Life's Greatest Secret
Page 5
Von Neumann adopted this approach after attending the Ninth Washington Conference on Theoretical Physics, which had taken place at the end of October 1946. The subject of the small conference, ‘The physics of living matter’, was inspired by Schrödinger’s What is Life? and had been chosen by an eccentric physicist and lifelong friend of Max Delbrück’s, George Gamow (pronounced Gam-off).27 The conference dealt with many of the points outlined by von Neumann in his letter to Wiener. As the rather excitable conference press release put it:
During the past three days, a group of theoretical physicists and biologists have been meeting at The Carnegie Institution of Washington and The George Washington University here to discuss problems relating to ‘the physics of living matter.’ Much of the discussion has concerned problems of heredity and the mechanisms by which the almost fantastic gene is able to imprint its characteristics on the cell constituents in a hereditary fashion. … It was clear from the discussions this year that the borderline area between physics and biology will see a great deal of research activity during the next few years.28
The excitement may have been heightened by the fact that at the beginning of the meeting, it was announced that one of the thirty-six attendees, Hermann Muller, had been awarded the Nobel Prize in Physiology or Medicine for his work on genetics. Also at the meeting were two representatives of the new wave of geneticists: George Beadle and Max Delbrück. The question of the material basis of heredity – what genes are actually made of – was at the centre of everyone’s attention. This interest was reinforced by a dramatic discovery that had been made more than two years previously in New York by a man who was not a geneticist and who would never have dreamt of going to any of the speculative conferences on the link between physics and biology that took place at this time.
* Most of that federal money was poured into the private sector. Before the war, 70 per cent of government-sponsored research was undertaken by federal organisations, 30 per cent by companies and universities. By 1944, those figures had been reversed. There was plenty of cash to go round: both universities and the private sector were drowning in money from government contracts focused on military problems (Noble, 1986).
–THREE–
THE TRANSFORMATION OF GENES
In December 1943, the Australian virologist Macfarlane Burnet disembarked in San Francisco after a three-week crossing of the Pacific. He was on his way to Harvard, where he had been invited to give a lecture – despite the war, academic life continued, for some. In his mid-forties, handsome and with wavy hair, Burnet had made his reputation working on influenza and other viral diseases; in 1960 he received the Nobel Prize in Physiology or Medicine for his work on the immune response to infection. After the Harvard lectures were over, Burnet travelled to Chicago and then New York, where he had an astonishing discussion with Oswald Avery – a small, bald microbiologist in his mid-sixties, whose quiet manner impressed those who met him. Salvador Luria, a pioneer of virus genetics, recalled:
Talking with Avery was a marvellous experience. He was a wonderful, short man. Very unpompous … He had the dignity of the nondignified people, very simple; and as he was talking he would close his eyes and rub his bald head. And always very precise.1
Avery had spent the whole of his academic life studying pneumococci – the bacteria that cause pneumonia – and had gained an international reputation for his work using immunological responses to characterise different pneumococcal strains. But the story that Avery told Burnet had nothing to do with immunology. As Burnet explained to his fiancée, Avery ‘has just made an extremely exciting discovery which, put rather crudely, is nothing less than the isolation of a pure gene in the form of desoxyribonucleic acid’ or DNA.2*
Avery’s claim was amazing for several reasons. First, it was not accepted that bacteria actually had genes; second, most scientists thought that genes were probably made of proteins, not DNA; finally, Avery was not a geneticist and had no experience in the field. He was nearing retirement, and seemed an unlikely revolutionary. But revolutions can arise in many ways.
