Lecture 2: A Brief History of DNA Flashcards
When and by whom was DNA first discovered?
In 1869, Friedrich Miescher discovered ‘nucelin’ from the nuclei of pus (white blood cells). He showed it was in the nuclei of many other cells. He thought it was a store of phosphorus.
What was Phoebus Levene’s contribution to solving the structure of DNA?
Phoebus Levene discovered the chemical constituents of DNA, including the four ‘letters’ A, C, G and T.
He was a leading biochemist at the time and misled the biochemistry community by concluding that DNA had too simple a structure to be the hereditary molecule, because it only has four ‘letters’, whereas proteins have twenty ‘letters’ (the amino acids).
What did Frederick Griffith discover in 1928?
In 1928, Frederick Griffith shows that bacteria can be transformed from one type to another. He suggested that there is a transforming principle, which can change heredity.
He was working with Streptococcus pneumoniae, which can be transformed from the ‘rough’ R to the ‘smooth’ S phenotype.
What did Avery, McCarty and Macleod discover about the smooth and rough strains of Streptococcus pneumoniae?
- Smooth colonies of Streptococcus pneumoniae have a glycoprotein coat and cause disease and death in mice.
- Rough colonies of Streptococcus pneumoniae do not have a glycoprotein coat and do not cause disease or death in mice.
Avery found that if smooth bacteria are injected into a mouse it dies, if rough bacteria is injected it is fine and if heat-killed smooth bacteria are injected the mouse is also fine.
However, if a mixture of heat-killed smooth bacteria and living rough bacteria are injected, the mouse dies. This means something in the heat-killed smooth bacterial extract is taken up by the rough bacteria, transforming it and making them the pathogenic smooth bacteria.
If bacteria are extracted from the mouse, they are seen to have the smooth phenotype.
How did Avery, McCarty and Macleod use Streptococcus pneumoniae to prove that DNA is the hereditary molecule?
They conducted an experiment:
1) Add detergent to heat-killed smooth bacteria, producing a lysate (soluble) and cellular debris (insoluble).
2) Does the lysate contain the transforming principle? Yes, because the lysate can transform rough bacteria to smooth bacteria.
3) Remove the glycoprotein coat by adding enzyme SIII. This ensures that the coat isn’t just moving from the dead smooth bacteria to the rough bacteria to make them smooth. The lysate without the glycoprotein coat still transforms rough to smooth bacteria.
4) Remove protein by adding trypsin and chymotrypsin (proteases). The lysate now lacks the glycoprotein coat and protein, and still transforms the rough bacteria to smooth.
5) He now suspects nucleic acids are the transforming factor. So he uses alcohol precipitation: increase the alcohol in a solution, which decreases the water in the solution and makes soluble things less soluble. He added alcohol to the lysate, so nucleic acids precipitate and the solution turns very cloudy. The precipitate can be removed with a glass rod and be dissolved in water to give a relatively pure nucleic acid sample.
6) He knows that there are two types of nucleic acid, DNA and RNA. He removes the RNA using RNAase enzyme. This leaves him with a pure DNA sample. This pure DNA sample can transform rough to smooth bacteria.
7) He now believes that DNA is the transforming principle. He tests this by removing the DNA from new lysate using DNAase enzymes. The transforming principle is lost and the rough bacteria are not transformed to smooth.
8) He published his results in 1944, but his results were rejected.
Who eventually convinced the scientific world that DNA is the hereditary molecule and how did they do it?
In 1952, Alfred Hershey and Martha Chase used a kitchen blender to prove the genetic role of DNA in Bacteriophage.
They radioactively labelled DNA and the protein coats (recognises host cell and injects genome into it) of bacteriophage viruses. The DNA was labelled using radioactive phosphorus 32P and the protein was labelled with radioactive sulfur 35S. They allowed the bacteriophage to produce progeny inside the host cell then made the parent viruses fall off the outside of the bacterial host cells using the blender. Then the fallen-off parent viruses as separated from the bacteria containing the progeny viruses by centrifugation. In the progeny only the phosphorus was still labelled, not the sulfur, showing that it is the DNA, not proteins, which is the hereditary molecule.
What are Chargaff’s rules?
- the amount of A = T and the amount of C = G
- the C/G content of different species varies (different species cannot interbreed to produce fertile young)
What was wrong with the structure than Linus Pauling suggested for DNA?
Linus Pauling, the most respected biochemist at the time, suggested a triple helix with phosphates in the middle. This must be wrong because the phosphate groups are excessively electronegative, so repel each other very strongly. Pauling’s structure would fly apart.
How was the structure of DNA solved?
Watson and Crick uses other people’s data, like Rosalind Franklin’s x ray diffraction data, to conclude that an anti-parallel double helix with the bases in the middle in pairs (A and T, C and G) was the structure of DNA. This structure explained Chargaff’s rules and the x ray diffraction data. In their paper Watson and Crick understated the importance of their findings in a very British manner.
