CONCEPTS IN BIOLOGY

PART III. MOLECULAR BIOLOGY, CELL DIVISION, AND GENETICS

 

11. Applications of Biotechnology

 

11.3. The Genetic Modification of Organisms

 

For thousands of years, civilizations have attempted to improve the quality of their livestock and crops. Cows that produce more milk or more tender meat were valued over those that produced little milk or had tough meat. Initial attempts to develop improved agricultural stocks were limited to selective breeding programs, in which only the organisms with the desired characteristics were allowed to breed. As scientists asked more sophisticated questions about genetic systems, they developed ways to create and study mutations.

Although this approach was a very informative way to learn about the genetics of an organism, it lacked the ability to create a specific desired change. Creating mutations is a very haphazard process. However, today the results are achieved in a much more directed manner using biotechnology’s ability to transfer DNA from one organism to another. Transformation takes place when a cell gains new genetic information from its environment. Once new DNA sequences are transferred into a host cell, the cell is genetically altered and begins to read the new DNA and produce new cell products, such as enzymes. The resulting new form of DNA is called recombinant DNA.

A clone is an exact copy of biological entities, such as genes, organisms, or cells. The term refers to the outcome, not the way the results are achieved. Many whole organisms “clone” themselves simply by how they reproduce; bacteria divide by cell division and produce two genetically identical cells. Strawberry plants clone themselves by sending out runners and establishing new plants. Many varieties of fruit trees and other plants are cloned by making cuttings of the plant and rooting the cuttings. With the development of advanced biotechnology techniques, it is now possible to clone specific genes from an organism. It is possible to put that cloned gene into the cell of an entirely different species.

 

Genetically Modified Organisms

Genetically modified (GM) organisms contain recombinant DNA. Viruses, bacteria, fungi, plants, and animals are examples of organisms that have been engineered so that they contain genes from at least one unrelated organism.

As this highly sophisticated procedure has been refined, it has become possible to splice genes quickly and accurately from a variety of species into host bacteria or other host cells by a process called gene cloning (How Science Works 11.4). Genetically modified organisms are capable of expressing the protein-coding regions found on recombinant DNA. Thus, the organisms with the recombinant DNA can make products they were previously unable to make. Since they can rapidly reproduce to large numbers, industrial-sized cultures of bacteria can synthesize large quantities of proteins. For example, recombinant DNA procedures are responsible for the production of:

• Human insulin, used in the control of diabetes (figure 11.6)

• Nutritionally enriched “golden rice,” capable of supplying poor people in less developed nations with beta-carotene, which is missing from normal rice

• Interferon, used as an antiviral agent

• Human growth hormone, used to stimulate growth in children lacking this hormone

• Somatostatin, a brain hormone implicated in growth.

 

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The primary application of GM technology is to put herbicide-resistance or pest-resistance genes into crop plants. Edible GM crops are used mainly for animal feed. In agricultural practice, two kinds of genetically modified organisms have received particular attention. One involves the insertion of genes from a specific kind of bacterium called Bacillus thuringiensis israeliensis (Bti). Bti produces a protein that causes the destruction of the lining of the gut of insects that eat it. It is a natural insecticide. To date, the gene has been inserted into the genetic makeup of several crop plants, including corn. In field tests, the genetically engineered corn was protected against some of its insect pests, but there was some concern that pollen grains from the corn might be blown to neighboring areas and affect nontarget insect populations. In particular, a study of monarch butterflies indicated that populations of butterflies adjacent to fields of this genetically engineered corn were negatively affected. One could argue that since the use of Bti corn results in less spraying of insecticides in cornfields, this is just a trade-off.

 

 

FIGURE 11.6. Human Insulin from Bacteria

The gene-cloning process is used to place a copy of the human insulin gene into a bacterial cell. As the bacterial cell reproduces, the human DNA it contains is replicated along with the bacterial DNA. The insulin gene is expressed along with the bacterial genes and the colony of bacteria produces insulin. This bacteria-produced human insulin is both more effective and cheaper than previous therapies, which involved obtaining insulin from the pancreas of slaughtered animals.

