We have seen in the previous post that in 1928, Frederick Griffith discovered the process of transformation in Streptococcus pneumoniae. He found that the dead S bacteria, which was encapsulated and virulent, could transform the non-virulent, non-encapsulated live R bacteria into encapsulated and virulent S bacteria. However, Griffith could not exactly find the nature of the transforming particle.
It was found that the transforming principle could be precipitated with alcohol, which showed that it was not a carbohydrate like the polysaccharide coat itself. At this point, the scientists were unsure if the transforming factor was a protein or DNA. Eventually, Avery and team‘s experiment revealed the nature of the principle of the transforming principle.
Avery had a medical degree but later was drawn to the bacteriological research. In 1913, he began research on the Streptococcus pneumoniae, the causative agent of lobar pneumonia. Avery and his colleagues isolated polysaccharide, which makes up the capsular envelope of the pneumococcus. Avery’s research contributed in the classification of pneumococci into different types. He also found that the polysaccharide could stimulate an immune response and lead production of antibodies. Hence revealing that a substance other than a protein could stimulate immune system and contributed to the development of immunochemistry as well.
After Griffith demonstrated transformation in vivo in the mice, Sia and Dawson (1931) induced transformation in vitro. Avery and his colleagues studied transformation for around 15 years. They began working on the purification and determining the chemical nature of the transforming principle in 1934.
(Just for info: Have a look at the original paper of Sia and Dawson (1931) titled In vitro transformation of pneumococcal cells.
For purification, Avery first heat-killed the bacteria and extracted the saline-soluble components. Then the cells were treated with Na-deoxycholate (a detergent to lyse cells and solubilise cellular and membrane components) followed by protein removal using chloroform and hydrolysing capsular polysaccharide using enzymes. The alcohol fractionation was done to extract the transforming principle in form of fibrous precipitation.
(Just for info: Have a look at the original paper published by Avery et al. In 1944)
They carried out series of experiments and studied various parameters of the purified material. They studied the general properties like solubility, storage and effect of heating. They found that the purified material was soluble in physiological salt solutions and could be stored for a long time under low temperatures without losing its ability to transform. It could withstand high temperature but below pH 5 became inactive.
Chemical tests like Biuret test and Millon tests for proteins, Dische diphenylamine reaction for DNA, Orcinol test for RNA were conducted. Tests for nuclei acids were seen to be positive.
Analysis of content of nitrogen, phosphorus, carbon and hydrogen were similar to that of DNA.
Enzymatic analysis: They tested the compound using crystalline enzymes, sera and preparations of enzymes to determine their effect on the transforming activity of the purified compound. The enzymes like ribonuclease, trypsin and chymotrypsin were also used for testing their effect on the transforming ability of the purified material. Sera and enzymatic preparation tested included those including phosphatases, depolymerases for DNA and esterases. It was observed that the preparations containing DNA depolymerases brought about inactivation of the purified material to transform.
Serological Analysis: Avery and team conducted tests using the polysaccharide capsule antisera, and found that the purification steps decrease the serological activity. Hence, they concluded that the purified material was not polysaccharide and that it induced the production of the capsule.
Physicochemical studies: The studies done using analytical ultracentrifuge and electrophoresis also showed that the results were similar to the DNA. Ultraviolet absorption curves showed maxima and minima in the region of 2600 and 2350 Angstrom respectively.
Avery and colleagues, hence, concluded that the purified material was DNA, in fact, they were the first to purify DNA from the pneumococci and suggested that it was the molecule responsible for the transformation. They also said that the change was permanent (similar to mutation). However, they were doubtful if any undetectable protein tightly bound with the DNA could be responsible for the change.
The main conclusion of the experiment, that DNA could be the transforming material was criticised by a number of scientists until the study conducted by Alfred Hershey and Martha Chase, in 1952 clearly showed that DNA is genetic material.
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Read other posts by The Biotech Notes:
Cobb (2014) Oswald Avery, DNA, and the transformation of biology. Current Biology 24 (2): R55-R60.
Avery et al. (1944) Studies on the chemical nature of the substance inducing transformation of pneumococcal types. JEM. 79 (2): 137.
Sia and Dawson (1931) In vitro transformation of Pneumococcal types. J Exp Med. 54(5): 701–710.
Hershey and Chase (1952) Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage. The Journal of General Physiology 36(1): 39–56.