The Mysterious World of the Human Genome

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 conversation into an artistic performance with spirited gestures, mimicry, pithy remarks and verbal pyrotechnics. Avery might have been somewhat reticent in manner (he could be silently introspective), but in his own quintessential way he was vulnerably human, and that made him all the more interesting and enchanting.

I would suggest that creativity in science is every bit as intertwined with personality as one finds in a writer, artist, or musically gifted composer or performer. It would seem unsurprising in an artist if he appeared unusually ascetic, withdrawn from the hurly-burly world of the surrounding New York, ensuring that he lived close enough to the Rockefeller Institute so he could walk to work. In his ways, Avery could seem curiously ambivalent. He suffered mood swings at times, when alone in the lab, when he would appear to be dejected by the difficulties facing him. Afterwards he would declaim, clearly referring to himself, that resentment hurts the person who resents much more than the person who is resented. He left many letters unanswered and refused to have a secretary. He refused to review, or sponsor, any scientific paper in which he had made no contribution. In Dubos’ words, ‘Graciousness and toughness when it came to what he himself was determined to do, was part of his nature.’ Avery was a very successful teacher during his early medical career, yet in his later years he appears to have resented being expected to lecture on his own research. In this respect, he bore some interesting similarities to Charles Darwin. Avery scrupulously avoided any discussion of his own health and any intrusion, however small, into his private life – which was devoted to his younger brother, Roy, and to an orphaned first cousin whom he supported all through his life. He never expressed resentment about criticisms of his work, even when these were unjustified. He left no record of his private thoughts, other than the letters to his brother. A single experience struck Dubos as being significant.

One day, in early 1934, the same year that Avery suffered the onset of his thyrotoxicosis, Dubos told Avery that he was about to be married. The lady in question was a Frenchwoman living in New York, named Marie Louise Bonnet. Avery immediately rejoiced at the news. They were conversing in the laboratory on the sixth floor of the Rockefeller hospital building. During the subsequent animated conversation, Avery climbed out of his chair, walked to the window and looked out, as if lost for a moment in deep reflection. Returning to his chair, he mentioned that he had contemplated marriage years before, but that circumstances had not proved favourable to his plans. It seems likely that the lady in question was a nurse that Avery had met during the course he had taught to student nurses at the Hoagland Laboratory. Avery would have been about 32 years old at the time. For a moment or two the older man’s eyes were full of longing.

‘One of the great joys of life,’ he remarked to Dubos, ‘is to go home to someone who would rather see you than anybody else.’

Fate would prove cruel to both men. Marie Louise Bonnet subsequently died from tuberculosis at a time when Dubos was pioneering the very antibiotics that would eventually help to cure the same illness. The marriage was childless and the effects of his wife’s death on Dubos were devastating. He resigned, forthwith, from his antibiotic researches, which were later taken up by his former teacher, Selman Waksman at Rutgers Agricultural College, now Rutgers University, and which led to the discovery of a series of important antibiotics, including streptomycin. This breakthrough resulted in Waksman being awarded the Nobel Prize in Medicine or Physiology in 1952.

Much of what Dubos witnessed of Avery spoke of an intense focus and purity of purpose in science and his work. But, increasingly, his devotion to his research appeared to be accompanied by insularity bordering on reclusiveness.

Scientists who have laboured long and hard at a difficult but eventually rewarding line of research are usually happy to talk about it – if not to the media or ordinary social channels, certainly to colleagues. They travel to scientific symposia. They take part in conferences. They enjoy the camaraderie that comes from sharing the same interests. In the words of Frank Portugal, ‘wide-ranging discussions with peers both individually and at meetings are part and parcel of the scientific process. It is an important component of how collaborations are formed and scientific advances are made and respected.’ Most scientists are only too glad to accept the, often rare, honours and distinction their work brings their way. Not so Oswald Avery.

