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It is little wonder that Avery was astonished by Griffith’s findings. Like Robert Koch before him, Avery subscribed to the view that bacterial strains were immutable in terms of their heredity. The very concept of a mutation – that heredity was capable of an experimentally induced change – was a highly controversial issue within biology and medicine at this time. To understand why, we need to grasp the concept of what a mutation means.
By the late nineteenth century Darwinian theory had entered a crisis. Darwin himself had been well aware that natural selection relied on some additional mechanism, or mechanisms, capable of changing heredity, so that natural selection would have a range of ‘hereditable variation’ to choose between. Generations later, in the opening chapters of his innovative book Evolution: The Modern Synthesis,Julian Huxley put his finger on the nub of the problem. ‘The really important criticisms have fallen upon Natural Selection as an evolutionary principle and centred round the nature of inheritable variation.’ In 1900, a Dutch biologist, Hugo de Vries, put forward a novel mechanism that would be capable of providing the necessary variation: the concept of a random change in a unit of inheritance. Opportunity for change exists when genes are copied during reproduction, when a random change in the coding of a gene might arise from an error in copying the hereditary information. De Vries called this source of hereditary change a ‘mutation’. It was only with what Julian Huxley termed ‘the synthesis’ of Mendelian genetics – the potential for change in the inherited genes through mutation – and Darwinian natural selection operating on the hereditary choices presented within a species, that Darwinian theory became credible again to the great majority of scientists.
In time Griffith’s finding would be confirmed to be what Avery was now wondering about: it was a mutation. Geneticists would show that the change from the R to the S strain of pneumococcus involved the transfer of a gene from the dead S-type II bacteria to the living R-type I bacteria, which was incorporated into subsequent bacterial reproductive cycles, transforming the cells of the R-type I bacterium into the cells of the S-type II bacterium. It was indeed the bacterial equivalent of a change of species. And Griffith was proven right in inferring that Darwinian natural selection had operated even in the short time frame of the infection of a cohort of laboratory mice.
Griffith’s experimental findings galvanised bacteriologists and immunologists around the world. His discovery was confirmed in several different research centres, including the Robert Koch Institute in Berlin, where the pneumococcal types had first been classified. The news was inevitably a hot topic of discussion in Avery’s department, as Dubos would recount: ‘but we did not even try to repeat them at first, as if we had been stunned and almost paralysed intellectually by the shocking nature of the findings’.
At first Avery simply couldn’t believe that bacterial types could be transformed. Indeed, he had been one of the authoritative figures who had settled the fixity of bacterial reproduction being true to type years before. But from 1926 Avery encouraged a young Canadian physician working in the Rockefeller Laboratory, M. H. Dawson, to investigate the situation. According to Dubos, Dawson, unlike Avery, was convinced from the start that Griffith’s conclusion must be correct because he believed that ‘work done in the British Ministry of Health had to be right’.
Dawson began by confirming Griffith’s findings in laboratory mice. His results suggested that the majority of non-virulent bacteria – the R types – had the ability in certain circumstances to revert to the virulent S type. By 1930 the young Canadian was joined by a Chinese colleague, Richard P. Sia, and between them they took the experimental observations further by confirming that the hereditary transformation could be brought about in culture media, without the need for passage through mice. At this stage, Dawson left the department and Avery encouraged another young physician, J. L. Alloway, to take the investigation further. Alloway discovered that all he needed to bring about the transformation was a soluble fraction derived from the S pneumococci by dissolving the living cells in sodium deoxycholate, then passing the resultant solution through filters to remove the bits of broken-up cells. When he added alcohol to the filtered solution, the active material precipitated out as sticky syrup. Throughout the laboratory this sticky syrup was referred to as the ‘transforming principle’. So the work continued, experiment following experiment, year by year.
