Year In Science

Sunday, January 13, 2002

The Power of Proteins
When the rough draft of the human genome was released in June 2000, scientists expected a count of roughly 100,000 genes. Yet when scientists refined it last spring, the count had dropped to little more than 30,000, only a third more genes than a simple worm like Caenorhabditis elegans. How can so few genes create human complexity? The answer lies in proteins, and the map of the genome does not reveal what proteins genes make to carry out their work, what the proteins do, or how the proteins interact. So researchers are turning their attention to the proteome, the array of human proteins and their various interactions. "In the case of the genome, you know when you're done," says Paul Bartel, vice president of proteomics at Myriad Genetics Inc. in Salt Lake City. "With the proteome, we don't even know how many protein interactions we need to find." And the number of proteins humans make could be as large as a million.
— Rabiya S. Tuma

Monkeys That Glow in the Dark
First it was bacteria. Then it was mice. Then it was plants. Now, after nearly a quarter century of efforts to transfer genes between organisms in the lab, comes the world's first transgenic primate. Last January a research team at the Oregon Regional Primate Research Center in Beaverton announced the birth of a rhesus monkey engineered to contain a jellyfish gene that encodes a green fluorescent protein.

The team did not set out to create a glowing monkey. They simply used the jellyfish gene, which is easily detected, to test whether inserting foreign genes into eggs (so-called germline gene alteration) can produce viable monkeys. The team's ultimate goal is to create strains of laboratory monkeys carrying genes that confer the risk of diseases such as Alzheimer's, Parkinson's, diabetes, and other illnesses. Mice are bred in a similar fashion for specific kinds of research and for drug development.

After failing in several attempts, the team succeeded using a technique developed for gene therapy. They slipped the jellyfish gene into a disabled virus, then injected the altered virus into a batch of monkey eggs. "The viruses are very good at getting inside cells and are an excellent delivery mechanism," says team member Anthony Chan.

The next step was in vitro fertilization of the eggs, which resulted in more than 100 embryos. Finally, 40 of the embryos were transferred to 20 rhesus monkeys—two per surrogate mother.

More than five months later, three healthy monkeys were born. Only one has been found to carry the jellyfish gene. "We have found the gene in the monkey, but so far there has been no sign of fluorescence," says Chan, indicating that the gene has not yet been used to create proteins. "The significant thing for us isn't whether or not the gene is expressed but whether it can be detected at all." (A set of miscarried twins did show such signs; one of them had a finger that glowed when placed under ultraviolet light.) As an homage to the gene-delivery technique, they named the monkey ANDi, or "inserted DNA" written backward.

Some critics argue that tampering with the genes of primates is wrong because it brings us one step closer to human "designer babies." But Chan defends the goals of the project. Medical researchers now rely on transgenic mice to develop drugs, he says, "but it is often very difficult to infer what will happen in humans based on what happens in mice, because there are so many differences between the two." Chan believes that using transgenic primates would narrow the gap and allow researchers to develop more effective treatments for human diseases. "We've shown that it can be done," he says. "Primates can tell you more about what affects humans than mice ever will."

In any case, model strains of monkeys à la transgenic mice could be developed only if the added human gene is passed along to offspring—and that will remain an open question for some time. Rhesus monkeys don't reach sexual maturity until they are four years old.
— Curtis Rist

The Beginning of Life?
Unlike sports fans dissecting a pivotal football play, scientists studying evolution don't have the luxury of instant replay. But last year molecular biologists at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, figured out a way to replay the most mysterious stage in life's evolution. Although no one knows how life began, one popular scenario holds that at some early stage life was based on ribonucleic acids, which spontaneously developed the ability not only to direct specific chemical reactions but also to store and copy a cell's genetic information. The problem with this idea is that nobody had ever shown that RNA molecules could accurately copy other strands of RNA.

David Bartel and his colleagues at the Whitehead Institute set out to see if it could happen. They started with a bit of RNA that had shown promise in early tests. Then they joined this portion to each of 1015 randomly mutated RNA strands. They exposed all the strands to the building blocks for RNA and to an RNA sequence to be copied. Any RNA strands that showed promising results were randomly mutated and retested. After 10 cycles of selection and mutation, the researchers came up with RNA enzymes capable of copying various short RNA sequences—albeit far more slowly than a cell would require. "We need something more efficient, but the foundation is there," says Bartel. "Evolution had much more time and a much larger test tube."
— Rabiya S. Tuma

It Takes Three
Not so long ago making a baby was simply about the birds and the bees. Then technological advances brought a host of new aids. The latest, cytoplasmic transfer, offers an infertile woman one last chance to have a baby genetically her own. Since its introduction in 1996, some 30 children worldwide have been born with the help of donated cytoplasm. But this year doctors discovered the technique had inadvertently introduced new genes. It may also have led to chromosomal abnormalities.

