We've seen these visions glinting in the distance for some time--the prospect that one day parents will be able to browse through gene catalogs to special-order a hazel-eyed, redheaded extrovert with perfect pitch. Leave aside for the moment whether scientists actually found an "IQ gene" last week or the argument over what really constitutes intelligence. Every new discovery gives shape and bracing focus to a debate we have barely begun. Even skeptics admit it's only a matter of time before these issues become real. If you could make your kids smarter, would you? If everyone else did, would it be fair not to?

It's an ethical quandary and an economic one, about fairness and fate, about vanity and values. Which side effects would we tolerate? What if making kids smarter also made them meaner? What if only the rich could afford the advantage? Does God give us both the power to re-create ourselves and the moral muscles to resist? "The time to talk about it in schools and churches and magazines and debate societies is now," says bioethicist Arthur Caplan of the University of Pennsylvania. "If you wait, five years from now the gene doctor will be hanging out the MAKE A SMARTER BABY sign down the street."

What makes the conversation tricky is that we're already on the slippery slope. Doctors can screen fetuses for genetic diseases like cystic fibrosis and Duchenn muscular dystrophy; one day they may be able to treat them in utero. But correcting is one thing, perfecting is another. If doctors can someday tinker with a gene to help children with autism, what's to prevent them from tinkering with other genes to make "normal" children smarter? Technology always adapts to demand; prenatal sex-selection tests designed to weed out inherited diseases that strike one gender or the other--hemophilia,

for instance--are being used to help families have the sonor daughter they always wanted. Human-growth hormone was intended for children with a proven severe deficiency, but it came to be used on self-conscious short kids--if their parents could afford as much as $30,000 for a year's injections.

Self-improvement has forever been an American religion, but the norms about what is normal keep changing. Many parents don't think twice about straightening their kids' crooked teeth but stop short of fixing a crooked nose, and yet, in just the past seven years, plastic surgery performed on teens has doubled. As for intellectual advantages, parents soak their babies in Mozart with dubious effect, put a toy computer in the crib, elbow their way into the best preschools to speed them on their path to Harvard. Infertile couples advertise for an egg donor in the Yale Daily News, while entrepreneurs sold the sperm of Nobel laureates.

"What, if anything, is the difference between getting one's child a better school and getting one's child a better gene?" asks Erik Parens of the Hastings Center, a bioethics think tank. "I think the answer has to do with the difference between cultivating and purchasing capacities." Buying a Harvard education may enhance a child's natural gifts, he argues, but it's not the same as buying the gifts.

Every novel, every movie that updates Frankenstein provides a cautionary tale: these experiments may not turn out as we expect. Genetic engineering is more permanent than a pill or a summer-school class. Parents would be making decisions over which their children had no control and whose long-term impact would be uncertain. "Human organisms are not things you hang ornaments on like a Christmas tree," says Thomas Murray, Hastings' director. "If you make a change in one area, it may cause very subtle changes in some other area. Will there be an imbalance that the scientists are not looking for, not testing for, and might not even show up in mice?"

What if it turned out that by enhancing intellectual ability, some other personality trait changed as well? "Everything comes at a price," argues UCLA neurobiologist Alcino

Silva. "Very often when there's a genetic change where we improve something, something else gets hit by it, so it's never a clean thing." The alarmists, like longtime biotech critic Jeremy Rifkin, go further. "How do you know you're not going to create a mental monster?" he asks. "We may be on the road to programming our own extinction."

The broader concern is one of fairness. Will such enhancement be available to everyone or only to those who can afford it? "Every parent in the world is going to want this," says Rifkin. "But who will have access to it? It will create a new form of discrimination. How will we look at those who are not enhanced, the child with the low IQ?" Who would have the right to know whether your smarts were natural or turbo-charged? How would it affect whom we choose to marry--those with altered genes or those without? If, as a parent, you haven't mortgaged the house to enhance your children, what sort of parent does that make you? Will a child one day be able to sue her parents for failing to do everything they could for her?

But just for the sake of argument, suppose raising IQ didn't require any permanent, expensive genetic engineering at all. Scientists are studying brain-boosting compounds. Suppose they found something as cheap and easy as aspirin; one pill and you wake up the next morning a little bit brighter. Who could argue with that?

Some people are worried about the trend toward making people more alike--taller, thinner, smarter. Maybe it's best for society as a whole to include those with a range of needs and talents and predispositions, warts and all "As someone who morally values diversity," says ethicist Elizabeth Bounds of Emory University's Candler

School of Theology, "I find this frightening. We run the risk of shaping a much more mogeneous community around certain dominant values, a far more engineered community." What sort of lottery would decide who is to leap ahead, who is to be held back for an overall balance? At the moment, nature orchestrates our diversity. But human nature resists leaving so much to chance, if there is actually a choice.

The debate raises an even more basic question: Why would we want to enhance memory in the first place? We may imagine that it would make us happier, except that we all know smart, sad people; or richer, except that there are wildly successful people who can't remember their phone number. Perhaps it would help us get better grades, land a better job, but it might also take us down a road we'd prefer not to travel. "You might say yes, it would be wonderful if we could all have better memories," muses Stanford University neuropsychiatrist, Dr. Robert Malenka. "But there's a great adaptive value to being able to forget things. If your memory improves too much, you might not be a happier person. I'm thinking of rape victims and soldiers coming back from war. There's a reason the brain has evolved to forget certain things."

In the end it is the scientists who both offer the vision and raise the alarms. People with exceptional, photographic memories, they note, sometimes complain of mental overload. "Such people," says University of Iowa neurologist Dr. Antonio Damasio, "have enormous difficulty making decisions, because every time they can think of 20 different options to choose from." There is luxury and peace in forgetting, sometimes; it literally clears the mind, allows us to focus on the general rather than the specific and immediate evidence in front of us. Maybe it even makes room for reflection on questions like when better is not necessarily good.

--Reported by David Bjerklie and Emily Mitchell/New York, J. Madeleine Nash/Chicago and Dick Thompson/Washington

-QUOT-

"Every parent is going to want this. But who will have access to it?"

"There's a reason the brain has evolved to forget certain things." --Dr. Robert Malenka

Article 2

Don't look for the southern French town of Montredon on your globe. It isn't even on local road maps, perhaps because it has only 20 inhabitants. But one of them, a Parisian intellectual turned activist-farmer named Jose Bove, may change that. He's the leader of the mobs of farmers who've trashed several McDonald's in France lately. Last week, with 200 supporters chanting outside the jail, Bove declined a Montpellier court's offer of bail and remained behind bars, the better to spotlight his cause. And that would be? "To fight against globalization and advance the right of people to eat as they see fit," he explained.

Grievance No. 1: the U.S. desire to export genetically modified crops and foods. So far, so French, right? But spin that same globe to Peoria, Ill., home of U.S. agribusiness giant Archer Daniels Midland. There, even as Bove's judges readied their decision, the self-styled "supermarket to the world" was demonstrating that the customer is, indeed, always right. In a fax to grain elevators throughout the Midwest, ADM told its suppliers that they should start segregating their genetically modified crops from conventional ones, because that's what foreign buyers want. It didn't matter that GM crops are widely grown by U.S. farmers, and that there's no evidence that the taco chips and soda you're enjoying right now are anything worse than fattening.

ADM had noticed something new sprouting under the bright, warm sun of economic interdependence: a strange hybrid of cultural and economic fears. So it decided to act before the problem got any bigger. Public opposition to GM foods in Europe has been mounting for more than two years, especially in Britain and France. Both Prince Charles and Paul McCartney have come out against the stuff. Now the protests and the tabloid headlines about "Frankenstein Foods" have reached such a pitch that they're reverberating across the Atlantic. Secretary of Agriculture Dan Glickman, a longtime backer of biotechnology, admitted as much in a key speech in July. So did Heinz and Gerber when they announced the same month that they'll go to the considerable trouble of making their baby foods free of genetically modified organisms.

Groups such as Greenpeace, which have long fought biotech on both continents, are crowing. U.S. trade officials, who face a tough fight keeping markets open for American agricultural products, are worrying. And U.S. consumers, who have never really thought much about genetically modified foods, are just plain confused. As well they might be, given the vastly different experiences the United States and Europe have had. In the United States, the FDA issued a key ruling in 1992 that brought foods containing GM ingredients to market quickly, and without labels. Companies such as Monsanto introduced herbicide-resistant soybeans and corn that makes its own insecticide. U.S. farmers loved the products; by 1998, 40 percent of America's corn crop and 45 percent of its soybeans were genetically modified.

In Europe, meanwhile, there was no real central regulator to green-light the technology and allay public concerns, and many more small farmers for whom biotech represented not an opportunity but a threat. Leaders have tried to steer a course between encouraging a new industry and giving the voters what they want, including labeling rules. So, to each his own, right? Not in 1999. If Europe is selling America Chanel perfume and Land Rovers, America will want to sell Europe its soybeans and corn--and maybe even its fervent faith in progress. While European biotech companies such as Novartis avoided the limelight, St. Louis-based Monsanto decided to press its case. The timing was terrible. GM fears were already running high last summer when Monsanto ran an informational campaign; Britain's 1996 bout with mad-cow disease, though unrelated, had weakened European confidence in regulators and industrial-strength agriculture.

Monsanto's PR effort only made the mood worse, as have a string of bad-news food headlines since then: dioxin-contaminated chicken in Belgium last spring; tainted Coke in Belgium and France this summer, and a punitive U.S. tariff on imports of foie gras and other products, imposed in July because Europe won't accept American hormone-fed beef. That last, also nongenetic, dispute actually triggered the vandalism at McDonald's last month. But to many of France's famously irascible small farmers, it's all of a piece. Even among the broader public in France and Britain, the GM-foods issue seems to be intersecting with second thoughts about globalization. French farmers protest American imperialism. But just last week their biggest customers, grocery giants Carrefour and Promodes, announced a $16.5 billion merger that will position them well in a global battle with America's Wal-Mart--and put further cost pressures on farmers.

Britain is a hotbed for Internet start-ups. But Brits still tune in to the BBC radio soap "The Archers" to see if young Tommy will go to jail for helping a group of eco-warriors wreck a GM-crop trial site on his uncle's land. Would an American jury let Tommy go? Probably not. Consumers Union, whose Consumer Reports magazine features a big piece on GM foods this month, has put together an array of poll data suggesting Americans would like to see GM food labeled, but remain interested in its benefits. Of course, if Tommy's trial were held in Berkeley, Calif., where the school board has announced a ban on GM foods, he might walk.

