News this Week

Science  07 Mar 1997:
Vol. 275, Issue 5305, pp. 1415
  1. Embryology

    Will Dolly Send in the Clones?

    1. Elizabeth Pennisi,
    2. Nigel Williams

    The first mammalian clone, produced from an adult sheep, took the world by storm, but leaves a rash of unanswered scientific questions in her wake

    No longer will the name Dolly bring to mind Carol Channing or Barbra Streisand, leading ladies in the musical, “Hello, Dolly,” or even the vivacious country western singer, Dolly Parton. Last week, a new Dolly—a 6-month-old lamb cloned from the udder cell of an adult ewe—made her debut. And even though animal scientists have been cloning sheep and cattle from embryos for a decade, the media went wild over Dolly, the first animal ever cloned from an adult cell.

    The page-one headlines heralding Dolly's creation ignited worldwide concerns about the potential of this approach for cloning people. Within days, there were calls for ethics inquiries and new laws to ban human cloning (see sidebar). Concerns that human cloning would soon follow were further heightened when The Washington Post reported on 2 March that Don Wolf's team at the Oregon Regional Primate Research Center in Beaverton had cloned two rhesus monkeys, although from embryonic cells. But even as the media frenzy continues, researchers say it's still unclear how practical cloning of animals, let alone humans, will be.

    Clone craze.

    Dolly became an instant media star.

    AP/Newsflash

    As reported in the 27 February issue of Nature, Ian Wilmut and Keith Campbell at the Roslin Institute in Edinburgh, Scotland, cloned Dolly by transferring the nucleus from an udder cell into an egg whose DNA had been removed—an approach that could lead to flocks of prize animals with a genetic makeup guaranteed to match that of the adult donating the cell or of animals that produce valuable human proteins for therapeutic use.

    But the procedure is quite inefficient. The Roslin group made 277 attempts in order to succeed with Dolly. And no one knows either how DNA from the udder cell was able to direct the development of an entire new organism, or whether the same will prove true in other species. “There are lots of questions to sit down and look at,” says embryologist David Whittingham, director of the Medical Research Council's (MRC's) Experimental Embryology and Teratology Unit in London.

    Until this report by Wilmut and Campbell, a great deal of evidence had indicated that while species ranging from frogs to mice to cattle and now monkeys can be cloned by transferring nuclei from embryonic cells, the DNA of older cells was irreversibly altered. Presumably, because of chemical changes and structural modifications, those genomes were supposed not to be “totipotent,” that is, capable of supporting the development of all the different cell types needed to build an animal. For example, no one could get mice to develop reliably when they used nuclei from anything but one-, two-, or four-cell mouse embryos.

    The trick behind the Roslin team's success, Wilmut says, was to make the DNA of donor cells behave more like the inactive DNA of a sperm or unfertilized egg. They did this by reducing the nutrient-laden serum supplied to the cells, in effect starving them into the dormant G0 or G1 stages of the cell cycle. The deprivation caused many genes to shut down and ensured that the DNA had not just replicated when it was transferred. The researchers then administered an electric current to fuse this donor cell with an egg whose own chromosomes had been extracted.

    The fusion provided the egg with a full complement of new DNA and triggered the development of the egg. The first three divisions of the sheep egg replicate its DNA without expressing any of the new genes: Proteins and messenger RNAs already in the cytoplasm do all the work required for division. While the DNA goes along for the ride, Wilmut says, it loses the proteins that came attached to it and takes up others from the cytoplasm. At the same time, it apparently becomes “reprogrammed” so that the embryo can develop normally.

    Those multiple replications, and the several days it takes for them to occur, may be the reason nuclear transfer works in sheep but not very well in mice, suggests Richard Schultz of the University of Pennsylvania, whose own work focuses on gene expression in early development. In mice, all DNA remodeling takes place in the first cell division and the new DNA takes over by the two-cell stage, rather than in the eight-cell stage as in sheep. “Maybe in rodents there's just not enough time [for reprogramming],” he says. (In humans embryos, the new DNA apparently takes charge after the four-cell stage, in between mice and sheep or cows.)

    Or it may be that Dolly's DNA didn't require much reprogramming. Her DNA came from cultured mammary cells, which are normally capable of developing into lactating tissue. Wilmut and his colleagues acknowledge that the collection of cells may have included a stem cell—an undifferentiated progenitor cell of many different tissue types—which has a higher developmental potential than an ordinary epithelial cell from the mammary gland. “The udder cells are a mixed population, and we don't know which are able to be totipotent,” comments human geneticist Nick Hastie of the MRC's Human Genetics Unit in Edinburgh, U.K.

    But assuming reprogramming did occur, its efficiency was low. The sole successful transfer out of 277 attempts “may say this is still a very difficult task in terms of successfully completing the reprogramming,” Schultz points out.

    Efforts to increase that success rate may run into another barrier, he adds: “We don't really know” how programming occurs normally during development. This makes understanding deprogramming difficult, although it likely involves reversal of chemical modifications, such as methylation and acetylation, that the DNA and its associated proteins undergo as cells take on specialized functions. Also, some reprogramming may occur when DNA is stripped of its old packaging proteins and repacked with new ones in the egg's cytoplasm—a process that also occurs with the DNA of a fertilizing sperm. “We need to spend a significant amount of effort in the near future in understanding that mechanism [of how the egg interacts with its new DNA],” says James Robl, a developmental biologist at the University of Massachusetts, Amherst. However, it may now be possible to study how that programming occurs by examining the molecular conversation that goes on between the egg and the transferred nuclei.