In 1944 Avery was proposed for an honorary degree at Cambridge University, a recognition most scientists would cherish. The following year he was awarded the Copley Medal by the Royal Society of London. Avery’s roots were English – in the late nineteenth century his family had emigrated to Canada from the city of Norwich – but he refused to visit England even on such prestigious occasions, putting forward the excuse that his state of health did not permit it except by travelling first class. In Dubos’ opinion, this was disingenuous, since the respective foundations would have funded the flights. That he might have felt nervous, claustrophobic, on the lengthy flight is possible. Recalling those dark moods in which Avery might mumble to himself about the damaging inflictions of resentment, it seemed more than likely to Dubos that he might have been unable to suppress lingering anger at the hurtful controversy provoked years ago by his polysaccharide typing of pneumococci. Whatever his reasons, Avery refused both honours.

An incident highlighted just how strong was Avery’s aversion to such formal acknowledgement of his work. Sir Henry Dale, who was President of the Royal Society in England, took it upon himself to bring the Copley Medal to the Rockefeller Institute, there to confer it on the shy and retiring Avery in person. Dale was accompanied by a Dr Todd, who knew Avery personally. The two highly respected English visitors arrived at the Institute in New York unannounced and went directly to Avery’s department in the main hospital building. But when they saw Avery working in his lab, through the ever-open door, they retreated without intruding on his presence.

Dr Todd would later recount how Sir Henry Dale said simply: ‘Now I understand everything.’

Bizarre as this behaviour would appear, it was in keeping with Avery’s increasingly reclusive personality: a man who avoided any of the normal personal contacts outside of immediate family and work colleagues. Genius can be strange. Yet such idiosyncratic behaviour apart, it was this son of an evangelical Baptist preacher who first discovered that DNA was the molecule of heredity. And putting such personal matters aside, the question remains: why was such a fundamental discovery not recognised by the awarding of the Nobel Prize?

In his letters to his brother, Avery retained a modest outlook. Could it be that a combination of Avery’s innate conservatism, his tendency to over-caution, and his downplaying of the implications of his discovery in the paper of 1944 might have contributed to his being overlooked? In Dubos’ words, the paper … ‘did not make it obvious that the findings opened the door to a new era of biology’. Dubos wondered if the Nobel Committee, unaccustomed to such restraint and self-criticism ‘bordering on the neurotic’ might have caused them to wait a while for both confirmation of the discovery and to see what the broader implications might be. To put it another way, Dubos questioned if the paper might have been a failure not in its own merits, as a scientific communication, but from the public relations point of view.

This lack of recognition is made all the more puzzling by the fact that, if the importance of the 1944 paper was not universally recognised when it was published, it became more and more obvious with the passage of time. The Hershey and Chase paper was published in 1952. And although he was retired by the time Crick and Watson published their famous discovery of the three-dimensional chemical structure of DNA in 1953, Avery was still alive. He wouldn’t die until two years later, in 1955.

More recently the Nobel authorities have allowed open access to their earlier thinking, and this has confirmed much of what Dubos had concluded. As part of the system for deciding who should get Nobel Prizes, the Nobel Committee receives proposals from leading experts around the world. In the words of Portugal, who reviewed their working and archives, ‘It seems that key biological chemists were not convinced by Avery’s claim that DNA was the basis of heredity.’ Not a single geneticist nominated Avery for the Nobel Prize. In part this may have reflected a difficulty in extrapolating his discovery in a single type of bacterium to genetics more widely, but even those colleagues who did nominate him for the Nobel Prize tended to overlook his work on DNA in favour of his immunological typing of the pneumococcal capsule. Portugal also wondered if Avery’s own idiosyncratic behaviour, including his reluctance to meet with and exchange findings with colleagues, and in particular geneticists, at scientific meetings had unintentionally confounded the acceptance of his groundbreaking discovery.

We are left with a lingering sense of regret that Avery was not accorded the recognition he deserved. He was 67 years old when his iconoclastic paper was published. It was, in the words of the eminent biochemist Erwin Chargaff, the rare instance of an old man making a major scientific discovery. ‘He was a quiet man: and it would have honoured the world more, had it honoured him more.’

But there is a greater acknowledgement of discovery than the awarding of a prize, no matter how respected and prestigious. In the words of Freeland Judson, ‘Avery opened up a new space in biologists’ minds.’ By space he meant he had unravelled a major truth, revealing new unknowns and raising important new questions. Avery himself had, with quintessential modesty, touched upon those important new questions in his letter to his brother:

If we are right, and of course that is 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 … Sounds like a virus – may be a gene. But with mechanisms I am not now concerned – one step at a time – and the first is, what is the chemical nature of the transforming principle? Someone else can work out the rest …

three

The Story in the Picture

You look at science (or at least talk of it) as some sort of demoralising invention of man, something apart from real life, and which must be cautiously guarded and kept separate from everyday existence. But science and everyday life cannot and should not be separated.

