The Avery-MacLeod-McCarty Experiment: DNA Is the Transforming Principle

In 1944, a quiet bacteriologist named Oswald Avery published a paper that should have been one of the most celebrated in the history of science. Working with Colin MacLeod and Maclyn McCarty at Rockefeller University in New York, he demonstrated — through meticulous elimination of every possible alternative — that the molecule responsible for transferring hereditary information between bacteria was deoxyribonucleic acid: DNA. The experiment did not directly answer how DNA works or what it looks like. It answered the prior question, the one nobody had confidently resolved: what is the material of heredity? The answer reorganized biology.

The Problem Avery Was Solving: Griffith's Unfinished Business

To understand the Avery-MacLeod-McCarty experiment, you need to start with an experiment it was designed to explain. In 1928, British bacteriologist Frederick Griffith published results showing that a non-lethal strain of Streptococcus pneumoniae could be converted into a lethal strain by mixing it with heat-killed material from the lethal strain.

Source: Griffith, F. (1928), "The significance of pneumococcal types," Journal of Hygiene, 27(2), 113–159 — the original transformation paper.

The two strains of Streptococcus pneumoniae that Griffith worked with had been characterized since the early 1920s:

The S (smooth) strain — virulent, enclosed in a polysaccharide capsule that gives colonies a smooth, glistening appearance on agar plates. The capsule protects the bacteria from the host immune system. In mice, infection with S-strain bacteria is lethal.

The R (rough) strain — non-virulent, lacks the polysaccharide capsule, produces rough-edged colonies. Infection with R-strain bacteria causes no disease in mice.

Griffith's finding was this: if he killed S-strain bacteria with heat (destroying their ability to reproduce), mixed the heat-killed S-strain material with living R-strain bacteria, and injected the mixture into mice, the mice died. When he recovered bacteria from the dead mice, they were live S-strain bacteria — encapsulated, virulent, reproducing.

Something in the heat-killed S-strain material had permanently converted the R-strain bacteria into S-strain bacteria. The converted bacteria produced progeny with the same S-strain characteristics: the transformation was heritable. Griffith called the agent responsible the "transforming principle."

Source: Griffith (1928) — "The possibility that the 'S' form does not arise anew but that the R forms may be modified in the manner described, seems to require consideration." Griffith declined to speculate further on the chemical nature of the transforming principle.

Griffith died in the London Blitz in 1941, before seeing his question answered. The question he left behind — what is the transforming principle? — was exactly the question Avery spent the next decade answering.

For a detailed account of Griffith's experiment itself, see The Griffith Transformation Experiment.

What the Transforming Principle Could Have Been

By 1940, when Avery's team was deep into the identification work, the scientific community was divided about the nature of hereditary material — and the dominant hypothesis was wrong.

The prevailing view was that proteins were the most likely candidates for hereditary information. Proteins are enormously diverse: they are constructed from 20 different amino acids in chains that can be thousands of units long, giving a near-unlimited number of possible structures. By contrast, DNA was thought to be a simple, repetitive molecule — built from only four types of nucleotide bases (adenine, thymine, guanine, cytosine) and widely believed to alternate in a monotonous regular pattern that could not encode complex information.

Protein hypothesis — the pre-1944 consensus that proteins, due to their structural diversity, were the molecules responsible for encoding and transmitting genetic information.

Tetranucleotide hypothesis — the widely held (and incorrect) belief, associated with the biochemist Phoebus Levene, that DNA consisted of a repeating unit of all four nucleotide bases in equal proportion, making it structurally too simple and uniform to encode information.

Source: Levene, P. A. (1919), "The structure of yeast nucleic acid," Journal of Biological Chemistry, 40(2), 415–424 — the original tetranucleotide hypothesis.

The tetranucleotide hypothesis was not unreasonable given the analytical techniques available before the 1940s. It was wrong — Erwin Chargaff would later demonstrate in 1950 that nucleotide base ratios vary between species in a way inconsistent with regular tetranucleotide repetition — but it shaped the scientific environment in which Avery worked.