    And the reprogramming is just one aspect of cloning that can go wrong. Many subtle differences exist between mammalian species in how they develop during those first few days. Not only do they differ in how quickly the new DNA takes charge, but they also vary in how they decide to implant in the uterus and develop a placental connection. These differences could make nuclear transfer from adult cells harder, if not impossible, in animals other than cows or sheep, suggests Zena Werb, a developmental cell biologist at the University of California, San Francisco. Also, in livestock, past efforts to clone embryonic cells have tended to produce oversized, delicate young that required extra care if they were to survive, notes embryologist George Seidel of Colorado State University in Fort Collins. The same may prove true of the new procedure.

    But the tantalizing possibility of making identical copies of prized livestock, or even of animals used for research, will be too exciting to pass up, says Robl, who helped form a company 3 years ago to take advantage of these advances in cloning technology. Add to that the prospect of cloning genetically modified animals that can produce drugs or better milk, meat, or wool, Robl says, and “tomorrow, next year, this field is going to be so crowded.”

    Yet, even if few or none of the potential applications come to pass, Dolly will forever have her place in history. As Werb points out, this lamb's creation “is the category of experiment that bends your mind.”

  2. Embryology

    Cloning Sparks Calls for New Laws

    1. Nigel Williams

    The news of the first successful cloning of an adult mammal, a sheep (see main text), has sent ethical shock waves around the world. As a result, in many countries, officials and even some scientists are calling for new or strengthened legislation to outlaw human cloning, although at this early date, no concrete measures have been proposed.

    In the United States, which currently has no law prohibiting the procedure, President Clinton announced an urgent inquiry into the potential ethical and legal implications by the National Bioethics Advisory Commission, which will report its conclusions by the end of May. Meanwhile, Clinton has banned federal funding for human cloning research and asked for a moratorium on nonfederally funded efforts. In addition, both houses of the U.S. Congress are holding hearings on the issue. In China, geneticist Zhang Jiaming, who was a delegate to last week's annual meeting of China's parliament, says he and other scientists in the legislature agree that new laws are needed to ban the cloning of humans.

    Many European countries already have detailed legislation covering human embryo experiments, but even there, the cloning success throws up potential new problems. Take the United Kingdom, which passed some of the most comprehensive legislation in this area in 1990, but has now found that changes may be needed. “We have prohibited the transplant of nuclei into embryos, but in these sheep experiments, the nuclei were transferred into eggs,” says Bea Heales, a policy manager at the Human Fertilization and Embryology Authority, which licenses research and treatment in Britain. “We may need a new policy that states such experiments in humans will not be licensed.” And Germany's current law on human experimentation may also have a loophole that permits cloning, some legal experts say. At the European level, Jacques Santer, president of the European Commission, has ordered an inquiry.

    Such is the scale of worldwide concern that moves have already begun to draw up international guidelines. The 40-nation Council of Europe, which includes countries outside the European Union, is currently developing a convention on human rights and bioethics. The council says work will start soon on a specific protocol on the protection of the embryo and the human fetus, which could include a ban on human cloning. “The cloning of an adult sheep may be an impressive scientific achievement, but it also demonstrates the need for firmer rules on bioethics,” says Daniel Tarschys, secretary-general of the council.

  3. Astronomy

    Farsighted Gravity Lens Sees Stars

    1. James Glanz

    Almost all stars are so distant that, even with the largest conventional telescopes, they appear as unresolved points of light, as featureless as the twinkling dots seen by an unaided observer on a clear night. Now, by using the gravity of one star as a huge magnifying glass, a team of astronomers has been able to make out features on the face of a second star located 30,000 light-years from Earth. The team found that the gravitational lens, which bends light rays as predicted by Einstein's theory of relativity, was aimed so precisely that it scanned across the face of the distant star, revealing details of its structure. “We have, in essence, obtained more than 8000 times better spatial resolution than the Hubble Space Telescope [HST],” says Andrew Becker of the University of Washington, Seattle, one of 57 astronomers from nine countries who collaborated in the study. The team announced its findings at a conference this week at the University of Notre Dame in Indiana.

    Becker quickly adds that gravitational lensing requires the chance, near-perfect alignment of Earth and two stars, and so is much less versatile than the orbiting HST or conventional ground-based telescopes, which can be pointed anywhere in the sky. Still, says astronomer Virginia Trimble of the University of California, Irvine, who is not part of the collaboration, this use of gravity to peer at an object so far away “is obviously enormously exciting.” The detail it reveals on distant stars, say other researchers, should help astronomers firm up computer models of how stars grow old and die.

    The multinational collaboration that first noticed the event goes by the acronym MACHO, for Massive Compact Halo Object. MACHO's principal aim is to use gravitational lensing to search for dark blobs of matter, such as burnt-out stars or black holes, that might be swarming in a shadowy halo around our galaxy and making up most of its overall mass, as some theories predict. The project does this by constantly monitoring stars in a nearby galaxy called the Large Magellanic Cloud with a telescope at the Mount Stromlo Observatory in Australia, seeking sudden brightenings—a signal that a star has been magnified by the gravity of an unseen object in the halo. By keeping track of these events, MACHO hopes to estimate the overall amount of this kind of dark matter.

    So far, MACHO has reported eight such brightenings, and is sitting on “a few” new ones for which the analysis hasn't been completed, says David Bennett, a team member at the University of Notre Dame. Settling the dark matter issue could take years, but while that program inches forward, the team also monitors stars near the Milky Way's more crowded central bulge. Here, the chance alignment of two stars and Earth in a straight line—the prerequisite for observing a gravitational lens—is more common. Becker says the team has collected roughly 120 of these events, gathering statistics on how matter is distributed in the bulge and therefore on its shape.

