Why is Cesium chloride used? Why can't the centrifugation be done without it?

The density of the cesium chloride solution increased along a gradient down the tube.

what causes the double helix of the DNA to "unzip"

During DNA replication, the enzyme helicase unwinds the DNA double helix by disrupting the hydrogen bonds that keep it together. Different proteins are also involved in the unzipping of the double helix such as single strand binding proteins that keep the two strands from reforming hydrogen bonds.

Could someone explain to me the results of the 3rd and 4th generation please? I don't quite understand that part, why isn't all DNA semi-conservative? Why is there a N^14 light strand?

1st we know: Bacteria is grown in N15 , then placed in N14 . (remember N14 is the light isotope and N15 is the heavy one.) So initially all nitrogenous bases of each nucleotide will have contained N15 isotope. So once place in N14 medium ,DNA replication will use the N14 Isotope only. No more N15 available. Thats why in the 3rd and 4th generation , the N15 strand disappears over time So what remains are DNA Strands that undergo Semi conservative replication with the fixed amount of N15 and an increasing amount of N14, as N14 is available in the medium. The chart in Fig. 2 kind of explains it.

What would have happened if they grew the bacteria in the light isotope and then introduced it to the heavy isotope? What would the vial layers and densities look like? (Essentially, what would happen if you did the opposite of what they did.)

I think the exact same thing would have happened, but instead of the number of light 14N DNA increasing over successive generations, the heavy 15N DNA would increase with each generation.

If it is concluded that each strand serves as a template for the synthesis of a new complementary strand, how come a hybrid strand does not produce two hybrid molecules. For example, one light and one heavy template strand, produces a complementary heavy and light strand respectively.

Good question! Keep in mind that the DNA is grown in N15 (heavy) but the next generations are in N14 (light). So a hybrid strand cannot produce hybrid molecules because there aren't N15-labelled nucleotides available! The question and response below might help clarify too :)

Why are bacteria like Escherichia coli good for studying DNA replication (as opposed to using other types of cells)?

E. coli is *model organism* that's why. Why E. coli model organism? E. coli is a perfect model for several reasons: E. coli is a single-celled organism that can be manipulated and killed with no ethical concerns. It has a rapid growth rate and is very easy to culture and grow. https://study.com/academy/lesson/escherichia-coli-e-coli-as-a-model-organism-or-host-cell.html The main reasons why E. coli is the organism of choice extends and is not limited to its fast growth in chemically defined media; relative cheap culture media; does not form aggregates; industrial scalability; several molecular tools for manipulation; extensive knowledge of its genetics and genomics; extensive knowledge on its transcriptome, proteome, and metabolome, and several strains are considered biosafety 1, which renders it ideal even for teaching and school demonstrations. In terms of ecology, E. coli is a facultative aerobe (either respiration takes place in the presence of oxygen or fermentation in its absence), which bears a sensor for oxygen presence (redox state in the quinone pool) and can activate or repress the required metabolic enzymes, depending on oxygen levels. The building blocks of E. coli consists of about 55% protein, 25% nucleic acids, 9% lipids, 6% cell wall, 2.5% glycogen, and 3% other metabolites [16–18], which for biotechnological applications is important since carbon flux is often a problematic issue to address to generate a novel metabolic pathway or to enhance a current functioning pathway. E. coli is part of the normal microbiota of mammals, rendering the predominant facultative microbe of the gastrointestinal tract and is currently a hot debate on the impact on normal microflora establishment and their role in disease. E. coli harbours a genome with particular features such as a strikingly organized structure, remnants of many phages, and insertion sequences (IS) and a high transport capacity toward the cytoplasm. https://www.intechopen.com/books/-i-escherichia-coli-i-recent-advances-on-physiology-pathogenesis-and-biotechnological-applications/-i-escherichia-coli-i-as-a-model-organism-and-its-application-in-biotechnology

Would the results be different if they didn't use E. Coli but something else instead?

No, because DNA replicates semi-conservatively. :) Maybe the experiment would be set up slightly different but results must be the same. Replication is semi-conservative in both: Eukaryotes and Prokaryotes meaning that it is the same for _any living organism_.

when its splitting is there every a case when it doesn't split correctly

Absolutely! There are numerous syndromes, diseases and conditions based on the improper splitting. This is something that is easily googled and you can find tons of information on!

