Meselson and Stahl’s Experiment

In 1953, after Watson and Crick came up with the shape of DNA, the double helix. The
question “how does DNA replicate?” lingered.

There were 3 theories that were proposed – the conservative, dispersive and semi-
conservative models of replication which were introduced earlier. To figure out which of
these three models answers the question of DNA replication, the two scientists -
Matthew Meselson and Franklin Stahl conducted an experiment that will be soon known
as one of the "most beautiful experiments in biology" which we will be going to explore
at this juncture.

Matthew Meselson and Franklin Stahl conducted an experiment to determine which of

the three replication models applied to E.coli cells. They used the bacteria E. coli in this
experiment since it could be easily grown in the laboratory.

In 1958, their findings were published in their article entitled "The replication of DNA in
Escherichia coli" in the Proceedings of the National Academy of Sciences.

So what did they do?

Meselson and Stahl grew a culture of E. coli in a medium that contained 15N as the sole
nitrogen source; after many generations, all the E. coli cells had 15N incorporated into
all the purine and pyrimidine bases of their DNA (see Figure 8.8). Meselson and Stahl
took a sample of these bacteria, switched the rest of the bacteria to a medium that
contained only 14N, and then took additional samples of bacteria over the next few
cellular generations. In each sample, the bacterial DNA that was synthesized before the
change in medium contained 15N and was relatively heavy, whereas any DNA
synthesized after the switch contained 14N and was relatively light.
Meselson and Stahl distinguished between the heavy 15N-laden DNA and the light
14N-containing DNA with the use of equilibrium density gradient centrifugation (Figure
9.2).
They grew E. coli cells in a medium for many generations where the only nitrogen
source is 15NH4Cl (ammonium chloride).

The scientists Meselson and Stahl grew a culture of E. coli in a medium for many
generations in a medium that had 15NH4Cl (ammonium chloride) as the only nitrogen
source.
A “heavy” isotope of nitrogen, 15N contains one more neutron than the naturally
occurring 14N isotope; thus, molecules containing 15N are denser than those
containing 14N.

Typically, nitrogen has an atomic or mass number of 14. That means it has 7 protons,
and 7 neutrons but here we have 15N, which means that the nitrogen contained an
extra neutron. So, it is heavier and denser than those molecules containing 14N.

Unlike radioactive isotopes, 15N is stable. After many generations in this medium,
almost all nitrogen-containing molecules in the E. coli cells, including the nitrogenous
bases of DNA, contained the heavier isotope.

During the experimental process, the sedimentation equilibrium centrifugation, also

known as buoyant density gradient centrifugation is utilized to distinguish DNA
containing 15N from DNA containing 14N, which is crucial for the success of this
experiment.
The experimental process uses sedimentation equilibrium centrifugation, also known as
buoyant density gradient centrifugation, to distinguish DNA containing 15N from DNA
containing 14N, which is essential to the success of this experiment.
In this technique, a centrifuge tube is filled with a heavy salt solution and DNA
fragments. The tube is then rapidly spun in a centrifuge. After several days of spinning,
a gradient of density forms with low-density material at the top and high-density material
at the bottom within the tube. The density of the DNA fragments is similar to that of salt,
with light molecules rising and heavy molecules sinking. In this case, the 14N-DNA
remains nearer to the top while 15N-DNA will reach closer to the bottom of the tube.

They used centrifugation, which can separate things according to their weight. Initially,
all of the DNA in the cell was heavy and was at the bottom of the tube since it was
grown in heavy nitrogen. Then they started growing these cells in the presence of light
nitrogen. So all of the new DNA made in subsequent cell divisions would be lighter.

In this experiment, Meselson and Stahl discovered that DNA from bacteria grown
exclusively on media containing 15N formed a single band at the location expected of
DNA containing only 15N (Figure 9.3a).

DNA from bacteria was transferred to the medium with 14N, it similarly formed a single
band after one round of replication, but at a position intermediate between that expected
of DNA containing only 15N and that expected of DNA containing only 14N (Figure
9.3b). This result is incompatible with the conservative replication model, which predicts
one heavy band for the original DNA molecules and one light band for the new DNA
molecules. However, a single band of intermediate density is predicted by both the
semiconservative and the dispersive models.

After one cell division, the DNA was half as heavy: so half of the DNA molecule
contained heavy nitrogen and the other half didn't. This is not in line with the
conservative DNA replication model, which would predict that one molecule
would be all light and the other all heavy. However, this was still in line with the
semi-conservative or the dispersive model.

Meselson and Stahl grew the bacteria in media containing 14N for a second generation
in order to differentiate between these two models. After a second round of replication in
medium with 14N, two bands of equal intensity appeared, one in the intermediate
position and the other at the position expected of DNA containing only 14N (Figure
9.3c).

