Tower: DNA History, Structure and Replication

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On levels 2-4 of the Tower are a series of experiments and activities related to DNA, including history of the identification of DNA as the archival molecule for genetic information, DNA composition, development of the double helix model, and DNA replication.

Genes and DNA

On level 2 of the Tower are reconstructions of two experiments that first established DNA as the molecule that carried genetic information, the 1928 Griffith experiment with pneumococcal transformation and the 1952 Hershey and Chase experiment with bacteriophage. Each experiment includes an explanatory sign giving background information and instructions and several interactive objects that produce the experimental data.

Bacterial Transformation:

This historical experiment, performed in 1928 by Frederich Griffith, was the first demonstration that genetic information could be extracted from nonliving materials, and that genes had a chemical basis. The experiment includes an informational sign that provides background and instructions, 4 test tubes containing living or dead cells and four mice that can be “injected” with the contents of the tubes to test their properties. Mice killed by the contents of the tube will fall over, while mice that survive their injection will run around happily.

• Living S cells: This tube represents a live culture of a virulent (deadly) pneumococcus (Streptococcus pneumoniae). The pneumococcus is one of the causative agents of bacterial pneumonia. S cells are virulent because they produce a sticky carbohydrate capsule that protects them from attack by the mouse's immune system. When these bacteria are grown on agar plate cultures, they produce a smooth, shiny bacterial colony due to the presence of the capsule. S = Smooth.
• Living R cells: This tube represents a live culture of an avirulent (not deadly) strain of the pneumococcus. R cells are mutants that have lost the ability to produce the carbohydrate capsule. The “bare” cells can be successfully defeated by the mouse's immune system. When these cells are grown on agar cultures, the colonies have a matte surface. R = Rough.
• Killed S cells: This tube represents a suspension of S cells that have been killed by heating. Injecting these cells into a mouse tests the ability of the cellular contents – including the capsular material -- to make the mouse sick when the cells can no longer reproduce.
• Living R cells + Killed S cells: This mixture of dead S cells and living R cells produced an astonishing result. Note that neither material alone harms the mouse. But what happens to the mouse injected with this material?
• Four Mice:  Clicking on each of the four mice gives the results of injecting the mouse with the contents of the bacterial culture behind it.  The mice will live or die depending on the presences or absence of virulent cells in the culture. 
• Cage of Mice: The cage of mice produces a notecard describing some of the work that extended Griffith's experiments and suggested that the active substance leading to the death of some of the mice was DNA. 

Hershey-Chase Experiment:

The Hershey-Chase experiment was necessary because of some continued ambivalence in the scientific community about accepting DNA with the potential to hold genetic information. The difficulty was that the structure of DNA, with its four bases, seemed to be far too simple to contain the information required for producing and sustaining the life of an organism. In addition, proteins were known to be structurally complex and were thus the favored candidate for the gene. This is an excellent example of how a strong intellectual commitment to a concept can blind scientists to the appropriate interpretation of data.

The experiment that overcame all residual resistance to the acceptance of DNA as an informational reservoir was performed by Alfred Hershey and Martha Chase in 1952. The experimental organism was a bacteriophage, or bacterial virus. Phage are composed of a DNA core contained in a protein coat or capsid. In the T2 phage, one or more phage bind to the surface of their host bacterial cell, E. coli. A protein tube is then used to inject the phage DNA into the interior of the cell, where it replicates and then directs the synthesis of new phage particles. But but could protein as an information source be entirely excluded from the infection process? This was the question that the Hershey and Chase experiment was designed to answer.

The experiment includes an informational sign, three flasks of phage and bacterial cultures, a blender and four test tubes representing the results of four different experimental manipulations. Clicking on each of these items provides information or data to interpret.

• S35-labelled phage: When phage are grown up in the presence of S-35, the sulfur is taken into sulfur-containing amino acids of the phage proteins. There is no sulfur in DNA, so DNA does not incorporate an radioactivity from this culture.
  
