GENETIC ENGINEERING

CONCEPT

Genetic engineering is the alteration of genetic material by direct intervention in genetic processes with the purpose of producing new substances or improving functions of existing organisms. It is a very young, exciting, and controversial branch of the biological sciences. On the one hand, it offers the possibility of cures for diseases and countless material improvements to daily life. Hopes for the benefits of genetic engineering are symbolized by the Human Genome Project, a vast international effort to categorize all the genes in the human species. On the other hand, genetic engineering frightens many with its potential for misuse, either in Nazi-style schemes for population control or through simple bungling that might produce a biological holocaust caused by a man-made virus. Symbolic of the alarming possibilities is the furor inspired by a single concept on the cutting edge of genetic engineering: cloning.

HOW IT WORKS

DNA

Any discussion of genetics makes reference to DNA (deoxyribonucleic acid), a molecule that contains genetic codes for inheritance. DNA resides in chromosomes, threadlike structures found in the nucleus, or control center, of every cell in every living thing. Chromosomes themselves are made up of genes, which carry codes for the production of proteins. The latter, of which there are many thousands of different varieties, make up the majority of the human body's dry weight.

Although it is central to the latest advances in modern genetic research, DNA was discovered more than 130 years ago. In 1869 the Swiss biochemist Johann Friedrich Miescher (1844-1895) isolated a substance, containing both nitrogen and phosphorus, that separated into a protein and an acid molecule. He called it nucleic acid, and in this material he discovered DNA. Some 74 years would pass, however, before scientists recognized the function of the nucleic acid Miescher had discovered. Then, in 1944, a research team led by the Canadian-born American bacteriologist Oswald Avery (1877-1955) found that by taking DNA from one type of bacterium and inserting it into another, the second bacterium took on certain traits of the first. This experiment, along with other experiments and research, proved that DNA serves as a blueprint for the characteristics and functions of organisms.

THE DOUBLE HELIX.

Nine years later, in 1953, the American biochemist James D. Watson (1928-) and the English biochemist Francis Crick (1916-) solved the mystery of DNA's structure and explained the means by which it provides necessary instructions at critical moments in the course of cell division and growth. They proposed a double helix, or spiral staircase, model, which linked the chemical bases of DNA in definite pairs. Using this twisted-ladder model, they were able to explain how the DNA molecule could duplicate itself, since each side of the ladder is identical to the other; if separated, each would serve as the template for the formation of its mirror image.

The sides of the DNA ladder are composed of alternating sugar and phosphate molecules, like links in a chain, and consist of four different chemical bases: adenine, guanine, cytosine, and thymine. The four letters designating these bases—A, G, C, and T—are the alphabet of the genetic code, and each rung of the DNA molecule is made up of a combination of two of these letters. Owing to specific chemical affinities, A always combines with T and C with G, to form what is called a base pair. Specific sequences of these base pairs, which are bonded to each other by atoms of hydrogen, constitute the genes.

ENDLESS COMBINATIONS.

A four-letter alphabet may seem rather small for constructing the extensive vocabulary that defines the myriad life-forms on Earth. If one stops to consider the exponential operations involved, however, it is easy to understand how large the range of possibilities can become. For any sequence, there are four possibilities for the first two letters (AT, TA, CG, or GC) and four more possibilities for the second two letters. Thus, just for a four-letter sequence, there are 16 possibilities, and for each pair of letters added to the sequence, the total is multiplied by four.

To see where this might lead, imagine that you started with a penny and tried to quadruple your funds every day. The first day there would not be a dramatic increase, since you would have to earn only $0.04, and even by day 4 you would need only $2.56 to meet your goal. But as the quadrupling process continued, day by day the sums of money would get bigger ($655.36 on day 8) and bigger ($16,772.16 on day 12) and bigger ($687,194,767.36 on day 18). Given the fact that the human body contains an almost unfathomable number of genes, each of which may be between 2,000 and 200,000 base pairs long, one can begin to imagine just how large the number of possibilities would become.

Each one of these combinations has a different meaning, providing the code for all manner of specific traits, such as brown hair and blue eyes, dimples, unattached earlobes, and so on. Except for identical twins, no two humans have exactly the same genetic information. What follows are just a few facts about the human genome—that is, all of the genetic material in the chromosomes of the human organism:

Some Facts About the Human Genome

• The human body contains about 100 trillion cells.
• Each cell has a DNA code consisting of some 1.5 billion base pairs.
• The DNA in each cell, if stretched to its full length, would be 6 ft. (1.8 m) long—yet it fits into a space about 0.0004 in. (0.0001 cm) across, smaller than the head of a pin.
• If all of the DNA in the human body were stretched end to end, it would reach to the Sun and back more than 600 times.
• If a person attempted to recite the entire human genome, with all its base pairs, at the rate of one letter per second, 24 hours a day, it would take a century.
• Every second scientists working on the Human Genome Project are decoding some 12,000 letters of DNA.
• Our DNA is 98% identical to that of chimpanzees.
• Only 0.2% of all human DNA differs between individuals; in other words, people are 99.8% the same, and all the vast differences between people are a product of just 1/500th of the total DNA.
• Despite all that scientists know about DNA, a staggering 97% of all human DNA has no known function.

Principles of Genetic Engineering

Just as DNA is at the core of studies in genetics, recombinant DNA (rDNA)—that is, DNA that has been genetically altered through a process known as gene splicing —is the focal point of genetic engineering. In gene splicing, a DNA strand is cut in half lengthwise and joined with a strand from another organism or perhaps even another species. Use of gene splicing makes possible two other highly significant techniques. Gene transfer, or incorporation of new DNA into an organism's cells, usually is carried out with the help of a microorganism that serves as a vector, or carrier. Gene therapy is the introduction of normal or genetically altered genes to cells, generally to replace defective genes involved in genetic disorders.

DNA also can be cut into shorter fragments through the use of restriction enzymes. (An enzyme is a type of protein that speeds up chemical reactions.) The ends of these fragments have an affinity for complementary ends on other DNA fragments and will seek those out in the target DNA. By looking at the size of the fragment created by a restriction enzyme, investigators can determine whether the gene has the proper genetic code. This technique has been used to analyze genetic structures in fetal cells and to diagnose certain blood disorders, such as sickle cell anemia.

GENE TRANSFER.

Suppose that a particular base-pair sequence carries the instruction "make insulin"; if a way could be found to insert that base sequence into the DNA of bacteria, for example, those bacteria would be capable of manufacturing insulin. This, in turn, would greatly improve the lives of people with type 1 diabetes, who depend on insulin shots to aid their bodies in processing blood sugar. (See Non-infectious Diseases for more about diabetes.)

Although the concept of gene transfer is relatively simple, its execution presents considerable technical obstacles. The first person to surmount these obstacles was the American biochemist Paul Berg (1926-), often referred to as the "father of genetic engineering." In 1973 Berg developed a method for joining the DNA from two different organisms, a monkey virus known as SV40 and a virus called lambda phage. Although the accomplishment was clearly a breakthrough, Berg's method was difficult. Then, later that year, the American biochemists Stanley Cohen (1922-) at Stanford University, and Herbert Boyer (1936-) at the University of California at San Francisco discovered an enzyme that greatly increased the efficiency of the Berg procedure. The gene-transfer technique developed by Berg, Boyer, and Cohen formed the basis for much of the ensuing progress in genetic engineering.

REAL-LIFE APPLICATIONS

Big Business in DNA

Ever since the breakthrough discoveries of Watson, Crick, and others in the 1950s made genetic engineering a possibility, the new field has promised increasingly bigger payoffs. These payoffs take the form of improvements to human life and profits to those who facilitate those improvements. The possible applications of genetic engineering are virtually limitless—as are the profits to be made from genetic engineering as a business. As early as the 1970s, entrepreneurs (independent businesspeople) recognized the commercial potential of genetically engineered products, which promised to revolutionize life, technology, and commerce as computers also were doing. Thus was born one of the great buzzwords of the late twentieth century: biotechnology, or the use of genetic engineering for commercial purposes.

Several early biotechnology firms were founded by scientists involved in fundamental research: Boyer, for example, teamed up with the venture capitalist Robert Swanson in 1976 to form Genentech (Genetic Engineering Technology). Other pioneering companies, including Cetus, Biogen, and Genex, likewise were founded through the collaboration of scientists and businesspeople. Today biotechnology promises a revolution in numerous areas, such as agriculture. Recombinant DNA techniques enable scientists to produce plants that are resistant to freezing temperatures, that will take longer to ripen, that will develop their own resistance to pests, and so on. By 1988 scientists had tested more than two dozen kinds of plants engineered to have special properties such as these. Yet no field of biotechnology and genetic engineering is as significant as the applications to health and the cures for diseases.

MEDICINES AND CURES.

The use of rDNA allows scientists to produce many products that were previously available only in limited quantities: for example, insulin, which we referred to earlier. Until the 1980s the only source of insulin for people with diabetes came from animals slaughtered for meat and other purposes. The supply was never high enough to meet demand, and this drove up prices. Then, in 1982, the U.S. Food and Drug Administration (FDA) approved the sale of insulin produced by genetically altered organisms—the first such product to become available. Since 1982 several additional products, such as human growth hormone, have been made with rDNA techniques.

One of the most exciting potential applications of genetic engineering is the treatment of genetic disorders, which are discussed in Heredity, through the use of gene therapy. Among the more than 3,000 such disorders, quite a few of which are quite serious or even fatal, many are the result of relatively minor errors in DNA sequencing. Genetic engineering offers the potential to provide individuals with correct copies of a gene, which could make possible a cure for that condition. In the 1980s scientists began clinical trials of a procedure known as human gene therapy to replace defective genes. The technique, still very much in the developmental stage, offers the hope of cures for diseases that medicine has long been powerless to combat.

In 2001 scientists at the Weizmann Institute in Israel brought together two of the most exciting fields of research, biotechnology and computers, to produce the DNA-processing nanocomputer. It is an actual computer, but it is so small that a trillion of them would fit in a test tube. It consists of DNA and DNA-processing enzymes, both dissolved in liquid; thus its input, output, and software are all in the form of DNA molecules. The purpose of the nanocomputer is to analyze DNA, detecting abnormalities in the human body and creating remedies for them.