*
Oswald T. Avery – generally known as ‘Fess’ (short for ‘Professor’, although he never actually held the title) – had worked at the Rockefeller Institute Hospital in New York since 1913, apart from a brief period as a soldier during the First World War. His laboratory was on the fifth floor of the hospital; the lab had once been a hospital ward and the original partitions were still in place. The lab desks were covered with microbiological paraphernalia – Petri dishes, Bunsen burners, wooden-handled wire loops and needles, microscopes, incubators – while sinks and a fume hood were placed around the edge of the room. The whole place had the distinctive smell of a lab working on pneumonia – the microbes are bred in a blood-based broth. Avery’s private lab had once been the ward kitchen; behind the swing door there was a roll-top desk that was generally crammed full of unanswered letters – Avery hated his routine to be disturbed, and even important invitations to travel to conferences would be left for weeks without reply. When he did respond, he almost always declined.
Before antibiotics became widely available in the 1940s, pneumonia was a major killer – in the US more than 50,000 people died each year of the disease. Physicians were powerless: treatment had little or no effect on survival rates. Some strains of the pneumonia microbe caused disease – they were ‘virulent’ – while others did not; Avery’s approach to finding a cure was to understand why there were these differences between strains. Much of Avery’s early work was carried out with his colleague, friend and flatmate, the opera-loving Alphonse Dochez. As a colleague recalled,
not infrequently he [Dochez] returned from the Metropolitan Opera, discovered Dr. Avery, with whom he shared an apartment, reading quietly in bed, and then would sit down in full evening dress and with vast animation describe to his old friend some of the illuminating thoughts on the subject of microbiology which had occurred to him during the second act of La Traviata.3
Together with Dochez, Avery showed that it was possible to detect differences between types of pneumococci by injecting bacteria into a mouse and then observing the presence of specific antibodies in the animal’s blood serum. Avery’s technique was soon widely adopted as a way of identifying pneumococci and other infectious bacteria.
Insight into the origins of differences in virulence between strains of bacteria came in 1921, when the British microbiologist Joseph Arkwright noticed that colonies of virulent dysentery bacteria had a smooth surface, whereas non-virulent bacteria formed small colonies that appeared rough when inspected under a microscope. Rather obviously, the virulent strains were called S (smooth) and the non-virulent were called R (rough). Two years later, Fred Griffith, a medical officer with the Ministry of Health in London, showed that in pneumococci, too, virulent strains were smooth, whereas avirulent strains were rough. Avery studied the differences between the S and R strains and discovered that when pneumococci became virulent and smooth they produced a capsule that was up to four times the size of the bacterium itself. Avery showed that the capsule consisted of a layer of complex sugars or polysaccharides, which protects the bacterial cell from the body’s defence mechanisms and gives the virulent colonies their smooth appearance.
Back in London, Griffith was exploring the mysterious fact that rough bacterial colonies could change into smooth colonies if they were mixed with smooth bacteria, a phenomenon that had first been described by Arkwright. Arkwright thought that this process was the outcome of competition between the two kinds of microbe; Griffith began to suspect that the avirulent R bacteria had actually changed into virulent S bacteria in a process he called transformation. Astoundingly, Griffith discovered that transformation could occur even if dead S bacteria were mixed with R colonies. Griffith injected mice with live, avirulent R bacteria together with killed S pneumococci. Some of the mice died; they were found to be full of S bacteria, even though the only living bacteria that had been injected were of the R strain. Griffith repor
ted these and many other findings in a dense 45-page article that was published in the Journal of Hygiene in 1928.4
Griffith noted that similar effects had previously been observed in anthrax; the author of the anthrax study, O. Bail, had suggested that the effect involved ‘the inheritance of the capsule-forming substance’. Griffith thought instead that the polysaccharide capsule carried by the dead S cells was being used as a kind of template by the R bacteria to make more capsules. Rather then being inherited, he argued, the ‘specific protein structure’ of the virulent pneumococcus was the cause.
