By the 1930s, the chromosomal theory of heredity was an established scientific dogma. Thomas Hunt Morgan’s school had mapped hundreds of genes to linear coordinates along chromosomes. Yet this triumph provoked an even deeper, agonizing biophysical mystery: What was the gene made of? Chemical analysis revealed that eukaryotic chromosomes were composed of two primary molecular species: basic nuclear proteins called histones, and an acidic, phosphorus-rich macromolecule named deoxyribonucleic acid (DNA). For almost half a century, the overwhelming consensus of biochemistry favored proteins. Proteins were constructed from twenty distinct amino acids with complex, folded three-dimensional architectures capable of vast informational diversity. DNA, by contrast, was presumed under Phoebus Levene’s flawed tetranucleotide hypothesis to be an inert, repetitive polymer of four nucleotides (adenine, thymine, guanine, cytosine) in monotonous equimolar sequence—a structural scaffold, biochemists assumed, upon which the informational proteins were mounted.
The fracture in this consensus began in 1928 within the modest laboratory of a British Ministry of Health medical officer named Frederick Griffith (1879–1941), who was investigating the epidemiology of lobar pneumonia.
Griffith’s Inoculation Experiments
Griffith worked with two distinct strains of the bacterium Streptococcus pneumoniae:
- Smooth (S) Strain: Formed glistening, smooth colonies on agar plates. These bacteria synthesized a protective polysaccharide capsule that shielded them from host white blood cells, rendering them lethally virulent. Inoculating mice with live S-strain bacteria caused rapid, fatal septicemia.
- Rough (R) Strain: Formed dull, granular colonies. Due to a mutation, they lacked the enzymatic machinery to construct the polysaccharide capsule, allowing the mouse immune system to destroy them immediately. Inoculating mice with live R-strain produced zero illness.
When Griffith injected mice with heat-killed S-strain bacteria, the heat had destroyed their viability, and the mice survived normally. But then Griffith performed a radical control experiment: he co-injected mice with heat-killed virulent S bacteria mixed together with harmless live R bacteria.
To Griffith’s astonishment, the mice succumbed to fatal pneumonia. When he autopsied the deceased animals, their blood was teeming not with R bacteria, but with millions of living, encapsulated Smooth bacteria. The live non-virulent R strain had somehow acquired the permanent, heritable capacity to synthesize the protective capsule from the corpses of the heat-killed S strain. Griffith termed the mysterious agent responsible the Transforming Principle. When cultured through subsequent generations, these transformed bacteria bred true, demonstrating that the change was an authentic genetic alteration.
Avery, MacLeod, and McCarty (1944)
Griffith was killed in his London laboratory in 1941 during the Nazi Blitz, never knowing the nature of his transforming agent. The task of isolating and identifying the substance fell upon a meticulous, ascetic bacteriologist named Oswald T. Avery (1877–1955) and his younger associates, Colin MacLeod and Maclyn McCarty, at the Rockefeller Institute for Medical Research in New York.
For more than a decade, Avery’s team purified large batches of heat-killed S-strain pneumococci. They removed the capsular polysaccharides through enzymatic digestion, extracted lipids with chloroform, and precipitated the active biological fraction with alcohol. The resulting active transforming principle was biologically potent at concentrations of one part in tens of millions.
To decisively identify which class of macromolecule mediated the transformation, Avery, MacLeod, and McCarty subjected the purified extract to systematic enzymatic interrogation:
- Trypsin and Chymotrypsin (Proteases): Destroyed all proteins. The extract retained full transforming activity.
- Ribonuclease (RNase): Completely degraded RNA. Transforming activity was completely unaffected.
- Deoxyribonuclease (DNase): An enzyme newly isolated that selectively hydrolyzes DNA into mononucleotides. Treatment with DNase completely, instantaneously, and irreversibly abolished all transforming capacity.
Elemental chemical analysis confirmed that the purified substance had a nitrogen-to-phosphorus ratio of 1.67:1, perfectly matching the theoretical composition of deoxyribonucleic acid and incompatible with proteins.
“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.”
— Oswald T. Avery, letter to his brother Roy Avery (1943)
Published in the Journal of Experimental Medicine in February 1944, Avery, MacLeod, and McCarty’s paper provided the first rigorous chemical evidence that the physical substrate of heredity was DNA. Yet the scientific community, conditioned by decades of protein-centric thinking, resisted. Critics claimed that Avery’s preparations might still contain 0.1% undetected protein contaminants that performed the real work. The genetic world demanded an even more dramatic, unassailable demonstration.