It means the other two models don't exist?

Models are proposed explanations for or descriptions of something. All three of the models discussed in this article "exist", but one of them is a better description of how DNA replication actually occurs. Does that help?

Main content

Course: Biology library > Unit 17

Lesson 3: DNA replication

Mode of DNA replication: Meselson-Stahl experiment

Google Classroom

A key historical experiment that demonstrated the semi-conservative mechanism of DNA replication.

Key points:

There were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive.
The semi-conservative model, in which each strand of DNA serves as a template to make a new, complementary strand, seemed most likely based on DNA's structure.
The models were tested by Meselson and Stahl, who labeled the DNA of bacteria across generations using isotopes of nitrogen.
From the patterns of DNA labeling they saw, Meselson and Stahl confirmed that DNA is replicated semi-conservatively.

Mode of DNA replication

Imagine yourself in 1953, after the double helix structure of DNA has just been discovered

^{1}

. What burning questions might be on your mind, and on the minds of other scientists?

One big question concerned DNA replication. The structure of the DNA double helix provided a tantalizing hint about how copying might take place

^{1, 2}

. It seemed likely that the two complementary strands of the helix might separate during replication, each serving as a template for the construction of a new, matching strand.

But was this actually the case? Spoiler alert: The answer is yes! In this article, we'll look at a famous experiment, sometimes called "the most beautiful experiment in biology," that established the basic mechanism of DNA replication as semi-conservative—that is, as producing DNA molecules containing one new and one old strand

^{3}

The three models for DNA replication

There were three basic models for DNA replication that had been proposed by the scientific community after the discovery of DNA's structure. These models are illustrated in the diagram below:

Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.
Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).
Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.

Most biologists at the time would likely have put their money on the semi-conservative model. This model made a lot of sense given the structure of the DNA double helix, in which the two DNA strands are perfectly, predictably complementary to one another (where one has a T, the other has an A; where one has a G, the other has a C; and so forth)

^{2, 4}

. This relationship made it easy to imagine each strand acting as a template for the synthesis of a new partner.

However, biology is also full of examples in which the “obvious” solution turns out not to be the correct one. (Protein as the genetic material, anyone?). So, it was key to experimentally determine which model was actually used by cells when they replicated their DNA.

Meselson and Stahl cracked the puzzle

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. Although the two researchers had different research interests, they became intrigued by the question of DNA replication and decided to team up and take a crack at determining the replication mechanism

^{5}

The Meselson-Stahl experiment

Meselson and Stahl conducted their famous experiments on DNA replication using E. coli bacteria as a model system.

They began by growing E. coli in medium, or nutrient broth, containing a "heavy" isotope of nitrogen,

^{15} N

. (An isotope is just a version of an element that differs from other versions by the number of neutrons in its nucleus.) When grown on medium containing heavy

^{15} N

, the bacteria took up the nitrogen and used it to synthesize new biological molecules, including DNA.

After many generations growing in the

^{15} N

medium, the nitrogenous bases of the bacteria's DNA were all labeled with heavy

^{15} N

. Then, the bacteria were switched to medium containing a "light"

^{14} N

isotope and allowed to grow for several generations. DNA made after the switch would have to be made up of

^{14} N

, as this would have been the only nitrogen available for DNA synthesis.

Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. They then measured the density of the DNA (and, indirectly, its

^{15} N

and

^{14} N

content) using density gradient centrifugation.

This method separates molecules such as DNA into bands by spinning them at high speeds in the presence of another molecule, such as cesium chloride, that forms a density gradient from the top to the bottom of the spinning tube. Density gradient centrifugation allows very small differences—like those between

^{15} N

- and

^{14} N

-labeled DNA—to be detected.

_Image modified from "Meselson-Stahl experiment diagram en," by Mariana Ruiz Villareal (public domain)._

Results of the experiment

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

_Image modified from "Basics of DNA replication: Figure 2," by OpenStax College, Biology (CC BY 3.0). Original artwork from "Meselson-Stahl experiment diagram en," by Mariana Ruiz Villareal (public domain)._

What did this result tell Meselson and Stahl? Let's walk through the first few generations, which provide the key information.