To prove DNA was the transforming principle, Avery's team had to do two things: first isolate a pure transforming extract, and then systematically demonstrate that destroying every molecular component except DNA left the transforming activity intact, while destroying DNA eliminated it.

Oswald Avery and the Rockefeller Team

Oswald Avery was born in Halifax, Nova Scotia, in 1877 and moved to New York with his family as a child. He received his medical degree from Columbia University in 1904 and joined the Rockefeller Institute for Medical Research (now Rockefeller University) in 1913, where he spent his entire research career.

He was a microbiologist, not a geneticist. His original interest in pneumococcus was clinical: understanding the different serological types of the bacterium was relevant to treating pneumonia, which was a leading cause of death in the early 20th century. The transformation phenomenon drew his attention because it was a tool for understanding the biology of the capsule — not initially because of its genetic implications.

Source: Dubos, R. J. (1976), The Professor, the Institute, and DNA, Rockefeller University Press — biography of Avery and account of the Rockefeller Institute pneumococcus research program.

Colin MacLeod joined Avery's lab in 1934 as a young Canadian physician-scientist. Maclyn McCarty joined in 1941. The critical experimental work identifying DNA as the transforming principle was done primarily by MacLeod and McCarty, guided by Avery's scientific direction. By the time the paper was published in 1944, MacLeod had left for war service, but remained an author on the work.

Avery was cautious to a fault. He had been pursuing the transforming principle since the early 1930s. He held the DNA paper back until he was confident of the result — confident enough to have examined the question from every angle the techniques of the time allowed.

The Experiment: Systematic Elimination

The methodological strategy of the Avery-MacLeod-McCarty experiment is sometimes called "differential destruction" or enzyme elimination. The logic runs as follows: if a specific molecule X is responsible for transformation, then destroying X should abolish transformation while leaving all other properties of the extract intact. If destroying everything except X leaves transformation intact, you have strong evidence that X is sufficient for transformation.

The team worked with large-scale cultures of encapsulated (virulent) S-strain pneumococcus and produced cell extracts — material released from broken-down bacterial cells. They then purified and characterized the extract using the biochemical tools available in the 1940s.

Step 1: Initial characterization. The raw extract was treated with chloroform (which killed any residual live bacteria), heat, and alcohol precipitation. The purified material was tested for its ability to transform R-strain bacteria in culture.

Step 2: Enzymatic treatment. The team subjected the transforming extract to a series of specific enzymes, each designed to destroy a particular class of biological molecule:

Enzyme Used	Target Molecule	Effect on Transformation
Protease (trypsin, chymotrypsin)	Destroys proteins	No effect — transformation intact
Ribonuclease (RNase)	Destroys RNA	No effect — transformation intact
Lipases	Destroys lipids	No effect — transformation intact
Hyaluronidase	Destroys polysaccharides	No effect — transformation intact
Deoxyribonuclease (DNase)	Destroys DNA	Transformation abolished

Source: Avery, O. T., MacLeod, C. M., & McCarty, M. (1944), "Studies on the chemical nature of the substance inducing transformation of pneumococcal types," Journal of Experimental Medicine, 79(2), 137–158 — the primary paper.

Step 3: Chemical analysis. Independent of the enzymatic work, the team used chemical analysis methods to characterize the purified transforming material directly. Elemental analysis — measuring the ratios of carbon, hydrogen, nitrogen, oxygen, and phosphorus — gave values consistent with a nucleic acid, specifically DNA, rather than a protein or polysaccharide. The material's ultraviolet light absorption spectrum showed a peak at 260 nm, characteristic of nucleic acids (proteins absorb at 280 nm).

Step 4: Serological tests. Anti-protein antibodies did not precipitate the transforming material, providing further evidence that it was not a protein.

The DNase result was the decisive finding. DNase (deoxyribonuclease) is an enzyme that breaks the phosphodiester bonds in the DNA backbone, cleaving it into fragments. Even trace amounts of DNase added to the transforming extract completely abolished the ability to transform R-strain bacteria. No other enzyme had any effect.

