Neanderthal Man (10 page)

Things got better again, but only for a while. It was time for paranoia, and the paranoia led not only to my mania for cleanliness in the clean room but to my establishment of several firm rules for how to work in a clean room—rules that to this day remain the standard. First of all, access was limited to the select group who did experiments there—in this case, my first two graduate students, Oliva and Matthias. Before they entered the clean room, they each donned a special lab coat, hairnet, special shoes, gloves, and a face shield. After some additional frustrations with contaminated blank extracts, I decided that they were allowed into the clean room only when they came directly from home in the morning. If they first walked through rooms where PCR products might be present, they were banned from the clean room for the rest of the workday. All chemicals had to be delivered directly to the clean room, and we bought new equipment that also went directly there. Slowly, things got better. Still, all new solutions and chemicals needed to be tested by the PCR for traces of human DNA, and it was not uncommon for a batch to have to be discarded. All of this was taxing work for Oliva and Matthias, who had joined me in hopes of studying ancient humans and extinct animals and found themselves instead vetting chemicals and fretting about contagion.

But the lab’s general mood improved as our efforts began to pay off. As our extracts became clean, we could start working on other methodological issues. So far, all our work had been on soft tissues, such as skin and muscle. But I remembered that one of my DNA-yielding mummy  samples in Uppsala had come from cartilage, a tissue not very different from bone. If DNA could be extracted from ancient bones rather than just soft tissues, this would obviously open up great opportunities, as bones are what generally remain from ancient individuals. In 1991, Erika Hagelberg and J. B. Clegg of Oxford University had published a paper describing the extraction of DNA from ancient human and animal bones.
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So when the contamination issue was under control, Matthias tried many methods for getting DNA out of bones, focusing on animals where the risk of contamination was much smaller (as DNA from most animals was rare in our laboratory). Among them was a protocol described in the literature for DNA extraction from microorganisms. It relied on the fact that DNA binds to silica particles—essentially a very fine glass powder—in solutions that contain high salt concentrations. The silica particles could then be thoroughly washed to get rid of all kinds of unknown components that were in many of the samples and that could interfere with the PCR. Finally, the DNA could be released from the silica particles by lowering the salt concentration. This extraction procedure was an arduous process, but it worked and so represented a major step forward.

Matthias and I published the silica extraction method in 1993; the experiment used Pleistocene horse bones, and the mtDNA sequence they yielded was proof that we could retrieve DNA from bones that were 25,000 years old—the first time that reliable DNA sequences from before the last Ice Age were presented.
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With small modifications, this is still the extraction protocol used in most ancient DNA extractions today. The many frustrations that preceded this paper were evident from our opening remark that our young field was “marred by problems.” But this was slowly changing. In fact, without realizing it at the time, Matthias and Oliva had laid the foundations for much of what was to come in the next few years. In 1994, Matthias retrieved the first DNA sequences from Siberian mammoths, working with four individuals between 9,700 and more than 50,000 years old. We sent this work to
Nature,
where it was published together with similar results from Erika Hagelberg, who had isolated DNA from the bones of two mammoths.
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Although these mtDNA sequences were very short, they hinted at what would be possible if more sequences could be retrieved. We saw, for example, that there were many differences among the DNA sequences from the four mammoths. So we could imagine not only clarifying the relationship of mammoths to the two living members of the same order—the Indian and African elephants—but also tracing the history of mammoths in the Late Pleistocene and on up to their  extinction some 4,000 years ago. Things were finally looking brighter for ancient DNA.

This was also a time when our skills in extracting DNA and doing the PCR were applied to other, rather less conventional biological materials. Felix Knauer, a wildlife biologist at the university, showed up one day in my office and asked about the application of our DNA techniques to “conservation genetics,” the field that tries to apply genetics to the question of how best to protect endangered species. Felix had collected feces from the last surviving wild population of Italian bears, who lived on the southern slopes of the Alps. I invited Felix and a few other students to try our silica extraction method and PCR from the bear feces. We showed that we could amplify bear mtDNA from such droppings. Previously, the only way to get DNA from an animal in the wild was either to kill it or to shoot it with a tranquilizing dart and draw blood, a risky (and for the animal obviously very disturbing) procedure. We could now study the genetic relationship of the Italian bears to other European bear populations without bothering the bears at all. We published this work as a small paper in
Nature,
in which we also showed that we could retrieve DNA from the plants that the bears had eaten and thereby reconstruct aspects of their diet.
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Extraction of DNA from droppings collected in the wild has since become common practice in wildlife biology and conservation genetics.