The Human Genome Project

At the center of genetic studies, with vast potential applications to genetic engineering, is the Human Genome Project (HGP), an international effort to analyze and map the DNA of humans and several other organisms. As discussed in the essay Genetics, the HGP began with efforts by the Atomic Energy Commission, a predecessor to the U.S. Department of Energy, to study the genetic effects of radioactive nuclear fallout. In 1990 the Department of Energy in cooperation with the National Institutes of Health (NIH), launched the project. At about the same time, the governments of the United Kingdom, Japan, Russia, France, and Italy initiated their own, similar undertakings, which are coordinated with American efforts.

The purpose of the project is to locate each human gene and determine its specific structure and function. Such knowledge will provide the framework for studies in health, disease, biology, and medicine during the twenty-first century and no doubt will make possible the cures for countless diseases. Although great strides have been made in gene therapy in a relatively short time, its potential usefulness has been limited by lack of scientific data concerning the multitude of functions that genes control in the human body. For gene therapy to advance to its full potential, scientists must discover the biological role of each of these genes and locate each base pair of which they are comprised.

A PROGRESS REPORT.

Scientists participating in the project have identified an average of one new gene a day, and this rate of discovery has increased. At the time of its establishment in 1990 (under the leadership of James D. Watson, who served as director until 1992), HGP was expected to reach completion by 2005. In 2002, however, the project's leadership predicted completion by some time in the following year. Along the way, they had discovered that the human genome, originally believed to include 100,000 to as many as 150,000 genes, actually consists of about 30,000 to 40,000 genes.

Both HGP and a private firm, Celera Genomics (founded 1998), had undertaken the study of the human genome, and in June 2000 the entities jointly reported that they had finished the initial sequencing of the three billion-odd base pairs in the human genome. By that point, researchers also had completed thorough DNA sequences for many other organisms. The basis for the latter undertaking is that humans share many genes with other life-forms. With the completion of initial sequencing, scientists working on the HGP undertook the effort of determining the exact sequence of the base pairs that make them up all human genes. Long before completion, the project had yielded some information. Some of the genes identified through the HGP include one that guides reproduction of the human immunodeficiency virus (HIV), which causes the acquired immunodeficiency syndrome, better known as AIDS (see Infectious Diseases). Researchers also have located a gene that predisposes people to obesity as well as genes associated with such inherited disorders as Huntington disease, Lou Gehrig disease (also called amyotrophic lateral sclerosis, or ALS), and some colon and breast cancers.

A World of Controversy

The HGP has numerous implications—and not all of them, in the minds of some critics, are positive. The NIH inadvertently created cause for concern when, in 1991, it attempted to patent certain forms of DNA. While patenting is touted as a necessary financial incentive for research initiatives, critics maintain that it restricts access to the information generated and to the use of those discoveries. That places a great deal of control in the hands of the private firm that funded the research and may limit the spread of real benefits that result from discovery.

Some scientists and politicians have raised the concern that the ability to produce detailed genetic information on people could give too much power to the people who possess that knowledge. Most states do not have laws protecting citizens against the misuse of genetic information, for instance, by employers and insurers. In the absence of effective legal remedies, genetic testing may be used to bar people from employment or insurance coverage. Insurers may even make mandatory testing a requirement for coverage. Existing laws may not be adequate to protect people's privacy: whereas the individual may be protected from having to provide potentially damaging genetic information, such information still can be obtained by testing the individual's relatives.

NIGHTMARISH IMAGES.

The idea of genetic information being used to control a person's destiny calls to mind all sorts of nightmarish images, such as those raised by the movie Gattaca (1997). Set in a dystopian, or anti-utopian, future, the film depicts a character who employs elaborate means to conceal his true identity in order to hold on to a job that otherwise would be forbidden to him because of his DNA. In fact, one does not have to go to fiction to find examples of societies and movements that have used genetics as a form of social control.

The most frightening example of this was Nazi Germany, which practiced mass murder not only of Jews and other ethnic and social groups but also of people who suffered from mental retardation or other "undesirable" traits. The purpose of DNA was discovered only a year before the collapse of the Nazi empire in 1945, and one can only imagine what Adolf Hitler and his minions would have done with this knowledge if they had had access to it. (DNA is one of several scientific and technological concepts that came to fruition at the end of World War II but which Hitler, fortunately, was unable to use to his advantage. Others include rocketry, nuclear weaponry, radar, computers, and television.)

Nazism was actually an especially repugnant version of a movement known as eugenics, which had its origins in the late nineteenth and early twentieth centuries. Based on the idea that populations could be improved by encouraging people with "positive" traits to reproduce while discouraging reproduction among those with less desirable traits, eugenics was at one time a mainstream movement whose adherents included the distinguished U.S. Supreme Court justice Oliver Wendell Holmes, Jr. (1841-1935).

OTHER CONCERNS.

The specter of eugenics raises the threat that a single human, or a group of humans, could "play God" with the lives of others. Another dramatic fear associated with genetic engineering is the threat that a genetically re-engineered virus could turn out to be extremely virulent, or deadly, and spread. There are other, more mundane questions of ethics: for instance, is it appropriate for scientists to establish private, for-profit corporations to benefit from discoveries they made while working for public-sponsored research institutions? No wonder, then, that the budget for the HGP in the United States includes a small allocation (3% of its total) toward study of the ethical, legal, and social implications (ELSI) of the project. The ELSI Working Group is charged with studying the issues of fairness, privacy, delivery of health care, and education. Meanwhile, there is a vast body of opposition to genetic engineering, biotechnology, and the HGP. And no aspect of the larger subject is more upsetting to certain individuals, as well as special interest groups, as that of cloning.

Cloning

A clone is a cell, group of cells, or organism that contains genetic information identical to that of the parent cell or organism. It is a form of asexual reproduction (see Reproduction), and as such it is not as new as it seems; what is new, however, is humans' ability to manipulate cloning at the genetic level. The first clones produced by humans as long as 2,000 years ago were plants developed from grafts and stem cuttings. By cloning—a process that calls into play complex laboratory techniques and the use of DNA replication—people usually mean a relatively recent scientific advance. Among these techniques is the ability to isolate and copy (that is, to clone) individual genes that direct an organism's development.

THE PROMISE OF CLONING.

The cloning of specific genes can provide large numbers of copies of that gene for use in genetic and taxonomic research as well as in the practical areas of medicine and farming. In the latter field, the goal is to clone plants with specific traits that make them superior to naturally occurring organisms. For example, in 1985 scientists conducted field tests using clones of plants whose genes had been altered in the laboratory to generate resistance to insects, viruses, and bacteria. New strains of plants resulting from cloning could produce crops that can grow in poor soil or even underwater and fruits and vegetables with improved nutritional qualities and longer shelf lives. A cloning technique known as twinning could induce livestock to give birth to twins or even triplets, and on the environmental front cloning might help save endangered species from extinction.

In the realm of medicine and health, cloning has been used to make vaccines and hormones. It has become possible, by combining two different kinds of cells (such as mouse and human cancer cells), to produce large quantities of specific antibodies, via the immune system, to fight off disease. When injected into the bloodstream, these cloned antibodies seek out and attack disease-causing cells anywhere in the body. By attaching a tracer element to the cloned antibodies, scientists can locate hidden cancers, and by attaching specific cancer-fighting drugs, the treatment dose can be transported directly to the cancer cells.

EXPERIMENTS IN CLONING.

The modern era of laboratory cloning began in 1958 when the British plant physiologist F. C. Steward (1904-1993) cloned carrot plants from mature single cells placed in a nutrient culture containing hormones. The first cloning of animal cells took place in 1964, when the British molecular biologist John B. Gurdon (1933-1989) took nuclei from intestinal cells of toad tadpoles and injected them into unfertilized eggs. The cell nuclei in the eggs had been destroyed with ultra-violet light, but when the eggs were incubated, Gurdon found that 1-2% of the eggs developed into fertile, adult toads.

The first successful cloning of mammals occurred nearly 20 years later, when scientists in Switzerland and the United States successfully cloned mice using a method similar to Gurdon's approach. Their method required one extra step, however: after taking the nuclei from the embryos of one type of mouse, they transferred them into the embryos of another type of mouse. The latter served as a surrogate, or replacement, mother. The cloning of cattle livestock was tried first in 1988, when embryos from prize cows were transplanted to unfertilized cow eggs whose own nuclei had been removed. An even greater breakthrough transpired on February 24, 1997, with the birth of a lamb named Dolly in Edinburgh, Scotland. Dolly was no ordinary sheep: she was the first mammal born from the cloning of an adult cell. Thus, she had been produced by asexual reproduction in the form of genetically engineered cloning rather than by anything resembling a normal process. Nonetheless, she proved her own ability to reproduce the old-fashioned way when, on April 23, 1998, she gave birth to a daughter named Bonnie.

ARE HUMANS NEXT?

Though Dolly's and Bonnie's births excited hopes, they also inspired fears. If large mammals could be cloned, could humans? As early as 1993 an attempt had been made at cloning human embryos as part of studies on in vitro (out of the body) fertilization. The purpose was to develop fertilized eggs in test tubes and then to implant them into the wombs of women having difficulty becoming pregnant. These fertilized eggs, however, did not develop to a stage that was suitable for transplantation into a human uterus. Then, on October 13, 2001, scientists at Advanced Cell Technology in Worcester, Massachusetts, successfully cloned a human embryo. They had not created human life, as it might sound; what they had developed instead was a source for nerve and other tissues that could be harvested for use in medicine and research. Still, the news—overshadowed though it was in America, where people were still reeling from the September 11 terrorist attacks—was earth-shattering. Human cells had been reproduced, and once again it appeared that the production of human clones might be possible.

It is easy to understand how people might respond with alarm to such frightening news with alarm. Such fears have a great deal more to do with Hollywood than they do with science. In fact, the accomplishment of the Massachusetts firm, while impressive from a scientific standpoint, was fairly modest compared with the Frankenstein-like image presented by anti-genetic engineering scaremongers. "Cloned an embryo" actually sounds a great deal more dramatic than what the Massachusetts scientists achieved, with just one embryo reaching the size of six cells before the cells stopped dividing. This is hardly the beginnings of a clone army.