U.S. activists, encouraged by the successes of their European brethren, hope to build on such sentiments. Some of the rhetoric is extreme, and one group--or perhaps it's just one person--has resorted to vandalism, trashing a test-bed of GM corn at the University of Maine last month and crediting the act to "Seeds of Resistance." But there's science going on, too. A Cornell University study published in the journal Nature in May found that half of a group of monarch-butterfly caterpillars that ate the pollen of insecticide-producing Bt corn died after four days. What if the pollen spreads to the milkweed the monarchs lay their eggs in? "The arguments aren't enough to say we shouldn't have any biotechnology," says Rebecca Goldburg of the Environmental Defense Fund. "But they are enough to say we should be looking before we leap." Of course we should, says Gordon Conway, president of the Rockefeller Foundation and an agricultural ecologist. Invited to speak to the Monsanto board in June, he used the forum to talk about the need to go a little slower. But, he adds, don't worry about the monarch. Bioengineers can stop the pesticide (which is supposed to kill caterpillars; they eat the corn) from being expressed in pollen. "There are always problems in the first generation of a new technology," he says. And, he adds, successes.

The foundation just unveiled a genetically modified rice grain it funded to improve nutrition in the developing world. If a shouting match over GM foods should derail such not-for-profit efforts, he says, "that would be a tragedy." Agriculture Secretary Glickman doesn't see Americans growing as fearful as Europeans, mainly because he thinks Americans have more faith in their regulators. He also thinks that labeling of GM foods is a big part of the answer--not mandatory labeling, which industry opposes and activists demand, but voluntary labeling. "I'm not going to mandate this from national government level," he told News-week, "but I believe that more and more companies are going to find that some sort of labeling is in their own best interest." Especially companies that want to export.

Because, as ADM showed with its heartland-stopping announcement on Thursday, it isn't only up to Americans anymore. Brian Kemp, a Sibley, Iowa, farmer, made an urgent call to his elevator on Thursday to see if it would still buy his GM corn. It will--this year. "Europe is so important to the industry that it could mean we'll really have to pull back on growing GM crops in this country," says Walt Fehr, head of Iowa State University's biotech department. "Given the choice, who wants to grow GM?" Glickman says the trade issue--which is sure to generate plenty of heat at the November World Trade Organization meeting in Seattle--will be a tough one to resolve. "But I think over the next five years or so we can get it done." That's a mighty slow pace, considering how quickly the industry came along in the previous half decade. But then, you generally do travel faster when you travel alone. With John Barry in Washington, Scott Johnson in Montpellier, Jay Wagner in Des Moines, William Underhill in London and Elizabeth Angell in New York Mag.Coll.: 100A0624

Organic Gardening, Sept 1999 v46 i5 p16

Article 3

According to a growing number of studies that have been conducted around the world. In Canada, the herbicide 2,4-D was found in rainfall at levels that could harm plants. And Swiss scientists have shown that much of Europe's rainwater is so contaminated with pesticides that it would be illegal to use as drinking water. Don't believe it? Full details on the Swiss research are available in the Summer 1999 issue of the journal Analytical Chemistry.

An international committee has found that use of Monsanto's genetically engineered bovine growth hormone (rBGH) may produce chemicals in cows' milk that could cause breast or prostate cancer in humans who drink the milk, as well as reproductive disorders and other diseases in treated cows. Canadian health officials have refused to recommend approval of the hormone injections, recognizing that more long-term research is needed to assure the hormone's safety. Meanwhile, millions of U.S. consumers are unknowingly drinking milk from cows treated with rBGH, which was approved several years ago in a controversial decision made by the U.S. Food and

Drug Administration.

Genetically engineered (GE) corn pollen contains a toxin that can poison the caterpillars of monarch butterflies, according to a new study from Cornell University.

Millions of acres of corn and other GE crops are now being grown in the United States, but their use is still heavily restricted in most other countries.

The U.S. Department of Agriculture has dropped a policy that had barred organic-meat producers from using the term organic on their products. Look for certified-organic

meats and poultry in stores soon.

Ninety percent of all nonorganic beef raised in the United States contains up to six growth hormones that are banned in Europe because of health concerns. At least one of those hormones, 17 beta-oestradiol, is considered "a complete carcinogen," the European Union's Scientific Committee on Veterinary Measures reported. And all of the banned hormones "may cause a variety of health problems, including cancer, developmental problems, harm to immune systems, and brain disease." The 139-page report added: "Even exposure to small levels of residues in meat and meat products carries risks."

U.S. Secretary of Agriculture Dan Glickman dismissed the panel's concerns as "unsubstantiated." But Samuel Epstein, M.D., a professor of environmental medicine at the University of Illinois, told the Los Angeles Times, "The question we ought to be asking is not why Europe won't buy our hormone-treated meat, but why we allow beef from hormone-treated cattle to be sold to American and Canadian consumers." We've been wondering the same thing. To learn more about this issue, see the Campaign for Food Safety News (item number 19) at www.purefood.org.

Organic groceries and suppliers across Europe were hustling to keep up with soaring demand this summer, after cancer-causing dioxin was found to be widely spread through the nonorganic Belgian food chain. The contamination occurred when fat tainted with dioxin--a by-product of the manufacture of some chemical herbicides and pesticides--was used to make animal feed. According to The Associated Press, initial tests showed that dioxin levels in some chickens were 1,000 times the accepted limit. The poisoned feed also spread to beef cattle and pig farms, and recalled foods eventually included such items as waffles, pork chops, and ice cream.

The British Medical Association has called for the government to require that all genetically engineered (GE) foods be labeled so that consumers can make informed choices, especially until the foods can be proved safe. Britain's chief medical and science officers have called for a special committee to study whether GE foods could cause birth defects, cancer, or immune-system damage. Michael Antoniou, a scientist at Guy's Hospital in London, warned: "Each genetic engineering event holds its own dangers. You could have acute toxicity or something that sneaks up over many years. Any of these things are possible." Meanwhile, a group of government scientists in Britain has issued a warning that some genetically altered plants could unleash antibiotic-resistant strains of meningitis and gonorrhea on the human population. The scientists are looking at modified genes that are already in use on some biotech corn and cotton crops. The genes have proved resistant to antibiotic treatment, and scientists fear that resistance could be transferred to people, rendering established medical treatments ineffective.

The Orkin Exterminating Company has been ordered to pay $2 million to a Florida couple whose home was sprayed in 1993 with the toxic pesticide chlordane. The house was condemned by health officials because of the contamination, according to the Tampa Tribune. Chlordane is a highly toxic and persistent organochloride insecticide that can affect nerve, reproductive, and immune systems. It was widely used in the United States from 1948 until 1988, when it was banned from use on food crops. It has continued to be used in and around homes, and a 1998 study of yard-waste composts found chlordane residues in every sample.

Giant agribusinesses manufacture and market more than 95 percent of the food in the United States, and they also control more than 80 percent of the land around the world that is cultivated for export crops, according to The , Green Guide, the newsletter of Mothers & Others for a Livable Planet. The good news is that organic farming practices continue to grow worldwide.

Corporations are trying to mislead consumers into believing household pesticides are

harmless, according to Marion Moses, M.D., director of the Pesticide Education Center in San Francisco. Take Raid insecticides, marketed by S.C. Johnson Corporation for killing roaches and ants, for example. Dr. Moses points out that the largest words on the Raid label read, "Made With Pyrethrins: Pyrethrin Insecticide Is Made From Flowers." This sounds reassuring, but the fine print reveals that pyrethrins make up only a tiny amount of the product (.08 percent). The dominant active ingredient is actually the controversial pesticide Dursban (.24 percent). In addition, more than 98 percent of Raid's ingredients are so-called inert ingredients, which do not even have to be identified on the label.

Article 4

Dept. of Energy researchers are taking a new approach to plants as pharmaceutical components. They are genetically modifying plants to produce human blood proteins and tissue growth agents.

Researchers at Pacific Northwest National Laboratory, Richland, Wash. (509-375-2561), are using genetic engineering technology to transplant applicable human genes into tobacco plants and produce blood factors. So far, they have produced two blood factors that are used to treat most patients with blood clotting disorders. The first, coagulation factor VIII, is critical to hemophilia therapies. The second, factor XIII and a substance called thrombin, are clotting enzymes that aid in healing wounds and offer an alternative to sutures and other surgical sealants.

Using plants to produce human blood proteins eliminates the possibilities of transmitting disease during lifesaving treatments such as blood transfusions.

PNNL researchers are using a similar technique to grow valuable industrial enzymes in non-edible portions of common agricultural crops. They developed a method to get the desired proteins to express or grow in specific areas of a plant. This can result in two profitable crops in one plant. For instance, potatoes could produce food and cellulases, which are used to produce ethanol.

Article 5

W. Wick and colleagues from the University of Tubingen, Germany, noted that the tumor suppressor gene PTEN (MMAC1, TEP1) encodes a dual-specificity phosphatase and is considered a progression-associated target of genetic alterations in human gliomas. Further analysis revealed that gene transfer of PTEN has therapeutic potential. Results were reported in the journal Oncogene ("PTEN Gene Transfer in Human Malignant Glioma: Sensitization to Irradiation and CD95L-Induced Apoptosis," Oncogene, July 8, 1999;18(27):3936-3943).

"Recently, it has been reported that the introduction of wild type PTEN into glioma cells containing endogenous mutant PTEN alleles (U87MC;, LN-308), but not in those which retain wild-type PTEN (LN-18, LN-229), causes growth suppression and inhibits cellular migration, spreading and focal adhesion," wrote Wick et al. "Here, we show that PTEN gene transfer has no effect on the chemosensitivity of the four cell lines."

Further, upon a correlational analysis of the endogenous PTEN status of 12 human glioma cell lines with their sensitivity to seven different cancer chemotherapy drugs, the researchers found no link between PTEN and chemosensitivity. In contrast, they found that ectopic expression of wild-type PTEN, but not the PTENG129 mutant, in PTEN-mutant gliomas markedly sensitized those cells to irradiation and to CD95-ligand

(CD95L)-induced apoptosis.

According to the researchers, PTEN-mediated facilitation of CD95L-induced apoptosis is associated with enhanced CD95L-evoked caspase 3 activity. They noted that protei kinase B (PKB/Akt), which has previously been shown to inhibit CD95L-induced apoptosis in nonglial COS7 cells, is inactivated by dephosphorylation.

Interestingly, noted the researchers, both PTEN-mutant U87MG and PTEN-wild-type LN-229 cells contain phosphorylated PKB constitutively. They demonstrated that wild-type PTEN gene transfer promotes dephosphorylation of PKB specifically in U87MG cell but not in LN-229 cells. Further, sensitization of U87NG cells to CD95L-apoptosis by wild-type PTEN could be blocked by insulin-like growth factor-1 (IGF-1).

The protection by IGF-1 could be inhibited by the phosphoinositide 3-OH (PI 3) kinase inhibitor, wort-mannin, the researchers found. Although PKB is a downstream target of PI 3 kinase, they found that the protection by IGF-1 was not associated with the reconstitution of PKB phosphorylation.

