MODE OF DNA REPLICATION: MESELSON-STAHL EXPERIMENT

There were three models for how organisms might replicate their DNA: semi-
conservative, conservative, and dispersive.

1. The semi-conservative model, in which each strand of DNA serves as a template to

make a new, complementary strand, seemed most likely based on DNA's structure.

Point of difference Semi-Conservative conservative Dispersive

Prediction for Parental strands One helix is made Two DNA
replication acts as template for Entirely of old molecules are a
the synthesis of a DNA and one mixture of both
new daughter helix made entirely parent and
DNA molecule of new DNA daughter DNA
molecule

2. The models were tested by Meselson and Stahl, who labeled the DNA of bacteria
across generations using isotopes of nitrogen.

3. From the patterns of DNA labeling they saw, Meselson and Stahl confirmed that
DNA is replicated semi-conservatively.
Mode of DNA replication

Imagine yourself in 1953, after the double helix structure of DNA has just been
discovered^11start superscript, 1, end superscript. What burning questions might
be on your mind, and on the minds of other scientists?

One big question concerned DNA replication. The structure of the DNA double
helix provided a tantalizing hint about how copying might take place 1,2. It seemed
likely that the two complementary strands of the helix might separate during
replication, each serving as a template for the construction of a new, matching
strand.

But was this actually the case? Spoiler alert: The answer is yes! Meselson and
Stahl conducted the famous experiment, sometimes called "the most beautiful
experiment in biology," that established the basic mechanism of DNA replication
as semi-conservative that is, as producing DNA molecules containing one new and
one old strand3.

The three models for DNA replication

There were three basic models for DNA replication that had been proposed by the
scientific community after the discovery of DNA's structure. These models are
illustrated in the diagram below:
 Schematic representation of models of DNA replication.

1. Conservative. Replication produces one helix made entirely of old DNA and one
helix made entirely of new DNA.

2. Semi-conservative. Replication produces two helices that contain one old and one
new DNA strand.
3. Dispersive. Replication produces two helices in which the individual strands are
patchworks of old and new DNA.

 Semi-conservative replication. In this model, the two strands of DNA unwind

from each other, and each acts as a template for synthesis of a new, complementary
strand. This results in two DNA molecules with one original strand and one new
strand.

 Conservative replication. In this model, DNA replication results in one molecule

that consists of both original DNA strands (identical to the original DNA
molecule) and another molecule that consists of two new strands (with exactly the
same sequences as the original molecule).

 Dispersive replication. In the dispersive model, DNA replication results in two

DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In
this model, each individual strand is a patchwork of original and new DNA.
However, biology is also full of examples in which the “obvious” solution turns
out not to be the correct one. So, it was key to experimentally determine which
model was actually used by cells when they replicated their DNA.

Meselson and Stahl cracked the puzzle

Matt Meselson and Franklin Stahl originally met in the summer of 1954, the year
after Watson and Crick published their paper on the structure of DNA. Although
the two researchers had different research interests, they became intrigued by the
question of DNA replication and decided to team up and take a crack at
determining the replication mechanism5
The Meselson-Stahl experiment

Meselson and Stahl conducted their famous experiments on DNA replication

using E. coli bacteria as a model system.

They began by growing E. coli in medium, or nutrient broth, containing a "heavy"

isotope of nitrogen, 15N. (An isotope is just a version of an element that differs
from other versions by the number of neutrons in its nucleus.) When grown on
medium containing heavy 15N, the bacteria took up the nitrogen and used it to
synthesize new biological molecules, including DNA.

After many generations growing in the 15N medium, the nitrogenous bases of the
bacteria's DNA were all labeled with heavy 15N. Then, the bacteria were switched
to medium containing a "light" 14N isotope and allowed to grow for several
generations. DNA made after the switch would have to be made up of 14N, as this
would have been the only nitrogen available for DNA synthesis.

Meselson and Stahl knew how often E. coli cells divided, so they were able to
collect small samples in each generation and extract and purify the DNA. They
then measured the density of the DNA (and, indirectly, its 15N and14N content)
using density gradient centrifugation.
This method separates molecules such as DNA into bands by spinning them at high
speeds in the presence of another molecule, such as cesium chloride, that forms a
density gradient from the top to the bottom of the spinning tube. Density gradient
15
centrifugation allows very small differences—like those between N-and 14N-
labeled DNA—to be detected.
Diagram of a test tube containing CsCl, nitrogen-14-labeled DNA, and nitrogen-
15-labeled DNA following high-speed centrifugation.