When Alloway left the department, in 1932, Avery began to devote some of his own time to the pneumococcus transformation, in particular aiming to improve the extraction and preparation of the transforming substance. Frustration followed frustration. He focused on its chemical nature. Discussion took place with other members of the department, ranging from the ‘plamagene’ that was thought to induce cancer in chickens (now known to be a retrovirus), or to the genetic alterations in bacteria that were thought to be caused by viruses. According to Dubos, Alloway suggested the transforming agent might be a protein-polysaccharide complex. But by 1935 Avery was beginning to think along other lines. In his annual departmental report that year he indicated that he had obtained the transforming material in a form that was essentially clear of any capsular polysaccharide. In 1936, Rollin Hotchkiss, a biochemist who had now arrived to work in the department, wrote a historic comment in his personal notes:
‘Avery outlined to me that the transforming agent could hardly be a carbohydrate, did not match very well with a protein and wistfully suggested it might be a nucleic acid!’ At this stage, Dubos, who many years later would write a book about Avery and his work, dismissed this as no more than a surmise. There were good reasons for his caution.
That year few researchers throughout the world believed that the answer to heredity lay with nucleic acids. These chemical entities had been discovered by a Swiss biochemist, Johann Friedrich Miescher, back in the late 1800s. Fascinated by the chemistry of the nucleus, Miescher had broken open the nuclei of white blood cells in pus, and subsequently the heads of salmon sperm, to discover a new chemical compound which was acidic to pH testing, rich in phosphorus and comprised of enormously large molecules. After a lifetime of experimentation on the discovery, Miescher’s pupil, Richard Altmann, would introduce the term nucleic acid to describe Miescher’s discovery. By the 1920s, biochemists and geneticists were aware that there were two kinds of nucleic acids. One was called ribonucleic acid, or RNA, which contained four structural chemicals: guanine, adenine, cytosine and uracil, or GACU. The other was called ‘desoxyribonucleic acid’, or DNA, which was a major component of the chromosomes. They had deciphered its four bases – three identical to RNA, guanine, adenine and cytosine, but with the uracil replaced by thymine – making the acronym GACT. They knew that these four bases consisted of two different pairs of organic chemicals; adenine and guanine being purines, and cytosine and thymine being pyrimidines. They also knew that they were strung together to form very long molecules. At first they thought that RNA was confined to plants while DNA was confined to animals, but by the early thirties this was dismissed when it was found that both RNA and DNA were universally distributed throughout the animal and plant kingdoms. Still they had no knowledge of what nucleic acids actually did in the nuclei of cells.
A distinguished organic chemist based at the Rockefeller Institute, Phoebus Aaron Levene, proposed that the structures of DNA and RNA were exceedingly boring – they formed groups of four bases that repeated themselves in the identical repetitive formation throughout the molecule, like a four-letter word, repeated ad nauseam. This was called ‘the tetranucleotide hypothesis’. Such a banal molecule couldn’t possibly underlie the exceedingly complex basis of heredity. In the words of Horace Freeland Judson, ‘the belief was held with dogmatic tenacity that DNA could only be some sort of structural stiffening, the laundry cardboard in the shirt, the wooden stretcher behind the Rembrandt, since the genetic material would have to be protein’.
Proteins are lengthy molecules made up of smaller organic chemical units known as amino acids. There are 20 amino acids in the make-up of proteins, reminiscent of the number of letters that make up alphabets. If genes were the hereditary equivalents of words, only the complexity of proteins could fashion the words capable of spelling out the narratives. Chemists, and through extrapolation geneticists, not unnaturally assumed that only this level of complexity could possibly accommodate the incredible memory template that the complexity of heredity demanded – a line of thought that Judson labelled ‘The Protein Version of the Central Dogma’.
This was the contentious zeitgeist that Avery now confronted. As early as 1935, in his annual reports to the Board of the Institute, he indicated that he had growing evidence that the ‘transforming substance’ appeared free of capsular polysaccharide and it did not appear to be a protein.
