History of DNA Discovery (7): Oswald Avery proved DNA was Genetic Material

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Although Frederick Griffith discovered that R bacteria could transform into S bacteria within mice, he did not delve into what substance facilitated this change. Most scientists believed that the substance promoting bacterial transformation was protein. However, some scientists were skeptical and embarked on the path of exploring genetic material (DNA), among whom Oswald Avery was a notable figure. His most remarkable achievement was proving that the transforming substance in bacteria was DNA. It was a decade of research by him and his team, and it is also referred to as the Avery–MacLeod–McCarty experiment.

Background: conclusion of Griffith experiment was confirmed by other scientists.

Oswald Avery was a researcher at the Rockefeller institution engaged in the isolation, classification and serum treatment of pneumococcus. He identified the R bacteria without polysaccharide capsule and the S bacteria covered by polysaccharide capsule. He also delved into the chemical composition of these polysaccharide capsules, as well as the relationship between the capsule and virulence, immunity. However, Oswald Avery did not observe the transformation of bacteria in his studies. The first time he heard about the bacterial transformation experiment, he was skeptical.

Shortly after Frederick Griffith published his paper, Fred Neufeld from Germany and colleagues at the Rockefeller institution replicated the bacterial transformation experiment. Oswald Avery's colleagues even improved the experiment to explore the transformation phenomenon more deeply. In 1929, Michael Dawson discovered that under proper conditions, R bacteria could also transform into S bacteria in a petri dish.James Alloway tried to expand on his colleagues' findings in 1930. He crushed S bacteria to release their internal substances. Subsequently, he filtered out the cell debris to obtain a solution—the cell free extract, which could still transform R bacteria into S bacteria in vitro. When he added alcohol to the cell free extract, he observed a flocculent precipitate. James Alloway believed that the substance changing the bacterial hereditary trait neither from mice nor from the polysaccharide shell, but from an active substance inside the bacteria—the cell free extraction.

How Oswald Avery cultured bacteria and extracted nucleic acids: a decade-long experiment

These experiments made Oswald Avery realize the importance of transformation phenomenon in bacteriology and even genetics. Therefore, he refocused on the active substance that carried hereditary material, and its purifying became his primary goal. However, the active substances (cell-free extract) produced by Alloway method were unstable and easily deactivated, and his method was unreliable for quantitative detection of the active substances. Consequently, the experiments suffered from poor reproducibility and unreliable results for a long time. These problems were solved with the help of Colin MacLeod and Maclyn McCarty. MacLeod joined Avery's team in 1934 until he took a position at New York University in 1940.

Sometimes, R bacteria transform into S bacteria spontaneously even without the aid of external substances. Obtaining a stable strain of R bacteria was crucial for the experiment. Colin MacLeod identified a stable R strain from a type II S strain, which was widely used in the study of the transformation phenomenon. He also made progress in acquiring extracts and in the measurement of their activity. McCarty joined the laboratory in 1941 to assist Oswald Avery in exploring the transforming factor. At this time, MacLeod often came to the laboratory to help them.

Steps of transforming principle extraction by Oswald Avery's team

1. S bacteria were cultured on a large scale in 50 to 75 liters of beef heart infusion broth at 37℃ for 16 to 18 hours and then separated by a steam-driven Sharples centrifuge.

2. These microbes were maintained at 65℃ for 30 minutes to inactivate enzymes within the cells that could destroy the transforming principle.

3. The heated cells were washed three times with a saline solution to remove debris of polysaccharides, RNA, and proteins.

4. The washed cells were extracted with 0.5% sodium desoxycholate by shaking for 30-60 minutes to extract the active substances.

5. Alcohol was added to the crude extract of the active substances to dissolve the sodium desoxycholate. The fibrous active substance floated on the surface of the alcohol and was collected with a spatula directly.

6. The active substance was dissolved in a saline solution and then shaken with chloroform to remove most of the remaining proteins.

7. The active substance was treated with enzymes capable of hydrolyzing proteins, polysaccharides, and RNA. Immunoprecipitation induced by antibodies was used to verify the complete destruction of the polysaccharide capsule. Since these enzymes are proteins, the previous step was repeated to remove the enzymes and traces of remaining proteins.

8. The final step involved repeated precipitation by alcohol to get the pure active substance. Alcohol was added dropwise to the solution, and the fibrous active substance appeared and wound around the stirring rod. These collected fibrous materials were dissolved in saline and repeated step 8 several times to obtain a high-purity transforming principle.

The composition and chemical properties of the transforming principle indicated that it is DNA.

After obtaining a pure active substance that was free of proteins and lipids, Oswald Avery and his team analyzed its chemical composition and properties. They found that the transforming principle had good thermal stability. It retains its activity even exposed to 65°C for 30 to 60 minutes. It was soluble in alkaline salt solutions but would precipitate in organic solvents. It lost its activity at pH 5 and below (this is why Avery did not use pepsin in his tests, as it requires activation around pH 1.5, where DNA would be inactivated). Chemical analysis showed that the amount of carbon, hydrogen, nitrogen and phosphorus was close to the theoretical values of DNA.

The active substance did not produce a blue precipitate when mixed with Biuret reagent for detecting peptide chains. It did not appear a red precipitate when mixed with Millon's reagent for phenolic tyrosine detection. It turned blue when treated with diphenylamine in a boiling water bath (a test for DNA). It almost did not react with orcinol reagent for RNA.

R bacteria were cultured for 36 generations to achieve stable genetic inheritance, and they almost never transformed back to S bacteria spontaneously. These bacteria still transformed into S bacteria when exposed to the transforming principle treated with trypsin, chymotrypsin (which hydrolyzes proteins), and RNase (which hydrolyzes RNA). However, DNase (a crude preparation from animal organs that degrades DNA) destroyed its genetic transformation capability. These analyses and tests showed that the transforming principle was neither protein nor RNA. It also was not lipids or polysaccharides, as during the extraction process, lipids would be dissolved by alcohol and chloroform, and the polysaccharide capsule was hydrolyzed by enzymes. Oswald Avery and his team were convinced that the transforming principle was DNA that played a fundamental role in heredity.

Frequently Asked Questions

Why Oswald Avery did not receive the Nobel Prize? His conclusions were doubted.

Although the conclusions of the Avery-MacLeod-McCarty experiment were astonishing, they were not universally accepted by others. Avery faced far more criticism, skepticism than praise and support.

1. Phoebus Levene's Tetranucleotide hypothesis was widely accepted at that time. Nucleic acids were considered to be simple linear arrangements of four nucleotides, incapable of carrying the diverse genetic information. Biologists favored proteins as the carriers of genetic material, since the 20 amino acids seemed to provide endless combinations to encode life's information. Nucleic acids were thought to play only a secondary role in the hereditary traits. The crystallization of the Tobacco Mosaic Virus by Wendell Stanley serves as a prime example. He believed these crystals to be self-assembling proteins and was awarded the Nobel Prize in Chemistry. At that time, it seemed only those conclusions that aligned with mainstream were awarded.

2. They declared that a very pure nucleic acid was made and protein was removed as much as possible, there was still about 0.02% protein residue. Opponents argued that the protein contaminated experiment could not prove nucleic acids were the hereditary material definitively. It was likely that the protein facilitated bacteria to produce their polysaccharide coat.

3. The modest and unassuming Oswald Avery did not actively promote his results. He continued to seek more evidence at the Rockefeller institution. Ten years later, when the view that proteins were the genetic material was thoroughly abandoned, Oswald Avery's life was also coming to an end (the Nobel Prize is not awarded posthumously).

Anec  > Biology > Genetic material