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 the substance was protein. However, some scientists were skeptical to Griffith report, so they embarked on the road to exploring genetic material, among whom Oswald Avery was a notable figure. It was a decade of research by him and his team to prove transforming substance was DNA.
Background: conclusion of Griffith experiment was confirmed by other scientists.
Oswald Avery was a researcher at the Rockefeller institution engaged in isolation, classification and serum therapy for pneumococcus. He also identified R bacteria and S bacteria. The chemistry of polysaccharide capsule was carefully explored by him, as well as its relationship to virulence and immunity. However, the transformation of bacteria wasn’t observed by him. Therefore, he was skeptical about Griffith experiment at the first time.
Soon after Frederick Griffith published his paper, Fred Neufeld from Germany and colleagues of Avery at the Rockefeller institution replicated his experiment, but it was not known whether they could mutate in culture dish. In 1929, Griffith's experiment was improved by Michael Dawson. The transforming phenomenon still occurs in a petri dish. James Alloway tried to expand on his colleagues' findings in 1930. He crushed S bacteria to release their internal substances. Then, the cell debris were filtered out to obtain a cell free extract, which could still transform R bacteria into S bacteria in vitro. Thus, James Alloway believed that substance about hereditary trait neither from mice nor polysaccharide shell, but from an active substance inside bacteria.
How Oswald Avery cultured bacteria and extracted nucleic acids
Sometimes, transformation phenomenon occurs spontaneously even in the absence of external factors, so obtaining a stable R strain was crucial. Colin MacLeod joined Avery's team in 1934. A strain without polysaccharides coat was identified by him from a type II S strain. Then, it was cultured for 36 generations to achieve stable genetic inheritance. He also made progress in acquiring reliable extracts and determining their activity. In 1941, McCarty joined Avery's team to replace MacLeod who took a position at New York University in 1940, but still came to the lab frequently.
Bacteria are inactivated at 65°C. They are washed with saline to remove cell wall and cytoplasm. Nucleic acids are dissolved by sodium desoxycholate and alcohol. The researchers used chloroform and enzymes to remove proteins, polysaccharides, lipids and RNA from nucleic acids to obtain a pure transforming principle.
Oswald Avery and his team analyzed chemical composition and properties of transforming principle. The excellent thermal stability 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. The proportions of carbon, hydrogen, nitrogen and phosphorus were close to the theoretical values of DNA. Transforming principle didn’t react with reagents for detecting proteins and RNA, but it turned blue when treated with DNA detectors in a boiling water bath.
Oswald Avery Experiment: Transforming principle was DNA.
These stable R bacteria still became S bacteria when exposed to transforming principle treated by trypsin, chymotrypsin (which hydrolyzes proteins), and RNase. When Avery mixed transforming principle with DNase, no S-type bacteria were found in culture dishes. Hence, it was neither protein nor RNA. Lipids and polysaccharides were also excluded from the candidates, as lipids would be dissolved by alcohol and chloroform, and polysaccharide capsule was hydrolyzed by enzymes during extraction. Oswald Avery and his team convinced DNA played a fundamental role in heredity.