Cytoplasmic transfer involves injecting a bit of cytoplasm—the jellylike substance that surrounds the nucleus of an egg—from a healthy donor egg into the egg of an infertile woman before the egg is fertilized. Female infertility is often linked to abnormalities in cytoplasmic components that help ensure proper sorting of chromosomes during cell division. Infusing the problem egg with a shot of healthy cytoplasm replaces the defective machinery. Most important of all seem to be the tiny powerhouses called mitochondria that fuel cellular development. Because mitochondria carry their own genetic sequence, however, the resulting baby could theoretically have DNA from both women and the man, which means that, technically, it would have three parents.

In May biologists at the Institute for Reproductive Medicine and Science of St. Barnabas Medical Center in Livingston, New Jersey, reported that three of 16 babies born through cytoplasmic transfer at their center indeed carried mitochondrial DNA from the donor cytoplasm. So far, the babies are developing normally. But their altered genetic makeup raises concerns about incompatibilities between the recipient's nuclear genome and the donor's mitochondrial genome. "If it's not a successful combination, it could cause developmental and physiological problems," says John Eppig, a reproductive and developmental biologist at Jackson Laboratory in Maine. "And these could become evident only much later in life." Moreover, since the mixture of mitochondrial genes is passed down the maternal line, the altered genetic blueprint of such female babies could affect future generations.

Even if the added mitochondrial DNA is not harmful, there may be other problems with the method. At St. Barnabas, genetic defects were discovered in two additional fetuses (one miscarried; the other was aborted). Both had Turner's syndrome, which occurs when one of a girl's two X chromosomes is missing. Girls with this syndrome are short, do not develop sexually, and suffer from heart and kidney problems. The condition occurs naturally in two of every 100 conceptions, and the vast majority of such fetuses miscarry. Among 18 conceptions aided by cytoplasmic transfer at St. Barnabas, two had Turner's—about six times the natural rate. Some experts suspect the fertility technique is to blame. One possibility is that the infusion of healthy donor cytoplasm might allow development to proceed when it otherwise would not.

Some researchers think demand from infertile patients is prodding new treatments into the clinic too soon. "These babies are an experiment," says Carol Brenner, director of molecular biology and genetic research at Reproductive Medicine Associates in Morristown, New Jersey, and a former team member at St. Barnabas. "Should we be doing this? We don't know."
— Diane Martindale

Why Human Clones Won't Work — Yet

Noah, one of the first interspecies clones, receives oxygen through a plastic tether after birth.
Photographs courtesy of Advanced Cell Technology
Baby Noah, heralded as the first clone of an endangered species, came kicking and mooing into the world on January 8, 2001. An ordinary cow delivered Noah, but his genes came from a long-dead male gaur, an increasingly rare type of ox native to India and Southeast Asia, whose frozen tissue had been preserved at the San Diego Zoo. To create Noah, the nucleus was removed from a domestic cow's egg and replaced by a single skin cell of Dad's that contained his gaur DNA. That mingling produced the first living interspecies clone.

Two days after his birth, Noah died of clostridial enteritis, a common bacterial infection almost always fatal to newborn livestock. Something as simple as an unsterilized milk bottle may have transmitted the bug, say researchers at Advanced Cell Technology in Worcester, Massachusetts, which created Noah. Scientists at the company don't believe Noah's death was related to cloning, because an extensive autopsy showed that the animal's internal organs were normal and his immune system functioning.

By summer the team had resumed attempts to create another gaur clone. And in a surprise announcement in October, Pasqualino Loi at Italy's University of Teramo reported that his team had ushered in a clone of another endangered species a year earlier, in October 2000. The clone is a baby mouflon, a wild sheep native to Sardinia and other Mediterranean islands. Begotten from a cell of a female mouflon found dead at a Sardinian wildlife refuge, the mouflon lamb was gestated by a domestic sheep.

Nonetheless, cloning is still poorly understood. In most cases, dozens of clone embryos must be created before one yields a viable pregnancy. Of those clones born alive, many are abnormally large or grossly deformed. They often die shortly after birth. And survivors tend to be sickly. Even when clones appear healthy at birth, they often develop mysterious illnesses later. For example, the clone Dolly was a dainty lamb but has since become morbidly obese. Her creators have no idea why.