    Most of those brightenings follow a standard “light curve” of increasing and then decreasing brightness with time. Such a curve suggests that the degree of alignment—and of magnification—was slight, causing a modest jump in the star's apparent brightness, but not enough to resolve it beyond a point source. But in the case of the event called MACHO Alert 95-30, says Becker, deviations from that curve showed that the alignment was so perfect that “the lens passed across the face of the star,” like a detective scanning a body with a magnifying glass.

    As the brightness started increasing and MACHO issued the worldwide alert, says Bohdan Paczynski of Princeton University, a “spectacular and successful” coordination of telescopic observations came into play. Called the Global Microlensing Alert Network, or GMAN, the program, whose astronomers were co-authors on the paper, collected spectra and light curves (see graphic) of the days-long brightening almost 24 hours a day. The result was a kind of transect, indicating how the spectrum of the starlight—a clue to composition and structure—changed from point to point across the star, which had been identified as a bloated red giant.

    A gravitational spyglass.

    A source star's brightness shoots up, then drops again as the gravity of a second star magnifies the signal through gravitational lensing.

    Source: MACHO-GMAN

    In this case, the MACHO team found that although the orientation of the stars was nearly perfect, the lens's focus passed along a line closer to the edge than the center of the red giant's face. But that was enough to make the first-ever comparisons between the calculated structure of an old, bloated star and actual observations, says Dimitar Sasselov of the Harvard-Smithsonian Center for Astrophysics (CfA) in Cambridge, Massachusetts. Calculations by Sasselov and CfA's Abraham Loeb showed how the spectral signatures of stellar constituents like hydrogen and titanium oxide should vary from the center of the star to its visible edge, where the line of sight passes through more of the star's atmosphere, “and that's exactly what's observed,” says Sasselov. Gravitational lensing, he says, “is giving us a fairly cheap and amazing way of studying the surfaces of distant stars”—and in greater detail than possible even for nearby stars with lengthy exposures on ordinary telescopes.

    That kind of sleuthing could help firm up calculations of the age of the Milky Way's oldest stars, which seem to be paradoxically older than the universe itself. The MACHO group may eventually achieve its original goal of finding the galaxy's dark matter. Meanwhile, says Trimble, “it's impressive not only that the thing works, but that it's doing things it wasn't even designed for.”

  4. Wildlife Biology

    In Search of Africa's Forgotten Forest Elephant

    1. Laura Tangley
    1. Laura Tangley is a science writer in Washington, D.C.

    Say “African elephant,” and one pictures a vast savanna, where the largest land mammal mingles with lions, giraffes, gazelles, and zebras. In fact, about one-third of the continent's elephants live in its dark, often inaccessible rain forests, where the creatures are difficult for researchers to spot, let alone study. As biologist Claude Martin wrote in The Rainforests of West Africa, “Whoever is lucky enough to spy a forest elephant with his own eyes must satisfy himself with a few seconds of gray skin or a bit of white tusk shimmering behind the leaves.”

    But now a wealth of new studies is bringing these huge, elusive creatures out of the shadows. Scientists analyzing everything from elephant dung to DNA are piecing together the basics of forest elephant biology. With the forest elephant's rounder ears and thinner, straighter tusks, most taxonomists have long thought of it as a separate subspecies from its larger savanna cousin. But new analyses of mitochondrial DNA suggest that the two may be different species entirely (see sidebar). Studies of the forest elephant's diet suggest that the animal plays a crucial role in rain-forest ecology, dispersing the seeds of many fruiting trees. Ongoing research also is unraveling the secrets of their social behavior. Biologists have found, for instance, that unlike savanna elephants, the forest dwellers live in small groups. But like savanna elephants, they maintain dominance hierarchies and seem to mourn a family member's death, an event that scientists fear is becoming ever more common as an upsurge in commercial logging in Africa opens once-remote forests to poachers.

    It's hard to blame scientists for overlooking the forest elephant. Why hike into an uncomfortably wet, hot habitat, when savanna elephants—one of the world's best studied mammals—can be easily observed from a jeep? The debate that preceded the 1990 ban on international ivory trade helped spark this new wave of research. Without knowing how many elephants were living in the forests of Africa, scientists found it hard to assess the overall impact of the trade. “Our ignorance of forest elephants was appalling,” says Richard Barnes, a visiting scholar at the University of California, San Diego, and a member of the World Conservation Union's African Elephant Specialist Group.

    Forest elephants range across more than a million square kilometers of dense forest and cannot be seen from airplanes, so estimating their numbers was a daunting task. But with support from the Bronx, New York-based Wildlife Conservation Society (WCS), Barnes and his wife, Karen Barnes, developed the first standardized method for gauging elephant populations by counting dung piles along transects and inserting the results into a mathematical formula that considers rates of defecation and dung decay.

    The researchers first used the methodology in Gabon, where in 1993—after 2 years of hard, sweaty labor deep in the forest—they concluded that about 60,000 elephants lived. Over the next few years, WCS sponsored additional forest elephant surveys, and “today we have a pretty good idea where they live and of their relative abundance,” says Richard Barnes. Of the continent's roughly half-a-million elephants, between a quarter and a third are forest elephants, the vast majority living in central Africa. The rest hang on in West Africa's very fragmented forests.

    Elephant dung has also provided clues to what the animals eat and their role in forest ecology. Lee White, a WCS conservation scientist, says that unlike savanna elephants, which feed mostly on grass, forest elephants subsist on the leaves, twigs, bark, and fruit of rain-forest trees: “Their diet is much more like that of a gorilla than a savanna elephant.” In Gabon's Lopé Reserve, White found that elephants eat 80 different kinds of fruit and will travel 50 kilometers or more to feed at a favored tree.

