Dr. Israa ayoub alwan Lec -4- AL-Ma’moon University College Medical Laberatory techniques Department Molecular biology/ Second stage Dr. Israa ayoub alwan Lec -4-

Maintaining Genetic Information (Replication Is Semiconservative) Watson and Crick envisioned the two halves of the DNA double helix unwinding and separating, exposing unpaired bases that would attract their complements. In this way, two double helices would form from one. This route to replication is called semiconservative, because each new DNA double helix conserves half of the original. However, separating the long strands posed a huge physical challenge, a little like having to keep two pieces of thread the length of a football field from tangling.

At first, some researchers suggested that DNA might replicate in any of three possible ways: ■ semiconservative, ■ conservative, with one double helix specifying creation of a second double helix, and ■ dispersive, with a double helix shattering into pieces that would join with newly synthesized DNA pieces to form two molecules.  

Meselson and Stahl experiment :- Matthew Meselson and Franklin Stahl, tried Haldane’s experiment using bacteria. Their experimental results not only supported one hypothesis (semiconservative replication), but disproved the other two (conservative replication and dispersive replication). Meselson and Stahl labeled DNA newly synthesized by bacteria with heavy nitrogen ( 15 N) in the media. The DNA could then be distinguished from older DNA that had been synthesized with the more common lighter form, 14 N. The idea was that DNA that incorporated the heavy nitrogen could be separated from newly synthesized DNA that incorporated the normal lighter nitrogen by its greater density. DNA in which one-half of the double helix was light and one-half heavy would be of intermediate density.

In their “density shift” experiments, Meselson and Stahl grew cells on media with heavy nitrogen and then shifted the cells to media with light nitrogen. They traced replicating DNA through several cell divisions. The researchers grew cells, broke them open, extracted DNA, and spun it in a centrifuge. The heavier DNA sank to the bottom of the centrifuge tube, the light DNA rose to the top, and the heavy-light double helices settled in the middle area of the tube. Meselson and Stahl grew E. coli on media containing 15 N for several generations, making all of the DNA heavy. They knew this because only “heavy-heavy” molecules appeared in the tube after centrifugation. They then shifted the bacteria to media containing 14 N, allowing enough time for the bacteria to divide only once (about 30 minutes).

When Meselson and Stahl collected the DNA after one generation and centrifuged it, the double helices were all of intermediate density. The DNA settled in the middle of the tube, indicating that the molecules contained half 14 N and half 15 N. This pattern was consistent with either semiconservative DNA replication or a dispersive mechanism. In contrast, the result of conservative replication would have been one band of material in the tube completely labeled with 15 N, corresponding to one double helix, and another totally “light” band containing 14 N only, corresponding to the other double helix. This did not happen, eliminating conservative replication.

To definitively distinguish among the three routes to DNA replication, Meselson and Stahl extended the experiment one more generation. If the semiconservative mechanism held up, each hybrid (half 14 N and half 15 N) double helix present after the first generation following the shift to 14 N medium would separate and assemble a new half from bases labeled only with 14 N. This would produce two double helices with one 15 N (heavy) and one 14 N (light) chain, plus two double helices containing only 14 N. The tube would have one heavy-light band and one light-light band.

The conservative mechanism would have yielded two bands in the tube in the third generation, indicating three completely light double helices for every completely heavy one, as the bottom portion of the hypothesis 2 column . The third generation for the dispersive model would have been a single large band, somewhat higher than the second-generation band because additional 14 N would have been randomly incorporated into the DNA.

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