1. DNA: How do we know, what we know?

2. Discovery of DNA…
Many people believe that scientists James Watson and Francis Crick discovered DNA in the 1953, however in reality it was the combination of the work of several scientists that truly lead to our understanding on the structure of DNA. Most notably Rosalind Franklin who doesn’t get the full credit she deserves.

3. Model Building
Watson and Crick used evidence of the possible structures of DNA and tested them by model-building.
They knew the general atomic composition of DNA (because of Erwin Chargaff’s experiments) and X-Ray diffraction patterns by Rosalind Franklin gave insight into the shape of DNA

4. Erwin Chargraff
Experiments showed the abundance of the nitrogenous bases in DNA
Determined that the amount of adenine in DNA is equal to the amount of thymine, and the amount of cytosine is equal to guanine
Led to the understanding of base pairing

5. Rosalind Franklin
The cross in the center indicates a
helical shape

6. Franklin- X-ray Diffraction
A beam of X-rays is directed at a material
Most of if passes through but some is scattered by particles in the material
This scattering is called diffraction.
An X-Ray detector is placed close to the sample to collect the scattered rays, and rotated to determine a 3-D structure, and recorded using X-ray film

7. Watson and Crick’s 1st Model
Their first model consisted of a triple helix with bases on the outside and magnesium holding the strands together and ionic bonds to phosphate groups.
The helical pattern and spacing between subunits fit the diffraction pattern
However,
It was difficult to get all the parts of the model to fit together.
There was not enough magnesium to link the strands
It did not take into account Chargaff’s findings

8. Model Building DNA
So, they made cardboard cut outs to represent the shapes of the 4 bases.
They determined that A-T and C-G base pairs could be formed with hydrogen bonds
This insured the base pairs would be the same length and could fit between two outer sugar-phosphate backbones
They got the idea the helices must be antiparallel
They created another model using metal rods and sheet metal cut to scale

10. Model Building DNA The model convinced everyone who saw it.
The comment was, “It just looked right”
This structure led to the suggested mechanism for copying DNA, and the realization that genetic code was read in triplets.

11. Meselson and Stahl
The semi-conservative replication of DNA intuitively makes sense, however, it still needed to be backed up with evidence.
In 1958, Matthew Meselson and Franklin Stahl published results of their experiment , using a technique known as Cesium Chloride Density Centrifugation

12. Caesium Chloride Density Gradient Centrifugation
STEP #1
A solution of caesium chloride is spun in an a centrifuge ~ revolutions per minute for 20 hours
This separates the cesium ions by density by creating a gradient with the greatest cesium concentration at the bottom of the tube and the lowest at the top of the tube
This means that any substance centrifuged with the CsCl solution with become concentrated at a level corresponding with its density

13. Meselson and Stahl STEP #2
N-15 is a rare isotope of nitrogen (has 1 more neutron than N-14)
They grew E.coli for several generations in a N-15 medium, insuring that almost all the nitrogen atoms in DNA of the bacteria was N-15

14. STEP #3
The bacteria was then transferred to a N-14 medium and cultured.

15. STEP #4
The DNA was extracted and its density was measured using the cesium chloride density gradient centrifugation.
The DNA could be detected because it absorbs ultraviolet light.

16. Meselson and Stahl
The results show the composition of nitrogen in the DNA molecules
half N-15 (from the original parent DNA) and half N-14 (from the second medium)
This proved that DNA replication was semiconservative:
One strand from the original parent strand and one newly synthesized

18. Units of Heredity
From the late 1800s, scientist were convinced that chromosomes played a role in heredity
They were aware that chromosomes were composed of both protein and nucleic acids and so both materials were contenders to be the genetic material of cells.
Until the late 1940s protein was favoured to be the hereditary material
In the late, 1950s, Alfred Hershey and Martha Chase set out to determine if the genetic material of viruses was protein or DNA

20. The Hershey-Chase Experiment
1. They cultured viruses that contained proteins with radioactive S-35 and separately cultured viruses that contained DNA with radioactive P-32
(Remember, DNA contains phosphorus but not sulfur, and proteins contain sulfur but not phosphorus)
2. They infected bacteria separately with the two types of viruses

21. Side Note - bacteriophages
A bacteriophage is a virus that infects bacterial cells
Remember, a virus injects its genetic material into a host cell.
The host cell will replicated the genetic material and make more viruses

23. Hershey-Chase Experiment
3. They used a blender to separate the non-genetic components of the virus (the capsule of the bacteriophage) from the cell 4. The cultured solution was centrifuged to concentrate the cells in a pellet and the non-genetic material of the bacteriophage in the supernatant. 5. The radioactivity of the pellet and the supernatant were measured.

24. Hershey-Chase Experiment
Where should the genetic material be found? In the supernatant or the pellet?
The results showed that 80% of S-35 was in the supernatant compared to 35% of P-32.
This means there was more P-32 in the cell and DNA must be the genetic material of cells.