• P32-labelled phage: When phage are grown up in the presence of P-32, the phosphorus is taken into the phosphate group of DNA. When you study the structure of DNA in the next room, you will see that each subunit of which DNA is composed contains a phosphorus atom, so P-32 is an excellent material for labelling DNA.
• Uninfected E coli cells: The cells infected with the radioactively labelled phage had no previous exposure to either phage or to radioactive compounds. Therefore the labelled phage was the only source for radioactivity that might appear in these cells.
  
• Blender: Clicking on the blender will make the contents spin and also passes an informational note explaining the importance of the blender in this experiment. Since the phage must attach to the surface of the host bacterial cell to inject their DNA and infect the cell, spinning the cells in a blender knocks the phage off of the cell surface. If the phage are left undisturbed long enough, the cells become infected and spinning cannot affect the process of infection. The cells are separated from the medium containing the infecting phage after blending and examined. The results of these manipulations are seen in the four test tubes. In each case you will be asked about the success of the infection and the evidence for the entry of one of the two labelled materials into the host cell.
  
• S-5 tube: This tube contains cells infected with S-35 labelled phage and left undisturbed for only five minutes before the mixture was spun in the blender. Click on the tube to see if the cells were infected or if either of the two radioactive substances got into the cell.
  
• S-20 tube: This tube contains cells infected with S-35 labelled phage and left undisturbed for 20 minutes before the mixture was spun in the blender. Click on the tube to see if the cells were infected or if either of the two radioactive substances got into the cell.
  
• P-5 tube: This tube contains cells infected with P-32 labelled phage and left undisturbed for only five minutes before the mixture was spun in the blender. Click on the tube to see if the cells were infected or if either of the two radioactive substances got into the cell.
  
• P-20 tube: This tube contains cells infected with P-32 labelled phage and left undisturbed for 20 minutes before the mixture was spun in the blender. Click on the tube to see if the cells were infected or if either of the two radioactive substances got into the cell.
  
• After examining the four preparations, visitors can draw conclusions about whether the DNA or the protein carried the information for making a new generation of phage into the bacterial cells. 

Extract Your Own DNA:

Clicking on the flask gives an informational notecard and a link to the Nova website that describes how DNA can be extracted from human cells using simple household materials. The URL of the instructional site is: http://www.pbs.org/wgbh/nova/teachers/activities/2809_genome_01.html. The materials used in the DNA extraction are a salt solution, dishwashing detergent, and chilled alcohol. Ethanol is most effective, but isopropyl alcohol will work if the alcohol concentration is high enough. The DNA precipitates out at the interface between the cold alcohol and the salt-detergent solution used to extract the DNA, and can be wound up on a glass or acrylic rod.

Level : DNA Structure and Replication:

This level includes information about the evidence leading to the construction of the Watson and Crick double helix model for DNA, structures of the four DNA bases, a model of the helix and a reconstruction of the Meselson and Stahl experiment verifying the semiconservative model of DNA replication. Information is given both from the signs in each area and from many of the objects themselves.

Base Ball: The Four Bases of DNA

Near the entrance to this room is a ball that pops up models of each of the four bases in DNA. Clicking on the ball offers a menu, from which you can select “Adenine”, Guanine”, “Cytosine”, “Thymine”, " Nucleotide", "Basics" or “Info”.

Visitors with little chemistry background should select "Basics", which provides a quick introduction to the composition of organic molecules.  The Basics module has its own menu, including "Atoms," "Small molecules," "Functional Groups," "Complex Molecules" and "Macromolecules."

Selecting “Info” gives a notecard describing the structure of nucleotides, identifying adenine and guanine as purines and cytosine and guanine as pyrimidines. Selecting "Nucleotide" produces a model of a nucleotide. The notecard also explains how adjacent nucleotides are connected by phosphodiester bonds.

Selecting any of the four base names produces a model of the base on the next click. Visitors can compare the purines and pyrimidines and see the side chains that differentiate adenine from guanine and thymine from cytosine. The bases are arranged in space in their pairing positions so that visitors can return to the models to examine the groups involved in hydrogen bonding.

