Identification of the transforming principle (The Avery, MacLeod, and McCarty Experiment)
- Griffith experiment was a turning point towards the discovery of hereditary material. However, it failed to explain the biochemistry of genetic material. Hence, a group of scientists, Oswald Avery, Colin MacLeod and Maclyn McCarty continued the Griffith experiment in search of biochemical nature of the hereditary material.
- After 10 years of research, Avery, Colin MacLeod, and Maclyn McCarty succeeded in isolating and purifying the transforming substance.
- In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty reported that transforming principle is DNA.
Experimental protocol used by Avery, MacLeod and MacCarty to identify the transforming principle
Avery, MacLeod and McCarty’s experiment was set up very similarly to Griffith’s, except using test tubes instead of mice.
Step 1: Type IIIS strain of Pneumococci were grown in liquid culture medium
Step 2: Centrifugation
Type IIIS cells were settle down to bottom of the centrifuge tube
Step 3: Heat killing of type IIIS bacteria
Heat will kill virulent bacteria
Step 4: Homogenize cells
- Detergent was used to lyse heat killed type IIIS cells.
Example: Deoxycholate (DOC)
- This process is used to destruct the cell membrane of the present cells, releasing the cell’s contents.
- This solution known as lysate contained the RNA, DNA, sugar coat, and proteins of the heat killed S cells.
Step 5: filtration (get Type IIIS bacterial filtrate)
- Heat killed type IIIS cells were filtered out from the lysate.
- Soluble filtrate retained the ability to induce transformation of type IIR avirulent cells.
- This meant that the “transforming factor” was in the filtrate and had to be isolated.
Step 6: Removal of lipids and carbohydrate from the filtrate
- Polysaccharides were enzymatically digested and removed. (The sugar coat was not the “transforming factor)
- Proteins, RNA and DNA remains in the filtrate
Step 7: Removal of proteins from the filtrate
- Protein was removed from the active filtrate by several chloroform extractions.
- When they removed the protein from the extract with organic solvents like chloroform they found that the extract still transformed.
(Some amount of proteins remained in the filtrate after extraction)
Step 8: Treat samples with enzymes that destroy protein, RNA, or DNA
Protease (trypsin, chymotrypsin): destroys proteins
After protease treatment, the two components remaining in the filtrate were RNA and DNA which were precipitated with alcohol. The precipitate was then removed from the lysate and dissolved in water.
The filtrate was then subjected to enzymatic digestion. (First with RNase and then with DNase)
DNases: Destroy DNA
RNase: Destroys RNA
Step 9: Assay for transformation
- Add the treated sample to cultures of type II R bacteria in separate flasks.
- Allow sufficient time for DNA/RNA/Protein to be taken up by the typeII R bacteria
Flask 1: Type IIR cells + Type IIIS filtrate
Flask 2: Type IIR cells +Protease treated type IIIS filtrate
Flask 3: Type IIR cells + RNase treated type IIIS filtrate
Flask 4: Type IIR cells + DNase treated type IIIS filtrate
Step 10: Addition of antibody that aggregates type IIR bacteria
- After incubation cells were exposed to antibodies, which are molecules that can specifically recognize the molecular structure of the macromolecules.
- In this experiment, the antibodies recognized the cell surface receptors of type R bacteria and caused them to clump together.
- The clumped bacteria were removed by gentle centrifugation step.
- Only the bacteria that were not recognized by the antibody (type S bacteria) remained in the supernatant
Step 11: Observe whether transformation has occurred by testing for the presence of virulent S strains of Streptococcus pneumoniae
- The cells in the supernatant were plated on solid growth media.
- After overnight incubation visible colonies of type S bacteria may be observed
Results of the transformation assay
Flask 1: Transformation occurs (Control for transformation assay)
(They already knew that Type IIIS filtrate contains the active factor. So in control flask transformation should occur because it doesn’t treated with any enzyme)
Flask 2: Transformation occurs
Result: Cultures treated with protease contained type IIIS bacteria
Conclusion: Active factor is not protein
Flask 3: Transformation occurs
Result: Cultures treated with RNase contained type IIIS bacteria
Conclusion: Active factor is not RNA
Flask 4: No transformation occurs
Result: Cultures treated with DNase not contained type IIIS bacteria
Conclusion: Active factor is DNA
Conclusions from the experiment
These experiments ruled out protein or RNA as the transforming material. Avery and his coworkers found that the enzyme DNase, which breaks down DNA, destroyed the transforming ability of the virulent cell extract. These results suggested that the transforming substance was DNA.
Further testing clearly established that the transforming principle was DNA. The purified substance gave a negative result in chemical tests known to detect proteins, but a strongly positive result in a chemical test known to detect DNA.
The analytical tools Avery and his colleagues used were the followiing
They spun the transforming substance in an ultracentrifuge (a very high-speed centrifuge) to estimate its size. The material with transforming activity sedimented rapidly (moved rapidly toward the bottom of the centrifuge tube), suggesting a very high molecular weight, characteristic of DNA.
They placed the transforming substance in an electric field to see how rapidly it moved. The transforming activity had a relatively high mobility, also characteristic of DNA because of its high charge-to-mass ratio.
Ultraviolet Absorption Spectrophotometry
They placed a solution of the transforming substance in a spectrophotometer to see what kind of ultraviolet light it absorbed most strongly. Its absorption spectrum matched that of DNA. That is, the light it absorbed most strongly had a wavelength of about 260 nanometers (nm), in contrast to protein, which absorbs maximally at 280 nm.
Elementary Chemical Analysis
This yielded an average nitrogen-to-phosphorus ratio of 1.67, about what one would expect for DNA, which is rich in both elements, but vastly lower than the value expected for protein, which is rich in nitrogen but poor in phosphorus.
Even a slight protein contamination would have raised the nitrogen-to-phosphorus ratio.