Neanderthal Man (7 page)

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.

____________________________

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,
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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.

The extinct South African zebra, the quagga, from which Russell Higuchi had cloned two pieces of mtDNA, offered itself as a first step. Russ had left  Allan’s lab for Cetus, but some of his quagga samples were still there. I extracted DNA from a piece of quagga skin, had primers synthesized for the same mitochondrial sequences he had cloned, and started a PCR in the new machine. It worked! I amplified beautiful pieces of quagga DNA, and when I sequenced them, they were very similar to what Russell had determined by cloning in bacteria. The big advance was that I could do it again and again. Bacterial cloning was so inefficient that replication of the findings would have been next to impossible, because the process was unlikely to produce the same stretch of DNA. The quagga sequences I retrieved were very similar to the sequences cloned in bacteria but they differed in two places from Russell’s sequences, probably because of molecular damage that had induced errors when the bacteria took up and replicated his samples. With the PCR, I could now retry the same sequence multiple times to ensure that it could be exactly replicated. This was what science was supposed to be about: reproducibility of results!