Unlocking the Past

by Martin Jones

  Back in England, some of us were having more success in persuading the UK research councils to follow this avenue of research. We were keen to shift the agenda away from the race for headlines that had begun to characterize the quest for ancient DNA, to support research into the dynamics of the various biomolecules involved, and to look more carefully at what they could tell us about the past. It was another eighteen months before the Ancient Biomolecules Initiative (ABI) was under way, funding projects in a number of different universities and institutions around the country, several of which were directing their attentions to ancient DNA.

  getting the science right

  One of the first issues the ABI programme needed to tackle was that of the DNA from deposits far too old to tally with Lindahl’s rates of DNA breakdown. By this time, Tomas Lindahl had addressed the issue of ancient DNA in his own publications. Now that he had entered the debate, one of the critical variables to emerge was water. One might think that water was quite an easy thing to assess. Either an object has remained wet or dry, and that is that. Things are, however, rather more complicated, largely because biological structures are more complicated. As Eglinton’s work reminded us, they contain lipid barriers that stop water moving around, though it is very difficult to establish whether such barriers have remained intact–especially the microscopic barriers around cells and within tissue. Water’s impact also depends on what is dissolved in it. Anyone who has lived by the sea and seen the relative corrosive impacts of salt water and rainwater will know that. The situation is yet more complex underground where dissolved minerals can get mopped up from the soil water by complex organic molecules. In sum, just because we can see that an ancient object is surrounded by wet sediment, as in the case of the Clarkia leaves, and indeed of the Windover bodies, it does not follow that all the contained biomolecules have been exposed to the effects of water.

  An alternative approach was developed by Hendrik Poinar, by now the front runner in the ‘oldest DNA race’, thanks to his 130-million-year-old weevil. With great foresight, Hendrik crossed the board to join the sceptics in Svante Pääbo’s new lab at Munich. Here, he developed an elegantly simple approach to the problem of water. It is difficult to establish whether water has successfully reached the inner recesses of a complex organic object, so rather than look for the water itself to discover whether it had reached the DNA, we could look instead at a neighbouring molecule that was also affected by water, to see whether water had left its imprint. Poinar turned his attention to the proteins surviving alongside the DNA.

  The amino acids that make up any protein chain have a rather interesting property. As with any molecule, it is possible to make a three-dimensional model of an amino acid, displaying its atoms and the bonds between them as a kind of geometrical construct. In the case of amino acids, a mirror image can be taken to produce the model in reverse. So two different geometrical models of an amino acid share the same atoms, and the same links between them, but each is the mirror image of the other. For convenience, we can refer to these as the left-handed and right-handed forms of the amino acid. If we artificially prepare amino acids in the lab, the two forms occur in equal amounts. One of the remarkable, and indeed little understood, features of the living cell is that one of the two forms, the left-handed form, predominates. It is only after death that the balance gradually moves back to the more stable, even proportions of left- and right-handed forms. This happens by the amino acids spontaneously ‘flipping over’ or ‘racemizing’ at a rate that can be measured, but only in the presence of water. If the proteins retained contact with water after death, we would expect racemization to proceed. Otherwise, the marked imbalance between left- and right-handed forms of amino acid would persist.

  In amino acid racemization, Hendrik Poinar found a test to assess the internal dryness of an ancient biomolecule. It was not just a measure of how dry the specimens were now, but constituted a kind of cumulative record of any tiny quantities of water that might have reached the biomolecules in the past. If any of the geological specimens really did contain ancient DNA, we would expect the molecules to have been sufficiently protected from water for their neighbouring amino acids to show minimal signs of racemization. Poinar assessed and disposed of various of the geological samples, but one interesting result came from the amber-embedded insects from which he, with his father and Raul Cano, had amplified DNA. These specimens displayed very low levels of racemization. Here, perhaps, was one place where their geological DNA findings would stand up to close scrutiny. The terpenes within these amber prisons might have kept the water sufficiently at bay for at least a small element of Jurassic Park to shift from fiction to factual possibility. At the first workshop of the Ancient Biomolecules Initiative, Tomas Lindahl agreed this was the right place to look for the longest surviving DNA. Andrew Smith and colleagues at the London Natural History Museum set to work scrutinizing this possibility.