The Chargaff Rules:

This poster is located on the next level, just behind the Base Ball and presents data collected by Erwin Chargaff on the base composition of the DNA extracted from various species. Clicking on the poster gives a notecard that points out important features of the data.

Earlier models of the structure of DNA suggested that it was a tetranucleotide, with one copy of each of the four bases. Chargaff's work showed that the four bases were not present in equal concentrations. Chargaff found that, as anticipated, the DNA of different species had different base compositions, but that the DNA of different tissues of the same species had effectively the same base composition. Of particular interest in this data are the ratios among the various bases.

Within the limits of measurement error, the percentage composition of the two purines (A + G) was equal to that of the two pyrimidines (T + C), but the percentage composition of one of the two purines (or one of the two pyrimidines) could be different from that of the other purine (or pyrimidine). So whereas for all species, the A+G / T+C ratio was 1:1, the ratio of A + T/ G + C was species-specific. Finally, there was a 1:1 relationship between certain purine-pyrimidine pairs, with the concentration of A equal to the concentration of T and the concentration of G equal to the concentration of C. These regularities became known as the Chargaff Rules and were critical clues in the development of the Watson-Crick model of DNA structure.

Watson and Crick, 1953:

Across the room is a section discussing the development of the Watson and Crick model for DNA structure, and the features of the model itself. Active objects in this section include an informational sign, a poster showing the arrangement of the A=T and G=C base pairs, a photo of Rosalind Franklin’s X-ray diffraction image of DNA crystals, a photo of Watson and Crick with their model, a -D model of DNA showing its major features, and a facsimile of the paper published by Watson and Crick in 1953 in the journal Nature.The article is short and can be read within the Second Life viewer. 

Franklin X-Ray Diffraction Photo:

When Watson and Crick were working on a model for DNA structure, studies on the structure of proteins had established the alpha helix as an important structural element in proteins. Alpha helix is a regular folding pattern in which the backbone of the molecule winds clockwise to produce a tubelike structure with a constant diameter. If you wind pipe-cleaner or a similar item in the right direction around a pencil, you can see what an alpha helix looks like. Because photomicrographs of DNA indicated that it also had a uniform diameter, the possibility that DNA might be also helical seemed reasonable.

Rosalind Franklin succeeded in producing DNA crystals that could be examined using the technique of X-ray crystallography. X-rays have a short wave length and so when a beam of X-rays is passed through a crystal, the light bounces off of individual atoms in the molecule and will produce a pattern on X-Ray film. In crystals, light waves that hit the same atom at the same angle will combine to produce an interference pattern that is recorded as light and dark patches on the film. The pattern produced by DNA was that predicated for a helical molecule. Franklin also determined that the periodicity, or repeating pattern, for the molecule was 3.4 nm and that the width of the molecule was about 2 nm.

ATCG Base Pairs:

This poster contains a diagram of the A=T and C=G base pairs as they might be viewed looking down into the helix from above. Clicking on the poster produces a notecard explaining the diagram.

From the A:T and G:C equivalence proposed by the Chargaff Rules, Watson and Crick began building models in which the DNA contained two polynucleotide chains and pairs of bases were fitted together in various configurations. Ultimately the solution that worked required that the phosphate and sugar backbone was on the outside of the helix, the base pairs were arranged across the middle, and the two chains were arranged antiparallel to each other, that is, the two chains of the duplex pointed in opposite directions.

DNA Model:

A rotating double helix illustrates the main features of the Watson-Crick model. The two ribbons on the outside represent the alternating phosphates and sugars of the backbone. The bars laid across the middle of the model represent the base pairs. The individual bases are represented by letters in large (for purines) or small (for pyrimidines) blocks. One full turn of the helix includes 10 base pairs. The width of the helix is nm (nanometers: 1 nm = .000001 mm). The distance covered by a full turn of the helix is 3.4 nm, so the distance from one base pair to the next is 0.34 nm. Knowing this, how far would all of the DNA in the human genome (3 billion nucleotides) stretch?