As we were painstakingly developing methods to detect and eliminate contamination, we were frustrated by flashy publications in
Nature
and
Science
whose authors, on the surface of things, were much more successful than we were and whose accomplishments dwarfed the scant products of our cumbersome efforts to retrieve DNA sequences “only” a few tens of thousands of years old. This trend had begun in 1990, when I was still at Berkeley. Scientists at UC Irvine published a DNA sequence from leaves of
Magnolia latahensis
that had been found in a Miocene deposit in Clarkia, Idaho, and were 17 million years old.
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This was a breathtaking achievement, seeming to suggest that one could study DNA evolution on a time scale of millions of years, perhaps even going back to the dinosaurs! But I was skeptical. From what I had learned in Tomas Lindahl’s laboratory in 1985, I had concluded that it was possible for DNA fragments to survive for thousands of years, but millions seemed out of the question. Allan Wilson and I did some simple extrapolations, based on Lindahl’s work, in which we determined how long DNA would survive if water were present  and conditions were neither too hot nor too cold, neither too acid nor too basic. We concluded that after some tens of thousands of years—and perhaps, under extraordinary circumstances, a few hundreds of thousands of years—the last molecules would be gone. But who knew? Perhaps there was something very special about those fossil beds in Idaho. Before going to Germany, I visited the site. The deposits were formed of dark clay, which was removed by a bulldozer. Upon being pried open, the blocks of clay revealed green magnolia leaves, which rapidly turned black when exposed to air. I collected many of these leaves and brought them with me to Munich. In my new lab, I tried extracting DNA from the leaves and found that they contained many long DNA fragments. But I could amplify no plant DNA by PCR. Suspecting that the long DNA was from bacteria, I tried primers for bacterial DNA instead, and was immediately successful. Obviously, bacteria had been growing in the clay. The only reasonable explanation was that the Irvine group, who worked on plant genes and did not use a separate “clean lab” for their ancient work, had amplified some contaminating DNA and thought it came from the fossil leaves. In 1991, Allan and I published our theoretical calculations in an article about the stability of DNA,
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and in a second paper we described my failed attempts to get DNA from the plant fossils from Idaho.
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This was a sad time, since Allan had fallen severely ill with leukemia the year before. Nevertheless, he made substantial contributions to both papers. He died in July of that year at the young age of fifty-six.

Naïve as always, I thought our paper pointing out the chemical impossibility of DNA survival over millions of years would halt the search for such super-old DNA. But rather than being the end of things, the Idaho plant fossils were the beginning of a whole new area of research. The next super-old DNAs to pop up were found in amber. Amber is resin that was exuded from trees millions of years ago and solidified into translucent golden clumps; it is found in large quantities in quarries in the Dominican Republic and on the shores of the Baltic Sea, among other places. Not uncommonly, insects, leaves, and even small animals such as tree frogs can be found entombed in resin. Such inclusions often preserve exquisite details of organisms that lived millions of years ago, and many investigators hoped that the same would be true for their DNA. One of the first such papers came in 1992, when a group at the American Museum of Natural History published a paper in
Science
presenting DNA sequences from a 30-million-year-old termite encased in a piece of Dominican amber.
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This was followed in 1993 by a whole series of papers from a lab headed by  Raul Cano at California Polytechnic State University, in San Luis Obispo, including one on DNA from a weevil between 120 million and 135 million years old found in Lebanese amber
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and another on DNA from a 35- to 40-million-year-old leaf from the Dominican tree that produced the amber.
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Cano went on to found a company that claims to have isolated more than twelve hundred organisms from amber, including nine ancient strains of live yeast. Leaving such outlandish claims aside, it seemed to me that one could not rule out the possibility that DNA might be preserved for an extraordinarily long time in amber, since it was probably protected from water and oxygen, the two factors most destructive to the chemistry of DNA. That supposition, however, didn’t necessarily mean protection from the effects of background radiation over millions of years, nor did it explain why we had struggled so mightily to amplify traces of DNA a thousand times younger.

