Neanderthal Man (17 page)

We wrote to several museums all over Europe to collect Neanderthal and early modern human bones. After our success with the Neanderthal type specimen it had become somewhat easier to convince curators to allow us to sample their collections, and we ended up with bits of bones from twenty-four Neanderthals and forty early modern humans. David analyzed the amino acids in all sixty-four samples. Just four Neanderthal and five early modern human samples were well enough preserved to suggest the presence of mtDNA—a grim but typical proportion. He extracted DNA from these nine bones and tried to do PCRs using primers that could amplify mitochondrial DNA from the great apes, Neanderthals, as well as humans. David got amplification products from all nine samples. When  sequenced, they turned out to be similar or identical to those found in people today. These results were disturbing. Maybe Trinkaus was right after all.

I asked David to do the experiment again, this time including samples from five cave bears from Vindija and one from Austria. When he amplified these, they also yielded human sequences! This strengthened my suspicion that we were simply obtaining contaminating DNA sequences from modern humans who had handled these bones. David then carefully designed primers that would amplify Neanderthal-like mtDNA but not present-­day human mtDNA. After using mixtures of DNAs in the lab to test that these primers were indeed specific for Neanderthal mtDNA, he tried them on the cave bears. He could amplify nothing. This was reassuring. The primers were indeed specific for Neanderthal mtDNA. He then used these primers on the extracts from the Neanderthal and modern human bones. Now all the Neanderthal bones yielded mtDNA sequences similar to that of the Neanderthal type specimen, suggesting once again that Neanderthals did not carry mtDNAs similar to those of present-day humans. In contrast, none of the five early modern humans yielded any products, suggesting that Trinkaus was wrong.

We next turned to theory to explore this topic further. We designed a population model in which we assumed that Neanderthals bred with anatomically modern humans 30,000 years ago and that those modern humans had descendants living today. We then asked what the biggest genetic contribution to present-day humans could be, given our findings that neither any humans today nor five early modern humans some 30,000 years ago carried any Neanderthal mtDNA. According to this model (which we made tractable by using simplifying assumptions, such as not incorporating modern human population growth), Neanderthals could have contributed no more than 25 percent to the nuclear genome of people living today. However, because we saw no direct evidence for a genetic contribution from Neanderthals, I felt that the most reasonable hypothesis, unless new data showed something different, was that Neanderthals had made no genetic contribution to people alive today.

I found that this result nicely illustrated the strength of our approach as compared to a typical paleontological analysis. We used clearly defined assumptions and drew conclusions that were bounded by probabilities. Nothing approaching this in rigor could be done using morphological features of bones. Many paleontologists liked to portray what they did as rigorous science, but the very fact that they had been unable to agree on the occurrence of a genetic contribution from Neanderthals to present-day  humans despite at least two decades of debate illustrated that their approach had big limitations.

After we published David’s results,
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a theoretical group in Switzerland, led by the population geneticist Laurent Excoffier, developed a much more plausible model than ours for how Neanderthals and modern humans might have interacted. They assumed that when anatomically modern humans moved across Europe, any interbreeding with Neanderthals would have taken place in the areas at the front of the modern human advance. This initial invasion would have been characterized by small modern human populations that would then increase in size. The Swiss group showed that under this model even rare instances of interbreeding would have been likely to leave traces in today’s mitochondrial gene pool, because on average females in a growing population have multiple daughters who will transmit their mother’s mtDNA. So under such circumstances, any Neanderthal mtDNA that had ended up in the modern human population would run much less risk of getting lost than if this population were of constant size. Since we had seen no Neanderthal mtDNA in either the five early modern humans or the thousands of living humans we and others had studied, Excoffier’s group concluded that our data suggested “an almost complete sterility between Neanderthal females and modern human males, implying that the two populations were probably distinct biological species.”

I had nothing against this conclusion from the Swiss group, but it was of course still possible that something special, not captured by their model, had gone on when Neanderthals and humans met. For example, if all children of mixed Neanderthal-modern human ancestry ended up in Neanderthal communities, they would not have contributed to our gene pool and the result would look like “almost complete sterility” as that group described it. Also, if all interbreeding events involved Neanderthal males and modern human females, they would not be detectable in today’s mtDNA gene pool, since males do not contribute mitochondrial DNA to their children. Such mixtures would be seen only in the nuclear genome. To fully understand how interactions between our ancestors and the Neanderthals may have impacted our genomes, we clearly needed to study the Neanderthal nuclear genome.

