Assume that a culture of E. coli was grown for approximately 50 generations in 1

Question

Assume that a culture of E. coli was grown for approximately 50 generations in 15N (provided in the medium in the ammonium ion), which is a heavy isotope of nitrogen (14N). You extract the DNA from the culture, and it has a density of 1.723 gm/cm3 (water = 1.00 gm/cm3). From the literature, you determine that DNA containing only the common form of nitrogen, 14N, has a density of 1.700 gm/cm3. Bacteria from the 15N culture were washed in buffer and transferred to 14N medium for one generation immediately after which the DNA was extracted and its density determined.

What would be the expected density of the extracted DNA?
What band(s) would be expected if subjected to ultracentrifugation through a CsCl gradient?
(b) How would your answer to (a) above differ if replication occurred in a conservative fashion?
(c) Assume that the extracted DNA was completely denatured (separated into two single strands by heating at 90oC for 15 minutes), what would be the density (or densities) of the DNA (s) in the heated extract?

Explanation / Answer

Since the cuture was grown for 50 generations in N15 the entire DNA has N15 in place of N14 and the density observed is only for N15. DNA replication follows semiconservative mode of replication (Meselson and Stahl, 1958). The DNA extracted from generation one grown in 14N medium will be intermediate to the density of 14N and 15N DNA. Since semi-conservative mode of replication suggests that exactly one strand is old and one is new, the density will be (1.723+1.700)/2 = 1.7115 g/cm3. It will show only one density band (as observed in Meselson-Stahl experiment) while doing CsCl density gradient centrifugation. (b) In conservative fashion of replication you will observe two bands of densities 1.723 and 1.700 g/cm3 in CsCl gradient centrifugation. (c) the heated extract will have DNA of two differnent densities, corresponding to 14N ssDNA and 15N ssDNA. (In alkaline CsCl the buoyant density of denatured DNA is higher than that of native DNA due to the titration of guanine and thymine. )

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