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Science  11 Jul 1997:
Vol. 277, Issue 5323, pp. 176

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    DNA From an Extinct Human

    1. Patricia Kahn,
    2. Ann Gibbons


    MUNICH, GERMANY—A chance discovery by quarry workers who blasted their way into a limestone cave one summer day in 1856 is making scientific history once again. The unusual skeleton they found in the cave's rubble soon became the first recognized example of a now-vanished type of human. Named Neandertal, after the Neander Tal (Neander Valley) in Germany where it was found, the skeleton was dramatic proof that humans evolved, just like all other living things. Ever since, researchers have fiercely debated what these ancient beings were like, how they lived, and whether they are ancestors of modern humans or an evolutionary dead end.

    Out on a limb.

    Neandertals were not the ancestors of modern humans, according to new mitochondrial DNA data. The single Neandertal DNA sequence is distinct from all those known for humans and chimps.


    Now, 141 years later, this same skeleton is yielding the world's first glimpse into the genome of an ancient type of human. In today's issue of Cell, Matthias Krings and Svante Pääbo at the University of Munich and Anne Stone and Mark Stoneking of Pennsylvania State University report that they have recovered and analyzed a tiny snippet of DNA from the skeleton. Its sequence, says Pääbo, is “very different from [the corresponding region of] modern humans.” While one sequence from a single individual is not definitive proof, the data lend a new kind of support to the now-favored view of Neandertals: that they were a side branch of the human family tree, not our direct ancestors.

    The work is creating a stir among human origins researchers, not only for its implications for their field but because it demonstrates the feasibility of recovering DNA from at least some human fossils. The emerging field of ancient DNA has been badly tainted in recent years by spectacular claims, such as DNA from a dinosaur, that were later discredited. But researchers familiar with the new work are convinced that this Neandertal sequence is the real thing. “I really looked for holes in the methodology, but I just couldn't find any. It seems to be an authentic sequence and certainly as far as I can tell the most rigorous ancient DNA study I've ever seen,” says evolutionary biologist and ancient DNA researcher Blair Hedges of Penn State.

    Although duplicating the Pääbo group's achievement will be difficult for many reasons, Hedges and others say that this gives the whole field a needed shot in the arm. “It's one of my dreams that this would be possible,” rejoices paleoanthropologist Christopher Stringer of The Natural History Museum in London, who belongs to one of several other teams that have tried—unsuccessfully—to recover Neandertal DNA. “It's a marvelous achievement. … For human evolution, this is as exciting as the Mars landing. … It's the biggest breakthrough in Neandertal studies.”

    Stringer is particularly excited because the data tend to support his view in the debate about modern human origins, although they won't put the argument to rest. He is the leading advocate of the currently favored idea called “Out of Africa”—that modern humans arose recently in Africa and then replaced existing human populations around the world, including the European Neandertals, without interbreeding with them. That theory predicts that Neandertals were a separate species, as the differences between the Neandertal DNA and that of modern humans now suggest. “You can't prove [Neandertals] were a separate species from just this sequence, but it's very unlikely they contributed to the modern gene pool,” says molecular anthropologist Maryellen Ruvolo of Harvard University.

    Technical triumphs

    The groundwork for this astonishing success goes back several years. Archaeologists Hans-Ekhard Joachim of the Rheinisches Landesmuseum (where the Neandertal type specimen is kept), and paleontologist Ralf Schmitz of the Rhine State Department of Archaeology organized a committee to study what new research could be done on their precious skeleton, thought to be somewhere between 30,000 and 100,000 years old. They approached Pääbo, a pioneer in the ancient DNA field, about trying to recover DNA. After extensive studies of the preservation of various bones, Schmitz and Heike Krainitzki of the Technical College for Preparators in Bachum sent a sample of an upper arm bone to Pääbo's Munich lab last July.

    Pääbo decided to search for a particular sequence called the control region in the DNA of mitochondria, cells' energy-producing organelles, which have their own tiny genome. Over the past decade, this inconspicuous region, which doesn't code for any protein, has become a crucial tool for inferring evolutionary relationships among species and populations (Science, 16 May, p. 1032). That's because mitochondria are inherited only from the mother and do not undergo genetic recombination, so their sequence stays the same from one generation to the next—except when mutations change it.

    As a result, the number of nucleotide differences between two mitochondrial sequences generally reflects the degree of evolutionary divergence between them. The control region is particularly useful because it mutates faster than most other parts of the mitochondrial genome and so has more power to reveal differences between populations. Coincidentally, mitochondrial DNA (mtDNA) is usually the only type to survive in long-dead specimens, thanks to its abundance—500 to 1000 copies per cell rather than only two copies, as in DNA of the nucleus.

    But the difficulties in recovering any DNA from ancient bone are vast. The biggest problem is that DNA begins to degrade from the moment of death as water, oxygen, and microbes attack it. In most cases, all the DNA is probably gone after 50,000 to 100,000 years. So the best Pääbo's team could hope for in their Neandertal samples was trace amounts of highly damaged, short fragments. Indeed, they later calculated that their standard sample of bone extract contained only about 50 copies of the mtDNA piece they were after, in fragments of about 100 base pairs. Moreover, the damaged state of ancient DNA makes the method used to amplify it—the polymerase chain reaction (PCR)—far more error prone.

    Worst, it's tough to distinguish DNA intrinsic to an ancient sample from the modern DNA that unavoidably contaminates it—the source of many false claims in the past. Ancient human samples are especially tricky, because their sequences might not differ much from that of contaminating modern human DNA, so it's hard to get a believable result.

    Past failures had left Pääbo skeptical that recovering Neandertal DNA was even possible. Krings, the graduate student who took on the project, was skeptical, too. But initial tests on the bone's amino acids, which require only minuscule amounts of sample and measure the extent of DNA-killing water damage, were surprisingly promising.

    Krings then went after the DNA, strenuously trying to reduce contamination. He repeated what he had done for the amino acid tests—bleaching the outside of the bone to help rid it of contaminating DNA from past handlers, sterilizing instruments, and working in a room where no modern DNA work is done. He ground part of the sample to powder and extracted DNA from it; then he used PCR to amplify part of the control region and cloned the products, which allows more careful sequencing.