"Thus, PTEN may sensitize human malignant glioma cells to CD95L-induced apoptosis in a PI 3 kinase-dependent manner that may not require PKB phosphorylation,"

concluded Wick et al.

The corresponding author for this study is M. Weller, University Tubingen, Department Neurology, Hoppe Seyler Str 3, D-72076 Tubingen, Germany.

Article 6

continue to be significant, such as a death

rate of almost 50% for cloned

AFTER THE CIRCUS PROcession of cloned sheep, cows, mice, and goats in the past couple years, humans seemed likely to join the list soon. Now this sobering news: A cloned calf in France dropped dead seven weeks

after its birth.

The calf appeared healthy until days before her death; then she developed severe anemia and collapsed. An autopsy revealed a withered thymus gland, where white blood cells mature, suggesting that her immune system never started working. Jean-Paul Renard of the National Institute for Agronomic Research in Jouy-enJosas, who cloned her, thinks a defective donor cell might be at fault. He points out that the cloning process can work fine--other clones produced with his technique are thriving--but concedes that 30 to 50 percent of cloned calves die shortly before and immediately after birth. "If we want to apply this technique outside of research," he says, "such a high rate of abortion and mortality will not be acceptable."

Failure is actually the norm in the cloning business. Ryuzo Yanagimachi at the University of Hawaii has had perhaps the greatest success, producing five generations of cloned mice. Recently he created the first male clone, also a mouse. Yet Yanagimachi and his team had to transplant 274 embryos just to produce three live male mice, two of which died almost immediately That's not much of an improvement over the 276 failures that preceded Dolly the sheep, the first mammal clone.

There are other signs of trouble. The cloned mice were born with mild breathing problems. More disturbing is that Dolly's chromosomes are worn down at the edges,

possibly showing signs of premature aging. It is not clear if she will die early For now, she appears healthy and has given birth to four lambs, also doing fine. But Margaret

Mellon of the Washington, D.C.-based Union of Concerned Scientists warns: "There are many things that could be seriously wrong with her that would be very difficult to detect."

Article 7

A gene has been identified that allows plants to detoxify heavy metals that are hazardous to human health and the environment, according to newly published research.

It is not a new discovery that plants produce peptides called phytochelatins that naturally bind and detoxify dangerous toxic metals such as lead, mercury and cadmium. Phytochelatins mediate the accumulation of the bonded peptide-metal mix in the leaves of the plant, where they can be safely harvested. Researchers have known and publicized that fact for years.

Low-tech cleanup methods such as mustard plants can suck up toxins. And poplars, willows and cottonwoods can help prevent contamination of water resources and encourage the growth of natural soil bacteria that decompose poolutants.

Now, however, according to the article in the June 15 issue of the European Molecular Biology Organization Journal, researchers from the University of California at San Diego have identified the gene family responsible for producing phytochelatins: phytochelatins synthase, or PCS. They hope to repeat and duplicate its naturally cleansing mechanisms to help clean up places like Superfund sites and certain brownfields.

"We initially identified a PCS gene from wheat roots, but by looking into genome databases, we found a sequence similar to PCS in the mustard plant Arabidopsis," said university biologist Julian Schroeder, quoted in an article by the Environmental News Network. "We then tested the gene in Arabidopsis and, sure enough, it also detoxified the hazardous metal cadmium."

The researchers further used genome databases to successfully locate a PCS homologous sequence in a yeast species, called S. pombe. When the PCS gene was deleted from the genome of S. pombe, yeast growth was much more sensitive to cadmium. Much to their surprise, the investigation also turned up a similar sequence in the genome of a worm, indicating that certain animals might also use PCS genes for detoxification of hazardous metals. Researchers have sought the identities of gene families such as PCS in an effort to ability of plants to detoxify metals at hazardous waste sites, a process known as bioremediation.

Of the 10 leading Superfund toxic site contaminants, four are metals that interact with phytochelatins: lead, arsenic, mercury and cadmium.

"I believe that this gene, together with other genes that help detoxify metals in plants, will optimize the removal of metals in the future," Schroeder said.

"You will never remove the metals completely out of hazardous waste sites, but these genes can dramatically bring down the levels of toxicity, hopefully to below hazard levels determined by the EPA, which will reduce the health and environmental risks at these sites."

Article 8

Peter Lachmann; Alan D B Malcom; Carl

B Feldbaum; H Schellekens; Eric

Brunner; Erik Millstone.

Sir--It is profoundly depressing to follow the public debate on genetically modified (GM) crops. As the passion of the arguments increase, their scientific content diminishes correspondingly. It is a sad day for UK medicine when first the BMA and then The Lancet, in your May 29 editorial,(1) align themselves with the tabloid press in opposition to the Royal Society and the Nuffield Council on Bioethics. It is also disturbing and unusual for

an editorial in The Lancet to be factually so inaccurate.

I imagine the first paragraph refers to maize made resistant to stem boring insects. This crop is not sterile and there is no difficulty in planting the GM seeds. The "Gene Use

Restriction Technology", called the "terminator gene" by the press is so far merely a patent claim and has not yet been produced. This device to prevent the formation of fertile seed from a GM crop, would also prevent the spread of the inserted gene to other plants. Surprisingly, this idea has found no favour with the opponents of GM organisms who concentrate entirely on the rights of the farmer to save seed.

Farmers currently have to use new seed with the conventionally bred F1-hybrid crops that are increasingly used worldwide, because F1-hybrid seeds do not produce F1 progeny. Their yield is so much higher that, in many cropping systems, F1-hybrids are highly advantageous.

There is no experimntal evidence nor any plausible mechanism by which the process of genetic modification can make plants hazardous to human beings. Individual introduced genes may not be a great idea. For example, the use of a nut protein to enhance the protein content of a cereal may be a hazard to people who are allergic to nuts, but this danger would be the same if the nut protein were simply added to the cereal. The practice of leaving antibiotic-resistant markers in the GM plant has attracted criticism from the Royal Society among others since there is a hypothetical risk that antibiotic resistance could spread o gut flora.

The Scottish Crop Research Institute initiated an entirely sensible study to see whether lectins which make some plants unpalatable to insects could be introduced into other plants for the same purpose. The study used potatoes only to make the experimentation easier. These particular potatoes were never intended to be developed as a food crop. Some assessment of the transgenic pota-toes was made at the Rowett Institute. One of their scientists announced on television last autumn that feeding thes transgenic potatoes to rats had caused abnormalities of organ growth and had damaged their immune systems. These remarks were seized upon by the tabloid press and engendered an hysterical reaction that has not died down. The Royal Society produced a careful peer review of all the avilable data on this work and concluded that the experi-ments were badly designed, poorly carried out, and inaccurately inter-preted. Your editorial's comment that it is impertinent of the Royal Society to review the data because they may not be in their final form is incomprehensible. A scientist invites expert scrutiny by making his work public through the media and the worldwide web.

One reason for welcoming GM technology is that intensive agriculture, on which the world's food supply now depends, is in the long term both unsustainable and potentially harmful. It is unsustainable because it relies on the consumption of fossil fuels and consumes more energy than the food produces. The high levels of nitrogen and phosphate fertilisers used are a potential hazard to human health when these ions appe

ar in the water supply. There are also potential concerns about the residues of pesticides and herbicides. Any technology that may enable better yields to be obtained with less external input should be welcomed.

It is also an illusion that there ever was a time when the food supply was entirely safe. All those who, in previous centuries, died of ergot poisoning and those who still develop liver disease from aflatoxin in their food are forgotten, especially by the enthusiasts for organic farming. Bacterial food poisoning always was a serious problem.

It is wrong to regard the introduction of resistance to insects or herbicides as the only long-term goals of genetic manipulation. This is the "horse-less carriage" stage of development of this new technology. Looking rather further into the future, other goals include developing plants that can grow in saline-polluted soil, a major issue in some parts of the world. Similarly, it may be possible to develop plants that need less water input. Finally, there is the pros-pect of developing plants with an in-creased efficiency of the photosynthetic process itself. If this efficiency could be increased several fold agriculture would be able to meet not just the food but also the energy

needs of the world.

The attempt of single interest groups, supported by the tabloid press and now by others who should know better, to declare this whole technology as dangerous and immoral is sad for the UK, but is also absurd. 300 million Americans and a billion Chinese eat genetically modified food with neither ill effects nor hysteria. On the world scale, what happens in the UK may not be of overwhelming importance. However, what this campaign of vilification does to the science base and the prosperity

of the UK may be serious.

Peter Lachmann

UK

Sir--Your editorial(1) on genetically modified (GM) crops would never have passed the rigorous refereeing which you normally use for the contents of the rest of your journal. Of course the motive behind the commercial production of anything is added shareholder value. No farmer is compelled to buy any seed from any company They can always continue to use traditional crops if they feel it is to their benefit. Farmers are just as driven by the profit motive as anybody else, whether in developing countries or not. There are many parts of India where cotton farmers have gone bankrupt through insect depredation, whereas North American cotton growers are now able to guarantee increased yields of cotton accompanied by a 70% reduction of chemical-based insecticides.

Although antibiotic-resistance markers are used for some plant biotechnology products to "select" for the presence of a desired trait, the antibiotic markers in these products were selected on the basis of their frequent occurrence in nature, their efficacy as a marker, and the limited clinical importance of the antibiotics which they inactivate. The potential effect on human health of these markers is carefully evaluated during the safety assessment regulatory review process in the UK and other countries. Further, it is recognised that the increased frequency of bacterial resistance to antibiotics is mainly attributable to the widespread use and misuse of antibiotics in human and veterinary applications, not to genetically modified crops. Experts and regulatory bodies that have assessed the probability that antibiotic markers used in genetically modified plants will impact antibiotic efficacy have concluded that this likelihood is remote. Nonetheless, alternative markers should be and are being developed for use in future genetically modified crops.

It is unusual for the Royal Society to take a judgment on an independent scientist's unpublished data. Unfortunately, it was clear that the mythology surrounding these experiments was having a major impact on public perception of this technology. Although the data were not refereed by the usual channels, they had been made available to the world via the internet. It was therefore perfectly appropriate that any scientist or group of scientists should comment on the false conclusions being drawn.

The British Medical Association did not need to recommend a moratorium on the commercial planting of GM crops. Such a moratorium already exists. No permission for commercial planting has been given nor will it be given until ACRE is satisfied on the various issues which have been widely discussed.

Of course the Government should take an interest in any possible health risks associated with any new food and indeed this is exactly the role which the Advisory Committee on Novel Foods and Processes takes. Nobody, however, has been able to identify any potential health risk associated with consumption of lecithin, or soya oil, or soya starch obtained from a GM crop, compared with traditional crop. In the absence of any

hypothesis, the design of a sensible experiment is impossible.