In testimony last spring before a congressional committee investigating human cloning, Rudolf Jaenisch, a cloning specialist at the Massachusetts Institute of Technology's Whitehead Institute, said: "I believe there is no normal clone anywhere." In July, he and researchers from the University of Hawaii published evidence that apparently normal mouse clones can possess fundamental defects. Several studies had already pinned some of the visible abnormalities in clones on the absence of vital molecules that turn gene expression on and off during fetal development. Jaenisch's team showed that even healthy clones lack some of these crucial molecules. "It may not be just a technical problem; it may be biological," says William Rideout III, a postdoctoral fellow at the Whitehead Institute and coauthor of the study. "There may be little we can do to manipulate the process." As yet, there is no reliable way to screen clone embryos for the regulatory-molecule deficiency. And even if that screening were possible, there's no way to repair defective embryos. These questions about the cloning process itself, Rideout warns, "should put fear in the hearts of anyone planning to clone a human."
— Christine Soares

The Waiting List for Clones
Somewhere on the web is a site soliciting signatures for an appeal to clone Elvis Presley. You can't add your name, however, because the sponsor couldn't keep up with the overwhelming volume of signatures received. Meanwhile, a trio that intends to clone more ordinary mortals within the next few years had their say in a debate at the National Academy of Sciences in Washington, D.C., last August. One speaker was Brigitte Boisselier, the scientific director of Clonaid, a private company that plans to offer cloning as a reproductive option to a waiting list of 2,000 people. Boisselier, a French biochemist, also happens to be a bishop in the Raelian sect, a Clonaid sponsor. Raelians believe that extraterrestrials brought life to Earth; their founder considers cloning a means of eternal life. The other two pro-cloners who spoke hailed from more familiar realms: Panos Zavos, a former professor of reproductive physiology at the University of Kentucky and director of the Andrology Institute of America in Lexington, Kentucky, and Severino Antinori, the Italian fertility specialist who has pioneered methods that allow postmenopausal women to give birth. Cloning, the three argued, should be regarded as a promising technique for assisted reproduction that further research would refine and make safe. Boisselier contends that choosing how to reproduce is a basic human right; she regards clones as "belated twins."

Researchers who have developed cloning techniques in animals argued that cloning humans—even were it acceptable, in principle, to create children in this unorthodox manner—would be too risky in practice. As yet little is known of how the genome of the clone turns back the clock and reprograms itself for development. The few animal clones that survive to term often die shortly after birth, probably from abnormalities that have not yet been characterized. As cloning pioneers Rudolf Jaenisch and Ian Wilmut have argued, "if human cloning is attempted, those embryos that do not die early may live to become abnormal children and adults; both are troubling outcomes."

Germany, France, Japan, and Australia have already banned cloning humans. In August the House of Representatives voted 265-162 to outlaw cloning, both for reproduction and research. The Senate vote on the bill was stalled in the aftermath of the September 11 attacks. President Bush's position on stem-cell research prohibits the use of federal funds to create embryonic clones, the cells of which may hold clues to the treatment of disease.
— Sarah Richardson

Genetic Tinkering Makes Bioterror Worse
Last year an experimental mouse vaccine raised worries about an entirely new and surprisingly simple method of creating bioterrorist weapons. A team at the Cooperative Research Center for the Biological Control of Pest Animals in Canberra, Australia, was using an altered mousepox virus, a distant kin of smallpox, to create a contraceptive vaccine for mice. In January they reported that their efforts had unexpectedly created a lethal new strain of mousepox. "This is the public's worst fears about genetically modified organisms come true," says Bob Seamark, the center's former CEO. "We have inadvertently shown that something we thought was difficult—increasing the pathogenicity of a virus—is in fact quite easy."

The team's idea was to use the mousepox virus to induce an immune response that would target a mouse's eggs. They took a fairly benign strain of mousepox virus and added genes for proteins carried on the surface of mouse eggs. Any mouse cells infected with the virus would produce both viral proteins and egg proteins, thus arousing antibodies that would attack proteins on the mouse eggs. The vaccine worked well in one strain of mouse, but to increase its effectiveness, researchers inserted another gene into the virus. The added gene encodes for the protein interleukin-4, which boosts antibody production. The researchers were surprised to find that the added gene rendered the mice incapable of fighting off the mousepox. Within nine days, every mouse inoculated with the vaccine died. "Even when we tried to protect them with vaccination, we found we could not," says Seamark.

Nobody knows if adding the interleukin-4 gene would have the same effect in a different pathogen, but "the question instantly became what would happen if somebody tried this with smallpox or other human viruses," says Seamark. The group debated among themselves for 18 months about whether or not to keep the results to themselves. They also consulted with various government agencies before making their decision. In the end, they opted to publish their findings. "In the wrong hands, these could become biological-warfare weapons of terrible proportions, and we were able to demonstrate how easily they could be produced," Seamark says. The discovery took on greater significance in light of the September 11 attacks, with the ensuing fears of crop duster-borne pathogens and the swift, unexpected deaths from mail-distributed anthrax. Now even the most far-fetched schemes seem plausible. "To be forewarned is to be empowered," Seamark says. "If we understand that this is a grave risk, we can develop policies needed to prevent it."
— Curtis Rist

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