    He and others contend that many fruiting trees depend on elephants to disperse their seeds. Seedlings of the makore (Tieghemella heckelii), for example—found only in Liberia, Côte d'Ivoire, and Ghana—were abundant along elephant trails in Ghana in the 1950s. Botanists who returned to the same sites in the 1970s, however, found no young makore trees, and many believe that as the elephants were killed off by hunters, the tree could no longer disperse its seeds. White has identified a total of 25 species he believes rely exclusively on elephants for seed dispersal, and he estimates that in central African forests as a whole, there may be at least 50 such trees.

    One example is Omphalocarpum sp., whose fruit, says White, is “the size of a human head and as hard as a human skull—nothing but an elephant could break it open.” Like many fruits consumed by elephants, this one is dull in color (elephants have poor vision), but it emits a strong, garlicky odor and makes a tremendous thud when it falls, appealing to an elephant's two most important senses. The animals gobble up virtually all Omphalocarpum fruit in Lopé, and a single dung pile may contain hundreds of intact or germinating seeds.

    The patchy distribution of fruit trees in a forest may help explain differences in group sizes between forest and savanna elephants. Savanna elephants live in groups ranging in size from about eight to more than a hundred, but White and other researchers rarely see more than two or three elephants together in the forest. “A hundred elephants cannot feed under one rain-forest tree, but two to three easily can,” says White.

    More clues about the elephants' social behavior are emerging from the dense rain forests of southwestern Central African Republic. Since 1990, Andrea Turkalo, a research fellow with WCS, has been observing elephants at a 250- by 500-meter clearing, known as Dzanga Bai, which attracts large numbers of animals that come to eat the soil's rich mineral salts. Turkalo identifies individual forest elephants by physical features such as scars, ear markings, and tusk lengths and then records their comings and goings. Fully 40 to 100 animals appear daily, and as of September 1996, she had identified and collected data on 2300 individuals.

    Sitting high above the clearing on a wooden platform, Turkalo has found that while forests elephants typically travel in smaller groups, they love to socialize. The animals enter the clearing in twos and threes, but they quickly disperse to greet and interact with others.

    Like savanna elephants, the forest males seem to be governed by a strict dominance hierarchy, and Turkalo believes that Dzanga Bai may be an important place for establishing and maintaining that hierarchy. “If a dominant male comes up to a water hole, a smaller one will get out of the way fast,” she says. In contrast to savanna elephant males, which form small bachelor groups, however, forest elephant males tend to be loners. Also, young males leave their mothers sooner in the forest. Turkalo attributes this difference to the presence of large predators in the savanna, such as lions, which may make solitary life dangerous for young males.

    Turkalo and others raise the provocative possibility that forest elephants, despite appearances, form large social groups whose members keep in touch over long distances through low-frequency sounds. Support for this notion comes from Turkalo's observations that some small elephant groups always seem to arrive at the clearing at the same time—apparently coordinating their movements—and from new research on elephant infrasound vocalizations. Previous studies in east Africa have revealed that infrasound is an important form of communication among savanna elephants. And recently, Steve Gulick, a WCS researcher based in northern Congo, recorded a forest elephant call with a very low frequency of 5 hertz. “It is possible that forest elephants rely on low-frequency sounds even more than do savanna elephants,” he says.

    Turkalo also says that, like their savanna cousins, forest elephants seem to form strong attachments. Mothers are very protective of their calves. And, last year, Turkalo saw one young female standing over the body of a dead male, “stroking him with her trunk,” for the better part of a day. The male had been shot.

    Death has been a rare event at Dzanga, but Turkalo counts the vulnerability of the elephants to poachers as a serious threat to the long-term success of her study. Thus far, a shortage of firearms locally, and perhaps her own presence, has kept poaching at Dzanga to a minimum. But just across the border in Congo, Turkalo's husband, WCS researcher Michael Fay, found 300 carcasses of illegally slaughtered forest elephants near a national park last September.

    Conservationists say a recent upsurge in commercial logging in central Africa also poses a serious danger to forest elephants. According to a report released this week by the Washington, D.C.-based environmental organization World Resources Institute, logging endangers 79% of Africa's large, intact forests. In West Africa, 90% of the rain forests have already been destroyed, and elephants there are confined to small, isolated populations that are vulnerable to inbreeding and to natural disasters such as drought and disease. According to Richard Barnes, these small populations would also be at risk should countries fall prey to civil unrest.

    Logging not only destroys forest habitat, however; it opens up once-remote areas to farmers and miners. And when this occurs, elephants can run into trouble with their neighbors. Throughout Africa, “crop raiding” has turned elephants into feared and hated pests. Last July, for example, 13 village chiefs from around Ghana's Kakum National Park marched on the nation's capital to protest park-dwelling, forest elephants that foraged in nearby fields.

    Efforts to find solutions are bringing together unusual allies. At a meeting last fall in Washington, D.C., at Conservation International, an environmental group, Ben Asamoah-Boateng, director of Kakum, met with Richard Barnes and Jack Birochak of the Pennsylvania-based Counter Assault Tactical Systems, a company specializing in personal safety devices. Birochak has designed a special capsicum pepper spray—a larger version of what city dwellers carry to deter muggers—that will be tested on the park's crop-raiding elephants.

    Some researchers contend that, in certain areas, crop-raiding claims may be exaggerated. In Gabon, cane rats and porcupines cause far more damage, White says, but are not nearly so hated by farmers. The difference, he says, is psychological. “An elephant may have raided your farm [only] once 10 years ago, but you never forget an elephant.”

  5. Wildlife Biology

    One Species or Two (or Three)?

    1. Laura Tangley

    The more researchers learn about the diet and social habits of Africa's forest elephant, the less it looks like its larger savanna cousin (see main text). Now, molecular genetics is adding to the differences: Recent studies of elephant DNA suggest that forest and savanna elephants, generally thought of as two distinct subspecies, may actually be entirely different species.