 

A second kind of genetically engineered plant involves inserting a gene for herbicide resistance into the genome of certain crop plants (figure 11.7a). The value of this to farmers is significant. For example, a farmer could plant cotton with very little preparation of the field to rid it of weeds. When both the cotton and the weeds begin to grow, the field could be sprayed with a specific herbicide that would kill the weeds but not harm the herbicide-resistant cotton. This has been field-tested and it works. Critics have warned that the genes possibly could escape from the crop plants and become part of the genome of the weeds that we are trying to control, thus creating “super-weeds.”

Many more products have been manufactured using these methods. Genetically modified cells are not only used as factories to produce chemicals but also for their ability to break down many toxic chemicals. Bioremediation is the use of living organisms to remove toxic agents from the environment. There has been great success in using genetically modified bacteria to clean up oil spills and toxic waste dumps.

 

 

FIGURE 11.7. Application of Genetically Modified Organisms

Soybeans, corn, cotton, Hawaiian papaya, tomatoes, rapeseed, sugarcane, sugar beets, sweet corn, and rice are a short list of GM crops being grown and sold. (a) One of the most important applications of this technology involves the insertion of genes that make a crop plant resistant to herbicides. Therefore, the field can be sprayed with an herbicide and kill the weeds without harming the crop plant. (b) Normal rice does not produce significant amounts of beta-carotene. Beta-carotene is a yellow-orange compound needed in the diet to produce vitamin A. (c) Genetically modified “golden rice” can provide beta-carotene to populations that have no other sources of this nutrient.

 

HOW SCIENCE WORKS 11.4

Cloning Genes

Cloning a specific gene begins with cutting the source DNA into smaller, manageable pieces with restriction enzymes. Next, there are several basic steps that occur in the transfer of DNA from one organism to another:

1. The source DNA is cut into a usable size by using restriction enzymes.

The source DNA is usually isolated from a large number of cells. Therefore, It consists of many copies of an organism's genome. The source DNA is cut into many small fragments with restriction enzymes. Isolating the small portion of DNA that contains the gene of interest can be difficult because the gene of interest is found on only a few of these fragments. To identify the desired fragments, scientists must search the entire collection. The search involves several steps.

2. The DNA fragments are attached to a carrier DNA molecule.

The first step is to attach every fragment of source DNA to a carrier DNA molecule. A vector is the term scientists use to describe a carrier DNA molecule. Vectors usually contain special DNA sequences that facilitate attachment to the fragments of source DNA. Vectors also contain sequences that promote DNA replication and gene expression.

A plasmid is one example of a vector that is used to carry DNA into bacterial cells. A plasmid is a circular piece of DNA that is found free in the cytoplasm of some bacteria. Therefore, the plasmid must be cut with a restriction enzyme, so that the plasmid DNA will have sticky ends, which can attach to the source DNA. The enzyme ligase creates the covalent bonds between the plasmid DNA and the source DNA, so that a new plasmid ring is formed with the source DNA inserted into the ring. The plasmid and its inserted source DNA is recombinant DNA. Because there are many different source DNA fragments, this process results in many different plasmids, each with a different piece of source DNA. All of these recombinant DNA plasmids constitute a DNA library for the entire source genome.

3. The carrier DNA molecule, with its attached source DNA, is moved into an appropriate cell for the carrier DNA. In the cell, the new DNA is replicated or expressed.

 

Cutting Genomic DNA

The first step in cloning a specific gene is to cut the source DNA into smaller, manageable pieces with restriction enzymes.

 

 

Creating Recombinant DNA

The source DNA is cut with restriction enzymes to create sticky ends. The vector DNA (orange) has compatible sticky ends, because it was cut with the same restriction enzyme. The enzyme ligase is used to bond the source DNA to the vector DNA.

