by Pbo, Svante
Pettersson asked me to join his group’s efforts to study a protein encoded by an adenovirus, a virus that causes diarrhea, cold-like symptoms, and other unpleasant features of our lives. It was thought that this viral protein became bound by the transplantation antigens inside the cell, so that, once transported to the cell surface, it could be recognized by immune-system cells, which would then become active and kill other infected cells in the body. Over the next three years, I and the others working on this protein came to realize that this idea of what the protein did was utterly wrong. We found that rather than becoming a hapless target of the immune system, the viral protein seeks out the transplantation antigens inside the cell, binds to them, and blocks their transport out to the cell surface. Since the infected cell thus ends up having no transplantation antigens on its surface, the immune system cannot recognize that it is infected. This protein camouflages the adenovirus, so to speak. In fact, it leads to the creation of a cell within which the adenovirus can probably survive for a long time, perhaps even as long as the infected person lives. That viruses could foil the immune system of their hosts in this way was a revelation, and our work resulted in a number of high-profile papers in the best journals. Indeed, it turns out that other viruses, too, use similar mechanisms to evade the immune system.
This was my first taste of cutting-edge science, and it was fascinating. It was also the first (but not the last) time I saw that progress in science often entails a painful process of realizing that your ideas and those of your peers are wrong, and an even longer struggle to persuade your closest associates and then the world at large to consider a new idea.
But somehow, in the midst of all the biological excitement, I could not quite shake off my romantic fascination with ancient Egypt. Whenever I had time, I went to lectures at the Institute of Egyptology, and I continued to take classes in Coptic, the language of pharaonic Egypt as spoken during the Christian era. I befriended Rostislav Holthoer, a jovial Finnish Egyptologist with an immense capacity for friendships across social, political, and cultural boundaries. During long dinners and evenings at Rosti’s home in Uppsala in the late 1970s and early ’80s, I often complained that I loved Egyptology but saw little future in it, while I also loved molecular biology, with its apparently boundless promise of advances in the welfare of humankind. I was torn between two equally alluring career paths—a conundrum no less painful because it was doubtless viewed without much sympathy as the fretting of a young man faced with nothing but good choices.
But Rosti was patient with me. He listened when I explained how scientists could now take DNA from any organism (be it a fungus, a virus, a plant, an animal, or a human), join it to a plasmid (a carrier molecule made of DNA from a bacterial virus), and introduce the plasmid into bacteria, where it would replicate along with its host, making hundreds or thousands of copies of the foreign DNA. I explained how we could then determine the sequence of the foreign DNA’s four nucleotides and find differences in the sequences between the DNAs of two individuals or two species. The more similar two sequences were—that is, the fewer the number of differences between them—the more closely related they were. In fact, from the number of shared mutations we could infer not only how the particular sequences had evolved from common ancestral DNA sequences over thousands and millions of years but also approximately when those ancestral DNA sequences had existed. For example, in a 1981 study the British molecular biologist Alec Jeffreys analyzed the DNA sequence of a gene that encodes a protein in the red pigment in the blood of both humans and apes and deduced when the genes began evolving independently in humans and apes. This, I explained, could soon be done for many genes, from many individuals of any species. In this way, scientists would be able to determine how different species were related to one another in the past, as well as when they began their separate histories, with much greater accuracy than was possible from the study of morphology or fossils.