Further progress on this line of research appeared to drag. In part this was because Dubos, working in the same department, had made a breakthrough in his search for antibiotic drugs. In 1925, Alexander Fleming, at St Mary’s Hospital in London, had discovered a potential antibiotic, penicillin, but he had been unable to take his work to the stage of useful production for medical purposes. Now, working on the philosophical principle encapsulated by the biblical saying ‘dust to dust’, Dubos had pioneered the search for microbes in soil that would potentially attack the polysaccharide coat of the pneumococcus. By the early 1930s he was making progress. From a cranberry bog in New Jersey he found a bacillus that dissolved the thick polysaccharide capsule that coated the pneumococcus with its armour-like outer covering. Dubos went on to extract the enzyme that the Cranberry Bog bacillus produced. He and Avery had reported their discovery in a paper in the journal, Science, in 1930. In a further series of papers the two scientists would report further experiments, all aimed at extrapolating the discovery to human trials of the Cranberry Bog enzyme in treating the potentially fatal pneumonia and meningitis caused by the pneumococcus.
But their researches encountered difficulty after difficulty. In part these arose from a predictable ignorance in a field of such pioneering research. A more personal, and devastating, problem arose when, under the stress of it all, Avery developed thyrotoxicosis – a debilitating autoimmune illness in which his thyroid gland became overactive.
Thyrotoxicosis causes the system to be flooded by thyroid hormones, which would have inappropriately switched his metabolism into a dangerous overdrive. He would have felt shaky, agitated, physically and mentally restless, suffering difficulties with relaxation and sleep – an impossible situation for a creative person. Avery had to spend time away from the lab undergoing surgery to remove the bulk of the ‘toxic goitre’, a procedure that carried risk of side-effects, even fatality in a minority of cases. His surgeon advised him against any early activity, physical or mental, that provoked stress. Dubos later recalled how Avery was away from his work for as long as six months. And while Avery was away, the laboratory stagnated. In Dubos’ own words, ‘I … pursued [the research] for three or four years. However I could not carry the work very far because there were serious gaps in both my knowledge of genetics and biochemistry and in the [prevailing] states of these sciences themselves.’
Dubos would continue his researches against such difficulties, to be rewarded, in 1939, with the discovery of the first soil-derived antibiotic. He called it ‘gramicidin’. But gramicidin could not be taken by mouth or administered by injection because it was too toxic. It could only be applied to skin conditions. The research continued. But then, all of a sudden, the hopes of Avery and Dubos were overtaken by a rival breakthrough. Working in the pharmaceutical research laboratories of the Bayer Company in Elberfeld, Germany, doctor Gerhard Domagk reported the discovery of a new antibacterial agent called prontosil. The first of what would come to be known as the sulphonamide drugs, it immediately entered the medical formulary, pioneering the treatment of a number of hitherto untreatable infectious diseases.
Today we are apt to forget how little we could do to control infection in the 1930s. Epidemics such as scarlet fever, measles, pneumonia, meningitis and poliomyelitis swept through the population in regular, sometimes annual, cycles. Other notorious infections were everyday threats, including tuberculosis, which ravaged entire families, or boils, septic arthritis, septic osteomyelitis, which caused agonising abscesses in bone, and the commonplace but potentially deadly streptococci capable of breaking through a septic throat to cause abscesses in the brain. Most of the human population, whether in developed or developing countries, died from infections, including the insidious pneumonias that hit those whose immunity was depressed. The treatment of infections was the most urgent problem then facing humanity. For Dubos, and even more so Avery, the disappointment of failing in their line of research would have been shattering.
When, in due course, Avery returned to work, he switched the emphasis of his research to the ‘transforming substance’. Colin MacLeod improved the techniques of extraction so they could now produce sizeable amounts for assay and further testing. They began to make more rapid progress so that, in a report to the Rockefeller Board for the year 1940–41, they were more confident in stating that even a highly purified extract of the transforming substance appeared to be protein-free.
