If Colin MacLeod had happened to call in then, he might have remembered the startling china blue colorimetric reaction which flags up the presence of desoxyribose. By then, though, MacLeod had been sucked into his new job and had dropped any ambitions to continue hunting the transforming principle. And so the chemical nature of the peculiar white precipitate was destined to remain a mystery for several months more.
Upstairs, downstairs
Very few scientists will ever find themselves teetering on the brink of making ‘perhaps the pivotal discovery in biology of the century’. We might suppose that the brilliance of that discovery would light up all the landmarks on the trail that had led them there. However, disappointment awaits anyone hoping to hear from the lips of Maclyn McCarty how it feels to find something that will rewrite a large chunk of biology.
His book, Discovering that Genes are made of DNA, was published nearly forty years after the event. McCarty ‘tried as best I could to reconstruct the sequence of events’, but after ransacking his memory and his lab notebooks, he could not identify any ‘flash of insight’ when everything magically clicked into place. This gravity-defying penny was agonisingly slow to drop. He and Avery were biochemically well read, but only in their areas of interest. Neither was aware that, in 1865, the great Felix Hoppe-Seyler had obtained a fibrous precipitate on adding distilled water to the slimy mess produced by stirring pus into a strong salt solution. The precipitate reminded Hoppe-Seyler of myosin, the contractile protein in muscle, but he did not try to analyse it. During the 1930s, various researchers found a similar precipitate when water was added to strong-salt extracts of kidney and liver. It was named ‘plasmosin’, in the belief that it came from the cytoplasm of cells.
In early 1941, around the time that McCarty first visited Avery’s lab, plasmosin came to the attention of an energetic biochemist who knew all about muscle proteins. He isolated ‘plasmosin’ by winding the precipitated fibres around a wooden rod and pulling them out of the liquid, like a swirl of candy floss on a stick. On analysis, it was nothing like myosin and turned out to be something that would not surprise readers who remember that pus was the starting-point for this whole story. The discovery was made by Alfred E. Mirsky, a biochemist famous for his ‘rigorous chemical orientation’ – which meant that he was at the top of his field, but also that he had no patience for the chemically uneducated. By an astonishing coincidence, initially happy, Mirsky was another of the Rockefeller’s stars. And to crown it all, his labs occupied the top floor of the hospital, just two levels above Avery’s pneumococcal emporium.
Alfred Mirsky (see Figure 18.1) was born in New York, ten months into the twentieth century. He attended Harvard and then 40 per cent of the five-year medical course at Columbia University, abandoning medicine for a scholarship to Cambridge to study haemoglobin with the legendary Joseph Barcroft. Mirsky started his forty-year stint at the Rockefeller in 1922 as an assistant working on heart-muscle proteins. His interest in the structure of proteins led in 1936 to a sabbatical year in California with the protein guru Linus Pauling, when they wrote a landmark paper on how proteins unfold as they denature.
In 1940, Mirsky joined forces with Arthur Pollister, a zoologist at Columbia University, to tackle plasmosin. They showed that it came from the nucleus, by using a gentle extraction method with distilled water; under the microscope, intact nuclei could be seen sitting like marbles in the ruins of liver cells. The nuclei were filtered through very fine muslin to separate them from cellular debris, then concentrated by centrifugation and dissolved in strong sodium chloride.* Adding water caused a mass of ‘beautiful’ white fibres to precipitate. Because ‘plasmosin’ came from the nucleus, Mirsky and Pollister renamed it ‘chromosin’, in line with ‘chromatin’ and ‘chromosomes’.
On analysis, the fibres were found to consist of desoxyribonucleic acid bound to the protamine and histone proteins to which Albrecht Kossel had devoted his opus magnum. Mirsky and Pollister did not comment on the possible role of desoxyribonucleic acid in the nucleus, but they had inherited a belief from an old friend of Mirsky which locked them into the assumption that it could not do anything interesting. In noting that plasmosin must have a high molecular weight, they suggested that it was based on long chains of ‘desoxyribose tetranucleotides’. A couple of years after the death of Phoebus Levene, his tetranucleotide hypothesis was still very much alive. And before long, it would destroy a collaboration that could have been as beautiful as the silky precipitated fibres of desoxyribonucleic acid.
