Biology: Replication: Meselson and Stahl

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Molecular Genetics
Introduction to DNA Replication
Replication: Meselson and Stahl

Remember the four things that genetic information needs. These are important. It needs to carry information. Well we’re okay with that. We know that DNA has this whole sequence of A, G, C and T. Even though we don’t know how it’s going to communicate that information to the rest of the cell, so the talking thing we’re not so positive about. We know that DNA, any kind of genetic information has to have the ability to change, either willingly or unwillingly. And Watson and Crick proposed that a change of an A to a T or a G to a C, in some way, would cause a mutation. The next question we had that was answered was one that Watson and Crick actually proposed an answer to themselves in their original paper. And it’s this. “We imagined that prior to the duplication the hydrogen bonds are broken, and the two chains unwind and separate. Each chain then acts as a template for the formation onto itself of a new companion chain. So that eventually we shall have two pairs of chains, where we only had one before. Moreover, the sequence of the pairs of bases will have been duplicated exactly.” Watson and Crick, in that paper, proposed a semiconservative method of replication. And I’ll show you what that means in a second.
What good is a proposition without demonstration? And that was a nice idea, but it was left to Meselson and Stahl to figure out the answer to this. And that’s the work that I want to talk to you about right now, the work of Meselson and Stahl. Now I just mentioned something to you called the semi-conservative method of replication. Let’s talk about
that. What the semi-conservative method of replication looks like is something like this. Imagine that you have a DNA strand that’s a double strand. And what Watson and Crick proposed was that the DNA strand would separate into two, one, two. And then somehow new nucleotides would come in there and use the original as a template giving you
two new strands. Okay, nice story, how are you going to demonstrate that?
Well, Meselson and Stahl set up an experiment. And their experiment was to show that the way the DNA was replicated. Now there were three theories going around at that time. One was called the semi-conservative, which I just showed you. So we’ll call that number one, semi. The other was the conservative method of DNA replication. Let me show you how that one would work. That one would work something like this. You have your original template. And instead of it separating, what’s going to happen there is you’re going to use the original template and replicate it in a conservative fashion, meaning that the template
would remain intact and you would make new DNA from that, not a bad thought.
And the last one was called the dispersive method. And the dispersive method is exactly what we’re saying it is. In the dispersive method, it would disperse. In other words, the DNA would break up. We’ll give you the original template here. And it would break up into hunks. And those hunks would somehow be reunited with the new template. Now this one, if you think about it, is probably the least likely, because you have to put those hunks back
together in the proper sequence, because remember sequence is going to be everything here. So what they're suggesting here is that somehow the new ones are going to be hybrids of the new and the old.
Well looking at these three things, Meselson and Stahl came up with a technique that could clearly demonstrate which of these three were right. And here’s what they did. I have to give you a little background about some of the biochemistry here. Number one, they used isotopes. Now I want you to imagine that an isotope can be either
radioactive or it can be heavy. And one of the heavy isotopes we know of is an isotope of regular nitrogen called N15. Now I want you to imagine that I grew bacteria. Let’s say I have all of these normal bacteria growing on a culture. And I put them in this culture. And to this culture I added N15. So we’re going to put N15 in here. Now the bacteria are
going to take up N15, and they’re going to end up heavy bacteria. We’re going to have some fat bacteria. We’re going to have some bacteria that are heavier than normal bacteria. So we’re going to put some normal bacteria growing in N14 here. Okay, now let’s just say I took these bacteria. I filtered them out, and I put them in a centrifuge tube.
And now we come to the second thing I have to tell you about, differential centrifugation. You guys probably know that a centrifuge spins things and settles it by its density. Well let’s think about it. If we put these things in identical conditions, except these are heavy bacteria and these are light bacteria, well when you centrifuge them through a medium, say like a sucrose gradient, something thick and gooey, the heavy bacteria will settle deeper than the light ones. And in fact, if I put them in the very same centrifuge tube just for fun, I get a band of light bacteria, the N14, and
these would be the 15s, the heavy ones.
With that principle, Meselson and Stahl were able to answer the question of DNA replication. Let’s see how they did it. Okay, ready? Now you’ve got some bacteria. And you’re putting the bacteria in a petri dish. So here’s what you’re going to do. The bacteria start to grow. There are my bacteria. And I am going to add to that petri dish N15.