It is not true that the population of the USA had been eating genetically modified ingredients. The ingredients they have been eating have not been modified in any way whatsoever. It is the crops from which the ingredients were derived that have been modified. There has not been one example of any identifiable medical condition induced in the 250000000 Americans who have consumed such material during the past 3 years.

Alan D B Malcom

Institute of Biology, 20 Queensberry Place, London SW7

2DZ, UK

Sir--Your May 29 editorial(1) misleads readers by saying biotechnology companies and government officials "have paid little evident attention to the potential hazards to health of genetically modified foods". You ignore thousands of scientific studies, environmental risk assessments, and the field trials undertaken worldwide before the com-mercial introduction of transgenic crops.

Before the approval of the first transgenic Bacillus thuringiensis (BT) corn in the USA, the Department of Agriculture (USDA) conducted an environmental assessment in 1995 that analysed data on risks to insects beneficial to agriculture and other non-target insects as risks to endangered organisms--from bobwhite quail to certain species of butterflies. Tests to find out if endangered aquatic organisms were threatened examined

the impact of BT corn pollen blown into water. The USDA concluded the data showed no significant potential to adversely affect organisms other than the targeted pest

that destroys corn.

Scientific inquiry, however, has not ended there. Researchers continued to examine transgenic corn, such as scientists who explored the impact on beetles, flower bugs, and lacewings, all of which feed on corn borers. These predators also eat corn pollen. In a paper published in April 1997, the researchers reported they found no detrimental effects on the beneficial insects. In fact, they observed more of them in BT corn fields than in non-BT corn fields.(2)

As for the safety of transgenic corn and other biotech crops in food, the US Food and Drug Administration (FDA) undertook its own exhaustive studies and concluded in 1992 and again in 1995, "It is not aware of information that would distinguish genetically engineered foods as a class from foods developed through other methods of plant breeding".(3) The fact is, the FDA observed, because recombinant DNA techniques are used to introduce only one or a few genes into a crop, agricultural scientists avoid a major difficulty of conventional cross hybridisation, which is the multiple introduction of undesirable genes.

Results of all studies by the USDA, FDA, and the Environmental Protection Agency--the three US agencies charged with monitoring biotech crops and foods--are produced with public input and available for perusal when completed.

A 1996 report from the Food and Agricultural Organisation of the United Nations and WHO also explored in depth the safety of foods derived from biotech crops. Those two groups concluded: "Food safety considerations regarding organisms produced by techniques that change the heritable traits of an organism, such as recom-binant DNA technology, are basically of the same nature as those that might arise from other ways of altering the genome of an organism, such as conventional breeding."(4) The report also noted, "The presence in foods of new and introduced genes per se was not considered to present a unique food safety risk."

It is one thing for The Lancet to urge caution in introducing genetically modified foods. Everyone agrees continued vigilance is necessary. It is an egregious error however, for you to imply potential health hazards have been overlooked by industry and regulatory agencies. In doing so, it dismisses decades of dedicated scientific work that clearly proves otherwise.

Carl B Feldbaum

20006, USA

Lancet 1999; 353: 1811.

(2) Pilcher CD, Obrycki JJ, Rice ME, Lewis LC. Preimaginal development, survival, and field abundance on insect predators on transgenic Bacillus thuringiensis corn. Environmental Entomology April 1997; Vol. 26, no 2, 446-54.

(3) FDA's policy for foods developed by biotechnology contained in the proceedings of the American Chemical Society Symposium, series no. 605, 1995, by Maryanski JH, Strategic Manager for Biotechnology. Centre for Food Safety and Applied Nutrition, FDA.

(4) Biotechnology and food safety, report of a joint consultation of the food and agricultural organisation of the United Nations and WHO, Rome, Italy, 30 September to 4 October 1996.

Sir--As chairman of the Dutch Committee on Genetic Modification (COGEM), the main adviser to the government in our country on the safety of genetic modifications, I and others stand accused in your editorial of May 29(1) of badly mishandling important health issues.

The safety of genetic modification is a serious topic and the quality of the decision-making process can only improve by the input from as many sources a possible. However, these contributions should be based on the data collected by the cautious step-by-step approach during the 20 years of genetic modification. Although uncer-tainties remain, there is no reason to ignore the information that is available. Your editorial fails to make a continuing argument against the opinion of the US Food and Drug Administration that genetic modification does not constitute a risk in itself.

Phenotypic characteristics such as the presence of an antibiotic resistance in plants may be considered harmful, but you ignore the reports and other scientific evidence on this topic. It would for example, be interesting to know your reaction to the criteria used by the Dutch COGEM to assess the risks of antibiotic resistance gene in crops. Our approach is based on the assumption that antibiotic resistance may spread from plants to microorganisms.

You also fall short of supporting the moratorium on introduction of genetically modified crops, as advocated by the British Medical Association and other organisations. In my opinion there is at present no rationale for a moratorium. Those who argue in its favour have to formulate the specific reasons why and what they want to achieve. They have to quantify the current risks and to what extent these risks have to be reduced to be acceptable and to terminate the moratorium.

Learned societies and journals, mainly in the UK, seem to have lower scientific standards with regard to the genetic modification of plants than other topics. If the assumption is correct that the reasons for this unbalanced approach lie outside science, there is more at stake than the future of genetic modification of plants.

H Schellekens

Nieuwerk, Netherlands

Sir--Peter Mitchell and Jane Bradbury's May 22 news item (p 1769)(1) reports the Royal Society's judgment that Arpad Puztai's study on the potential toxic effects of genetically modified (GM) potatoes is "flawed in many

aspects of design, execution and analysis and that no conclusions should be drawn from it". The society rightly says that research scientists should expose their new results to peer review before releasing them.

The episode at the Rowett Research Institute highlights just how important the integrity of the peer-review process is to the maintenance of high standards in science. Our experience(1) with trial data on recombinant bovine somatotropin (rBST, an injectable hormone which raises milk yield in dairy cows) suggests that increased vigilance, perhaps through some mechanism of formal audit, may be needed to preserve such standards.

In that case, official regulatory authorities accepted the manufacturer's unpublished analysis. We previously identified the shortcomings in this analysis, and did our own, but we were unable to publish it because the company concerned withheld consent. Here the peer-review process was compromised, partly by the pressures on the existing regulatory process and partly by the requirement for commercial scientists to deliver th product to market. In general, if preapproval studies are not published, any questionable conclusions may go unchallenged. Put another way, if applicants are able to argue successfully that disclosure would cause commercial harm, then peer scrutiny may be restricted.

A process that bears the hallmarks of these difficulties led to the approval of rBST for farm use by the US Food and Drug Administration in 1994. rBST is unlicensed in Canada and the European Union. The company seeking to market this productivity aid gave us data from eight randomised controlled trials. We did a meta-analysis and found evidence for a pro-mastitic effect due to rBST. The report was sent to the company and to a UK peer-review journal. Although our report passed the peer review, the company refused permission for its publication on the basis that the trial investigators would soon submit their own analysis for publication. In the following 3 years, no such report appeared. We resubmitted our paper to a US journal and then to another UK journal. On each occasion the paper received peer approval but could not be published because the company alleged they had copyright over our analysis of their data. We were eventually able to publish our findings as a response to a public accusation of plagiarism by a company representative, but only after FDA approval for rBST had been given.

There is no method of recourse in such situations. We therefore support the recent recommendation contained in the first report of the Commons Select Committee on Science and Technology2 for further openness in the regulatory process in the UK and elsewhere for new foods, with the rapid publication of papers and data (http://www.publications.parliament. the-stationery-office.co.uk/pa/ cm199899/cmselect/cmsctech/286/ 28602.htm; accessed June 10, 1999).(2) This approach would substantially strengthen the peer-review process.

[*] Eric Brunner, Erik Millstone

[*] Department of Epidemiology and Public Health, University College London Medical

School, London WC1E 6BT, UK; and Science Policy Research Unit, Sussex University,

Brighton

(1) Millstone EP, Brunner EJ, White IR. Plagiarism or protecting public health. Nature

1994; 371: 647-48.

(2) House of Commons Select Committee on Science and Technology. Scientific Advisory

System: genetically modified foods. First Report of Session 1998-99 (HC 286). London:

HM Stationery Office, 1999.

Article 9

game of evolutionary

one-upmanship, plants and

viruses wrangle over gene

silencing. (includes related article on

gene splicing) Elia T. Ben-Ari.

Abstract: Post-transcriptional gene silencing (PTGS) is a mechanism where cosuppression occurs in plants. Studies showed that PTGS supports an antiviral function and that viruses that support nucleic acid sequences homologous to transgenes can promote the silencing of genes.

In a game of evolutionary one-upmanship, plants and viruses wrangle over gene silencing

In a way, it started with petunias. In the late 1980s, plant geneticist Richard Jorgensen and his colleagues at DNA Plant Technologies, in Oakland, California, were attempting to create petunias with more deeply colored flowers by over-expressing the gene coding for chalcone synthase, an enzyme that plays a key role in producing the pigment protein anthocyanin. Because chalcone synthase was known to control the rate of anthocyanin synthesis in purple corn seeds, the researchers reasoned that boosting the levels of chalcone synthase in petunia flowers might lead to increased amounts of anthocyanin and thus to flowers of deeper hues.

To try to maximize chalcone synthase expression, Jorgensen, now at the University of Arizona, and his research team created transgenic Petunia hybrida plants containing a "hybrid" chalcone synthase gene. This hybrid gene consisted of the strong promoter region of cauliflower mosaic virus, which drives high expression of downstream genes, hooked up to the petunia chalcone synthase coding sequences. But the result of these genetic engineering efforts, Jorgensen says, "was the opposite of what we intended." Instead of producing deeper purple flowers, many of the transgenic plants had white, pigment-free flowers, and others had flowers with white patterns on a purple background.

The reason for these seemingly paradoxical results? Further experiments showed that, despite the strong promoter, the chalcone synthase transgene was not expressed; moreover, the researchers were surprised to find, the endogenous chalcone synthase gene was also inactivated, or silenced, in the white flower tissues.

Jorgensen coined the term cosuppression to describe the phenomenon in which two or more homologous gene sequences are specifically silenced. Cosuppression has since been observed in many transgenic plants. Research by several groups has shown that, in many of these plants - including the chalcone synthase-transgenic petunias - cosuppression occurs via a mechanism known as post-transcriptional gene silencing, or PTGS, in which silenced genes are still actively transcribed but the resulting messengerNAs are degraded before they can be translated into proteins. Gene silencing due to the presence of multiple homologous nucleic acid sequences can also occur at the transcriptional level (via homology between promoters rather than between transcribed sequences), through a mechanism whereby a gene's promoter is inactivated, preventing transcription.