    When he saw the sequences, “something started to crawl up my spine,” he says. Mixed among a few clones that looked like modern human DNA—and were most likely contamination—were some unlike any in living humans. “I can't describe how exciting it was,” says Krings. Then came 3 months of painstaking work to piece together the total sequence of 379 base pairs.

    The clincher came when Stone repeated the entire procedure in Stoneking's lab at Penn State—and came up with the identical sequence. “That's when we opened the champagne,” says Krings. That sense of triumph was fully justified, say other researchers. “It's really on the edge of what's possible technically,” says Richard H. Thomas, a molecular systematist at The Natural History Museum in London. Adds paleoanthropologist Dan Lieberman of Rutgers University in New Brunswick, New Jersey, “The fact that they managed to find DNA from a region of prime importance is proof that there is a God who likes paleoanthropology.”

    Finally, the team compared the Neandertal sequence with 986 distinct sequences from living humans. They found, on average, three times more differences between the Neandertal and modern human sequences than between pairs of modern humans. Specifically, pairs of modern human sequences differed at an average of only eight positions, while human-Neandertal pairs differed at an average of 25.6 positions. And the range only barely overlapped: The most divergent modern humans differed in only 24 nucleotides, while the closest modern-Neandertal pair had 20 differences. Also, the type of nucleotide substitutions and their locations were different. These data put the Neandertal sequence outside the statistical range of modern human variation and, says Pääbo, make it “highly unlikely that Neandertals contributed to the human mtDNA pool.”

    To find out when the mitochondrial DNA lineages of Neandertals and our own ancestors must have separated, the Pääbo team also used the sequence data as a molecular clock. They used the estimated date when the human and chimp lineages diverged (4 million to 5 million years ago) and the sequence differences between humans and chimps (taking into account the types of changes that occur in this sequence) to come up with a rate at which the control region accumulated mutations over time. With other assumptions and correction factors, they calculated that the sequence ancestral to both modern and Neandertal mitochondria began to diverge some 550,000 to 690,000 years ago, compared to only 120,000 to 150,000 years ago for the ancestral sequence of all modern humans. To put it in relative terms, the last common ancestor of Neandertals and modern humans is four times older than the last ancestor of all modern humans.

    Such molecular clocks are full of questionable assumptions, but that relative difference is significant, says evolutionary geneticist Ryk Ward of Oxford University. Specifically, it suggests a deeply rooted split in the human family tree and implies that the two lineages diverged before the first known Neandertal at about 300,000 years ago, and long before the first modern humans at less than 200,000 years ago, says Stringer.

    Out of Africa supported

    All this has major implications for how modern humans arose, although this one study is unlikely to end the debate. Although recent fossil and genetic evidence has tended to support the Out of Africa theory, an opposing theory holds that modern humans evolved continuously in many parts of the world. In this view, called regional continuity, modern humans interbred with archaic people in Europe and Asia, including with Neandertals during the roughly 70,000 years they apparently coexisted in the Middle East and parts of Europe and Asia. Recently, each of these models has inched toward the other: Some Out of Africa contenders have begun to admit that populations on continents other than Africa could have contributed genes to modern populations, and multiregional proponents now advocate something short of widespread mixing.

    But the new data suggest no mixing at all, at least in mitochondrial genes. “Neandertals in Europe could not have contributed to the modern human mitochondrial genome,” says Stanford University geneticist Luca Cavalli-Sforza. That “destroys one of the fortresses of the regional continuity model,” he says, which postulates that Neandertals in Europe are among the ancestors of living Europeans.

    Indeed, one of the paper's most important findings is that Neandertal DNA shows no particular similarity to that of Europeans. “If regional continuity were correct, “we'd assume that Europeans would be closest [in their sequence] to Neandertals. But the results show Neandertals are equidistant to all races,” says Stringer. As a result, most researchers who spoke with Science consider the new data as support for the idea that modern humans replaced, rather than intermingled with, Neandertals. “The multiregional guys will have a hard time wriggling out of this one,” says Ward.

    But there is some wriggle room left. For starters, the fact that Neandertal sequences differ from those in modern humans doesn't by itself settle the tricky question of whether some members of the two groups interbred in the distant past. The genetic variation seen between the modern and Neandertal sequences is within the range of other single species of primates, notes Ruvolo, who says “there isn't a yardstick for genetic difference upon which you can define a species.”

    It's true that the Neandertal mtDNA is different from that of living humans. But that may simply support what geneticists have long known: Living humans are strangely homogeneous genetically, presumably because they originated recently from a small group or their ancestors underwent a population bottleneck that wiped out many variations. Thus, genetically diverse ancient populations could have intermingled long ago, then over time modern humans lost many of those genetic variants.

    That's why multiregional partisans say it's not possible to rule out their theory with data on a single sequence from one individual. “This is an extremely important piece of work. They're first. But we just don't have the data to answer the question of whether it supports one hypothesis or another,” says paleoanthropologist Milford Wolpoff of the University of Michigan, Ann Arbor. He argues that Neandertals may have contributed to the modern gene pool, but their sequences disappeared through random genetic loss, selection, or both. Or the particular Neandertal sequence analyzed might be at one extreme of a diverse spectrum in Neandertals that includes other, more modernlike sequences. But most population geneticists consider these possibilities remote, says anthropological geneticist John Relethford at the State University of New York College at Oneonta.

    And of course, because mtDNA comes only from the mother, it's possible that Neandertal fathers—but not mothers—contributed nuclear genes to the modern gene pool. Most researchers think such a one-sided genetic interchange is quite unlikely or “odd,” as Ruvolo puts it. But it's impossible to test directly, as the chances of recovering nuclear DNA are basically nil, says Pääbo—a fact sure to disappoint potential entrepreneurs dreaming of “Neandertal Park,” as resurrecting any extinct creature would require intact nuclear DNA, among other impossibilities.

    Other population geneticists say they would like to see more data to be sure. “The icing on the cake would be Neandertal number two,” says Penn State's Blair. “Get one of those North African Neandertals or something really far away, and see if it clusters with this one.” Genetic data from archaic moderns would also be helpful. Says Relethford, “I'd like to see DNA from the first undisputed early modern Europeans, the Cro Magnon from about 30,000 years ago. That's a real good test. Their mtDNA should look more like us.”

