Avery, McLeod and McCarty set out to identify the transforming principle described by Griffith. The background of their experiment is therefore the work of Griffith, and his discovery that something can be passed on from heat-killed virulent bacteria to living avirulent bacteria to transform them into virulent bacteria.
The idea is to prepare a filtrate of the heat-killed 'S' bacteria, submit it to enzymatic treatment prior to incubating it with the live 'R' bacteria. The ability to transform is then analysed. Four enzyme treatments are used : Dubos SIII enzyme destroys polysaccharide; trypsin and chymotrypsin destroy proteins; ribonuclease destroys RNA, and deoxyribonuclease destroys DNA.
|1||Dubos SIII enzyme||Mouse dies|
|2||Trypsin / Chymotrypsin||Mouse dies|
1) What steps are involved between the treatment with enzymes and the observation on the mouse?
After treating the 'S' form filtrate with enzymes, there are probably some purification steps involved to get rid of the enzymes. Then an important step is to incubate with the 'R' form bacteria, prior to injecting to a mouse. ('S' and 'R' forms are explained in the previous chapter: the Griffith experiment.
2) What is destroyed by the Dubos SIII enzyme?
Dubos SIII enzyme destroys polysaccharide which are the major part of the capsule of the 'S' form bacteria. The fact that the filtrate keeps its ability to transform the 'R' form after treatment with Dubos SIII shows that the polysaccharide capsule is not the transforming agent. This confirms the hypothesis made by Griffith.
3) What is RNA?
RNA stands for ribonucleic acid. More is explained in a special chapter about RNA. The fact that the filtrate doesn't loose its ability to transform after treatment with ribonuclease shows that RNA is not the transforming agent.
4) What is the conclusion of this experiment?
The destruction of DNA by deoxyribonuclease in the filtrate leads to the loss of its ability to transform the 'R' form into an 'S' form. It becomes clear that DNA is the transforming agent. This idea is also supported by the analysis of the transforming principle by electrophoresis, ultraviolet spectroscopy, and ultracentrifugation.
5) Avery was quite surprised at the result of experiment 2. Why?
The fact that the filtrate doesn't lose its ability to transform after treatment with proteases shows that proteins are not the transforming agent. Doubts were raised about the efficiency of the enzymatic degradation, because proteins were thought to be the genetic material. It was suggested that as much as 2% residual protein could be enough to transform the 'R' form into a 'S' form if genes were made of proteins. These doubts prevented Avery's work from being properly recognised: another experiment was needed.