The detailed mechanisms underlying PTGS remain to be worked out and are the subject of a great deal of investigation and debate (see box page 435). Nevertheless, plant biologists - not only molecular geneticists but also virologists - have learned a great deal

about cosuppression and PTGS in the decade since cosuppression was first reported in petunias.

Presumably, plants did not evolve the ability to silence homologous genes as a means of frustrating the efforts of molecular biologists hoping to create flowers of a deeper hue or improve food crops through transgenic technology. Indeed, a growing body of evidence suggests that the evolution of PTGS may be related, at least in part, to plants' need to defend against viruses.

The first evidence hinting that PTGS might have an antiviral function came from studies by John Lindbo, William Dougherty, and their colleagues at Oregon State University, and subsequent studies by David Baulcombe and his colleagues at The Sainsbury Laboratory, in Norwich, UK. These researchers all showed that viruses can be the targets of gene silencing. For example, if transgenic plants that were exhibiting PTGS were infected with a virus into which nucleic acid sequences homologous to the silenced gene had been inserted, virus accumulation was inhibited. That is, the plants were immune to infection by the virus.

Furthermore, several research groups showed that viruses that contain nucleic acid sequences homologous to transgenes or to endogenous plant genes can trigger silencing of those genes. In one set of experiments, for example, Dougherty and his colleagues infected transgenic tobacco plants expressing all or part of the tobacco etch virus (TEV) coat protein (CP) with TEV. Several weeks after infection, the new stems and leaves that developed in the transgenic plants were virus free. The researchers found that the CP transgene was post-transcriptionally silenced in these "recovered" tissues, which also turned out to be immune to subsequent infection by TEV.

"Those separate sets of observations - that viruses could be targets of gene silencing and [that] viruses could also induce gene silencing - made one think that maybe this wasn't a coincidence," Baulcombe says. These findings led him and others to examine nontransgenic plants for examples of virus resistance that could be accounted for by a gene silencing - like mechanism.

The search paid off, yielding more persuasive evidence that PTGS in plants is involved in natural virus resistance. In 1997, research groups led by Baulcombe and by Simon Covey, at the John Innes Center in Norwich, reported that infecting nontransgenic plants with tomato black ring nepovirus (an RNA virus) or cauliflower mosaic virus (a. DNA pararetrovirus) can trigger a viral resistance mechanism in the plants that resembles transgene-induced gene silencing. However, in contrast to the cosuppression phenomenon, silencing of these viruses occurs even though the viral genomes are not homologous to any host genes.

Just like in transgenic plants infected by certain viruses, virus-infected nontransgenic plants initially showed symptoms of viral infection, but new leaves that developed after systemic infection were symptom-free and largely lacked virus. The accumulation of viral transcripts was greatly reduced in newly developed tissues in the "recovered" plants. Furthermore, Baulcombe's group showed that recovered tissues were resistant to infection by a second virus if that virus contained nucleic acid sequences that were similar to sequences from the first virus. This work extended the early work of plant virologist R. E. F. Matthews, which had shown that "dark green islands" in virus-infected leaves are immune to superinfection by related viruses.

More recently, Frank Ratcliff, in Baulcombe's group, has shown that the ability to induce a PTGS-like defense is not restricted to nepoviruses and pararetroviruses. Other, more frequently studied viruses, such as potato virus X and tobacco rattle virus (the latter being similar to tobacco mosaic virus) are also able to activate this mechanism, and Baulcombe and his colleagues think that most viruses are activators of a PTGS-like defense.

Although these results indicate that plants can use gene silencing to fight viral infection, it turns out that viruses are not entirely passive in the face

of PTGS. Three independent research groups have now reported that plant viruses encode proteins that can suppress post-transcriptional gene silencing.

These reports, from groups led by Vicki Vance, at the University of South Carolina; James Carrington, at Washington State University; and Baulcombe (and published, respectively, in Proceedings of the National Academy of Sciences 95: 13079-13084; Cell 95: 461-470; and EMBO Journal 17:6739-6746) - and the discovery of other silencing suppressors yet to be reported in the literature-lend further credence to the hypothesis that PTGS is a natural antiviral defense mechanism in plants. "You can postulate that the reason [viruses make these suppressors of silencing] is that the silencing response is likely an antiviral response" and that the production of silencing suppressors by viruses "serves as an effective counter-defensive mechanism," Carrington says. In this sense, he and Kristin Kasschau noted in their Cell article, "plant viruses join their animal-infecting counterparts in being promoters of their own infection through suppression of innate or adaptive host responses."

All three research groups provided evidence that a protein known as viral helper component-proteinase (HC-Pro), which is made by a family of plant RNA viruses known as potyviruses, suppresses PTGS in plants infected with these viruses. In particular, they found that HC-Pro can reverse existing transgene-induced gene silencing and prevent virus-induced silencing in tobacco (Nicotiana tabacum) and Nicotiana benthamiana.

Like many other viral proteins, HC-Pro has multiple functions. Results of earlier studies showed that HC-Pro aids in viral spread and replication. For example, studies by Carrington's lab showed that HC-Pro is necessary for long-distance movement of TEV, a potyvirus, through a plant's vascular system. Studies by Carrington and others also showed that HC-Pro allows TEV to continue replicating in plant cells for relatively long periods of time. By contrast, "in the absence of functional HC-Pro, the plant appears to mount an effective defense response that shuts off [replication of] the virus after a relatively short period of time," Carrington says. The latter finding, he says, suggested that perhaps HC-Pro was not stimulating replication and spread of the virus directly but rather was exerting an indirect effect by suppressing some type of plant defense response.

Strong support for this hypothesis was provided by work from Vance's lab and from collaborations between Vance and Carrington. This work showed that when the HC-Pro coding sequence from TEV was inserted into the genome of a virus that does not encode HC-Pro, that virus was able to replicate for a longer period of time than normal.

Moreover, HC-Pro was found to be responsible for the ability of TEV and other potyviruses to mediate a phenomenon known as synergistic viral disease (or synergistic viral infection). In synergistic viral disease, plants infected with TEV together with an unrelated virus develop much more severe symptoms than plants inoculated with either virus alone. The ability of HC-Pro to suppress PTGS, thereby limiting the general plant defense response, could explain this protein's facilitation of the replication and spread of other viruses in synergistic viral infection.

Baulcombe's group also reported that a second viral protein known to stimulate long-distance movement of viruses in plants acts as a silencing suppressor in N. benthamiana. This protein, the 2b protein from cucumber mosaic virus (which is a cucumovirus), is smaller than HC-Pro and has no sequence similarity to it, and it appears to work by a completely different mechanism. "HC-Pro is able to reverse silencing wherever it's happened, whereas 2b is only able to reverse silencing in the leaves that emerge from the growing point of the plant after the virus starts spreading," Baulcombe says. These findings suggest that, whereas HC-Pro blocks a reversible step in the gene silencing mechanism, the 2b protein blocks an irreversible step.

Carrington notes that the ability of the 2b protein to suppress silencing only in newly developing plant tissues suggests that it may inhibit a systemic signaling mechanism that mediates the spread of gene silencing throughout a plant (see box page 436). By contrast, HC-Pro seems to act at an earlier stage in the establishment of PTGS. For instance, it might interact with a component of the silencing apparatus or with some cellular factor that in turn regulates a component of the silencing machinery.

Silencing suppressors have now been found in several different viruses, and Carrington suspects that "most viruses probably have ways to deal with the silencing response in at least certain hosts." Indeed, he notes, one reason for the limited host range of certain plant viruses may be that a particular silencing suppressor may be effective only in certain plant species.

The finding that HC-Pro enables viruses to suppress PTGS points to an apparent paradox: The first experiments (by Dougherty's lab) demonstrating the ability of silenced transgenes to confer immunity against viruses containing homologous gene sequences and the ability of transgenic plants to "recover" after viral infection through a mechanism involving PTGS were done with TEV - the very same virus in which the HC-Pro silencing suppressor was discovered.

So if the virus produces HC-Pro, how can plants silence the virus? Conversely, if the plant silences the virus, how can enough HC-Pro accumulate to suppress silencing? One possible explanation, Baulcombe says, "is that you get some activation of silencing by the virus, even though it produces the suppressor." Perhaps a signal (see box page 436) carries the silencing response from cell to cell through the plant ahead of the advancing front of viral infection, he says, "so the gene silencing is basically switched on before the virus gets into those cells, and then as a result of the silencing the ability of the virus to accumulate - the ability of the virus to produce the suppressor of silencing - is compromised."

In essence, it seems that plants and viruses are engaged in a sort of tug-of-war, in which the balance between gene silencing and suppression of silencing can shift back and forth. "You can think of [silencing] as a sort of basal defense mechanism against viruses," Baulcombe says. "If the gene silencing mechanism didn't exist...then the virus would just go through the roof and kill its host."

Indeed, Baulcombe's group found that when they inserted a gene coding for HC-Pro or 2b into a virus that contained a different silencing suppressor gene, "these viruses killed the infected plant quite rapidly and spectacularly," he says. And Baulcombe notes that killing the host would not be advantageous for a parasitic organism such as a virus, "because what the virus needs to do is to spread within a plant and also hang out in the plant" until the opportunity to infect another plant comes along.

The discovery of viral suppressors of gene silencing opens new doors for plant biologists. For one thing, plant geneticists may be able to use HC-Pro to prevent gene silencing in transgenic plants, thereby ensuring that transgenes are expressed at high levels. Conversely, researchers might exploit newfound knowledge about the mechanisms of silencing suppression to deliberately silence specific genes.

The silencing suppressor proteins will also be useful in dissecting the mechanism responsible for the plant silencing response. If researchers can identify cellular factors that interact with, or bind to, HC-Pro or 2b, they will almost certainly uncover key components in the PTGS pathway. Identifying these components should shed some light on the mechanisms involved in the silencing response. But to gain further insights into the mechanistic details of PTGS, Carrington notes, "we need genetics to come through on model systems." For example, he says, "we need silencing-defective mutants, and using those mutants we need to isolate the genes and identify components" of PTGS. Indeed, researchers are actively pursuing this tack, and reports identifying some of these genes have begun to appear.

The finding that PTGS is likely to be part of the "immune system" of plants is "a fundamental shift in how we view plants' response to pathogens," Carrington says. One of the main ways in which gene silencing differs from other plant defense systems is that it is adaptive, he says. In other words, "plants can recognize a new invading virus, they can elicit a specific antiviral response to that viral pathogen, and [they can] therefore suppress it specifically." Although radically different in its mechanism, PTGS in plants is analogous to the mammalian immune system in that it can generate a "customized" defense response to pathogens.

In contrast to gene silencing, Carrington says, other plant defense mechanisms are "carried out by pre-existing components that do not change." For example, wild plants have dozens of resistance, or R, genes, each of which is specific for a given pathotype, or race, of pathogen. "If that particular race lands on a particular plant that has a dominant R gene that can recognize that pathogen, then you have a resistance response...that results in limitation of the pathogen," Carrington says. However, other races of that pathogen would be virulent in the plant.