    No one has that, quite, but Bryan Sykes of Oxford University and Stringer think they have isolated mtDNA from a 10,000-year-old late Cro Magnon from Cheddar, England—and it shows only one base pair difference from that of modern humans. This as yet unpublished work shows that “we can put Cro Magnons at 10,000 nicely in the present variation,” says Stringer.

    But although Pääbo's group has shown that it is possible to get believable results with ancient, human DNA, he and others call for caution in going after more. Fossils are precious, and it's crucial to test the preservation of tiny samples or of animal bones found with human fossils before grinding them up, says Ward. What's more, adds London's Thomas, “all [researchers] have to do is read the paper closely to see it's a vast amount of work to do this right.”

    Even if the new result doesn't quite settle the debate about whether Neandertals mixed with modern humans, it does underscore how different they were from our own lineage. And that implies that “up to 200,000 or 300,000 years ago, humans evolved just like gorillas and everything else,” says Ward, perhaps with several contemporaneous, diverse human species. That picture is emerging from other new lines of evidence, too. For example, new dates on Homo erectus fossils in Asia show that this species also coexisted with modern humans until as recently as 30,000 years ago. These results are forcing paleoanthropologists to renounce the once-preferred linear model of human evolution in which a single primitive species gradually gave rise to the most advanced form—us. Rather, it seems, there was once a bushy human family tree—and all the branches but one went extinct.

    “It's what happens to [other kinds of] populations,” says paleontologist Rob Foley of Cambridge University in the United Kingdom. “But in the case of Neandertals, we get excited about it.” So it seems that the Neandertal sequence reinforces what this skeleton told the world when it was first discovered: that humans evolve just like everything else.


    Interference Sharpens the View

    1. Gretchen Vogel

    Last week, astronomers released the first images made with the help of a new orbiting radio telescope. Although the images—which show a powerful jet of subatomic particles spewing from a quasar—are not groundbreaking themselves, they are a powerful demonstration of the new system, indicating that it can provide detailed views of some of the most energetic and mysterious objects in the universe.

    The radio antenna, called HALCA, is the first space-based one designed for interferometry, a technique that allows scientists to combine data from far-flung telescopes and create the equivalent of an enormous collecting dish. The larger the dish, the more detailed the images. HALCA works in concert with ground-based telescopes, allowing astronomers to simulate a dish with a diameter greater than 30,000 kilometers and a resolving power—the ability to detect fine details—more than 100 times that of the Hubble Space Telescope.

    The image to the left (reduced in size for comparison) was taken by the Very Long Baseline Array (VLBA), a collection of ground-based radio telescopes. It shows quasar 1156+295, which is 6.5 billion light-years from Earth. The image to the right is a view of the same quasar in which data from HALCA are combined with those from the VLBA. It shows in unprecedented detail the particle jet emanating from the quasar, which may harbor a black hole, says astronomer Jonathan Romney of the National Radio Astronomy Observatory in Socorro, New Mexico, which is part of the international team that uses HALCA. No one is sure how the jets are formed, he says, but a clearer picture of their source may help scientists solve that mystery.

    Astronomers hope that HALCA, built by scientists at the Japanese Institute for Space and Astronautical Science and launched in February (Science, 31 January, p. 620), will allow them to make the most precise observations yet of objects that emit radio waves, including other quasars and black holes.


    Amino Acid Alchemy Transmutes Sheets to Coils

    1. Robert F. Service

    BALTIMORE—In January 1994, George Rose and Trevor Creamer threw down the gauntlet. They challenged their fellow protein researchers to try their hands at a bit of alchemy: radically transforming the basic three-dimensional (3D) structure of a protein while altering no more than 50% of its amino acid building blocks. As a reward, they offered $1000 of their own money. While that may seem like a rash bet—50%, after all, is a substantial change—Rose and Creamer knew that pairs of natural proteins differing in up to 70% of their amino acid sequences virtually always fold up into the same general 3D structure. “Realistically, we didn't expect to have to pay this for a long time,” says Rose, a biophysicist at the Johns Hopkins University School of Medicine in Baltimore. “It turned out we were not safe at all.”

    Reforming a protein.

    Splicing pieces of a coil-forming sequence (red) into a sheetlike protein (right) transforms its structure to coils. Blue indicates sequence retained in the hybrid protein.


    Late last month, Yale University biochemist Lynne Regan gave a symposium here, presenting her team's work to Rose and a roomful of Hopkins faculty, postdocs, and graduate students. She walked away with a cool grand. Regan and her Yale colleagues Seema Dalal and Suganthi Balasubramanian earned the prize by converting the 3D structure of a small protein from one that resembles a sheet with four flat strips lying side by side to another that resembles a collection of four helical telephone cords bunched together.

    The result, published in this month's issue of Nature Structural Biology, “is very impressive,” says Peter Kim, a protein designer at the Massachusetts Institute of Technology's Whitehead Institute for Biomedical Research, who saw the work presented at a recent Gordon Conference. The unexpectedly small number of amino acid differences between the two structures, he says, could have implications for efforts to infer the shapes of new proteins by comparing their sequences to those of known ones. The result is also a step toward the long-sought goal of being able to predict a protein's 3D structure from its sequence, which would be a boon to genome researchers and drug designers.

    Rose and Creamer launched their competition—dubbed the “Paracelsus Challenge” after the 16th century Swiss physician and alchemist—in hopes of sparking greater understanding of why proteins fold into particular patterns. At least two other groups beside Regan's had sought the prize. One team—Johns Hopkins structural biologists Neil Clarke and Shao-Min Yuan—even worked with the same two basic protein structures, a sheet and a set of coils. Clarke and Yuan started with the coiled structure and tried to transform it into a sheet. That change had the best chance of success, they thought, because “we knew the β sheet was a very stable structure,” says Clarke. As a result, he and Yuan hoped that a small complement of altered amino acids would hold the sheet together. “But we clearly didn't succeed,” says Clarke.

    Clarke and Yuan's work was what inspired Regan and her colleagues to try their hand. After hearing Clarke give a talk at a protein-design meeting in the spring of last year, says Regan, the trio got to talking about how the prize could be won. They decided to try going the other way: altering a sheetlike section of a molecule called protein G, which is found on the surface of Streptococcus bacteria, into a set of coils resembling those found in an RNA-binding protein called Rop. “We knew the properties of both these proteins very well,” says Regan. “But we knew a lot more about what stabilizes helixes than sheets.”

