Replicación adn

Classic Experiment

4.3

PROVING THAT DNA REPLICATION IS SEMICONSERVATIVE

T

he discovery that the structure of DNA is a double helix, containing two complementary strands of DNA, led to a number of hypotheses about how

DNA might be replicated. Although the possible replication mechanisms were relatively easy to deduce, proving which occurs in vivo was a more difficult task. In 1958,Mathew Meselson and Franklin Stahl used the newly developed techniques of density-gradient centrifugation, to show that DNA replication proceeds in a semiconservative fashion.

Background
During the 1950s, scientists uncovered many of biological facts we now take for granted, beginning with the discovery that genetic information is passed on through deoxyribonucleic acid (DNA), and continuingthrough the elucidation of DNA’s three-dimensional structure. As the decade neared a close, biologists were ready to study how DNA passed on genetic information from the parental to the progeny generation. James Watson and Francis Crick had hypothesized, based on their double-helical model of DNA, that replication occurs in a semiconservative fashion. That is, the double helix unwinds, the originalparental DNA stands serve as templates to direct the synthesis of the progeny strand, and each of the replicated DNA duplexes contains one old (parental) strand, and one newly synthesized strand, often called the “daughter” strand. Another hypothesis proposed at the time was conservative replication, whereby after replication the parental strands formed one DNA duplex and the two daughter standsformed the second duplex. When these hypotheses were first proposed, little experimental evidence was available to support one over another. In 1957, however, Messelson and Stahl, along with Jerome Vinograd, developed density-gradient centrifugation, a technique that can separate macromolecules

exhibiting very small differences in density. The tools were now available for a definitive test todetermine whether DNA replication occurs by a semiconservative or conservative mechanism.

The Experiment
Meselson and Stahl reasoned that if one could label the parental DNA in such a way that it could be distinguished from the daughter DNA, the replication mechanisms could be distinguished. If DNA replication is semiconservative, then after a single round of replication, all DNA moleculesshould be hybrids of parental and daughter DNA strands. If replication is conservative, then after a single round of replication, half of the DNA molecules should be composed only of parental strands and half of daughter strands. To differentiate parental DNA from daughter DNA, Messelson and Stahl used “heavy” nitrogen (15N). This isotope contains an extra neutron in its nucleus, giving it a higheratomic mass than the more abundant “light” nitrogen (14N). Since nitrogen atoms make up part of the purine and pyrimidine bases in DNA, it was easy to label E. coli DNA with 15N by growing bacteria in a medium containing 15N ammonium salts as the sole nitrogen source. After several generations of growth, the bacteria contained only 15N-labeled DNA. Now that the parental

DNA was labeled, Meselsonand Stahl abruptly changed the medium to one containing 14N as the sole nitrogen source. From this point on, all the DNA synthesized by the bacteria would incorporate 14N, rather than 15N, so that the daughter DNA strands would contain only 14N. As the bacteria continued to grow and replicate their DNA in the 14 N-containing medium, samples were taken periodically and the bacterial DNA wasanalyzed with the newly developed technique of equilibrium density-gradient centrifugation. In this type of analysis, a DNA sample is mixed with a solution of cesium chloride (CsCl2). During long periods of high-speed centrifugation the CsCl2 forms a gradient, and the DNA migrates to the position where the density of the DNA is equal to that of the CsCl2. If the DNA sample contains molecules of...