That summer MacLeod left the Institute to become Professor of Bacteriology at the New York University School of Medicine. But he still took an interest in the project and frequently returned to the Institute to add his advice. A young paediatrician, Maclyn McCarty, took MacLeod’s place in the transforming experiment. McCarty brought a useful level of biochemical training to the laboratory. And now they had the transforming substance in quantity and in stable form, he applied his chemical skills to further process and identify the active material. He began to culture the pneumococci in large batches of 50 to 75 litres, developing a series of steps that increased the yield of transforming substance while removing proteins, polysaccharides and ribonucleic acid. The prevailing beliefs about the hereditary principle claimed that nucleoproteins were the answer. Thus the topmost priority in all of this effort was to ensure that the final test material contained no protein.
By now McCarty had extracted concentrated solutions of the active material. He treated this with a series of protein-digesting enzymes, such as the gut-derived trypsin and chymotrypsin, which were known to destroy proteins, ribonucleic acid and pneumococcal capsular polysaccharide. What remained was once more shaken with chloroform in a final effort to remove even the finest traces of protein.
By late 1942, after repeated extraction and experiment, McCarty had come to the conclusion that the transforming activity was confined to a highly viscous fraction that consisted almost exclusively of polymerised deoxyribonucleic acid. When he precipitated this fraction in a flask by adding absolute ethyl alcohol, drop by drop, all the while stirring the solution with a glass rod, the active material separated out of the solution in the form of long, white and extremely fine fibrous strands that wound themselves around the stirring rod. Dubos would recall the excitement felt within the lab by all those who witnessed the sight of the beautiful fibres, which were the pure form of the transforming substance.
In early 1943, Avery, MacLeod and McCarty presented their findings to distinguished chemists at the Princeton section of the Rockefeller Institute for Medical Research. The chemists must have been astonished, perhaps even nonplussed, but they offered no contradiction of the evidence nor asked for further proof. The researchers summed up the evidence for the Board of the Rockefeller in April of that year. Avery, MacLeod and McCarty, all three medical doctors rather than geneticists, were now ready to inform the world in a paper submitted to the Journal of Experimental Medicine in November the same year, which would be published early the following year. The title of the paper was long-winded and cautious: ‘Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III’.
In the words of Dubos, this paper ‘had staggering implications’. The sense of excitement, tempered by caution, was captured in a letter that Avery wrote to his brother, Roy, dated 26 May 1943:
… For the past two years, first with MacLeod and now with Dr McCarty, I have been trying to find out what is the chemical nature of the substance in the bacterial extracts which induces this specific change … Some job – and full of heartaches and heartbreaks. But at last perhaps we have it … In short, the substance … conforms very closely to the theoretical values of pure desoxyribose 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.
two (#ulink_a8f9dc67-6bfa-5215-bbf6-a95d6a68aef6)
DNA Is Confirmed as the Code (#ulink_a8f9dc67-6bfa-5215-bbf6-a95d6a68aef6)
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 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 (#ulink_1f573ad9-98b3-558a-8d1f-470d1b6d5745)
The Story in the Picture (#ulink_1f573ad9-98b3-558a-8d1f-470d1b6d5745)
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’.
Schrödinger subsequently extrapolated the three lectures into a book of less than a hundred pages with the same title: What Is Life? This was published the following year. In what is now a very famous book, Schrödinger popularised a quantum mechanics interpretation of the gene that had been proposed earlier by another distinguished physicist, the previously mentioned Max Delbrück.
In the opening pages of the first chapter, Schrödinger posed the question: ‘How can the events which take place within a living organism be accounted for by physics and chemistry?’ Admitting that at the time of writing the prevailing knowledge within the disciplines of physics and chemistry was inadequate to explain this, he nevertheless hazarded the opinion that ‘the most essential part of a living cell – the chromosome fibre – may suitably be called an aperiodic crystal’. The italicisation is Schrödinger’s to emphasise, as he further explained, that the physics up to this time had only concerned itself with periodic crystals, the kind of repetitive atomic structures seen, for example, in very obvious crystalline compounds such as gemstones.
