by Pbo, Svante
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.
I got a piece of a gene I’d studied in the lab that contained an Alu element, incorporated radioactivity in it, and hybridized it to my filters. Several of the clones took up the radioactivity, as one would expect if some of the DNA was human. I picked the most strongly hybridizing clone. It contained a piece of DNA consisting of about 3,400 nucleotides. With the help of Dan Larhammar, a graduate student who was the master of DNA sequencing in our group, I sequenced a part of the clone. It did indeed contain an Alu element. I was very happy. There was human DNA among my clones, and it could be cloned in bacteria.
As I was grappling with my sequencing gels in November 1984, a paper appeared in Nature that was of great relevance for me. Russell Higuchi, who worked at UC Berkeley with Allan Wilson, the primary architect of the out-of-Africa theory of modern human origins and one of the most famous evolutionary biologists of the time, had extracted and cloned DNA from the 100-year-old skin of a quagga, an extinct subspecies of zebra that had existed in southern Africa until about a hundred years ago. Russell Higuchi had isolated two fragments of mitochondrial DNA and shown that the quagga was, as expected, more closely related to zebras than to horses. This work inspired me greatly. If Allan Wilson was studying ancient DNA, and if Nature considered an article about 120-year-old DNA interesting enough to publish, then surely what I was doing was neither crazy nor uninteresting.
For the first time, I sat down to write a paper of my own that I believed many people in the world would be interested in. Inspired by Allan Wilson’s example, I wrote it for Nature. I described what I had done with the mummy from Berlin. One of my first references was to the paper that had appeared in the East German journal. However, before I sent the manuscript off to London, where Nature had its office, there was something I needed to do. I needed to talk to my thesis adviser, Per Pettersson, and show him the manuscript, now ready to submit. With some trepidation, I entered his office and told him what I had done. I asked if he might perhaps want to be a co-author with me on the paper, in his capacity as my adviser. Obviously, I had underestimated the man. Rather than scolding me for what could have been seen as misappropriation of research funds and valuable time, he seemed amused.
He promised to read the manuscript and said that, no, obviously he should not be the co-author of work that he hadn’t even been aware of.
A few weeks later, I received a letter from Nature, with a promise from the editor to publish my manuscript if I could respond to some minor comments from reviewers. Shortly thereafter, the proofs arrived. At that point, I thought about how to approach Allan Wilson—a demigod, in my view—to ask if I might work with him at Berkeley after my PhD defense. Not knowing exactly how to broach this topic, I mailed him a copy of the proofs without any comment whatsoever, thinking he might appreciate seeing the paper before it appeared. I thought that I would then later write to him about job opportunities in his laboratory. Nature progressed rapidly toward publication and even solicited a cover illustration of a mummy with DNA sequences artfully wrapped around it. Even more rapidly, I received a response from Allan Wilson, who addressed me as “Professor Pääbo”—this was before both the Internet and Google, so there was no obvious way for him to find out who I was. The rest of his letter was even more amazing. He asked if he could spend his upcoming sabbatical year in “my” laboratory! This was a hilarious misunderstanding, resulting from my insecurity about knowing what to write to him. I joked with my lab mates that I would have Allan Wilson, perhaps the most famous molecular evolutionist of the time, wash gel plates for me for a year. Then I settled down to write him back—explaining that I was not a professor, not even a PhD, and certainly did not have a lab where he could spend his sabbatical. Rather, I wondered if there might be a chance for me to spend my postdoc in his Berkeley lab.
Chapter 3
Amplifying the Past
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Allan Wilson wrote me a gracious reply, inviting me to work in his group as a postdoctoral fellow. This would prove to be a turning point in my career. Once I had earned my PhD, I had three choices: finishing my medical studies at the hospital (a boring prospect after the excitement I had just experienced); following up on my successful PhD work on viruses and immune defense at some world-class lab; or accepting Allan’s offer to spend my postdoc trying to retrieve ancient genes. Most of my peers and the professors with whom I discussed these choices suggested the second alternative, arguing that my interest in mummy DNA was a quaint hobby but ultimately a distraction from the serious work on which a solid future in research could be built. I, of course, was tempted by the third option but still felt hesitant, wondering whether mainstream research in virology, with “molecular archaeology” as a hobby, was not the more realistic course. What changed it all was the 1986 Cold Spring Harbor Symposium.
Cold Spring Harbor Laboratory, on Long Island, New York, is the Mecca of molecular genetics. The laboratory organizes many well-respected meetings, in particular a yearly Symposium on Quantitative Biology. Thanks to my paper in Nature,{5} I was invited to the 1986 symposium, where I presented, for the first time, a lecture on my mummy work. As if this were not already exciting enough, in the audience were many people I knew only from the literature, including Allan Wilson himself and Kary Mullis, who in the same session described the polymerase chain reaction. The PCR was a real technical breakthrough, since it did away with most of the cumbersome cloning of DNA in bacteria, and it was immediately obvious to me that it might be used to study ancient DNA. In principle, the PCR would enable me to target and multiply DNA segments of interest even if just a few survived. In fact, referring to my presentation, Kary ended his talk by noting that the PCR would be ideally suited for studying mummies! I could hardly wait to get back to the lab and try it out.
The meeting was electrifying in another way as well: it was the first time that a coordinated and publicly funded effort to sequence the entire human genome was on the agenda. Although the meeting made me feel much like the novice I was, I was elated to be present as the big guys discussed the millions of dollars, the thousands of machines, and the new technologies needed for this endeavor. In lively debates, some well-known scientists denounced the proposed project as technically impossible, unlikely to yield interesting results, and likely to divert valuable funding from more worthwhile research by small groups led by single investigators. To me, it was all very exciting; I wanted to be part of the genomic adventure.