At any rate, the cloning practiced at the Massachusetts firm was therapeutic cloning, involving the production of genetic material for the treatment of specific conditions. It is a far cry from reproductive cloning, which entails implanting a cloned embryo in a uterus—and even that is still a long way from the clichéd image of clones produced in a test tube without any parents other than the biological material used to create them.

Such ideas are related much more closely to those highlighted in Aldous Huxley's 1932 novel Brave New World than they are to scientific realities. And even if humans wanted to develop such technology, it would be many, many years in the future. As for "creating life," to do so is probably not even possible; if it is, such an achievement is about as far off as travel to another solar system. This is not to say that all fears of cloning and genetic engineering are unwarranted; on the contrary, it is good to have a healthy level of skepticism. But it is also good to be an equal-opportunity skeptic and therefore to question ideas in the popular culture—including opposition to genetic engineering.

WHERE TO LEARN MORE

Barash, David P. Revolutionary Biology: The New, Gene-Centered View of Life. New Brunswick, NJ: Transaction Publishers, 2001.

Chadwick, Ruth F. The Concise Encyclopedia of the Ethics of New Technologies. San Diego: Academic Press, 2001.

"Cloning." New Scientist (Web site). <http://www.newscientist.com/hottopics/cloning/>.

Department of Energy Human Genome Program (Web site). <http://www.ornl.gov/hgmis/>.

Genetic Engineering and Cloning: Improving Nature or Uncorking the Genie? (Web site). <http://library.thinkquest.org/19697/>.

Genetics Education Center, University of Kansas Medical Center (Web site). <http://www.kumc.edu/gec/>.

Hyde, Margaret O., and John F. Setaro. Medicine's Brave New World: Bioengineering and the New Genetics. Brookfield, CT: Twenty-First Century Books, 2001.

Judson, Karen. Genetic Engineering: Debating the Benefits and Concerns. Berkeley Heights, NJ: Enslow Publishers, 2001.

National Human Genome Research Institute (Web site). <http://www.nhgri.nih.gov>.

Twenty Facts About the Human Genome. Wellcome Trust (Web site). <http://www.wellcome.ac.uk/en/genome/thgfac.htm>.

Wade, Nicholas. Life Script: How the Human Genome Discoveries Will Transform Medicine and Enhance Your Health. New York: Simon & Schuster, 2001.

KEY TERMS

AMINO ACIDS:

Organic compounds made of carbon, hydrogen, oxygen, nitrogen, and (in some cases) sulfur bonded in characteristic formations. Strings of amino acids make up proteins.

BASE PAIR:

A pair of chemicals that form the "rungs" on a DNA molecule, which has the shape of a spiral staircase.

BIOTECHNOLOGY:

A name for the industry built around the application of genetic engineering techniques.

CHROMOSOME:

A DNA-containing body, located in the cells of most living things, that holds most of the organism's genes.

CLONE:

A cell, group of cells, or organism that contains genetic in formationidentical to that of its parent cell or organism.

CLONING:

A specialized genetic process whereby clones are produced. Cloning is a form of asexual reproduction.

DNA:

Deoxyribonucleic acid, a molecule in all cells, and many viruses, that contains genetic codes for inheritance.

ENZYME:

A protein material that speeds up chemical reactions in the bodies of plants and animals without itself taking part in or being consumed by those reactions.

GENE:

A unit of information about a particular heritable trait. Usually stored on chromosomes, genes contain specifications for the structure of a particular polypeptide or protein.

GENE SPLICING:

A process wherebyrecombinant DNA is formed by cutting a DNA strand in half lengthwise and joining it with a strand from another organism or perhaps even another species.

GENE THERAPY:

The introduction of normal or genetically altered genes to cells, typically to replace defective genes involved in genetic disorders.

GENE TRANSFER:

Incorporation of new DNA into an organism's cells, usually with the help of a micro organism that serves as a vector.

GENETIC DISORDER:

A condition, such as a hereditary disease, that can be traced to an individual's genetic makeup.

GENETIC ENGINEERING:

The alteration of genetic material by direct intervention in genetic processes with the purpose of producing new substances or improving functions of existing organisms.

NUCLEUS:

The control center of a cell, where DNA is stored.

PROTEINS:

Large molecules built from long chains of 50 or more amino acids. Proteins serve the functions of promoting normal growth, repairing damaged tissue, contributing to the body's immune system, and making enzymes.

rDNA:

Recombinant DNA, or DNA that has been genetically altered through gene splicing.

RESTRICTION ENZYMES:

Enzymes that break DNA into fragments at particular sites.

VECTOR:

In the context of genetics, a vector is a microorganism or virus that is used to transfer DNA from one organism to another.

Chapter 3
Genetic Engineering

Genes contain coded information that leads to the production of proteins. Proteins, in turn, are responsible for creating the traits that characterize individual organisms. Therefore, if a way could be found to transfer genes from one organism to another, creatures could be manufactured with traits that they had never before exhibited. Based on the description of the structure of DNA provided by Watson and Crick, researchers began to search for a way to cut genes from the DNA of one organism and paste them into another. By the 1970s, they had the answer, and the science of genetic engineering was born. It was a giant step forward. Now, a mere thirty years later, it is possible to exchange genes between one plant and another and one animal and another. It is even possible to transpose genes between plants and animals. No organism—from primitive life-forms, like bacteria, to higher order animals, like human beings—is exempt from this genetic swap meet. Genetic engineering has led to monumental advances in medicine and agriculture, but it has also given rise to a storm of controversy and debate over the limits on humankind's intrusion into the natural order of things.

Restriction Enzymes and Plasmids

The first major breakthrough on the road to genetic engineering came with work done on restriction endonucleases by Herbert Boyer of the University of California at San Francisco. As defined by Karl Drlica in Understanding DNA and Gene Cloning: A Guide for the Curious, restriction endonucleases "are a group of enzymes [a special type of protein] that . . . occur naturally in a large number of different bacterial species, serving as part of the natural defense mechanism that protects bacterial cells against invasion by foreign DNA molecules such as those contained in viruses."¹⁵

When, for example, a virus attacks a single-celled bacterium, restriction endonucleases are unleashed and go to work, cutting the invading DNA into small, nonthreatening pieces. "Crucial to this protective device is the ability of the nuclease to discriminate between its own DNA and the invading DNA; otherwise the cell would destroy its own DNA," Drlica says.

This recognition process involves two elements. First there are specific nucleotide sequences [As and Ts, Cs and Gs] that act as targets for the nuclease. These are called the restriction sites. Second, there is a protective chemical signal that can be placed by the cell on all the target sequences that happen to occur in its own DNA. The signal modifies the DNA and prevents the nuclease from cutting. Invading DNAs, lacking the protective signal, would be chopped by the nuclease.¹⁶

Thus, restriction enzymes have the remarkable ability to recognize specific arrangements of DNA base pairs—As and Ts, Gs and Cs. They also have the capacity to act like a molecular scalpel, severing the DNA at exactly the spot where they detect this sequence of genetic letters. Restriction enzymes are a powerful tool because there are thousands of them, and each one acts only on a unique arrangement of As and Ts and Cs and Gs.

A second piece of the genetic engineering puzzle fell into place when it was discovered that bacteria have another interesting property. Under the right conditions, small, circular pieces of DNA can be transferred from one bacterial cell to another. These DNA structures, called plasmids, are not located on the bacterium's solitary chromosome, but float freely in other parts of the organism. Single-cell bacteria duplicate when the cell divides, producing an exact copy of itself. During this process, its plasmids, as well as its chromosomal DNA, are also reproduced. "In 1959, Japanese doctors found that the ineffectiveness of antibiotics as a cure for dysentery with some patients was due to the fact that the bacteria with which the patients were infected carried a plasmid containing several genes of resistance to different antibiotics," says geneticist Maxim D. Frank-Kamenetskii. "It was discovered that genes of resistance to antibiotics are always carried by plasmids. An ability to move freely between bacteria enables the plasmids carrying such genes to spread rapidly among bacteria immediately in the wake of a broad application of the antibiotic."¹⁷

Recombinant DNA Technology

Boyer and Stanley Cohen, another scientist at the University of California who was working on plasmids, pooled their knowledge to conduct a series of experiments on two different strains of the E. coli bacteria. Some forms of E. coli live in the intestines of humans and other animals, where they aid the body's digestive processes. Boyer and Cohen marshaled restriction endonucleases to cut some E. coli plasmids. When plasmids are cut, they leave what researchers call "sticky ends," to which other plasmid segments can easily attach themselves. The point at which the pieces of the two plasmids join is cemented by the activity of an enzyme called ligase, which can be described as molecular glue, to form a stable chemical bond. Then, the two scientists severed particular genes from another type of bacteria, one that was resistant to antibiotics, and spliced them to the sticky ends of the cut E. coli plasmids. The result: a hybrid form of antibiotic-resistant
E. coli.

One big question remained to be answered. Thus far, genes had been successfully exchanged between two types of bacteria, but would the same cut-and-paste technique work when the genes came from two radically different life-forms? In other words, could genes cross species boundaries? Cohen's and Boyer's first attempt to transplant genes from one form of life into another involved a tadpole and E. coli bacteria. The scientists removed a gene from one of the tadpole's cells and transplanted it into an
E. coli bacterial cell. When the bacterial cell started to multiply, the scientists analyzed each successive generation and found that they all contained the tadpole gene. The first gene transfer between species had been accomplished, and the door was now open to a wide range of similar experiments—many of them far more controversial. It had been practically demonstrated that genes from fish, even genes from plants, could be transplanted into humans.

The new technique was called recombinant DNA technology—just another name for genetic engineering—because the procedure recombined genes that originated in different organisms. The popular media gave it another name that has been responsible for a great deal of confusion. They called it gene cloning, creating the belief that science could duplicate entire organisms, an achievement that was not at that point even distantly attainable. But what the technique did allow scientists to do was create specific types of proteins in large quantities. "Usually, a specific protein is produced by a cell in very small quantities, sometimes a mere one or two molecules per cell," says Frank-Kamenetskii.

As a result, the production of proteins needed for particular research was an arduous and costly undertaking. One had to process dozens of kilograms [a unit of weight equal to 2.2 pounds], nay tons, of biomass to obtain milligrams of protein.

