DNA Replication, Part II

Watson and Crick’s semi-conservative model contrasted with a couple of other possibilities for how DNA could possibly replicate. There is the conservative model, which suggests that both strands in the original DNA double helix stay together during replication. Then there is the dispersive model, which suggests that both strands are not only separated, but even broken up into smaller pieces during replication. Deciding which of these models was the correct one seemed to be pretty easy, because they make very different predictions. It was not obvious how to prove it in the laboratory, however.

The prediction of Watson and Crick’s semi-conservative hypothesis is that each of the two daughter double helixes, after one round of replication, should be made up of one old strand and one new strand. The conservative model predicts that after replication, one of the double helixes that result would be entirely old. The daughter helixes would be entirely new. Finally, the dispersive model predicts that both the daughter and parental helixes would be made up of just a mixture of old and new DNA.

This sounds simple, but the difficulty was figuring how to actually test that. We need some way to be able to determine what’s old and what’s new DNA after replication. It took several years before anybody figured out how to do this.


Meselson-Stahl Experiment


This brings us to a pair of researchers, Matthew Meselson and Franklin Stahl. In 1957, a few years after Watson and Crick’s work, they came up with a novel method for distinguishing new and old DNA during replication. Let me explain how they did that.

First, they grew bacteria in two different kinds of culture media. One of these culture media had normal nitrogen in it (N14). The other media had a heavier isotope of nitrogen in it (N15). This isotope of nitrogen is not radioactive, it’s just a little bit heavier. Not much heavier, just a little bit.

The point of culturing bacteria in these two different media is that the nitrogen in those media would be taken up and incorporated into any new biological molecules that were being synthesized. Specifically, the nitrogen would be taken up and incorporated into any new DNA that was being synthesized.

If you culture bacteria for some period of time, what would be many generations, then you can assume that all of the nitrogen that is incorporated in that DNA would either have N14 or N15 depending on the culture media in which you are growing it. In this way, Meselson and Stahl could essentially label old and new DNA by how heavy (dense, really) that DNA was.

As you can imagine, the density difference between DNA that had been made with N15, as compared to DNA that was made with N14, is really small. The really clever part was figuring out how to very accurately measure the densities of these kinds of DNA.


The Clever Part


To do this, Meselson and Stahl devised a new kind of procedure called “density gradient centrifugation”. This density gradient centrifugation allowed them literally to sort out DNA according to how dense it was.

The idea behind this is actually similar to the reason why swimmers don’t sink in the Great Salt Lake. If the density of a liquid and an object in it are more or less the same, then the object would neither sink nor float, it would just sort of stay where you put it. So, if you add a lot of salt to water, actually it becomes of the same density as our own tissues, and you don’t sink in it, you just sort of stay there.

It is more interesting, though, if you have a gradient of densities. In other words, if you have some range from high to low densities in some liquid mediums, then objects of slightly different densities would sort themselves out. The objects would end up at that gradient at exactly where their own density matches the density of that point in the density gradient.

That’s the idea that Meselson and Stahl had. How do you create a density gradient? After trying a number of different kinds of solutions, they found a compound called Caesium Chloride. This is a salt that when it is put into a solution has approximately the same density as DNA. What they then did was take a tube of caesium chloride solution and centrifuge it. If you centrifuge a tube, what happens is that the heavier stuff goes to the end of the tube, and the lighter stuff stays at the top.

What Meselson and Stahl had to do with this experiment was centrifuge the ceasium chloride solution enormously quickly. They actually spun it around so fast that they created a 100000 g-forces. This is really fast, so it is called ultra-centrifugation. They did it for a number of days. At the end, they would get as a density gradient of the caesium chloride along the tube. Remember, caesium chloride is about the same density as DNA.

That means that if you then take some DNA and put it in that tube and spin it around, the DNA would ordinarily just be dispersed in that tube because is more or less the same density as the ceasium chloride. As the density of ceasium chloride develops, however, the DNA would all coalesce in a single band. That band would be in a position along the length of the tube that corresponds exactly to the density of DNA at that point in the tube.

This method was so sensitive, that Meselson and Stahl determined that they could tell the difference between N15 and N14 DNA.


Watson and Crick Had it Right


So, that’s the technique. Armed with this technique, Meselson and Stahl then did the following experiment, which should be sort of obvious bases on what we talked about. They took a culture of N15 bacteria and transferred them to a culture flask that had N14. Now, those bacteria, when they started to replicate their DNA, would start incorporating the lighter nitrogen. Any new DNA produced by those bacteria would be lighter than the old DNA that they had.

They waited for about 20 minutes, which is long enough for just one round of DNA replication. Then they took the bacteria out, extracted the DNA from them and used their density centrifugation method to determine what the densities of the DNA in the sample was.

The conservative model predicts that at this point there should be two separate sets of DNA. There should be lighter DNA and heavier DNA. The new DNA is going to be lighter and the old DNA is going to be heavier. This is because, according to this model, the parent strand stays intact. After one replication, you should have some DNA that is heavy, and some DNA that is all light.

This is not the result they observed. What they saw was just one intermediate band. So, they could rule out the conservative model directly. They couldn’t rule out the dispersive model, thought. After just one round of replication, both the dispersive and semi-conservative models made the same prediction. Each daughter double helix should be composed of half old(heavy) DNA and half new(light) DNA. All of the DNA in the sample, after one replication, should be at some intermediate weight. This is what they saw. The dispersive hypothesis made exactly the same prediction.

If you wait for two replications, however, all of a sudden you get a distinct difference in the predictions made by the semi-conservative and dispersive models. After two replications (about 40 minutes), the dispersive model would still predict there would be only one band, all of new and old DNA is all mixed up. Therefore, all of the DNA would be about the same density. The thing that should change is the position of that band along the gradient.

What Meselson and Stahl saw was the creation of two bands after two rounds of replication, which confirmed the prediction of the semi-conservative model.

Well, it took some years to prove it, but Watson and Crick had it right.

Be the first to leave a comment

Copyright © 2010
Template by bloggertheme