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Components of Cells
The Macromolecules
SemiConservative Synthesis
Semiconservative

Living and growing cells can be fed isotopes of atoms that are heavier than those they normally encounter. As a result, all the molecules they make (proteins, polysaccharides, and of course, their DNA molecules) are also heavier and denser than normal.

These artificially "heavy" cells can be gently broken open, their "heavy" molecules can be extracted, cleaned up a bit, and then placed into a solution of cesium chloride, which is itself a heavy, dense molecule. When this mixture of DNA molecules and the solution of heavy cesium ions is rotated very, very fast in an ultracentrifuge, the cesium ions form a gradient of density; light density at the top of the tube and heavy density at the bottom of the tube.


The DNA molecules also move in the massive gravity field generated by the centrifuge. They slowly sink through the cesium solution to the place in the gradient of cesium ions where their density is the same as that of the local concentration of cesium ions. Light molecules stay near the top, and heavier, denser molecules sink lower and lower.

It's a bit like water, cork and lead. Cork is less dense than water, so it floats whereas lead is much more dense than water, so it sinks to the bottom of the vessel. Molecules which are less dense than the local cesium ion concentration in the gradient float upwards, whereas the molecules that are more dense than the local cesium ion concentration sink downwards.

In this way it is possible to separate out substances if different density. Imagine a mixture of tiny particles of cork and lead; how could you easily separate them?

The answer would be to pour them into water; the cork particles would float and the lead particles would sink. This method makes use of the different densities of the cork, water and lead. Separating molecules of different density in cesium chloride gradients makes use of the same principle.

This technique was used to investigate how DNA molecules were replicated by living cells.


  • cells were grown in the presence of the heavy isotopes of those atoms used in the making of DNA.

  • the "heavy" DNA was extracted from these cells and centrifuged in cesium chloride gradients and the exact position at which the heavy-DNA "floated" was noted.
    (position 1 in the diagram)

  • living cells with heavy-DNA were transferred to a growth environment containing normal, lighter isotopes of those atoms used to make DNA, and the cells allowed to grow, replicate their DNA and divide once.

  • the new DNA was then extracted and subjected to centrifugation in the cesium chloride density gradients. Once again the DNA molecules moved through the gradient of cesium ions until they "floated" at the position where their new density was the same as the local density of cesium ions.
    (position 2 in the diagram)

  • other cells with heavy-DNA were transferred to normal, light growth conditions and allowed to make DNA once, for the first division, and then allowed to make DNA a second time during a second round of growth and division.

  • the mixture of DNA molecules from these cells were extracted and centrifuged as before, and the positions at which all the different DNA molecules "floated" in the gradients of cesium ions was recorded.
    (position 3 in the diagram)

  • the results showed that the original (heavy) parental DNA molecules banded towards the bottom of the tube and the totally light DNA floated closer to the top of the tube. In-between was a band of DNA which was half-heavy and half-light that had been made during one round of DNA replication.

Interpretation of results
During replication of DNA molecules the two complementary strands are separated from one another.

Enzyme machinery within the cells then makes a new strand (using light atoms) on the separated original strands. This produces a hybrid-DNA molecule which is half-heavy and half-light. These hybrid-DNA molecules float higher in the cesium density gradients, and are easily seen in a different place in the tube from that of the original, totally heavy-DNA parental molecules.

This result would indicate that the cells are replicating their DNA molecules by saving half the original molecule and making a brand new half. This method of "saving half" the molecule is called semiconservative.

This is confirmed when these hybrid-DNA-containing cells carry out a second round of DNA replication in presence of light atoms.

The hybrid-DNA molecules split into two halves yet again and two more "light" halves are made. The mixture of DNA molecules now contains totally light-DNA (made by making a light-half on an already light-half) and a hybrid-DNA molecules (made by making light-half on a heavy-half). These different DNA molecules are easily seen as two different bands in the cesium chloride density gradients.

Conclusion
These experiments suggested that all DNA molecules were made by first separating the two complementary halves of the parental DNA molecule and then making two new complementary DNA halves onto the original halves. DNA replication was semiconservative.

BIOdotEDU
© 2001, Professor John Blamire