Hello. I am going to talk to you about the semi-conservative
replication of DNA. Not so much about the technical details but about how Frank Stahl and I
ended up doing the experiment that showed that DNA
replicates by the two chains coming apart,
each making a new copy and then you get two,
each of which has one old chain and one new one.
It is hard to know where to start any particular story
but let me start with the publication in 1953
of the paper by Watson and Crick. There were actually two papers
in Nature. The first was about the structure
which was based on model building and a little bit of information from X-ray diffraction
The X-ray diffraction certainly did not tell you the structure.
It told you something about the repeat distance along the helix.
It told you that it was a helix and very little more.
The model building was model building,
so the structure certainly wasn't proven.
This was a proposal and many people didn't believe it
or maybe didn't even pay any attention to it.
The second paper proposed how the molecule might replicate. The two chains would separate, each would guide on its surface
the formation of a new chain, so you would
end up with two double helices, each one has
one of the old chains and one brand new chain.
That's called semi-conservative replication.
At Caltech, Max Delbruck who knew Jim Watson and was in correspondence with him
was pessimistic about this replication scheme.
Pessimistic about semi-conservative replication.
The chains were wound around each other, so to get them apart
unless you break them, you would have to unwind the basic double helix
to get the two arms separate.
Max thought this would be impossible, hydronamically impossible.
And so he proposed that maybe the two chains separated by a system of
breaking every several nucleotides along
the chain. And he and Gunther Stent published a paper
which proposed three different methods for DNA replication.
Semi-conservative, as Watson and Crick had predicted,
conservative, in which at least conceptually maybe there was
a way that a double helix could guide the formation of
another double helix just like it nearby
and so you would never have to separate the chains at all.
You'd have a brand new double helix and a completely old double helix, and that would
be the act of replication.
And then dispersive, the third way, break the single chains, separate the pieces, and then put everything back together again.
I visited Max in his office. I was in Chemistry.
I was a student of Linus Pauling.
Max was over in Biology, and he told me
about this problem, and it occurred to me that maybe one could
do an experiment to find out the mode of replication of DNA
based on the use of heavy isotopes.
I wish I had time to tell you about why I thought about heavy isotopes
but it did have to do with taking a course which was a piece of luck
Linus Pauling's course on the nature of the chemical bond.
in which deuterium and hydrogen bonds played an important role.
Nevertheless, the idea was to label DNA with something heavy,
I don't think I thought about, oh yes, it was deuterium I thought about at the time.
Then to label it by growing bacteria or phages
in heavy medium, deuterium medium,
and then switch the growth to light medium
and then put all this in the centrifuge and look so see where the
DNA went-up to the top if you adjusted the density right
if it was all light, down to the bottom if it was all heavy,
and in the middle if it was half heavy and half light.
That's an oversimplification, but that's the way I thought about the experiment
at that time. This was around 1954 sometime.
I then went to Woods Hole as a teaching assistant
for Jim Watson (he had lived at Caltech the previous term)
in the physiology course.
And one day as a teaching assistant in that course
Jim Watson and Sydney Brenner, who were teaching the course,
were in an upstairs room in a building called the Lillie building, the Marine Biological Lab,
and Jim looked through the window and pointed down at a tree
under which was sitting a man who was, you couldn't tell from a distance,
but he was selling gin and tonic.
He had a big jug of gin and a big thing of tonic, and some ice and some glasses and some limes,
and he would sell gin and tonic to passersby, and with the profits he could buy some gin and tonic for himself.
It was called the gin and tonic tree.
But Jim was saying that this guy thought pretty much of himself,
so let's give him a really tough experiment to do in the Physiology class,
the famous Hershey-Chase experiment done by two people over a period of time,
Hershey and Chase, and see if Stahl could do it all in one afternoon
by himself. Well I thought that was pretty rotten to gang up on this poor guy
So I went down and introduced myself to him and told him what lay in store for him.
And he told me what he was doing in phage genetics
and that he would be at Caltech the next year.
So we agreed to try to do this experiment together, ourselves, on how DNA replicates
when he got to Caltech. I had to finish my X-ray crystallography first
before Frank would let us start because he said it would be bad for my character to go ahead and start some new project
when I hadn't finished my thesis work,
which was X-ray crystallography.
Finally I got the X-ray crystallography done
and we could start the experiment.
Frank and I decided that we should develop the method
for doing this density gradient centrifugation
and so we spent quite a long time, more than a year,
developing a method that could separate macromolecules in a density gradient.
And to do that we put in pure DNA
into a centrifuge and turned it on to see what was happening
and to our amazement a density gradient was forming
before our very eyes.
This was because the cesium ion is quite heavy, quite dense, and it
tends to settle to the bottom in a powerful centrifugal field
and the diffusion wants it to go back up to redistribute it
and at equilibrium you have a density gradient in which there is more cesium chloride
near the bottom than near the top.