Generation 0

DNA isolated from cells at the start of the experiment (“generation 0,” just before the switch to

^{14} N

medium) produced a single band after centrifugation. This result made sense because the DNA should have contained only heavy

^{15} N

at that time.

Generation 1

DNA isolated after one generation (one round of DNA replication) also produced a single band when centrifuged. However, this band was higher, intermediate in density between the heavy

^{15} N

DNA and the light

^{14} N

DNA.

The intermediate band told Meselson and Stahl that the DNA molecules made in the first round of replication was a hybrid of light and heavy DNA. This result fit with the dispersive and semi-conservative models, but not with the conservative model.

The conservative model would have predicted two distinct bands in this generation (a band for the heavy original molecule and a band for the light, newly made molecule).

Generation 2

Information from the second generation let Meselson and Stahl determine which of the remaining models (semi-conservative or dispersive) was actually correct.

When second-generation DNA was centrifuged, it produced two bands. One was in the same position as the intermediate band from the first generation, while the second was higher (appeared to be labeled only with

^{14} N

This result told Meselson and Stahl that the DNA was being replicated semi-conservatively. The pattern of two distinct bands—one at the position of a hybrid molecule and one at the position of a light molecule—is just what we'd expect for semi-conservative replication (as illustrated in the diagram below). In contrast, in dispersive replication, all the molecules should have bits of old and new DNA, making it impossible to get a "purely light" molecule.

Generations 3 and 4

In the semi-conservative model, each hybrid DNA molecule from the second generation would be expected to give rise to a hybrid molecule and a light molecule in the third generation, while each light DNA molecule would only yield more light molecules.

Thus, over the third and fourth generations, we'd expect the hybrid band to become progressively fainter (because it would represent a smaller fraction of the total DNA) and the light band to become progressively stronger (because it would represent a larger fraction).

As we can see in the figure, Meselson and Stahl saw just this pattern in their results, confirming a semi-conservative replication model.

The dispersive model predicts that each replicated DNA molecule should be a patchwork of parental DNA (from the previous generation) and daughter DNA (newly synthesized during replication). Mechanistically, it's as if the old and new DNA were chopped up during replication, swapped with one another, and then put back together to from helices. Thus, every round of replication under the dispersive model would produce patchwork molecules with both heavy and light sections. The patchwork DNAs would contain a larger and larger fraction of

^{14} N

with each successive replication, so the density of the DNA would gradually decrease over time, resulting in a band (or fuzzy band/smear) that moved higher with each generation.

Under the conservative model, if we started with a single “heavy” (

^{15} N

) DNA molecule, after one round of replication we’d have the original heavy DNA molecule along with one new light molecule. If these DNAs went through a second round of replication, what would we see? There would be one heavy DNA molecule and three light molecules. And after a third round of replication, we'd end up with one heavy and seven light molecules.

This pattern illustrates that, under the conservative model, the heavy DNA never completely disappears but is quickly outnumbered by newly synthesized molecules of light DNA. You can also see that conservative replication never produces the hybrid molecules seen in the other two models. This distinction allowed Meselson and Stahl to eliminate the conservative model after a single generation.

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 your cells are replicating their DNA semi-conservatively right now!

Attribution:

This article is a modified derivative of "Basics of DNA replication," by OpenStax College, Biology, CC BY 4.0. Download the original article for free at http://cnx.org/contents/185cbf87-c72e-48f5-b51e-f14f21b5eabd@10.53.

The modified article is licensed under a CC BY-NC-SA 4.0 license.

Works cited:

Watson, J. D. and Crick, F. H. C. (1953). A structure for deoxyribose nucleic acid. Nature, 171(4356), 737-738. Retrieved from http://www.nature.com/nature/dna50/watsoncrick.pdf.
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.
American Institute of Biological Sciences. (2003). Biology's most beautiful. http://www.aibs.org/about-aibs/030712_take_the_bioscience_challenge.html.
Watson, J. D., and Crick, F. H. C. (1953). Genetical implications of the structure of deoxyribonucleic acid. Nature, 171, 740-741.
Davis, T. H. (2004). Meselson and Stahl: The art of DNA replication. PNAS, 101(52), 17895-17896. http://dx.doi.org/10.1073/pnas.0407540101.

References:

Carr, S. M. (2015). Preparative density-gradient ultracentrifugation of DNA. Retrieved from https://www.mun.ca/biology/scarr/CsCl_density-gradient_centrifugation.html.