Although it is becoming clear that PTGS serves as a natural defense mechanism against viruses, some researchers think that plants may use gene silencing as a more general mechanism to regulate the expression of endogenous genes. This hypothesis is still speculative, but the petunia may once again offer researchers some clues. Jorgensen notes that when German plant breeders first created the species Petunia hybrida in the 830s from crosses among wild petunia species from South America, "they produced many of these same flower colors or patterns that we see" in the cosuppressed transgenic petunias. Some of the patterns in these hybrid strains have turned out to result from post-transcriptional silencing of duplicate copies of the chalcone synthase gene in the hybrid plants' genomes. Although Jorgensen points out that this lone example of cosuppression amon endogenous plant genes was produced by breeding, not by natural selection, he says that "certainly if you can have that in a breeding population it can happen in nature."

Whether gene silencing indeed serves a broader gene regulatory purpose in plants remains to be determined. It is interesting to note, however, that gene silencing responses similar to the cosuppression phenomenon in plants have also been reported in fungi and, more recently, in nematodes and fruit flies. If these phenomena turn out to be mechanistically similar to gene silencing in plants. PTGS may ultimately be found to be part of a universal gene regulation system.

Researchers have suggested that post-transcriptional gene silencing (PTGS) can be broken down into three phases: initiation, systemic spread (see also box page 436), and maintenance. Several types of models have been suggested for the initiation phase of gene silencing:

* Threshold models propose that gene silencing is a response to inappropriately high levels of expression of the targeted gene(s). These models suggest that the plant can somehow "sense" the levels of specific RNA species and will activate the silencing response when these levels exceed a certain threshold.

* Aberrant RNA models propose that PTGS is activated by the presence of RNA that is qualitatively "different" from most other RNAs. The nature of these "aberrant" RNAs is also a matter of debate, but they might include double-stranded sequences or truncated sequences resulting from premature termination of transcription.

* Ectopic pairing models suggest that PTGS is triggered by pairing between homologous DNA sequences (e.g., between a transgene and a related endogenous gene) or by DNA-RNA interactions (e.g., between introduced DNA sequences and transcripts in the cytoplasm).

The three types of models are not necessarily mutually exclusive. For example, a threshold of aberrant RNA may be required to trigger silencing, or an ectopic DNA interaction might in turn lead to transcription of aberrant RNA species that elicit the silencing response.

Researchers have discovered that, at least in some cases, cosuppression due to post-transcriptional gene silencing (PTGS) can spread through a plant over significant distances. The results of two studies in particular convincingly demonstrated the systemic spread of gene silencing.

In one set of experiments, Olivier Voinnet and David Baulcombe, of The Sainsbury Laboratory in Norwich, England, studied Nicotiana benthamiana plants that were expressing the green fluorescent protein (GFP) from jellyfish. The GFP gene serves as a "reporter gene" that, when expressed, produces a protein that lends plants a green glow under ultraviolet light. When the researchers infiltrated the lower leaves of the GFP-expressing plants with strains of Agrobacterium designed to deliver a second copy of the GFP gene into the leaves, newly developing leaves far from the infiltrated leaf did not show GFP fluorescence. This loss of fluorescence resulted from the post-transcriptional silencing of the GFP genes. Silencing occurred even though the newly introduced GFP DNA apparently did not enter the silenced leaves.

Voinnet and Baulcombe also found that when they inoculated the GFP-silenced leaves with an RNA virus (potato virus X, or PVX) containing the GFP gene, the virus failed to accumulate in the plant - that is, the plant was resistant to the virus. By contrast, GFP-transgenic plants were not resistant to infection by PVX carrying a different reporter gene. The difference suggests that viral resistance was sequence specific.

In another series of experiments, Jean-Christophe Palauqui, Herve Vaucheret, and colleagues at INRA in Versailles grafted the upper, growing shoots from plants expressing a nitrate reductase transgene (as well as the endogenous nitrate reductase gene) onto the lower parts (stocks) of plants whose endogenous nitrate reductase gene was cosuppressed via PTGS. These researchers found that the nitrate reductase gene in the upper shoots (scions) grafted from the non-silenced plants rapidly became silenced as the scions grew on the cosuppressed stocks. Moreover, transmission of silencing in the grafte plants occurred in a sequence-specific manner. That is silencing occurred only in grafted scions that expressed the nitrate reductase transgene; other transgenes were not affected.

Together, these observations suggest that some type of signaling molecule can transmit silencing throughout a plant. The pattern and timing of the spread of silencing seen in these and subsequent experiments are consistent with a signal that travels over long distances through the plant via the phloem and that moves from cell to cell via the specialized intercellular channels called plasmodesmata. The plasmodesmata may play a role in regulating plant growth and development and physiological function by permitting the selective intercellular trafficking of proteins and their transcripts.

This putative pathway of systemic PTGS signaling is similar to that of viruses. Viruses spread via the use of movement proteins that exploit existing routes for molecular trafficking in plants by allowing viruses to squeeze through the narrow plasmodesmata and thereby move from cell to cell and into the phloem for long-distance transport.

Some researchers have proposed that the as-yet-unidentified silencing signal (which is widely assumed to be an RNA molecule) is carried through the plant by an accessory protein that is analogous to virus-encoded movement proteins. Indeed, William Lucas, at the University of California-Davis, and his colleagues recently reported the cloning of a protein that may turn out to be a plant version of the viral movement proteins, and they hypothesize that one function of this protein, which appears to bind to RNA, could be to mediate the spread of a gene silencing signal in plants. Jorgensen and Lucas postulate that plants may have evolved such movement proteins, which travel the same paths as viruses, to track viruses down and stop their spread by turning on the gene silencing response throughout the plant.

Article 10

The Lancet, May 29, 1999 v353 i9167 p1873

Paul R Billings; Ruth Hubbard; Stuart A

Newman.

Abstract: Genetic manipulation of germ cells should be prohibited. Alterations of the genetic code in sperm cells, ova, and fertilized ova could be used to prevent disease or to induce desired traits. In comparison to gene transfer in other body cells, germ cell modification may produce effects that were not predicted, and may not reveal themselves until the child is grown, or in future generations. Germ cell modification not only influence the single offspring, but create unique DNA that will persist into subsequent generations. Prenatal diagnosis permits specific pregnancies to be evaluated for genetic fitness. Manipulation of germ-cell DNA is unnecessary and potentially dangerous.

Human germline gene modification has been foreseen but not yet accomplished.1-6 It can be defined as the genetic manipulation of human germ cells, or of a conceptus, resulting in inherited changes in DNA. With the development of advanced in-vitro fertilisation (IVF) methods, preimplantation DNA analysis, improved techniques for gene transfer, insertion, or conversion, and of embryo implantation procedures, the technical barriers to such an intervention seem easily surmountable. Unintended changes in DNA may occur when gametes are manipulated or stored.7,8 Inadvertent germline mutations. therefore, may have already occurred as a result of reproductive technologies in current use, such as artificial insemination and IVF. There are unpublished reports that researchers in the USA have already carried out a manipulation involving the exchange of a mitrochondrial genome in an IVF protocol. If true, this human experimentation involving intentional hereditary changes was probably conducted without federal oversight of safety, since there are no discussions of this protocol in the available public record.

Tsukui and colleagues9 used viral vectors in somatic gene therapy protocols to infect mouse eggs in vitro, leading to germline transmission of a transgene in the progeny. Although removal of the zona pellucida is a prerequisite for infection of the eggs in vitro, the early oocytes of postnatal ovaries also lack zonas. These experiments thus raise the possibility that modification of gametes may occur in vivo, and constitute a germline hazard in the 200 or more somatic gene therapy protocols now in use. Any such alterations would be difficult to detect. Intentional or inadvertent germline modifications may pose significant burdens. Although there are restrictions on experimentation that might result in human modifications,10 and opposition to its implementation has been voiced,11-15 some leading scientists and other commentators have begun to advocate the development and application both of techniques that may increase the risk of inadvertent alteration of the germline, and of methods that would alter it deliberately.6,16-18

W French Anderson and his colleagues have developed an experimental protocol for the treatment of adenosine deaminase deficiency during fetal development; although their therapeutic intent is directed towards somatic cells, they acknowledge that the technique may modify germ cells as well. They have submitted this proposal to the National Institutes of Health (NIH) for review (panel). By introducing a genetic construct in utero, which knowingly allows for the alteration of germinal tissue, their attempt at a potentially transmissible correction could be used to erode opposition to germline genetic manipulation since germline modification would be achieved, though unintentionally.

Opposition to germline modification is based on several lines of reasoning.19-22 First, as we have already suggested, germline DNA modifications may affect gene function in ways that are not immediately apparent, so their occurrence may not be recognised for a generation or more-for example, germline introduction in mice of an improperly regulated normal gene resulted in progeny with unaffected development but high tumour incidence during adult life.23 Furthermore, interactions among genes and their products are highly integrated, have been refined over evolutionary time scales, and often serve to stabilise developmental pathways and physiological homoeostasis.24-26 Through experimental error, unanticipated allelic interactions, or poorly understood regulatory mechanisms such as imprinting, there is a risk that germline genetic manipulation will alter sensitive biological equilibria. Disruption of these interactive systems is likely to have complex and uncertain biological effects, including some that appear only during the development or functioning of specific cells or tissues.27 Many of these effects could be undesirable.

Second, this sort of intervention is not needed. With available methods of prenatal diagnosis, virtually all interested couples can choose not to transmit specific identifiable genes. Other reproductive options (artificial insemination, egg donation) and adoption are available to those not able or willing to use prenatal or preimplantation selection methods. An exception might be when, rarely, two individuals have the same recessively inherited disorder. If such couples chose to reproduce, it could be argued that they would "need" germline or very early genetic interventions since all their progeny might inherit a disease-associated genotype. Yet, even these children may differ genotypically and phenotypically from their parents and the development of a new mode of treatment for this unusual occurrence does not seem justifiable. Although available alternative procedures are invasive, germline modifications would also require similar interventions since they would probably involve IVF. Moreover, the associated risks with existing procedures are not as serious as those created by introducing a hereditary genetic "error" into a family. People who oppose prenatal diagnosis on philosophical or religious grounds would be unlikely to want to take part in germline modification if they were aware of its intrinsically experimental nature and of the numbers of human embryos that would have to be expended during the development of the technology. No unmet need balances the risks of germline interventions to mothers, fetuses, and future generations. Moreover, the costs associated with the general development and implementation of germline manipulation would be formidable.