Source: Avery, MacLeod, & McCarty (1944), Journal of Experimental Medicine, 79(2), 137–158 — the original paper reports: "Transforming activity was completely abolished by treatment with desoxyribonuclease."

The convergence of multiple independent lines of evidence — enzymatic destruction, chemical composition, spectroscopic characterization, serological tests — all pointing to DNA constituted an unusually strong case for a biological identification.

What the Paper Said and What It Carefully Did Not Say

The Avery-MacLeod-McCarty paper is notable for its restraint. Avery was aware that the claim — that DNA carries hereditary information — contradicted the prevailing protein hypothesis and the tetranucleotide hypothesis. He structured the paper to present the evidence and then carefully hedge the interpretation.

The paper's conclusion reads: "The evidence presented supports the belief that a nucleic acid of the deoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type III."

Not: "DNA is the molecule of heredity." Not: "DNA carries genetic information in all organisms." The specific, careful claim is that a DNA fraction is the transforming principle in this specific bacterial system. The generalization — which was what made the finding revolutionary — was left to the reader to draw.

In a letter to his brother Roy written in 1943, before the paper was published, Avery was less guarded: "If we are right, and of course that's not yet proven, then it means that nucleic acids are not merely structurally important but functionally active substances in determining the biochemical activities and specific characteristics of cells — and that by means of a known chemical substance it is possible to induce predictable and hereditary changes in cells. This is something that has long been the dream of geneticists."

Source: Avery, O. T. (1943), letter to Roy Avery, February 26, 1943, reproduced in Dubos (1976) and in McCarty, M. (1985), The Transforming Principle: Discovering That Genes Are Made of DNA, W. W. Norton.

The Reception: Contested, Not Ignored

The popular narrative that Avery's work was completely ignored is not quite accurate. The paper received significant attention in microbiology and biochemistry circles immediately after publication. What it did not receive was universal acceptance, and the resistance to the DNA hypothesis was substantial and prolonged.

Alfred Mirsky, a protein chemist at Rockefeller — in the same institution as Avery — became the most persistent critic. Mirsky argued that the transforming principle was not pure DNA but contaminated with small amounts of protein, and that the proteins were the active ingredient. The trace-protein argument was difficult to definitively refute with 1940s purification technology.

Source: Mirsky, A. E. & Pollister, A. W. (1946), "Chromosin, a desoxyribose nucleoprotein complex of the cell nucleus," Journal of General Physiology, 30(2), 117–148 — Mirsky's counter-argument.

The objection was methodologically reasonable given the available tools. DNase preparations in the 1940s were not certified pure; they might have contained proteases. The purification methods available could not completely exclude trace protein contamination. The argument was never fully resolved experimentally during this period.

What resolved the question was not any single experiment but the accumulation of evidence from multiple directions: Chargaff's base-ratio rules in 1950, the Hershey-Chase experiment in 1952 (see The Hershey-Chase Experiment), and finally the Watson-Crick double helix structure in 1953 — all pointed to DNA as the information-carrying molecule.

Source: Chargaff, E. (1950), "Chemical specificity of nucleic acids and mechanism of their enzymatic degradation," Experientia, 6(6), 201–209 — demonstration that A=T and G=C in DNA, inconsistent with tetranucleotide hypothesis.

The Nobel Prize That Never Came

Oswald Avery is one of the most frequently cited examples of a scientist who deserved a Nobel Prize and did not receive one.

The Nobel Committee's records, made public 50 years after the relevant deliberations under Swedish law, show that Avery was nominated for the Nobel Prize in Physiology or Medicine multiple times in the late 1940s and early 1950s. The committee's concern, reflected in the assessors' reports, was the ongoing controversy about whether the transforming principle was truly pure DNA or a DNA-protein complex — the Mirsky objection. They waited for the controversy to resolve before acting.

Source: Liljestrand, G. (Nobel Committee assessor reports, made public 1995) — discussed in detail by Van Helvoort, T. (1995), "Historical neglect of Avery's work," in History and Philosophy of the Life Sciences, 17, 91–118.