Despite such meager quantities, it was still not possible to ensure the necessary purity of the protein. Hence, the cost of many protein preparations was exorbitant and their purity was substandard. Genetic engineering brought about a radical change in this situation. Genetic-engineering strains now exist—superproducers of many proteins with high standards of purity—that were undreamed of before. Molecular biology firms have sharply diversified the production of enzymes and other protein preparations and have reduced the prices of these products. Thus, molecular biology received a powerful new impetus, resulting in an unheard of acceleration in the pace of scientific research.¹⁸

In accomplishing this goal, bacteria, especially E. coli bacteria, have proved to be the most effective host for transplanted genes because they reproduce rapidly. For example, if scientists wish to produce a certain kind of protein, they snip the required genes from an animal that produces the protein naturally and transplant them into an E. coli cell. They then put the cell in an environment that encourages it to divide and just let nature take its course until they have millions of cells all producing the desired protein. Finally, the scientists extract the protein from the cells and use it for whatever purpose they have in mind.

Biofoods

The first area in which the new science of genetic engineering took hold was agriculture. It quickly became apparent that food plants could be genetically altered so they were more resistant to pests, needed less water to grow, and provided more nutrition than in their natural states. Since human beings first began to till the land, farmers have been trying to produce hardier, more profitable crops. The method at their disposal was called selective breeding. In the same way that Gregor Mendel bred pea plants to yield other pea plants with certain traits, agriculturists crossbred the healthiest plants with each other to create the most productive varieties possible. But the process took a long time and, because the relationship between genes and traits is complex, often led to unwanted results.

Genetic engineering takes the guesswork out of this effort and greatly reduces the time it takes to produce a plant with the desired traits. It also—for the first time—makes it possible to breed entirely different types or species of plants with each other to create some truly novel hybrids. Previously, selective breeding limited farmers to experiments with plants of the same or very closely related species. By cutting and pasting genes from one plant to another, genetic engineers are able to do all the things that crossbreeding can do, and do them faster and more accurately.

The technique also allows scientists to do things that nature alone is incapable of doing. A report prepared at University of Virginia on the state of currently available genetically engineered, or transgenic, plants states:

Many plants have been commercialized, including tomatoes and squash and commodity crops like corn and soybeans. Most have been engineered for one of three traits: herbicide [weed killer] tolerance, insect resistance, or virus tolerance. This is the fastest growing area of biotechnology in agriculture. Genetically engineered cotton has been approved for commercial use. There are between 10 and 12 million acres of cotton in the U.S. and estimates are that all of this acreage will be planted to transgenic varieties within the next 10 years. One of the newest innovations in cotton is the development of naturally-colored cotton fibers where the pigments have come from inserting color genes from flowers into cotton.¹⁹

As a further indication of the widespread use of genetic engineering in farming, the Food and Agriculture Organization of the United Nations, citing figures for the year 1999 (the process of gathering such information accurately is slow when developing countries are involved), reports:

Transgenic plants . . . now cover large areas in certain parts of the world. Estimates for 1999 indicate that 39.9 million hectares [a unit of measurement equivalent to 2.47 acres] were planted with transgenic crops. . . . Of the 39.9 million hectares, 28.1 million (i.e. 71%) were modified for tolerance to a specific herbicide (which could be sprayed on the field, killing weeds while leaving the crop undamaged); 8.9 million hectares (22%) were modified to include a toxin-producing gene from a soil bacterium . . . which poisons insects feeding on the plants, while 2.9 million hectares (7%) were planted with crops having both herbicide tolerance and insect resistance.²⁰

The bioengineering of plants has become big business. Hundreds of millions of dollars of research money are being poured into a diverse range of projects. Among the most promising are creating plants that produce their own fertilizer and modifying plants to be delivery systems for medicines and nutrients they do not naturally produce. For example, work is under way to produce a banana that contains in its DNA a vaccine for hepatitis B, a highly contagious disease that damages the livers of people who contract it. The banana is also being turned into a megavitamin to deliver much-needed nutrients to children in the underdeveloped countries of Africa and Asia.

Transgenic Animals

Plants are not the only organisms that genetic engineers are working on. Turning their attention to animals, scientists have produced a number of transgenic creatures they hope will bring major benefits to mankind. For example, human genes have been put into pigs to allow the pigs to produce human insulin, a substance needed to control diabetes, one of the fastest-growing diseases in affluent countries.

The applications are wide-ranging. Goats are being genetically modified to produce a protein that aids in blood clotting. Other experiments with goats aim to find cures for multiple sclerosis and some forms of cancer. Sheep are being altered to generate a protein that may fight the lung disease emphysema. Designer dairy products are also on the drawing boards. Geneticists hope to end up with a breed of cow that produces, for example, only low-fat milk.

Other genetically engineered animals are being designed to contract human diseases so that experimental treatments can be explored. In these cases, healthy genes are replaced with malfunctioning counterparts, using a technique similar to the cut-and-paste procedure used with plasmids.

Finally, researchers are optimistic that they will be able to turn animals, principally pigs, into sources of organs for human transplants. To accomplish this, they are transferring human genes into pigs so that the resulting organs will more closely resemble those found in humans and thus be less likely to be rejected.

Cloning

Work on transgenic animals has also led to the cloning of entire organisms. A clone is an identical genetic copy of an organism—its DNA is the same as that of the original from which the copy was made. In humans, identical twins are naturally occurring clones. In these cases, the original fertilized egg divides into two genetically identical halves and proceeds to develop into two distinct babies. Since the babies originated from the same egg fertilized by the same sperm, they have exactly the same DNA. By contrast, fraternal twins come from two separate eggs, each of which is fertilized by a different sperm cell. Even though these children are born at the same time, they are as genetically different from each other as any other pair of siblings born years apart would be.

The cloning of organisms must be carefully distinguished from the cloning of genes—a distinction that the popular media have not always succeeded in making. The cloning of single genes, using plasmids, is an established procedure; the cloning of organisms is still experimental and highly controversial. Although several fringe groups claim to have successfully cloned human beings, they have failed—as of the writing of this book—to produce any evidence to support their contentions.

To this date, the most famous cloned mammal remains Dolly the sheep. A close look at how Dolly was created will provide a good description of the techniques required to clone any higher animal, including humans. In announcing Dolly's birth in 1997, Scientific American magazine reported:

Dolly, unlike any other mammal that has ever lived, is an identical copy of another adult and has no father. She is a clone, the creation of a group of veterinary researchers. That work, performed by Ian Wilmut and his colleagues at the Roslin Institute in Edinburgh, Scotland, has provided an important new research tool and has shattered a belief widespread among biologists that cells from adult mammals cannot be persuaded to regenerate a whole animal.²¹

Previously, researchers had cloned mammals and other animals using embryonic cells as a starting point. Embryonic cells, taken from an undeveloped fertilized egg, are different from adult cells in that they are undifferentiated. When an egg cell is fertilized, it starts to divide. Up to a certain point, the cells in each succeeding generation have the ability to develop into specialized cells that will make up the various parts (organs, bones, skin, etc.) of the mature organism. After that point, cells become differentiated, or specialized—some of them begin to turn into liver cells, others into brain cells, and so on. Until Dolly, it was thought that clones could be produced only from undifferentiated cells that would divide and grow to maturity as the cloned organism developed.

Promise and Problems of Cloning

Dolly, however, was cloned from a cell taken from the udder of a six-year-old female sheep, a fully developed adult. Dolly was the 277th attempt made by Wilmut and his fellow researchers. The other attempts had failed, but the same technique was used in all of them. "Wilmut and his co-workers accomplished their feat by transferring the nuclei from various types of sheep cells into unfertilized sheep eggs from which the natural nuclei had been removed by microsurgery," the Scientific American article continues.

Once the transfer was complete, the recipient eggs contained a complete set of genes, just as they would do if they had been fertilized by sperm. The eggs were then cultured for a period before being implanted into sheep that carried them to term, one of which culminated in a successful birth. The resulting lamb was, as expected, an exact genetic copy, or clone, of the sheep that provided the transferred nucleus, not of [the sheep] that provided the egg.²²

Wilmut used a pipette many times thinner than a human hair to remove the DNA from the host egg. Then the empty egg was placed next to a cell taken from the donor sheep's udder and the two were fused together using a tiny jolt of electricity. Another pulse of electricity caused the egg cell, with its new DNA, to start dividing. The cell was now behaving just like a normal egg cell would if it had been fertilized by sperm from a male sheep. It was cultured for a few days in a laboratory dish and then implanted into the uterus of a third sheep, which carried it to term and gave birth to Dolly.

From the beginning, Wilmut and other geneticists were concerned that since Dolly's DNA came from a six-year-old sheep she might age prematurely. At first, she seemed to be perfectly healthy and gave birth to a lamb of her own in 1998. But then Dolly began to develop medical problems frequently associated with aging. "Early in life Dolly had a weight problem. Then in 1999, it emerged that caps at her chromosome ends called telomeres, which get shorter each time a cell divides, were 20 percent shorter than was normal for a sheep her age," says science journalist John Whitfield, writing in the journal Nature. "This led to speculation that Dolly's biological age might equal that of her and her mother combined."²³

In 2002, Dolly was diagnosed with arthritis, another disease associated with old age, and then she came down with lung cancer. Dolly was humanely put to death at the age of six and a half years, half the normal life span of a sheep of her kind. Cloned animals since Dolly, including cows, rabbits, mice, cats, goats, and pigs, have experienced similar problems. "Dolly's premature death is typical of cloned animals," Whitfield writes.

From conception onwards, clones suffer a higher mortality rate than non-clones. Studies in mice seem to show that this bad health persists throughout life. Some seized upon Dolly's ailments as evidence that clones are invariably sickly and age prematurely. Although it can't be ruled out that her origins made her less robust than other sheep, it is not possible to make generalizations about clones' health from the fate of a single animal. . . . But the process of genetic reprogramming seems too complex and haphazard to control tightly, and its success rate has not improved much since Dolly's day.²⁴

Although many questions about animal cloning remain to be answered, scientists are hopeful that, as with genetically engineered plants, there are many benefits in store. Among these are creating oversized cattle to improve beef yield and the production of stem cells, multipurpose cells found in embryos, which may prove to have a wide range of medical applications. Like transgenic plants, transgenic animals can be created to produce greater amounts of nutrients, and some scientists also claim that cloning can be used to preserve endangered species from extinction.