After two cell divisions, the DNA molecule was now either half-heavy and half-light or
entirely light. So this is now not in line with the dispersive model which will predict that
the DNA after two cell divisions will contain a mixture of heavy and light DNA.
Instead, this experiment agrees with the semi-conservative DNA replication model:
every cell gets one old DNA strand and one new one.
All samples collected after successive replication rounds produced two bands, and the
band representing light DNA became progressively stronger (Figure 9.3d). Meselson
and Stahl's findings were incompatible with those predicted by both conservative and
dispersive replication, and they were exactly what was expected for semiconservative
replication.

By discovering that two DNA strands can separate and provide a template for the
production of two new strands. The possibility of this was once viewed with great
skepticism by scientists, but Meselson and Stahl demonstrated it through a brilliantly
executed experiment. They proceeded by showing that heating DNA can also separate
the DNA strands. This simple concept serves as the foundation for an important
laboratory technique known as a polymerase chain reaction, which is utilized in genetic
testing, forensics, and paternity tests.

Meselson and Stahl identified the fundamental mechanism by which our DNA is passed
from cell to cell. This is the underlying principle of heredity and a crucial step in human
evolution from a single cell to a fully developed organism with the same DNA sequence
in every cell. This fascinating discovery leads in many new doors and research avenues
for further exploration about this topic.
And so after Watson and Crick came up with the shape of DNA, this characteristic
double helix, the next thing to unlock was how does it make a copy of itself?

And so there were immediately three proposals as to how it might copy itself. Number
one, it could kind of unfold in the middle and then it could copy new templates on either
side. This is kind of what Watson and Crick proposed. There was another what's called
the conservative theory that somehow the DNA was kind of wrapped around these
histone proteins. And then it would make an exact duplicate of itself, almost like a
photocopier. And then there's the dispersive model that maybe it would chunk itself into
little ten, twelve, nucleotide segments. And then each of those would be copied. And so
what Meselson and Stahl did is they figured out which of these three is right. And
in this picture, down here at the bottom is, it was later figured out that this is Martha
Chase, who's also famous for that Hershey-Chase experiment.

And so what did they do? Well basically what they did was they grew e. coli, so this
is Escheriichia coli, or a bacteria. And they grew it in heavy nitrogen. What does
that mean? Well nitrogen normally has an atomic or mass number, excuse me, of 14.
That means it has 7 protons, 7 neutrons. But heavy nitrogen is going to have an extra
neutron. And so it's slightly heavier. And when you spin it in an ultra centrifuge, DNA
that contains this heavy nitrogen is going to be moved to the bottom. And those
that contain lighter nitrogen is going to stay up near the top. And so we can look at
the density of the DNA to figure out what nitrogen is actually being added. And so if you
think about it this way, thought experiment.

Let's say we started with heavy nitrogen. And then we were to copy that. So as the
bacteria make copies of themselves, they're going to unzip that DNA in the middle. And
since they're just in nitrogen 15, you're going to have two strands of DNA. But
each of those are going to be all heavy nitrogen. So it would go to the bottom of a
centrifuge. If they were to copy themselves again, the e.coli and make new template
strands, 100% of them are going to be of heavy nitrogen. And so what they did is they
bred them in there until the point at which they were all nitrogen, all heavy nitrogen.

They then switched them to e.coli living in the lighter normal nitrogen. And then they
observed what happened with each generation. And so basically the lighter nitrogen in
my little simulation, we'll color in orange color. And so bacteria on generation 0, we'll
say, are going to be 100% heavy nitrogen or 100% N15. As they unzip and make copies
of themselves, that new template strand that they're going to add is going to contain
nitrogen 14. And so at this point, so kind of to keep track of everything, we had 100%
heavy nitrogen the first time. On this first generation, now we're going to have 100%
of the strands are a hybrid between the two. Half of them are heavy nitrogen and half
of them are not.

We're then going to go through another copying process. And so at this point we've got
one hybrid strand. Another light strand. Another light strand and then a hybrid strand.
And so at this point it would be 50%, if this semiconservative theory is right, 50% light
nitrogen and 50% of the hybrid.

Now think in your head, if we were to do one more copy, which I don't
have room for over here on my simulation, well this strand would create a hybrid. But
the next six strands as they split in half are going to form light nitrogen and this
would form a hybrid. So now we'd have a 75% to 25%.

