News this Week

Science  25 May 2007:
Vol. 316, Issue 5828, pp. 1108
  1. AVIAN INFLUENZA

    Indonesia Earns Flu Accord at World Health Assembly

    1. Martin Enserink*
    1. With reporting by Dennis Normile.

    Indonesia's battle to ensure access to flu vaccines that could save the lives of millions of its citizens during a pandemic reached a fevered climax earlier this week at the World Health Assembly (WHA), the annual meeting of member states of the World Health Organization (WHO) in Geneva, Switzerland. Supported by other developing countries, Indonesia demanded action and once again employed its valuable bargaining chip: cooperation in a 55-year-old global network of virus sample sharing that acts as the cornerstone of the world's defenses against flu.

    As Science went to press, a WHA committee had approved a draft resolution, hammered out in 5 days of long and often tense meetings, that called on WHO to do more to help developing nations obtain access to vaccines and proposed establishing an international working group to change the rules of the virus-sharing system. (The draft was widely expected to be approved by the entire WHA on Wednesday.) “It was very, very, very difficult to reach an agreement,” Indonesian Health Minister Siti Fadilah Supari, who participated in the negotiations, told Science.

    Under the Global Influenza Surveillance Network, countries send virus samples from the field to one of four WHO centers in London, Australia, Tokyo, and Melbourne. At these sites, analyses of the viruses help track viral evolution and resistance to drugs, judge the risk of a pandemic, and, most critically, guide the development of vaccines.

    Indonesia, a continuing H5N1 hot spot, has rebelled against the system, which Supari describes as “very unfair” because Indonesia receives no guarantees about access to pandemic vaccines in return for participating in the surveillance network. Nine Western countries currently have influenza vaccine factories, but experts say they won't be able to produce nearly enough vaccine for the entire world. Indonesia is also angered that researchers in other countries were taking out patents based in part on Indonesian viruses.

    In January, Indonesia pulled out of the flu-sharing system, denying WHO new influenza strains. That led to intensive talks between the country and WHO officials—and failed promises from Indonesia to resume sharing. WHO, which shares Indonesia's concerns but says the country's actions are a “threat to global health security,” has put forth several proposals to improve access to vaccines. For instance, it has developed a technology-transfer plan that could eventually give some developing nations their own flu vaccine manufacturing capacity; in April, it awarded six countries—Brazil, India, Indonesia, Mexico, Thailand, and Vietnam—a total of $18 million in seed money to develop the necessary plans. WHO has also proposed to form a stockpile of H5N1 vaccine that could be used in developing countries as needed, but its size is uncertain.

    Standing her ground.

    Indonesian Health Minister Siti Fadilah Supari says the current virus-sharing system is “very unfair.”

    CREDIT: DENIS BALIBOUSE/REUTERS

    At the start of the WHA, Supari announced that Indonesia had resumed sharing influenza viruses, and WHO confirms that the network has recently received three samples. But Indonesia also jumped on the opportunity of the WHA to press its case. It pointed to the 1992 Convention on Biological Diversity, which stipulates that a country has to share in the benefits if others make use of its genetic resources. Carlos Correa, an intellectual-property expert at the University of Buenos Aires, agrees that the convention applies to all genetic resources, including viruses. “Indonesia has a fair claim,” he concludes. WHO is still consulting legal experts about the issue, says Assistant Director-General David Heymann.

    The arguments about exactly what developing nations should get in return for their participation in the flu surveillance network took place behind closed doors in a “drafting group” composed of several dozen countries. Supari says the United States in particular opposed Indonesia's demands. (David Hohman, the health attaché at the U.S. mission in Geneva, was not available for comment.)

    The draft resolution that finally emerged late Tuesday afternoon calls on WHO member states to keep sharing their viruses, but it also asks WHO to take a range of measures to ensure that developing countries can produce their own vaccine and to guarantee “fair and equitable distribution” if a pandemic occurs. The resolution also calls for representatives from 24 countries around the world to propose changes to the rules of the global surveillance system that would benefit the developing world. To address another sore point, the group would have to ensure increased participation of scientists from developing countries in flu research and wider recognition of their role.

    Supari says Indonesia got most of what it wanted. But David Fedson, a retired pharma executive and a longtime advocate for pandemic preparedness, says the resolution doesn't do enough to address the fundamental problem: the scarcity of vaccine production capacity. “If I were the minister of health of Indonesia, I would not be satisfied,” Fedson says.

  2. INFECTIOUS DISEASE

    Resurgence of Yellow Fever in Africa Prompts a Counterattack

    1. Leslie Roberts

    Experts tracking the resurgence of yellow fever across Africa worry about one scenario in particular: simultaneous outbreaks in several of the continent's teeming mega-cities. In Lagos, Nigeria, with its population of 15 million, an estimated 4.5 million could be infected, says Sylvie Briand of the World Health Organization (WHO), and international stockpiles of vaccine would be rapidly exhausted fighting the outbreak. And that's just for one city.

    An urban outbreak in Africa would be “a catastrophe,” says David Heymann, WHO's assistant director-general for communicable diseases.

    That's why WHO and partner agencies are launching a major initiative to protect the populations at highest risk for yellow fever epidemics. Announced at the World Health Assembly last week and kick-started with $58 million from the Global Alliance for Vaccines and Immunization (GAVI)—a public-private partnership established in 1999 to strengthen immunization and boost the use of new and underused vaccines—the goal is to immunize more than 48 million people in 12 West African countries over the next 5 years. That should be enough, they hope, to reestablish an immune barrier against this often-fatal hemorrhagic fever.

    An ounce of prevention.

    A man receives a vaccination against yellow fever during a mass campaign in Agoto, 150 km northwest of Togo's capital, on 23 February 2007.

    CREDIT: NOEL KOKOU TADEGNON/REUTERS

    To yellow fever expert Thomas Monath, it's a no-brainer. “This is a very small expenditure that could save a great number of lives,” says Monath, a partner with the Kleiner Perkins Caufield & Byers venture-capital group.

    Transmitted by the bite of an infected mosquito, yellow fever decimated New Orleans and other cities in the early 1900s. But thanks largely to vector control and the introduction of a remarkably safe and effective vaccine, known as 17D, yellow fever has disappeared from the developed world. But within the tropical belt in Africa and South America, it remains “a very dangerous disease, with a high lethality,” says Monath.

    At first, the fever and chills can be relatively mild and easily confused with other tropical diseases. But about 15% to 20% of patients progress to the so-called toxic phase, when jaundice rapidly sets in, the kidneys fail, and massive hemorrhaging from the mouth, nose, and eyes begins. Roughly 20% to 50% of those with severe disease die. It is a “horrible thing” to witness, says Monath.

    In the French-speaking West African countries now considered at highest risk, widespread vaccination campaigns between 1940 and 1960 virtually wiped out yellow fever. But it came back with a vengeance in the 1990s, after the campaigns had halted and a generation had grown up without immunity, says Briand, project manager of the yellow fever initiative at WHO. At the same time, rapid urbanization and population movements have brought susceptible people into closer proximity with infected mosquitoes such as Aedes aegypti, an urban mosquito that breeds in water jugs, discarded tires, and other urban detritus.

