The "Meselson-Stahl Experiment”

The results of the first critical test of Watson and Crick's proposal that DNA replicates
semiconservatively were published in 1958 by M. S. Meselson and F. W. Stahl. Their results
showed that the chromosome (now known to contain a single Watson-Crick double helix of
DNA) of the common colon bacillus Escherichia coli replicated semiconservatively.

Meselson and Stahl grew E coli cells for many generations in a medium in which the
15 14
heavy isotope of nitrogen, N, had been substituted for the normal, light isotope, N. The
purine and pyrimidine bases in DNA contain nitrogen; thus, the DNA of cells grown on
medium containing 15N will have a greater density (weight per unit volume) than the DNA of
14
cells grown on medium containing N. Since molecules of different densities can be
separated by a procedure called equilibrium density-gradient centrifugation, Meselson and
Stahl were able to distinguish between the three possible modes of DNA replication by
15
following the changes in the density of DNA of cells grown on N medium and then
14
transferred to N medium for various periods of time (so-called density transfer
experiments).

The density of most DNAs is about the same as the density of concentrated solutions of
heavy salts such as cesium chloride (CsCl). For example, the density of 6 M CsCl is about 1.7
14
g/cm3. Escherichia coli DNA containing N has a density of 1.710 g/cm3. Substitution of
15 14
N for N increases the density of E coli DNA to 1.724 g/cm3.

When a heavy salt solution such as 6 M CsCl is e centrifuged at very high speeds
(30,000- 50,000 revolutions per minute) for 48-72 hours, an equilibrium density gradient is
formed.(Fig. 5.14). The centrifugal force caused by spinning the solution at high speeds
sediments the salt toward the bottom of the tube. Diffusion, on the other hand, results in
movemenit of salt molecules back toward the top (low salt concentration) of the tube. After a
sufficient period of high speed centrifugation, an equilibrium between sedimentation and
diffusion is reached, at which time a linear gradient of increasing density exists from the top
of the tube to the bottom of the tube (Fig. 5.14). If DNA is present in such a gradient, it will
move to a position where the density of the salt solution is equal to its own density. Thus, if a
15 14
mixture of E. coli DNA containing N ("heavy" DNA) and E. coli DNA containing N
("light" DNA) is subjected to CsCl equilibrium density-gradient centrifugation, the DNA
molecules will separate into two "bands," one containing "heavy" DNA and one containing
"light" DNA (Fig. 5.14).
 Meselson and Stahl took cells that had been growing in medium containing 15N for
15
several generations (and thus contained "heavy" DNA), washed them to remove the N-
containing medium, and transferred them to medium containing 14N. After allowing the cells
14
to grow in the presence of N for varying periods of time, the DNA was extracted and
analyzed in CsCl equilibrium-density gradients. The results of their experiment (Fig. 5.15)
are only consistent with semiconservative replication, excluding both conservative and
dispersive models of DNA synthesis. All the DNA isolated from cells after one generation of
14
growth in medium containing N had a density halfway between the densities of "heavy"
DNA and "light" DNA. This intermediate density is usually referred to as "hybrid" density.
After two generations of growth in medium containing 14N, half of the DNA was of "hybrid"
density and half was "light." These results are precisely those predicted by the Watson and
Crick semiconservative mode of replication (Fig. 5.15). One generation of semiconservative
15 14
replication of a parental double helix containing N in medium containing only N would
15
produce two progeny double helices both of which had N in one strand (the "old" strand)
and 14N in the other strand (the "new" strand). Such molecules would be of "hybrid" density.

Conservative replication would not produce any DNA molecules with "hybrid" density;
after one generation of conservative replication of "heavy" DNA in "light" medium, half of
the DNA would still be "heavy" and the other half would be "light." If replication were
dispersive, Meselson and Stahl would have observed a shift of the DNA from "heavy" toward
"light" in each generation (i.e., "half heavy" ar "hybrid" after one generation, "quarter heavy"
after two generations, etc). Meselson and Stahl result are clearly inconsistent with either of
thesepossibilities.

Subsequent studies have verified Meselson and Stahl's conclusion that DNA replication
is semiconservative and have extended it to many other organisms, including higher plants
and animals.

Autoradiography of Replicating Bacterial Chromosomes

The visualization of replicating chromosomes was first accomplished by J. Cairns in

1963 using the technique called autoradiography. Autoradiography is a method for detecting
and localizing radioactive isotopes in cytological preparations or macromolecules by expo
sure to a photographic emulsion that is sensitive to low energy radiation. Autoradiography is
particularly useful in studying DNA metabolism because DNA can be specifically labeled by
growing cells on [3H] thymidine, the tritiated deoxyribonucleoside of thymine. Thymidine is
incorporated exclusively into DNA, it is not present in any other major component of the cell.