    In their studies of Rop, for example, they had found that substituting the amino acids alanine and leucine for other amino acids in spots where the four coils all face a central core made the protein even more stable than the standard version. Likewise, they found that they could destabilize the sheetlike region of protein G—a section known as the B1 domain—by removing key amino acids such as threonine.

    By plucking out such amino acids from the B1 structure and replacing them with α-helix formers such as alanine and leucine, the Yale team came up with a preliminary design for their transformed protein. They then plugged the substitutions into a computer program used to predict how amino acid sequences will fold and weeded out amino acid placements that clearly wouldn't allow the protein to fold into a bundle of helixes. In the end, they settled on a sequence that retained 50% of the amino acids in B1 but was identical to Rop in another 41%. They called their hybrid protein Janus, after the Roman god of new beginnings, who bears two faces.

    With Janus's blueprint complete, the researchers synthesized a stretch of DNA encoding the new protein's amino acid sequence and inserted it in an Escherichia coli bacterium, which expressed the DNA sequence and built the protein. Finally, the researchers purified the protein and characterized it via a variety of methods, including one called circular dichroism, which relies on the scattering of polarized light to differentiate protein structures such as helixes and sheets. “We were very encouraged,” says Regan. “Right from the beginning, it looked really helical.”

    That was enough to win the team the Paracelsus laurels, but they didn't stop tinkering with Janus. They have begun working backward, restoring more and more of B1's sheet-forming sequence to Janus in an attempt to see just how much of the original sequence they can add back and still get a helical fold. Already, the group has been able to design Janus analogs that share as much as 60% of the sequence of the β sheet-forming B1, but still fold into a cluster of helixes.

    “This is something that should give people at least some pause,” says Kim. He explains that genomics researchers commonly compare newly discovered gene sequences to those of genes that code for proteins with a known structure. If the new gene's protein product resembles the known one at 30% of its amino acid positions or more, it is considered very likely to have a similar shape and function. But the new Janus analogs share twice that number of amino acids with B1, yet form a very different structure. For genomics researchers working with the 30% standard, says Kim, “there are times we will be fooled.”

    Kim and others say the study also has important implications for researchers trying to learn the rules of protein folding. Most important, it underscores the growing understanding that not all amino acid residues have equal influence over the structure of a protein. Learning all the rules that govern how those structures are formed will take more protein alchemy studies, and perhaps a few more competitions. But as for the next competition's prize, Rose says he's tapped out. “Virtue,” he says, “will have to be its own reward.”


    Newfound Gene Holds Key to Cell's Cholesterol Traffic

    1. Elizabeth Pennisi

    At age 4, Marcia Parseghian was the most coordinated student in her dance class. Now 8, “she's having a real hard time keeping up with the other girls,” says her mother, Cindy Parseghian. “She stumbles a lot.” Her stumbling is just one sign that Marcia's nervous system is decaying. Like an older brother who died 4 months ago from a seizure and a sister who is 2 years younger, Marcia suffers from a rare genetic disorder called Niemann-Pick type C disease. Because the cells of Niemann-Pick patients cannot process cholesterol, they become glutted with the fatlike molecule. Nerve cells are the first to die, causing problems in seeing, walking, hearing, and swallowing. Like their brother, the two Parseghian girls are not expected to reach adulthood.

    Cholesterol clutter.

    Cholesterol (blue) builds up in cells that have a defective NPC1 gene.


    When the parents learned in 1994 that three of their four children were affected by this disease, “we were devastated,” Cindy Parseghian recalls—but not defeated. Together with Ara Parseghian, the grandfather and a well-known college football coach, they formed a foundation that has raised $6 million to support research on this obscure disease. That support has begun to pay off.

    On page 228, a team of some three dozen investigators—funded in part by the Ara Parseghian Medical Research Foundation and another parents' organization called the National Niemann-Pick Disease Foundation—report that they have tracked down the gene that causes the disease. The discovery “is a very big step forward,” says Yvonne Lange, a cell biologist at Rush-Presbyterian-St. Luke's Medical Center in Chicago. Already, the gene's nucleotide sequence is yielding some early clues to the function of its protein, says Peter Pentchev, a cell biologist at the National Institute of Neurological Disorders and Stroke (NINDS) in Bethesda, Maryland, who is one of the leaders of the research team. The Niemann-Pick protein, called NPC1, seems to sense a cell's level of cholesterol and help shuttle it from one part of the cell to another.

    A second result, reported on page 232, should help researchers test those ideas. Molecular biologist William Pavan of the National Human Genome Research Institute (NHGRI) in Bethesda, Maryland, and his colleagues have found that an identical gene is at fault in a mouse model of the disease. The availability of a precise mouse model should open the way for detailed study of how the NPC1 protein causes disease when it is disabled and could lead to the development of a treatment.

    The Niemann-Pick protein should also shed some light on how normal cells manage cholesterol, which is a component of cell membranes, an essential building block for steroid hormones—and an ingredient of the artery-clogging deposits of atherosclerosis. “[The gene] is going to be relevant to all problems in cholesterol homeostasis, especially atherosclerosis,” Lange predicts. “[These] are important and elegant studies,” agrees Joseph Goldstein of the University of Texas Southwestern Medical Center in Dallas who, with his colleague Michael Brown, won a Nobel Prize for studies of cholesterol regulation.

    Pentchev has been on the trail of the Niemann-Pick gene for some 20 years, ever since he began studying a mutant mouse strain that mimicked the human symptoms. Five years ago, Eugene Carstea, now at St. Mary's Hospital and Medical Center in Grand Junction, Colorado, joined Pentchev's lab, and the team's interest escalated into a full-scale gene hunt.

    With the help of Danilo Tagle and other colleagues at NHGRI, Carstea and NINDS's Jill Morris first narrowed their search to a 1.5-million-base-pair region along chromosome 18 by linkage analysis in families with the disease, including a large Bedouin family from Israel. To close in on the gene, Jessie Gu and Melissa Rosenfeld of the NHGRI obtained yeast artificial chromosomes (YACs) that contained different pieces of that entire chromosome region. They added each YAC to a set of cultured cells that had the Niemann-Pick defect and looked for cells that could then process cholesterol correctly. Those cells must have received a YAC containing a normal copy of the gene.