    Since 2000, 18 countries in Africa have reported cases of yellow fever. West Africa is the hardest hit region, with worrisome outbreaks occurring in four major cities. A 2001 outbreak in Abidjan, Côte d'Ivoire, required immunizing 2.6 million people in 12 days, a huge logistical challenge, says Briand. Yellow fever's exact toll is hard to measure, but WHO estimates there are now up to 200,000 cases a year, with 30,000 deaths.

    With the increasing threat, many at-risk countries added yellow fever vaccine to their package of routine childhood immunizations. But as outbreaks continued, it quickly became clear that a strategy relying on routine immunization alone would take too long to build up sufficient population immunity, says Michel Zaffran, deputy executive secretary of the GAVI Alliance. What's more, adds Zaffran, limited supplies of vaccine have been diverted from routine immunization programs to deal with emergencies.

    As part of the new initiative, the GAVI Alliance will increase the emergency stockpile from 6 million to about 11 million doses a year. UNICEF will negotiate with vaccine manufacturers to ensure production of the 50 million or so doses for the 12-country prevention campaign. The GAVI Alliance will foot the bill for the vaccine, syringes, and half of the operational costs; the 12 countries have committed to raising the rest of the money for the vaccination campaigns, says Zaffran.

    It will take commitment, as these countries face other health problems including cholera and meningitis, not to mention malaria, tuberculosis, and AIDS, says Briand. But yellow fever vaccine is one of the best buys out there, she adds, as a single dose of vaccine can confer immunity for decades, perhaps a lifetime. “It is the most effective vaccine we have,” agrees Monath.

    Already, the 12 countries, with technical assistance from WHO, have begun conducting risk assessments to help determine which districts in each country are at greatest risk and should receive priority vaccination. At the World Health Assembly, other countries were asking whether they could be included in the plan. That may be a possibility later on, says Zaffran, but first, “we need to show it is working.”

  3. GEOCHEMISTRY

    Isotopes Suggest Solar System Formed in a Rough Neighborhood

    1. Richard A. Kerr

    Astrophysicists have long assumed that a supernova played midwife to the solar system. An exploding star could have collapsed wispy interstellar gas and dust into a dense swirling disk to get things started and loaded it with the intensely radioactive aluminum that cooked up chunks of the nascent solar system. But on page 1178, a group of cosmo-chemists presents evidence that the sun was born into an even more brutal environment.

    What's rougher than a supernova next door? A supernova that, before detonating, blasts its neighborhood with eons' worth of energy in an astrophysical instant. Astrophysicists think such behavior is typical of stars dozens of times as massive as the sun. And if one of those massive stars was so close, our home system must have formed in a dense, swirling cluster of stars. The newborn solar system's neighborhood would have been “a much more violent and turbulent” place than had been assumed, says theoretical astrophysicist Alan Boss of the Carnegie Institution of Washington's Department of Terrestrial Magnetism.

    The evidence for our violent beginnings comes from some of the most precise isotopic measurements yet of nickel in samples of Earth, Mars, and meteorites. Martin Bizzarro of the University of Copenhagen in Denmark and colleagues had gone looking for signs of radioactive iron-60 in the oldest meteorite from an asteroid that had melted in the earliest solar system. The iron-60 itself wouldn't be there. It was forged in the heart of a star and spewed into the material that would become the solar system after the star went supernova. Then the iron-60 promptly decayed away into nickel-60. So the researchers looked for the nickel “ash” using a type of mass spectrometer that can ionize all the nickel in a sample. That allows sensitive detection of the isotopes following magnetic separation. They also analyzed each sample many times to drive down the analytical error.

    To their surprise, Bizzarro and colleagues did not find the expected extra dose of the iron-60 marker. Instead, the samples contained less nickel-60 than found in younger meteorites. Apparently, the solar system's shot of iron-60 had not arrived when this old meteorite solidified about a million years after the solar system's start. Yet radioactive aluminum-26—also made in stars—had been there all along.

    Just like home?

    As massive stars (off top of image) blast the Eagle nebula's gas and dust, they may be triggering formation of planetary systems.

    CREDIT: NASA, ESA, AND THE HUBBLE HERITAGE TEAM STSCI/AURA

    “Iron-60 and aluminum-26 don't seem to be coming into the solar system at the same time,” says Bizzarro. “There's only one stellar environment that can do that: very, very massive stars.” The bigger the star, the faster it burns its hydrogen fuel. If it has more than 30 times the mass of the sun, a star will blow away much of its outer layers—including its aluminum-26—in the last million years of its brief life of 4 million years or so. That stellar wind could have driven the collapse of interstellar gas and dust to form our sun and the protoplanetary disk that once surrounded it. Later, the massive star exploded, spewing iron-60 from its deep interior.

    The Bizzarro paper “has a great story to tell … based on some truly spectacular nickel-isotope data,” says cosmochemist Meenakshi Wadhwa of Arizona State University in Tempe. There is a caveat, however. Three other labs, including her own, have analyzed similar samples with similar levels of precision—albeit using a different data-analysis approach—without finding a deficit of nickel-60 in the oldest samples. Wadhwa still believes the authors make “a pretty good case for the accuracy and precision of their data.” But you can bet that “pretty good” won't stop competing labs from gearing up for more analytical runs.

  4. U.S POLITICS

    Nobelist Eyes Minnesota Senate Seat

    1. Eliot Marshall
    New experiment.

    Chemist Peter Agre may test his popularity in one of the hottest U.S. races.

    CREDIT: DUKE UNIVERSITY MEDICAL CENTER

    Peter Agre wants to do something no Nobelist has done before: get elected to the U.S. Senate. After a colleague disclosed it in a newspaper op-ed column last week, the 2003 chemistry laureate confirmed that he is considering a run for the Senate seat now held by Minnesota Republican Norm Coleman.

    Agre, 58, plans to take leave this summer from Duke University in Durham, North Carolina, where he is a professor of cell biology and vice chancellor for science and technology. He aims to test his welcome among Democrats in Minnesota, his childhood home, which he left 3 decades ago to pursue a career on the U.S. East Coast. With no campaign kitty, little public visibility, and no political experience, he concedes that the odds of winning a Democratic primary, much less the general election in November 2008, are long. But he says that Minnesota has promoted “some very unusual candidates over the years,” including another student at his high school, Jesse Ventura, the professional wrestler who was elected governor.

    The news that Agre is weighing a run for the Senate came as a surprise to science policy leaders, some of whom were pleased. “That he would even consider this is an extraordinary public service, for which he should be applauded,” said Neal Lane, former director of the National Science Foundation and science adviser to President Bill Clinton. “It's pretty clear … that we're in dire need of serious leadership by people who think that facts are important, that evidence should be considered.” Lewis Branscomb, professor emeritus of the John F. Kennedy School of Government at Harvard University, thinks that Agre's Nobel credentials would enable him to “fight for rational, fact-based policy decisions so essential to the survival of democracy.” Former Republican House Science Committee chair Robert Walker says, “It's always positive to have scientists involved,” adding that many are uncomfortable with the compromises required in public life.