The density of the medium in the test tube is greatest at the bottom and least at the
top, thanks to the formation of the CsCl gradient. The nitrogen-14-labeled DNA
forms a band relatively close to the top of the test tube, while the nitrogen-15-
labeled DNA forms a band closer to the bottom of the test tube. The positions of
the bands reflect their relative densities.

Results of the experiment

When DNA from the first four generations of E. coli was analyzed, it produced the
pattern of bands shown in the figure below:
Meselson and Stahl used the first few generations, to provide key information’s.

Generation 0

DNA isolated from cells at the start of the experiment (“generation 0,” just before
the switch to 14N medium) produced a single band after centrifugation. This result
made sense because the DNA should have contained only heavy 15N at that time.

Generation 1

DNA isolated after one generation (one round of DNA replication) also produced a
single band when centrifuged. However, this band was higher, intermediate in
density between the heavy 15N DNA and the light 14N DNA.

The intermediate band told Meselson and Stahl that the DNA molecules made in
the first round of replication was a hybrid of light and heavy DNA. This result fit
with the dispersive and semi-conservative models, but not with the conservative
model.
The conservative model would have predicted two distinct bands in this generation
(a band for the heavy original molecule and a band for the light, newly made
molecule).

Generation 2

Information from the second generation let Meselson and Stahl determine which of
the remaining models (semi-conservative or dispersive) was actually correct.

When second-generation DNA was centrifuged, it produced two bands. One was in
the same position as the intermediate band from the first generation, while the
second was higher (appeared to be labeled only with light nitrogen ).

This result told Meselson and Stahl that the DNA was being replicated semi-
conservatively. The pattern of two distinct bands—one at the position of a hybrid
molecule and one at the position of a light molecule—is just what we'd expect for
semi-conservative replication (as illustrated in the diagram below). In contrast, in
dispersive replication, all the molecules should have bits of old and new DNA,
making it impossible to get a "purely light" molecule.

Diagram of the Meselson-Stahl experiment.

 All DNA is initially nitrogen-15-labeled. A DNA sample is taken prior to adding
the bacteria to nitrogen-14 medium, and when centrifuged in a CsCl gradient, the
DNA forms a single band low in the tube (indicating DNA labeled entirely with
nitrogen-15). This is labeled as "generation 0."

The bacteria are then added to nitrogen-14 medium and grown for four generations.
At each generation (which takes about 20 minutes to grow), a DNA sample is taken
and analyzed by centrifugation in a CsCl gradient.

 Generation 0 (see above). 100% of DNA in nitrogen-15 band.

 Generation 1. 100% of DNA in a band intermediate in position between nitrogen-

14 and nitrogen-15 bands.

 Generation 2. 50% of DNA in a band intermediate in position between nitrogen-14

and nitrogen-15 bands. 50% of DNA in nitrogen-14 band.

 Generation 3. 25% of DNA in a band intermediate in position between nitrogen-14

and nitrogen-15 bands. 75% of DNA in nitrogen-14 band.

 Generation 4. 12% of DNA in a band intermediate in position between nitrogen-14

and nitrogen-15 bands. 88% of DNA in nitrogen-14 band.

The right panel of the figure is a cartoon illustrating how these results can be
explained by the semiconservative model. The starting double helix is fully labeled
by nitrogen-15 (generation 0). Replication of this helix produces two helices that
each contain one nitrogen-15 (old) and one nitrogen-14 (new) strand (generation
1). Replication of these two helices produces four helices, two which are also
nitrogen-15/nitrogen-14 hybrids and two which are purely made of nitrogen-14
(generation 2). Replication of the generation 2 helices produces eight helices, two
of which are nitrogen-15/nitrogen-14 hybids and six of which are purely made of
nitrogen-14 (generation 3). Replication of the generation 3 helices produces sixteen
helices, two of which are nitrogen-15/nitrogen-14 hybrids and fourteen of which
are purely made of nitrogen-14 (generation 4).

Generations 3 and 4

In the semi-conservative model, each hybrid DNA molecule from the second
generation would be expected to give rise to a hybrid molecule and a light
molecule in the third generation, while each light DNA molecule would only yield
more light molecules.