    The overlap between the human DNA in the YAC and the region defined by linkage analyses was 300 kilobases. The researchers further subdivided this region and picked out the protein-coding regions within each piece. By comparing those coding sequences to fragments of unknown genes archived in the public database called GenBank, they were able to piece together and sequence a complete gene. That gene turned out to be the one mutated in cells of patients with the disease, confirming that the search was over.

    This hunt gave a boost to another quest that had been under way for 2 years: finding the gene defect in mutant mice with the same symptoms as human sufferers. Already, several research teams had shown that the defective gene was on mouse chromosome 18—the same chromosome as in people. By combining linkage data from mouse-breeding studies with information about the gene's location in humans, Pavan, NHGRI's Stacie Loftus, and their colleagues narrowed the candidates to two possible genes.

    Of the two, one was much less active—as indicated by the amount of its messenger RNA—in liver and brain cells of mice with Niemann-Pick disease than in those cells of normal mice, suggesting that it was the defective gene. When Pavan, Tagle, and their colleagues compared the suspect gene's sequence to that of the human NPC1, they confirmed that the two are identical. “The mouse paper is a terrific confirmation of the human gene defect,” concludes Laura Liscum, a cell biologist at Tufts School of Medicine in Boston.

    Because the mouse and human genes are so similar, researchers will have an easier time sorting out the role of the NPC1 protein, which “will be central to understanding the process of how cells deal with cholesterol,” says Goldstein. In particular, the protein could help fill out a picture that Goldstein and Brown began developing 25 years ago. Their studies of another rare disease, familial hypercholesterolemia, helped them work out how the body maintains the proper amounts of cholesterol in cells. They showed that cholesterol from the bloodstream binds to a docking site called the LDL receptor on the cell surface. The receptor is then drawn into the cell's interior and the cholesterol freed in a compartmentlike cell organelle called a lysosome.

    Brown and Goldstein found that cholesterol ultimately gets incorporated into the cell membrane or repackaged for storage or breakdown. They and others also showed that when a cell is glutted with cholesterol, feedback mechanisms kick into action: The cell stops making LDL receptors and shuts down its own cholesterol-synthesizing processes.

    Even though Brown and Goldstein worked out much of the beginning and end of cholesterol's journey, “how free cholesterol moves out of the lysosome and into other parts of the cell [remained] a black box,” explains George Rothblat, a cell biologist at the MCP-Hahnemann School of Medicine in Philadelphia. “This is where the defect is in Niemann-Pick disease.” Somehow, the defective protein hampers the cholesterol's movement through the cells and the feedback mechanisms that control its level. “It's a protein that's screaming for attention,” says Liscum.

    Already, an examination of the NPC1 protein has revealed a stretch of amino acids where it resembles other proteins known to interact with cholesterol to regulate its level. Another section of the NPC1 protein looks as if it may somehow interact with the lysosome. In between are 16 regions with sequences that indicate, says Carstea, that “this [protein] is locked in a membrane” somewhere inside the cell.

    Making sense of those clues will take more study, but in the meantime, the gene may lead to other trafficking molecules. “There could be a whole family of these proteins,” says Goldstein. For example, a very small subset of people with Niemann-Pick type C disease have a different genetic defect, located on another chromosome. It affects a protein that seems to function as a trafficking molecule, much like the NPC1 protein.

    The identification of the NPC1 gene may also be a step toward developing a treatment. “We're hopeful that having the gene will let us get at a therapy faster,” says Liscum, who hopes to use cell cultures and mice to screen for drugs that might slow cholesterol buildup in Niemann-Pick children. She cautions, however, that “it's going to be a long time coming in having an impact on kids.”

    While Cindy Parseghian realizes that the new gene find may not help her daughters, she thinks it may help other family members, by leading to a way to test for the mutant gene in those who do not have symptoms, including her one healthy son. In the meantime, she and her husband make the best of the time they have with their daughters. “We can look at our children as if they were dying, or we can look at them as though they are living,” she points out. “They look at life as if they are living, and that's what we try to do.”


    Accelerator Probes the Private Lives of Nucleons

    1. Andrew Watson
    1. Andrew Watson is a science writer in Norwich, U.K.

    Census data can tell us much about how the average person lives, but they inevitably miss the interplay between individuals that ultimately makes our societies tick. Nuclear physicists face similar difficulties in their attempts to fathom the crowds of particles that populate the atomic nucleus. Nuclei contain as many as a few hundred protons and neutrons, collectively known as nucleons, as well as a sea of other particles that bind the nucleons together. The interaction of so many particles is immensely complicated, so physicists have had to content themselves with models that average out nucleon behavior.

    Hard knock.

    An electron dislodges a close-knit pair of protons from a nucleus.

    Now, however, a team at NIKHEF, the Netherlands's National Institute for Nuclear and High-Energy Physics in Amsterdam, has been able to coax pairs of protons out of the nucleus just at the moment when their interactions are at their most intense, revealing the intimate relationships that determine how the nucleus works. “What we are trying to learn is how the fact that the two nucleons are bound in the nucleus influences their mutual interaction,” says NIKHEF's Gerco Onderwater.

    Other nuclear physicists are thrilled to have this intimate glimpse of nucleons, which the Dutch team offers in the 30 June issue of Physical Review Letters. “The NIKHEF experiment demonstrates that it will be possible to learn about how protons interact with each other inside the nucleus,” says Wim Dickhoff of Washington University in St. Louis. Adds Carlotta Giusti of the University of Pavia in Italy: “This is a fundamental milestone in the understanding of nuclear structure.”

    Each nucleon is made up of three quarks bound by the strong nuclear force. This force also holds nucleons together as they exchange force-carrying particles called mesons. However, calculating the details of how the hundreds of nucleons and mesons interact is impossibly complex, so physicists fall back on statistics. They define a single, averaged force field, or mean field, which describes how a nucleon interacts with its averaged neighbors. Mean-field models “are able to reconstruct and explain a lot of details with enormous accuracy in nuclear structure,” says Onderwater. “Until now, these mean-field approximations worked fine.”