The opportunity to find out came in 1994, when Hendrik Poinar joined our lab. Hendrik was a jovial Californian and the son of George Poinar, then a professor at Berkeley and a well-respected expert on amber and the creatures found in it. Hendrik had published some of the amber DNA sequences with Raul Cano, and his father had access to the best amber in the world. Hendrik came to Munich and went to work in our new clean room. But he could not repeat what had been done in San Luis Obispo. In fact, as long as his blank extracts were clean, he got no DNA sequences at all out of the amber—regardless of whether he tried insects or plants. I grew more and more skeptical, and I was in good company. In 1993, Tomas Lindahl, who had been interested in ancient DNA ever since my 1985 visit to his lab, published a highly influential review on DNA stability and decay in
Nature,
in which he devoted a section to ancient DNA.
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He pointed out—as I had with Allan earlier—that survival beyond a few hundred thousand years was unlikely. He left open the possibility that DNA from specimens encased in amber was an exception; in the meantime, however, I had given up even on the amber.

Tomas had also found the perfect term for super-old DNA:
antediluvian DNA.
We loved it, applied it, and it stuck. But this ridicule could not, of course, deter the enthusiasts. The inevitable happened in 1994, when Scott Woodward of Brigham Young University in Utah published DNA sequences that he and his colleagues had extracted from 80-million-year-old bone fragments—bone that “likely” came from a dinosaur or dinosaurs.
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Not unexpectedly, this paper appeared in one of the two journals that compete for headline-worthy work and enjoy an often undeserved scientific  prestige. This time it was
Science.
The authors had determined many different mtDNA sequences from the bone fragments, and some of them seemed to the authors to be as distant from birds and reptiles as from mammals. They suggested that these might be dinosaur DNA sequences. This seemed ludicrous to me. Thoroughly frustrated by the way the field had developed, Hans Zischler, a meticulous, even slightly pedantic, postdoc in my lab, decided to go after this particular piece of work. When we did a more rigorous analysis of the DNA sequences that the Utah group had published, they seemed closer to mammalian—and indeed human—mtDNA than to birds or reptiles.

Still, they didn’t quite seem to be human mtDNA. Explaining what they were takes a bit more explanation of the nature of mtDNA. Recall that mitochondrial genomes are circular DNA molecules of 16,500 nucleotides that reside in mitochondria, organelles located outside the cell nucleus in almost all animal cells. These organelles, as well as their genomes, derive originally from bacteria that almost 2 billion years ago entered primordial animal cells and were hijacked by those cells to produce energy. Over time, the hijacked bacteria transferred most of their DNA to the cell nucleus, where the DNA became integrated into the major part of the genome, situated on chromosomes. Even today in the human germ line, when eggs and sperm cells are formed, a mitochondrion will occasionally break, and fragments of its DNA will end up in the cell nucleus. There, efficient repair mechanisms recognize the ends of broken DNA strands and join them to other DNA ends that may exist if the nuclear genome also happens to carry a break. Thus, now and again, pieces of mtDNA become integrated in our nuclear genome, where, without having any function, they are passed on to new generations. Each of us carries hundreds if not thousands of such misplaced mitochondrial DNA fragments in our cell nuclei that have integrated into our genome at various times in the past. These fragments have different degrees of similarity to our real mitochondrial mtDNA; although they resemble ancestral mtDNA sequences, they have accumulated mutations, unconstrained as they are by any functional requirements in their new life as genetic garbage embedded in nuclear DNA. Hans Zischler had worked in our lab on detecting such integrations of mtDNA into the nuclear genome, and as we considered the putative dinosaur DNA, we wondered whether such mtDNA fragments might be what the Utah group had found. Indeed, given our experience with contaminating human DNA, it seemed probable to us that they had found nuclear versions of human mtDNA with unusual mutations. We decided to look in the human nuclear  genome for the sequences they had published. The problem with our plan was that any normal preparation of DNA from human cells ended up containing a mix of both nuclear and mtDNA, and the hundreds and thousands of copies of real mtDNA in the mitochondria of most cells would get in the way of our attempts to detect any mtDNA segments that had left the mitochondrion and settled among the nuclear DNA. Here we were helped by biology. As noted in Chapter 1, we inherit our mtDNA exclusively from our mothers, via the egg, and get no mtDNA from our fathers. This is because the heads of the sperm, which penetrate the egg, contain no mitochondria. So to get nuclear DNA without accompanying mtDNA, our lab simply needed to isolate sperm heads.