nucleic acid. Who could have guessed it?
In the letter, ‘desoxyribose nucleic acid’, in the paper, ‘desoxyribonucleic acid’: these are older names for what we now call deoxyribonucleic acid – commonly reduced to its acronym, DNA.
Looking back at his own failure to appreciate Avery’s discovery at the time, Stent came to the conclusion ‘in some respects Avery et al’s paper is a more dramatic example of prematurity than Mendel’s’.
UTI DEICHMANN
Scientists, in the opinion of the Nobel Prize-winning Linus Pauling, were fortunate because their world was so much the richer for its mysteries than those not interested in science could possibly appreciate. Certainly in those days Avery’s lab at the Rockefeller Medical Institute for Research was filled with a mood of expectation and excitement. In 1943 Oswald Avery was 65 years old. He had planned to retire and join his brother Roy’s family in Nashville, Tennessee, but there was no question of his leaving the lab at this time. He needed to continue his work on the transforming substance. In particular he needed to convince his colleagues throughout the world of microbiology and, more widely, the sceptical world of biochemists and geneticists, of the validity of their discovery.
Avery was conservative by nature. A generation earlier he and a colleague had proposed that complex sugar molecules, called polysaccharides, and not proteins determined the immunological differences between different types of pneumococcal bacteria. Although this theory was eventually confirmed to be true, at the time of discovery it provoked a storm of controversy that had haunted this nervous and sensitive man. In a long and rambling letter to his brother Avery had repeatedly referred to his worry about the reaction to the new discovery. ‘It’s hazardous to go off half-cocked … It’s lots of fun to blow bubbles – but it’s wiser to prick them yourself before someone else tries to.’
Avery had an adversary closer to home. Alfred E. Mirsky, a distinguished biochemist and geneticist also working at the Rockefeller Institute, had reacted to Avery’s discovery with incredulity. To make matters worse, Mirsky was widely regarded as an expert on DNA. He had discovered that the quantity of DNA in every cell nucleus remained the same, establishing a principle called ‘DNA constancy’. He now doubted the efficacy of McCarty’s DNA extraction. A stickler for ‘clean’ biochemical experiment, Mirsky believed that protein found in the nucleus, called nucleoprotein, must be the basis of heredity. Even as late as 1946, Mirsky insisted that the two enzymes McCarty had used in his extractions would not digest away all of the protein. Mirsky was very influential in genetic circles and his argument impressed the leading geneticist of the time, Hermann J. Muller, who had been awarded the Nobel Prize that same year for his discovery, made two decades earlier, that X-rays caused mutations in the genes of the fruit fly. In a letter to a geneticist colleague, Muller stated ‘Avery’s so-called nucleic acid is probably nucleoprotein after all, with the protein too tightly bound to be detected by ordinary method.’
To some extent such disagreement was typical of the situation one might find anywhere in science when various groups from different scientific backgrounds are investigating a major unknown. Never is the argument more acrimonious than when a new discovery confounds the accepted paradigm. But the vociferous opposition of Mirsky from within Avery’s home research foundation must have been particularly damaging. In 1947 Muller published his ‘Pilgrim’s Lecture’ as a scientific paper in which he concluded that whether nucleic acid or protein was the answer ‘must as yet be regarded as an open question’. In the words of Robert Olby, a historian and philosopher of science, ‘Through Muller’s widely read Pilgrim Lecture, this [sceptical] influence was spread to a wide audience.’
In a new series of extractions, with stringent quality checking, Avery attempted to confound his critics. McCarty left the laboratory in 1946, which was left in the hands of, amongst others, the meticulous Rollin Hotchkiss. Hotchkiss added several new chemical explorations of the extract, all further confirming that it was DNA. He disproved Mirsky’s objection by purifying the extract to the extent that the protein content was below 0.02 per cent and he showed that it was inactivated by a newly discovered crystalline enzyme specific to DNA: DNase. While many geneticists remained obdurate in their opposition, some were beginning to take notice.
In a subsequent interview with the biophysicist and future Nobel Laureate, the German-born physicist Max Delbrück, Horace F. Judson would discover that some distinguished researchers were aware of the potential importance of Avery’s discovery. ‘Certainly there was scepticism,’ Delbrück recalled. ‘Everybody who looked at it was confronted by this paradox. It was believed that DNA was a stupid substance … which couldn’t do anything specific. So one of these premises had to be wrong. Either DNA was not a stupid molecule, or the thing that did the transformation was not DNA.’ Avery had raised a monumentally important question and the only way of resolving the dilemma was for other researchers to probe it through some form of alternative experimentation to find out if he was right or wrong.
In 1951, two American microbiologists, Alfred Hershey and Martha Chase, undertook such an alternative experiment while studying the way that certain viruses use bacteria as a factory to make daughter viruses. These viruses are called ‘bacteriophages’, or ‘phages’ for short – from the Greek phago, which means to eat, because they devour cultures of host bacteria. The presence, and number, of viruses could be measured if you seeded your host bacteria into heat-softened agar and then added the viruses in various dilutions to the agar before spreading it over a laboratory plate. When the agar cooled it formed a thin, even layer of jelly in the plate, which, on overnight culture, would become clouded by growth of bacteria within the agar. Wherever a virus landed among the bacteria there would be a round window of transparency caused by the dissolving (lysis) of the bacteria which was easily visible, and thus countable. This ‘plaque-counting technique’, which I myself learnt from my microbiology professor as a medical student and later made use of in experiments on the nature of autoimmunity as a hospital doctor, is easily learnt and thus put to use by thousands of scientists in a great variety of experiments.
What interested Hershey and Chase was the fact that phage viruses were known to compose a core of genetic material surrounded by a capsule of protein. In fact, each virus closely resembled a medical syringe in structure, so that when it infected the bacterial cell of its host, it appeared to squeeze out the genetic material from the body of the syringe, leaving the empty protein coat attached to the outer bacterial cell wall. Meanwhile, the genetic material was injected into the bacterial cell interior, where the viral genome would be replicated as part of its reproduction. Hershey and Chase invented an ingenious experiment that would decide whether protein or DNA was the basis of the viral reproductive system. This would involve adding radioactive phosphorus and radioactive sulphur to the media in which separate batches of the host bacteria were growing. After four hours, to allow the radioactive element to be taken up by the bacteria, they introduced the phage viruses.
To understand the basis of the experiment we need to grasp that DNA contains phosphorus as part of its make-up but no sulphur, meanwhile the amino acids that make up proteins contain sulphur but no phosphorus.
By inoculating each of these two groups of bacteria with viruses, Hershey and Chase derived two populations of phage viruses – one containing the radioactive phosphorus and the other containing the radioactive sulphur. When the viruses infected the bacteria, they left their empty viral coats, mostly made up of protein, attached to the outside of the bacterial cell walls, having injected their core material, known to comprise DNA, into the bacterial bodies. Hershey and Chase used centrifugation