The second step in the cloning process is to mix the DNA library with bacterial cells that will take up the DNA molecules. Transformation occurs when a cell gains genetic information from its environment. Each transformed bacterial cell carries a different portion of the source DNA from the DNA library. These cells can be grown and isolated from one another.

 

 

Transformation

Bacterial cells pick up the plasmids with recombinant DNA and are transformed. Different cells pick up plasmids with different genomic DNA inserts.

The third step is to screen the DNA library contained within the many different transformed bacterial cells to find those that contain the DNA fragment of interest. Once the bacterial cells with the desired recombinant DNA are identified, the selected cells can be reproduced and, in the process, the desired DNA is cloned.

 

 

Screening the DNA Library

A number of techniques are used to eliminate cells that do not carry plasmids with attached source DNA. Once these cells are eliminated from consideration, the remaining cells are screened to find those that contain the genes of interest.

 

Genetically Modified Foods

Although some chemicals have been produced in small amounts from genetically engineered microorganisms, crops such as turnips, rice, soybeans, potatoes, cotton, corn, and tobacco can generate tens or hundreds of kilograms of specialty chemicals per year. Such crops have the potential of supplying the essential amino acids, fatty acids, and other nutrients now lacking in the diets of people in underdeveloped and developing nations. Researchers have also shown, for example, that turnips can produce interferon (an antiviral agent), tobacco can create antibodies to fight human disease, oilseed rape plants can serve as a source of human brain hormones, and potatoes can synthesize human serum albumin that is indistinguishable from the genuine human blood protein (figure 11.7b and c).

Many GM crops also have increased nutritional value yet can be cultivated using traditional methods. There are many concerns regarding the development, growth, and use of GM foods. Although genetically modified foods are made of the same building blocks as any other type of food, the public is generally wary. Countries have refused entire shipments of GM foods that were targeted for hunger relief. However, we may eventually come to a point where we can no longer choose to avoid GM foods. As the world human population continues to grow, GM foods may be an important part of meeting the human population’s need for food. The following are some of the questions being raised about genetically modified food:

• Is tampering with the genetic information of an organism ethical?

• Is someone or an agency monitoring these crops to determine if they are moving beyond their controlled ranges?

• What safety precautions should be exercised to avoid damaging the ecosystems in which GM crops are grown?

• What type of approval should these products require before they are sold to the public?

• Is it necessary to label these foods as genetically modified?

 

Gene Therapy

The field of biotechnology allows scientists and medical doctors to work together and potentially cure genetic disorders. Unlike contagious diseases, genetic diseases cannot be transmitted, because they are caused by a genetic predisposition for a particular disorder—not separate, disease-causing organisms, such as bacteria and viruses. Gene therapy involves inserting genes, deleting genes, and manipulating the action of genes in order to cure or lessen the effect of genetic diseases. These therapies are very new and experimental. While these lines of investigation create hope, many problems must be addressed before gene therapy becomes a reliable treatment for many disorders.

The strategy for treating someone with gene therapy varies, depending on the disorder. When designing a gene therapy treatment, scientists have to ask exactly what the problem is. Is the mutant gene not working at all? Is it working normally but there is too little activity? Is there too much protein being made? Or is the gene acting in a unique, new manner? If there is no gene activity or too little gene activity, the scientists need to introduce a more active version of the gene. If there is too much activity or if the gene is engaging in a new activity, this excess activity must first be stopped and then the normal activity restored.

To stop a mutant gene from working, scientists must change it. This typically involves inserting a mutation into the protein-coding region of the gene or the region that is necessary to activate the gene. Scientists have used some types of viruses to do this in organisms other than humans. The difficulty in this technique is to mutate only that one gene without disturbing the other genes and creating more mutations in other genes. Developing reliable methods to accomplish this is a major focus of gene therapy. Once the mutant gene is silenced, the scientists begin the work of introducing a “good” copy of the gene. Again, there are many difficulties in this process:

• Scientists must find a way of returning the corrected DNA to the cell.