    Agre drew some media attention several years ago when he helped his friend Thomas Butler, a microbiologist at Texas Tech University in Lubbock, Texas, fight charges that he had violated biohazard and accounting regulations (Science, 19 March 2004, p. 1743). Butler was convicted and sent to prison—a “disgrace,” says Agre, who figures that he spent a large part of his Nobel Prize money on Butler's legal defense. Agre also endorsed a 2004 report by the Union of Concerned Scientists accusing the Bush Administration of manipulating U.S. science for political aims. His political views are left of center, although within the mainstream for Minnesota; for example, he favors universal health insurance, strong action to control carbon emissions, and more public aid for poor women seeking abortions.

    Political strategists say that the Minnesota Senate race could cost candidates more than $30 million. And two well-heeled candidates are already seeking the Democratic nomination: political comic Al Franken and a popular Minneapolis attorney, Michael Ciresi. Agre says he will spend the summer with Minnesota voters to see whether his own candidacy “has traction or not.”

  5. NONPROLIFERATION

    Gloomier Prospects for Indo-U.S. Nuclear Pact

    1. Richard Stone,
    2. Pallava Bagla

    NEW DELHI— A landmark civilian nuclear deal between India and the United States has hit “a possibly fatal impasse,” says a U.S. official. Sources in both capitals say that negotiations to implement the agreement are deadlocked over long-standing sticking points, including India's desire to retain the right to reprocess spent nuclear fuel and to conduct future nuclear weapons tests.

    “We're far apart, and the gap is far from closing,” says a U.S. State Department official. On 1 May, after the most recent round of negotiations, State announced that its top negotiator, Under Secretary R. Nicholas Burns, would fly to India “in the second half of May to reach a final agreement.” The U.S. Embassy in New Delhi now says that Burns has no imminent travel plans.

    The pact seeks to end India's nuclear pariah status. Under the deal's terms, India has designated civilian nuclear facilities to be placed under international safeguards by 2014, in exchange for clearance to import technology and fuel for its civilian nuclear program. In March 2006, Indian Prime Minister Manmohan Singh and U.S. President George W. Bush hailed the accord as the anchor of a new “strategic partnership.”

    The mood on both sides has soured since then. Indian scientists have assailed plans to segregate the nuclear establishment into civilian and military facilities. Critics also contend that U.S. legislation would penalize India over further nuclear tests (Science, 22 December 2006, p. 1863). Subsequent negotiations on the “123 Agreement,” a bilateral treaty that would spell out how to implement the pact, have hit several snags. The biggest bone of contention, sources say, is India's demand for an explicit acknowledgment of its right to reprocess spent nuclear fuel.

    A coup de grâce for the pact may come this summer, when India's Supreme Court hears a case from a metallurgist challenging the legal basis of the 123 Agreement. The petitioner is seeking full disclosure of all agreement drafts on the grounds that “the security, sovereignty, dignity and honour of the country [are] likely to be jeopardized and compromised” by the agreement. No matter how the court decides, a happy ending looks more elusive than ever. “There is no place for change in the Indian position,” says a top official at India's atomic agency. “If the twain does not meet, so be it.”

  6. GENETICS

    Working the (Gene Count) Numbers: Finally, a Firm Answer?

    1. Elizabeth Pennisi

    COLD SPRING HARBOR, NEW YORK—How many genes are in the human genome? Seven years ago, researchers were predicting that our genetic code was anywhere from 28,000 to 150,000 genes strong. Those were the outliers in a betting pool organized by Ewan Birney of the European Bioinformatics Institute in Hinxton, U.K. Birney predicted the answer would be in by 2003, when the human genome was due to be finished (Science, 19 May 2000, p. 1146).

    He was wrong—and so was everybody who bet.

    Today, the gene number is still “a mess,” according to Michele Clamp, a computational biologist at the Broad Institute of the Massachusetts Institute of Technology and Harvard in Cambridge, Massachusetts, who spoke at the Biology of Genomes meeting here earlier this month. The three databases that track protein-coding genes can't seem to agree, giving totals of 23,000, 19,000, and 18,000 genes. The real answer is 20,488—well below the lowest guess—with perhaps 100 more yet to be discovered, Clamp reported.

    This count may hold up. “I've looked at her data very carefully,” says Francis Collins, director of the U.S. National Human Genome Research Institute in Bethesda, Maryland. “It's a pretty good number.”

    In the classical sense, a gene is a sequence of DNA that codes for a particular protein. For proteins to be produced, a gene must first be transcribed, a process in which the cell makes a matching RNA molecule that carries the gene's instructions to the centers of protein production. Gene-prediction programs rely heavily on identifying the so-called open reading frames between the three-base codes that start and stop transcription. But there's been an explosion of discoveries of confusing RNA “genes”: transcribed sequences that have a biological function but don't produce a protein. And at the meeting, Birney and his colleagues reported finding several thousand other genes that also don't code for proteins, but researchers have no clues as to what they do.

    Thus an open reading frame “is not enough” to identify a gene that codes for a protein, said Clamp: “It's time to produce an integrated catalog of protein-coding genes based on the comparative evidence.”

    Clamp compared all the human genes in a database called Ensembl with those cataloged for dog and mouse. In all, 19,209 were the real, protein-coding McCoy, 3009 had been erroneously put on the gene list, and 1177 remained ambiguous, she reported.

    Not even close.

    For a betting pool set up in 2000, genome experts estimated the number of human genes. Even the winning—and lowest—number, 26,000, was 6000 genes too high.

    CREDIT: ILLUSTRATION BY C. FABER SMITH/SCIENCE

    She rated the “geneness” of these leftovers by comparing them to random stretches of DNA. Almost all made the grade with respect to a genelike proportion of the bases G and C, but not for features such as the distribution of short insertions and deletions in their sequences. Overall, 1167 were “bogus” and lacked any independent evidence that they coded for proteins, she reported. She did a similar analysis with the other gene databases, then summed the unique genes of all of them to get her final count.

    For Clamp to take a firm stand and call for a reconciliation of differences among the official gene-counters “was kind of brave and a lot of hard work,” says Jim Kent of the University of California, Santa Cruz. Now, says Stephen Richards, a genomicist at Baylor College of Medicine in Houston, Texas, anyone who disagrees with this number “will have to prove her wrong.”

  7. AGBIOTECH

    A Growing Threat Down on the Farm

    1. Robert F. Service

    Farmers have become dependent on a herbicide called glyphosate and on crops engineered to resist it. Now, weeds are becoming resistant, and researchers are scrambling for alternatives

    Superweed.

    Glyphosate-resistant Johnsongrass in a soybean field in northern Argentina.

    CREDIT: M. VILA-AIUB/UNIVERSITY OF BUENOS AIRES

    Conventional wisdom has it that biotech drugs have flourished while genetically modified (GM) crops have foundered because of protests in Europe and elsewhere. Not so. Biotech drugs are doing just fine and, it turns out, so are GM crops. Last year, 10 million farmers in 22 countries planted more than 100 million hectares with GM crops. Over the past 11 years, biotech crop area has increased more than 60-fold, making GM crops one of the most quickly adopted farming technologies in modern history (see figure below). Even the European Union is beginning to embrace them, with six E.U. countries now planting GM crops.