The Human Genome Project

The human genome is composed of about 3 billion base pairs of As and Ts, Gs and Cs. In 1990, an international consortium of scientists set out to create a map that would show exactly where on our twenty-three pairs of chromosomes every one of those base pairs is located. The effort, called the Human Genome Project, is the most extensive scientific enterprise ever undertaken.

Genetic mapping is the first step in isolating a gene. There are two basic approaches to this complex endeavor. The first is called linkage mapping, and its goal is to show where on each chromosome each gene is located relative to other genes. The method is to compare the genetic makeup of members of a family or closely related group of people who have a history of a specific disease. By finding similar base pairs on the chromosomes of family members in succeeding generations of this group who have the disease, scientists can isolate the responsible gene and begin to build a map of the entire genome. The second type of map is far more ambitious. It is called a physical map and its goal is to show the absolute (not relative) location of the base pairs that make up every gene.

In both cases, the cut-and-paste techniques developed by genetic engineering are indispensable. Restriction enzymes are used to cut the chromosomes into small segments, which are then cut into even smaller pieces until the sought-after gene is found. It is a painstaking process, made possible not only by the methods of genetic engineering, but also by powerful supercomputers that enable researchers to compare various overlapping segments to weed out duplicate base pairs and base pairs that appear not to play any role in the process of genetic inheritance. Only about 2 percent of the 3 billion base pairs actually make up functional genes. The rest, called junk DNA, help to locate the genes and may play other roles that remain to be determined.

The Human Genome Project was virtually completed in the year 2003. It yielded many interesting—and surprising—insights into the genetic composition of human beings. Among them: the total number of genes in a human being lies between 30,000 and 35,000, far fewer than earlier estimates of 80,000 to 140,000; the average gene consists of about 3,000 base pairs, but sizes vary greatly, the longest being 2.4 million base pairs; 99.9 percent of base pairs are identical in all people; the function of more than 50 percent of human genes has not yet been determined. While work continues on tracking down the roles played by these mystery genes, the next step is the mapping of the human proteome—the complete array of proteins operating in the human body. This includes pinpointing all the proteins coded for by the genes and describing the specific role these proteins play in the human organism.

GENETIC ENGINEERING

GENETIC ENGINEERING is the deliberate manipulation of an organism's genetic makeup to achieve a planned and desired result. Proponents of genetic engineering consider it an extension of the selective breeding practiced for thousands of years in the domestication of agricultural products and animals. The genesis of modern biotechnology, most scholars agree, came in the early 1970s with the advent of recombinant DNA (rDNA). Since biotechnology often refers to the use of organisms in agriculture, industry, or medicine, its origins can be traced back to the use of yeast for baking bread and the fermentation of alcohol. The impact of contemporary genetic engineering and biotechnology affects nearly every area of human activity. The introduction of rDNA engineering has revolutionized our relationship to the organic world and to ourselves, demanding a reconsideration of our values, our notion of progress, and the morality of scientific research.

The History of Genetic Engineering

Genetic engineering owes its existence to the developments in molecular genetics, virology, and cytology that culminated in the determination of the structure of DNA by James Watson and Francis Crick in 1953. Building on research involving bacteriophages (a bacterial virus), Joshua Lederberg, a geneticist at the University of Wisconsin, found that bacteria can transfer genetic information through plasmids, small mobile pieces of DNA that exist independent of the chromosomes. In the 1950s, Lederberg pioneered the earliest techniques in genetic engineering, shuffling genetic material between bacterial cells. After the identification of restriction enzymes capable of "cutting" DNA in specific locations in 1968, scientists were able to insert foreign DNA directly into bacterial cells. The discovery that the foreign DNA would naturally bond with the host DNA, made it possible to splice together genes from multiple organisms, the technique used in recombinant DNA engineering. Although highly complicated, rDNA engineering can be simply explained: genetic material from the donor source is isolated and "cut" using a restriction enzyme and then recombined or "pasted" into the genetic material of the receiver. By 1971, advanced transplantation techniques had been developed and rDNA techniques using the restriction enzyme EcoRi were operable the following year, leading to the first experiments in genetic engineering.

In 1973, Stanford biochemist Stanley Cohen under-took one of the first rDNA experiments, inserting a piece of bacterial DNA into Escherichia coli (E. coli), a bacterium found in the human intestine. However, the research soon became controversial, particularly when American molecular biologist Paul Berg designed an experiment to insert DNA from simian virus #40 (sv40)—a known cancer-causing agent—into E. coli. As word of the daring procedure spread, the public was captivated and fearful, afraid that a genetically engineered virus, inured to antibiotics and carried in a common bacterium, could escape and cause an epidemic. Hoping to diffuse fears of a potential biohazard and maintain control of their research, over one hundred and fifty molecular biologists and related specialists met at the Asilomar Conference Center in Monterey, California, in late February 1975. The conference represented an extraordinary moment in the history of science, as the research community, recognizing its social responsibility, officially adopted a moratorium until appropriately safe procedures and guidelines could be developed. The conference ultimately resulted in the "National Institutes of Health Guidelines for Research Involving rDNA Molecules" and an ongoing National Institute of Health rDNA Advisory Committee (RAC)founded in 1974.

Yet the guidelines only increased public concern over genetic engineering. Critics charged that attempts to splice genes together from different organisms were akin to "playing God" and could result in dangerous and immoral hybrids. Adopting the literary example of "Dr. Frankenstein's monster" as an appropriate symbol of misguided science, opponents of rDNA engineering converged on research laboratories and public meetings. An attempt to build a recombinant laboratory at Harvard University set off such a firestorm that local politicians created a review board to assess potential risks, eventually requiring more stringent controls than those set by the NIH. By 1977, protests of rDNA facilities had spread to other campuses—the University of California San Diego, the University of Wisconsin, the University of Michigan, and the University of Indiana—while the state legislatures of New York, New Jersey, and California held public hearings. However, it was the resolution of an old court case and the introduction of a new form of rDNA engineering that ultimately created the greatest controversy.

In a monumental decision handed down on 16 June 1980, the United States Supreme Court held in Diamond v. Chakrabarty that man-made life forms were subject to patent laws and protection. The decision resolved a longstanding issue on patents and organic material, as the case dated to 1972, when Ananda Chakrabarty, a researcher at General Electric, applied for a patent on a form of Pseudomonas bacteria bred (but not genetically engineered)to digest oil slicks. By a narrow five to four margin the court construed the Patent Act, originally drafted by Thomas Jefferson, so as to include all products of human invention, relying on a 1952 Senate report that recognized as patentable "anything under the sun that is made by man." More than any other single event, the ruling galvanized many mainstream religious communities and environmental groups, eventually resulting in a letter of protest to President Carter and an indepth review by the President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research (1980–1983). The commission's report, issued in 1982 and entitled Splicing Life: The Social and Ethical Issues of Genetic Engineering with Human Beings, emphasized the importance of rDNA engineering to biomedical progress and American industries, arguing that it was best that the research be conducted under the auspices of government regulation and control. However, while the study resolved anxiety over rDNA engineering and patenting, proponents of genetic engineering still had to address concerns over the development of "germ-line" engineering, a controversial procedure that allowed scientists to literally create new strains of organisms.

Germ-line engineering differs from rDNA engineering in that the donor genes are inserted into a "germ," or reproductive cell, thereby permanently altering the genetic makeup of the organism's descendants. For example, in 1982, Ralph Brinster of the University of Pennsylvania Veterinary School inserted the gene that produces rat growth hormone into mouse embryos. The resulting strain of mice, dubbed "super mice" by the press, expressed the gene and thus grew into a substantially larger and more powerful new breed of mouse. Critics of germ-line engineering quickly denounced the technique as immoral and argued it was a form of "anthropomorphic Lamarckism." Jean-Baptiste de Lamarck, a nineteenth-century French naturalist, had proposed that traits acquired during an organism's lifetime were passed on to its progeny—an idea refuted by Darwinian evolutionary theory. Yet, in germ-line engineering, traits acquired during the organism's lifetime are passed on, but only those traits deemed necessary or desirous by man. Environmental groups also denounced germ-line engineering because of "biosafety" concerns, fearing that genetically engineered species, which would possess a distinct advantage over nonengineered species, could upset the globe's finely tuned ecological systems. However, because most politicians, scientists, and manufacturers believed the potential benefits from rDNA and germ-line engineering outweighed its potential dangers, the protests were overshadowed by the development of a biotechnology industry based on genetic engineering.

Contemporary Applications of Genetic Engineering

The decision to allow patents on genetically engineered organisms, combined with the commission's sanction of rDNA engineering, and a national commitment to biomedical progress, led to tremendous growth in the biotechnology industry. In 1975, only five biotech companies participated in the Asilomar conference, by 1980 the number of similar companies had increased to one hundred. Today there are over 1,300 companies involved in genetic engineering, many of which are located in the United States, a clear indication of the rapid growth of the American biotechnology sector and the applicability of the powerful new techniques. Indeed, genetic engineering influences nearly every area of human activity, including agriculture and aquaculture, industry and environmental remediation, and the development of medicines and therapies.

Although agriculture has been one of the most successful industries in utilizing genetic engineering, the techniques have also made an impact in other areas of food production. In 1990, Chymosin, an enzyme necessary for cheese production, became the first genetically engineered food product to go to market. A few years later, in 1994, the Monsanto Company created a bovine growth hormone designed to stimulate milk production, a hormone now estimated to be given to 30 percent of dairy cows. The same year, the "Flavr-Savr" tomato developed by Calgene passed the Food and Drug Administration standards for genetically engineered foods and also went to market. Like many transgenic foods, the "Flavr-Savr" was designed to have increased shelf life and resist spoilage, although disputes regarding labeling and advertisements combined with high production costs caused the company to discontinue the product in the late 1990s. Nonetheless, genetic engineering is integrated into agriculture production; researchers estimate that as of 2001, nearly one-third of the corn and one half of the soybeans grown in the United States were transgenic. A study conducted in 2000 by the Grocery Manufacturers of America reported that the majority of processed foods sold in America contained transgenic ingredients. To help develop aquaculture, researchers at Johns Hopkins University have taken a gene from flounder and inserted it into both trout and bass in the hopes of making the fish more resistant to cold climates, thus increasing commercial and sport fishing.