Whatever the explanation – and there was no real evidence as to what was going on – Griffith’s wealth of data and the rigour of his experiments were overwhelming. Almost straight away, Neufeld in Germany replicated the result.* Avery’s group also began to study the effect, getting transformation to occur in a Petri dish rather than in a mouse. By the early 1930s they had made a breakthrough, extracting a substance from S pneumococci that could transform R bacteria. The Avery group called this substance ‘the transforming principle’, and the rest of Avery’s working life was focused on identifying its nature. Shortly after he began studying transformation, Avery received the first of many international awards, the Paul Ehrlich Gold Medal, but severe illness prevented him from attending the ceremony in Germany. For years, Avery had been suffering from Graves’ disease, or hyperthyroidism. This made his eyes bulge out, left him feeling tired and depressed, and gave him a tremor that made it difficult to carry out the delicate and precise microbiological procedures that were his stock in trade. In 1934, Avery went into hospital and had his thyroid removed. It took him months to recover, and it was more than a year before he regained the weight he had lost.
During the summer of 1934 a Canadian physician called Colin MacLeod joined the Rockefeller Institute Hospital, attached to the pneumonia service. When Avery returned from his sick leave, the pair began investigating the chemical nature of the transforming principle. A little more than a year later, Avery explained to his new colleague Rollin Hotchkiss where he thought their study might be going. Hotchkiss recalled:
Avery outlined to me that the transforming agent could hardly be carbohydrate, did not match very well with protein and wistfully suggested that it might be a nucleic acid.5
There were no clear results to back up Avery’s hunch, as MacLeod’s work had not been conclusive. This caused a problem – the young Canadian needed to strengthen his curriculum vitae with some published articles, so he worked instead on the effectiveness of the new sulphonamide antibiotics. The Avery group did no further research on transformation until 1940.
Despite the fact that the method for separating the transforming principle from bacterial cells had been published, no scientists took up the challenge. This was not because people did not know about or appreciate the significance of pneumococcal transformation. In 1941, the leading evolutionary geneticist Theodosius Dobzhansky published the second edition of his influential book Genetics and the Origin of Species. In a chapter entitled ‘Gene mutation’, Dobzhansky described the work of Griffith and Avery and claimed that their findings were ‘not unduly surprising from the standpoint of genetics’, as the change from the R to the S form could be understood in terms of a mutation. More challenging was Griffith and Avery’s demonstration that transformation could take place through contact with a killed sample – Dobzhansky reassured his readers that this ‘extravagant’ finding was ‘conclusively proved’.6 Dobzhansky emphasised that the transformed strains did not merely acquire ‘a temporary polysaccharide envelope of a kind different from that which their ancestors have had, but are able to synthesize the new polysaccharide indefinitely.’ Dobzhansky’s conclusion was that contact with the transforming principle had somehow induced a mutation in the R bacteria, and that this could lead to the use of targeted mutation to study gene function:
If this transformation is described as a genetic mutation – and it is difficult to avoid so describing it – we are dealing with authentic cases of induction of specific mutations by specific treatments – a feat which geneticists have vainly tried to accomplish in higher organisms … geneticists may profit by devising experiments along the lines suggested by the results of the pneumococcus studies.7
Dobzhansky was not claiming that the transforming principle was a gene, but the attention he paid to it showed that Avery’s research was widely known and was seen as important.
*
In October 1940, MacLeod and Avery returned to the problem of identifying the nature of the transforming principle. To help with their analyses, they needed a powerful ultracentrifuge that could separate bacterial contents from the rearing medium – as the sample was spun round at high speeds, the heavier molecules sank to the bottom more quickly, concentrating compounds with a similar weight into a narrow band. The Rockefeller Institute had built some of these devices, using a design developed by the Swedish scientist Theodor ‘The’ Svedberg.8 Avery’s everyday needs were not so demanding – initially his group simply needed to obtain large quantities of bacteria. The solution was to adapt a kitchen cream separator made by the Sharples company. The Sharples, as it was called in the lab, consisted of a tube that was the size of a thick cucumber – about 5 cm in diameter and 25 cm long. There was one problem: the tube was not tightly sealed, and tiny gaps in the apparatus meant that every time it was used, the room became full of an invisible aerosol of potentially lethal bacteria. Sharples was therefore placed in a specially constructed containment device that could be sterilised before opening.9 Even so, using the equipment safely was no easy matter. After centrifugation, the cake of bacteria that had accumulated at the bottom of the tube had to be removed – this was impossible to do cleanly and ‘one would see small flecks of white material fly in one direction or another’, recalled a lab member.10 All of the cake was handled with towels soaked in germicide and then heated at 65°C before it was studied further, in an attempt to reduce the risk to lab members.11 This messy and dangerous procedure so distressed the fastidious Avery that he would leave the lab when the Sharples was in action.