    What's behind this blossoming of transgenics? Oddly enough, a herbicide called glyphosate. The compound is the world's bestselling herbicide by far, prized by farmers for its safety and effectiveness at wiping out hundreds of different kinds of weeds. That effectiveness has not only convinced farmers to make the switch but also prompted seed companies to engineer crops to be impervious to glyphosate's effects. That has allowed farmers to spray their growing crops to wipe out encroaching weeds without fear of wiping out their livelihood. The model has proven so successful that of the transgenic crops planted worldwide last year, approximately 80% were engineered to be glyphosate-resistant (GR). “The rate at which this technology has been adopted floors me,” says Donald Weeks, a plant biochemist at the University of Nebraska, Lincoln.

    But this success has sown the seeds of its own potential demise. Much of modern agriculture is now dependent on a single chemical. “Glyphosate is as important to world agriculture as penicillin is to human health,” says Stephen Powles, who directs the Western Australian Herbicide Resistance Initiative in Perth. It's an apt comparison, because just as pathogens have grown resistant to penicillin and other antibiotics, weeds resistant to glyphosate have recently begun sprouting and spreading around the globe. For now, the scale of the outbreak remains small. But agricultural experts worry that herbicide-resistant weeds are poised for their own takeover. “There is going to be an epidemic of glyphosate-resistant weeds,” Powles says. “In 3 to 4 years, it will be a major problem.” If farmers and seed companies lose their ability to rely on glyphosate, it could cost them billions of dollars in lost productivity. But the damage will likely be more than monetary, as it could also have a major environmental consequence as well (see sidebar, p. 1116).

    Success story.

    Over the past decade, herbicide-resistant varieties have come to dominate the world market for genetically engineered crops.

    CREDIT: C. JAMES/ISAAA

    In the face of this threat, agricultural researchers are mounting a multipronged campaign to safeguard glyphosate and come up with other options in case its effectiveness withers. On page 1185, for example, Weeks and his colleagues at Nebraska report that they have developed the first transgenic crops resistant to an alternative herbicide called dicamba. Down the road, growers may soon switch transgenic crops much as doctors select antibiotics to stay one step ahead of pathogens. But for now, the fight is on to save glyphosate.

    Fantasy league

    The love affair between farmers and glyphosate was kindled long before biotech crops hit the fields. In 1970, John Franz, a chemist at Monsanto, discovered that the compound acted as a broad-spectrum herbicide, capable of killing an enormous variety of plants when deposited on the leaves of young seedlings. Later, researchers found that glyphosate wreaks its havoc by inhibiting an essential plant enzyme known as 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS). The enzyme catalyzes an intermediate step in the construction of a trio of aromatic amino acids, which in turn are vital for the production of key plant metabolites. Without EPSPS, the plants are starved of these metabolites and quickly wither and die.

    Just as enticing was what glyphosate does not do. Although concerns have been raised about the surfactants that are used alongside glyphosate in most formulations, glyphosate itself does not appear to affect animals and insects, which don't have EPSPS and rely on their diet for the amino acids the enzyme helps produce. And when sprayed on fields, glyphosate doesn't readily leach into water systems. Instead, it latches tightly to soil particles and degrades within weeks into harmless byproducts. By contrast, herbicides such as atrazine have been widely implicated in contaminating groundwater.

    Monsanto began selling glyphosate in 1974 under the trade name Roundup. Sales remained modest for years—until researchers engineered GR crops to use in combination with the herbicide. By 1983, researchers had isolated a gene known as CP4 in bacteria that synthesized aromatic amino acids through a different route from that of the EPSPS in plants. By 1986, they had spliced CP4 into plants and shown that the plants could withstand the effects of glyphosate with no apparent damage.

    It was another 10 years before Roundup Ready soybeans hit the market, but their impact was dramatic. In 1995, U.S. farmers used 4.5 million kilograms of glyphosate; they now use 10 times that amount. “If I were playing in an herbicide fantasy league, my first pick would be Roundup Ready cropping systems with glyphosate, and I would let you have the next three selections,” says John Wilcut, a crop scientist at North Carolina State University (NCSU) in Raleigh.

    Since 1996, Monsanto and other seed companies have introduced GR canola, cotton, corn, sugar beets, and alfalfa. The popularity of the herbicide was further fueled when the compound went off patent in 2000, which has triggered a 40% price drop in the years since. That combination produced a massive shift from traditional crop varieties to GR versions. In just a 5-year span, GR soybeans commanded 50% of the land cultivated for soy in the United States, and GR corn a 40% share. Today, GR soybeans make up more than 90% of soybeans planted in the United States, and corn more than 60%. By comparison, organic agriculture accounts for about 1% of cultivated land. “Farmers are normally very conservative,” says Weeks. “Clearly, this was a real winner.”

    Awaiting the inevitable

    One effect of that winning combination has been to slash the market for competing herbicides. According to data from the U.S. Department of Agriculture (USDA), the prices of two popular herbicides—chlorimuron and trifluralin—have dropped 20% to 40% since 1998. Over the same period, U.S. sales of all herbicides, including glyphosate, have declined by about $1 billion, nearly 20% of the industry total. Faced with this shrinking market and the glyphosate juggernaut, herbicide companies have been backing out of the market. Nearly 20 herbicides with different mechanisms of killing plants were sprayed on soybeans a decade ago; now, farmers are increasingly relying on glyphosate for most or all of their herbicide needs. In a survey of 400 farmers in the U.S. Midwest, for example, researchers at Syngenta found that 56% of soybean growers in northern states and 42% in southern states use glyphosate as their sole herbicide. As a result, “the selective pressure for weeds to develop resistance has been huge,” says Stephen Duke, a plant physiologist at USDA's Agricultural Research Service in Oxford, Mississippi. “From a biological perspective, this is inevitable,” adds Jerry Green, a weed scientist with DuPont Crop Protection in Newark, Delaware.

    For years, many researchers doubted that plants would be able to overcome their vulnerability to glyphosate, because EPSPS plays such a vital role in plant metabolism. One 1997 paper in the journal Weed Technology even stated that “the complex mutations required for the development of glyphosate-resistant crops are unlikely to be duplicated in nature to evolve glyphosate-resistant weeds.” Unfortunately, that was written just after the first GR weeds were discovered in 1996. Today, about a dozen different varieties of weeds are known to have developed resistance. And the spread of resistance to new weed species is increasing. Resistant weeds have now been spotted in countries around the globe, including the United States, Argentina, South Africa, Israel, and Australia. According to WeedScience.com, an international herbicide-resistance tracking service, GR “horseweed” was first identified in a Delaware field of GR soybeans in 2000, and since then it has turned up in 14 states as well as in Brazil and China.

    Again, like many microbes that evolve to outwit antibiotics, it now appears that GR weeds don't make a frontal attack on glyphosate. According to Christopher Preston, a weed-management scientist at the University of Adelaide in Australia, one common resistance mechanism centers on the way glyphosate moves within plants. In a presentation at a symposium on glyphosate resistance held as part of the American Chemical Society (ACS) meeting in March in Chicago, Illinois, Preston noted that when glyphosate is sprayed on the leaves of a susceptible plant, it is normally absorbed quickly and moves readily throughout its tissues. Once inside, it accumulates at the growth point in roots and stems and kills the plants. However, when Preston and his colleagues looked at a resistant form of rigid ryegrass, they found that the glyphosate accumulated in the leaf tips. The plant was essentially steering the compound away from areas where it could inflict lethal damage. Preston's team found a similar mechanism of resistance in two populations of horseweed as well, suggesting that glyphosate sequestering could be a mode of resistance common to many weeds.

