Genetic engineering

Genetic engineering is the manipulation of the hereditary material of organisms at the molecular level. The hereditary material of most cells is found in the chromosomes, and it is made of deoxyribose nucleic acid (DNA). The total DNA of an organism is referred to as its genome. In the 1950s, scientists first discovered how the structure of DNA molecules worked and how they stored and transmitted genetic information.

Genetic engineering relies on recombinant DNA technology to manipulate genes. Methods are now available for rapidly sequencing the nucleotides of pieces of DNA, as well as for identifying particular genes of interest, and for isolating individual genes from complex genomes. This allows genetic engineers to alter genetic materials to produce new substances or create new functions.

The biochemical tools used by genetic engineers or molecular biologists include a series of enzymes that can "cut and paste" genes. Enzymes are used to cut a piece of DNA, insert into it a new piece of DNA from another organism, and then seal the joint.

One important group of these are restriction enzymes, of which well over 500 are known. Most restriction enzymes are endonucleases—enzymes that break the double helix of DNA within the molecule, rather than attacking the ends of the helix. Every restriction enzyme is given a specific name to identify it uniquely. The first three letters, in italics, indicate the biological source of the enzyme, the first letter being the initial of the genus, the second and third letters being the first two letters of the species name. Thus restriction enzymes from Escherichia coli are called Eco, those from Haemophilus influenzae are Hin, from Diplococcus pneumoniae comes Dpn, and so on.

The genetic engineer can use a restriction enzyme to locate and cut almost any sequence of bases. Cuts can be made anywhere along the DNA, dividing it into many small fragments or a few longer ones. The results are repeatable: cuts made by the same enzyme on a given sort of DNA will always be the same. Some enzymes recognize sequences as long as six or seven bases; these are used for opening a circular strand of DNA at just one point. Other enzymes have a smaller recognition site, three or four bases long; these produce small fragments that can then be used to determine the sequence of bases along the DNA.

The cut that each enzyme makes varies from enzyme to enzyme. Some, like Hin dII, make a clean cut straight across the double helix, leaving DNA fragments with ends that are flush. Other enzymes (Eco RI) make a staggered break, leaving single strands with protruding cohesive ends ("sticky ends") that are complementary in base sequence. Following breakage, and under the right conditions, the complementary bases from different sources can be rejoined to form recombinant DNA.

Another important biochemical tool used by genetic engineers is DNA polymerase, an enzyme that normally catalyses the growth of a nucleic acid chain. DNA polymerase is used by genetic engineers to seal the gaps between the two sets of fragments in newly joined chimera molecules of recombinant DNA. DNA polymerase is also used to label DNA fragments, for DNA polymerase labels practically every base, allowing minute quantities of DNA to be studied in detail. If a piece of ribonucleic acid (RNA) of the target gene is the starting point, then the enzyme reverse transcriptase is used to produce a strand of complementary DNA (cDNA).

Genetic engineers usually need large numbers of genetically identical copies of the DNA fragment of interest. One way of doing this is to insert the gene into a suitable gene carrier, called a cloning vector. Common cloning vectors are bacterial plasmids or viruses such as the bacteriophage lambda, which are small circles of DNA found in bacterial cells independently of the main DNA molecule. When the cloning vectors divide, they replicate both themselves and the foreign DNA segment linked to it.

In the plasmid insertion method, restriction enzymes are used to cleave the plasmid double helix so that a stretch of DNA (previously cleaved with the same enzyme) can be inserted into the plasmid. As a result, the "sticky ends" of the plasmid DNA and the foreign DNA are complementary and base-pair when mixed together. The fragments held together by base pairing are permanently joined by DNA ligase. The host bacterium, with its 20- to 30-minute reproductive cycle, is like a manufacturing plant. With repeated doublings of its offspring on a controlled culture medium, millions of clones of the purified DNA fragments can be produced overnight.

Similarly, if viruses (bacteriophages) are used as cloning vectors, the gene of interest is inserted into the phage DNA, and the virus is allowed to enter the host bacterial cell where it multiplies. A single parental lambda phage particle containing recombinant DNA can multiply to several hundred progeny particles inside the bacterial cell (E. coli ) within roughly 20 minutes.

Cosmids are another type of viral cloning vehicle that attaches foreign DNA to the packaging sites of a virus and thus introduces the foreign DNA into an infective viral particle. Cosmids allow researchers to insert very long stretches of DNA into host cells where cell multiplication amplifies the amount of DNA available. Large artificial chromosomes of yeast (called megaYACs) are also used as cloning vehicles, since they can store even larger pieces of DNA, 35 times more than can be stored conventionally in bacteria.

The polymerase chain reaction (PCR) technique is an important new development in the field of genetic engineering, since it allows the mass production of short segments of DNA directly, and offers the advantage of bypassing the several steps involved in using bacterial and viruses as cloning vectors.