    But experiments are beginning to reveal cracks in these models. According to Onderwater, experiments using electrons to knock out single protons only seem to see about 65% of the proton population in the nucleus. Moreover, the momentum of many of the knocked-out protons is unacceptably large according to mean-field models, which predict that such boisterous nucleons should spontaneously fly out of the nucleus. “A proton in the nucleus spends only a fraction—albeit a sizable one—of its time in this average field,” says Dickhoff. “In fact, because it interacts strongly and collides with other nucleons, it spends about 35% of its time doing ‘other’ things.”

    Theorists are now attempting to patch up the mean-field model by incorporating short-range correlations (SRCs) between nucleons, describing how nucleons influence only their nearest neighbors and not the nucleus as a whole. The main short-range effect is a switch from attractive to repulsive forces when nucleons are very close together. The repulsion ultimately keeps nucleons apart and prevents nuclei from collapsing to a fraction of their normal size.

    In this picture, nucleons consist of a repulsive quark core surrounded by a cloud of attracting mesons. “The high energies and momenta arise from violent collisions in which the quark cores touch each other,” says Derek Branford of the University of Edinburgh in the United Kingdom. The NIKHEF experiment, he says, sets out to test this image. “It is designed to catch two protons at the time they are so close that the quark cores are touching, and knock them out of the nucleus.”

    To do this, the NIKHEF team modified its Amsterdam Pulse Stretcher electron accelerator, transforming its beam of brief electron pulses into a more or less continuous beam, to increase the chances of catching close interactions. The beam, which has an energy of 584 mega-electron volts, is trained on a water target surrounded by three detectors. One captures electrons that bounce from the target, while two newly built proton detectors track protons knocked from oxygen nuclei in the target. A scattered electron and two protons emitted all at once signal a rare double knockout event; the group spotted about 5000 of these in all.

    From the momenta and energies of the scattered electron and emitted protons, the researchers can determine the protons' original relationship inside the nucleus. Particular patterns of momentum mark proton pairs that were originally close neighbors, on the point of being flung apart by the repulsion between their quark cores.

    Focusing on those events that involved closely interacting protons, Onderwater and his colleagues were able to reconstruct the energy states of the original nuclei, which correspond to the energy of the closely interacting proton pairs. “This is the first experiment of this kind that is really able to make a distinction between the different states,” he says.

    By comparing their results with recent calculations that attempt to accommodate SRCs, Onderwater's team concludes that about 70% of the proton pairs they detected were originally interacting very closely. The finding supports a picture of the nucleus in which many nucleons have a higher momentum than a mean-field approach alone would predict. That would explain why earlier single-proton knockout experiments, looking at only low momenta, could not see many of the protons.

    The technique should eventually reveal even more details about SRCs, says Onderwater. “It's aimed at providing more information on how the mean-field model should be extended in order to incorporate this strong repulsive core in [nucleons],” he says. Soon, the private lives of nucleons may be a mystery no more.


    One Molecule Orchestrates Amoebae

    1. Trisha Gura
    1. Trisha Gura is a science writer in Cleveland.

    For a mere soil-dwelling amoeba, Dictyostelium manages some impressive feats of organization. When food is scarce, individual cells band together with their comrades to form a mound that crawls to better pastures and disperses by building a stalk and a spore body. Researchers have struggled for years to understand the signals that coordinate this multifarious behavior, hoping to gain insights into the development of more complex organisms. Now, with a paper in this issue, the picture has been dramatically simplified.

    Migratory slugs.

    Dictyostelium mutants lacking cAMP (top) behaved like normal organisms. (Bar equals 0.25 millimeter.)


    On page 251, Bin Wang and Adam Kuspa of Baylor College of Medicine in Houston show that a single molecule—an enzyme called PKA—can single-handedly drive all steps of development: clumping together; forming the cell cluster or slug; and differentiating into stalk or spore cells. The finding narrows the role of another molecule, cyclic AMP (cAMP), which activates PKA and can also act as a signal in its own right. Researchers had suspected that cAMP might work via many other pathways, says longtime Dictyostelium researcher William Loomis of the University of California, San Diego (UCSD). Now, “the paper shows that essentially all of the internal cAMP responses are mediated by PKA.”

    Besides telling Dictyostelium researchers that they needn't look for alternative cAMP signaling pathways to drive development, the finding also has an important message for researchers studying higher organisms. There, PKA and cAMP are key actors in everything from embryogenesis in fruit flies to memory in mice, and the new finding suggests that PKA may have the leading role. Says Loomis, “The universality of this is very important.”

    For decades, cAMP has been the center of attention in the study of Dictyostelium development. The molecule, synthesized by an enzyme called adenylyl cyclase, initially seemed to have many faces. Inside individual cells, it binds to PKA and sparks it into action to trigger cell differentiation. Outside the cells, it acts as a molecular magnet, drawing single cells into well-integrated structures. Many other roles for cAMP had been proposed, but no one had been able to pin them down or gauge their importance.

    Kuspa and Wang had run across mutant amoebas that hinted that cAMP might be less crucial than many researchers had thought. These mutants had inactive adenylyl cyclase enzymes, yet they managed to develop normally. Given the many open questions about cAMP's roles, Kuspa says he and Wang, a graduate student in his lab, decided to take a closer look at such mutants. “We had been staring at the data for 2 years, and we just had to test this the best way we knew how.”

    They began with a well-characterized adenylyl cyclase mutant—one that essentially made no traces of the enzyme—provided by Peter Devreotes at Johns Hopkins University in Baltimore. Next, the Baylor researchers genetically engineered the mutant so that its cells produced high levels of a mutant PKA that was permanently turned “on,” even without cAMP to activate it. The Baylor team then watched how these microorganisms developed over the course of 24 to 30 hours.

    To their surprise, the mutants looked and behaved virtually the same as normal organisms. Moreover, when the researchers tested the activity of specific genes that are turned on and off at particular times in Dictyostelium development, they observed a normal pattern. “The cell-type specific genes were turned on at the same time as wild-type,” says Kuspa. “That was probably the most shocking thing.”

