of Meselson and Stahl was the start of understanding the replication of DNA, there was still so much more to understand and discover. It also highlights how vital E. coli, an organism responsible for disease was vital in understanding DNA replication. E. coli is still used in laboratories today. 

Whilst it is easy to say that the 1953 DNA model was the most important development in molecular biology- after all, it identified the structure of DNA, it can be argued that the semi conservative experiment was the most important development. Of course, it can be also argued that Meselson and Stahl only conducted their experiment due to the theories made by Watson and Crick.

Semi conservative replication was a theory of Watson and Crick that was proven to be correct. Later, Francis Crick suggested that RNA acts as the intermediary between DNA and Protein; This was later proven by Arthur Kornberg, confirming the ‘Central Dogma of molecular biology’ first suggested by Crick. This Central Dogma still stands.

Nitrogen-15 is still used in laboratories but is now used in a technique called Nuclear Magnetic Resonance (NMR). This technique provides structural information at the atomic level but is much more challenging when being used on nucleic acids than on proteins (Nelissen et al, 2016). This highlights that Nitrogen 15 is still extremely useful in current research on RNA and DNA.

                                        

                                             Meselson and Stahl, 2020.

As every scientist knows, science only progresses due to the efforts of those that came before us. It is impossible to keep going forward, without keeping an eye backward. The ‘Modern Biotechnology Age’ would not have been at all possible without PCR, and PCR would not have been possible without first understanding how DNA replicates. So, keeping that in mind, it is only right that Meselson and Stahl remain an essential aspect of Biology A-level!