DNA fragments can be introduced into mammalian cells, but a different method must be used. Here, genes packed in solid calcium phosphate are placed next to a cell membrane that surrounds the fragment and transfers it to the cytoplasm. The gene is delivered to the nucleus during mitosis (when the nuclear membrane has disappeared) and the DNA fragments are incorporated into daughter nuclei, then into daughter cells. A mouse containing human cancer genes (the onchomouse) was patented in 1988.

The potential benefits of recombinant DNA research are enormous. In recent years, scientists have identified the genetic basis of a number of medical disorders. Genetic engineering helps scientists replace a particular missing or defective gene with correct copies of that gene. If that gene then begins functioning in an individual, a genetic disorder may be cured. Researchers have already met with great success in finding the single genes that are responsible for common diseases like cystic fibrosis and hemochromatosis. In the early twenty-first century, scientists were applying knowledge from the human genome project to try to map multiple genes responsible for diseases like diabetes, hypertension, and schizophrenia.

Genetic engineering has also led to a great deal of controversy, however. Since scientists can duplicate genes, they can also duplicate, or "clone" animals and humans. In December 2001, scientists announced the birth of the first cloned female cat, although the 1990s saw big headlines for the first big mammal cloning of sheep. The announcement was made in April 2002 that the first human baby produced by a human cloning program would be born in November, sparking considerable medical and ethical controversy. In August 2001, President George W. Bush struggled with the debate over allowing federal funding for embryonic stem cell research. He allowed funding for only existing lines of cells, leaving further research in the hands of those who could seek private funding.

Genetic engineering also presents positive and negative controversy in its application to agriculture and the environment . Recombinant DNA techniques help scientists produce plants that offer medicinal value too. For example, calcium-fortified orange juice or vitamin-enriched milk boost the nutritional value of these foods. However, scientists can now further genetically modify foods, offering benefits and risks to the environment. The benefits mean possible cheaper and easier production of certain medicines, while the risks include unnatural introduction of plants and animals produced in a laboratory environment. At the University of Arizona, scientists have modified tomatoes to produce vaccines for diarrhea and hepatitis B, and that these vaccines will likely be much cheaper to produce than current drugs. Some critics worry that once the new crops move out of the safety of locked greenhouses and into crop fields, them may cross-pollinate with conventional tomato crops and contaminate them with modified genes.

A poll reported on in 2002 showed that Americans were fairly evenly split on their feelings about the risks and benefits of genetically modified foods and biotechnology . Most like the idea that scientists can create plants that will help clean up toxic soils, reduce soil erosion and reduce fertilizer run-off into streams and lakes. They also favor production of genetically engineered methods to reduce the amount of water used to grow crops and development of disease-resistant tree varieties to replace those that might be threatened or endangered. Americans also favor use of genetic engineering to reduce the need to log in native forests and to reduce the amount of chemical pesticides used by farmers. On the other hand, Americans express concern over possible environmental effects of genetically modified plants or fish contaminating ordinary plants and fish, reducing genetic diversity, increasing the number of insects that might become resistant to pesticides, or changing the ecosystem .

See also Gene bank; Gene pool

[Neil Cumberlidge Ph.D. ]

RESOURCES

BOOKS

Cherfas, J. Man Made Life: An Overview of the Science and Technology and Commerce of Genetic Engineering. New York: Pantheon Books, 1982.

Watson, J. D, J. Tooze, and D. T. Kurtz. Recombinant DNA: A Short Course. San Francisco: W. H. Freeman, 1983.

Wheale, P. R., and R. M. McNally. Genetic Engineering: Catastrophe or Utopia? New York: St. Martin's Press, 1988.

PERIODICALS

"Americans Evenly Divided Over Environmental Risks, Benefits of Genetically Modified Food and Biotech." Health and Medicine Week, March 4, 2002, 18.

Coghlan, A. "Engineering the Therapies of Tomorrow." New Scientist 137 (April 24, 1993): 26–31.

Kahn, P. "Genome on the Production Line." New Scientist 137 (April 24, 1993): 32–36.

Miller, S. K. "To Catch a Killer Gene." New Scientist 137 (April 24, 1993): 37–40.

Primedia Intertec, Inc. "Cloning Controversy."Better Nutrition 64 (July 2002): 18–21.

Verma, I. M. "Gene Therapy." Scientific American 263 (November 1990): 68–84.

OTHER

"Genetic Engineers Blurring Lines Between Kitchen Pantry and Medicine Cabinet." Pew Initiative on Food and Biotechnology Newsroom. [cited July 9, 2002]. <http://pewagbiotech.org/newsroom/summaries/display.php3?NewsID=170>.

ORGANIZATIONS

Pew Initiative on Food and Biotechnology, 1331 H Street, Suite 900, Washington, DC USA 20005 (202) 347-9044, Fax: (202) 347-9047, Email: [email protected], <http://pewagbiotech.org