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Science  06 Jun 1997:
Vol. 276, Issue 5318, pp. 1495

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    How the Nucleus Gets It Together

    1. Elizabeth Pennisi

    Cytological and biochemical studies both indicate that the synthesis of finished messenger RNAs requires a high degree of coordination among reactions in the nucleus

    Much like a train station at rush hour, the nucleus seems chaotic at first glance—packed with a jumble of chromosomes, RNA molecules, and proteins. Yet, just as careful observation of a train station reveals an orderly flow of commuters to platforms at particular times, research over the past few years has revealed that the molecular movements needed to carry out one of the nucleus' main functions—the conversion of the information carried by the DNA of the genes into the messenger RNAs (mRNAs) that direct protein synthesis—are far more coordinated than had been suspected.

    In the loop.

    Splicing factors (green) occur in granules and along chromosomal loops—the sites of transcription and, presumably, splicing.

    Joseph Gall

    To produce an mRNA, a gene first has to be transcribed into a raw copy called a pre-mRNA. But to make the finished products, pre-mRNAs have to undergo certain “processing” reactions, such as removal of the noncoding sequences, called introns, that were copied directly from the gene. At one time, molecular biologists thought that these reactions could take place anywhere in the nucleus, wherever the RNAs and their associated molecules happen to meet up. Now, two converging lines of evidence are making it clear that the production and processing of RNA are closely linked in time and space.

    Cytological studies, in which researchers observe cells to track the movements of the molecules involved, show that gene transcription occurs in very specific places, with intron removal and other processing reactions apparently taking place either at the same spots or very close by. And while there are still uncertainties about exactly how the transcribing and processing machinery get together, test-tube studies are bolstering the cellular observations by uncovering biochemical connections between key components of the two types of reaction. In particular, they suggest that RNA polymerase II (Pol II), the enzyme that copies genes into pre-mRNAs, also plays a critical role in seeing that the machinery that splices introns out of the pre-RNAs is assembled appropriately.

    “What's really coming to the fore is [the need for] viewing mRNA synthesis as a whole,” says David Bentley, a molecular biologist at the Amgen Institute, which is funded by the biotech company Amgen in Toronto. “It seems to be a highly integrated process.” Indeed, the high level of coordination and organization prompts cell biologist Alan Wolffe of the National Institute of Child Health and Human Development to describe the nucleus as “very much like a Swiss watch.”

    Understanding how the gears in this nuclear “watch” mesh is essential, not just for understanding how mRNAs are formed but also for understanding the functioning of the cell as a whole. Recently, for example, researchers have shown that mechanical forces transmitted through the protein filaments of the cell cytoskeleton alter nuclear structures, possibly leading to changes in gene expression (Science, 2 May, p. 678). Because the proteins involved in RNA synthesis and processing may be among the structures rearranged, figuring out where and when they do their jobs may help provide a clearer view of how that cytoskeletal network works.

    What's more, there are indications that disorganization of certain nuclear proteins, including those involved in RNA processing, can lead to diseases such as a leukemia and spinal muscular atrophy, an often fatal genetic disease in which muscles waste away and the spinal cord degenerates. “From the point of view of cell biology and disease, it's important to understand the relationship between nuclear structure and function,” concludes Angus Lamond, a biochemist from the University of Dundee in Scotland.

    On to splicing

    The “Swiss watch” picture of the nucleus began developing in cytological studies performed in the early 1980s. In one experiment, cell biologist Jeanne Lawrence and her colleagues at the University of Massachusetts Medical Center in Worcester found a link between gene transcription and the splicing of RNAs. They observed what appeared to be mRNAs within domains that correspond to small structures called interchromatin granular clusters (IGCs).

    Those clusters had already been implicated in RNA processing, as previous research had shown that these clusters are packed full of splicing factors, proteins needed to cut the introns out of newly synthesized RNAs. Some newly made RNA was known to exist along the rim of these clusters, entwined with molecular tangles called perichromatin fibrils, but these researchers were the first to see it inside clusters.

    The Lawrence team also went a step further. Using fluorescent-labeled nucleic acids to tag both a gene and its newly made RNA, they showed that the gene and the clusters are juxtaposed, and that transcription apparently takes place during this liaison (Science, 26 February 1993, pp. 1326 and 1330).

    Results from cell biologist David Spector and his colleagues at Cold Spring Harbor Laboratory in New York have also fingered the IGCs, in particular their associated fibrils, as important sites for transcription and splicing. These researchers have a different picture of how the processes are coordinated, however. They think the clusters keep their distance from the genes, instead serving as storage depots and assembly sites for splicing factors that move to transcription sites to work on newly made RNA.

    Spector and his postdocs Luis Jiménez-García and Sui Huang came to that conclusion in 1993 after putting RNA, either with or without introns, into cells with splicing factors labeled with fluorescing antibodies. When they then localized the RNAs by tagging them with fluorescent stains, they found that the splicing factors gathered around the intron-containing RNA but not around RNA lacking introns. “The splicing factors seem to be dynamic,” Spector explains—capable of making their way from the IGCs to the genes.

    Developmental biologist Joseph Gall of the Carnegie Institution in Baltimore saw a similar picture in the nucleus of the unfertilized eggs of various amphibians, including the toad Xenopus laevis. Because these eggs are very large compared to the ordinary somatic cells other researchers are studying, it's much easier to distinguish one site from another in the nucleus, Gall says. “What people who study somatic nuclei argue endlessly about, we can answer with a quick look,” he maintains.