    For now, however, resistant weeds are still the minority. According to the Syngenta survey, 24% of farmers in the northern portion of the Midwestern United States and 29% in the south say they have GR weeds. But only 8% say it's a problem across all of their acreage. Still, Syngenta's Chuck Foresman, who presented the data at the ACS meeting, says, “the resistance issue is across the Midwest, South, and Southeast. Nobody is exempt.” Crop scientists from Argentina, Brazil, and Australia echoed growing concerns about the problem in their countries as well.

    Backlash.

    Weeds that tolerate glyphosate are starting to appear throughout the world.

    CREDIT: I. HEAP/WEEDSMART LLC

    What to do?

    Fighting resistance is something of an uphill battle, says Duke. At the moment, not all farmers see resistance as a major issue, but by the time they do, resistance may be so widespread that it will be hard to combat. In recent decades, when resistance to one herbicide has spread, farmers have simply switched to another. But glyphosate's recent dominance of the herbicide market has reduced work on alternatives just when they are needed most. “Weed control is shifting to herbicide-resistant crops, and so are the research budgets,” Green says. That's bad news, NCSU's Wilcut says: “We need to have more of a diversity of herbicides out there.” But there are no new silver-bullet herbicides that are safe and broadly effective waiting in the wings. “We are not likely to get additional herbicide modes of action,” Wilcut says.

    With a multibillion-dollar market for herbicides and transgenic seeds at risk, agricultural researchers underscore the need to educate farmers to use long-standard methods of combating weeds, to preserve glyphosate's effectiveness as long as possible. Among these, says Weeks, are traditional resistance-management strategies of rotating crops and using a variety of different herbicides to combat weeds, practices that hinder resistant organisms from gaining a foothold in their fields. In many cases, that's likely to mean rotating in crops that don't rely on using glyphosate.

    Aside from proper stewardship practices, most researchers feel that the best hope for combating herbicide-resistant weeds is the continued development of transgenic crops. Nicholas Duck and colleagues at Athenix, a crop sciences start-up in Durham, North Carolina, for example, are developing crop varieties that are resistant to even higher levels of glyphosate. Planting them may allow farmers to buy some time by applying heavier doses of the herbicide to their crops, but it could add to the selective pressure on weeds to develop resistance.

    Other companies, meanwhile, are pushing crops resistant to herbicides other than glyphosate. Bayer Crop Sciences, for example, has already commercialized soybean and corn seeds resistant to glufosinate, a herbicide that kills plants by a different mechanism from glyphosate's. These crops, sold under the trade name Liberty Link, have not done as well in the market as glyphosate has because the herbicide is more expensive yet less effective at killing a broad range of weeds. But if GR crops continue to falter, Bayer could find itself a beneficiary.

    Dicamba, another cheap herbicide that has been on the market for 4 decades, could also emerge as a successor. Researchers in Texas created dicamba-resistant plants in 2003 by adding the gene for an enzyme that deactivates the herbicide. Seed companies have never managed to develop varieties that expressed enough of the enzyme to fully protect the crops. But in their report in this issue, Weeks and his colleagues managed to do just that, developing soybeans that in 3 years of field trials proved highly resistant to dicamba.

    As with previous herbicide-resistant crops, Weeks's team engineered their soybeans to express a bacterial gene that confers resistance, in this case by breaking down the herbicide. But in an ingenious twist, the Nebraska researchers targeted the engineered gene to be expressed in the plants' photosynthetic chloroplasts. The move offers two benefits, Weeks explains. First, the resistance-conferring enzyme works better because it can swipe the electrons it needs from the steady stream generated during photosynthesis. Also, like mitochondrial DNA, chloroplast DNA is inherited through the maternal side. That means a GM crop can't spread resistance through wind- or insect-carried pollen, which comes from the male side.

    New front.

    Soybeans resistant to the herbicide dicamba may help farmers diversify their antiweed arsenal.

    CREDIT: D. WEEKS/UNIVERSITY OF NEBRASKA

    Weeks says Monsanto has licensed the technology and that it could be commercially available within 3 to 4 years. If so, he says, it could allow growers to rotate their crops between varieties resistant to two different herbicides. “It gives farmers an alternative to the continual use of glyphosate-resistant crops,” Weeks says. And the development of herbicide-resistant crops won't stop with dicamba. “We have the technology today to develop herbicide resistance to about anything we want to,” Green says.

    Another approach being pursued at Monsanto and elsewhere is to combine, or “stack,” genes for resistance to multiple herbicides in the same plants. Researchers at Pioneer HiBred, a division of DuPont, for example, are working to create crops that are resistant to both glyphosate and herbicides that target a plant enzyme called acetolactate synthase. ALS inhibitors have also been on the market for years and face resistant weeds of their own. And scientists elsewhere announced last year that they plan to create crops resistant to herbicides that inhibit ACCase, an initial enzymatic step in lipid synthesis that is critical to grasses.

    In addition to stacking traits for resistance to multiple herbicides, researchers at Pioneer and elsewhere are looking to add other traits to crops, such as heat and drought resistance, increased yield, and insect resistance. In some cases, they hope to add genes for novel nutrsients and even pharmaceutical compounds. “There is a tremendous opportunity to do this for the next generation of traits,” Duck says. Although such efforts are still in the early stages, he adds, “in the future, everything is going toward product stacks.” The question is whether crops resistant to multiple herbicides will prolong the life of one of the farming community's favorite herbicides.

  8. AGBIOTECH

    Glyphosate--The Conservationist's Friend?

    1. Robert F. Service
    Stop loss.

    Plowed fields (left) suffer much more soil erosion than their no-till counterparts.

    CREDITS: P. CHRISTOFFOLOTI/UNIVERSITY OF SÃO PAULO

    Weeds resistant to the powerhouse herbicide glyphosate not only threaten the livelihoods of farmers worldwide, but they could have environmental downsides as well. Among the worst, glyphosate's disappearance could increase the loss of topsoil, require farmers to switch to more harmful herbicides, and force them to use more fuel to rid their fields of weeds.

    The current combination of herbicide-resistant crops and herbicide use is hardly an environmental panacea. A 2003 farm-scale evaluation in the United Kingdom, for example, found that the combination contributed to a loss of biodiversity both by reducing the numbers of weeds and by indirectly affecting insects that rely on those weeds for food. Many governments have also been cautious about allowing the use of herbicide-resistant crops for fear that genes that confer herbicide resistance could spread far beyond agricultural fields.

    Despite such concerns, many agricultural researchers now say glyphosate-resistant (GR) crops have had widespread environmental benefits, at least compared with the previously used alternatives. “Glyphosate-resistant crop weed management systems are generally safer to the environment than what they replace, and in many cases much safer," says Stephen Duke, a plant physiologist at the U.S. Department of Agriculture's Agricultural Research Service in Oxford, Mississippi.