Avery died in February 1955, before the Nobel Committee could act. The Prize is not awarded posthumously. Watson and Crick received the Nobel in 1962 for the double helix structure, along with Maurice Wilkins. Franklin had died in 1958. Avery, MacLeod, and McCarty received no Nobel recognition.

Watson and Crick's famous 1953 paper in Nature does not cite Avery, MacLeod, and McCarty. In 2003, on the 50th anniversary of the double helix paper, Maclyn McCarty stated in a Nature essay that he was "not so pleased" about the omission.

Source: McCarty, M. (2003), "Discovering genes are made of DNA," Nature, 421, 406 — McCarty's 50th-anniversary reflection.

Watson later acknowledged the omission as an error. The failure to cite Avery's group was not merely a discourtesy to the researchers; it also contributed to the historical pattern of the 1944 paper being underweighted in the standard history of DNA's discovery.

From Transforming Principle to Double Helix: The Direct Connection

The connection between the Avery-MacLeod-McCarty experiment and the Watson-Crick structure is direct, even if the citations were missing.

The 1944 paper established that DNA, not protein, carries hereditary information. This reoriented research on the chemistry of the gene: if DNA is the information molecule, then its structure must encode information, and understanding that structure becomes a central scientific goal.

Erwin Chargaff, a biochemist at Columbia University, read the Avery paper carefully and changed the direction of his research as a result. He applied paper chromatography — a new technique — to analyze the base composition of DNA from many different species. In 1950, he reported that in all species examined, the amount of adenine equaled the amount of thymine, and the amount of guanine equaled the amount of cytosine (the base-pairing rules now known as Chargaff's rules). This was the first strong empirical hint at the complementary base-pairing structure of DNA.

Source: Chargaff, E. (1950), Experientia, 6(6), 201–209 — Chargaff explicitly states that Avery's paper motivated his analytical work.

Watson and Crick used Chargaff's rules, along with Rosalind Franklin's X-ray diffraction data, to construct the double-helix model. The chain from Griffith (1928) to Avery (1944) to Chargaff (1950) to Hershey-Chase (1952) to Watson-Crick (1953) is a nearly continuous line of experimental questions and answers.

What People Get Wrong About the Avery-MacLeod-McCarty Experiment

"Avery discovered DNA." DNA was discovered and named in the 1860s and 1870s by Friedrich Miescher, who called it "nuclein." Avery's contribution was demonstrating DNA's biological function — that it carries hereditary information — not its existence.

"The experiment was completely ignored." It was published in a major journal (Journal of Experimental Medicine), was widely read, generated significant debate, and was cited by Chargaff as motivation for his work. The controversy was about interpretation, not visibility. The debate over whether the transforming principle was truly pure DNA was scientifically reasonable given the available evidence.

"Protein was always a crazy hypothesis." Before 1944, the protein hypothesis for the genetic material was the scientifically defensible position. Proteins are enormously structurally diverse; DNA was believed to be a simple repeating structure. The evidence that changed this was experimental, not obvious from first principles.

"Watson and Crick's omission of Avery was deliberate slight." Watson has described it as an oversight rather than a deliberate act. The paper was produced under intense competitive pressure and submitted quickly. Whether this excuses the omission is a separate question from its motivation.

The Timeline

Year	Event
1877	Oswald Avery born in Halifax, Nova Scotia
1923	Griffith's colleagues differentiate S (smooth/virulent) and R (rough/non-virulent) pneumococcal strains
1928	Frederick Griffith publishes transformation experiment — "transforming principle" identified
1931	Avery begins systematic pursuit of the transforming principle at Rockefeller
1934	Colin MacLeod joins Avery's lab
1941	Maclyn McCarty joins the team; concentrated experimental effort begins
February 1944	Paper published: Journal of Experimental Medicine, 79(2), 137–158
1944–1952	Mirsky controversy; DNA hypothesis contested; evidence accumulates
1950	Chargaff publishes base-pairing rules (A=T, G=C), directly motivated by Avery's work
1952	Hershey-Chase experiment confirms DNA (not protein) enters host cells during phage infection
1953	Watson and Crick propose double-helix structure; do not cite Avery
February 20, 1955	Avery dies in Nashville, Tennessee, aged 77
1962	Watson, Crick, and Wilkins receive Nobel Prize; Avery, MacLeod, McCarty receive none