Home front
For Lieutenant Maclyn McCarty, 1942 began with uncertainty and frustration. He and Avery knew that the fibrous precipitate was the transforming principle, but still had no idea what it consisted of. If Avery was ever aware that MacLeod had detected desoxyribose a year earlier, he had forgotten. They would also have been thrown off the scent if they had peered into the neglected bottle labelled ‘Thymus nucleic acid’, which MacLeod had used to standardise his colorimetric test. Inside was a dirty brownish powder which looked nothing like the silky white transforming principle, but which had to be authentic because it had been provided by the late Phoebus Levene.
The impasse broke when someone spotted that two sets of experiments, which had begun on different continents and ended up in the same building in New York, produced white fibrous precipitates that looked remarkably similar. We shall never know who wondered if they might be identical, because McCarty, the only one to write anything down, was frustratingly vague: ‘By January 1942, we were aware that the transforming principle was usually associated with the stringy, fibrous precipitate . . . and before long we realised that most of the DNA in our extracts occurred in this same fibrous fraction . . . Sometime in the late winter or early spring of 1942, Mirsky gave us some preparations of his mammalian DNA.’
On 30 March 1942, McCarty set up a milestone experiment – ‘to determine the relationship of the “stringy” material to the thymus nucleic acid and also to the transforming principle’. He harvested the ‘stringy material’ using Mirsky’s method of stirring with a wooden rod, confirmed that it could transform R pneumococci – and (with the same colorimetric test that MacLeod had used) showed that it was loaded with desoxyribose.
They reached the same conclusion after spending several weeks in a room in the hospital basement with a massive new ultracentrifuge which spun its samples so fast (50,000 rpm) that it could separate substances in solution. Those with the highest molecular weight were dragged outwards most rapidly; the bands of migrating molecules could be tracked in real time with an ingenious optical interference system, and their molecular weights could be calculated from the speed of sedimentation. This showed that the transforming principle weighed in at between 500,000 and 1 million – around the value estimated for desoxyribonucleic acid. And when they dug the gelatinous pellet out of the bottom of the centrifuge chamber after spinning for several days, they found that it contained virtually all the transforming activity in the original sample, and that it consisted of desoxyribonucleic acid – or DNA, as we can now call it.
The ultracentrifuge experiments were still running when Avery prepared his routine report for April 1942, the penultimate of his thirty-five years at the Rockefeller. He decided to keep the chemical identity of the transforming principle under wraps until the evidence was rock-solid, and mentioned McCarty only briefly; an external observer might have assumed that the transformation research, now silent for seven years, was dead in the water. In fact, McCarty was going flat out, swept along by the ‘increasing tempo of the research’ and the mounting excitement because ‘all roads seemed to lead to DNA’. He had found that enzymes which broke down protein, Type III SSS or RNA affected neither authentic DNA (prepared from animal tissues by Mirsky) nor the transforming principle.
In July, Avery and McCarty joined forces with the group upstairs to see whether authentic DNA isolated by Mirsky from Type III S pneumococci could transform R36 into virulent Type III. McCarty presented Mirsky wit
h heat-killed Type III S harvested from 75 litres of culture medium, and Mirsky got to work with his salt extraction method. The yield was low, because pneumococci were tougher than liver cells, but he obtained small amounts of a fibrous white precipitate. Some of this remained on the top floor, where Mirsky showed that it contained DNA and some protein; the rest went down to Avery’s lab, where McCarty confirmed that it had the power to transform.