And here is the key. I am going to take those bacteria out after one generation. And in that one generation, what am I expecting? I am expecting, if it’s one generation and the bacteria have doubled, I am expecting the bacteria to have N15 incorporated into them. They will have incorporated N15. Now I am then going to let that thing go, that’s one
generation. I am now going to let it go a second generation again with the N15. So I have one experiment that is going to have one generation. I’m going to take some of them out. And then I’m going to let it go a second generation, two generations, again with the N15. So in other words, the first generation has incorporated N15, the
second generation has incorporated N15. What have they incorporated it into? And that’s the key, my friends. What they have incorporated this N15 into is their DNA. They have made heavy DNA. Now think about this. One generation with heavy DNA, two generations with heavy DNA, let’s look at some diagrams. Let’s talk about the conservative method of replication. The conservative method of replication, my first DNA is N14.
Why? I put my bacteria in the dish and there’s only N14. Right? Now I’m going to add N15 to the dish. What’s going to happen? If the conservative method of replication happens after one generation, watch this. After one generation, I’m going to have two DNAs, right? Because they’re going to double their DNA. Are you with me? When they double
their DNA, some of the DNA is going to be the original DNA. And if it’s conservative, the DNA will be like that. And if I let it go still another generation, let’s see what’s going to happen next. In my next generation, I am going to have my original, because it’s never been broken open, and now to my original I’m going to make a second. And from this one I’m going to copy. And so what’s happening is it’s very clear to me what’s going on here. I’m getting a whole lot of heavy, and my original, light.
Now I have to ask you a question. Suppose I took this second generation, and I ground it up. And I put it in a centrifuge tube, and I spun it down. What would you expect? And then I have another question for you. What would you expect if I did this? I’ll show you, here’s the thing. If I spun it down, you are going to expect from this, let’s draw
the tube. I’ve got some better artwork for you in a second. You would expect a heavy band and a light band. And from this you would expect that. You would expect a heavy band and a light band. Let me tell you something. Let’s take a look at what this would look like here. Here we go. Parent cell, first replication, second replication, what would that look like? Well I’ll tell you what. I’ll tell you what they got. When they did this experiment, and they said, “We’re going to see if it’s the conservative method.” And they ground those babies up, they knew after the first generation what would have happened. Because what would you have expected? Remember? You expect two bands. What they got was one band. The conservative method, forget it. They got one band. They didn’t get two. There’s no way this explained it. They got one band. Let’s see what that explains. Here we go. Imagine the semi-conservative, one DNA. But now we’re going to let it go
through one generation. I should show you how it does it. Let’s not be chintzy here. So we’re going to have my first generation. We’re going to feed it N15. Let me get some N15 here. It’s going to open up. You’re going to get separate strands. And then the separate strands are going to form half heavy, half lights. What are you going to get when you
centrifuge that? When you centrifuge that, you’re going to get a band in the middle, neither heavy nor light. That
sounds pretty good.
And let’s see what you’re going to get after the second one. So what we’re going to get here, let’s draw this. So this
is going to open, and we’re going to get my original strands, one, two, three, four. And look what we’re going to get.
Remember heavy, heavy, heavy only. We’re going to get that, and that, and that, and that. Now we’re going to get, in
the first generation, two bands. But where are those bands going to be? One is going to be a hybrid band right there,
the same height as that one. And these are going to be heavy only. How cool is that?
Guess what. When Meselson and Stahl did this as they were expected, do you know what they got? Is that it? Oh
wait, we’ve got to discount everything. I’m excited about this. This looks great. Look, it looks so great. I even made
it for you in a special artistic rendition here. See there’s the first generation, and there’s the second generation. You
see? How cool is that?
Let’s not forget the dispersive. The dispersive, here’s my band. Watch this. You predict, ready? I’ll give you the first
generation. Nah, you predict the first generation. We’ll feed it some heavy nitrogen. We expect this. The thing is
going to break apart. And you’re going to get a little of this, and a little of this, and a little of this. And they’re going to
come together in a hybrid. Well what do you expect from here? Well what we expect from here when we run it
through the centrifuge is kind of goop in the middle, which is kind of what we expected from the semi-conservative.

Wasn’t it? Remember the semi-conservative we expected a band in the middle? So that’s why we can’t discount
from our first generation. We can’t discount the semi-conservative. And we can’t eliminate this dispersive.
But watch this. Generation two to the rescue, when we do this very same thing, what are we going to get? Once
again, it’s going to disperse. We’re going to get a little of the original strand in there, and we’re going to get a mix of
the DNAs. But once again, what do you expect? You expect perhaps that band to widen out a little bit, but you don’t
expect what you got. Because what you got, indeed, was a band in the middle. But what you got was two bands.
Dispersive out, conservative out, it’s a victory for the semi-conservatives of the world.