Oswald Avery’s Pneumococcus Experiments: Forerunner of the DNA Story

Portrait of Oswald T. Avery, ca. 1940s.

DNA, although now known to be extremely important, was overlooked for quite some time. Until early 1953, around when the Watson and Crick structure of DNA was published, most major scientists thought that proteins, rather than DNA, were probably the site of the gene.

In the early 1940s however, experiments performed by Oswald T. Avery and his colleagues at the Rockefeller Institute for Medical Research made a strong argument for DNA as the source of the genetic material. Unfortunately, for many years not much attention was paid to Avery’s work.

Streptococcus pneumoniae, also called pneumococcus, was the subject of Avery’s experiment. This bacterium causes a variety of diseases, including pneumonia and peritonitis. The organism can be found in two forms, smooth (S) and rough (R) which are designated as such simply because of their appearance when viewed microscopically. The smooth appearance is a result of the formation of a polysaccharide capsule that encases the bacterial cell. This capsule protects the cell from immunological defenses, which makes the S form virulent. The R form, on the other hand, is mutated so that it does not synthesize the enzyme that creates the polysaccharide capsule, and is therefore not virulent.

The pneucmococcus bacteria can be further characterized into types, which are designated by roman numerals. Although an S form bacterial cell can be experimentally changed to an R form (and vice versa) provided the cell is not too far degraded, change of type never suddenly occurs. For example, a type III S cell can be converted to a type III R cell, but a type II cell will never spontaneously convert to a type III cell.

Although a spontaneous change of type is not possible, a specific experiment had been done showing that a transformation of type can be induced. This experiment was first performed by injecting a live culture of the Type II R form into mice along with a dead culture of the Type III S form. Theoretically, none of the mice should have died because they hadn’t been exposed to a virulent form of the bacteria. However, many of the mice did actually die, and living Type III S form bacteria was extracted from their blood. Later, the same experiment was accomplished by growing the bacteria in a glass dish rather than in mice.

Understandably, this transformation from a non-virulent bacteria to a virulent bacteria was troubling to the medical community. Although some scientists may have been concerned with the mechanism of transformation, Oswald Avery was more concerned with the identity of the agent performing the transformation. He went to work on devising an experiment that would allow him to isolate the transforming agent from the rest of the bacterial cell. Although DNA extraction is now considered a simple process, it was just beginning to emerge during the time when Avery began his work. The fact that Avery did not know that the transforming agent was in fact DNA complicated matters even further.

Nevertheless, after years of hard work, Avery and his colleagues were able to develop an experiment that effectively isolated the transforming agent from the bacterial cells. Type III S form bacteria were grown in large vats of broth made from beef hearts. The bacteria was then killed, and washed with brine in order to remove the polysaccharide capsule and whatever protein would come off in the process. The remainder of the bacteria was then precipitated in pure grain alcohol. After this, the precipitate was washed with chloroform and subjected to a digestive enzyme, both of which functioned to remove the remaining protein. Finally, after no trace of protein was evident, pure grain alcohol was once again added, which allowed the transforming agent to be separated. The process was long and difficult, and in the end only yielded approximately ten to twenty-five milligrams of the agent per seventy-five liters of culture.

Transformation of pneumococcal types, from Avery's 1944 paper.

After obtaining enough of the active transforming agent to conduct his tests, Avery and his colleagues set out to show exactly what the substance was. First, standard qualitative tests for proteins were performed, which came back negative. Qualitative tests for DNA, however, were strongly positive. Chemical analysis of the substance also showed that the ratio of nitrogen atoms to phosphorous atoms was approximately 1.67 to 1. This number is very close to the DNA ratio, and would have been different had there been a significant amount of protein present.

Next, tests with digestive enzymes were performed. The addition of enzymes that digest proteins and RNA left the agent intact, while enzymes that digest DNA completely destroyed the substance.

Finally, immunological tests involving centrifugation and electrophoresis were performed, which also showed that proteins and polysaccharides weren’t present, but that DNA was.

Once Avery was satisfied with the results of his tests, he began writing a manuscript that explained the experiment. In January of 1944 “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types” by Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty was published. Although it may seem that Avery and his colleagues had proven that DNA was the site of the gene, this was not entirely the case. There was still a possibility that, as Avery puts it in the manuscript, “the biological activity of the substance described is not an inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so intimately associated with it as to escape detection. . .”

Avery was clearly being very cautious in his conclusions, never stating that he was certain that DNA was the transforming agent. It is possible that his cautiousness with the matter contributed to his lack of attention received. However, there were other reasons why Avery wasn’t given serious attention. As James Watson later stated in 1983:

Both Francis and I had no doubts that DNA was the gene. But most people did. And again, you might say, ‘Why didn’t Avery get the Nobel Prize?’ Because most people didn’t take him seriously. Because you could always argue that his observations were limited to bacteria, or that [the transformation of pneumococcus that he described was caused by] a protein resistant to proteases and that the DNA was just scaffolding.

Although Avery’s manuscript may not have been received with high praise at the time of its publication, it is now considered to be a very thorough account of an expertly accomplished experiment. For more information on DNA, please visit the Race for DNA website. For information on Linus Pauling, a major player in the DNA story, visit the Linus Pauling Online portal.