    Under the microscope, the oocyte's chromatin looks like a bottle brush in which the “bristles” are loops of DNA that extend out from the chromosome axis. And Gall says that when he tags the molecules he wants to track with labeled antibodies, he can see that both transcription and the RNA-processing components occur along these loops. Thus, he thinks RNA processing takes place there, not near the clusters, which are some distance away.

    But perhaps the best evidence for splicing-factor migration comes from Spector and his postdoctoral fellow Tom Misteli, who have actually followed the movements of a splicing factor in living cells, not just in the dead, fixed cells used in previous experiments. For this work, the researchers merged the gene for green fluorescent protein with the gene for a splicing factor and then put this genetic construct into living cells. The fusion protein it produced glowed green, enabling the researchers to film the actual splicing-factor movements.

    These images revealed that clusters basically stay put. But as the Spector team's previous experiments had indicated, splicing factors move out from the clusters and head to a transcription site, identified by subsequent studies in cells fixed right after these observations were made. The fluorescent green arms could be seen “extending out [from the IGCs], twisting and contorting,” Spector explains. (These results appear in the 29 May issue of Nature.)

    The Lawrence team, too, finds that certain genes don't move to the clusters. Last December at the annual meeting of the American Society for Cell Biology, Lawrence's collaborator Phillip Moen Jr., now at NEN Life Science Products Inc. in Boston, described how the team studied 14 genes to see whether they are transcribed at the site of a cluster, and they find that the answer varies from gene to gene. For example, the gene for the myosin heavy chain, which becomes active only in mature muscle cells, apparently moves close to the clusters of splicing factors as the immature muscle cell precursors differentiate. “Its position in relationship with the [clusters] changed in correlation with its activation,” says Moen.

    The story is different for the gene for dystrophin, the muscle protein that's defective in muscular dystrophy. Even though its RNA requires splicing, the gene “is a very dramatic case of something that appears to avoid the [clusters] like the plague,” says Moen. The splicing factors must somehow move to its location. Moen does not know why the two genes differ in this regard. But the lesson from all this, Wolffe concludes, is that “what everyone is seeing may be true. It just depends on the particular gene.”

    Linchpin polymerase

    In spite of these differences about just how RNA synthesis and processing are choreographed, the conclusion that these events are linked is gaining further support from another quarter: biochemical data on the proteins involved. One is the RNA-synthesizing enzyme Pol II. Until a few years ago, most researchers had not considered any role for the enzyme beyond transcription. But in 1990, biochemist Jeffrey Corden of the Johns Hopkins University School of Medicine proposed that Pol II might be a key actor in RNA processing as well as in transcription. He noted that the last several hundred amino acids on the carboxyl end of the enzyme, a sequence known as the carboxyl terminal repeat domain (CTD), might be a loading site for proteins involved in processing RNA.

    The CTD is rich in serine and threonine, amino acids that are potential targets for the addition of phosphate groups. In 1993, biochemist Arno Greenleaf of Duke University Medical Center in Durham, North Carolina, suggested that by taking on several phosphates, the CTD would become negatively charged and attractive to splicing factors that happen to be rich in positively charged arginine amino acids. Once attached, these factors would then have access to the newly formed RNA the enzyme was producing.

    By 1995, Steve Warren, now a cell biologist at NeXstar Pharmaceuticals Inc. in Boulder, Colorado, had evidence supporting this picture. Using antibodies to tag both phosphorylated Pol II and the IGC proteins, he showed that much of Pol II and these proteins collected in large, rounded clusters when the cell was not actively transcribing genes. But these clusters seemed to break up when the nucleus became active, and Pol II and the proteins dispersed. These data suggested that Pol II and these proteins were working in concert, Warren says.

    Likewise, Spector and Misteli had observed that in cells that are actively transcribing DNA, splicing factors are found both in the IGCs and at the transcription sites, suggesting they moved between these two places. But when they treated cells with a specific Pol II inhibitor, they could no longer see arms extending from the clusters of splicing factors, an indication that the transport of splicing factors had stopped. While Spector hesitates to assign Pol II a role in shuttling splicing factors, it's clear that the arms are important to their movement, he notes.

    Other researchers have evidence that phosphorylation of the Pol II CTD helps the enzyme associate with splicing factors. In July of last year, for example, Corden and his colleagues reported that they had used a yeast screen to fish out molecules that interact with the CTD and had found four proteins with structures that suggested they were splicing factors. Then, when they used antibodies against the phosphorylated form of Pol II to extract the enzyme from cells, the enzyme came out in a complex with these proteins. These biochemical studies suggested these proteins and Pol II interacted in cells.

    That suggestion was borne out a month later by Ronald Berezney of the State University of New York, Buffalo, and his colleagues. They used fluorescing antibodies to label both these proteins and the phosphate-laden Pol II in rat kidney cells and found that the two colocalized. And the association apparently does have functional consequences, because Corden's team found that antibodies against either the CTD or the splicing proteins inhibit splicing reactions in a test-tube assay.

    All these data point to the CTD end of Pol II as having a role in the nucleus very similar to that of a dispatcher at a train station. It may help coordinate the movements necessary for transcription and RNA processing to occur. And just as commuters might eventually be able to find their way to the right train without being told where to go, splicing factors may still link up with their target RNA without Pol II. “But what [Pol II] may do is greatly accelerate the rate of splicing,” Corden suggests.