This was still not enough for Avery, who wanted independent analyses by ‘physical chemist friends’. McCarty spent July and August with the trusty Sharples, preparing highly purified transforming principle on a near-industrial scale. After extraction and purification, 200 litres of incubation medium bursting with Type III pneumococci yielded just 45 milligrams of transforming principle.† This was enough for the friendly physical chemists to confirm in November 1942 that the transforming principle had the same nitrogen and phosphorus contents as DNA.
Through the winter, McCarty worked hard to refine the recipe, and by mid-February 1943 could squeeze 100 milligrams of transforming principle/DNA out of 300 litres of incubation medium. The end product was extraordinarily potent: pneumococci could be transformed with just 3 thousand-millionths of a gram of the precipitate, roughly one part in 600 million of the harvested fibres.
Grand finale
In April 1943, Oswald Avery submitted his last annual report just twelve weeks before he was due to slip into the afterlife of retirement. His swansong could have been a celebration of his lab’s glories during the previous thirty-five years; instead, he looked forward to the post-Avery era, in which lobar pneumonia would be eclipsed by viral pneumonia and other infections of the new age.
The report’s top spot, however, was a ten-page account of ‘the chemical nature of the substance inducing transformation of specific types of pneumococci’. Until very recently, this had been the least productive topic of Avery’s career, but now – just in time – he could report a major breakthrough.
He first set the scene: ‘Biologists, especially geneticists, have long attempted by chemical means to induce in higher organisms predictable and specific changes which thereafter could be transmitted in series as hereditary characters.’ This was exactly what transformation achieved and now, ‘after numerous experimental approaches carried out over a period of years’, he and McCarty had reached the extraordinary conclusion that the transforming principle ‘might be desoxyribonucleic acid’. The implications were staggering, but Avery confined himself to the lowly version of life in bacteria:
Assuming that desoxyribonucleic acid and the active principle are one and the same substance, the transformation represents a change that is chemically induced and specifically directed by a known chemical compound . . .
The transforming principle – a nucleic acid – has been likened to a gene . . . If the present studies are confirmed, then nucleic acid of this type must be regarded not merely as structurally important, but as functionally active in determining the biological activities and specific characteristics of pneumococcus cells.
Professional geneticists might have cut to the chase and simply said that ‘these experiments strongly suggest that genes are made of DNA’. But Avery was cautious and on unfamiliar terrain and stuck to the conclusions that the evidence permitted. He finished by quoting a British physiologist, B.J. Leathes, who in 1926 had speculated that the ‘vital importance’ of proteins might be rivalled by the nucleic acids – which were, after all, major components of the chromosomes. Now, it seemed, the pneumococcus might be the first living organism to give substance to that speculation.
Avery’s last report contained something that any research institution on the planet would have been proud to claim: the first ever statement that genes were likely to consist of DNA. There is no indication that Tom Rivers, Director of the Rockefeller Institute Hospital, realised its significance then, and he made no comment about it in his biography nineteen years later, by which time the double helical structure of DNA and the genetic code had been worked out. Rivers’s biography, transcribed from conversations recorded in the months before his death in 1962, dwells in detail on the Rockefeller’s many achievements – but ‘DNA’ does not even figure in the index.
Rivers was not the only big name at the Rockefeller to be unmoved by Avery’s revolutionary finding – nor the only person guilty of the crime of omission. In his report, Avery had failed to mention a collaborator who had been instrumental in identifying the transforming principle as DNA – and who was now nursing a grievance and planning revenge.
Letter home
In mid-May, with just seven weeks of gainful employment left, Avery realised that retirement was not what he wanted. On the evening of 13 May, he sat down to write to Roy, younger brother and Professor of Microbiology at Nashville, to update him with progress and to drop a bombshell. He broke off that night, having taken three pages to say that he would not be moving to Nashville after all. It took another two weeks for him to finish the letter (five pages more) and to explain why: not cold feet, but an acute case of excitement.