• The corrected DNA must be made a part of the cell’s DNA, so that it is passed on with each cell division, it doesn’t interfere with other genes, and it can be transcribed by the cell as needed (figure 11.8).

• Cells containing the corrected DNA must be reintroduced to the patient.

 

 

FIGURE 11.8. Gene Therapy

One method of introducing the correct genetic information to a cell is to use a virus as a vector. Here, a dog is treated for a degenerative disorder of the retina. The normal gene is spliced into the viral genome. The virus is then used to infect the defective retinal cells. When the virus infects the retinal cells, it carries the functional gene into the cell.

 

The Cloning of Organisms

Cloning does not always refer to exchanging just a gene. Another type of cloning is the cloning of an entire organism. In this case, the goal is to create a new organism that is genetically identical to the previous organism. Cloning of multicellular organisms, such as Protists, plants, fungi and many kinds of invertebrate animals, often occur naturally during asexual reproduction and is duplicated easily in laboratories. The technique used to accomplish cloning in vertebrates is called somatic cell nuclear transfer. Somatic cell nuclear transfer removes a nucleus from a cell of the organism that will be cloned. After chemical treatment, that nucleus is placed into an egg cell that has had it original nucleus removed. The egg cell will use the new nucleus as genetic information. In successful cloning experiments with mammals, an electrical shock is used to stimulate the egg to begin to divide as if it were a normal embryo. After transferring the egg with its new nucleus into a uterus, the embryo grows normally. The resulting organism is genetically identical to the organism that donated the nucleus.

In 1996, a team of scientists from Scotland successfully carried out somatic cell nuclear transfer for the first time in sheep. The nucleus was taken from the mammary cell of an adult sheep. The embryo was transplanted into a female sheep’s uterus, where it developed normally and was born (figure 11.9). This cloned offspring was named Dolly. This technique has been applied to many other animals, such as monkeys, goats, pigs, cows, mice, mules, and horses, and has been used successfully on humans. However, for ethical reasons, the human embryo was purposely created with a mutation that prevented the embryo from developing fully. The success rate of cloning animals is still very low for any animal, however; only 3-5% of the transplanted eggs develop into adults (figure 11.10).

 

 

FIGURE 11.9. Cloning an Organism

The nucleus from the donor sheep is combined with an egg from another sheep. The egg’s nucleus had previously been removed. The egg, with its new nucleus, is stimulated to grow by an electrical shock. After several cell divisions, the embryo is artificially implanted in the uterus of a sheep, which will carry the developing embryo to term.

 

 

FIGURE 11.10. Success Rate in Cloning Cats

Out of 87 implanted cloned embryos, CC (Copy Cat) is the only one to survive. This is comparable to the success rate in sheep, mice, cows, goats, and pigs. (a) Notice that CC is completely unlike her tabby surrogate mother. (b) “Rainbow” is her genetic donor, and both are female calico domestic shorthair cats.

 

A cloning experiment has great scientific importance, because it represents an advance in scientists’ understanding of the processes of determination and differentiation. Recall that determination is the process a cell goes through to select which genes it will express. A differentiated cell has become a particular cell type because of the proteins that it expresses. Differentiation is more or less a permanent condition. The techniques that produced Dolly and other cloned animals use a differentiated cell and reverse the determination process, so that this cell is able to express all the genes necessary to create an entirely new organism. Until this point, scientists were not sure that this was possible.

 

11.3. CONCEPT REVIEW

9. A scientist can clone a gene. An organism can be a clone. How is the use of the word clone different in these instances? How is the use of the word clone the same in both uses?

10. What are some of the advantages of creating genetically modified (GM) foods? What are some of the concerns?

11. Describe how viruses are used in gene therapy.