DNA replication: Meselson and Stahl experiment – Semiconservative DNA replication

 DNA synthesis Is Semiconservative

Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semiconservative replication.

  • Shortly after the publication of their 1953 paper on DNA structure, Watson and Crick proposed the hypothesis of semiconservative replication
  • The experiment by Meselson and Stahl was an experimental proof for semi conservative DNA replication hypothesis.
  • In 1958, Matthew Meselson and Franklin Stahl conducted an experiment on E.coli which divides in 20 minutes, to study the replication of DNA.

To identify which of the three models of replication applied to E. coli cells, Matthew Meselson and Franklin Stahl needed a way to distinguish old and new DNA.

They did so by using two isotopes of nitrogen: 14N (the common form) and 15N (a rare, heavy form).

(15N is the nonradioactive isotope of nitrogen that makes molecules containing it denser than chemically identical molecules containing the common isotope 14N. Two isotopes of nitrogen which can be distinguished based on their densities by centrifugation in CsCl solution).

Experiment performed by Meselson and Stahl

Question in their mind: Which model of DNA replication – conservative, semiconservative or dispersive applies to E.coli?

Control 1: E.coli grown for many generations solely in 15N containing medium

DNA of E.coli grown in 15N had a higher density (because heavier isotope of nitrogen incorporated into its nitrogenous bases).

Control 2: E.coli grown for many generations solely in 14N containing medium

DNA of E.coli grown in 14N had a lower density (because lighter isotope of nitrogen incorporated into its nitrogen bases)

Figure 1: Controls used for Meselson and Stahl experiment

Method they followed in their experiment

Step 1: Culturing of E.coli cells in 15N containing medium

  • Meselson and Stahl grew a culture of E.coli for many generations in a medium that contained 15N as the sole nitrogen source. (15NH4Cl).
  • After many generations, all the E.coli cells had 15N incorporated into the purine and pyrimidine bases of their DNA.

(since 15N is a heavy isotope of nitrogen the DNA synthesized will be of heavy density).

Step 2: Transfer to 14N containing medium and allowed to replicate

  • Meselson and Stahl took a sample of these bacteria and switched the rest of the bacteria to a medium that contained only 14N (washed them before transferring to remove the medium containing 15N).
  • They purified the DNA sample collected from the bacteria just before transfer to the 14N containing medium.

Step 3: Collection of E.coli samples at regular intervals and DNA extraction

  • Meselson and Stahl collected some of the bacteria after each division and extracted DNA from the bacterial cells (Collected samples of bacteria over the next few cellular generations).
  • Under the conditions they used, E.coli replicates its DNA every 20 minutes. They took samples at an interval of 20 minutes.

Step 4: Density measurement using CsCl density gradient centrifugation

  • After extracting DNA from bacteria they checked for the density of DNA

Meselson and Stahl distinguished between the heavy 15N containing DNA and the light 14N containing DNA with the use of equilibrium density gradient centrifugation

 Why they chosen CsCl density gradient for seperation: The density of most DNAs is approximately same as the density of concentrated solutions cesium chloride (CsCl) which is a heavy salt.

For example:

The density of 6M CsCl is about 1.7 g/cm3. E. coli DNA containing 14N has a density of 1.710 g/cm3.

Substitution of 15N for 14N increases the density of E. coli DNA to 1.724 g/cm3.

Method: CsCl density gradient centrifugation

  • In this technique, an ultracentrifuge tube is filled with a heavy salt solution (CsCl) and a substance whose density is to be measured (DNA fragments).
  • The tube is then spun in a centrifuge at high speeds (50,000 to 60,000 rpm).
  • After several days of spinning, the cesium chloride salt created a density gradient  within the ultracentrifuge tube.

(High CsCl density at the bottom of the tube and low CsCl density at the top of the tube).

  • Under these circumstances, the DNA was pulled down by centrifugal force from the ultracentrifuge tube during the spin until it reached the place where the density of the DNA molecules matched that of the surrounding salt solution.
  • At that point, the DNA formed a stable band.
  • The molecules that form the lowest bands have the highest densities (Example: 15N containing DNA).

Observation

They found that the DNA banding pattern in the density gradient was different in each bacterial generation:

Control 1:  Band with heavier density (Band at the bottom of the tube)

Control 2: Band with lighter density (Band at the top of the tube)

(Control tubes are used for comparing the position of the different bands)

Sample no 1 (Generation 0): At the time of the transfer to the 14N medium, the DNA was uniformly labeled with 15N.

Location of band: Bottom of the tube (same as control 1)

Sample no. 2 (after 20 minutes – generation 1): The sample had all bacterial DNA with an intermediate density.

Location of band: Single band occupied an intermediate position between  DNA of cells grown exclusively in 15N and DNA of cells grown exclusively in 14N conataining medium.

Sample no. 3 (after 40 minutes – generation 2): There were two equally large DNA bands: one of low density and one of intermediate density.

 Location of bands: First band is similar to control 2 (at the top of the tube)

Second band occupy intermediate position between control 1 and control 2 bands

Sample no. 4 (generation 3): The sample contained DNA with both intermediate and light densities. The proportion of low-density DNA increased steadily and proportion of intermediate density DNA goes on decreased.

Location of the bands: First band is similar to control 2 (at the top of the tube)

Second band occupy intermediate position between control 1 and control 2 bands

Figure 2: Meselson Stahl experiment demonstrating that DNA replication is semiconservative

Conclusion from the above observations

By observing the bands generated after first round of cell division (DNA had been duplicated once), They ruled out the hypothesis of conservative replication.

Why conservative replication hypothesis is rejected?

  • In conservative mode of DNA replication, one progeny DNA molecule would consist of two newly synthesized DNA strands and the other would contain the two parent strands. 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 still would be heavy and the other half would be light. Conservative replication would have resulted in two bands.

What does the band with intermediate density appeared after first generation indicates?

  • In the first round of DNA replication, the strands of the double helix (both heavy with 15N) separated.
  • Each strand then acted as the template for the synthesis of second strand, which contained only 14N and hence was less dense.
  • Therefore each double helical DNA of the daughter cells were hybrids containing one new 14N strand and one parent 15N strand and was of intermediate density.

Appearance of a single band of intermediate density after first generation suggested either a semi-conservative or dispersive mode of replication.

To distinguish between these two models, Meselson and Stahl grew the bacteria in medium containing 14N for a second generation.

Reason for appearing two bands in second generation: In the second replication, the 14N-containing strands directed the synthesis of partners with 14N, creating low-density DNA, and the 15N strands formed new 14N partners.

How dispersive mode of replication rejected?

  • The DNA collected from cells grown in 14N containing medium for two generations formed two bands: one band of DNA was in the intermediate position between 15N and 14N and  the other band occupied a position corresponds to 14N DNA only.
  • These type of results will be obtained  if DNA replicates in a semi-conservative manner.
  • Dispersive replication would always result in single band. In each generation, the band will move move up closer to the  14N DNA band.
  • It may therefore also be possible to rule out dispersive replication.

Meselson and Stahl repeated the experiment for few more generation for concluding semi conservative mode of replication. They found that the proportion of low-density DNA increased steadily and proportion of intermediate density DNA goes on decreased.

Meselson and Stahl concluded on the basis of observations and experimental results that molecules of DNA can replicate semi conservatively.

 

 

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