Thus, over the third and fourth generations, we'd expect the hybrid band to become
progressively fainter (because it would represent a smaller fraction of the total
DNA) and the light band to become progressively stronger (because it would
represent a larger fraction).

As we can see in the figure, Meselson and Stahl saw just this pattern in their
results, confirming a semi-conservative replication model.

Conclusion

The experiment done by Meselson and Stahl demonstrated that DNA replicated
semi-conservatively, meaning that each strand in a DNA molecule serves as a
template for synthesis of a new, complementary strand.
 REFERENCES
Carr, S. M. (2015). Preparative density-gradient ultracentrifugation of DNA.
Retrieved from https://www.mun.ca/biology/scarr/CsCl_density-
gradient_centrifugation.html(Opens in a new window)(Opens in a new window).

Davis, T. H. (2004). Meselson and Stahl: The art of DNA replication. PNAS,
101(52), 17895-17896. http://dx.doi.org/10.1073/pnas.0407540101.

Hanawalt, P. C. (2004). Density matters: The semiconservative replication of

DNA. PNAS, 101(52), 17889-17894. http://dx.doi.org/10.1073/pnas.0407539101.

Meselson, M. and Stahl, F. W. (1958). The replication of DNA in Escherichia coli.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC528642/pdf/pnas00686-0041.pdf.

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Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and
Jackson, R. B. (2011). The basic principle: Base pairing to a template strand. In
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2016 from https://www.dnalc.org/view/15879-Semi-conservative-replication.html.

DNA Replication is Semiconservative

The structure of DNA suggested to Watson and Crick the mechanism by which
DNA — hence genes — could be copied faithfully. They proposed that when the
time came for DNA to be replicated, the two strands of the molecule
 separated from each other but
 remained intact as each served as the template for the synthesis of
  a complementary strand.
When the replication process is complete, two DNA molecules — identical to each
other and identical to the original — have been produced. This mode of replication
is described as semiconservative: one-half of each new molecule of DNA is old;
one-half new.
While Watson and Crick had suggested that this was the way the DNA would turn
out to be replicated, proof of the model came from the experiments of M. S.
Meselson and F. W. Stahl. They grew E. coli is a medium using ammonium ions
(NH4+) as the source of nitrogen for DNA (as well as protein) synthesis. 14N is the
common isotope of nitrogen, but they could also use ammonium ions that were
enriched for a rare heavy isotope of nitrogen, 15N.
After growing E. coli for several generations in a medium containing 15NH4+, they
found that the DNA of the cells was heavier than normal because of the 15N atoms
in it. The difference could be detected by extracting DNA from the E. coli cells and
spinning it in an ultracentrifuge. The density of the DNA determines where it
accumulates in the tube. Then they transferred more living cells that had been
growing in 15NH4+ to a medium containing ordinary ammonium ions (14NH4+) and
allowed them to divide just once. The DNA in this new generation of cells was
exactly intermediate in density between that of the previous generation and the
normal. This, in itself, is not surprising. It tells us no more than that half the
nitrogen atoms in the new DNA are 14N and half are 15N. It tells us nothing about
their arrangement in the molecules. However, when the bacteria were allowed to
divide again in normal ammonium ions (14NH4+), two distinct densities of DNA
were formed half the DNA was normal and half was intermediate.
 Figure 5.6.1 Meselson - Stahl experiment interpretation
As this interpretative figure indicates, their results show that DNA molecules are
not degraded and reformed from free nucleotides between cell divisions, but
instead, each original strand remains intact as it builds a complementary strand
from the nucleotides available to it. This is called semiconservative replication
because each daughter DNA molecule is one-half "old" and one-half "new".
Immortal strands. Note that the "old" strand (the red one in the top half of the
figure) is immortal because — barring mutations or genetic recombination — it
will continue to serve as an unchanging template down through the generations.
E. coli is a bacterium, but semiconservative replication of DNA also occurs in
eukaryotes. And because each DNA molecule in a eukaryote is incorporated in one
chromosome, the replication of entire chromosomes is semiconservative as well.
This also means that the eukaryotic chromosome contains one "immortal strand" of
DNA.

Contributors
 John W. Kimball. This content is distributed under a Creative Commons
Attribution 3.0 Unported (CC BY 3.0) license and made possible by funding
from The Saylor Foundation.