    One of the biggest benefits of GR crops is their indirect impact on topsoil. Modern farming encourages heavy topsoil losses because farmers traditionally plow fields before planting seeds. Turning over the topsoil buries many weed seeds that were present under 4 to 6 inches of dirt. Although that reduces the likelihood that weeds will compete with emerging crop plants, it also dramatically increases the amount of topsoil that washes away with rain and irrigation.

    By contrast, many farmers don't plow their fields before planting GR crops. Instead, they simply plant seeds and spray glyphosate on their fields shortly after their crops have emerged, wiping out their weedy competitors. The upshot is that herbicide-resistant crops often require minimal tilling or no tilling at all. In March, at a symposium on glyphosate at the American Chemical Society meeting in Chicago, Illinois, Pedro Christoffoleti of the University of São Paolo in Brazil reported a recent study in South America that found that growing soybeans with conventional tillage produced topsoil losses of 1.2 tons per hectare. With GR crops planted with no-till practices, those losses shrank to 0.2 tons per hectare, a reduction of more than 80%.

    No-till agriculture saves farmers time and money, and for that reason the practice has grown dramatically with the rise of GR crops. In one recent study, the American Soybean Association in Washington, D.C., found that in just 5 years from 1996 to 2001 when herbicide-resistant soybeans first came on the market, the area of soybean land farmed by no-till agriculture in the United States increased from about 5 million hectares to more than 11 million hectares, whereas conventional tillage dropped from close to 8 million hectares to under 4 million hectares. By 2001, almost all no-tillage soybeans were GR varieties. What is more, because no-till agriculture requires less tractor use, the practice reduces soil compaction and cuts fuel use on farms. All those benefits could take big hits should the emergence of GR weeds prompt farmers to abandon glyphosate, Duke says.

    SOURCE: S. DUKE, NPURU/USDA/ARS

    Additional impacts could come as farmers switch to herbicides that are more toxic to mammals. Gerald Nelson, an agricultural economist at the University of Illinois, Urbana-Champaign, and his colleagues have recently begun looking at the likely impact of that shift. To do so, they used a common yardstick, known as the LD50 dose, to compare the toxicity of various herbicides. The LD50 dose is a widely available measurement of the amount of a particular compound required to kill half of a population of rats in lab studies. When the researchers looked at the effect of switching from GR crops to conventional seeds with other herbicides, they found that the switch would require farmers to increase the LD50 doses applied to the average U.S. farm by about 10% per hectare in soybeans and 25% per hectare in cotton. Nelson says it's not yet clear how such changes will translate into impacts on organisms other than mammals, such as insects and birds. However, Nelson adds, “there will be some more effects on anything else susceptible to these [alternative] herbicides.”

  9. ANTONIO GIORDANO PROFILE

    'Dr. Hustle' Sells His Dream for Italian Medical Research

    1. Elisabeth Pain*
    1. Elisabeth Pain is a contributing editor for ScienceCareers.org and a freelance science writer based in Barcelona, Spain.

    After making his mark in the United States, an Italian cancer researcher with a knack for raising private money seeks to inject new life into biomedical science back home

    Special delivery.

    Antonio Giordano (left) shows off a lab to one of his backers, pizza magnate Mario Sbarro.

    CREDIT: SBARRO HEALTH RESEARCH ORGANIZATION

    It may not look like much now, but a dilapidated mansion in the green and hilly region of Umbria, far from any major research university or institution, is being touted as the future birthplace of an Italian renaissance in biomedical science. That's the dream of Antonio Giordano, who 20 years ago left Naples to make his scientific name in the United States. Giordano now runs his own cancer research institute in Philadelphia, thanks in part to a relentless pursuit of support from corporate and private donors. The magazine Philadelphia even dubbed Giordano “Dr. Hustle” in a profile that detailed how he obtained a large donation for his institute after wooing a pizza magnate during weekly strolls.

    By supplementing grants from the National Institutes of Health (NIH) with privately raised money, the 44-year-old Giordano has gained some freedom in the United States to pursue his own research agenda; he even established a private foundation that funds graduate students and postdocs at his institute. Now, after securing commitments for more than €60 million from Italian financial services institutions, Giordano would like to help a generation of young Italians back home pursue biomedical research. And by offering an alternative to the charity and governmental funding systems that he believes are narrow-minded and stifle Italy's science, Giordano hopes to persuade many of his protégés to stay there. “In Italy, there are not many possibilities for research, and many Italians … look at me not only as an example but [also] as a person that can help them,” he says.

    Giordano's reputation in Italy is such that when politicians in the Umbrian town of Terni, 108 kilometers north of Rome, heard of his plans to set up a biomedical research institute, they called to offer a city-owned mansion. The building, currently being refurbished, should open its doors in 2009.

    That wasn't fast enough for the impatient Giordano and his sponsors, however. So an interim laboratory is being built not far from the mansion. This fall, 20 to 30 students and postdocs should be working there. When the mansion is ready, the new institute will ultimately provide lab space for another 50 young researchers to work, primarily on cancer, but on cardiovascular disease and diabetes too. It will also include a facility to treat cancer patients, run trials of therapies, and develop research in cancer prevention.

    Paul Fisher, a cancer researcher at Columbia University in New York, notes that what really set Giordano apart from other good scientists are his entrepreneurial spirit and capacity to exploit nontraditional avenues of funding. “His personality makes it possible to integrate the picture of research into something that is sellable,” says Fisher.

    Breaking the mold

    Giordano initially set his heart on a career in medicine. But while training at the University of Naples back in the 1980s, he had second thoughts. “I realized that medicine can be very routine [and] that there were too many devastating illnesses that needed more … research work,” Giordano says. After earning his medical degree in 1986, Giordano decided to swap medicine for genetics and cancer research.

    In 1987, he came to America to be a postdoc, at New York Medical College in Valhalla, and then under Nobel Prize winner James Watson at Cold Spring Harbor Laboratory. There he won recognition for isolating the cyclin A protein, a cell growth regulator. That discovery, says Giordano, provided “the first physical evidence of a link between cell division and cancer.”

    Since then, Giordano has had other successes, including cloning the Rb2/p130 tumor-suppressor gene, which was subsequently found to be involved in many cancers. He's had “an outstanding career with some very exciting findings … that really helped launch a number of fields within cell cycle research,” says Fisher.

    Giordano moved in 1992 to Temple University in Philadelphia, Pennsylvania, and set up a 10-person lab conducting cell cycle and cancer research with an initial 3-year NIH grant. It quickly became clear to him, however, that private sources of support were also needed. “I saw colleagues, also very good, who disappeared because they didn't realize how it was important to be independent and search for your own funding,” he says.

    As Giordano began to envision a research institute of his own, he got lucky. His wife-to-be, whom he met during his time at Cold Spring Harbor, lived in the same New York neighborhood as the owners of Sbarro, a U.S.-based chain of fast-food restaurants that sells pizza and Italian dishes. Giordano soon encountered fellow Neapolitan Mario Sbarro and after almost a year of Sunday-morning walks on Long Island won from him an initial donation of about $1 million to create the Sbarro Institute for Cancer Research and Molecular Medicine. Sbarro says he was impressed by Giordano, particularly his vision of “creating an environment where talented [young] people … could work together … free of bureaucracy.”