    He and his colleagues all agree that much more needs to be learned about the traffic patterns in the nucleus and about the interactions of the many proteins involved in making RNA. But the convergence of the biochemical and cellular results is finally making sense of the cellular train station.

    Additional Reading

    1. 113.
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    3. 115.
    4. 116.
    5. 117.
    6. 118.

    Making Plants Aluminum Tolerant

    1. Marcia Barinaga

    Among all the high-profile pests and blights that plague the world's agriculture, there is a culprit less well known: aluminum. The most common metal in soils, aluminum is a problem on 30% to 40% of the world's arable lands, where acid soil releases aluminum ions into the ground water. Indeed, for some important crops, such as corn, it is second only to drought as an impediment to crop yields, reducing production by up to 80%. Now, Mexican researchers have come up with a possible genetic-engineering fix.

    On page 1566, molecular biologist Luis Herrera-Estrella and his team at the Center for Research and Advanced Studies of the National Polytechnic Institute in Irapuato, Mexico, report that they were able to make tobacco and papaya plants aluminum tolerant. They did so by genetically engineering them to pump out citric acid from their roots. This organic acid ties up aluminum ions in the soil, preventing them from entering and damaging the plants' roots.

    Crop researchers using traditional plant-breeding methods have boosted the aluminum tolerance of some food crops, notably wheat. But they would like to have a gene for aluminum tolerance that they could introduce into a wide variety of crop strains already bred for high yield and pest resistance. Herrera-Estrella's work is “a powerful first step” toward that goal, says Leon Kochian, who studies aluminum tolerance in crop plants at the U.S. Department of Agriculture's (USDA's) Agricultural Research Service laboratory in Ithaca, New York.

    Farmers around the world would reap the benefits of such a gene, but the payoff would be especially high in developing countries. Although aluminum is present virtually everywhere, in nonacid soils it is locked up in insoluble compounds. But in acidic soils, which are most common in the tropics (where heavy rains leach alkaline materials from the land), the aluminum becomes soluble. It can slip into the cells of plant roots, where it poisons cell metabolism and prevents healthy root growth.

    One solution is to plow lime into the soil, but lime must be added every few years, can treat only the top layer of soil, and is too expensive for many farmers in developing countries, says plant geneticist Shivaji Pandey, who directs the aluminum-tolerance breeding program for corn at the International Research Institute for Breeding of Maize and Wheat in Mexico City. As a result, farmers in much of the world settle for poor crop yields on acidic soils. The toll is huge: Strains of corn that would yield 10 tons per hectare in neutral soil may produce only 2 tons in acidic soils, says Pandey.

    In their effort to create aluminum-tolerant crops that could boost these yields without extra cost, Herrera-Estrella and his colleagues built on prior work by Emmanuel Delhaize and his colleagues at the Commonwealth Scientific and Industrial Research Organization in Canberra, Australia. Delhaize's team found that some naturally aluminum-resistant plant strains have roots that secrete citric or malic acid, which binds to aluminum and prevents it from entering the roots. Several labs are chasing down the mutant gene responsible for the acid secretion, but Herrera-Estrella decided to take a different approach. “It occurred to us that because organic acid biosynthesis is a general phenomenon,” he says, “we could use genes from other organisms to produce organic acids in plants.”

    The organism the researchers turned to was the bacterium Pseudomonas aeruginosa. They introduced the bacterial gene for citrate synthase, the enzyme that makes citric acid, into two plant species: tobacco, a popular plant for laboratory work because it is easy to transform with foreign DNA, and papaya, an important crop in tropical Mexico that is highly sensitive to aluminum.

    The gene transfers had the desired effect. The plants carrying the citrate synthase gene secreted five to six times more citrate from their roots than control plants did. And that extra citrate translated into aluminum tolerance: The citrate-producing plants could grow well in aluminum concentrations 10-fold higher than those tolerated by control plants. “That is a significant increase,” says Kochian.

    This degree of tolerance could well allow some crop plants to be planted where they couldn't grow before. For example, Mexico's papaya crop, estimated at $97 million a year, comes from 20,000 hectares of land in the tropics, says Jose Garzon of the National Institute of Forestry, Livestock, and Agricultural Research in Celaya, Mexico. That crop could be expanded, he adds, if the new aluminum-tolerant plants could be grown on some of the 3 million hectares of tropical Mexican land where aluminum toxicity has prevented papaya cultivation.

    With tobacco and papaya as a first step, Herrera-Estrella's team has recently moved on to put the citrate synthase gene into two major food crops: rice and corn. They found that the engineered plants make extra citrate, although the results aren't in yet on whether they are aluminum tolerant.

    Still, all the work so far has been in the lab, and Kochian and plant physiologist Michael Grusak of the USDA laboratory in Houston caution that the transferred gene may impose physiological costs on the recipient plants; those could show up in the field and offset some of the benefit. All that extra citrate the plants are churning out means that “fixed carbon … is lost from the plant,” Kochian says. And that, he says, will impose an extra energy demand that could reduce the plants' productivity. “It is a trade-off,” says Grusak, but one that might prove well worthwhile in areas where aluminum takes a serious toll.


    The Search for Mr. Goodfile Generates New Online Tools

    1. Dennis Normile

    HATOYAMA, JAPAN—Swimming alone in an ocean of data may be fine for cybersharks. But most of us would appreciate help as we splash about in the seemingly bottomless pool of information that's available online.

