Neanderthal Man (6 page)

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 suggesting 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.

During my time in East Germany, I had developed some understanding of the sensitivities of people living under socialism. In particular, I knew that the museum curator and other museum officials who hosted my visit would be very disappointed with just a perfunctory expression of gratitude at the end of my paper. I wanted to do the right thing, so after talking to Rosti and conferring with Stephan Grunert, a young and ambitious East German Egyptologist whom I had befriended in Berlin, I decided to publish my first paper on mummy DNA in an East German scientific journal. Struggling with my high school German, I wrote up my findings, including photographs of the mummy itself and of the tissue stained for DNA. In the meantime, I had also extracted DNA from the mummy. This time, the extracts contained DNA that I could demonstrate in a gel, and I included a picture of such an experiment in the paper. Most of the DNA was degraded, but a small fraction of it was several thousand nucleotides long, similar in length to the DNA one could extract from fresh blood samples.

This, I wrote, seemed to indicate that some DNA molecules from ancient tissues might well be large enough to allow the study of individual genes. I speculated wildly about what might be possible if DNA from ancient Egyptian mummies could be systematically studied. The paper ended on a hopeful note: “Work over the next few years will show if these expectations will be fulfilled.” I sent the manuscript to Stephan in Berlin. He fixed up my German, and in 1984 the article appeared in
Das Altertum,
a journal published by the East German Academy of Sciences.
{3}
And nothing happened. Not a single person wrote to me about it, much less asked for a reprint. I was excited, but no one else seemed to be.

Having realized that the world at large did not make a habit of reading East German publications, I had written up similar results from the fragment of the mummified head of a man and, in October of the same year, had sent them to a Western journal that seemed appropriate—the
Journal of Archaeological Science.
But here the frustration turned out to be the unbelievable slowness of the journal, even compared with the delay my manuscript had experienced in East Germany, where it needed to be fixed up linguistically by Stephan and then presumably scrutinized by the political censors. This was, I felt, a reflection of the glacial speed with which the disciplines concerned with ancient things were moving. The
Journal of Archaeological Science
finally published my paper at the end of 1985
{4}
—
by which time the results it described had been largely overtaken by events.

The next step—now that I had some mummy DNA—was obvious. I needed to clone it in bacteria. So I treated it with enzymes that make the ends of the DNA amenable to being joined to other pieces of DNA, mixed it with a bacterial plasmid, and added an enzyme that joins DNA fragments together. If successful, this would create hybrid molecules in which pieces of DNA from the mummy were joined to the plasmid DNA. When these plasmids were introduced into bacteria, they would not only allow the hybrid molecules to replicate to high copy numbers in bacterial cells but would also make the bacteria resistant to an antibiotic I would add to my culture medium, so that the bacteria would survive only if they contained a functioning plasmid. When seeded on growth plates containing the antibiotic, colonies of bacteria would appear if the experiment was successful. Each such colony would derive from a single bacterium that now carried  one particular piece of mummy DNA. To check on my experiment, I did controls—an essential thing in any laboratory experiment. For example, I repeated the exact process in parallel but added no mummy DNA to the plasmid, and also repeated the process but added modern human DNA. After making the bacteria take up the DNA solutions from these experiments, I plated them on agar plates containing the antibiotic and put them in an incubator at 37°C overnight. The next morning I opened the incubator and, with anticipation, inhaled the puff of moist air smelling of rich culture media. The plate with the modern DNA yielded thousands of colonies, so many that it was almost totally covered with bacteria. This showed that my plasmid had worked: the bacteria survived because they had taken up the plasmid. The plate where no DNA had been added to the plasmid yielded hardly any colonies, indicating that I did not have DNA from some unknown source in my experiment. The experiment itself, where I had added the DNA from the Berlin mummy, yielded several hundreds of colonies. I was ecstatic. I had apparently replicated 2,400-year-old DNA! But could it have come from bacteria in the child’s tissues, rather than from the child herself? How could I show that at least some of the DNA I had cloned in the bacteria was human?

I needed to determine the DNA sequence from some of the DNA in order to show that it was human rather than bacterial. But if I merely sequenced random clones, they would be likely to contain DNA sequences that could have come either from the human genome—which in 1984 was not yet decoded, except for some tiny parts that had been sequenced with great effort—or from some microorganism whose DNA sequences were even less likely to be known. So instead of sequencing random clones, I needed to identify some clone of interest. The answer lay in a technique whereby one could identify clones that carried DNA similar in sequence to something one wanted to find. This technique involved transferring some of the bacteria from each of hundreds of colonies to cellulose filters, where the bacteria were broken open and their DNA were bound to the filter. I then used a radioactively labeled piece of DNA, a “probe,” that was single-­stranded and then hybridized to complementary sequences from the single-stranded DNA on the filters. I chose to use a piece of DNA that contains a repeated DNA element—the so-called
Alu
element—of about 300 nucleotides that occurs almost a million times in the human genome and in no organisms besides humans, apes, and monkeys. In fact, these
Alu
elements are so numerous that more than 10 percent of the human genome is made up of them. If I could find an
Alu
element among my clones, it would  show that at least some of the DNA I had extracted from the mummy came from a human being.