The group soon found that adding calcium chloride to the liquid transforming principle produced a white precipitate that contained most, if not all, of the transforming activity: adding white precipitate from smooth bacteria to a rough colony would transform it into a smooth colony. This white substance was very powerful – even at 1/1,000 dilution it could still transform a rough colony. At the beginning of 1941, MacLeod noted that the white precipitate contained both the polysaccharides typical of the smooth capsule and nucleic acids – DNA and its close relative, ribonucleic acid or RNA. When MacLeod added an enzyme that was known to destroy RNA, this had no effect on the transforming activity of the extract, strongly suggesting that RNA played no role in producing the power of the white material. In April 1941, in his six-monthly report to the Rockefeller Institute, Avery described the progress he and MacLeod had made and hinted at the potential implications:
This study is being continued with the hope that knowledge of this important cellular mechanism may lead to a better understanding of the principles involved in certain induced variations of living cells, not only of the pneumococcus, but also those of other biological systems.12
*
In the summer, MacLeod left the Institute and another young physician, Maclyn McCarty, joined the Avery group. By the end of November 1941, McCarty had shown that if he used an enzyme to remove the polysaccharide, the extract nevertheless retained its transforming activity, showing that – as expected – the polysaccharide was not involved. That apparently left just two possibilities: proteins or DNA.
In December 1941 the Japanese attacked Pearl Harbor and the US entered the war. The Avery group shifted its work towards more practical aspects of pneumonia as the disease began to appear among US troops. Nevertheless, McCarty continued with his research, and in January 1942 he found that if alcohol was added to the transforming principle, a stringy white material appeared tha
t contained 99.9 per cent of the transforming activity. It soon became apparent that this stringy stuff also contained most of the DNA that was present in the sample.
Two floors above Avery’s office was the laboratory of Alfred E. Mirsky, one of the world’s leading experts on nucleic acids. Mirsky gave the Avery lab some mammalian DNA extracted from the thymus gland, the traditional source of DNA, and they compared it with the white stringy stuff produced by alcohol precipitation of the transforming principle. The two substances seemed to be very similar. McCarty took an extract of transforming principle that had been treated with enzymes to remove both proteins and polysaccharides, and placed it in an ultracentrifuge. After spinning the sample for a few hours at 30,000 r.p.m., a gelatinous ‘pellet’ appeared at the bottom of the tube, containing the heaviest components of the extract. This contained all the transforming activity of the original solution and was apparently composed entirely of DNA.
In the summer of 1942, the suggestion that the transforming principle was made of DNA became stronger when McCarty and Avery showed that enzymes that destroyed transforming activity also affected Mirsky’s DNA samples, and enzymes that had no effect on DNA did not affect the activity of the extract. This should have led to great excitement, but Mirsky was unimpressed. As he explained to the Avery group, the transforming principle could not be made of DNA because nucleic acids were all alike. As their late Rockefeller Institute colleague Phoebus Levene had argued more than three decades earlier in his tetranucleotide hypothesis, the components of nucleic acids – the two kinds of base, the purines (adenine and guanine) and the pyrimidines (cytosine and thymine; thymine is replaced by uracil in RNA) – were present at similar levels. Although DNA was known to be a component of cell nuclei, its apparently boring nature meant that it was not thought to have biological ‘specificity’ – the term used at the time to describe the unique effects of a particular molecule. Proteins, in contrast, were extremely varied, and could be active even at very low levels. It was quite possible that despite all the treatments to remove proteins from their extracts, minute amounts of very powerful protein molecules remained, Mirsky explained.