And so the density gradient forms automatically.
We didn't expect this.
We saw it happen, we thought we would have to make a preformed density gradient
in the centrifuge cell
but the centrifuge itself makes the density gradient.
So to make a long story short, we grew bacteria in heavy nitrogen medium
and then after many generation of growth, so that everything would
be labeled with heavy nitrogen, we switched the bacteria, centrifuging them and resuspending them
in a light medium and took samples at various times.
The first experiment Frank had warned me I would mix it up if I did both directions at once,
if I did heavy to light and light to heavy.
And I said, "no, I'll color code the tubes," and I mixed it up completely.
So it was unclear exactly which tubes were which. The second experiment
we labeled experiment number one and that is what we published,
along with experiment number 2, which was a repeat.
And what we found of course was that
the bacteria at one generation had only one kind of DNA.
It had a density half way between heavy and light.
And at the second bacterial generation, there were two kinds of DNA,
half heavy, and fully light.
Now we had to write this paper up. Actually we were rather sluggish in writing it up
and so Max Delbruck took us to the marine lab
at Corona del Mar, and locked us up in a tower room.
He literally did. Mary Delbruck, Max's wife, would bring us meals,
but then locked the door again,
until we- and there was a typewriter-
until we produced a draft manuscript, which we did.
But there was a question how to write this up.
There were two ways that we discussed.
One way is to start with a hypothesis, the Watson-Crick hypothesis,
and say here's a test, we do this experiment.
and see if it works out the way they said it should.
And that is certainly one way to do an experiment, and Richard Feynman, who was very close to students in those days
He would come over to our parties and so on. He thought that we should write it up that way.
The other way would be to write up exactly what your experiment said, no more, no less
without reference to any hypotheses at all,
and then at the very end say whether it may agree with some hypothesis.
We chose the last way, partly because we thought, well, if you are trying to test a hypothesis,
the only way to really be sure that you are going to be right
is to know all possible hypotheses.
And since nobody can know all possible hypotheses
just because your experiment might agree with one of them
doesn't prove that that is the right hypothesis.
There could be another one that your experiment agrees with
So it seemed to us more elegant to write our paper
up in terms of subunits, and only at the end say,
"ah, these subunits could be the single chains of the Watson-Crick model,"
which of course they were. So that is what we did.
And then this diagram here shows the result in terms of subunits
not DNA chains. What we did was blessed by a lot of accidents.
The accident of being at Caltech,
the accident of meeting each other at Woods Hole,
the accident of having Max Delbruck impress you
with his deep pessimism that DNA couldn't possibly replicate
the Watson and Crick work the way it does.
And finally the accident of finding out that the centrifuge itself
will make a density gradient, you don't have to
make a pre-existing one.
The effect of this experiment is worth saying something about.
When the DNA structure was proposed, a lot
of people didn't believe it.
And there were not a lot of references to it in the literature.
for the first several years after 1953.
After all, it was based on model building, which doesn't prove anything.
It looked so beautiful that some people were convinced
that it had to be right because it looked so right.
Other people I think were convinced that it couldn't be right because it looked too good to be true.
In any case, it was just a model.
The evidence from the X-ray diffraction was really not very supportive.
It showed that the repeat distance was a certain
distance and that it was helical, but you couldn't
deduce any of the details from those early X-ray pictures.
I think the effect of our experiment wasn't really a discovery,
It was a psychological effect. It made the DNA seem real. Suddenly you could
see bands in a centrifuge.
that were behaving just in the way Watson and Crick said they should.
And I think that the main value of the experiment was
that it had the psychological value of convincing a lot of people
that the DNA structure had to be right.
Let me say something about that molecule.
This molecule essentially gave orders to a whole period in the development of molecular biology.
Here is this double helix, standing there,
and it's saying, "Here I am. I have two chains. Go find out how I replicate."
"Here I am. I have four kinds of bases."
"Go figure out how that is converted into making proteins."
"I sit in the nucleus. Proteins are made out in the cytoplasm in eukaryotes."
"Go figure out what it is that takes this information from me out to the cytoplasm."
"My information being made up of these bases changes once in a while."
"That's called mutation. Go figure out how that happens."
"Every once in a while there is something called genetic recombination."
"Go figure out how I am..." What I am trying to say here
is that if I showed you a model of say a polysaccharide,
would it tell you what experiment you should do next?
This molecule, DNA, is like the wizard of Oz
standing there (except unlike the wizard, this is a true molecule)
standing there and telling you essentially the whole agenda
for the future of science for the next 20 years.
Now that's a completely different mood and attitude, I think,
from the science that went before it
and the biological science that came after it.
So that was currency of this time.
It was the DNA molecule telling you what the problems were, and what you had to go out and solve.