    One promising source of such aid is a new wave of research that combines the fields of library science, natural language processing, linguistics, and computer science. At a recent conference here,* information scientists described systems to link the physical resources of traditional libraries to the online digital world. They debated whether traditional indexing and cataloging efforts could be adapted to the World Wide Web. And they presented glimpses of ways to simplify information retrieval.

    These efforts are forcing major changes in the world of scholarly information. Stanford University is making the World Wide Web the primary gateway into the library's catalogs and indices of its holdings, notes Michael Keller, director of Stanford University Libraries and Academic Information Resources, because of the user-friendly quality of Web browsers, including their hyperlinks and global accessibility. Keller says the idea is to equip the library's physical resources with the same searching and linking capabilities that are available online.

    In addition to the challenge of digitizing and linking catalog information for 7 million books, plus recordings, rare manuscripts, and art, Stanford (which is responsible for the development and maintenance of the Science Online Web site) is also developing search and retrieval engines to manipulate and present the information. Starting with dozens of search engines and more than 100 indices in the various Stanford libraries, Keller would like to provide one-stop shopping for users. In addition to returning the requested information, the system would allow the user to review the resources tapped and get information on how deeply they were searched. “These are not trivial goals,” Keller says.

    An example of what can be done is Stanford's Web-based catalog interface, Socrates II, which goes beyond simple searches by title or subject to include sorting by publisher, place of publication, and language of the holding. Retrieved records also can be displayed in various sequences, and card records in the Web's hypertext markup language (HTML) mean that additional cross-referenced information is only a mouse click away. Bibliographic data can be extracted and formatted for use in a list of references.

    Socrates II, now being tested, has been developed by some of the same people involved in the Digital Libraries Project, a 4-year program begun in 1994 and funded by three federal agencies (Science, 7 October 1994, p. 20). Six university-led consortia are tackling the challenges of collecting, storing, and organizing digital information and making it easily accessible. While most have a particular focus, typically by discipline or type of material, Stanford's task is to develop a single interface that would shift the burden of navigating databases with different structures and various search engines from the user to the software. The tools would apply both within the Stanford system and on the Web.

    Although a library setting provides abundant challenges, Keller says that the real test is taming the Internet, which lacks the theoretical and taxonomic structures that allow controlled, systematic retrieval of information. Whether such structures could be created for online resources was a matter of some debate. Keizo Oyama, an information scientist at Japan's National Center for Science Information Systems, proposed standard sets of key words that authors would attach to documents. Internet service providers could then have indexes of material on their systems. Better software tools to automatically generate the key words and indexes would help make all this voluntary work easy, he says.

    However, relying on authors or service providers poses a serious problem, says Karen Sparck-Jones, a linguist at the University of Cambridge. Authors are not necessarily the best judges of how to index or catalog their own works, she says, because of their limited knowledge of how the documents might be accessed and used. In addition, she notes, few people who put information on the Internet are interested in cataloging or indexing the material. A better approach in this era of full-text retrieval, she suggests, is improved search engines and techniques.

    Better retrieval methods is one of the promises held out by developments in natural language processing. NLP, which grew out of attempts in the 1950s to automate library cataloging and indexing, uses computational methods to try to discern meaning from a text. It may count the number of times words appear, or look at the relationships among phrases in a sentence. The goal is for computers to recognize meaning in human language and to transmit messages for humans to read or hear.

    Researchers had hoped that NLP would improve information retrieval through an increased understanding of queries and better filtering of documents. For a question about legal suits, for example, NLP would realize that the user was interested in litigation and lawsuits and reject documents related to clothing. But progress has been slow. Sparck-Jones says NLP techniques have not proven significantly better than search techniques based on multiple simple terms. “The generally good information-retrieval strategy is just to use more single terms in the query,” she says.

    NLP enthusiasts hope that the increased availability of uncataloged, full-text documents will raise demand for solutions, and the workshop offered examples of such advances in NLP techniques as term recognition and parsing of sentences. But commercial search engines now make only rudimentary use of NLP techniques. Sparck-Jones believes that NLP's greatest contribution may come in summarizing documents once they are retrieved.

    While the payoff from natural language processing may be far off, work continues on refining the dominant method of searching the Web through an iterative cycle involving request-review-modification. Yoshiki Niwa, of Hitachi Ltd.'s Advanced Research Laboratory here, showed an interactive search scheme, called Dual-Navi, that presents search results both concretely and abstractly in side-by-side windows.

    Starting with a search string, Dual-Navi presents the typical list of retrieved titles on the left side of the screen while a graph of key words extracted from those retrieved documents appears on the right side. The key words are displayed according to their frequency of occurrence, and associated words are joined by solid lines.

    Although the popular AltaVista search engine uses a similar graph, Dual-Navi provides interactive links between the two views. To narrow the search, users select additional key words in the graph view and click a button. The documents containing those words will come to the top of the title list. Conversely, click on a title, and the key words found in that document are highlighted in the graph. Additional titles with similar characteristics can then be gathered. These processes can be repeated, with the graph and list views constantly changing to reflect the latest stage of the search.

    Even more user-friendly, however, would be a system tailored to a person's needs. Stanley Peters, a mathematical linguist at Stanford's Center for the Study of Language and Information, presented one approach based on concepts, or groups of synonymous words, extracted from a person's e-mail. The idea, says Peters, is to exploit the “idiosyncratic associations” among words to come up with customized searches.