    Not only was the cells' internal signaling taking place normally, but the collective behavior of the cells also appeared normal. They bunched together and formed stalks and spores—events that researchers had thought could only be orchestrated by pulses of cAMP. Only when the density of cells was low did the lack of cAMP seem to prevent the mutants from building normal mounds.

    “The intriguing thing is that the organism can bypass a number of events that normally require cAMP in the wild,” says developmental geneticist Richard Firtel of UCSD. Kuspa believes the mutants can get around the need for the chemical signal by bumping into each other, triggering alternative signaling pathways that require only PKA.

    At first sight, the results appear at odds with Firtel's own findings. He and his colleagues had created similar mutants and found that they failed to develop normally. But Firtel thinks the discrepancy may stem from “too much of a good thing.” While Kuspa's mutants produced twice the normal levels of PKA, Firtel's organism overproduced the enzyme in much greater amounts. “It just means that you need a specific amount of PKA … to produce the observed phenotype,” he says.

    If PKA in just the right amount can serve as a master coordinator of development, “the subtleties go way beyond Wang and Kuspa,” says Loomis. “The findings could be used as a guideline for studies of all vertebrate and invertebrate development—in addition to being fascinating for Dictyostelium in and of itself.”


    Upstart Ice Age Theory Gets Attentive But Chilly Hearing

    1. Richard A. Kerr

    Selling a new gizmo to replace a longtime favorite is always a tough job—especially if few are convinced that the original is broken. Physicist Richard Muller of the University of California, Berkeley, knows what it's like: For several years, he has been pitching a new way to drive the comings and goings of the ice ages. He's trying to displace the cherished Milankovitch mechanism in which cyclical changes in the elliptical shape of Earth's orbit shift the pattern of solar heating, triggering the buildup or melting of ice sheets.

    Battling curves.

    The single cycle of Earth's changing orbital inclination (green) seems a better match to climate (red) than the multiple cycles of orbital eccentricity (blue).


    Now, on page 215 of this issue of Science, Muller and geophysicist Gordon MacDonald of the International Institute for Applied Systems Analysis in Laxenburg, Austria, present what they consider their strongest statistical evidence yet that the ice ages are instead driven by a different orbital mechanism—changes in the inclination of Earth's orbit relative to the plane of the solar system. These orbital shifts would periodically dip Earth into a climate-altering cosmic dust cloud, Muller theorizes. Applying a statistical technique called spectral analysis to new climate records, he and MacDonald now offer evidence that changing orbital inclination provides a better fit to the cyclical timing of the ice ages than traditional Milankovitch mechanisms.

    So far, their radical idea is winning only scattered, cautious support. Oceanographer John Imbrie of Brown University, who in the 1970s helped convert his colleagues to the Milankovitch mechanism, says, “The statistical evidence [for inclination] is very good. [It] might play a role.” But most of the paleoclimate community isn't ready to dethrone their well-established favorite without a fight. Some take issue with the statistical methods, while others point out that there's no known—or as yet imagined—way for interplanetary dust clouds to plunge Earth into an ice age. At a small workshop held last year to give Muller a platform, the general conclusion was that “his arguments would just not sell,” says geophysicist and climate modeler Richard Peltier of the University of Toronto. Even Muller sees a long road ahead: “I know it's an uphill battle,” he says.

    To fight that battle, Muller and MacDonald are enlisting the same climate records that paleoclimatologists have been dissecting for the past 15 years. These consist of oxygen-isotope compositions of microfossils deposited on the sea floor during the past 600,000 years. The changing ratio of oxygen-18 and oxygen-16 isotopes reflects the changing volume of glacial ice around the world. Muller and MacDonald applied a statistical technique called spectral analysis to the oxygen-isotope records from eight sediment cores at sites around the world's oceans; the method can pick out distinct cycles from a record jumbled by other cycles and noise. They found that ice volume waxed and waned with a single period of about 100,000 years—exactly the period over which the plane of Earth's orbit gently nods a few degrees above and below the plane of the solar system.

    A single climate cycle of 100,000 years doesn't quite match what the Serbian astronomer Milutin Milankovitch predicted in 1941. Milankovitch and later workers calculated that gravitational perturbations of the planets would cause the shape of Earth's orbit, or its eccentricity, to vary with three different periodicities: 400,000, 125,000, and 95,000 years. If these eccentricity variations translated directly into climate variations, spectral analysis of the climate records should reveal three cycles centered on those three frequencies. However, the 400,000-year peak—predicted to be the strongest—has never shown up in records of ice volume. Since the 1970s, other workers who saw signs of a 100,000-year cycle assumed that it was the signature of the other two eccentricity cycles blurred into one. But Muller says it's simply a clear signal of orbital inclination. “Inclination is a good match to the data and eccentricity really isn't,” says Muller. “That's just a devastating problem.”

    But not everyone is wowed by Muller and MacDonald's application of spectral analysis. “I was never impressed with that whole argument,” says mathematician David Thomson of Lucent Technologies' Bell Labs Innovations in Murray Hill, New Jersey, a specialist in time-series analysis. With a short climate record—and there are only six or seven 100,000-year cycles in the record—the power of spectral analysis is greatly diminished, he says. Pinning down precise frequencies or even distinguishing two cycles of similar frequency is problematic. Imbrie agrees. “Different people can come up with different spectra” from the same data, he says. “I don't think that's the best way to look at it.”

    Imbrie and others favor an ice-age-by-ice-age comparison of the isotopic record and orbital perturbations instead. Muller and MacDonald did just that last year in a paper in Nature and found that times of maximum inclination consistently preceded glacial maxima, albeit by a hefty 33,000 years. But a similar study by paleoceanographer Maureen Raymo of the Massachusetts Institute of Technology favors eccentricity.

    In work forthcoming in Paleoceanography, Raymo calculated the effect of eccentricity combined with that of two other Milankovitch orbital variations, the tilting of Earth's axis of rotation (which has a period of 41,000 years) and the wobbling of the axis (which has a period of 23,000 years). She compared 11 lengthy isotopic records with the predicted effects of these combined orbital variations on climate. Milankovitch had reasoned that summer temperatures in high northern latitudes, where the great ice sheets formed, should affect whether each year's snows melted away or accumulated into a glacier. Raymo found that the ice ages consistently ended after the combined Milankovitch variations created a period of warm summers in these latitudes. That convinces her that the Milankovitch cycles were indeed triggering the ice ages—and that “orbital inclination has nothing to do with it.”