  They wanted to give themselves the best possible chance of encountering the DNA locked up in amber. So they tried five different DNA extraction techniques, targeted a variety of gene fragments that were suitably short (down to no base-pairs in length) and looked at several parts of the entombed insect’s body. Their first attempt was made on those extinct resin-collecting bees from the Island of Dominica, from which Cano and De Salle had recovered DNA. The amber chosen showed no signs of cracking, and Hendrik Poinar’s amino acid racemization tests had shown the entombed insect tissue to be free of water. The museum team came up with a negative result. There seemed to be no amplifiable DNA within the amber. They needed to look further. Perhaps a smaller body would be buried more quickly and dry out more effectively, so they expanded the range of species to include smaller insects. It would be interesting to see whether younger samples were any different, and bees from African copal, a fossil resin less than 100,000 years old, were assessed. None of these satisfied the team that any insect DNA remained.

  This is not to say they amplified no DNA. In fact, of the 156 trials they made, seven samples produced PCR products of the expected number of base-pairs in length, but on closer scrutiny these revealed more about how easily the minutely sensitive technique of PCR could deceive even the most rigorous scientist. Although they were the right lengths, sequencing of base-pairs showed them to be artefacts of the method. The most interesting of these artefacts was a length of DNA built entirely from copying the short sequences on the primers, over and over again. This accounted for two of the seven. The others clearly had some input from mammals or fungi, showing how sensitive PCR is to contamination, even in very clean labs. None of the seven sequences came close to an appropriate insect sequence.

  After three years of careful work, replicable amplification eluded the team. Furthermore, it eluded other labs in America and Switzerland, who had set about addressing the same issue. Everything pointed to a negative result. To put the cap on it, another member of the ABI team, Derek Briggs at Bristol, looked at the survival of what should be far more durable molecules than DNA, molecules such as chitin and certain structural proteins. Even these were broken down within the specimen. Seeing them with the naked eye, and especially with the help of low-power magnification, we may admire the beauty and apparently intact state of these amber-embedded insects, seemingly as fresh as the day they undertook their task of collecting resin with a little too much gusto. Their form seems wonderfully intact, but their molecules are not.

  This was by now becoming a recurrent message of biomolecular analysis. In the early days of seeking out biomolecules, the visual state of preservation was the only clue to molecular preservation. If the cellular structure was visible, and especially if the nuclei were visible, then surely some of the molecules had survived. It remains a useful guide at the broader level, but not an invariable indicator. An increasing number of cases of seemingly intact tissue with greatly transformed molecules were coming to light. Geoff Eglinton had found that the content of the Clarkia leaves had migrated out of the leaf tissue into the surrou
nding sediment. The ABI team were revealing considerable breakdown in amber-embedded insects.

  from the very old and unusual to the commonplace

  This first episode in ancient DNA analysis moved between a series of rather unusual objects, with what has proved to be a flawed foray into older and older specimens. It progressed from the liver of an ancient Chinese corpse, to the skin of an extinct quagga, on to Egyptian mummies, and from there to some extraordinary brains recovered from Florida sinkholes. We then heard of compressed Miocene leaves and insects in amber. The assembly of sources was strangely reminiscent of the objects and curiosities with which the eighteenth-century antiquaries would surround themselves.

  Around the time that Svante Pääbo was producing exciting results from the preserved brains in the Windover Bog, members of a research group in Oxford, England, were turning their attention to a rather more commonplace archaeological find than any of the above. As ever, the team brought together researchers who had moved in interesting ways across the boundaries of different disciplines. Bryan Sykes was a medical geneticist; Robert Hedges was a physicist turned archaeological scientist, who had been at the forefront of Britain’s carbon dating and was very familiar with the chemistry of archaeological materials, particularly bone; Erika Hagelberg had a PhD in biochemistry but, wanting to expand her horizons, moved to the history and philosophy of science, and now hoped to bring together science and history in an altogether novel way. Having heard about these exciting advances with unusual archaeological specimens, the team decided to see what could be done with something very ordinary in archaeological terms, in circumstances where no soft tissue survived. They sought to find ancient DNA from archaeological bones.