of them was conservative replication where a DNA molecules would get copied and
make
a second DNA molecules. The second was dispersive where the DNA molecule would
be cut at various
parts, each of which would get copied and reattached to produce two DNA molecules.
And
the third was semi-conservative where the two strands of DNA would separate and
each
one would serve as a template to copy a second strand, thus producing two DNA
molecules.
But which one was it? Many scientists did not believe that two DNA strands could
separate
because of how strong a DNA molecule is, so they were skeptical of the semi-
conservative
model. However, other scientists were skeptical that there could be a way to simply
copy a
whole DNA molecule and thus did not believe the conservative model. The only way to
solve
this was to create an experiment to tell the new DNA strand apart from the old one.
So two scientists, Matthew Meselson and Franklin Stahl, had an idea to design an
experiment
that would be known as the "most beautiful experiment in biology". Their findings are
published in their article titled "The replication of DNA in Escherichia coli" published in
May
1958 in the Proceedings of National Academy of Sciences.
In DNA synthesis, new nucleotides are joined one at a time to the 3’ end of the newly
synthesized strand. DNA polymerases, the enzymes that synthesize DNA, can add
nucleotides only to the 3’ end of the growing strand (not the 5’ end), and so new DNA
strands always elongate in the same 5’-to-3’ direction.
Because the two single-stranded DNA templates are anti-parallel and strand elongation
is always 5’->3’, if synthesis on one template proceeds from, say, right to left, then
synthesis on the other template must proceed in the opposite direction, from left to right.
As DNA unwinds during replication, the antiparallel nature of the two DNA strands
means that one template is exposed in the 5’-> 3’ direction and the other template is
exposed in the 3’-> 5’ direction. So how can synthesis take place simultaneously on
both strands at the fork?
DNA polymerase can only add DNA bases in one direction, from the 5' end to the 3'
end. One of the new strands of DNA, the leading strand, is made continuously, the DNA
polymerase adding bases one by one in the 5' to 3' direction. The other strand, the
lagging strand, cannot be made in this continuous way because it runs in the opposite
direction the DNA polymerase can therefore only make this strand in a series of small
chunks called Okazaki fragments. Each fragment is started with an RNA primer. DNA
polymerase then adds a short row of DNA bases in the 5' to 3' direction. The next
primer is then added further down the lagging strand. Another Okazaki fragment is then
made and the process is repeated again. Once the new DNA has been made the
enzyme exonuclease removes all the RNA primers from both strands of DNA. Another
DNA polymerase enzyme then fills in the gaps that are left behind with DNA. Finally the
enzyme DNA ligase seals up the fragments of DNA in both strands to form a continuous
double strand.

CONTINUOUS AND DISCONTINUOUS REPLICATION

As the DNA unwinds, the template strand that is exposed in the 3’ -> 5’ direction (the
lower strand in Figures 9.7 and 9.8) allows the new strand to be synthesized
continuously, in the 5’-> 3’ direction. This new strand, which undergoes continuous
replication, is called the leading strand.

The other template strand is exposed in the 5’-> 3’ direction (the upper strand in Figures
9.7 and 9.8). After a short length of the DNA has been unwound, synthesis must
proceed 5’-> 3’; that is, in the direction opposite that of unwinding (Figure 9.8). Because
only a short length of DNA needs to be unwound before synthesis on this strand gets
started, the replication machinery soon runs out of template.
By that time, more DNA has unwound, providing new template at the 5’ end of the new
strand. DNA synthesis must start anew at the replication fork and proceed in the
direction opposite that of the movement of the fork until it runs into the previously
replicated segment of DNA. This process is repeated again and again, so synthesis of
this strand is in short, discontinuous bursts. The newly made strand that undergoes
discontinuous replication is called the lagging strand.

What do we mean by direction? In DNA, we don't say North and South. Each strand has
a 5' end and a 3' end. The two strands are anti-parallel so they don’t go in the same
direction. We say DNA either 5' to 3' or 3' to 5'. The sugar of DNA is part of the
backbone of DNA. It has carbons. The carbons on the sugar are numbered right after
the oxygen in a clockwise direction. 1'2' 3'4' and 5'. The 5' carbon is actually outside of
this ring structure. Now, we do the same on the other side but keep in mind that DNA
strands are anti-parallel to each other. This strand on the left runs 5’ to 3’ and the strand
here on the right runs 3’ to 5’ and is known as the top original strand. The bottom strand
5’ to 3’ is the original DNA that is going to be replicated. Here, DNA is unwinding due to
helicase. In this example, it will keep unwinding in this direction (pa right). Primase
places primers. DNA polymerase is building the new strands. Now the thing about DNA
polymerase is, when it’s building a new strand, it can only build the new strand in the 5’
to 3’ direction, meaning it adds new bases to the 3’ end on the new strand. This one is
called the leading strand (upper). In the Lagging strand (lower), primers have to keep
being placed in order for DNA polymerase to build. These fragments that result are
known as Okazaki fragments.

Before we continue, OKAZAKI FRAGMENTS was discovered by Reiji Okazaki. They

short lengths of DNA produced by the discontinuous replication of the lagging strand.
Primers have to get replaced with DNA bases since the primers were made of RNA.
Ligase, the sealing enzyme has to take care of the gaps between the Okazaki
fragments, sealing them together. At the end of replication, we have two identical
double helix DNA molecules from our one original double helix DNA molecule. We call it
semi conservative because the two copies each contain one old original strand and one
newly made. In this process, we don't want DNA polymerase to make errors. If it
matches the wrong DNA bases, then we could have an incorrectly coded gene, which
could ultimately end up in an incorrect protein or no protein at all.