As I explained all this to Rosti, a question gradually arose in my mind. Would this kind of investigation necessarily be restricted to DNA from blood samples or tissues from humans and animals that live today? What about those Egyptian mummies? Could DNA molecules have survived in them—and could they, too, be joined to plasmids and made to replicate in bacteria? Could it be possible to study ancient DNA sequences and thereby clarify how ancient Egyptians were related to one another and to people today? If that could be done, then we could answer questions that no one could answer by the conventional means of Egyptology. For example, how are present-day Egyptians related to Egyptians who lived when the Pharaohs ruled, some 2,000 to 5,000 years ago? Did great political and cultural changes, such as the conquest by Alexander the Great in the fourth century BCE, or by the Arabs in the seventh century AD, result in replacement of a large part of the Egyptian population? Alternatively, were these just military and political events that caused the native population to adopt new languages, new religions, and new ways of life? In essence, were the people who lived in Egypt today the same as those who built the pyramids, or had their ancestors mixed so much with invaders that modern Egyptians were now completely different from their country’s ancient population? Such questions were breathtaking. Surely they must have already occurred to someone else.
I went to the university library and searched in journals and books but found no report of any isolation of DNA in ancient materials. No one seemed even to have tried to isolate ancient DNA. Or if they had, they had not succeeded, because if so, surely they would have published their findings. I talked to the more experienced graduate students and postdocs in Pettersson’s lab. Given how sensitive DNA is, they argued, why would you expect it to last for thousands of years? The conversations were discouraging, but I didn’t give up hope. In my forays into the literature, I had found articles whose authors claimed to have detected proteins in hundred-year-old animal hides in museums—proteins that could still be detected by antibodies. I had also found studies claiming to have detected, under the microscope, the outlines of cells in ancient Egyptian mummies. So something did seem to survive, at least sometimes. I decided to do a few experiments.
The first question seemed to be whether DNA could survive for long in tissues after death. I speculated that if the tissue became desiccated, as was the case when a mummy was prepared by the embalmers in ancient Egypt, then DNA might well survive for a long period since the enzymes that degrade DNA need water to be active. This would be the first thing to test. So in the summer of 1981, when not too many people were around in the lab, I went to the supermarket and bought a piece of calf liver. I glued the receipt from the store onto the first page of a new lab book that I would use to record these experiments. I labeled the book with my name but nothing else, since I had decided to keep my experiments as secret as possible. Pettersson might forbid me to pursue them, if they struck him as an unnecessary distraction from the intensely competitive study of the molecular workings of the immune system that I was supposed to be working on. And, in any case, I wanted to keep all this under wraps to spare myself the ridicule of my lab colleagues in the likely event of failure.
To somewhat imitate ancient Egyptian mummification, I decided to artificially mummify the calf liver by sequestering it in an oven in the lab heated to 50°C. The first effect of this was that the secrecy of my project was compromised. By the second day, the repugnant smell elicited considerable comment, and I had to reveal my project before someone found the liver and disposed of it. Fortunately, the smell decreased as the desiccation progressed, and neither the smell nor word of what was putrefying in the lab made it to my professor.
After a few days, the liver had become hard, blackish-brown, and dry—just like an Egyptian mummy. I proceeded to extract DNA from it, with immediate success. The DNA was in small pieces of a few hundred nucleotide pairs instead of the many thousands of nucleotide pairs typical of DNA extracted from fresh tissue, but there was still lots of it. I felt vindicated. It was not totally ridiculous to think that DNA could survive
in a dead tissue—at least for some days or weeks. But what about thousands of years? The obvious next step was to try performing the same stunt with an Egyptian mummy. Now my friendship with Rosti came in handy.