    Nicholas Georgiadis, director of the Mpala Research Centre in Nanyuki, Kenya, used a dart gun to collect skin samples from elephants in nine countries across Africa. Collaborating with Alan Templeton of Washington University in St. Louis, he analyzed the elephants' mitochondrial DNA by cutting it up with so-called restriction enzymes and looking for the distinct cutting patterns that reflect differences in DNA sequences. According to Templeton, the results show that forest and savanna elephants “represent distinct genealogical lineages that diverged about 3.6 million years ago.”

    That's enough time for two populations to evolve into separate species. But more analysis is needed to determine whether the forest and savanna lineages really are different species. Templeton says the next step is to look at nuclear DNA, which is inherited from both parents, unlike mitochondrial DNA, which comes only from the mother. Still, asserts Templeton, “from a conservation point of view, whether savanna and forest elephants meet all species criteria is moot. The two animals are different enough that they cannot be managed the same [way].”

    Georgiadis, meanwhile, is on the trail of a possible third species of African elephant. He recently returned to Kenya from Namibia, where he was collecting tissue samples from elephants that live in the desert. Like forest elephants, desert elephants look quite different from savanna dwellers—they are larger, for instance—and may represent yet another distinct evolutionary lineage.

  6. Paleontology

    How Reptiles Took Wing

    1. Bernice Wuethrich
    1. Bernice Wuethrich is a science writer in Washington, D.C.

    In 1910, a copper miner in central Germany picked up a nearly flawless fossil: an unusual reptile with a winglike fan of bones spreading from each shoulder. When he sold the specimen to Otto Jaekel, the premier German paleontologist, the miner labeled it “Flying Reptile.” But Jaekel thought the animal too improbable and removed the bones of the wings, believing they were the fin rays of a fish superimposed on the reptile. In later years, it turned out that the miner was right and the expert wrong. Now, a report in this issue of Science (p. 1450) makes clear just how bizarre a beast Coelurosauravus jaekeli—the earliest known flying vertebrate—really was.

    Winging it.

    Coelurosauravus's wings were supported by new bones that formed in the skin, rather than by modifications of existing bones.

    E. Frey

    By examining several new, exquisitely preserved specimens collected by amateur fossil hunters, Eberhard Frey and Wolfgang Munk of the State Museum of Natural History in Karlsruhe, Germany, and Hans-Dieter Sues of the Royal Ontario Museum in Toronto, show that this 250-million-year-old animal glided on a unique set of wings, unlike any others known in living or extinct animals. The long, hollow bones that strengthened these wings formed directly in the skin itself. “Coelurosauravus is totally bizarre because in every other animal that flies, wing support draws on the normal skeleton,” says Sues. For example, the wing bones of birds and bats are converted forelimbs.

    Strange as Coelurosauravus's wings are, they teach an important evolutionary lesson, says vertebrate paleontologist Robert Carroll of McGill University in Montreal: “We typically think of evolution as taking an existing structure and making some new function of it, but this animal has taken the capacity to produce bone and elaborated it in a completely unique way.”

    Carroll was the first paleontologist to identify C. jaekeli as a flying reptile, back in 1978. Based on the partial fossils then available, he interpreted the reptile's wing rods to be extensions of its ribs, like the wing struts of the modern gliding lizard Draco. Later, researchers suggested that the animal had hinged, two-part ribs. But in the early 1990s, Sues and Frey began to question this assumption. They reasoned that such a reconstruction required C. jaekeli to have a rib and a vertebra for each of its 24 to 28 wing rods, creating an animal with an implausibly long, flat trunk, stretched into a kind of “reptilian pancake.”

    Looking for a more plausible picture, they examined several beautifully preserved specimens, most of them owned or discovered by private collectors. Two partial skeletons showed that “the [wing] rods had nothing to do with the ribs,” Sues says. A nearly complete skeleton sold by an amateur collector to the museum in Karlsruhe offered the final proof. This fossil had only 13 vertebrae, but 22 rods on each side, which splayed out in bundles and were unattached to any other skeletal elements.

    Thus, the wings of this precocious flyer opened like an old Japanese fan, explains Sues. The bundles of bony rods formed directly in the skin and radiated from the shoulder area. When spread, the bundles extended to form two curved wings that could carry Coelurosauravus tens of meters. The long tail, which made up half of the 30-centimeter body length, may have added stability in flight.

    Carroll himself is now persuaded of this unusual wing structure. “The new German material is good enough that it should settle most of the arguments,” he says. “This demonstrates how early flight, even if not active, flapping flight, was achieved by vertebrates.” And the new understanding of C. jaekeli could also spur new insights into the mechanics and evolution of flight in other animals, adds Kevin Padian, a paleontologist and pterosaur expert at the University of California, Berkeley: “It may now prove interesting to take another look at later flying vertebrates that evolved independently.”

    Frey and his colleagues owe their close look at C. jaekeli to the work of several amateur fossil hunters, including a German house painter and a baker. They are among the hundreds who pore over the spoils of abandoned copper mines in central Germany's Kupfershiefer formation every weekend, splitting open blocks of copper-shale and noting their finds in detail. Because most of the fossils from this region are already well described, no professional paleontologists work these mines, says Sues, and amateurs have gathered almost all the known Coelurosauravus specimens. “Paleontology, unlike particle physics, is something an amateur can make important contributions to,” says Sues.

    Of course, relations between amateurs and professionals are not always so cordial. For example, given the surprising results on C. jaekeli, paleontologists would like to see what else may be learned from little-known groups such as the kuehneosaurs, rare, 225-million-year-old reptiles whose wings are thought to be adaptations of ribs. But researchers say that the best specimen, once held by the American Museum of Natural History, is now back in private hands—and unavailable for scientific study.