What did he mean by an ‘aperiodic crystal’?
He explained this with a metaphor. If we examined the images within the pattern of a wallpaper, we could see how the pattern was repeated, over and over. This was the equivalent of a periodic crystal. But if we examined the complex elaboration of a Raphael tapestry, we saw a pattern of images that did not repeat themselves, yet the pattern was coherent and meaningful.
Schrödinger intuited further.
It was the chromosomes, or more likely an axial fibre much finer than what was visible under the microscope, that contained what he termed ‘some kind of code-script’ that determined the blueprint of the individual’s development from fertilised egg to birth – and further determined the functioning of what we would now term the genome throughout the lifetime of the individual.
That intuition would provide the drive for a naïve but highly inquisitive young American, called James Dewey Watson, to join forces with a slightly older but equally inquisitive Englishman, Francis Crick, and form what is now seen as one of the most famous partnerships in scientific history. Both men would take their inspiration from Schrödinger to search for the aperiodic crystal that coded for DNA.
*
Watson was an exceptionally bright child who lived at home with his family in Chicago while attending the local university. He enrolled when aged just 15 and he graduated, aged 19, in 1947 with a bachelor’s degree that included a year studying zoology. His teacher of embryology would remember him as a student who showed little interest in lectures and made no notes whatsoever, so it was all the more puzzling when he graduated top of his class. Watson would subsequently admit to a habitual laziness. Though vaguely interested in birds, he had deliberately avoided any courses that involved chemistry or physics of ‘even medium difficulty’. This self-indulgent student left Chicago with only a rudimentary knowledge of genetics or biochemistry. As part of his education he had attended lectures by the geneticist Sewall Wright, who had devised a mathematical system of studying population genetics. Wright’s course included a discussion of Avery’s work, but Watson would subsequently confess that he took little notice. He would also confess that the inspiration for his subsequent interest in the ‘mystery of the gene’ was Schrödinger’s book, What Is Life?
Inspired by this book, Watson landed a research fellowship at Indiana University, at Bloomington. He was delighted by the move because Nobel Laureate Hermann Joseph Muller was the local Professor of Zoology. As early as 1921 Muller had observed that the genes of the fruit fly underwent mutations – as did the genes of the bacteriophages – the viruses that had inspired Hershey and Chase. Watson was intrigued by the fact that phage viruses could be manipulated in test tubes. Their reproductive cycles were extremely brief – an important consideration for an impatient young scientist. There were simple test systems that could be employed to follow their life cycles, and numbers, in a way that would open up new angles from which to attack the gene problem. All you had to do was carefully design an experiment aimed at probing some particular aspect of the gene problem and the whole shebang could be completed in a matter of days. This intimate, if brutal, interplay between phage viruses and their host bacteria allowed scientists to figure the complex chemistry of genes, genetics and chromosomes.
Curiously it would not be Muller but another phage researcher, Salvador Luria, who would now give shape and direction to the young scientist’s growing infatuation with the gene.
The Italian-born Luria was another European scientist – a microbiologist, like Avery – who found refuge in America from the European war zone. By now he had entered into a working collaboration with Max Delbrück, who was Professor of Biology at the California Institute of Technology. In 1943 Luria and Delbrück designed and conducted an experiment that demonstrated that genetic inheritance in bacteria followed precise evolutionary principles. This experiment became one of the foundation stones of modern Darwinism. That same year Delbrück befriended another microbiologist called Alfred Hershey, who would subsequently write the key DNA paper with Martha Chase. In a letter to Luria, Delbrück summarised Hershey as follows: ‘Drinks whiskey but not tea. Likes living in a sailboat … Likes independence.’ The three scientists joined forces to become the nucleus of a cooperating and mutually supportive network of scientists that would become known as the ‘phage group’. Delbrück would subsequently explain that they would be a group only in the sense that they communicated freely on a regular basis, and that they told one another what they were thinking and doing. In this way a loose creative movement grew around the two European expatriate scientists, all working towards the common ambition of figuring out how genes worked.
