Question

Escherichia coli bacteria grown in a medium containing \({ }^{15}\) N-labeled ammonium chloride produce \({ }^{15} \mathrm{~N}\) -containing DNA. This can be distinguished from ordinary \({ }^{14} \mathrm{~N}-\mathrm{DNA}\) by ultracentrifugation in concentrated cesium chloride solutionthe heavier \({ }^{15}\) N-labeled DNA undergoing sedimentation more rapidly. When the bacteria grown in an \({ }^{15} \mathrm{~N}\) medium are transferred to an \({ }^{14} \mathrm{~N}\) medium, DNA replication continues but, after one generation, all the DNA present appears to be of one kind, containing equal amounts of \({ }^{15} \mathrm{~N}\) and \({ }^{14} \mathrm{~N}\); after two generations, the DNA is now of two kinds present in equal amounts \(-\) all \({ }^{14} \mathrm{~N}-\mathrm{DNA}\) and \({ }^{14} \mathrm{~N},{ }^{15} \mathrm{~N}-\mathrm{DNA}\). What do these results tell about the replication of DNA and its stability in the cell?

Short answer

The results indicate that DNA replication in Escherichia coli is semiconservative, with each original strand serving as a template for new strands. After one generation in the \({ }^{14}\mathrm{N}\) medium, a hybrid \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\) DNA molecule is formed. After two generations, equal amounts of \({ }^{14}\mathrm{N}\)-DNA and \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\)-DNA are present, while the original \({ }^{15}\mathrm{N}\) DNA is no longer found in the cell, suggesting its stability decreases over generations.

Step-by-step solution

1. Understand the Given Information

First, the bacteria are grown in a medium containing \({ }^{15}\mathrm{N}\) -labeled ammonium chloride, which leads to the production of \({ }^{15}\mathrm{N}\)-containing DNA. This can be distinguished from the ordinary \({ }^{14}\mathrm{N}\)-DNA by ultracentrifugation due to the heavier weight of the nitrogen isotope. When the bacteria are transferred to a \({ }^{14}\mathrm{N}\) medium: - After one generation, all the DNA present appears to contain equal amounts of \({ }^{15}\mathrm{N}\) and \({ }^{14}\mathrm{N}\). - After two generations, the DNA is now of two kinds present in equal amounts: all \({ }^{14}\mathrm{N}\)-DNA and \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\)-DNA.

2. Analyze the DNA Replication Process

Based on the given information, we can see that after one generation in the \({ }^{14}\mathrm{N}\) medium, the DNA present contains equal amounts of \({ }^{15}\mathrm{N}\) and \({ }^{14}\mathrm{N}\). This indicates that during replication, the original \({ }^{15}\mathrm{N}\) DNA was used as a template for synthesizing new DNA strands, forming a hybrid \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\) DNA molecule. After two generations, the equal presence of both \({ }^{14}\mathrm{N}\)-DNA and \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\)-DNA suggests that during the second replication, both the \({ }^{14}\mathrm{N}\)-DNA and the \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\)-DNA were used as templates. Since the amounts are equal, it indicates that the original \({ }^{15}\mathrm{N}\) DNA is no longer present in the cell.

3. Determine the Stability of DNA in Cells

The results show that after one generation of growth in the \({ }^{14}\mathrm{N}\) medium, the original \({ }^{15}\mathrm{N}\) DNA is still stable and functional, giving rise to a hybrid DNA molecule. However, after two generations, the original \({ }^{15}\mathrm{N}\) DNA is no longer present, indicating that it is no longer functional or stable in the cell.

4. Conclusions

The experiment demonstrates the semiconservative nature of DNA replication in Escherichia coli, where each original strand serves as a template for the synthesis of a new strand. Hence, after two generations, there is a combination of the \({ }^{14}\mathrm{N}\)-DNA and the \({ }^{14}\mathrm{N}-{ }^{15}\mathrm{N}\)-DNA present in equal amounts. Additionally, this experiment shows that the stability of DNA decreases over generations, with the original \({ }^{15}\mathrm{N}\) DNA being no longer present after two generations.

Key concepts

Semiconservative Replication

DNA replication is a critical process that ensures each new cell receives a complete copy of the DNA. A fascinating aspect of DNA replication is the semiconservative model, which was proposed by James Watson and Francis Crick. This model posits that each of the two strands of the DNA molecule serves as a template for the formation of a new, complementary strand.

Picture two intertwined spirals - the famous double helix. When a cell prepares to divide, these spirals unwind. Each original strand, now separate, guides the formation of a new, complementary strand by attracting the right mix of nucleotides. Eventually, two double helices form, each comprised of one of the original strands and a new, identical partner.

The experiment with E. coli and nitrogen isotopes, as shown in the exercise, provides a classic demonstration of semiconservative replication. After one bacterium generation in the ¹⁴N medium, DNA strings were hybrids with one strand containing ¹⁵N and the other ¹⁴N, confirming that the new strands form alongside conserving the old ones.

Nitrogen Isotope Labeling

Nitrogen isotope labeling is a powerful tool used to trace atoms through metabolic processes and, as in this case, DNA replication. By growing E. coli in a medium with ¹⁵N-labeled ammonium chloride, all the nitrogen in the DNA was in the heavier isotope form.

Isotopes are atoms of the same element that differ in the number of neutrons. While ¹⁴N is the most common nitrogen isotope, ¹⁵N is heavier due to the extra neutron. This small difference in mass allows scientists to differentiate between the DNAs that incorporate these isotopes. Thus, when the bacteria replicated their DNA in a ¹⁴N environment, the new DNA incorporated the lighter isotope, providing a clear distinction between 'old' and 'new' strands during replication.

This technique not only illuminated the replication mechanism but also enabled a better understanding of molecular dynamics within a cell.

Ultracentrifugation

Ultracentrifugation is a sophisticated laboratory technique used to separate molecules based on their size, shape, and density. This process involves spinning samples at very high speeds, generating a force thousands of times greater than gravity. Molecules in the sample migrate, forming layers according to their mass and density.

In the context of the isotope labeling experiment, scientists used ultracentrifugation to separate DNA containing ¹⁵N from that containing ¹⁴N. The heavier DNA with ¹⁵N sank faster to the bottom of the cesium chloride gradient during centrifugation compared to the lighter DNA with ¹⁴N.

As a result, distinctive bands representing DNA with different isotopic compositions emerged. The analysis of these bands provided crucial insight into the semi-conservative replication of DNA, allowing scientists to visualize the separation and distribution of the hybrid DNA strands formed during replication in successive generations of bacteria.