Unlike most of the testosterone-fueled, high-powered scientists dominating the meeting, Allan Wilson was low-key and soft-spoken, the personification of what I imagined a Berkeley don to be. A long-haired New Zealander with a warm gaze, he made me feel comfortable and encouraged me to follow my inclinations and do what seemed most promising to me. The meeting with him helped me overcome my indecision and I told him I wanted to come to Berkeley.
There was a hitch, though. Unable to come to “my” laboratory for his sabbatical, Allan had decided to spend the year at two labs in England and Scotland, which meant that I would have to find something else to do in the meantime. As a part of my PhD project, I had worked for a few weeks in the Zurich laboratory of Walter Schaffner, a famous molecular biologist who had discovered “enhancers,” crucial DNA elements that help drive the expression of genes. Walter, always full of unabashed enthusiasm for unorthodox ideas and projects, now invited me to spend the year in his lab working on ancient DNA. He was particularly interested in the thylacine, an extinct wolf-like marsupial from Australia. Could I not clone DNA from museum specimens of this creature? I agreed and moved to Zurich as soon as I had passed my PhD defense in Uppsala.
In the meantime, I had hoped that the attention generated by my Nature paper would allow me to obtain more mummy samples from East Germany, so that I could generate more clones and find interesting genes instead of the mundane Alu repeats. So when Rosti went to Berlin some months after the Nature publication to arrange for me to sample the mummies again, I expected clear sailing. Instead, he returned with disturbing news. None of his friends at the museum had had time to see him; in fact, they all seemed to avoid him. Eventually he had been able to corner one of them as the fellow was leaving the museum. It seems that after the publication of my Nature paper, the Stasi, the feared East German secret police, had appeared at the museum and interviewed each staff member in turn in a small room, asking them what they had been doing with me and Rosti. That I had published my first results in East Germany and had prominently referred to that publication in the Nature paper—none of this impressed the Stasi. Instead, they impressed upon the museum employees that, as they put it, Uppsala University is a well-known antisocialist propaganda center. No matter how ridiculous this characterization of the oldest university in Sweden was, no East German citizens in their right mind would of course have anything to do with us after being told this by the Stasi.
I was depressed by the futility of dealing with a totalitarian system. Having entertained visions that our two competing political systems might grow closer, perhaps catalyzed by scientific contacts, I had hoped that I might contribute to the process in some small way. Little did I know the role that East Germany would play in my life, but at that point neither samples nor cooperation seemed to be in the cards.
In Zurich, I set about extracting DNA both from the small mummy samples I had left in my possession and from specimens of the marsupial wolf. Despite my enthusiasm for the PCR, getting it to work following Kary Mullis’s protocol was no picnic. It involved heating the DNA in a 98°C water bath to separate the strands, then cooling it in a 55°C water bath to let the synthetic primers attach to their targets, then adding the heat-sensitive enzyme and incubating the mix in a 37°C water bath to try and coax it to make the new strands. For each experiment, this tedious cycle of manipulations needed to be done at least thirty times. I spent hours on end in front of steaming water baths wasting many test tubes of expensive enzyme in my attempts to amplify pieces of DNA. Sometimes I was able to generate a weak product from modern DNA, but I had no luck with the badly degraded DNA from the thylacine and the mummy samples. I did have some success in showing, by electron microscopy, that much of the mummy and thylacine DNA was in short pieces.
Some DNA molecules had even become linked to each other by chemical reactions, a feature that was sure to make them intractable to multiplication either in bacteria or by PCR in the test tube. This was not surprising, given some findings I had made in 1985, when I had visited Tomas Lindahl’s lab in Hertfordshire outside London for a few weeks. Tomas is originally of Swedish descent and one of the world’s experts on chemical damage to DNA and the systems that organisms have evolved to repair it. In his lab I had shown that there was evidence for several forms of damage in the DNA I had extracted from the old tissues. These results as well as my new Zurich findings constituted solid descriptive science, but they did not take me closer to my goal of reading DNA sequences from long-extinct creatures. Months passed in front of the water baths—as well as on the Alpine ski slopes—but no breakthroughs transpired, so it was with a distinct sense of relief in the spring of 1987 that I left Zurich for Berkeley, where Allan Wilson was back in residence.
Upon arriving in the Biochemistry Department at UC Berkeley, I soon realized that I was in the right place at the right time. Kary Mullis had been a graduate student there before he moved to the Cetus Corporation, down by the Bay, where he invented the PCR. Several of Allan’s previous graduate students and postdocs worked at Cetus. The result was that while I had been alone in my struggle to get the PCR to work in Zurich, in Berkeley many people worked on it, and as a result many improvements were made. At Cetus, they had cloned and expressed a version of DNA polymerase, the enzyme used in the PCR to make new DNA strands, from a bacterium that grows at high temperatures. Since this enzyme could survive high temperatures, there was no need to open the test tubes and add enzyme during each PCR cycle. This meant that now the whole process could be automated; indeed, one postdoc in the lab had already built a contraption in which a small water bath was fed water from three bigger water baths in cycles controlled by a computer. This allowed the PCR to be done automatically. After months in front of the water baths in Zurich, this was progress I could appreciate. I could start a PCR and leave for home in the evening (a practice my colleagues and I had to abandon after a major flood in the lab when a valve failed to close as expected). Our innovative but unreliable lab machinery was soon replaced by the first PCR machine produced by Cetus. Consisting of a metal block with holes for the test tubes, it would heat and cool our samples however we pleased, for as many cycles as we wanted, all of this computer-controlled. I remember the awe we all felt when it was wheeled in. I threw myself at this machine, booking it for as many runs as my lab mates would tolerate.