  7. Biophysics

    Double Helix Does Chemistry at a Distance—But How?

    1. Gary Taubes

    It's hard to be surprised anymore by DNA's repertoire of talents. It is a genetic archive with a remarkable combination of security and accessibility, a powerful probe that can seek out and bind to matching DNA molecules, even a potential computer. Now add yet another startling ability to the DNA résumé. In a paper in this issue of Science (p. 1465), chemists at the California Institute of Technology (Caltech) led by Jackie Barton present evidence that the DNA double helix can perform what they call chemistry at a distance. A DNA molecule with a chemical group artificially tethered to one end appears to mediate a chemical change far down the helix, causing a patch of damaged DNA to be mended.

    Wired?

    In DNA's core, base pairs form a so-called π stack (gray, shown in top and side views) along which electrons may tunnel from a distant site to an artificial electron acceptor (yellow).

    DANDLIKER ET AL.

    The DNA damage repaired in the experiment—a small kink in the helix known as a thymine dimer—is the kind of damage caused by the sun's ultraviolet rays, and it can be a first step toward the deadly skin cancer melanoma. While the chemical groups Barton and her colleagues used for their demonstration aren't found in the body, long-range DNA repair of some kind might play a role in normal cells, and Barton thinks the finding might point the way to therapies that could patch up damaged DNA after severe sun exposure.

    The result may point to an even more impressive attribute of DNA—if it means what Barton thinks it does. The ability of DNA to carry out long-range repair launches this paper into the heart of an already heated controversy over the possibility that DNA's unique structure allows it to behave like a conductive wire, utterly unlike the insulating behavior of proteins. The paper is the latest of four from Barton and her colleagues supporting the proposition that electrons can flow freely through the channel that runs down the center of the joined bases of the helix—in this case, traveling from the thymine dimers to the added chemical groups and repairing the dimers in the process. “There is no question that these results are saying DNA is a different system than proteins,” says Barton.

    If Barton is right and DNA readily transports electrons, the implications could go well beyond DNA repair. In living things, the transfer of electrons in DNA plays a crucial role in DNA regulation and other biological processes. And the technological possibilities are alluring as well: Knowing the precise electrical properties of DNA, says Georgia State University chemist Tom Netzel, could allow chemists to tailor artificial DNA molecules to serve as sensitive biological probes and minute photochemical machines.

    But Barton faces some determined skeptics. By taking a variety of different experimental tacks, her group has finally proved its case to the satisfaction of some colleagues. Columbia University's Nick Turro, for instance, who collaborated on the first of Barton's papers, says the four experiments taken together “show unambiguously that there's long-range chemistry that can be performed on DNA, and that electron transfer can be accomplished.” Stanford University biologist Philip Hanawalt, a leader in the study of DNA damage and repair, calls Barton's latest work “convincing.” Others, however, see loopholes in each of the earlier papers—interpretations equally consistent with the data that do not require a paradigm shift. As University of Pittsburgh theoretical chemist David Beratan puts it, “It's a mystery story. You have to decide what data are convincing and try to piece together a coherent story.”

    Easy as π. At issue is exactly how electrons move through large organic molecules. Twenty-five years of study have convinced chemists that in proteins, electrons move only by the laborious process of quantum-mechanical tunneling through pathways that connect one atom to the next along the protein's backbone. Researchers have suspected that DNA might be different. They have pointed out that the arrangement of bases on the complementary strands allows the electrons shared by multiple atoms to inhabit donut-shaped electron clouds above and below each ring of bases. The interior of the helix can be thought of as a stack of these π orbitals. If electrons could be injected into this stack, so the theory goes, they might easily tunnel from one end of the DNA to the other. While this would still be a quantum-mechanical effect, the electron transfer would be as effortless as moving current through a wire.

    But the π-stack conductivity theory has always been a minority opinion. DNA simply does not fit the expected criteria for conductors, says Beratan: “What we know about it from basic physical chemistry doesn't make it look like a wire.” Even biology has argued against the theory. If electrons could scoot around DNA with such facility, says Beratan simply, “we'd all be in a lot more trouble when we walk out in sunlight.”

    In 1993, however, Barton, Turro, and their colleagues in effect hooked a DNA strand up to a circuit, tested its conductivity, and came up with evidence that seemed inconsistent with the theory of DNA as a resistor (Science, 12 November 1993, p. 1025). They had created metal complexes that slipped between adjacent base pairs in the DNA. One arm of the complex would stick into the core of the DNA helix, or intercalate, “like one blade of a propeller,” says Barton, injecting an electron into the core or retrieving one, depending on the complex.

    The two chemists attached an electron-donating ruthenium complex near one end of a 15-base pair synthetic DNA helix and an electron-accepting rhodium complex near the other end. When hit by photons, the ruthenium would be excited and begin to glow until it could transfer an electron. If no rhodium acceptor was attached to the other end of the DNA, the ruthenium continued to glow. But if a rhodium-acceptor complex was in place, says Barton, “the glow was quenched because of the presence of electron transfer.”

    Indeed, Barton and Turro saw no detectable glow at all, which they interpreted as evidence that the DNA shuttled electrons between the metal compounds so fast that the quenching happened before it could be measured. The implication, they said, was that electrons could move huge distances through the DNA at speeds a million times faster than would be possible if the electrons had to tunnel laboriously from atom to atom, as they do in proteins.