    To retain control of his private money, Giordano felt he needed to break free from the university's authority. But he also wanted the university's administrative support and infrastructure to keep nonresearch costs minimal. Convincing Temple to go along was not easy. In fact, in 1994, Giordano moved his lab to Thomas Jefferson University, also in Philadelphia, where he was offered an agreement that included the university matching Sbarro's donation. “After 2 years, my lab had tripled in number of people and space,” Giordano says. But nearly a decade later, in 2002, Giordano returned to Temple after securing, in his words, “complete independence” in administering the funds, staff, research programs, and patent rights.

    Giordano's return also marked the launch of a nonprofit organization—the Sbarro Health Research Organization (SHRO)—to collect additional private funds for the institute. Sbarro, who had continued to support Giordano's work, kicked in another $200,000 a year for 3 years as seed money. To date, Giordano has raised $3 million in private funding, supplementing about $27 million that he and other investigators at his institute have obtained through NIH grants and earmarks from the state of Pennsylvania and the Department of Defense, which has also just awarded SHRO $2 million a year for 2 years for breast cancer research.

    The private money raised by SHRO comes with fewer entanglements than those attached to NIH grants, contends Giordano. As a result, he's free to dedicate a great part of these private research dollars to risky projects, such as the development of a novel gene-therapy approach for the treatment of lung, liver, and ovarian cancers. SHRO mainly funds young scientists, through research grants and 1- to 3-year fellowships of $25,000 a year for students and between $35,000 and $40,000 for postdocs. Although SHRO has an external scientific advisory board, Giordano has largely decided which areas are investigated and who gets funded. But then, he says, “we want these people to … pursue their own independent ideas and careers.”

    Going home

    With the project in Terni, Giordano is extending his reach into Italy, hoping eventually to use it as a springboard to fund scientists across Europe. He had in 2000 started to use his privately raised money to fund graduate students and postdocs in a few labs at places such as the University of Siena, University of Rome “La Sapienza,” and the University of Naples. As in Philadelphia, the universities offer the researchers access to equipment and other infrastructure.

    Extreme makeover.

    Once refurbished, this mansion in the Italian town of Terni will house a new biomedical research institute.

    CREDIT: HUMAN HEALTH FOUNDATION

    Once SHRO was established, it became Giordano's avenue for distributing funds abroad. SHRO's money is welcome because it is more difficult to find funding for cancer research in Europe than in the United States. In 2002-03, the whole of Europe spent €1.43 billion on public cancer research with the 25 E.U. Member States disbursing only one-seventh the per capita amount spent by the United States, according to the European Cancer Research Managers Forum. “European minds are excellent,” says Giordano, but they often do not flourish until they get to America, where there is better support.

    By creating the Terni institute, as well as a new nonprofit, the Human Health Foundation (HHF), Giordano says he's throwing a lifeline to researchers in Italy, where only a few institutions—“oases,” he calls them—typically receive money from the country's major research funding bodies. Already, HHF has collected €60 million from two Italian financial institutions, the Banca Popolare di Spoleto and Spoleto Credito e Servizi. Giovanni Antonini, the president of the Spoleto bank, has even agreed to head HHF; Giordano will head its scientific committee. By funding HHF, says Antonini, his bank hopes to encourage “the return of the Italian minds who were constrained to leave Italy to improve their professional careers.”

    Fifteen million euros will be used to refurbish the Terni mansion. The remainder of the HHF money will go directly into research projects and the creation of additional labs. And Giordano stresses that the foundation's funds will be awarded through a transparent process involving peer review. By this summer, between 10 and 15 early-career scientists will start working on HHF-funded projects in Siena and Philadelphia while the interim lab in Terni, which will cost €500,000, gets up and running.

    At the moment, Giordano supports about 100 young researchers across Italy and the United States together. About 70% of the students and postdocs Giordano has trained or funded so far, a network that today counts more than 250, are Italian. He has been able to aid that many in part thanks to complementary national and European funding programs.

    Giordano “acts as a role model and mentor,” says Alessandro Bovicelli, 39, who came to the Sbarro Institute in 2000 for a postdoc in gynecological oncology and still collaborates with Giordano. “He is … very focused on the objectives that the young doctor would like to pursue.” Now a faculty member at the Department of Obstetrics and Gynecology at the University of Bologna, Bovicelli says that Giordano's continuous encouragement was vital.

    Normally confident, Giordano admits uncertainty about whether he will be as successful in his new project as he's been in the United States. “In Italy, there is not the infrastructure there is in the U.S.,” he says, and building an institute from scratch is a major undertaking. Giordano notes that his mother asks why he can't be satisfied with what he has already done on the U.S. side of the Atlantic. His answer is simple: “I owe this to Italy. This is where I grew up and was trained.”

  10. DNA SEQUENCING

    A New Window on How Genomes Work

    1. Elizabeth Pennisi

    A deluge of discount, high-quality sequences made possible by new technologies has inspired researchers to use these data in new ways to understand DNA regulation

    DNA on glass.

    One new technology sequences DNA fragments anchored on slides, using bases tagged in four different colors (dots).

    CREDIT: OMEAD OSTADAN/SOLEXA

    COLD SPRING HARBOR, NEW YORK—Starting with the Human Genome Project, researchers and companies have been racing to make DNA sequencing faster and cheaper. The dream is to decipher a person's genome for $1000, a price that would open up a wealth of medical applications. Nobody is close yet, but recent successes in driving costs down have opened up a different application: New high-throughput sequencing machines are giving researchers unprecedented views of where and how proteins interact with DNA.

    Like viewing the planet through Google Earth, researchers are using these machines to swoop down on genomic neighborhoods to reveal details of the complex landscape of gene regulation: the places where proteins turn genes on or prevent them from being expressed. “It's showing us things we've never seen before,” says computational biologist Michele Clamp of the Broad Institute of the Massachusetts Institute of Technology and Harvard in Cambridge, Massachusetts.

    DNA is nothing without its proteins. At any one time, tens of thousands of proteins are latching onto or backing away from the genome, creating the dynamic biochemistry that fuels life. Transcription factors turn the appropriate genes on and off. Some proteins, in particular histones, shape chromosomes, grabbing onto and holding DNA in its characteristic spiral, closing genes down, or unwinding it to allow genes to function. Others cut DNA at specific locations. It's this control of gene expression that differentiates brain from liver, T cell from pancreatic islet cell. Researchers want to pin down the sites where all this action takes place, and the latest sequencing technologies—including the star of the moment, Illumina Inc. (Solexa)—are proving adept at doing just that. The new technologies, which began making their debut last year (Science, 17 March 2006, p. 1544), promise greater accuracy while reducing the costs of sequencing several-fold below those of the Human Genome Project. But they all share one potential drawback for sequencing whole genomes: They can only sequence short DNA fragments—so-called reads. Short reads are difficult to reassemble accurately into a completed genome. But for researchers studying genome function, short, inexpensive reads are just what they need to characterize—rapidly and cheaply—the sites where a particular protein binds to the genome.

    The process, dubbed tag sequencing, had a coming-out party at the Biology of Genomes meeting here last month. It drew rave reviews. “[These] sequencing technologies are really transforming the way we do things and what we are able to do,” says Bradley Bernstein, a pathologist at Massachusetts General Hospital in Boston. Tag sequencing has been limited because of its cost, but now “if you are not thinking about your experiments on a whole-genome level, you are going to be a dinosaur,” says molecular biologist John Stamatoyannopoulos of the University of Washington, Seattle.