Genetic engineering also has substantial applications in many other industries from plastics and energy to the new field of bioremediation. In 1993, Chris Sommerville, director of plant biology at the Carnegie Institute in Washington, D. C., successfully inserted plastic-making genes into a plant; the Monsanto Company hopes to market a cotton/polyester plant early in the twenty-first century. Scientists at numerous biotech companies are currently working on strains of E. coli bacteria capable of transforming agricultural refuse into ethanol, an efficient and clean source of energy. Genetic engineering is also aiding environmental clean-up through the emerging field of bioremediation—the use of organisms to reduce waste. Bacteria were employed to help with the Exxon Valdez oil spill in 1989, while scientists at the Institute for Genomic Research are among those hoping to engineer microbes that can detoxify waste, including radioactive materials. However, the fastest growing, and one of the most controversial, fields of biotechnology is applied human genetics, which includes transgenic medicines, xenotransplantation, and human gene therapy.

In 1982, Eli Lilly and Company began marketing bacterial-produced insulin, the first transgenic commercial product and an excellent marker of the industry's progress. Today, the vast majority of insulin used by Americans diabetics is genetically engineered and over 300 transgenic proteins and medicines are currently in production, many of which are made by animals. Indeed, animal "pharming" has been central to biomedical research and development since the introduction of genetic engineering; in 1988, Harvard University patented the "oncomouse," strains of mice missing or carrying specific genes and used in cancer research. In 1996, Genzyme Transgenics created a goat capable of producing anti-thrombin, an experimental anticancer drug; the following year PPL Therapeutics engineered a calf whose milk contains proteins necessary for nursing babies, including those born prematurely. Human hemoglobin, a protein essential for oxygen transportation in the bloodstream, can now be harvested from genetically engineered pigs. Transgenic pigs are also used in xenotransplantation, the transference of organs or parts from nonhuman species to humans. Nextran, a leading biotech company, hopes to use genetically engineered pig livers as temporary external reservoirs for patients suffering from acute liver failure. In the future, researchers hope that these transgenic medicines and proteins will help supplement human gene therapy, one of the boldest and most ethically and medically problematic areas of genetic engineering.

The history of human gene therapy is one of great promise and success mixed with controversy and stringent regulation. In the early 1980s, Martin Cline, a medical researcher at the University of California in Los Angeles, performed rDNA procedures in Italy and Israel on patients afflicted with hereditary blood disorders. Cline's unauthorized experimentation, although legal because the countries lacked genetic regulations, ultimately cost him funding and a department chairmanship. In response, the RAC established the Human Gene Therapy Subcommittee in 1984 to issue protocols and review applications. Years later, in 1990, researchers at the National Institute of Health (NIH)attempted the first approved human gene therapy for Ashanti DeSilva, a young girl forced to live inside a "bubble" because of severe combined immune deficiency, or ADA. As in most cases of human gene therapy, the researchers removed cells from the patient, genetically engineered the desired changes, and then replaced the cells. However, for ADA, as for most diseases, gene therapy offers only treatment, not a cure, as the procedure must be repeated periodically. Nonetheless, the success of Ashanti's procedure stimulated human gene therapy research; in 1992, Bernadine Healy, then director of the NIH, approved a "compassionate use exemption" to increase access to promising gene therapy trials for critically ill patients. Within a year, procedures had been approved for familial hypercholesterolemia, cystic fibrosis, and Gaucher's disease, and trials for cancer, AIDS, Parkinson's, Alzheimers, arthritis, and heart disease were being conducted. Unfortunately, the 1999 death from liver disease of Jesse Gelsinger, an eighteen-year-old student taking part in a University of Pennsylvania gene therapy trial, led to questions regarding the safety of established protocols, as the fatality resulted from a common immune reaction to the adenovirus vector (see Genetics) that the researchers could have easily anticipated.

Although genetic engineering remains in its infancy, the rapid development of the science and its related techniques has generated considerable disagreement in the attempt to address its moral and legal implications. The birth of the sheep "Dolly" in 1997, the first cloned adult mammal, led to debates over the sanctity of life and the individual, while the advent of human gene therapy has revived fears of eugenics programs and genetically engineered "designer" children. The marketing of transgenic foods stimulated the growth of an "organic" agricultural industry and created ongoing international disputes over patent rights, truth-in-labeling claims, and restrictions on genetically engineered imports. Some critics fear that xenotransplantation will promote the transference of animal diseases to humans, while others decry the use of animals simply for the benefit of mankind. The development of stem cell research, promising because the embryonic cells can be manipulated to become nearly any type of cell in the body, has led to protests by many pro-life organizations over the use of embryonic or fetal tissue; in August 2001, President Bush declared that only a limited number of cell lines were acceptable for federal research funding. Whether involved in human gene therapy, xenotransplantation, industry, or agriculture, genetic engineering and biotechnology will no doubt continue providing astounding advancements alongside heated controversy and debate well into the future.

BIBLIOGRAPHY

Bud, Robert. The Uses of Life: A History of Biotechnology. New York: Cambridge University Press, 1993.

Fiechter, A., ed. History of Modern Biotechnology. 2 vols. Berlin: Springer Verlag, 2000.

Rifkin, Jeremy. The Biotech Century: Harnessing the Gene and Remaking the World. New York: Putnam, 1998.

Shannon, Thomas A., ed. Genetic Engineering: A Documentary History. Westport, Conn. : Greenwood Press, 1999.

Von Wartburg, Walter P., and Julian Liew. Gene Technology and Social Acceptance. Lanham, Md. : University Press of America, 1999.

J. G.Whitesides

See alsoMicrobiology ; Molecular Biology .

Genetic engineering

Genetic engineering is any process by which genetic material (the building blocks of heredity) is changed in such a way as to make possible the production of new substances or new functions. As an example, biologists have now learned how to transplant the gene that produces light in a firefly into tobacco plants. The function of that gene—the production of light—has been added to the normal list of functions of the tobacco plants.

The chemical structure of genes

Genetic engineering became possible only when scientists had discovered exactly what is a gene. Prior to the 1950s, the term gene was used to stand for a unit by which some genetic characteristic was transmitted from one generation to the next. Biologists talked about a "gene" for hair color, although they really had no idea as to what that gene was or what it looked like.

That situation changed dramatically in 1953. The English chemist Francis Crick (1916– ) and the American biologist James Watson (1928– ) determined a chemical explanation for a gene. Crick and Watson discovered the chemical structure for large, complex molecules that occur in the nuclei of all living cells, known as deoxyribonucleic acid (DNA).

DNA molecules, Crick and Watson announced, are very long chains or units made of a combination of a simple sugar and a phosphate group.

Words to Know

Amino acid: An organic compound from which proteins are made.

DNA (deoxyribonucleic acid): A large, complex chemical compound that makes up the core of a chromosome and whose segments consist of genes.

Gene: A segment of a DNA molecule that acts as a kind of code for the production of some specific protein. Genes carry instructions for the formation, functioning, and transmission of specific traits from one generation to another.

Gene splicing: The process by which genes are cut apart and put back together to provide them with some new function.

Genetic code: A set of nitrogen base combinations that act as a code for the production of certain amino acids.

Host cell: The cell into which a new gene is transplanted in genetic engineering.

Human gene therapy (HGT): The application of genetic engineering technology for the cure of genetic disorders.

Nitrogen base: An organic compound consisting of carbon, hydrogen, oxygen, and nitrogen arranged in a ring that plays an essential role in the structure of DNA molecules.

Plasmid: A circular form of DNA often used as a vector in genetic engineering.

Protein: Large molecules that are essential to the structure and functioning of all living cells.

Recombinant DNA research (rDNA research): Genetic engineering; a technique for adding new instructions to the DNA of a host cell by combining genes from two different sources.

Vector: An organism or chemical used to transport a gene into a new host cell.

Attached at regular positions along this chain are nitrogen bases. Nitrogen bases are chemical compounds in which carbon, hydrogen, oxygen, and nitrogen atoms are arranged in rings. Four nitrogen bases occur in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T).

The way in which nitrogen bases are arranged along a DNA molecule represents a kind of genetic code for the cell in which the molecule occurs. For example, the sequence of nitrogen bases T-T-C tells a cell that it should make the amino acid known as lysine. The sequence C-C-G, on the other hand, instructs the cell to make the amino acid glycine.

A very long chain (tens of thousands of atoms long) of nitrogen bases tells a cell, therefore, what amino acids to make and in what sequence to arrange those amino acids. A very long chain of amino acids arranged in a particular sequence, however, is what we know of as a protein. The specific sequence of nitrogen bases, then, tells a cell what kind of protein it should be making.

Furthermore, the instructions stored in a DNA molecule can easily be passed on from generation to generation. When a cell divides (reproduces), the DNA within it also divides. Each DNA molecule separates into two identical parts. Each of the two parts then makes a copy of itself. Where once only one DNA molecule existed, now two identical copies of the molecule exist. That process is repeated over and over again, every time a cell divides.

This discovery gave a chemical meaning to the term gene. According to our current understanding, a specific arrangement of nitrogen bases forms a code, or set of instructions, for a cell to make a specific protein. The protein might be the protein needed to make red hair, blue eyes, or wrinkled skin (to simplify the possibilities). The sequence of bases, then, holds the code for some genetic trait.

Gene splicing

The Crick-Watson discovery opened up unlimited possibilities for biologists. If genes are chemical compounds, then they can be manipulated just as any other kind of chemical compound can be manipulated. Since DNA molecules are very large and complex, the actual task of manipulation may be difficult. However, the principles involved in working with DNA molecule genes is no different than the research principles with which all chemists are familiar.

For example, chemists know how to cut molecules apart and put them back together again. When these procedures are used with DNA molecules, the process is known as gene splicing. Gene splicing is a process that takes place naturally all the time in cells. In the process of division or repair, cells routinely have to take genes apart, rearrange their components, and put them back together again.

Scientists have discovered that cells contain certain kinds of enzymes that take DNA molecules apart and put them back together again. Endonucleases, for example, are enzymes that cut a DNA molecule at some given location. Exonucleases are enzymes that remove one nitrogen base unit at a time. Ligases are enzymes that join two DNA segments together.