It had been gruelling voyage of discovery: ‘Try to find in that complex mixture the active principle! Some job – full of heartaches and heart-breaks.’ Finally, though, they had pinned down the substance that conferred ‘aristocratic distinctions’ (i.e. the ability to kill mice) on dull pneumococci, and had proved that it ‘conforms very closely to the theoretical values of pure desoxyribonucleic acid’. What an unlikely candidate: ‘Who could have guessed it?’ Nonetheless, the induction of ‘predictable and irrevocable changes in cells’ was ‘something that has long been the dream of geneticists’. Avery added, ‘Maybe a gene.’
It had been ‘good fun and lots of work’, but Avery was not yet done. He had to polish off the remaining ultracentrifuge experiments before heading on holiday to Maine so that he would ‘come back refreshed & try to pick up the loose ends & write up the work’. After that, ‘Someone else can work out the rest.’
So it came to pass. The ultracentrifuge experiments proved without doubt that a solution of the purified transforming principle contained just one substance with a very high molecular weight, and that this was DNA. And on 1 July 1943, Dr Oswald Avery underwent a barely noticed transformation of his own, and became a Member Emeritus of the Rockefeller Institute.
By now, Colin MacLeod was once more taking an active interest from across town. He and McCarty thought that the ultracentrifuge experiments had tied up the last loose end but Avery continued to dither, possibly because he too had been a close friend of Phoebus Levene and had been put off DNA by the tetranucleotide hypothesis.
Things came to a head in early summer, when the trio made a ‘pilgrimage’ to seek advice from two wise old men at the Rockefeller’s research base in Princeton. John Northrop, famous for isolating enzymes and detecting mustard gas, and Wendell Stanley, who had crystallised viruses and worked out their structure, were both Nobel laureates in waiting. Both were intrigued by the notion that DNA could permanently alter the characteristics of bacteria, and told Avery to press ahead with publication. On the train home, an exasperated MacLeod asked, ‘What else do you want, Fess? What more evidence can we get?’
‘Fess’ could not provide a convincing answer. Shortly after, he and McCarty sat down separately to write up ‘the most interesting and portentous biological experiment of the twentieth century’. Avery, by now relaxing on Deer Isle, tackled the introduction and discussion. Back at base, McCarty filled in the methods, results and ‘loose ends’ such as taking a nice photograph to bring out the differences between the ‘smooth’ and ‘rough’ colonies of S and R pneumococci.
Avery made two executive decisions. Irrespective of who had done what, alphabetical order and time associated with the project were what mattered; Avery was therefore first author on the paper, followed by MacLeod, who bumped McCarty into last place. And despite all the contributions from the lab upstairs – where the notion of DNA might well have originated – Avery removed all mention of Mir
sky, except for a one-line acknowledgement as the supplier of DNA.
After much revision and polishing, Avery handed the manuscript to Peyton Rous, the editor of the Journal of Experimental Medicine, on 1 November 1943. Rous, another future Nobel laureate, was famously even more pernickety than Avery. He swiftly returned the typescript, heavily embellished with comments and suggestions. Avery and McCarty responded in kind, and on 1 December, Rous accepted the revised paper and dispatched it to the printer, to be published in February 1944.
Little bang
In the interim, Avery was cajoled by his co-authors into reporting their findings at the Institute’s regular Friday afternoon seminar on work in progress. Fred Griffith would have hated having to do this, and Avery – despite his long list of invited lectures – did not seem that keen either.
Nonetheless, he presented ‘Studies on the chemical nature of the substance inducing transformation of pneumococcal types’, by Drs Avery, MacLeod and McCarty, on 10 December 1943. Later, McCarty remembered a full room, with standing room only at the back – a typically slick and erudite Avery presentation – a ‘resounding round of applause’ – and then a deafening silence. The only reaction came from Michael Heidelberger, who chipped in some supportive but non-specific comments. After another long silence, the meeting broke up, leaving the chairman strangely unsettled by the instinct they had all been ‘witnesses to something important’.
Unravelling the Double Helix Page 23