    The result shook up the field. As Beratan says, “I don't even have to do much theory to tell you the Barton '93 result is extremely provocative.” Chemists were skeptical, and they were especially troubled by the lack of any glow from the ruthenium, says Tony Harriman, a spectroscopist at the University Louis Pasteur in Strasbourg, France. “[Barton and Turro] took a very, very negative result and converted it into an extremely positive conclusion. Many people would interpret not seeing the luminescence as failure of the experiment. To interpret it in a very spectacular way and a very positive way is going to raise a few eyebrows.”

    Indeed, Harriman promptly set off to do an experiment using organic molecules as donors and acceptors, spaced at random distances on the DNA. He was able to see his complexes luminesce and could measure the rate at which the glow was quenched, which indicated electron transfer rates “a little faster” than would be expected from a protein, but consistent with the mundane DNA-as-resistor theory. And Caltech chemist Tom Meade tethered donor and acceptor metal complexes to opposite ends of an 8-base pair DNA helix and found a similar, modest electron-transfer rate.

    Barton agrees that “there was no question that [the 1993 finding] was a surprising result and required lots of controls.” To show that the lack of luminescence in her experiments really was due to electron transfer and to measure its rate, she enlisted University of Minnesota chemist Paul Barbara, an expert in ultrafast spectroscopy. These experiments required concentrations of complexes too high for them to be tethered a fixed distance apart on the DNA helix. Instead, the two chemists simply mixed them with the DNA and allowed them to intercalate randomly along the helix, presumably some distance apart. The disappearance of the glow, as indicated by the spectroscopy, turned out to be as fast as Barton and Turro had reported in 1993, providing further evidence that the reaction was driven by rapid electron transfer down the helix (Science, 26 July 1996, p. 475).

    This result came with a caveat of its own, however. Because the metal groups weren't tethered to the DNA, as Barton explains, the distance the electrons had to travel could not be established. It was conceivable that the metal complexes were clustering together, in which case adjacent molecules would be swapping electrons over a very short distance. “Without tethering the complexes to the DNA,” says Barton, “we couldn't rule clustering out.” They did consider it unlikely, she adds.

    But Barbara reports in the 16 January issue of the Journal of Physical Chemistry B that he has now reanalyzed the data from his experiment with Barton and found that the “data are completely consistent with clustering combined with short-range electron transfer.” In the 16 February issue of the Journal of the American Chemical Society, Bengt Nordén and his colleagues at the Chalmers Institute of Technology in Sweden come to the same conclusion from a similar experiment. Clustering, he says, is “a much more plausible explanation” than conductive DNA.

    A charged issue. For Barton, the experience just underscored the need to lock the metal complexes onto the DNA. Her latest experiments rely on a chemical change in DNA, driven by charge transfer to a distant chemical group, to make a case for the electrical conductivity of the π stack. In the 22 August 1996 issue of Nature, she and her colleagues describe an experiment in which a metal complex, excited by a photon, stole an electron from a pair of guanine bases at a distant site on the DNA. The result was what is known as oxidative damage to the DNA, apparently triggered by the electron transfer down the double helix.

    The ratio of reactions to photons going into the system—the so-called quantum yield—was extremely low, however: just one in 10 million. That seemed to leave room for alternative explanations. “Maybe [the reaction happens] the one time the duplex opens up and something strange happens,” says Netzel. “If we were dealing with a quantum yield of 30%, we could be pretty sure we're dealing with a phenomenon with an intact helix. If we're dealing with 10−7 quantum yield, the room for nature to be fooling us is much greater.”

    In this week's Science paper, Barton presents a long-range chemistry experiment that she says isn't open to this objection, and now she's winning some converts. Barton and her colleagues fabricated DNA helices with a built-in thymine dimer, then intercalated an electron-accepting metal complex at the end of the DNA. Exposing the sample to light excites the rhodium compound, triggering it to absorb an electron from the thymine dimer and repair the DNA damage. “Even California sunlight works just fine,” says Barton. And because the rhodium complex can catalyze the repair reaction over and over, she says that the experiment “may represent a strategy to rationally design molecules that can accomplish this kind of repair therapeutically.”

    It also constitutes the first systematic measurement of how electron transfer in DNA changes with distance, says Barton. In a resistor, such as a protein, the rate or efficiency of electron transfer falls off very quickly with distance. In contrast, Barton and her colleagues found that the repair efficiency didn't change with the distance between the metal-acceptor complex and the thymine dimer. But specially fabricated disruptions in the base-pair stack did cut into the efficiency. “The bottom line,” she says, “is we were carrying out a long-range electron-transfer reaction that depended on the π stack.”

    Barton says that because the reaction was able to repair every one of the damaged strands, there is no room to argue that what is happening is a fluke that depends on some rare change in the double helix. And Claude Helene, a biophysical chemist at CNRS in Paris, says the data are convincing, and they “open up the possibility that people will be searching for [evidence of chemistry at a distance in DNA] in living organisms.”

    But even after this latest experiment, just how each electron makes its way down the double helix is still an open question. “We don't yet understand the mechanism,” says Barton. Instead of tunneling from the thymine dimer to the rhodium in one step, she says, “maybe it's hopping” down the helix. If the energies of the electron orbitals and that of the electron accepted by the intercalator are close enough, then the electron might easily tunnel from base to base down the π stack, virtually unaffected by distance.

    This mechanism would not work in proteins, where the gap between the energies of the orbitals and that of artificial electron donors or acceptors would be too large. And Barton's critics say it can't explain the results reported in her 1993 and 1996 Science papers. But it is consistent with prevailing theory, says University of North Carolina chemist Holden Thorp: “This may really be chemistry at a distance, with a believable mechanism. And there's a lot of cool stuff she could do with that.”

