by Frank Ryan
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 goiter,” 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's 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 research 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 sulfonamide 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 agonizing 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 pediatrician, 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 liters, 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 polymerized 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 fibers, 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 May 26, 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.
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 skeptical 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. Muller 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 Trust 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 [skeptical] influence was spread to a wide audience.”
In a new series of extractions, with stringent quality checking, Avery attempted to confound his critics. In 1946, McCarty left the laboratory, which was left in the hands of, among 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 percent, 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 skepticism,” 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 learned 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 learned 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 sulfur 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 makeup but no sulfur, meanwhile the amino acids that make up proteins contain sulfur 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 sulfur. 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 percent of the radioactive sulfur was left behind in the viral coats when the virus infected the cell, and 85 percent 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 skeptical 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.
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's 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 behavior. 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. Afterward 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's 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.