  Hedges gathered together a range of fairly typical archaeological bones, some just 300 years old, from an English Civil War cemetery, some first-millennium AD bones from an Anglo-Saxon cemetery, a child’s bones from a late prehistoric hillfort, and a 7,500-year-old human bone sample from a Judaean cave. Under Sykes’s guidance, Hagelberg set to work, grinding up bone samples and preparing them for PCR. By the end of 1988, she had succeeded. From these samples, a gramme of powdered bone yielded up to five microgrammes of ancient DNA. The analysis had shifted from the unusual finds and museum acquisitions to the commonplace material of archaeological excavations.

  In order to answer specific questions bone samples could be collected from a vast range of sites. Erika Hagelberg went on to amplify DNA from a mammoth tooth, deposited in the Siberian soils around 50,000 years ago. It was both a replicable result and consistent with Lindahl’s dynamics. Shortly after Hagelberg had her first success with bone, Terry Brown and I met up and discussed the equivalent commonplace plant tissue from archaeological sites. Terry was a crop plant molecular biologist whose marriage to a prehistorian had aroused his interest in the past. My research had been in early agriculture, based on the plant tissue that is encountered on numerous excavations. I selected for Terry a series of the archaeological crop remains from different but commonplace conditions of preservation, and Terry got positive results within each category. At the same time that the very ancient DNA assays going back millions of years were looking vulnerable, the last few thousand years was looking considerably more possible. No matter how well an ancient amber prison had been hermetically sealed, or how speedily a Miocene leaf had been compressed, the oldest DNA looked doubtful. However, within the relatively recent history of our own species, we did not have to confine our attentions to unusual samples. DNA was potentially extractable from the most commonplace organic materials on an archaeological site.

  As the doors to geological DNA began to close, those to archaeological DNA were opening up. The next chapters examine the stories that are unfolding as we pass through those doors. But before we leave geological time behind, we can look at one further attempt to take the methodology back into areas that were still the preserve of fiction.

  one last stab at Jurassic Park

  In the years leading up to this point, life and art had been having some difficulty keeping pace with one another. In April 1990, Nature published Ed Golenberg’s paper on Miocene Magnolia. Within a month, Michael Crichton’s publishers had passed the galley proofs of his forthcoming novel to Universal Pictures. By the time Steven Spielberg had released the movie Jurassic Park, Crichton’s source of inspiration, George Poinar Jr., had worked with his son Hendrik and Raul Cano to publish a series of papers on amber-preserved DNA going back 130 million years. In London’s Natural History Museum, the work that would sound the death knell of the amber story was soon to begin. However, Crichton had picked up the other possibility in his story, that the bones themselves might retain Jurassic period DNA.

  Early in 1992, Mary Schweitzer, who happened to be a graduate student of one of the blockbuster film’s palaeontological advisers, detected what seemed to be nucleated blood cells in a bone of the dinosaur Tyrannosaurus rex. The bone was 65 million years old. By the summer of the film’s release, she believed she had detected DNA within the bone. Schweitzer was wisely cautious about her find and well aware of the problems of contamination.

  In broad medical terms, the structure of bone was well understood. It was a living tissue in which the bio-mineral matrix was fully permeated with blood vessels. In other words, the outside world had plentiful access to a dead bone through its natural openings. Fungi and other decay organisms could make their way in and speedily digest most of the lingering DNA. However, PCR could work with tiny residues as well, so it was not just a question of the general survival. Some cells could become completely engulfed by bio-minerals, and their DNA isolated from water and biology even before they left the original body. So long as the bio-mineral remained intact, and engulfed at least some cells, then bone tissue would be a very effective protector for ancient DNA. This became increasingly evident as studies of ancient DNA from bone increased. Some of the best results came from Moabones preserved in New Zealand peat bogs. The DNA within the bone was clearly being kept isolated from the watery peat. Perhaps that dry enclosure could also work for geological bone.