    What's clear to everyone is that the field needs more data to shake out the reality. Perhaps half a dozen labs, Barton's among them, have experiments or papers in the works that might pin down DNA's electrical properties and what their mechanism might be. The double helix is not accepting its new accolades easily. “The burden of proof for such a startling result,” says Netzel, “is simply higher than for a boring result.”

  8. Cell Biology

    Parasites Shed Light on Cellular Evolution

    1. Gretchen Vogel

    By now, it's well established that the cells of higher organisms acquired some of their most important components—the energy-producing mitochondrion and the photosynthesizing chloroplast—when they engulfed much simpler bacterial cells, which then took up residence and now provide those key services. But now, it seems that even a complex cell can be engulfed by another cell and become an essential part of it.

    The alga inside.

    The parasite Toxoplasma and its relatives may have derived an organelle with multiple membranes (arrow, right) from an ingested alga.

    Source: Köhler et al.; Illustration: K. Sutliff

    On page 1485, molecular parasitologists Sabine Köhler and David Roos of the University of Pennsylvania, molecular evolutionist Jeff Palmer, and botanist Charles Delwiche of Indiana University and their colleagues report that the chloroplast-like organelles recently found in an important group of single-celled parasites apparently arose when one of the parasites' ancestors engulfed, and then retained, a chloroplast-containing algal cell. The work adds to the growing evidence suggesting that secondary endosymbiosis, as it is called, may have been a relatively common event in evolution: There is already strong evidence that it occurred in some of the commonest types of algae, perhaps on several different occasions.

    While no one yet knows exactly what function the plastids have in the parasite group—which includes Toxoplasma, a common cause of infections in AIDS patients, and the malaria-causing Plasmodium—the fact that they have been retained through evolutionary history suggests that they are essential. That would make them tempting targets for drug therapies, because humans and other mammalian hosts of the parasites don't have such organelles. Indeed, says molecular parasitologist Jean Feagin of the Seattle Biomedical Research Center, researchers are attracted “almost like flies to the potential that this could have.”

    The first clue that the parasites carry plastids came when researchers found that Plasmodium and its relatives carry three distinct sets of genetic material: the usual nuclear DNA, a small circular molecule, and an even shorter linear fragment. Scientists at first thought the circular piece belonged to the cells' mitochondria, which typically have circular remnants of the original bacterial genome. But on closer examination, they found that the circular DNA's sequence contained some key features of the plastid DNA of plants and algae, while the linear piece turned out to be the mitochondrial DNA. That was very puzzling, says Roos. “Here, we have a genome without a known function and without a known home [in the cell],” he says.

    Last year, while studying Toxoplasma, botanist Geoffrey McFadden of the University of Melbourne in Australia and his colleagues provided the first solid evidence for the misfit genome's home. They selectively tagged the plastidlike DNA and found that it indeed resides in a small membrane-bound organelle that had no obvious function. That finding only deepened the puzzle, says Roos: “Its DNA looks more like a chloroplast's than a mitochondrion's, but these are not plants. So, what are they doing with a chloroplast?”

    The current work, says McFadden, “provides that missing piece of the puzzle.” The team presents two lines of evidence that the parasites obtained their plastid when one of their ancestors attempted to eat a chloroplast-containing algal cell. First, electron-microscope images revealed that the Toxoplasma organelle is surrounded not just by two membranes, as chloroplasts and mitochondria normally are, but by four. The inner two, the researchers reason, are from the double-membraned plastid that existed inside the engulfed cell. The third derives from the outer membrane of the algal cell, while the outermost membrane came from the vacuole formed when the host cell surrounded and engulfed the potential prey. Second, a phylogenetic analysis of one of the plastid genes suggested that it is more closely related to a gene in the plastids of green algae than to the comparable gene in the photosynthetic bacteria that were the original source of chloroplasts.

    The scientists acknowledge that neither line of evidence is strong enough to stand alone. “I wouldn't bet my life on there being four membranes there,” says Roos, who notes that it is sometimes difficult to see all four clearly. And Palmer, whose laboratory conducted the phylogenetic analysis, acknowledges that the data, which apply to only one gene, do not yield “a strong answer.” But the two lines of evidence taken together, says McFadden, “provide the first explanation of parasite plastid origin,” one consistent with secondary endosymbiosis.

    The parasites' plastids add to growing evidence that endosymbiosis may have happened “at least a half-dozen times” during the early evolution of cells, says Palmer. Other researchers have found not only multiple membranes, but also the remnants of an engulfed cell's nucleus, in two groups of algae, the red Cryptomonas and the green chlorarachniophytes, implying that they got their chloroplasts in the same way. And based on an as-yet-unpublished genetic analysis of red and green algae, University of Washington botanists Benjamin Hall and John Stiller suggest that green algae themselves may have arisen from a cell that engulfed a plastid-containing alga.

    But even though the new results help explain how the plastid got into Toxoplasma and its relatives, they do not explain what it is doing there today. It apparently does not carry out photosynthesis, having lost the genes required to obtain energy from sunlight, as well as its chlorophyll. It does, however, retain genes that may be involved in such crucial metabolic processes as the manufacture of amino acids, which are the building blocks of proteins, and the breakdown of lipids, which produces energy needed by the cell.

    Biomedical researchers would like to pinpoint the plastid's critical functions, because it might be possible to design drugs that block them with minimal side effects for humans. Indeed, several drugs used to treat malaria and toxoplasmosis may already be targeting the plastids. The drugs are thought to inhibit protein synthesis, but researchers find no sign of inhibition in the usual places—the cytoplasm or the mitochondria. The plastid, therefore, may be the target, says Roos. To date, direct evidence that such drugs attack the plastid has not surfaced, he says, “but it sure smells like it. It's very close.” If researchers have their way, whatever benefits the ancestral cell derived from its potential dinner will end up as its Achilles' heel.

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