It should be obvious that enzymes such as these can be used by scientists as submicroscopic scissors and glue with which one or more DNA molecules can be cut apart, rearranged, and the put back together again.

Genetic engineering procedures

Genetic engineering requires three elements: the gene to be transferred, a host cell into which the gene is inserted, and a vector to bring about the transfer. Suppose, for example, that one wishes to insert the gene for making insulin into a bacterial cell. Insulin is a naturally occurring protein made by cells in the pancreas in humans and other mammals. It controls the breakdown of complex carbohydrates in the blood to glucose. People whose bodies have lost the ability to make insulin become diabetic.

The first step in the genetic engineering procedure is to obtain a copy of the insulin gene. This copy can be obtained from a natural source

(from the DNA in a pancreas, for example), or it can be manufactured in a laboratory.

The second step in the process is to insert the insulin gene into the vector. The term vector means any organism that will carry the gene from one place to another. The most common vector used in genetic engineering is a circular form of DNA known as a plasmid. Endonucleases are used to cut the plasmid molecule open at almost any point chosen by the scientist. Once the plasmid has been cut open, it is mixed with the insulin gene and a ligase enzyme. The goal is to make sure that the insulin gene attaches itself to the plasmid before the plasmid is reclosed.

The hybrid plasmid now contains the gene whose product (insulin) is desired. It can be inserted into the host cell, where it begins to function just like all the other genes that make up the cell. In this case, however, in addition to normal bacterial functions, the host cell also is producing insulin, as directed by the inserted gene.

Notice that the process described here involves nothing more in concept than taking DNA molecules apart and recombining them in a different arrangement. For that reason, the process also is referred to as recombinant DNA (rDNA) research.

Applications of genetic engineering

The possible applications of genetic engineering are virtually limitless. For example, rDNA methods now enable scientists to produce a number of products that were previously available only in limited quantities. Until the 1980s, for example, the only source of insulin available to diabetics was from animals slaughtered for meat and other purposes. The supply was never large enough to provide a sufficient amount of affordable insulin for everyone who needed insulin. In 1982, however, the U.S. Food and Drug Administration approved insulin produced by genetically altered organisms, the first such product to become available.

Since 1982, the number of additional products produced by rDNA techniques has greatly expanded. Among these products are human growth hormone (for children whose growth is insufficient because of genetic problems), alpha interferon (for the treatment of diseases), interleukin-2 (for the treatment of cancer), factor VIII (needed by hemophiliacs for blood clotting), erythropoietin (for the treatment of anemia), tumor necrosis factor (for the treatment of tumors), and tissue plasminogen activator (used to dissolve blood clots).

Genetic engineering also promises a revolution in agriculture. Recombinant DNA techniques enable scientists to produce plants that are resistant to herbicides and freezing temperatures, that will take longer to ripen, and that will manufacture a resistance to pests, among other characteristics.

Today, scientists have tested more than two dozen kinds of plants engineered to have special properties such as these. As with other aspects of genetic engineering, however, these advances have been controversial. The development of herbicide-resistant plants, for example, means that farmers are likely to use still larger quantities of herbicides. This trend is not a particularly desirable one, according to some critics. How sure can we be, others ask, about the risk to the environment posed by the introduction of "unnatural," engineered plants?

The science and art of animal breeding also are likely to be revolutionized by genetic engineering. For example, scientists have discovered that a gene in domestic cows is responsible for the production of milk. Genetic engineering makes it possible to extract that gene from cows who produce large volumes of milk or to manufacture that gene in the laboratory. The gene can then be inserted into other cows whose milk production may increase by dramatic amounts because of the presence of the new gene.

Human gene therapy

One of the most exciting potential applications of genetic engineering involves the treatment of human genetic disorders. Medical scientists know of about 3,000 disorders that arise because of errors in an individual's DNA. Conditions such as sickle-cell anemia, Tay-Sachs disease, Duchenne muscular dystrophy, Huntington's chorea, cystic fibrosis, and Lesch-Nyhan syndrome result from the loss, mistaken insertion, or change of a single nitrogen base in a DNA molecule. Genetic engineering enables scientists to provide individuals lacking a particular gene with correct copies of that gene. If and when the correct gene begins functioning, the genetic disorder may be cured. This procedure is known as human gene therapy (HGT).

The first approved trials of HGT with human patients began in the 1980s. One of the most promising sets of experiments involved a condition known as severe combined immune deficiency (SCID). Individuals with SCID have no immune systems. Exposure to microorganisms that would be harmless to the vast majority of people will result in diseases that can cause death. Untreated infants born with SCID who are not kept in a sterile bubble become ill within months and die before their first birthday.

In 1990, a research team at the National Institutes of Health (NIH) attempted HGT on a four-year-old SCID patient. The patient received about one billion cells containing a genetically engineered copy of the gene that his body lacked. Another instance of HGT was a procedure, approved in 1993 by NIH, to introduce normal genes into the airways of cystic fibrosis patients. By the end of the 1990s, according to the NIH, more than 390 gene therapy studies had been initiated. These studies involved more than 4,000 people and more than a dozen medical conditions.

In 2000, doctors in France claimed they had used HGT to treat three babies who suffered from SCID. Just ten months after being treated, the babies exhibited normal immune systems. This marked the first time that HGT had unequivocally succeeded.

Controversy remains. Human gene therapy is the source of great controversy among scientists and nonscientists alike. Few individuals maintain that the HGT should not be used. If we could wipe out sickle cell anemia, most agree, we should certainly make the effort. But HGT raises other concerns. If scientists can cure genetic disorders, they can also design individuals in accordance with the cultural and intellectual fashions of the day. Will humans know when to say "enough" to the changes that can be made with HGT?

Despite recent successes, most results in HGT since the first experiment was conducted in 1990 have been largely disappointing. And in 1999, research into HGT was dealt a blow when an eighteen-year-old from Tucson, Arizona, died in an experiment at the University of Pennsylvania. The young man, who suffered from a metabolic disorder, had volunteered for an experiment to test gene therapy for babies with a fatal form of that disease. Citing the spirit of this young man, researchers remain optimistic, vowing to continue work into the possible lifesaving opportunities offered by HGT.

The commercialization of genetic engineering

The commercial potential of genetically engineered products was not lost on entrepreneurs in the 1970s. A few individuals believed that the impact of rDNA on American technology would be comparable to that of computers in the 1950s. In many cases, the first genetic engineering firms were founded by scientists involved in fundamental research. The American biologist Herbert Boyer, for example, teamed up with the venture capitalist Robert Swanson in 1976 to form Genentech (Genetic Engineering Technology). Other early firms like Cetus, Biogen, and Genex were formed similarly through the collaboration of scientists and businesspeople.

The structure of genetic engineering (biotechnology) firms has, in fact, long been a source of controversy. Many observers have questioned the right of a scientist to make a personal profit by running companies that benefit from research that had been carried out at publicly funded universities. The early 1990s saw the creation of formalized working relations between universities, individual researchers, and the corporations founded by these individuals. Despite these arrangements, however, many ethical issues remain unresolved.

[See also Birth defects; Chromosome; Diabetes mellitus; Gene; Genetic disorders; Genetics; Human Genome Project; Nucleic acid ]

Genetic Engineering

Genetic engineering is the altering of an organism's deoxyribonucleic acid (DNA) to create a desired effect. Genetic engineers follow a set of techniques that allows them to remove genetic material from two or more species, recombine the genetic material (to create recombinant DNA), and integrate it into a host's genome, or genetic material. Genetic engineering is used for a variety of scientific, agricultural, and medical purposes.

Restriction Enzymes

All recombinant DNA technology requires the use of restriction enzymes. These enzymes are naturally occurring in bacteria that fight phage, or virus, DNA, but geneticists can use restriction enzymes as tools to cut DNA into manageable fragments.

Restriction enzymes recognize specific sequences along the DNA where it can be cut. These sequences contain four or more bases and occur randomly, and each enzyme cuts at a different sequence. Most restriction enzymes cut double-stranded DNA in a staggered fashion, so that a single strand extends from each end. These complementary "sticky" ends tend to bond to each other in solution.

Geneticists often need to isolate specific DNA molecules from a mixture. Restriction enzymes cut DNA into many small fragments. These fragments can be run through an electrophoretic gel, which distributes them according to size. In a technique known as Southern blotting, an absorbent membrane is then placed on the gel, transferring the ordered DNA fragments. Finally, a radioactive DNA probe is applied to the membrane, binding to any complementary DNA sequences and thereby labeling them.

Restriction enzymes can also be used to map genes. Restriction sites often vary by one or more nucleotides within a species. Genetic variation at a restriction site can produce a change in the length of a DNA sequence, also known as restriction fragment length polymorphism (RFLP). RFLP can then be measured, what is often called "DNA fingerprinting." Any genetic variation that is correlated with the RFLP is likely to be in the same part of the genetic material because correlations between distant genetic locations (loci) tend to break down over time as a result of recombination between chromosomes.

Recombinant DNA

Making recombinant DNA requires isolating the DNA that will be cut, donor DNA, and the DNA into which the donor DNA will be inserted, vector DNA. The combination of donor DNA and vector DNA is recombinant DNA.

A vector is a small piece of DNA that carries the donor DNA into host cells. A common vector is a plasmid, a small ring of bacterial DNA that replicates independently of the main chromosome. Plasmids are useful vectors because they easily enter bacterial cells. A plasmid vector must first be removed from the rest of the bacterial genome. The DNA is removed and centrifuged so that the smaller plasmids sink farther than the larger chromosomes. Alternatively, an alkaline pH degrades genomic DNA but not plasmids, and the genomic DNA can be precipitated out of the solution.

Donor DNA and vector DNA must be digested with a restriction enzyme so that they can be spliced together. Some sticky ends will anneal (bind) vector DNA to donor DNA. Although their initial bonding is temporary, adding DNA ligase creates stronger bonds between the joined ends to make a continuous molecule.

To improve the efficiency of this procedure, annealing between two vector molecules or two donor molecules can be prevented by adding complementary nucleotides to one strand of each molecule, for example, As to the donor molecules and Ts to the vector molecules. Furthermore, two different restriction enzymes may be used for donors and vectors, allowing greater flexibility in choosing splice sites.