If there is no clinical need for germline modifications, the primary reason for using this intervention would be human enhancement.28 Apart from the uncertainties about its ultimate outcome, enhancement is a form of eugenics. Though not a recrudescence of overtly coercive, public-health-based eugenics popular earlier this century, germline manipulations represent an individual or familial form. Seemingly private personal decisions and "choices" about medical or non-medical programmes for enhancement would, nevertheless, reflect prejudices, socioeconomic and political inequalities, and even current fashion. Though enhancement procedures now in use (eg, cosmetic surgery or orthodontics) also change according to fashion, germline intervention would intentionally subject later generations to modifications undertaken on the basis of existing values and conditions. The chance that "desirable" manipulations might later be viewed as disastrous makes germline enhancement "therapies" unacceptable.

Human germline interventions would necessarily alter the lives of individuals who are yet to be born. Informed consent by the affected individuals is not possible. Extension of the parental right to consent for minors would be required.29 Such legal permission to specifically alter the lives of generations of unborn individuals would be unprecedented and unjustified.

If germline manipulation is attempted, there will be mistakes or errors in its application. Neither social acceptance nor the necessary range of protections and care for accidentally damaged individuals can be guaranteed.30 Unexpected alterations in family relationships will occur, and "wrongful life" disputes could arise.31 Irrespective o whether such interventions were to take place in research or clinical settings, these issues mean that germline modifications cannot be approved by existing standards for the protection of human beings.32 No benefits to and future individual would justify abrogating or curtailing these restrictions.

For these biomedical reasons, as well as others based in legal,33 philosophical,19,34,35 cultural, and spiritual/religious traditions,36,37 human germline modifications should be opposed and prohibited. Experimentation that may gradually make human germline modification more feasible is under way; it may require further review. Further study is needed of the safety of somatic gene therapy protocols to ensure that they detect, with adequate sensitivity, germline alterations. Many individuals and groups that monitor developments in human genetics can be expected to mount vigorous opposition to the development of human germline protocols, involving direct action, legal manoeuvres, and organising among interested public groups. Unlike man other countries, including those of the EU, which have prohibited germline manipulation in principle,38,39 restrictions on the procedure in the USA are mainly based on practical considerations (see, for example, the summary of the January 1999, RAC-sponsored conference at http://www.nih.gov/od/orda/gtpcconc.htm. Site accessed March 20, 1999) and are subject to revision as the state of the science changes. Although debate about human germline modifications should continue and, indeed, be broadened to include representation of a diverse cross- section of viewpoints and backgrounds, such discussion should not be construed as suggesting that such a method would ever be appropriate or acceptable.

We thank Jacque Bradley for technical assistance, Jon Beckwith, Felipe Cabello, Suzanne Bodor, Vernon Chong and Parris Burd for helpful suggestions and critical

comments on drafts of this paper.

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Veterans Health Administration, Heart of Texas Health Care System, 1901 North HighwaY

360 Grand Prairie, TX 75050

(P R Billings MD); Biological Laboratories, Harvard University, Cambridge, MA (R

Hubbard PhD); and Department of Cell Biology and Anatomy, New York Medical

College, Valhalla, NY, USA (S A Newman PhD)

Correspondence to: Dr Stuart A Newman (e-mail:newman@nymc.edu)

Article 11

At birth, the female calf appeared blissfully normal, and the French scientists who had nurtured her since her start in a petri dish heaved a sigh of relief. But the celebration was short lived. A month later, tests revealed a sharp drop in the calf's hemoglobin. Iron supplements failed to remedy it. At seven weeks, the calf died suddenly from massive anemia.

This young calf was a clone, the product of a single skin cell from another animal's ear. And her mysterious death, reported this month in the Lancet, is just one of many failures in this dazzling new specialty. Cloned cows are at least 10 times more likely to be unhealthy than their naturally conceived counterparts. No one knows why.

"The problem," says James Robl, a developmental biologist and cloning pioneer at the University of Massachusetts- Amherst, "is the embryos all look fine. We don't see [abnormalities] until eight months of gestation." Still, scientists suspect that something goes awry in the embryos' first days.

One possible culprit is genetic imprinting, a poorly understood process in which maternal and paternal genes ensure that neither predominates in the offspring. With only one parent, the cloned fetus could be left without a balance of genes necessary to thrive. Scientists point to disorders triggered when imprinting goes wrong that resemble those they see in clones. One, Beckwith-Weidemann syndrome, produces an unnaturally large child with an oversized tongue and internal organs.

Clone defects also could be caused by the process itself. With so little known about early embryonic development, handling the embryo, or even creating it--which involves using an electric shock to fuse the donor's cell with an oocyte that lacks a nucleus--could disrupt crucial growth processes. It's also conceivable that scientists simply haven't mastered a complex and novel technology. Indeed, Ryuzo Yanagimachi, a professor of reproductive biology at the University of Hawaii Medical School and the first to clone mice, calls his 1 percent success rate "a rather encouraging figure."

But 1 percent success, coupled with many more disastrous outcomes, means that human cloning can't be considered until the process is almost foolproof. Most scientists are optimistic that a few years of toil is all it will take to make significant improvements. But it could be that without genes from two parents of their own, cloned mammals are largely doomed.

Article 12

Drumsticks, anyone? If you're partial to chicken legs, here's good news for you. Thanks to some clever genetic engineering, scientists at the Salk Institute in La Jolla,

Calif., have come up with a way to grow birds with an extra pair of legs.

The work, described in last week's Nature, centers on so-called T-box genes. Common to all vertebrates. including humans, they're important in the development of limbs in the embryo--determining, for example, whether they become hind- or forelimbs (or in chickens, legs or wings). But, says geneticist Juan Carlos Belmonte, the study's senior scientist, "we didn't know if one of these genes by itself was sufficient to send a limb down one pathway or the other."

To unravel the puzzle, the scientists infected the unformed wing region of day-old chicken embryos with a virus carrying a T-box gene known as Tbx-4. A day later, they transferred the tissue to other embryos still in their shells. The transplanted cells quickly grew into recognizable legs. By contrast, when the scientists transferred wing tissue without first infecting it with Tbx-4 genes, the tissue always grew into wings. That shows that Tbx-4 contains a full genetic blueprint for a leg, says Belmonte.

The scientists hope to learn how T-box genes turn on and off. That could give them clues to human birth disorders like Holt-Oram syndrome, which is characterized by stunted arms and hands and is linked to these genes. As for the chicks, the scientists didn't let them hatch, resisting the temptation to grow drumsticks for KFC.

--By Frederic Golden

Article 13

Eric B. Kmiec.

Abstract: Media reports create the impression that genetic technology has been making rapid progress in recent years. The problem with this portrayal is that it inevitably leads to a disappointed public whose expectations were raised by an almost daily report of genetic breakthroughs. The truth is genetic technology has yet to mature and many fundamental problems still have to be worked out to make the technology clinically valuable.

Investigators have been searching for ways to add corrective genes to cells harboring defective genes. A better strategy might be to correct the defects

In the middle of the 19th century, the now-famous monk Gregor Mendel performed his landmark experiments indicating that certain traits can be inherited, and he postulated a discrete unit of inheritance that we now call a gene. Since then, scientists have come to appreciate how much of an individual's constitution is determined by genes, and, in particular, they have focused on the link between genes and disease. Indeed, over the past 10 or so years, identifying disease-related genes has becom something of a cottage industry within the scientific community.

Any reader of newspapers knows just how fruitful this enterprise has been. Almost daily come reports about the discovery of a new gene that contributes to some disease or another, be it sickle cell disease, muscular dystrophy, familial hypercholesterolemia, Alzheimer's or some form of cancer. Right now, therapies directed toward these conditions can only alleviate the symptoms - the manifestations of the defective genes. Implicit, and sometimes explicit, in stories about genetic discoveries is the idea that new therapies can be created that directly address the source of the problem. These gene therapies seek treatments, even cures, that act at the level of the gene itself.

Most of the gene therapy techniques developed so far are of the gene-addition variety; that is, they attempt to provide a good copy of a gene to a cell that harbors a bad one. The hope is that the good, corrective gene will compensate for the bad one and restore the cell to its proper function. Gene addition has been achieved by a variety of means - not only in test-tube experiments, but in clinical trials involving real patients as well. Yet, to date, the results of these trials have been disappointing. Even the most successful clinical trial has fallen short of therapeutic efficacy.

Unfortunately, many of these trials have been widely publicized - and, in some cases. oversold - in popular books and magazine articles. Having failed to live up to the inflated expectations created by such publicity, these disappointing trial results have left a general impression that gene therapy cannot now or ever fulfill its initial promise. But these clinical trials may have been conducted before the technology was fully mature, driven in part by investor demands on biotechnology companies to rush products to market. Such clinical trials were almost certainly destined to fall short of the mark.

Many of the fundamental problems with gene therapies have not yet been worked out sufficiently to make the technology therapeutically viable. A number of vehicles have been developed to deliver corrective genes into cells. Some are more effective than others, but none is yet exactly right. The greater challenge, however, lies in the problem of how to make the therapeutic gene behave reliably and at clinically beneficial levels.

Making gene therapy a successful endeavor will require careful research to understand why traditional approaches have not produced the hoped-for results and, in turn, to improve them, while exploring new ways to deal with genetic defects. In my own laboratory, we are considering the possibility that inserting an entire gene into a cell and then expecting it to behave as a native gene may be overly ambitious at this point. It may also be unnecessary. Since the defects in many disease-related genes are fairly small, my colleagues and I are exploring ways to repair rather than replace them. Our initial results lead us to be cautiously optimistic that with adequate basic research this or some other approach will ultimately yield fruit. My own feeling is that pronouncements of gene therapy's imminent demise are as premature as were those overly optimistic pronouncements of its imminent success. At its core, the notion of gene therapy or gene correction is scientifically sound.

Abnormal cell behavior is often the result of an altered gene whose expression is either absent or unregulated. A mutation in just one gene can sometimes cause a cell to malfunction. The mutated gene directs the synthesis of a dysfunctional protein, with the consequence that the cell functions marginally or not at all.

In the case of sickle cell anemia, for example, a mutated hemoglobin molecule actually distorts the red blood cell in which it resides, causing the cell to assume a sickle shape instead of its usual disk shape. The shape change prohibits the cell from adequately performing its designated role of carrying oxygen to the body's organs and tissues.

Another example is presented by muscular dystrophy, which is linked to mutations in the dystrophin gene. This gene codes for the dystrophin protein, which is crucial for the strength and movement of normal muscle tissue. People lacking dystrophin experience the muscle weakness characteristic of the disease.

Finally some genetic mutations do not alter a cell's function as much as they interfere with the cell's normal life cycle, specifically its cell-division cycle. Such mutations can lead the cell to divide uncontrollably, as is the case in certain cancers.