Watson and Crick with their model:

One of the interesting things about the development of the double helix model is that it was done by two “young” investigators. Crick, although he was in his 30’s, was still in the process of completing his PhD. Watson had completed his PhD work a few years earlier, but was only 25. The story of their collaboration is told in Watson’s book The Double Helix, a copy of which lies on the table near the Nature publication.  The book offers a link to the Amazon book store. A copy of Rosalind Franklin's story, Rosalind Franklin and DNA, is on the adjacent couch. 

Semiconservative Replication:

This experiment represents the Meselson-Stahl experiment that verified the semiconservative model for DNA replication. The active objects include an informational sign, and a centrifuge with a monitor that displays data. The centrifuge has several buttons, each of which produces either information about the experiment or experimental data.

 

• Semiconservative Replication Sign:
  
  One of the great understatements of the scientific literature is near the end of the Watson and Crick 1953 paper on the structure of DNA. A copy of of the paper is on the table elsewhere in this room.

"It has not escaped our attention that the specific pair we have postulated
immediately suggests a possible copying mechanism for the genetic material. "

What Watson and Crick had noticed was that since each of the two DNA strands of the duplex was complementary to the other, each had the necessary information for determining the sequence of the other strand. Each individual strand could act as a template for aligning the nucleotides for its partner.

For example in the short duplexed sequence below

----A T G A C C G A G T T A C G -----
----T A C T G G C T C A A T G C -----

If the two strands are separated and then the base pair rules applied to select new nucleotides opposite each strand you would have:

A T G A C C G A G T T A C G

T A C T =======>
+

              <=====T A C G
T A C T G G C T C A A T G C

This mode of replication is called SEMICONSERVATIVE, because half of each old DNA duplex becomes part of a new DNA duplex. Put another way, each new DNA molecule contains one original strand and one newly assembled strand. In the diagram the old and newly synthesized DNA strands are different colors.

How can we be sure that DNA replication is semiconservative? Click on the INFO button for information on the test of this hypothesis.

• INFO Button:

Clicking on this button delivers a notecard explaning the rationale behind the Meselson and Stahl experiment. In 1957 Matthew Meselson and Franklin Stahl published an experiment in which they tested the hypothesis that DNA replicates semiconservatively. If you have not already read the notecard from the DNA replication chart, take a look at it now.

First they grew bacterial cells in a medium containing a heavy isotope of Nitrogen: N-15. Then they extracted the DNA and determined its density by centrifugation. Denser DNA migrates further during centrifugation than lighter DNA. DNA which had incorporated the heavier N-15 was denser than DNA grown with the lighter (and more common) isotope of Nitrogen, N-14.
After they had determined the density of the DNA from cells grown under the two conditions described above, they took cells that had been grown up in N-15 and then dropped them into a medium containing only the lighter N-14 and let them replicate several times. Under such conditions, the cells will replicate synchronously for a few generations.

Examine the poster above the centrifuge to figure out what the density of DNA ought to have been after shifting cells whose DNA already contained N-15 into fresh medium containing only N-14 for two cycles of DNA replication.

• SWITCH Button:

Click on the SWITCH button to turn the monitor light on, if it isn't on already.

• DATA Button:

Click on the DATA button to see the density results for the DNA from each experiment. Continue clicking until you get the message “End of Experiment.” In each graph, density is displayed on the X axis and the amount of DNA at various densities is displayed on the Y axis. Density increases along the X axis to the right. If you get close enough to the monitor, you can read the density values.

• The first screen shows the density data for DNA from cells grown in N-14 alone.
• The first screen shows the density data DNA from cells grown in N-15 alone.
• The first screen shows the density data DNA from cells grown in N-15, but then moved into medium with only N-14 for one cycle of replication.
• The first screen shows the density data DNA from cells grown in N-15, but then moved into medium with only N-14 for TWO cycles of replication.

Does the data match your predictions for the density of DNA replicating semiconservatively?

• OPERATION Button:

This one is just for fun. It gives the instructions for opening and closing the centrifuge (click on the lid) and for starting and stopping the centrifuge (click on the rotor).