  Schweitzer was not the only one with an eye open for DNA in fossil bone. A microbiologist from Utah who had been working on colon cancer decided to give it a go. Scott Woodward was impressed by various examples of excellent preservation within peat, and reasoned that very ancient peat eventually became coal. He had grown up in mining country and knew about the occasional dinosaur tracks and dinosaur bones that turned up within coal. In late 1992, he asked some of his friends in the coal-mining industry to look out for ancient bones. Soon after, they provided him with some large bone fragments from a sandstone band within coal measures believed to be 80 million years old. The bone was fragile, brittle and waxy, which seemed promising. It had not, apparently, been heavily replaced with minerals, as many fossils are. Under a microscope, cellular structures could be made out. Some of those structures picked up stains designed to attach preferentially to biomolecules. Woodward was optimistic.

  He decided to look for a certain gene connected with the way cells manage their energy supply, the so-called cytochrome b gene. These can be found on the sub-cellular powerhouses known as mitochondria, which carry their own DNA independently from the cell nucleus. The cytochrome b gene is widespread among animals, and common to vertebrates, with slight variations between taxonomic groups. Dinosaurs should therefore have them. If he could pick up sections of the gene from his ancient bones, and compare them with living vertebrates, then he might have a basis for sticking his neck out further than the more sceptical Schweitzer. He designed PCR primers for the cytochrome b gene, and applied them to forty-two different extracts from the two bones. With many replications, the number of amplifications attempted came to nearly 3,000. Among these, the great majority were blank, but nine came up trumps.

  The sequences he obtained were short, 174 base-pairs in length, but long enough to show a match with part of the cytochrome b sequence. Within this sequence there was a lot of variation, but v
ariation with a certain amount of structure. In particular, some of that variation was attributable to the difference between the two bones. Other variation was due to the age and state of preservation of the biomolecules. Woodward concluded that the result was worth publishing as a possible example of ancient DNA from Cretaceous bone. The number of differences between his sequences and those of living animals pointed to a remote and extinct group, and the difference between the two bones implied that they had amplified DNA from not one species of dinosaur, but two.

  Within six months of the publication in Science of Woodward’s exciting find of ‘dinosaur DNA’, the same journal carried three pages of dense and carefully constructed critique of his argument. In that short time, twelve scientists from both sides of the Atlantic, including geneticists, palaeontologists, zoologists and virologists, had carried out new PGR amplifications and analyses, built new phylogenetic trees with the data, and lined up a battery of arguments for Woodward to address. Mary Schweitzer was one of those arguing that Woodward needed to take the phylogenetic analyses further. When she herself did so, she found that the dinosaur sequences had marked affinities with the human sequence. Svante Pääbo, from his new position as Professor of Zoology at Munich, was still keeping a keen eye on adventurous ancient DNA claims. His team decided to mount their search for dinosaur DNA not in amber or the coal measures. They chose to search for it in fresh human semen.

  This seemingly eccentric quest did have a logic of its own. Sperm is an ideal medium for a separate examination of nuclear and non-nuclear DNA. The head of the sperm carries only nuclear DNA, whereas the tail needs those minuscule powerhouses within the cell, the mitochondria, to enable its movement. If a method can be devised to chop the head from the tail, then the separate examination can proceed. The gene targeted by Woodward was a gene on the mitochondrion, not the nucleus. It was this mitochondrial gene that was the basis of Woodward’s analysis. However, one of the complicated things about DNA is that a particular sequence can crop up more than once within the same genome. There are a number of reasons for this to happen, some functional and others historical accidents. One example of the latter is that at some time in the distant past, copies of the cytochrome b gene transferred themselves from the mitochondrion to the cell nucleus. There, as inactive ‘fossil’ genes, they could embark on their own mutation pathway, unfettered by the constraints of natural selection. Pääbo’s team suspected this might have something to do with Woodward’s results, and so embarked on the sperm test to look at the nuclear and mitochondrial versions of the human genome separately. They used direct copies of Woodward’s own primers to seek out the dinosaur DNA in the sperm fractions and in various other samples. They got a very good match between the Cretaceous dinosaur bones and the modern human sperm heads.