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Henrik’s work on the X chromosome had shown that the patterns of similarities and differences seen in the mitochondrial DNA of humans and apes were extendable to at least one part of the nuclear genome. Whether we would ever be able to study nuclear DNA from Neanderthals or be forever limited to their mitochondrial genome was not at all apparent. In my darker moments, I thought we were going to be stuck with mtDNA’s blurry, one-eyed view of human history. Certainly, if one disregarded results from animals and plants embedded in amber, dinosaurs, and other fanciful “antediluvian” studies (which I did), nobody had yet succeeded in retrieving any nuclear DNA from ancient remains. But in my more considered moments, I felt that we should give it a try.

It was at this point that Alex Greenwood, a diminutive but determined new postdoc from the United States, arrived in the lab. I told him about our hopes for retrieving nuclear DNA from Neanderthals, noting that it was a high-risk project but also a very important one. He was eager to take on the challenge.

I suggested a “brute-force” approach. My plan was to test samples of many bones to find those with the most mtDNA and then extract DNA from yet larger samples in an attempt to retrieve any nuclear DNA. This approach meant that we could not perform our initial experiments with the uncertain technique on Neanderthal remains; they were too scarce and valuable to use when the risk of failure was so high. Instead we resorted to animal bones, which were both considerably more abundant and less valuable to paleontologists. The cave-bear bones I had brought back from the dark basement of the Quaternary Institute in Zagreb now came in handy. They had been found in Vindija Cave, a limestone cave that had also produced some Neanderthal remains that contained mtDNA. So if we were able to retrieve nuclear DNA from the cave bears, we could hope to do the same with the Neanderthals.

Alex began by extracting DNA from the Croatian cave-bear bones, which were between 30,000 and 40,000 years old, and checked to see if they contained any bear-like mtDNA. Many of them did. He then took the extracts that seemed to contain the most mtDNA and tried to amplify short fragments of nuclear DNA. This failed. He was frustrated, and I was dismayed but not surprised. The problem he faced was a familiar one to me: because each cell in a living animal contains hundreds of mitochondrial genomes but only two nuclear genomes, any particular piece of nuclear DNA was present in 100- or 1,000-fold fewer copies in the extracts than any particular piece of mitochondrial DNA. So even if some nuclear DNA was present in minute amounts, the chances of amplifying it were a 100- or 1,000-fold lower.

One obvious way to overcome this problem was to simply use more bone. Alex made extracts of ever larger amounts of cave-bear bone and tried amplifying ever shorter pieces of nuclear DNA using primers flanking nucleotides where bears were known to differ from humans. That would enable him to discriminate between ancient bear DNA and contaminating human DNA. But in these mega-extracts,
nothing
could be amplified—not even bear mitochondrial DNA. He got no products at all.

After several weeks of repeated failures with multiple bones, we realized it was impossible to make useful DNA extracts from such large amounts of bone material. This was not because the bones contained nothing to amplify but because the extracts contained something that inhibited the enzyme used for the PCR; it became inactive and no amplification at all took place. We struggled to purify the unknown inhibitor away from the DNA in the extract but failed. We diluted the extracts in small steps until they started working again for the amplification of mtDNA. Then, at that dilution, we tried the nuclear amplification. It always failed. I tried to remain upbeat but as the months passed, Alex became more and more frustrated and anxious about whether he would ever produce any results that would justify a paper. We began to wonder if after a bear’s death the nuclear DNA might be degraded by enzymes leaking through the nuclear membrane of the decaying cells. Perhaps the DNA in mitochondria, having a double membrane, would have been better protected, making the mtDNA more likely to survive until the tissue dried out, froze, or was otherwise protected from enzymatic attack. This possibility made me wonder whether it would be possible to find nuclear DNA in ancient bones at all, even if we could overcome the inhibition of the PCR. I was slowly becoming as frustrated as Alex.

