M. Meselson and F. Stahl experiment

Know how M. Meselson and Franklin Stahl proved the semi-conservative model of DNA replication

1. BY RABBI C. BAIDOO UNIVERSITY OF ENERGY AND NATURAL RESOURCES-SUNYANI

2. PRESENTATION OBJECTIVES At the end, you should be able to; Explain DNA replication Describe the three models of DNA replication Explain how Meselson and Stahl established the basic mechanism of DNA replication as semi- conservative. Describe how Meselson and Stahl showed that each strand identified in the CsCl medium was either all heavy or all light

3. INTRODUCTION The base complementarity of Watson - Crick Model immediately suggested that DNA should replicate with each strand remaining intact and specifying a new complementary strand . This mode is called semi-conservative because each strand is conserved –kept intact-even though the double helix is not. Matt Meselson and Franklin Stahl originally met in the summer of 1954, the year after Watson and Crick published their paper on the structure of DNA.

4. In 1958, Mathew Meselson and Franklin W. Stahl found a superb way to test the prediction of the Watson - Crick Model regarding replication. The three models for DNA replication

5. Experiment begins Materials: Ultracentrifuge (60,000 rev/min ), E. coli bacterial cell, NH4Cl ( 15 N source), Cesium chloride (density = 1.7 g/ml ) STEPS ; The scientist grew E. coli in 15 N medium for 14 generations Bacteria took up the 15 N and used it to synthesize new biological molecules including DNA.

6. Cont …. At this time they isolated the first bacteria sample, prepared the DNA and added the CsCl for centrifugation . At the same time, some of the 15 N coated bacteria were switched to medium containing a "light" 14 N isotope and allowed to grow for several generations.

7. cont ….. They called the 1 st sample generation 0 After the transfer to light medium, A sample was taken every 20mins which is the generation time for E coli cells to grow under optimum temperature The DNA from the samples were prepared for high speed centrifugation. (60,000rev/min)

8. Cont … . They found that the salt ions ( CsCl ) form a density gradient, a solution whose density is low at the top of the tube and increases steadily toward the bottom. Large molecules in the solution will find an equilibrium position where their buoyant density equals that of the gradient.

9. EXPERIMENT RESULTS When DNA from the first four generations of E. coli was analyzed, it produced the pattern of bands shown in the figure below :

10. After the Experiment ; Meselson and Stahl then had to show that the units being separated in the cesium chloride ( CsCl ) gradient were double stranded molecule with each strand either all heavy or all light. Their evidence for this took advantage of a useful property of DNA: since the double helix is held together by weak hydrogen bonds, heat of 80-100 °C will separate the strand by disrupting or melting this bonds . Meselson and Stahl melted/heated some of their half-dense DNA and showed that is separates into one dense fraction and one light fraction as it should be as it should.

11. They concluded that each strand of half dense DNA consists of either totally dense or totally light nucleotides, not a mixture of dense and light nucleotides in one strand . This work provided a definitive confirmation of Watson - Crick Model.

12. CONCLUSION The experiment done by Meselson and Stahl demonstrated that DNA replicated semi-conservatively, meaning that each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand . Although Meselson and Stahl did their experiments in the bacterium E. coli , we know today that semi-conservative DNA replication is a universal mechanism shared by all organisms on planet Earth. Some of our cells are replicating their DNA semi-conservatively right now!

13. REFERENCES 1. Reece , J. B., Urry , L. A., Cain, M. L., Wasserman, S. A., Minorsky , P. V., and Jackson, R. B. (2011). The basic principle: Base pairing to a template strand. In Campbell biology (10th ed.). San Francisco, CA: Pearson, 318-319 . 2. Davis , T. H. (2004). Meselson and Stahl: The art of DNA replication. PNAS, 101(52), 17895-17896. 3. Watson , J. D., and Crick, F. H. C. (1953). Genetical implications of the structure of deoxyribonucleic acid. Nature, 171, 740-741 . 4. Davis , T. H. (2004). Meselson and Stahl: The art of DNA replication. PNAS, 101(52), 17895-17896. 5. Meselson , M. and Stahl, F. W. (1958). The replication of DNA in Escherichia coli. PNAS, 44(7), 671-682. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC528642/pdf/pnas00686-0041.pdf.