Meselson and Stahls Experiment

To determine 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). Meselson and Stahl grew a culture of E. coli in a medium that contained 15N as the sole nitrogen source; after many generations, all

A centrifuge tube is filled with a heavy salt solution and DNA fragments.

It is then spun in a centrifuge at high speeds for several days.

It is then spun in a centrifuge at high speeds for several days.

DNA with ,4N DNA with ,5N

A density gradient develops within the tube. DNA will move to where its own density matches that of salt. Heavy DNA (with 15N) will move toward the bottom; light DNA (with 14N) will remain closer to the top.

12.2 Meselson and Stahl used equilibrium density gradient centrifugation to distinguish between heavy, 15N-laden DNA and lighter, 14N-laden DNA.

the E. coli cells had 15N incorporated into the purine and pyrimidine bases of DNA (see Figure 10.10). 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 (I Figure 12.2). In this technique, a centrifuge tube is filled with a heavy salt solution and a substance whose density is to be measured—in this case, DNA fragments. The tube is then spun in a centrifuge at high speeds. After several days of spinning, a gradient of density develops within the tube, with high density at the bottom and low density at the top.

The density of the DNA fragments matches that of the salt: light molecules rise and heavy molecules sink.

Meselson and Stahl found that DNA from bacteria grown only on medium containing 15N produced a single band at the position expected of DNA containing only 15N (iFigure 12.3a). DNA from bacteria transferred to the medium with 14N and allowed one round of replication also produced a single band, but at a position intermediate between that expected of DNA with only 15N and that expected of DNA with only 14N (< Figure 12.3b).* After a second round of replication in medium with 14N, two bands of equal intensity appeared, one in the intermediate posi-

*This result is inconsistent with the conservative replication model, which predicts one heavy band (the original DNA molecules) and one light band (the new DNA molecules). A single band of intermediate density is predicted by both the semiconservative and dispersive models.

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

Question: Which model of DNA replication—conservative, dispersive or semiconservative—applies to E. coli?

Heavy (

Transfer to ,4N medium and replicate

Replication in ,4N medium

Replication in ,4N medium

Heavy (

2j After one round ofreplkatiön~M B^l After a second round of replication, the DNA appeared as a single band intermediate between that expected for DNA with 15N and that expected for DNA with 14N.

DNA appeared as two bands, one in the position of hybrid DNA (half 15N and half 14N) and the other in the position of DNA that contained only

Parental strand

New strand

Replication in ,4N medium

Replication in ,4N medium the DNA appeared as a single band intermediate between that expected for DNA with 15N and that expected for DNA with 14N.

Conclusion: DNA replication in E.coli is semiconservative.

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