Thwarted by the cave bears, and wondering whether the conditions in the cave may simply have been too unfavorable to preserve nuclear DNA, we decided to switch to material that we expected to show the very best preservation—permafrost remains of mammoths from Siberia and Alaska. These had been frozen ever since the animals died and freezing, of course, will slow down and even stop both bacterial growth and many chemical reactions, including those that degrade DNA over time. We also knew, from Matthias Höss’s work, that mammoths from the Siberian permafrost tended to contain large amounts of mtDNA. Of course, no Neanderthals had ever been found in the permafrost—so switching to mammoths meant taking a step away from my ultimate goal. But we needed to know whether nuclear DNA could survive over tens of thousands of years. If we found no nuclear DNA in the frozen remains of mammoths, then we could forget about finding it in Neanderthal bones preserved under much less ideal conditions.

Fortunately I had systematically collected ancient bones from different museums over the past few years, so Alex could immediately try remains of several mammoths. He found one mammoth tooth that contained particularly large amounts of mtDNA. It had been pulled out of the frozen ground when the Alaska Highway, extending from northeastern British Columbia to near Fairbanks, was built in great haste during World War II and stored ever since in a huge box in the American Museum of Natural History. To make the search for DNA a bit easier, we carefully targeted a segment of the nuclear genome containing part of the gene known as 28S rDNA, which encodes an RNA molecule that is part of the ribosome, a structure that synthesizes proteins in cells. For our purposes, this gene had the great advantage of existing in a few hundred copies per cell. It should thus have been about as abundant as mitochondrial DNA in the extracts, assuming that the nuclear DNA had not been degraded more than mitochondrial DNA after death. To my delight and profound relief, Alex could amplify the ribosomal gene. He sequenced clones of the mammoth PCR products and reconstructed the gene’s sequence using the overlapping-­segments approach we had established when studying Neanderthal mtDNA. He then wanted to compare the sequence to those from African and Asian elephants, the closest living relatives of mammoths. I had been so paranoid about contamination that, until Alex had the mammoth results, I had forbidden him or anyone else to work on elephants. But now, working outside our clean room, Alex used the same primers he had used for the mammoth work to amplify and sequence the 28S rDNA fragment from an African  and an Asian elephant. The mammoth sequences were identical to those of the Asian elephant but differed at two positions from the African elephant version, suggesting that mammoths were more closely related to Asian than to African elephants. But comparing mammoths to living elephants hadn’t been the point of the exercise: finding ancient nuclear DNA had been. To clinch it, we sent a bit of the tooth off for carbon dating. When the 14,000-year-old date came back, I felt satisfied for the first time in months. It was now official. These were the first nuclear DNA sequences ever determined from the late Pleistocene.

Encouraged by these results, Alex designed primers for amplifying two short pieces of a fragment of the von Willebrand factor gene, only one copy of which exists in the elephant genome. This gene, abbreviated vWF, encodes a blood protein that helps platelets stick to wounded blood vessels. We focused on it because others had already sequenced it from elephants (and many other extant mammals), so if we managed to determine a sequence from the mammoth, we could directly compare it to those present-­day sequences. I could hardly believe my eyes when, during our weekly lab meeting, Alex showed pictures of bands in a gel that suggested he was able to amplify these gene fragments from the mammoth. He repeated the experiment twice, using an independently prepared extract from the same mammoth bone. Among the many clones he sequenced, he saw errors in individual molecules, presumably due either to chemical damage in the old DNA or to the DNA polymerase’s addition of the incorrect nucleotide during the PCR cycles (see Figure 9.1). At one position, however, he saw an interesting pattern. He had sequenced a total of thirty clones from three independent PCR amplifications. At one position, fifteen of those clones carried a C, fourteen carried a T, and one carried an A. The single A, we assumed, was an error caused by the DNA polymerase, but the others represented something that made my heart beat a bit faster. This particular spot in the sequence was clearly what geneticists call a heterozygous position, or a single nucleotide polymorphism (SNP for short), a place where the two copies of the gene that this mammoth had received from its mama and papa mammoth differed. What we saw was the first heterozygous position or SNP from the Ice Age. This was, if you like, the essence of genetics—a nuclear gene that has two variants in a population. Things were looking up. If we could see both versions of this mammoth gene, then all parts of the genome must be potentially accessible. It should thus be possible, at least in theory, to retrieve any genetic information we wanted from a species that went extinct many thousands of years ago. To drive home this point, Alex  amplified pieces of two more single-copy genes: one encoding a protein regulating the release of neurotransmitters in the brain and one encoding a protein that binds vitamin A and is produced by the rods and cones in the eyes. He was successful in both cases.