Rosti had been primed by my fretting about Egyptology and molecular biology and was happy to abet my attempt to take Egyptology into the molecular age. The small university museum of which he was the curator had some mummies, and he consented to my request to sample them. He was, of course, not about to let me cut them open and remove their livers. But if a mummy was already unwrapped and its limbs had broken off, Rosti allowed me to remove small pieces of skin or muscle tissue from the area where the mummy had already been broken to try my DNA extraction. Three such mummies were available. As soon as I put the scalpel to what had once been the skin and muscles of a person who existed some 3,000 years ago, I realized that the texture of the tissue was different from that of the calf liver I had baked in the oven. The liver had been hard and a bit tough to slice up, whereas the mummies were brittle and their tissues tended to crumble to brown powder when cut. Undeterred, I submitted them to the same extraction procedure I had performed on the liver. The mummy extracts differed from the liver extract in that they were as brown as the mummies themselves, whereas the liver extract had been as clear as water. And when I looked for DNA in the mummy extracts by letting them migrate in a gel in an electric field and staining them with a dye that would fluoresce pink in ultraviolet light if it had bound to DNA, I saw nothing except the brown stuff, which indeed fluoresced in the ultraviolet light, but blue instead of pink, not what was expected if it was DNA. I repeated this process on the two other mummies. Again, there was no DNA; nothing but an undetermined brown substance had ended up in the extracts that I had hoped would contain DNA. My lab colleagues seemed to be right: How could the fragile DNA molecules survive for thousands of years, when even inside a cell it needed constant repair in order not to decompose?
I hid my secret lab book at the bottom of my desk drawer and returned to the virus that tricked the immune system with its clever little protein—but I could not get the mummies out of my mind. How could it be that others had seen what seemed to be remains of cells in some mummies? Perhaps that brown stuff was actually DNA, chemically modified in some way so as to look brown and fluoresce blue in UV light. Perhaps it was naïve to expect DNA to survive in every mummy. Perhaps one needed to analyze many mummies to find the rare good ones. The only way to find out was to convince museum curators to sacrifice pieces of many mummies in the perhaps vain hope that one of them would produce ancient DNA, and I had little idea how to get their permission. It seemed I needed a quick and minimally destructive way to analyze a lot of mummies. My medical education gave me a clue. Very small pieces of tissue, such as those removed with a biopsy needle from a suspected tumor, for example, could be fixed and stained and then studied under a microscope. The level of discernible detail was generally exquisite, allowing a trained pathologist to distinguish normal cells in the lining of the intestine or in a prostate or mammary gland, on the one hand, from cells that had started to change in ways that suggested they were early tumors, on the other. Moreover, there were dyes specific for DNA that could be applied to microscope slides to show whether DNA was present. What I needed to do was to collect small samples from a large number of mummies and analyze them by microscopy and DNA staining. The largest numbers of mummies, obviously, were to be found in the largest museums. But the curators could be expected to be skeptical about letting a slightly overexcited student from Sweden remove even tiny pieces for what seemed a pie-in-the-sky project.
Again, Rosti proved sympathetic; he pointed out that there was one large museum that had huge mummy collections and might be willing to cooperate. It was the Staatliche Museen zu Berlin, a complex of museums in East Berlin, the capital of the German Democratic Republic. Rosti had spent many weeks there working on its ancient Egyptian pottery collection. That Rosti came to East Germany as a professor from Sweden, which at the time was perceived as a country that attempted to find a “third way” between capitalism and communism, probably helped him gain permission to work in the museum. But it was his ability to develop warm friendships across borders that then allowed him to become close friends with several of the curators at the museum. And thus, in the summer of 1983, I found myself on a train that was driven onto a ferry in southern Sweden to arrive the next morning in communist East Germany.
I spent two weeks in Berlin. Every morning I had to pass several security controls to enter the storage facility of the Bode Museum, located on an island in the River Spree near the heart of Berlin. Almost forty years after the war, the museum was still clearly marked by it. On several of the facades, I could see bullet holes in the walls around the windows that had been targeted by machine guns as Berlin fell to the Soviet Army. On the first day, when I was taken to see the prewar Egyptological exhibition, I was handed a hard hat like the ones used by construction workers. It soon became clear why. The exhibition hall had huge holes in the roof from artillery shelling and bombs. Birds were flying in and out, and some were nesting in the pharaonic sarcophagi. Everything that was not of durable material was now sensibly stored elsewhere.