    In one test, researchers generated associations based on 3 months of an individual's e-mail and, for comparison, a database of 42,000 Associated Press (AP) news wire articles. They then searched a target database using four key words—race, identity, Asian, and dating. The results were strikingly different. The documents retrieved using the associations generated from the AP articles were primarily about black-white race relations, while those retrieved using the associations gleaned from the individual's e-mail were much more closely related to issues involving Asian race relations, the individual's primary research interest.

    Peters believes this approach could be extended. Civil engineers and stamp collectors, for example, could use sets of associations generated from databases of civil engineering journals or philatelic magazines to narrow the range of retrieved documents when searching something like the Web. But even this feature has its limitations. “There is not likely to be one approach that suits all particular needs,” Peters says. So, while trolling through the ocean of information may get easier, it is still going to take work to stay afloat.

    • * Workshop on New Challenges in Information Retrieval and Dissemination, 7-8 April, Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama, Japan.


    Physics, Biology Meet in Self-Assembling Bacterial Fibers

    1. Carol Potera
    1. Carol Potera is a science writer in Great Falls, Montana.

    Twenty years ago, when Neil Mendelson first described a mutant strain of bacteria that twisted itself into ropy helical fibers, his fellow microbiologists considered it just a curiosity, one of many in the world of microbes. As Mendelson, a professor at the University of Arizona, narrowed his research to focus on the quirky twists and turns of these bacteria, the scores awarded to his grant applications took a nose dive and so did the number of papers he published in peer-reviewed microbiology journals.


    Bacterial filaments (top) twist spontaneously into helical fibers (above).


    Lately, however, Mendelson's odd microbes have been making a name for themselves in some unexpected settings, far from microbiology. They have won devotees among mathematicians, engineers, and physicists who, collaborating with Mendelson, have used the microbial fibers to help solve long-standing problems in elasticity theory, model solar flares, and make a new siliceous material that could be used in medical implants. Mendelson was “a pioneer and out of the mainstream,” says Ralph Slepecky, a professor emeritus of microbiology at Syracuse University in New York. “But his work is proving useful.”

    What has sparked all this interdisciplinary effort is a mutant form of a common, rod-shaped bacterium called Bacillus subtilis, about 4 micrometers long and 0.7 micrometer in diameter. Back in 1975, Mendelson discovered a strain lacking the enzymes that normally cleave daughter cells after cell division, so that the daughters grow stuck to their parent cells like beads. Individual filaments of linked bacteria spontaneously twist and double back on themselves many times to form a thick, ropelike helical coil of up to 100 filaments (Science, 3 January 1992, p. 32). Mendelson dubbed these coils “macrofibers,” and while their unique penchant for self-assembly may have left some microbiologists cold, it piqued the interest of physical scientists.

    For example, when Michael Tabor, head of applied mathematics at the University of Arizona, first saw a film of macrofibers self-assembling in 1992, he realized that he had found a living, dynamic model for flexible elastic filaments. Mathematicians have been modeling the way these filaments twist for more than a century, but most models were static—describing only starting and ending structures, rather than the complex stages in between, says Tabor. His group, including postdoc Alain Goriely, spent several years observing B. subtilis—live and on video—and developed new dynamic equations to describe the twisting and coiling of macrofibers. Their analysis describes a seemingly unpredictable aspect of macrofiber coiling: how two-dimensional twisting causes the fibers to kink or rise up from a flat surface, adding a third dimension to their shape. For many natural systems, the spontaneous twisting of the bacterium is a better model than, say, a rubber band, which twists only in response to an outside force, says Tabor.

    Mathematicians call this move into a third dimension “writhe.” Tabor's model shows that writhe stems from subtle mathematical instabilities: When the researchers altered certain variables in their equations, the solutions required a shift into three dimensions. These mathematical instabilities are likely to be a general property of elastic filaments, Tabor says, although he doesn't know to what physical properties they might correspond. Because elasticity theory is used to model everything from the supercoiling of DNA to the twisting of magnetic field lines in a star, the new model will likely have plenty of applications, adds Mendelson's collaborator John Thwaites, a mechanical engineer at Cambridge University.

    In fact, Tabor's model of elastic filaments has already been applied to the behavior of so-called magnetic flux tubes in the sun. These structures, made up of bundles of magnetic field lines, cause sunspots and can trigger the enormous magnetic detonations on the surface of the sun known as solar flares. The tubes emerge from the sun's interior as narrow strands of magnetic field, which float to the surface and appear as sunspots. The long tails trailing back into the interior can be modeled as elastic filaments, because ionized gases associated with them have mass and anchor the magnetic flux tubes in the interior. Because one end is weighted down, the filaments acquire tension, explains physicist Dana Longcope of Montana State University (MSU) in Bozeman.

    But theorists have had a hard time explaining how this tension causes flux tubes to twist above the surface of the sun and trigger flares, says Longcope. Then, MSU mathematician Isaac Klapper, who worked with Tabor, showed Longcope the new elasticity model for supercoiling bacteria. “I knew these were the equations that could describe magnetic flux tubes,” says Longcope, and “it was a surprise that they came from bacteria.” When applied to the sun, the model showed that the twisted magnetic flux tubes release their stored-up energy by writhing out of their plane, thus triggering solar flares. Longcope and Klapper describe this in a paper in press at the Astrophysical Journal. Modeling solar flux tubes is the first step in understanding them, and perhaps eventually predicting the flares, which emit energetic particles that can damage satellites, says Klapper.