Davis, T. H. (2004). Meselson and Stahl: The art of DNA replication. PNAS, 101(52), 17895-17896. http://dx.doi.org/10.1073/pnas.0407540101.

Hanawalt, P. C. (2004). Density matters: The semiconservative replication of DNA. PNAS, 101(52), 17889-17894. http://dx.doi.org/10.1073/pnas.0407539101.

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.

Noles, S. R. (2008). Traditional methods for CsCl isolation of plasmid DNA by ultracentrifugation. In Thermo scientific. Retrieved from https://static.thermoscientific.com/images/D17309~.pdf.

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., pp. 318-320). San Francisco, CA: Pearson.

Semi-conservative replication. (n.d). In DNA learning center. Retrieved July 27, 2016 from https://www.dnalc.org/view/15879-Semi-conservative-replication.html.

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Shailu
Posted 7 years ago. Direct link to Shailu's post “Why is Cesium chloride us...”
Why is Cesium chloride used? Why can't the centrifugation be done without it?
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(22 votes)
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- Dipanshishah1998
  Posted 4 years ago. Direct link to Dipanshishah1998's post “The density of the cesium...”
  The density of the cesium chloride solution increased along a gradient down the tube.
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  (5 votes)
leatisha.phillips
Posted 8 years ago. Direct link to leatisha.phillips's post “what causes the double he...”
what causes the double helix of the DNA to "unzip"
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(8 votes)
Answer
- isaacboardman
  Posted 8 years ago. Direct link to isaacboardman's post “During DNA replication, t...”
  During DNA replication, the enzyme helicase unwinds the DNA double helix by disrupting the hydrogen bonds that keep it together. Different proteins are also involved in the unzipping of the double helix such as single strand binding proteins that keep the two strands from reforming hydrogen bonds.
  Comment on isaacboardman's post “During DNA replication, t...”
  (27 votes)
viegasmaya
Posted 6 years ago. Direct link to viegasmaya's post “Could someone explain to ...”
Could someone explain to me the results of the 3rd and 4th generation please? I don't quite understand that part, why isn't all DNA semi-conservative? Why is there a N^14 light strand?
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(6 votes)
Answer
- Taka Tanabe
  Posted 6 years ago. Direct link to Taka Tanabe's post “1st we know: Bacteria is ...”
  1st we know: Bacteria is grown in N15 ,
  then placed in N14 .
  (remember N14 is the light isotope and N15 is the heavy one.)
  So initially all nitrogenous bases of each nucleotide will have contained N15 isotope.
  So once place in N14 medium ,DNA replication will use the N14 Isotope only.
  No more N15 available.
  Thats why in the 3rd and 4th generation , the N15 strand disappears over time
  So what remains are DNA Strands that undergo Semi conservative replication with the fixed amount of N15 and an increasing amount of N14, as N14 is available in the medium.
  The chart in Fig. 2 kind of explains it.
  Button navigates to signup page
  (14 votes)
Julie S
Posted 6 years ago. Direct link to Julie S's post “What would have happened ...”
What would have happened if they grew the bacteria in the light isotope and then introduced it to the heavy isotope? What would the vial layers and densities look like? (Essentially, what would happen if you did the opposite of what they did.)
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(5 votes)
Answer
- Harine S
  Posted 6 years ago. Direct link to Harine S's post “I think the exact same th...”
  I think the exact same thing would have happened, but instead of the number of light 14N DNA increasing over successive generations, the heavy 15N DNA would increase with each generation.
  Button navigates to signup page
  (3 votes)
Bonin Sok
Posted 6 years ago. Direct link to Bonin Sok's post “If it is concluded that e...”
If it is concluded that each strand serves as a template for the synthesis of a new complementary strand, how come a hybrid strand does not produce two hybrid molecules. For example, one light and one heavy template strand, produces a complementary heavy and light strand respectively.
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(4 votes)
Answer
- Jen
  Posted 6 years ago. Direct link to Jen's post “Good question! Keep in mi...”
  Good question! Keep in mind that the DNA is grown in N15 (heavy) but the next generations are in N14 (light). So a hybrid strand cannot produce hybrid molecules because there aren't N15-labelled nucleotides available! The question and response below might help clarify too :)
  Button navigates to signup page
  (6 votes)
Hannah
Posted 4 years ago. Direct link to Hannah's post “Why are bacteria like Esc...”
Why are bacteria like Escherichia coli good for studying DNA replication (as opposed to using other types of cells)?
Button navigates to signup pageComment on Hannah's post “Why are bacteria like Esc...”
(4 votes)
Answer
- Ivana - Science trainee
  Posted 4 years ago. Direct link to Ivana - Science trainee's post “E. coli is *model organis...”
  E. coli is model organism that's why.
  
