DNA is the primary agent for all genetic material. This is common sense but it was not always that way.
In the early 1900's many people thought that protein must be the genetic material responsible for inherited characteristics. One of the reasons behind this belief was the knowledge that proteins were quite complex molecules and therefore, they must be specified by molecules of equal or greater complexity (i.e. other proteins). DNA was known to be a relatively simple molecule, in comparison to proteins, and therefore it was hard to understand how a complex molecule (a protein) could be determined by a simpler molecule (DNA). What were the key experiments which identified DNA as the primary genetic material?
F. Griffith (1928)
Diplococcus pneumoniae, or pneumococcus, is a nasty little bacteria which, when injected into mice, will cause pneumonia and death in the mouse. The bacteria contains a capsular polysaccharide on its surface which protects the bacteria from host defences. Occassionally, variants (mutants) of the bacteria arise which have a defect in the production of the capsular polysaccharide. The mutants have two characteristics: 1) They are avirulent, meaning that without proper capsular polysaccharide they are unable to mount an infection in the host (they are destroyed by the host defences), and 2) Due to the lack of capsular polysaccharide the surface of the mutant bacteria appears rough under the microscope and can be distinguished from the wild type bacteria (whose surface appears smooth).
The virulent smooth wild type pneumococcus can be heat treated and rendered avirulent (still appears smooth under the microscope however).
- w.t. (smooth) + mouse = dead mouse
- mutant (rough) + mouse = live mouse
- heat treated w.t. (smooth) + mouse = live mouse
- heat treated w.t. (smooth) + mutant (rough) + mouse = dead mouse
In this case when the bacteria were recovered from the cold lifeless mouse they were smooth virulent pneumococcus (i.e. indistinguishable from wild type).
The overall conclusions from these experiments was that there was a "transforming agent" in the the heat treated type I bacteria which transfomed the live mutant (rough) type II bacteria to be able to produce type I capsule polysaccharide. This "transforming agent" could have been the DNA or the protein or something completely different.
O.T. Avery (1944)
The experiment of Griffith could not be taken further until methods were developed to separate and purify DNA and protein cellular components. Avery utilized methods to extract relatively pure DNA from pneumococcus to determine whether it was the "transforming agent" observed in Griffith's experiments.
- w.t. (smooth) type I -> extract the DNA component
- mutant (rough) type II + type I DNA + mouse = dead mouse
Isolation of bacteria from the dead mouse showed that they were type I w.t. (smooth) bacteria
A more sophisticated experiment:
Purified type I DNA was divided into two aliquots. One aliquot was treated with DNAse - an enzyme which non-specifically degrades DNA. The other aliquot was treated with Trypsin - a protease which (relatively) non-specfically degrades proteins.
- Type I DNA + DNAse + mutant (rough) type II + mouse = live mouse
- Type I DNA + Trypsin + mutant (rough) type II + mouse = dead mouse
The work of Avery provided strong evidence that the "transforming agent" was in fact DNA (and not protein). However, not everyone was convinced. Some people felt that a residual amount of protein might remain in the purified DNA, even after Trypsin treatment, and could be the "transforming agent".
A.D. Hershey and M. Chase (1952)
T2 is a virus which attacks the bacteria E. coli. The virus, or phage, looks like a tiny lunar landing module.
The viral particles adsorb to the surface of the E. coli cells. It was known that some material then leaves the phage and enters the cell. The "empty" phage particles on the surface cells can be physically removed by putting the cells into a blender and whipping them up. In any case, some 20 minutes after the phage adsorb to the surface of the bacteria the bacteria bursts open (lysis) and releases a multitude of progeny virus.
If the media in which the bacteria grew (and were infected) included 32P labeled ATP, progeny phage could be recovered with this isotope incorporated into its DNA (normal proteins contain only hydrogen, nitrogen, carbon, oxygen, and sulfur atoms). Likewise if the media contained 35S labeled methionine the resulting progeny phage could be recovered with this isotope present only in its protein components (normal DNA contains only hydrogen, nitrogen, carbon, oxygen and phosphorous atoms).
Phage were grown in the presence of either 32P or 35S isotopic labels.
1) E. coli were infected with 35S labeled phage. After infection, but prior to cell lysis, the bacteria were whipped up in a blender and the phage particles were separated from the bacterial cells. The isolated bacterial cells were cultured further until lysis occurred. The released progeny phage were isolated.
Where the 35S label went:
- Adsorbed phage shells 85%
- Infected cells (prior to lysis) 15%
- Lysed cell debris 15%
- Progeny phage <1%
2) E. coli were infected with 32P labeled phage. The same steps as in 1) above were performed.
Where the 32P label went:
- Adsorbed phage shells 30%
- Infected cells (prior to lysis) 70%
- Lysed cell debris 40%
- Progeny phage 30%
The material which was being transfered from the phage to the bacteria during infection appeared to be mainly DNA. Although the results were not entirely unambiguous they provided additional support for the view that DNA was the "stuff" of genetic inheritance.
- Dr. Michael Blaber, Florida State University