Over the following days, the curator in charge of Egyptian antiquities showed me all his mummies. For a few hours before lunch in his dusty run-down office I removed small snippets of tissues from mummies that were unwrapped and broken. Lunch was a long affair that required exiting through all the security checks to reach a restaurant across the river, where we ate greasy food that needed lubrication with copious amounts of beer and schnapps. Back in the collections, we spent the afternoon over more schnapps, lamenting the fact that the only foreign travel the curator had ever been allowed was visits to Leningrad. It soon became clear that my host dreamed of visiting the capitalist West and that if he got the chance he would probably defect. To provide some perspective on working life in the West I suggested, as diplomatically as I could, that in the west if you drank on the job, you were likely to be fired—an unknown concept in socialism. Such sobering thoughts seemed not to detract from the allure of the opportunities my host imagined to abound in capitalism. In spite of the hours spent on these theoretical discussions, I managed to collect more than thirty mummy samples to take back to Sweden.
At Uppsala, I prepared the samples for microscopy by soaking them in a salt solution to rehydrate them, then mounting them on glass slides and staining them with dyes that permitted visualization of cells. Then I looked for preserved cells in the tissues. I did this work on weekends and late at night, so as not to let it be widely known what I was doing. As I peered through the microscope, the appearance of the ancient tissues depressed me. In muscle sections, I could barely discern the fibers, let alone any traces of cell nuclei where DNA might be preserved. I was almost despairing, until one night I looked at a section of cartilage from a mummified outer ear. In cartilage, as in bone, cells live in small holes, or lacunae, inside the compact, hard tissue. When I looked at the cartilage, I saw what appeared to be the remains of cells inside the lacunae. Excited, I stained the section for DNA. My hands were trembling as I put the slide under the microscope. Indeed, there was staining within the cellular remains in the cartilage (see Figure 2.1). There seemed to be DNA preserved inside!
With renewed energy, I went on to process all of the remaining samples from Berlin. A few looked promising. In particular, the skin from the left leg of the mummy of a child showed what were clearly cell nuclei. When I stained a section of the skin for DNA, the cell nuclei lit up. Since this DNA was in the cell nuclei, where the cellular DNA is stored, it could not possibly be from bacteria or fungi because such DNA would appear at random in the tissue where the bacteria or fungi were growing. This was unambiguous evidence that DNA from the child herself was preserved. I took many photos through the microscope.
Figure 2.1. Microscopic picture of cartilage tissue from an Egyptian mummy from Berlin. In some lacunae, cell remains light up sug
gesting that DNA may be preserved. Photo: S. Pääbo, Uppsala University.
I found three mummies with staining of the cell nuclei showing the presence of DNA. The child seemed to have the largest number of well-preserved cells. But now doubt started to gnaw at me. How could I be sure that this mummy was really old? Modern corpses were sometimes falsified to look like ancient Egyptian mummies so that the perpetrators might earn a few dollars from tourists and collectors. Some of these mummies might later be donated to museums. The staff of the museum in Berlin had been unable to give me any records of the provenance of this particular mummy, perhaps because the relevant parts of the catalog had been destroyed in the war. The question of its age could be resolved only through carbon dating. Fortunately, Göran Possnert, an expert on carbon dating, worked at Uppsala University. He used an accelerator to determine the ages of tiny samples of ancient remains by measuring the ratios of carbon isotopes present. I asked him how much it would cost to date my mummy, worrying that I would not be able to afford it on my meager student stipend. He took pity on me and offered to date it for free, considerately not even mentioning the price, doubtless because it would have been well out of my range. I delivered a small piece of the mummy to Göran and waited to hear the results. For me, this exemplified one of the most frustrating situations in science, when your work depends crucially on the work of someone else and you can do nothing to expedite it—just wait for a phone call that seems to never come. But finally, a few weeks later, I got the call I had been waiting for. The news was good. The mummy was 2,400 years old; it dated from about the time of the Alexandrian conquest of Egypt. I drew a sigh of relief. First I went out and bought a big box of chocolates, which I delivered to Göran. Then I started to think about publishing my findings.