  Why E. coli model organism?
  E. coli is a perfect model for several reasons: E. coli is a single-celled organism that can be manipulated and killed with no ethical concerns. It has a rapid growth rate and is very easy to culture and grow.
  
  https://study.com/academy/lesson/escherichia-coli-e-coli-as-a-model-organism-or-host-cell.html
  
  The main reasons why E. coli is the organism of choice extends and is not limited to its fast growth in chemically defined media; relative cheap culture media; does not form aggregates; industrial scalability; several molecular tools for manipulation; extensive knowledge of its genetics and genomics; extensive knowledge on its transcriptome, proteome, and metabolome, and several strains are considered biosafety 1, which renders it ideal even for teaching and school demonstrations.
  
  In terms of ecology, E. coli is a facultative aerobe (either respiration takes place in the presence of oxygen or fermentation in its absence), which bears a sensor for oxygen presence (redox state in the quinone pool) and can activate or repress the required metabolic enzymes, depending on oxygen levels.
  
  The building blocks of E. coli consists of about 55% protein, 25% nucleic acids, 9% lipids, 6% cell wall, 2.5% glycogen, and 3% other metabolites [16–18], which for biotechnological applications is important since carbon flux is often a problematic issue to address to generate a novel metabolic pathway or to enhance a current functioning pathway.
  
  E. coli is part of the normal microbiota of mammals, rendering the predominant facultative microbe of the gastrointestinal tract and is currently a hot debate on the impact on normal microflora establishment and their role in disease.
  
  E. coli harbours a genome with particular features such as a strikingly organized structure, remnants of many phages, and insertion sequences (IS) and a high transport capacity toward the cytoplasm.
  
  https://www.intechopen.com/books/-i-escherichia-coli-i-recent-advances-on-physiology-pathogenesis-and-biotechnological-applications/-i-escherichia-coli-i-as-a-model-organism-and-its-application-in-biotechnology
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  (5 votes)
Darya Behrooz
Posted 5 years ago. Direct link to Darya Behrooz's post “Would the results be diff...”
Would the results be different if they didn't use E. Coli but something else instead?
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(4 votes)
Answer
- Ivana - Science trainee
  Posted 5 years ago. Direct link to Ivana - Science trainee's post “No, because DNA replicate...”
  No, because DNA replicates semi-conservatively. :)
  
  Maybe the experiment would be set up slightly different but results must be the same.
  
  Replication is semi-conservative in both: Eukaryotes and Prokaryotes meaning that it is the same for any living organism.
  Button navigates to signup page
  (4 votes)
Adi.price
Posted 8 years ago. Direct link to Adi.price's post “when its splitting is the...”
when its splitting is there every a case when it doesn't split correctly
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(2 votes)
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- Emily
  Posted 8 years ago. Direct link to Emily's post “Absolutely! There are num...”
  Absolutely! There are numerous syndromes, diseases and conditions based on the improper splitting. This is something that is easily googled and you can find tons of information on!
  Button navigates to signup page
  (8 votes)
Sundus
Posted 6 years ago. Direct link to Sundus's post “It means the other two mo...”
It means the other two models don't exist?
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(2 votes)
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- tyersome
  Posted 6 years ago. Direct link to tyersome's post “Models are proposed expla...”
  Models are proposed explanations for or descriptions of something.
  
  All three of the models discussed in this article "exist", but one of them is a better description of how DNA replication actually occurs.
  
  Does that help?
  Button navigates to signup page
  (7 votes)
ThisIsAVeryMessedUpNickname
Posted 9 months ago. Direct link to ThisIsAVeryMessedUpNickname's post “Something that I can't ye...”
Something that I can't yet understand is how Cesium Chloride helps in creating a density gradient?
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(4 votes)
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