Cloning

Plasmid vectors are introduced into bacteria by transformation. The plasmid replicates within each bacterium, and the bacteria divide many times. Consequently, a single DNA donor molecule can be amplified into billions of copies. This set of copies is referred to as a clone.

Most plasmids used to make recombinant DNA carry genes for drug resistance. This feature allows geneticists to apply an antibiotic to select bacteria that have been transformed by the recombinant plasmid. The antibiotics destroy bacteria that do not carry the plasmid. Plasmids also have particular restriction target sites that tailor them to the use of certain restriction enzymes. Vectors can have two different drug resistance genes, one of which contains the restriction site. If an insertion of donor DNA occurs, drug resistance is lost, and application of the drug will destroy all bacteria carrying the insert. In this way bacterial colonies carrying the desired gene can be identified and allowed to grow.

Lambda phage, a bacterial virus, can also be used as a cloning vector. Phage heads selectively package chromosomes about fifty kilobases in length. If a piece of phage DNA is removed and replaced by a donor DNA fragment of approximately the same size, the recombinant DNA can still be packaged. However, if the insertion is not successful, the phage head will not form and the DNA cannot be injected into bacteria. Successful transfection of donor DNA into a bacterial colony can be detected as a plaque indicative of infection.

There are several other types of vectors. Cosmids are hybrids of lambda phages and plasmids. Because they are larger than either phages or plasmids, they are capable of inserting larger DNA fragments. Expression vectors allow foreign genes produce proteins in a bacterium. Yeast artificial chromosomes (YAC) can carry very large inserts into yeast cells for replication. Bacterial artificial chromosomes (BAC) are capable of carrying large inserts into bacteria.

DNA Libraries

One goal of cloning genetic material is to develop a DNA library. A library is a collection of clones covering part or all of the genome of interest. It may consist of phage, bacteria, or yeast, depending on the vector used. A library may be genomic or cDNA. A cDNA library contains only coding DNA because it is synthesized from messenger RNA, which is the template from which proteins are made. A cDNA library is useful to researchers interested in genes that are expressed in particular tissues, from which they can obtain mRNA. Because the donor cDNA encodes protein, what is known as a cDNA expression library can also transcribe and translate genes into proteins.

Once a library is created, it can be screened with a probe to find a gene of interest. A probe is a piece of single-stranded DNA or an antibody that is radioactively labeled. A DNA probe will bind to a complementary strand of DNA in the library, whereas an antibody probe will bind to a protein. cDNA probes can be synthesized as an oligonucleotide, a short piece of synthetic DNA, from the amino acid sequence of the protein of interest. Because of the redundancy of the genetic code, many different cDNA's can code for the same protein. Therefore, a cocktail is often made of many different cDNA oligonucleotides.

Positional cloning is a process of using information about the location of a gene so as to clone it more efficiently. Clones can be ordered by their positions along a chromosome by doing a chromosome walk. A chromo-some walk uses the ends of each clone to probe the rest of the library for overlapping clones. If another gene is closely linked to the gene of interest and its position is known, only the clones near the first gene need be probed to find the gene of interest.

Tagging is a process in which a known DNA sequence (tag) is allowed to insert at random places in a genome. If by chance an insertion occurs in the gene of interest, it will likely cause a mutation that limits the functioning of the gene's product. Mutant lines are selected and used to construct a genomic library, which is then tested with the tag as a probe to identify the gene of interest.

DNA Sequencing

Once a gene is cloned, its DNA sequence can be determined. First, the gene of interest is copied with a polymerase enzyme, a primer to direct the polymerase to the gene, and nucleotides. The nucleotides are not all the same, however. Some are missing an oxygen atom, which prevents them from adding another nucleotide to the end. Thus, the sequencing reaction results in an array of partial copies of the gene. If the reaction is performed with some deoxygenated thymine, then all of the copies will end in thymine. A different sequencing reaction can be performed for each deoxygenated base to generate four different sets of partial copies. The next step is to separate the partial copies according to size. Larger fragments travel more slowly through a gel, and mobility through an acrylamide gel is extremely sensitive to size. If one were to run two sets of fragments differing by just one base in size, the difference would be evident as two distinct bands on an acrylamide gel. Thus the products of a sequencing reaction will show up as an array of bands on an acrylamide gel, with each band representing the position of the deoxygenated nucleotide used in the sequencing reaction. Alternatively, just one sequencing reaction may be done with some of each nucleotide deoxygenated if each is labeled with a unique fluorescent dye, resulting in color-coded bands.

If the precise location of a gene is not known, sequencing can reveal its location. A functional gene usually appears as an open reading frame (ORF), which is a sequence of DNA uninterrupted by a stop signal. A sequence can be analyzed by a computer program, which examines all three possible reading frames on each strand. Unusually long ORFs indicate the presence of a gene, because otherwise stop codons are expected to appear at random.

If a gene has been cloned and sequenced, it can be copied from any individual in a population by polymerase chain reaction (PCR). Primers, short oligonucleotides that attach to complementary DNA and act as targets for the polymerase enzyme, are designed from the gene sequence. The strands of DNA are separated at a high temperature. The temperature is lowered again, one primer attaches to each strand of DNA, and the polymerase extends each one in opposite directions. This process is repeated many times by a machine, providing billions of copies of the gene in a few hours. PCR is useful for quickly amplifying DNA from many individuals in a population.

Gene Alteration and Expression

All of the above recombinant techniques allow geneticists to identify and describe genes. Once a gene has been sufficiently characterized, it can be manipulated to produce a desired outcome. In vitro mutagenesis can change a single target nucleotide to another nucleotide. First the gene is cloned into a single-stranded phage vector. An oligonucleotide primer is constructed from the gene's DNA sequence. The primer is complementary to the gene with the exception of the target site, which is changed to the desired nucleotide. (Although not completely complementary to the template DNA, hybridization occurs under certain conditions.) The phage is allowed to replicate so that its copy incorporates the new nucleotide. Continued copying will produce mostly mutant genes, which can be identified with the mutant primer as a probe under conditions that prevent hybridization with nonmutants.

Synthetic oligonucleotides can also be used to construct entire genes up to sixty bases in length. The automated reaction adds bases one at a time to the growing oligonucleotide, which is embedded in a resin. Overlapping oligonucleotides can be pieced together to synthesize longer sequences. Herbert Boyer's lab synthesized the gene for somatostatin, a human growth hormone, in this manner. The scientists added a restriction site to each end of the gene and a methionine codon to one end. Restriction enzymes were used to insert the gene into a bacterial plasmid that carried the same restriction sites. The insertion occurred in the middle of a bacterial beta-galactosidase gene, which the bacteria transcribed and translated. The resulting protein was a chimera (of genetically diverse tissue) of somatostatin and beta-galactosidase, separated by a methionine residue. Cyanogen bromide was used to cleave the protein at the methionine residue and recover the somatostatin.

There are other ways to produce eukaryotic (non-bacterial) gene products in bacteria. For example, phage T7 contains promoters that generate a large amount of protein during a late stage of infection. First, a gene is inserted next to a T7 promoter. The promoter interacts with T7 RNA polymerase, which is synthesized in the presence of lactose. Therefore, the addition of lactose to bacteria carrying the recombinant T7 will produce large quantities of the gene product. Insulin, somatostatin, and many pharmaceutical drugs are now produced with engineered bacteria and fungi.

Genetic engineering is not restricted to placing eukaryotic genes in bacteria. Eukaryotic cells can be altered by inserting foreign DNA into their genomes. An organism derived from such a cell is referred to as transgenic. There are several ways to produce transgenic organisms. For example, the gene of interest (the transgene) can be paired with a eukaryotic promoter in a vector and injected into the nucleus of the organism's gamete. The promoter will cause the gene to be expressed whenever the promoter's natural gene is expressed. This may be useful in determining the expression pattern of the natural gene if the product is not as easily detectable as that of the transgene. Or, the transgene's product alone may be valuable. For example, crops can be endowed with genes for pesticides or metabolizing nitrogen. Unfortunately, the ecological consequences of introducing transgenic organisms into nature are unknown.

Gene Therapy and Screening

Gene therapy is the insertion of a normal gene into a chromosome carrying a defective copy of the gene. The first case of gene therapy in a mammal was the cure of a growth hormone deficiency in mice. A gene was inserted into the ova of mice carrying two defective promoters for the gene. The gene was paired with a promoter-regulator for the metallothionein gene, which activates in the presence of heavy metals. Offspring carrying the transgene showed higher growth rates in the presence of heavy metals than did those without the transgene, indicating that a functional metal-lothionein promoter had taken control of the growth hormone gene.

Human gene therapy can be either germinal or somatic. Germinal gene therapy introduces transgenes into both somatic cells and germ line cells of the early embryo. This therapy has been performed on mice but not humans. Somatic gene therapy inserts transgenes only into affected tissues using a viral vector. This form of therapy has proven successful in treating severe combined immunodeficiency disease and atherosclerosis. Because most transgene vectors insert randomly throughout the genome, one potential side effect of gene therapy is a mutation caused by insertion into a healthy gene.

Although gene therapy for humans remains highly experimental, genetic screening for diseases is already being applied. Embryonic cells are collected from the amniotic fluid or the placenta. Some genetic abnormalities can be detected by their effects on restriction sites. A probe constructed from the cDNA of the gene of interest is used to identify fragments of DNA carrying the gene. These fragments are digested by a restriction enzyme whose target site contains the deleterious mutation. Mutant genes will not be cleaved by the restriction enzyme, whereas normal genes will. This can be detected as two different banding patterns on an electrophoretic gel. A related technique takes advantage of the often tight linkage between mutant genes and restriction fragment length polymorphisms (RFLP), which can serve as indicators for the presence of mutant genes.

PCR can also be used to amplify DNA for sequencing or another form of testing.

see also Genes; Genetically Engineered Foods; Genetics.

Brian R. West

Bibliography

Griffiths, Anthony J. F., et al. An Introduction to Genetic Analysis, 6th ed. New York: W. H. Freeman, 1996. Chapters 14 and 15.

Johnson, George B. Biology: Visualizing Life. New York: Holt, Rinehart and Winston, Inc., 1998.