The essence of gene therapy, then, is to deliver to a cell a correct version of a mutated gene, the expression of which will produce the normal protein and hence restore normal cellular function. This has been obvious for some time, but how to achieve this goal has not been. An initial problem centered on how to get a gene into a cell. The chromosomes of a mammalian cell are housed inside a membrane-bounded compartment, called a nucleus. It is not enough for a gene-delivery system to deposit the gene into the cell; the gene must be delivered to the nucleus.

This in itself is not difficult. Scientists have been able to do it for decades. Foreign DNA can be injected into a cell, or its entry can be facilitated by various chemical or electronic means. But these methods are not very efficient, and one requirement for gene therapy is that sufficient amounts of corrective DNA be delivered to enough cells to be therapeutically beneficial.

Under the best circumstances, one would also want the therapeutic DNA to become a permanent part of the host's chromosomes. This would ensure its stability and would meanhat the therapeutic gene would be replicated along with the host's chromosomes during each cell division. In contrast, DNA delivered to a cell by physical or chemical means can be placed in the cell's nucleus and can be expressed, but it does not become integrated into the chromosomes.

An ideal gene-delivery vehicle would be able to enter a large number of cells and integrate its DNA into the host's chromosomes. As it happens, some kinds of viruses are perfectly adapted to do just that. And, about 15 years ago, Richard Mulligan and Constance Cepko, who were then at Massachusetts Institute of Technology, along with colleagues at MIT and Harvard, made the important technological leap that initiated the modern era of gene therapy. Specifically they demonstrated that members of the retrovirus family could be engineered to carry foreign genes into mammalian cells and splice them into the host's chromosomes.

To create these gene-delivery vectors, Mulligan and his coworkers essentially gutted the virus of its genes, disposing of those that could be harmful to the host. At the same time, they retained those genes that enable the retrovirus to insert DNA into host chromosomes. By attaching this integrative machinery to the therapeutic gene, they created a retrovirus capable of infecting cells and splicing a corrective gene into chromosomes.

Inserting a gene, however, is only half of the problem. The vector must also contain a mechanism for activating the therapeutic gene, since this is not automatic. Genes have evolved a pattern of expression wherein certain levels of their product are required at specific times in the life cycle of the cell. Hence the corrective action of gene therap must include a timing and regulatory "device." Such devices are usually found at the start of a gene and constitute the gene's "on" switch, or promoter. But this leads to another problem.

Promoters are often exquisitely complex and sometimes quite large, so placing them into a therapeutic vector is difficult. When constructing their retroviral vectors, Mulligan and his colleagues opted to use promoters native to the virus, rather than the corrective gene's own promoter. In laboratory petri dishes, these vectors sometimes worked quite well, but not always.

In some cases, the therapeutic genes entered the cells as expected but were expressed at unpredictably low levels. Low levels of expression continue to dog gene-therapy efforts, and improving expression levels remains a major focus of research. Recent vectors include portions of the gene's own promoter. This has the added benefit that the therapeutic gene is expressed as naturally as possible - only during the times when its product is needed.

Other constructions attach promoters that can be externally controlled. For example, certain genes have promoters that are sensitive to the antibiotic tetracycline and are activated when the drug is present. A vector was recently constructed by Herman Bujold and colleagues at the University of Heidelberg that pairs a tetracycline-sensitive promoter with a corrective test gene. The test gene would be activated only if the patient ingests tetracycline.

The initial expectation was that cells would have to be removed from the body in order to be treated. This ex vivo approach would necessarily limit therapy to those cells, such as blood cells, that are easily removed and replaced. But more recently, retroviral vectors have been developed that can be infused directly into an organ, such as the liver, or placed into the lung by inhalation. This versatility is one of the great advantages of retroviral vectors.

There are also some considerable disadvantages to retroviral vectors that have made investigators cautious about using them. The same feature that makes the retroviruses so attractive to gene-therapy investigators has also been one of their greatest drawbacks - namely the ability to integrate genes into chromosomes. The problem is that scientists have no control over how many copies of the gene become integrated or where on the chromosome they insert. Since integration appears to be essentially random, the vector's genetic payload may become inserted within another important gene, disrupting or altering its expression. Or a gene may integrate within the regulatory region of a gene responsible for controlling cellular proliferation, thus putting the cell on the path towards cancerous growth. Although these are remote possibilities, they are real and must nevertheless be considered as a potential consequence of retroviral-based gene-delivery vectors.

Considering some of the safety issues surrounding the use of retroviral vectors, investigators have been casting about for other viruses that can deliver genes to cells without disrupting their normal chromosomal configuration. There has been much interest in the use of adenoviruses for this purpose. The bulk of the early work on adenoviral gene therapy was conducted by Ronald Crystal at Cornell Medical School and James Wilson at the University of Pennsylvania.

Like the retroviruses, adenoviruses deliver their genetic payload to the nucleus, but, except under rare circumstances, the genes do not integrate into the resident chromosomes. This, of course, relieves concern about random genetic integration, but it also means that the therapeutic gene is only transiently active. The adenoviral vectors have to be repeatedly administered in order to maintain a steady therapeutic dose.

Adenoviruses can infect a broad range of human cells, including those of the lung, liver, blood vessels and brain. In fact, brain tumors have been treated with adenoviral vectors carrying "suicide genes," whose expression leads to cell death only when its product interacts with a specific drug taken by the patient. These studies generated mixed results.

Adenoviral vectors have also been used in human trials to correct mutations in the cystic-fibrosis transmembrane receptor (CFTR) gene, which contributes to cystic fibrosis. The success of these trials, however, has been quite low. For one thing, the host's immune system registers the adenoviral vector as foreign and eliminates it from the system. In addition, some of the vectors cause an inflammatory response at the high levels required to achieve therapeutic doses.

One of the most promising vehicles to emerge from recent gene-therapy studies is adeno-associated virus (AAV). This virus infects a wide range of cells, including lung and muscle cells, and it integrates its genes within the host's. In addition, it can infect nondividing cells and does not elicit an immune response both of which are important advantages over retroviral and adenoviral vectors. The work on this virus has been pioneered by three investigators: Kenneth Berns and Nicholas Muzyczka at the University of Florida and R. Jude Samulski at the University of North Carolina at Chapel Hill. Significant advances in the use of AAV for gene therapy have recently been reported by Mark Kay and colleagues at Stanford University and Kathryn High and coworkers at the University of Pennsylvania. Both of these research groups used a modified AAV vector to achieve long-term expression and correction in animals of gene that contributes to hemophilia. This achievement required a detailed appreciation for the basic biology of AAV.

However, as expected, this virus has some drawbacks. First, it can carry only a small genetic payload, which considerably restricts its usefulness. Second, it, too, carries the risk of disrupting functioning genes by randomly inserting itself into the chromosomes. Finally, it is somewhat difficult to manufacture these vectors in sufficiently high quantities. Other viruses under study as potential vector candidates include Herpes simplex, Vaccinia and even the human immunodeficiency virus.

In addition to vital-based vectors, investigators are continuing to explore nonvital delivery systems. One system that holds some promise delivers drugs via liposomes, small vesicles artificially created from lipids that resemble those making up the membranes of mammalian cells. Because they are constructed of virtually identical materials, the liposomes can fuse with cell membranes and empty their contents - which can include drugs or corrective genes - inside the cell. Some of the DNA delivered by liposomes makes its way into the cell's nucleus.

[TABULAR DATA FOR FIGURE 6 OMITTED]

Ultimately, scientists would like to replace a dysfunctional gene with a functional one, within the normal context of the chromosome, an approach that could skirt the concerns about the number of genes delivered, the chromosomal location and the level of expression. Right now, homologous recombination, the only technique that comes close to this, is so inefficient that its success rate is 1 in 10,000. Needless to say, this is not adequate for human use.

But the idea of completely replacing a bad gene with a good one may be overreaching, especially when one considers how small are many of the mutations that contribute to disease. To understand how small, we must first consider a few basic facts about the composition of genes.

The gene is to inheritance what a word is to language; it is the basic trait of meaning.

In the genetic lexicon, the gene is a length of DNA that codes for a particular protein. The alphabet used by the genetic language contains only five letters, or nucleotides, named for the bases. These are adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U). The nucleotides A, T, C and G are found in DNA. (RNA, a chemical cousin to DNA and an important participant in genetic decoding, lacks thymine, but has uracil in its place.) The average human gene is a little over 1,000 nucleotides long. In many inherited disorders only one or a few of these nucleotides is incorrect.

For example, sickle cell anemia is the result of a single nucleotide substitution, a single letter misspelled, in the gene encoding the [Beta]-globin strand of hemoglobin. Yet this one-nucleotide substitution can cause the structural deformity of the molecule and the characteristically distorted shape of the sickled red blood cell. Over 70 percent of the cases of cystic fibrosis are attributable to the deletion of three nucleotides in the CFTR gene. Why should the entire gene be replaced when the error is so minimal? That strategy seems akin to remodeling the whole kitchen to repair a leaky faucet.

In 1993, while studying homologous recombination in mammalian cells, members of my laboratory began experimenting with ways to repair damaged genes, rather than replacing them. The cell's own repair mechanisms are extremely efficient, as evidenced by the simple and continual inheritance of normal genes through generations of cell divisions. If we could harness the cell's own power of DNA repair, we reasoned, we might be able to correct mutations.

Normal human chromosomes are actually made up of two strands of DNA complexed to each other in an interesting way. It turns out that the nucleotides of DNA can bind with each other in a specific pattern. Except in very rare cases, adenine always pairs with thymine, and guanine always pairs with cytosine. Each DNA strand carries a nucleotide sequence exactly complementary to the other, such that every adenine nucleotide on one strand is matched up with a thymine on the partner strand, and every guanine is matched with a cytosine on the complementary strand. A sequence of GATC on one strand would therefore bind to the sequence of CTAG on its partner, or so it should be.

Occasionally the wrong nucleotide is inserted into a spot, so that the corresponding nucleotide on the partner strand cannot properly bind in that position. In that case the mismatched nucleotides form a bulge. Usually this is not a problem, since the cell contains DNA repair mechanisms that actually scan the DNA and detect such bulges. When one is discovered, the repair systems work to remove the incorrect nucleotide and replace it with the correct one. But if the mismatch is overlooked by the cell's repair machinery, the error is retained, and the gene remains defective.

It was our idea to alert these repair mechanisms to the error. The principle is quite simple. We artificially create a short string of nucleotides, called an oligomer, that, with one exception, is exactly complementary to the section of the gene in which the error is located, the exception being at the site of the error. Here we insert the nucleotide complementary to the one that is supposed to be in the DNA sequence of the normal gene. The oligomer binds to its complementary sequence on the DNA, and by design creates a bulge at the site of the mismatch. This bulge is eventually detected by the cell's internal DNA-repair mechanisms. Repair enzymes remove the erroneous nucleotide and replace it with a nucleotide complementary to the one in that position in th