Cairns grew E coli cells in medium containing [3 H] tyhymidine for varying periods of
time, lysed the cells very gently so as not to break the chromasomes (long DNA molecules
are very shear sensitive), and carefully collected the chromosomes on membrane filkers.
These filters were afixed to glass slides, coated with emulsion sensitive to ß particles(the low
energy electrons emited during decay of tritium), and stored in the dark for a period of time to
allow sufficient radioactive deçays. The autoradiographs observed when the films were
developed (Fig. 5:16) showed that the chromosomes of E. coli are circular structuses that
exist as θ-shaped intermediates during replication. These autoradiographs further indicated
that the unwinding of the two complementary parental strands (which is necessary for their
separation) and their semiconsservative replication occur simultaneously or are closely
coupled. Since the parental double helix must rotate 360° to unwind each gyre of the helix,
this necessitates the existence of some kind of “swivel” in the chromosome. Present evidence
suggeststhat a transient single strand break (cleavage of one phosphodiester bond in one
strand of the double helix) provides an axis of rotation to allow unwinding.

Cairns interpretation of the autoradiographs was that semiconservative replication

strated at a site on the chromosome, which he called the “origin”, and proceeded sequentially
and unidirectionally around the circular structure (Fig 5.16). subsequent evidence has shown
his original interpretation to be incorrect on one point: replication actually proceeds
bidirectionally, not unidirectionally. Each Y shaped structure is a replication fork, and the
two replication forks move in opposite directions sequentlly around the circular chromosome
(FIG. 5.16).

Figure 5.13 Three theoritical modes of DNA replication: semiconservative,

conservative, and dispersive. During semiconservative DNA replication (left) , each single
strand of the parental double helix is conserved, acting as a template for the synthesis of a
new complementary strand. During conservative DNA replication (center), the entire
parental double helix is conserved and directs the synthesis if a new double helix. During
dispersive DNA replication (right) , both strands of the parental double helix are fragmented
and parental and newly synthesised segments of DNA become interspersed in each of the
strands of the two resulting DNA double helices.
 Figure 5.14 The use of cesium chloride (CsCl) in density gradients centrifugation to
separate DNAs of different densities. This procedure was used by Meselson and Stahl to
demonstrate that the chromosomes of E. coli replicate semiconservatively (see Fig 5.15). the
density of 6 M CsCl is about 1.7 g/cm3 / if such a solution is centrifuged at very high speeds
for a long enough period of time, a density gradient will be formed because of the
equilibrium between (1) sedimentation of the CsCl to the bottom of the centrifuge tube as a
result of the centrifugal force and (2) diffusion of the CsCl toward the top of the tube . the
densities of most naturally occuring nucleic acids fall within the range covered by such
gradients. CsCl density gradients have thus been very useful in the study of nucleic acids.

Figure 5.15 Results (right) and interpretation (left) of the Meselson and Stahl
experiment that demonstrated that the E coli chromosomes replicate semiconservatively. The
DNA of E coli was density-labeled by growing cells for several generations in medium
15
containing the heavy isotope of nitrogen, N. The cells were then transferred to medium
14
containing the normal isotope of nitrogen, N, and allowed to grow for varying periods of
time (one generation, two generations, etc.). The transfer of the density label (from parental
DNA molecules to progeny DNA molecules was followed by the extraction of DNA from
cells grown for varying periods in 14N and the analysis of the DNA in CsCl density gradients
(see Fig. 5.14). DNA that contains 15
N in both strands (“heavy” DNA) forms a band ini the
CsCl density gradient at a higher density position than DNA that contains 14 N in both strands
(“light” DNA ) . After one generation of growth in 14
N medium , the DNA bands at an
intermediate (“hybrid”) density. Such “hybrid” DNA contains 15
N in one strand and 14
N in
14
the other strand. After two gradients of growth in N medium , half of the DNA bands at
the “light” density. These results are precisely those predicted by semiconservative
replication.

Figure 5.16 Depiction of a replicating chromosome of E. coli by autoradiography. (a)

Cairns autoradiograph and an interpretative drawing (upper left inset) of a θ shaped
replicating chromoosme from a cell that had been grown for two generations in the presence
of 3 H tymidine ; section C remains to be replicated the seconds time. Cairns drawing (upper
left insert) show radioaktive strands , of DNA solid lines and nonradioactive strands as
dashed lines. note that autoradiograph loop B, with two radiactive strands, exhibits about
twice the grain density of loop A, with only one radiactive strand. (b) Simplified diagram of
Cairns autoradiograph ilustrating only the events of the replication in progress (1) and
analogous diagrams illustrating the two possible interpretations results, namely ,
unidirectional replication (2) and bidirectional replication (3) In these diagrams, the yellow
lines represent the two strands of the nascent (or daughter) strands. Originally, one of the two
branch points (X and Y) was believed to be a replication fork and the other a terminus
containing a “swivel” , which served as an axis of rotation for unwinding the double helix (2)
. Replication has subsequently been shown to be bidirectional in E. coli . Thus , both X and Y
are replication forks.