    Meanwhile, materials scientists have been pursuing another line of research on mutant B. subtilis.When cultured in crowded conditions, the separate strands become matted together into webs resembling spaghetti in a bowl. Mendelson invented a device to pull meter-long threads from webs; a thread contains up to 30,000 parallel bacterial filaments and “looks like nylon fishing line,” says materials chemist Stephen Mann of the University of Bath in England.

    Mendelson found that because the cell walls of B. subtilis consist of two negatively charged polymers that attract positively charged minerals, the threads bind mineral salts in solution, including those of iron, calcium, and copper. These encase and stiffen the threads, forming a crystalline fibrous framework—and opening a whole realm of potential new materials. Called bionites, these bacterial-mineral hybrids look like red, black, green, or silver fiberglass, depending on the mineral used. Weight for weight, bionites are stronger than steel, yet are also biodegradable.

    In the most recent bionite work, Mann, Mendelson, and colleagues created siliceous bionites that may be useful for medical implants. Ordinary silica is a good candidate for implants because it is fairly inert and doesn't react with tissue. But an ideal material would be porous, so that natural tissue could attach to it, explains Stuart Williams, chair of biomedical engineering at the University of Arizona. Silica naturally contains only nanometer-sized pores, too small for cells to gain a foothold, he says.

    Bionite researchers were able to improve on nature. Mann's team first made siliceous bionites, then burned out the bacterial fibers by heating them in an oven. The resulting white silica fibers contained ordered channels of two sizes: the tiny, natural pores, as well as larger channels 0.5 micrometer wide left by the fibers (Nature, 30 January). These large channels could provide a scaffolding for cell growth, as well as a route for antibiotics or growth factors added to the implant to leak slowly into surrounding tissue, says Williams, who calls this “a major improvement” in medical materials.

    Even in microbiology, the mutant B. subtilis is finally getting its due. The microbe's antics are making their way into microbiology texts and for the first time in 20 years, Mendelson spoke by invitation at last year's annual meeting of the American Society for Microbiology. Accepting his ideas took time because of his “unique approach” and focus on mechanics, says microbiologist Gerald Shockman of Temple University in Philadelphia. But whether the research is called microbiology, mathematics, or materials science, it's clear that Mendelson's microbes have come into their own.


    Mapping a Star's Magnetic Field

    1. Erik Stokstad

    Astronomers have enlisted a continent-wide array of radio telescopes to map the magnetic field of a bloated red giant star. This first magnetic map of another star, to be published this week in Astrophysical Journal Letters, suggests that at least some stars have magnetic fields resembling those of Earth and the sun. And the details of the map—about 50 times smaller than the finest features visible to the Hubble Space Telescope—could help theorists model how red giant stars shed gas and dust into interstellar space.

    Fine detail.

    Regions of intensified radio emission around a red giant star (top) enabled astronomers to map parts of its magnetic field.


    Astronomers can't observe magnetic fields directly, so Athol Kemball and Philip Diamond of the National Radio Astronomy Observatory in Socorro, New Mexico, relied on radio waves, which are polarized by magnetic fields. Normally, the radio waves from the star they observed—TX Camelopardalis (TX Cam), which lies about 1000 light-years away—would be too faint to see. But TX Cam is swathed in clouds of excited silicon monoxide molecules. These clouds act as an amplifying medium for radio waves, in much the same way that a laser amplifies light.

    To pick up the signals and map their polarization, Kemball and Diamond turned to the Very Long Baseline Array (VLBA), a network of 10 identical radio telescopes that stretches across the United States. By combining signals from its far-flung dishes, the VLBA mimics a single antenna the size of the continent, picking up details that would elude smaller telescopes.

    The VLBA's map of radio polarization across TX Cam is “strongly suggestive of order in the magnetic field,” says Kemball, although he cautions that the map shows only two dimensions and doesn't pin down the field's north and south poles. But Kemball says the field's most likely structure is similar to Earth's field, with magnetic field lines looping around the planet parallel to lines of longitude. In several spots, however, the field lines are twisted and kinked, much as they are around flares on the sun. The magnetic kinks could affect the way gas and dust escape from the star, says Kemball.

    He and Diamond hope to monitor TX Cam every 2 weeks, looking for changes in the field lines to see whether this giant star has a magnetic cycle. Meanwhile, astrophysicists are delighted by their first clear pictures of a star's field. The resolution, says Mark Reid of the Smithsonian Astrophysical Observatory in Cambridge, Massachusetts, “is truly astounding.”


    Fossilized Hatchling Heats Up the Bird-Dinosaur Debate

    1. Virginia Morell

    No children allowed. That's the way the history of life has looked in the fossil record, so few and far between are the remains of any youngsters. But that lopsided view has changed for one lineage of extinct birds. In this issue of Science, an international team of paleontologists reports the discovery, in the Pyrenees of northern Spain, of a remarkably well-preserved nestling bird dating from about 135 million years ago. It is the earliest hatchling bird yet discovered and comes just 10 million years after Archaeopteryx, the first undisputed bird. And for one so young, the fossil nestling has a surprising number of lessons to teach.

    Pretty baby.

    To some paleontologists, the nestling bird's well-preserved skull looks dinosaurian; other researchers link it with much earlier reptiles.


