1. Lecture 2
Structure of DNA

3. Discoveries Hereditary material exists – Mendel’s plant genetics
Chemical nature of DNA
Physical nature of DNA
Structure of DNA

4. Discoveries: The Chemical Nature of DNA
1869—Fredrich Miescher named the chemical nuclei contained nuclein. Other chemists discovered it was acidic and named it nucleic acid.
It was soon realized that there were two types of nucleic acids: DNA and RNA.
Early in the 20th century, 4 types of nucleotides were discovered.

5. Physical Nature of DNA – A Physical Unit
You can see it – experiments demonstrated that inheritance is on chromosomes
You can move it from one place to another – experiments demonstrated that DNA can carry new traits into an organism
You can change it with other physical processes – traits can be changed by physical processes

6. The Search for Genetic Material Leads to DNA
Once Morgan showed that genes are located on chromosomes, proteins and DNA were the candidates for the genetic material.
Until the 1940s, the specificity of function of proteins seemed to indicate that they were the genetic material.
However, this was not consistent with experiments with microorganisms, like bacteria and viruses.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

7. I. Bacterial Transformation is Mediated by DNA
Experiment by Frederick Griffith – 1928
Demonstrated first evidence that genes are molecules
Two different strains of Streptococcus pneumoniae
Non-pathogenic = Avirulent = ROUGH cells (R)
Pathogenic = virulent = SMOOTH (S)
Smooth outer covering = capsule
Capsule = slimy, polysaccharide
Encapsulated strains escape phagocytosis

9. Continued. The capsule alone did not cause pneumonia
Heat-killed S strain was avirulent
Ability to escape immune detection and multiply
When heat-killed S strain was mixed with living R strain  the mouse dies of pneumoniae
Encapsulated strain (S) recovedred from dead mouse  Now a live strain
The R strain had somehow acquired the ability to produce the polysaccharide capsule
Transformation
Ability to produce coat was an inherited trait  Daughter cells also produced capsule

10. The Experiment

11. First Demonstration of Transformation
Uptake of genetic material from an external source resulting in the acquisition of new traits (phenotype is changed)
Griffith’s expriment was the earliest document evidence of transformation

12. What was this transforming agent?
Avery, MacLeod and McCarty defined the transforming agent of Griffith’s experiment as DNA (1944)
Chemical components of heat-killed S strain bacteria were purified and co-injected with live R strain
Polysaccharide/Carbohydrate
Lipids
Protein
Nucleic acids
DNA
RNA

13. Oswald Avery, Maclyn McCarty and Colin MacLeod announced that they found that only DNA transformed the cells.
To replicate, a virus infects a host cell and takes over the cell’s metabolic machinery.
Viruses that specifically attack bacteria are called bacteriophages or just phages.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

14. II. Viral DNA is Transferred into Cells During Infection – The Hershey-Chase Experiment (1952)
T2 Bacteriophage studies
Bacteriophage = viruses that infect bacteria
Major chemical components = DNA and protein
Escherichia coli infected with T2 produce thousands of new viruses in the host cell
Host cell lyses and phage are released

15. Hershey Chase Experiment
Determination of whether DNA or protein was directing synthesis of new phage particles
Viral proteins were radioactively labeled with:
35S by growing T2-infected bacteria in 35S-methionine = 1st Batch
Amino acid labeling
DNA does not contain any sulfur atoms
32P by growing T2-infected bacteria in 32-P
Nucleic acid labeling
Amino acids do not contain phosphorous

16. Continued..
Radioactively labeled viruses were isolated from the culture and used to REINFECT new host cells
Batch 1 = protein labeled
Batch 2 = DNA labeled
Blender used to disrupt phage on surface of bacteria from cells and their cytoplasmic components  then centrifuged
Supernatant?? (Protein never entered the cell)
Pellet?? (DNA injected into the cell)

17. III. Chargaff’s Rules
Erwin Chargaff (1947) provides more evidence that DNA = genetic material
Analysis of base composition of DNA compared between different organisms
Nitrogenous bases
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
Conclusions of Chargaff
DNA composition is species specific
The amounts of A,G,C and T are not the same between species
Ratios of nitrogenous bases vary between species

18. This diversity strengthened argument that DNA is the molecular basis of inheritance
Chargaff’s Rules
Amount of A = T
Amount of G = C

19. IV. X-Ray Crystallography Data Provides James Watson and Francis Crick with Insight into DNA Structure
The Race is On
Linus Pauling
Maurice Wilkins and Rosalind Franklin
Watson and Crick
X-ray Crystallography defined
Diffracted X-rays as they pass through a crystallized substance
Patterns of spots are translated by mathematical equations to define 3-D shape

20. Watson and Crick Discovered the Structure of DNA by Building Models of X-ray Data
Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA.
X-rays are diffracted as they passed through purified, crystallized DNA.
The diffraction pattern can be used to deduce the three-dimensional shape of molecules.
James Watson learned from their research that DNA was helical in shape.

21. X-ray Chrystallograph of DNA
The diffraction pattern obtained by Franklin and Wilkins showed a X pattern which hinted of a 2 stranded helical form

22. The helical turn of DNA correlates to the horizontal lines in the picture which measures to 34 Angstroms.
They also calculated that the gap between based pairs was 3.4 A as measured on the distance from the center of the X to the ends.
Simple math deduced that there are 10 nucleotides per turn.

23. Franklin and Wilkins also showed that the sugar phosphate backbones were found to be on the outside of the helix.
The hydrated and dry forms of DNA showed that water could easily come in and bind to DNA, a fact that could only happen if the feature showed sugar phosphate backbones being on the outside.

24. Rosalind Franklin’s data provide clues about DNA’s 3-D shape
Helix
Width = 2 nm  probably two strands (DOUBLE HELIX)
Nitrogenous bases = 0.34 nM apart
One turn every 3.4 nM (10 base pairs per turn)

25. The arrangement of the three major components in nucleic acid polymers was already well known – but the 3-D shape was still unclear
Sugar phosphate backbone
Bases

26. Putting the hydrophobic nitrogenous bases on the inside, and the sugar-phosphate groups on the outside was a stable arrangement

27. Base pairing was worked out by trial and error
The distance between the sugar-phosphate backbone groups is constant
Therefore purine-purine or pyrimidine-pyrimidine were not allowed because spacing would be in inconsistent with data
Purines = A and G (two organic rings)
Pyrimidines – C and T ( one organic ring)

28. Purine-pyrimidine base pairing would be consistent with X-ray data

29. Hydrogen bonding between purines and pyrimidines established the appropriate pairs and reinforced Chargaff’s Rules
2 hydrogen bonds between
A and T
3 hydrogen bonds between
G and C

30. Nature 171: – April 1953
Watson JD and Crick FC (1953) Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid.
1962 – Nobel Prize awarded to three men – Watson, Crick and Wilkins