FAQs

Q: What exactly is bacterial transformation, and does it happen in the body?
A: Bacterial transformation is the uptake of external DNA by a bacterial cell, resulting in heritable changes to the cell's characteristics. Some bacteria are naturally "competent" — able to take up DNA from their environment — while others require specific conditions or laboratory manipulation. Transformation does occur in nature: it is one mechanism of horizontal gene transfer, through which antibiotic resistance genes spread between bacterial strains. The resistance gene was not inherited from the bacterium's ancestors; it was acquired from DNA in the environment.

Q: Why was DNA thought to be too simple to carry genetic information?
A: The tetranucleotide hypothesis, developed by Phoebus Levene at Rockefeller in the 1910s–1920s, proposed that DNA consisted of a regular repeating unit of all four nucleotide bases. If this were true, all DNA would have the same structure, making it impossible to encode the variable information required to specify different traits. We now know the hypothesis was wrong — the four bases vary in proportion between species and between regions of the genome — but the analytical methods of the era could not distinguish a regular from an irregular sequence.

Q: How certain were Avery, MacLeod, and McCarty that their DNA preparation was free of protein contamination?
A: They were as certain as 1940s technology allowed. The elemental analysis, spectroscopic data, and negative results with anti-protein antibodies all pointed to a DNA preparation with very low protein content. But they could not achieve the purity levels modern molecular biology takes for granted, and they acknowledged this limitation explicitly in the paper. The DNase result was their strongest evidence precisely because it did not depend on purity claims — even if trace proteins were present, the complete abolition of transformation by DNase implicated DNA as the essential component.

Q: Who were MacLeod and McCarty, and what happened to them after 1944?
A: Colin MacLeod (1909–1972) was a Canadian microbiologist who left Rockefeller for war service and went on to a distinguished career, eventually serving as science advisor to President John F. Kennedy. Maclyn McCarty (1911–2005) remained at Rockefeller his entire career and became its vice president for academic affairs. Both outlived Avery by decades and continued to write and speak about the 1944 experiment. McCarty's memoir, The Transforming Principle (1985), is the most accessible first-person account.

Q: What is the Hershey-Chase experiment, and how does it relate to Avery's work?
A: The Hershey-Chase experiment (1952) used bacteriophage — viruses that infect bacteria — to provide an independent confirmation that DNA, not protein, carries genetic information. Alfred Hershey and Martha Chase labeled phage DNA with radioactive phosphorus and phage protein with radioactive sulfur, then showed that after infection only the DNA entered the bacterial cell while the protein coat remained outside. The result independently confirmed Avery's conclusion using a completely different experimental system. See The Hershey-Chase Experiment for the full account.

Q: Did Avery know his work would lead to the double helix?
A: Avery did not live to see the double helix (he died two months after its publication). His 1943 letter to his brother suggests he understood the magnitude of what his team was finding — that it pointed toward a chemical understanding of heredity. He was professionally cautious about making large claims; his published work reflects this caution. The private letter suggests he understood the implications more fully than the published text admits.

The Takeaway

Avery, MacLeod, and McCarty answered the most fundamental question in mid-20th-century biology — what is the material of heredity — using the simplest possible experimental logic: destroy everything that is not DNA, and watch transformation persist; destroy DNA, and watch transformation disappear. The result contradicted the dominant hypothesis, fit no existing theory, and took nearly a decade to win general acceptance. Its relationship to the Watson-Crick double helix of 1953 is direct: without knowing that DNA is the hereditary molecule, there is no reason to ask what DNA looks like. The 1944 paper created the question the double helix answered.

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Read next:

• The Griffith Transformation Experiment
• The Hershey-Chase Experiment
• The Meselson-Stahl Experiment