    As José Sanz of the Universidad Autonoma in Madrid and his colleagues report on page 1543, the hatchling's bone structure shows that even at this early date, birds matured much as they do now. In other respects, though, the fossil is what Peter Wellnhofer, a paleontologist at the Bavarian State Collection of Paleontology in Munich, Germany, and an expert on Archaeopteryx, calls “a wonderful mosaic,” mixing primitive and advanced features. As a result, Sanz and his colleagues argue, their finding has much to say about the evolution of modern birds—as well as the hypothesized link between birds and dinosaurs.

    To some paleontologists, including the study's authors, the nestling's skull looks dinosaurian, while other features resemble those of modern birds. This provides “more evidence that birds do indeed have their origins in the small theropod [meat-eating] dinosaurs,” says Lawrence Witmer of Ohio University in Athens. It's the second boost for the bird-dinosaur link in 2 weeks, closely following a Nature paper in which a team of Argentine scientists announced the discovery of a 90-million-year-old dinosaur that folded up its forelimbs as if they were wings.

    But the bird-dinosaur connection is hotly disputed, and other scientists who believe birds evolved from earlier reptiles interpret both fossils differently. “Neither one of these fossils has anything to say about birds being related to dinosaurs,” says Alan Feduccia, an ornithologist at the University of North Carolina, Chapel Hill. “And it's a misrepresentation of the evidence to say they do.”

    Partisans on both sides, however, acknowledge the importance of this first glimpse of an ancient baby bird. “I knew when I first saw it that it was a hatchling, just from looking at the size of its orbits and comparing the size of the skull to the rest of the body,” says Sanz. He persuaded the amateur fossil hunter who unearthed the specimen nearly 8 years ago to donate it to a scientific institute in Catalonia some 6 years later. The institute then loaned it to Sanz for detailed study.

    That was when Sanz made what he rates as his most “amazing” discovery: foramina, tiny holes on the surface of the bird's bones that are “almost identical to those in extant nestling birds”—and a telltale sign of bird youth. “It shows that this kind of bone was present even in an extinct lineage of birds, one that is only distantly related to modern birds,” says Sanz. The nestling was up-to-date in other ways, too. Its wings, for example, are “almost as sophisticated as those of modern birds,” says Luis Chiappe, a paleontologist at the American Museum of Natural History and one of the paper's authors.

    The bird's beautifully preserved skull isn't nearly as modern, however. That supports an earlier suggestion that in bird evolution the wings led the way—just as in the evolution of humans the bipedal stance evolved before brain size changed. Indeed, says Chiappe, the skull “shows [the nestling's] dinosaurian ancestry.” For example, he and Witmer note, certain bony structures found behind the eye in small theropod dinosaurs but not in modern birds are still present in the nestling, although they show signs of breaking down. “That's a key innovation in avian evolution,” explains Witmer, “as it allows birds to raise their upper jaw as well as lower their mandible,” enabling them to eat a wide variety of food. “And here we see the first steps toward that change.”

    A different mosaic of birdlike and dinosaurian features appears in the Argentine fossil. It is no bird, but rather an adult two-legged dinosaur that probably stood as tall as a man but had winglike forearms, says Fernando Novas, a paleontologist at the Argentine Museum of Natural Sciences in Buenos Aires and an author of the Naturepaper. The dinosaur, which Novas and his colleague named Unenlagia comahuensis (half-bird from northwest Patagonia), “folded its arms close to its body” in a very birdlike manner, yet could have stretched them out as if taking flight—although it probably extended its arms for balance instead. That arm and shoulder anatomy, plus the dinosaur's very birdlike pelvic girdle, suggest the kind of changes that dinosaurs would have undergone during their transition to birds, Novas says.

    Both “missing-link” claims are coming under heavy fire. Linking the fossils to the origin of birds is “a complete non sequitur,” fumes Walter Bock, an ornithologist at Columbia University. “It just shows the fixation of these dinosaur paleontologists.”

    Feduccia argues, for example, that the nestling's skull, far from being dinosaurian, is more reminiscent of a primitive archosaur. Primitive archosaurs are the ancient reptilian group, more than 250 million years old, that predated both birds and dinosaurs and, he argues, gave rise to both groups via independent lines of descent. “Many of the features the authors claim indicate a dinosaur ancestry are actually characteristic of the basal archosaurs,” says Feduccia.

    Bock, Feduccia, and others are equally dismissive of Novas's claim that the Argentine fossil shows anything about how birds evolved. Chronologically, the fossil is simply too recent to be relevant, they say, because even if the bird-dinosaur link is real, birds would have diverged tens of millions of years earlier. “That dinosaur is 90 million years old!” exclaims Feduccia. “Birds have been around for at least 60 million years when this guy appears.”

    Novas and Witmer, however, think the 90-million-year-old dinosaur is a representative of a much older lineage that did give rise to birds, probably 170 million years ago in the Middle Jurassic. “Phylogenetically, birds are dinosaurs, just as humans are mammals, and Unenlagia is further proof that birds descended from theropod dinosaurs,” says Novas.

    Perhaps. Other scientists would like to see the hard evidence before they agree, and will simply wait until those earliest birds from the Middle Jurassic are found.


    No Bones About a Genetic Switch for Bone Growth

    1. Steven Dickman
    1. Steven Dickman is a science writer in Cambridge, Massachusetts.

    “Here was the ugliest man who ever came to Troy, … both shoulders humped together, curving over his caved-in chest, and bobbing above them his skull warped to a point. …” Thus, Homer describes Thersites, a Greek soldier in the Iliad who taunts his own leaders, Odysseus and Achilles. Now, geneticists have come up with their own account of Thersites's symptoms, which appear to reflect an extreme case of a skeletal disease known as cleidocranial dysplasia (CCD). They have identified a gene—some call it a master gene for bone growth—that, when mutated, causes this disease.