    Imbrie notes, however, that “the two theories are not mutually exclusive.” He too has done a termination-by-termination comparison and finds that both eccentricity and inclination have kept the same pace as the waxing and waning of the ice. “There is a surprisingly strong statistical match for inclination,” says Imbrie. “The problem is far from solved.”

    While the statistical arguments run on, other paleoclimatologists see an even larger problem with Muller's idea: How could a changing inclination send Earth into a deep freeze? If inclination is to be proven, says paleoclimatologist André Berger of the Catholic University of Louvain in Belgium, “we must find a physical process that will convince me that the inclination is the key factor in creating the 100,000-year cycle. The only answer for me is the physical mechanism.”

    The only possible agent of climatic change Muller can cite is the dust that asteroids and comets spew across the solar system. Asteroid dust remains largely in a disk across the solar system, and Earth periodically dips in and out of that disk as inclination varies. This interplanetary dust filters into Earth's upper atmosphere, but it has no known influence on climate. “I have to confess, I don't have a physical mechanism that works,” Muller admits. “But part of the problem is our ignorance of the atmosphere and [the dust in] the nearby solar system.”

    Muller also points out that the physical mechanism behind the Milankovitch cycles—the amount of sunshine reaching high northern latitudes—is far from perfect. “The only real defense I have,” he says, “is that if you look carefully at the Milankovitch model, the physical mechanism there was always hand waving, too.” Indeed, paleoclimatologists have long recognized that the amount of Milankovitch-induced change in solar heating is too small to melt glaciers or to send Earth into a deep freeze, unless some as yet unidentified part of the climate system amplifies it.

    Still, even a partial physical mechanism is better than none, researchers say. “Even though you can't pin down exactly why Earth's climate responds to Milankovitch [orbital] cycles, at least there is some physical connection, whereas Rich Muller has none,” responds oceanographer Wallace Broecker of Columbia University's Lament-Doherty Earth Observatory, who in 1970 first found a 100,000-year isotopic cycle and noted that it was roughly in step with eccentricity. “It would be hard to believe this tiny influx of dust could be having such a profound effect on climate.”

    “I think the chances that the 100,000-year cycles are due to inclination are exceedingly small,” says Broecker, but the challenge to Milankovitch “has been healthy, because [Muller] made everybody stand up and defend what they thought. It's a fascinating problem—but unresolved.”


    Sizing Up Dung Beetle Evolution

    1. Wade Roush

    BOULDER, COLORADO—Imagine 120-kilogram antlers on an 800-kilogram moose, and you will have an idea of the outlandish proportions of the horns on some male dung beetles. The horns, which males of the genus Onthophagus use to repel competing suitors, have awed evolutionary biologists since Charles Darwin. Among other things, biologists have wondered what keeps the horns from getting even bigger. New experiments on these beetles now confirm a long-standing suspicion about such exaggerated traits. Horn size is limited, the studies show, because horns exact a high cost during the beetles' development: the bigger the horns, the smaller other nearby body parts.

    Horns aplenty.

    Male dung beetles with short horns (left) have relatively big eyes, but big-horned beetles have tiny eyes.

    D. EMLEN

    Biologists from Darwin on have speculated about these kinds of trade-offs between body parts, but researchers had found few concrete examples. At the annual evolution meetings here,* however, evolutionary biologist Douglas Emlen of the University of Montana, Missoula, reported selective breeding and hormone experiments showing that boosting the development of Onthophagus males' horns causes the beetles to form smaller eyes; artificially restricting horn size leads to bigger eyes. This link, Emlen said in his talk, suggests that horns are indeed costly—and that beetles who squander too much on them are likely to be blind sided by natural selection.

    The work also supports the notion that some changes in development may be a “zero-sum game,” with every enhancement in one trait compensated by a reduction somewhere else, says Hugh Dingle, an evolutionary biologist at the University of California, Davis. “What Doug's work shows so nicely is that these [constraints on evolution] involve—as people have been predicting for a long time but haven't been able to show—clear trade-offs between structures.”

    Emlen, who won the American Society of Naturalists' Young Investigator Prize for his study, began his beetle work as a graduate student at Princeton University. He found that Onthophagus beetles have a developmental switch that turns on horn growth only if the larva has acquired enough reserves, such as body fat, before it metamorphoses into an adult beetle. Without enough fat, the larva has to save its resources to produce other adult structures and can't afford to make horns at all.

    To test Darwin's suspicion that neighboring structures compete for finite resources during development, Emlen restudied the beetle specimens of his graduate work while doing a postdoctoral stint in the lab of Duke University entomologist Frederik Nijhout. He found that in Onthophagus, which has horns that develop just above the eyes, males with big horns did tend to have proportionally smaller eyes. “But this was just a correlation,” as Emlen says; demonstrating an actual developmental trade-off would require manipulating the system.

    So he and Nijhout applied juvenile hormone, which delays metamorphosis in beetles, to the beetle larvae. This postponed the onset of horn growth, producing adult males with shorter horns than untreated males. The only other trait that differed systematically between treated and control beetles was eye size: Short-horned beetles had bigger eyes.

    Emlen had no comparable method to artificially make horns bigger, but he bred the beetles for seven generations and selected for large horns. The big-horned products of such selections had tiny eyes, he found, with fewer units in their compound eyes and, thus, a restricted field of vision. Carried to an extreme, Emlen says, big horns could even lead to blindness. That demonstration, says University of Maryland, College Park, evolutionary biologist Gerald Wilkinson, is “a neat trick. … People have been writing papers about these organisms for 100 or more years, but they have all been comparative” rather than experimental.

    Of course, this is just one case, and researchers exploring the links between evolution and development want more examples (Science, 4 July, p. 34-39). But Emlen and Nijhout say that dung beetles can likely help out. For almost every body part, there's a horn sprouting close by in one dung beetle species or another, says Emlen: “The huge variation in the morphology and location of horns should give us a big opportunity to predict how development constrains evolution.”

    • * Joint meetings of the Society for the Study of Evolution, the American Society of Naturalists, and the Society of Systematic Biologists, Boulder, Colorado, 14–18 June.

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