Luria, Delbrück and Hershey now posed some interesting questions. How does the phage virus actually get into the bacterium? How, once inside, does it multiply? Does it multiply like a bacterium, growing and budding off daughter viruses? Or does it multiply by an entirely different mechanism? Is this multiplication some complex physical or chemical process that could be understood in terms of known physical and chemical principles? Through making use of the phage reproductive system, they hoped to solve the mystery of the gene. To begin with it all seemed simple in principle, but as experiment followed experiment and year followed year, they found themselves no closer to the answer.
Up to 1940 or so, people like Delbrück and Luria had assumed that viruses were simple. They had little to go on since the majority of viruses were so minuscule they could not be seen with any clarity through the ordinary light microscope. They would even talk about them as if they were akin to protein molecules. Luria would come to define phage viruses, in a misleading oversimplification, as extensions of the bacterial genome. But with the invention of the electron microscope, by the German company Siemens, even the smallest viruses, including bacteriophages, would soon become visible for the first time. And when they did become visible, they proved to be more complex than the two scientists had initially conceived.
Many phages had a head that was cylindrical in shape, with a narrow sheath below it, as tall as the head, and a base plate with six spikes with fibres attached. Now that they could visualise phages in the process of infecting their host bacteria, something struck Delbrück and Luria as exceedingly odd. The viruses didn’t actually pass through the bacterial cell wall. What they appeared to do was to squat down against the wall and inject their hereditary material into the cell. In 1951 a phage researcher called Roger Herriott would write to Hershey, ‘I’ve been thinking that the virus may behave like a little hypodermic needle full of transforming principles.’ This became the background to Hershey and Chase’s experiment in which they confirmed that that was precisely what happened. The virus behaved exactly like a hypodermic syringe; the tail and its elongated fibrils would attach to the bacterial wall and the phage would then inject its viral DNA in through the bacterial wall to take over the bacterium’s own genetic machinery, the viral genome compelling the bacterial genome to construct what was necessary for the generation of daughter viruses. In effect, the infected bacterium became a factory for the production of daughter viruses.
It would be this discovery, together with many associated extrapolations to microbiology and genetics, that would lead to all three scientists – Delbrück, Luria and Hershey – sharing the Nobel Prize in 1969.
Meanwhile, back in 1947, it was the dynamic energy and infectious charm of Luria, and the innovative genius of Delbrück, that proved most influential to the youthful Watson after his arrival into Indiana University. Still fascinated by the mystery of the gene, it was his hope that the mystery might be solved without his bothering to learn any of the complex physics or chemistry.
It is instructive to discover, from conversations between Luria and Watson, that there was no ignorance at Bloomington about Avery’s discovery of DNA. Luria had visited Avery in 1943, prior to the publication of the key paper, when he had the opportunity of discussing Avery’s findings in detail. He would recall Avery to Watson as an utterly non-pompous scientist, precise in his language, with a tendency as he spoke to close his eyes and rub his bald head – ‘every bit of a chemist, even though he was an MD’. Watson would take his cue from Luria, writing, in The Double Helix, how Avery had shown that hereditary traits could be transmitted from one bacterial cell to another by purified DNA molecules. Given the fact that DNA was known to occur in the chromosomes of every type of living cell, ‘Avery’s experiments strongly suggested that … all genes were composed of DNA.’
In the autumn of 1947, Watson, still just 19, took Luria’s course in bacteriology and Muller’s in X-ray-induced gene mutation. Faced with the choice of entering into research with Muller on Drosophila or with Luria on microbes, he plumped for Luria, despite the fact that the Italian scientist had a reputation among the graduate students for having a short fuse with dimwits. Watson would subsequently adopt his patron’s example. Delbrück was a heroic figure to Watson because he had inspired Schrödinger’s ideas in the inspirational book. Watson was delighted when Luria introduced him to Delbrück when the eminent German physicist paid a visit to Bloomington.