    In the 30 May issue of Cell, four papers tell the tale of the gene CBFA-1, drawing on evidence from human families, knockout mice, and cell cultures. Taken together, the data show that this gene turns precursor cells into osteoblasts, the cells that actually secrete bony matrix, and switches on at least one bone protein and probably many more. Although there are other key bone-forming genes, “this is the first one reported which, when inactivated, leads to the development of a complete organism—with no bone,” says Rik Derynck, a cell and developmental biologist at the University of California, San Francisco.

    The identification of CBFA-1 is “one of the top discoveries of the year,” says Peter Lomedico, vice president for human genetics at Genome Therapeutics Corp. in Waltham, Massachusetts, a company active in bone research. The discovery “provides a powerful molecular insight to the regulation of osteoblast differentiation and bone formation,” processes crucial not only to CCD but to more common diseases such as osteoporosis, he says.

    Researchers had already uncovered a string of bone-forming proteins, the best known being the BMPs or “bone morphogenic proteins.” But these molecules act early in development and are “anything but skeleton-specific,” because they are expressed in all sorts of cells, says Patricia Ducy. A developmental geneticist in the laboratory of Gérard Karsenty at the M. D. Anderson Cancer Center of the University of Texas, Houston, she co-authored one of the papers. CBFA-1, in contrast, appears to exert its effects only in osteoblasts, the cells that actually deposit bone.

    At the molecular level, very little was known about the osteoblasts, except that only these cells produce a bone-matrix protein called osteocalcin. In 1994, Karsenty identified multiple mouse genes that code for osteocalcin; Ducy followed up that work by searching for the gene that switched on an osteocalcin gene in the bone-forming cells.

    In mid-1996, she found one—CBFA-1. To her “extreme surprise,” it was already known—as the source of a protein important in the thymus gland, where the T cells of the immune system develop. But “the signal in bone was a hundred times stronger than in thymus,” says cell biologist Bjorn Olsen of Harvard Medical and Dental Schools, another co-author. Ducy spliced the new gene into mouse skin cells and found that they develop into osteoclasts and express osteocalcin.

    Just as Ducy closed in on her discovery last fall, pediatric geneticist Stefan Mundlos of the University of Mainz in Germany, who was then in Olsen's laboratory, was using linkage analysis of CCD families to identify the gene involved in that syndrome. CCD patients rarely have the severe deformities of Thersites or “ugly” physiognomies, says Mundlos. But they often have a missing collarbone, allowing them to fold their shoulders together in front of their bodies—as in the Greek's “caved-in chest.” Furthermore, their skull sutures don't close, so adults retain the soft fontanel normally found only in babies. Some patients also have extra teeth, up to 60 in all, which usually have to be extracted. In addition, growth in other bones is stunted and average height is about 164 centimeters for men and 154 centimeters in women, says Mundlos. His team found genetic deletions on chromosome 6 in many of these patients. The deletions led them to the gene, and they subsequently found that people with the disease have only one intact copy of CBFA-1; the other copy is mutated.

    Meanwhile, Mike Owen at the Imperial Cancer Research Fund in London and postdoctoral fellows Florian Otto and Anders Thornell were exploring the role of CBFA-1 in thymus development by creating mutant mice lacking one or both copies of the mouse version of the gene, cbfa-1. But they were baffled when the mice showed no evidence of immunological problems or defects in the thymus. Then, they realized that mice with a single copy of the gene were two-thirds the size of normal mice. And when they stained the skeletons of mice missing both copies, they hit the jackpot, recalls Owen.

    These mice, which were born but survived only 10 minutes, gasping for breath, had no bone at all, only cartilage. Mice missing only one copy of the gene had no collarbone and other defects that exactly matched CCD in humans. At about the same time, molecular biologist Toshihisa Komori, in the research group of immunologist Tadamitsu Kishimoto at Osaka University Medical School in Japan, made other cbfa-1 knockout mice—and got very similar findings.

    All this adds up to a picture of CBFA-1 as a crucial transcription factor—a gene that turns on other genes—that causes preosteoblasts to become osteoblasts and begin producing osteocalcin. This essential role leads Ducy and others to call CBFA-1 a “master gene.” Hundreds of genes are thought to be active in converting cartilage to bone, says Mundlos, but this one “is the first that could potentially control the final step of the transformation.” Derynck agrees that CBFA-1 is a key regulator, although he is wary of the term “master gene” because the other genes in the pathway are unknown.

    The new one “is not a drug and never will be,” warns Karsenty. Transcription factors make notoriously poor drugs because they act within cells, not between them, and so present delivery problems. Still, understanding this molecular switch is likely to benefit research on bone diseases, says Lomedico. For example, one theory of osteoporosis holds that aging reduces the body's ability to form new osteoblasts. Says Karsenty, “If you could recreate [CBFA-1's] function later in life”—and cause precursor cells to become osteoblasts and secrete new bone matrix—“you could in principle increase bone formation and slow the progression of the disease.”

    The next goal is to find more genes that are turned on by CBFA-1, to see how bone is formed, gene by gene. All these questions “could not have been asked 6 months ago,” says Olsen. Mundlos agrees, adding, “It's not the discoveries that explain everything that are the most exciting. It's the ones that open new doors.”

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