ROSALIND FRANKLIN

The discovery of the ‘transforming substance’ by Avery, MacLeod and McCarty, confirmed by Hershey and Chase’s elegant experiment with the bacteriophage, proved that DNA was the molecule of heredity. But both groups were working with microbes, bacteria and viruses, which were known to be much simpler in their hereditary nature than, say, animals and plants. This left huge unknowns that needed to be explored. Was DNA the key to the heredity of all of life, or was it just relevant to bacteria and viruses? By the early 1950s, work in many different laboratories had confirmed that DNA was a major ingredient in the nuclei of animals and plants. This supported the idea that DNA was the coding molecule of life. But if so, how did it really work? How, for example, did a single chemical molecule code for the complex heredity of a living organism?

Biologists, doctors, molecular biochemists and geneticists were now asking themselves the same, or similar, questions. Critical to any such understanding was the precise molecular structure of DNA. If, for example, we were to regard the role of DNA as akin to a stored genetic memory, how did that molecular structure enable the quality of such a phenomenally complex memory? How was that genetic memory transferred from parents to offspring? How did the same stored memory explain embryological development, where a single cell arising from the genomic union of a paternal sperm and maternal ovum gives rise to the developing human embryo and future adult human being?

There was another profoundly important question.

Darwinian evolution lay at the heart of biology. To put it simply, Darwin’s idea of natural selection implied that nature selected from a range of variations in the heredity of different individuals within a species. The way in which it worked was exceedingly simple, if brutal. Those individuals, and by inference their variant heredities, who carried a small advantage for survival and thus a better chance of giving rise to offspring, would therefore be more likely to contribute to the species gene pool. In reality natural selection worked more through a process of attrition. Those less advantaged individuals who did not carry the advantage for survival, were more likely to perish in the struggle for existence, and thus they were less likely to contribute to the species gene pool.

This is what Darwinian evolutionary biologists refer to as ‘relative fitness’. It is the measure of the individual’s contribution to the species gene pool. Certainly it has nothing to do with racist notions of superiority and inferiority attached to ‘survival of the fittest’ – a term introduced not by Darwin but by the social philosopher Herbert Spencer. But if we take a pause and think about it, such variant heredity, essential for natural selection to work, must also come about through mechanisms involving this wonder molecule, DNA, which must lie not only at the heart of heredity but also at the absolute dead centre of evolution. All of these questions needed to be answered by the scientists now struggling to understand the structure and, assuming structure was function, the properties of this remarkable chemical, DNA.

In fact the first step towards answering these questions had already been taken back in 1943, in what might appear unlikely circumstances. It was taken not by a biochemist, biologist or geneticist, but by an Austrian physicist. The spark was lit when, at 4.30pm on Friday 5 February, Erwin Schrödinger stepped up to the podium in Dublin to deliver a lecture that is now seen as a landmark moment in the history of biology. Schrödinger had been awarded the Nobel Prize in 1933 for work in quantum physics that expanded our understanding of wave mechanics – but I won’t confuse myself or my readers by entering further into the physics. The simple facts were that Schrödinger had exiled himself from his native Austria in protest at human rights abuses and had been given sanctuary in neutral Ireland by its President, Eamon de Valera. In Dublin Schrödinger had helped found the Institute for Advanced Studies. As part of his duties in support of the Institute, he had agreed to give a series of three lectures in which he developed a central theme: ‘What Is Life?’

Such was Schrödinger’s fame that the lecture theatre, which had a seating capacity for 400, could not accommodate all who wished to attend the lectures – this despite the fact that they had been warned in advance that the subject matter was a difficult one and that the lecture was not going to be pitched at an easy or popular level, even though Schrödinger had promised to eschew mathematics. De Valera himself was present in the audience, as were his cabinet ministers and a reporter for Time magazine. One wonders what these politicians and journalists made of Schrödinger’s focus on ‘how the events in space and time which take place within the spatial boundary of a living organism can be accounted for by physics and chemistry’.