Luria set Watson a PhD dissertation on the pathological effects on phage of exposure to X-rays. The work proved so mundane that Watson would barely mention it in his biography. But his obsession with the gene was undimmed. By the summer of 1949, his thesis nearing completion, he had the itch to travel to Europe. Luria arranged a Merck Fellowship from the National Research Council – three thousand dollars for the first year, potentially renewable. In May the following year, with his PhD under his belt, he sailed for Denmark, where he had been assigned to study nucleotides with a biochemist named Herman Kalckar. Kalckar was a gifted scientist but his interest was neither the gene nor the bacteriophage. A disenchanted Watson switched his attentions to another Dane, and a member of the phage group, Ole Maaløe, who was working on the transfer of radioactively-tagged DNA from phages to their viral offspring.
Out of the blue, Kalckar accepted a short-term project in the Zoological Station in Naples. He suggested that Watson might tag along. Though he had little interest in marine biology, Watson was delighted to acquiesce. He hoped to warm himself in the Italian sun. But he was disappointed to find Naples chilly, with no heater in his room on the sixth floor of a nineteenth-century house. ‘Most of my time I spent walking the streets or reading journal articles … I daydreamed about discovering the secret of the gene, but not once did I have the faintest trace of a respectable idea.’
Here, by happenstance, he attended a lecture in the Zoological Station given by an English scientist named Maurice Wilkins. The lecture could hardly have excited him in prospect, knowing that most of it would be about the biochemistry of proteins. ‘Why should I get excited learning boring chemical facts as long as the chemists never provided anything incisive about the nucleic acids?’
But he took the risk and attended anyway.
Tall, bespectacled, asthenic and somewhat diffident in manner, you might have expected Wilkins’ presentation to bore the restless and impatient Watson. But it did not. To begin with, it was delivered in a language that Watson readily understood. And for all of his diffident manner, Wilkins kept to the point. Then suddenly, close to the end of the lecture, a projected slide jarred Watson to full attention. On the screen was a photograph that showed something Wilkins called an X-ray diffraction pattern of DNA that had been taken in the King’s College laboratory in London. Watson would subsequently admit that he knew nothing about X-ray crystallography. He hadn’t understood a word of what he had read about it in the scientific journals and he thought that much of what the ‘wild crystallographers’ were claiming was very likely baloney.
But now here was Wilkins mentioning in passing that this was the clearest picture of DNA that he and his colleagues had yet obtained from their X-ray studies. In the same audience was the Leeds-based English physicist, William Astbury, who had pioneered X-ray diffraction studies of biological molecules, and who had produced the first X-ray pictures of DNA. Astbury would subsequently confirm that no one had ever shown such a sharp, discrete set of reflections from the DNA molecule as Wilkins then projected onto the screen. ‘There was nothing like it in the literature.’ In explaining the picture, Wilkins suggested that DNA might be thought of as a crystalline substance.
Watson was electrified to hear Schrödinger’s prophecy confirmed. He sat in a daze of wonderment as Wilkins went on to explain that if and when we understood the structure of DNA, then we might be in a better position to understand how genes worked. Watson was now asking himself some pertinent questions. Who was this interesting English scientist, Wilkins? And how could he get to join his team at King’s College in London?
*
Maurice Hugh Frederick Wilkins was not, in fact, English, as Watson initially surmised. He was born in Pongaroa, New Zealand, where his father, Edgar Henry, was a practising doctor. The family were Anglo-Irish in origins, hailing from Dublin, where Maurice’s paternal grandfather had been headmaster of the high school and his maternal grandfather chief of police. On leaving New Zealand the family first returned to Ireland, then headed for London, where Dr Wilkins was later to do his pioneering work in public health.
