The Mysterious World of the Human Genome. Frank Ryan
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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 to separate and extract empty viral coats. Meanwhile, the infected bacteria were allowed to go through their normal reproductive cycle, which allowed the viral cores inside them to generate entire new phage viruses, rupturing the bacterial bodies and flooding the growth media with large numbers of fully formed viruses. Hershey and Chase now removed what was left of the host bacterial bodies to gather dense concentrations of fully formed viruses.
When they now compared the empty viral coats, made up of protein, with the fully formed viruses, with their cores full of genetic material, they found that 90 per cent of the radioactive sulphur was left behind in the viral coats when the virus infected the cell, and 85 per cent of the phosphorus was now part of DNA that had entered the bacterial cell to code for the future offspring of virus. This confirmed Avery’s findings: DNA, and not protein, was the code of heredity.
We might duly note that this separation of coat from core DNA of virus involves a much higher degree of protein impurity than Avery’s extractions. Yet the hitherto sceptical geneticists appeared to be more convinced by the phage experiment than by Avery’s work. Perhaps the strikingly visual nature of the experiment was a factor. Perhaps it was the additional, quite different, avenue of confirmation. It didn’t harm credibility that leading geneticists were within the ‘phage camp’, too.
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Today, with the advantage of retrospect, scientists by and large see the 1944 paper by Avery, MacLeod and McCarty as the pioneering discovery of DNA as the molecule of heredity. It has been portrayed as one of the most regrettable examples of a discovery that merited, but was not awarded, the Nobel Prize. There is ample evidence that Avery was recommended by senior colleagues, particularly within his own discipline of microbiology and immunology – indeed he was nominated twice, first in the late 1930s, for his work on the pneumococcal typing and its relevance to bacterial classification, and, after the 1944 paper was published, he was nominated yet again for his fundamental contribution to biology. But it would appear that the Nobel Committee was not sufficiently swayed. In retrospect, it is seen as a major omission that causes people to scratch their heads and wonder why.
Dubos worked for fifteen years in the lab next door to Avery’s and, in so much as the reticent Professor allowed it, he had plenty of opportunity to get to know Avery and to understand his approach to science and his reaction to the stresses involved in pioneering new concepts. In Dubos’ opinion, writing in 1976, the curious lack of recognition most likely derived from a combination of happenstance and Avery’s own personality. He would subsequently remark how, in all that time, Avery never closed the door of his lab, or the small office that led off it, allowing any of his staff to come and talk to him. This same eternally open door also allowed Dubos to witness ‘Fess’s’ activities at the bench, to listen in to his conversations with colleagues and to observe his interludes of introspective brooding.
This reserved, small and slender bachelor would inevitably arrive at work dressed in a neat and subdued style, his conservative attire somehow at one with the charm of his lively and affable behaviour. His eyes, under the domed bald head that seemed too voluminous for the frail body, were sparkling and always questioning, and he would transform the most ordinary