    One protein's reach

    Richard Myers and Ali Mortazavi of Stanford University in Palo Alto, California, are using tag sequencing to nail down where a transcription factor called neuron restrictive silencer factor (NRSF/REST) shuts down nerve-cell genes in non-nerve cells. Like many other researchers, the Stanford duo, along with Barbara Wold and David Johnson of the California Institute of Technology in Pasadena, start with a technique called chromatin immunoprecipitation to isolate the sites where the transcription factor binds to DNA. In this procedure, they break up the genome from an immortalized T cell and add antibodies to NRSF/REST to pick out the pieces of DNA with NRSF/REST attached.

    Until recently, they used microarrays—expensive chips studded with thousands of snippets of DNA from known locations on a reference genome—to identify the segments of DNA bound to NRSF/REST. But now they simply sequence all the DNA to which NRSF/REST is attached and map those sequences directly to the reference genome. “This represents an order of magnitude increase in resolution compared to [microarrays],” says Martin Hirst, a molecular biologist at the British Columbia Cancer Agency Genome Sciences Centre in Vancouver, Canada. “And this is achieved at a fraction of [the] cost.”

    Myers, Wold, and their colleagues found more than 1950 NRSF/REST binding sites—30% more than they were able to identify by microarrays—and pinned them down to within 50 bases. They also discovered that NRSF/REST has three types of DNA landing sites. One is a 21-base stretch already known to bind NRSF/REST. Another consists of those 21 bases split in half, with 17 other bases stuck in the middle. A third consists of just half the bases, the researchers reported at the meeting. By looking at more types of cells, they expect to learn about how these various landing sites evolved.

    Defining genes

    Bernstein had a different protein-DNA interaction in mind when he turned to tag sequencing. In the nucleus, DNA is wrapped around proteins called histones, which help control the DNA's state of readiness. Depending on where a methyl group sits on the histone, the protein can activate or silence a gene, in part by making the gene's regulatory DNA more or less accessible to transcription factors. Bernstein and his colleagues have used methylated histones—which are attached to their nearby DNA—to find active and inactive parts of chromosomes in mouse embryonic stem cells and cells that have differentiated into specific types.

    Bernstein used one set of antibodies against a histone with a methylation profile known to silence DNA and repeated the process with antibodies against histones whose methylation activates DNA. Tarjei Mikkelsen of the Broad Institute and his colleagues then sequenced and analyzed the DNA fragments attached to each type of histone.

    When they mapped the sequences back onto the mouse genome, a striking contrast emerged. In differentiated cells, the map of silencing histones was different from the map of activating ones—as one might expect. But in the stem cells, the maps overlapped in some places. Genes important, say, in turning that cell into a nerve cell tended to bear both a silencing and an activating histone, Bernstein reported. The former kept the gene quiet to enable the stem cell to keep its options open, but by having the activating histone on board as well, those genes “are poised for activation,” he suggested.

    Bernstein is also finding that gene boundaries can be defined by their histone companions, something that genomicists have had a lot of trouble doing. One type of histone is present at the beginning of a gene; another is attached all along the gene. The histone map “basically tells you not only where the exons are but where the gene starts and stops,” says Francis Collins, director of the U.S. National Human Genome Research Institute in Bethesda, Maryland. “It looks awfully good.”

    Hirst too has used histones to tag sequences, but in cancer cells. “We have been able to find functional classes of genes, which are enriched for specific combinations of [histone] modifications,” he reported. If he and others are able to pinpoint tagging patterns that are characteristic of cancer, “diagnostics could be designed to probe for these regions,” he pointed out.

    Broad overview

    Greg Crawford, a molecular biologist at Duke University in Durham, North Carolina, and, independently, Stamatoyannopoulos have even bigger plans for using sequencing as a tool to understand genome regulation. Instead of using chromatin immunoprecipitation, they depend on a biochemical trick—an enzyme called DNase I—to track down docking sites for the full gamut of regulatory proteins. “Those regions are hidden in the genome and historically have been very difficult to find,” says Crawford.

    Pushing limits.

    By combining cheap sequencing with the use of antibodies to pin down protein-DNA interactions, researchers are learning what turns genes on and off throughout the genome.

    CREDIT: C. BICKEL/SCIENCE, ADAPTED FROM J. D. WATSON, A. A. CAUDY, R. M. MYERS, AND J. A. WITKOWSKI, RECOMBINANT DNA: GENES AND GENOMES, 3RD EDITION (2007)

    Where regulatory proteins dock onto DNA, the chromosome begins to unwind and expose the DNA. Those are also sites where the DNA is hypersensitive to DNase I, which is now able to get to the DNA itself to cut it. For decades, molecular biologists have used this enzyme to track down docking sites a few at a time. More recently, microarrays have made it possible to search for them more broadly. However, the DNA probes on microarrays are not always reliable because they can't detect docking points buried in repetitive DNA and at times incorrectly flag other spots.

    At the meeting, Crawford described how, instead of using microarrays, he simply sequences all the spots that DNase I targets across the entire genome. Stamatoyannopoulos, too, is moving ahead with tag sequencing. Both are finding hundreds of thousands of docking sites, many in unexpected locations. Only about 40% are at the starts of genes, Crawford reported. Others are in the introns, the sequence in between a gene's protein-coding regions, and some are 200,000 bases from the nearest gene. “Some are mapping to gene deserts where people didn't think there was much going on,” he said.

    At the same time that Crawford, Stamatoyannopoulos, Bernstein, and Myers are mapping regulatory sites along DNA, Yijun Ruan and Chia-Lin Wei of the Genome Institute of Singapore are probing how DNA from different parts of a chromosome, or even different chromosomes, affect gene activity from afar. Some of the regulatory DNA associated with a particular gene can reside a long way from the gene itself. Sometimes a protein attached to DNA in one part of the genome contacts a protein attached at another location, causing DNA to form loops, and both play a role in the gene's activity.

    To “see” these long-distance interactions, Ruan and Wei have combined chromatin precipitation, sequencing, and a technique for freezing these DNA loops in place. In this way, they are able to track down exactly where these interacting proteins operate throughout the whole genome. “It's a high-throughput way of looking at these molecular interactions in three-dimensional space,” says Ross Hardison of Pennsylvania State University in State College. With this technique, the Singapore team has studied the effects of estrogen on gene regulation in breast cancer cells. Estrogen activates the estrogen receptor, which in turn activates genes. They found that more often than researchers have realized, the estrogen receptor binds to DNA quite far away from its target gene.

    Just as Google Earth can take you from the whole planet to a neighborhood, these new approaches are providing glimpses of gene regulation at different levels of resolution. DNase I provides a global view, and transcription factor studies a very focused view, with histones helping to tag a variety of regulatory landmarks. And Ruan's technique may reveal connections between remote regulatory regions of the genome. It's a complex network, but Collins is confident of rapid progress. By combining these levels, he predicts, “you can really start to figure out what's happening.”

Log in to view full text

Log in through your institution

Log in through your institution